Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

Domain: physics
Published: 2025-12-10
Editorial outcome: major_revision

Abstract
Toll-like receptor 3 (TLR3), an innate immune sensor for double-stranded RNA (dsRNA), traffics from the endoplasmic reticulum (ER) after synthesis to endolysosomes for proteolytic cleavage and activation. However, the molecular mechanisms governing TLR3 trafficking remain largely unclear. Here, we identify the ER-resident E3 ligase HMG-CoA reductase degradation protein 1 (HRD1), a core component of ER-associated degradation (ERAD), as a key regulator that promotes TLR3 trafficking and downstream signaling. HRD1 deficiency in macrophages significantly impairs poly(I:C)-induced TLR3 signaling and inflammatory responses in vitro and in vivo, caused by a marked reduction in TLR3 transport into endolysosomes and subsequent proteolytic processing. Mechanistically, HRD1 mediates ubiquitination of ER-localized TLR3 at lysine 813, which is required for its recognition and sorting by the endosomal sorting complex required for transport (ESCRT) machinery. This HRD1 function is decoupled from its canonical ERAD activity and the ER stress sensor inositol-requiring enzyme 1 alpha (IRE1α). Hence, our study identifies a previously unrecognized mechanism controlling TLR3 signaling and links HRD1-mediated ubiquitination to immune sensor trafficking during innate immune responses.

Introduction
Toll-like receptors (TLRs) are an evolutionarily ancient family of proteins operating as germline-encoded pattern recognition receptors (PRRs) to sense structurally conserved microbial components termed pathogen-associated molecular patterns (PAMPs)1. TLR3 detects dsRNA from genomes of dsRNA viruses or intermediates formed during replication of single-stranded RNA (ssRNA) viruses, to induce the transcription of type I interferons (IFNs) and proinflammatory cytokines and initiate antiviral immune response2. The full-length TLR3 is synthesized in the endoplasmic reticulum (ER) and transported through the Golgi apparatus to endosomes, where it undergoes cleavage by cathepsins to generate the activable form3–5. This strategically restricted activation of TLR3 in endolysosomes allow the receptor to mediate immune response against the internalized viral dsRNA but sequestering it away from self-nucleic acids6. Despite the vital role of TLR3 trafficking in initiating immune response, the molecular mechanisms governing this process remain largely unclear.

Proteins targeted to expression on membranes, including many immune sensors, are initially synthesized and folded in the ER7. The regulation of synthesis, modification and stability of membrane immune receptors within the ER can significantly impact the immune response. For example, folding and function of TLRs are dependent on the activity of ER-resident chaperones gp96 and Unc-93 homolog B1 (UNC93B1)8,9. Stimulator of interferon genes (STING) is an ER adaptor protein that mounts the body’s inflammatory response against cytosolic double-stranded DNA10, Toll interacting protein (TOLLIP), stromal interaction molecule 1 (STIM1) and STING ER exit protein (STEEP) control the stability, retention and exit of ER-resident STING to regulate its activation and signaling11–14. Thus, ER emerges as a central hub of immune regulation by monitoring the synthesis, maturation, degradation and trafficking of immune receptors. While TLR3 is synthesized in its precursor form and primarily resident in the ER under basal stage3,4, whether any ER factors can regulate TLR3 protein modification and thus its trafficking remains unknown.

Proteins synthesis in the ER is coupled with stringent quality control to ensure that only correctly folded proteins exit the ER for further maturation and trafficking. ERAD is a fundamental quality control mechanism responsible for the recognition and removal of misfolded and aberrant proteins in the ER for cytosolic proteasomal degradation15–17. HRD1, an ancient and highly conserved ER membrane-embedded E3 ubiquitin ligase, forms a complex with adaptor protein SEL1L to nucleate the ERAD machinery17–19. Recent studies have collectively demonstrated the pivotal functions of HRD1 ERAD in critical physiological processes, such as food digestion, nutrient storage, glucose homeostasis, food intake, water balance and thermogenic regulation20–27. In immune cells, we and others have showed HRD1-mediated ERAD activity downregulates the protein level of ER-resident pre-B cell receptor complex (pre-BCR) and STING, negatively regulates pre-BCR- and STING-mediated signaling, and exerts critical roles in controlling early B cell development and innate immune response28–30. While TLR3 is mostly resident in the ER of resting cells, whether HRD1 modifies TLR3 and regulates its signaling has not thoroughly examined.

In this article, we report that ubiquitination of TLR3 by HRD1 within the ER is a prerequisite for its trafficking into endolysosomes and subsequent processing. Hrd1 deficiency or its inhibition reduces the levels of cleaved and activable TLR3, significantly dampening TLR3-mediated signaling and inflammatory response after ligand stimulation. Interestingly, HRD1 ubiquitinates TLR3 to promote its trafficking, decoupled from its degradation by ERAD. Hence, HRD1 moonlights as a positive regulator for TLR3 innate immune signaling by directly modifying the innate immune sensor.

Results
Identification of the HRD1-TLR3 interaction and its enhancement upon TLR3 stimulation
Our previous study has demonstrated that ER-resident E3 ligase HRD1 can ubiquitinate and negatively regulate the level of ER-resident STING protein, and Hrd1 deficiency in macrophages amplifies production of pro-inflammatory cytokines and type I interferon (IFN) upon STING stimulation30. To further elucidate the role of HRD1 in macrophage function, we performed immunoprecipitation on protein lysates from the wild-type (WT) RAW 264.7 macrophage cell line and Hrd1−/− cells, generated via the CRISPR/Cas9 system (Fig. S1a), using an anti-HRD1 antibody, followed by mass spectrometry (CoIP-MS) to identify proteome-wide endogenous HRD1 substrates. MS analyses of endogenous HRD1 interactome identified 812 hits specially in WT macrophage cells but not in Hrd1-deficient cells (Fig. 1a). Among the top specific hits were HRD1 itself and known components of the endogenous ERAD complex, including SEL1L and OS9 proteins (Fig. 1a). Intriguingly, the ER-synthesized innate immune sensor TLR3 was identified as a top-ranking hit (Fig. 1a). Given that TLR3 primarily resides in the ER membrane, we speculated whether TLR3 and its signaling is regulated by HRD1. Protein level of HRD1, but not the ER chaperone Binding immunoglobulin protein (BiP) or the unfolded protein response (UPR) sensors IRE1α and PKR-like endoplasmic reticulum kinase (PERK), was increased in primary peritoneal macrophage upon treatment with the dsRNA analog poly(I:C) (Fig. 1b), further suggesting a possible role of HRD1 in TLR3 signaling pathway.

We then confirmed the interaction of HRD1 with TLR3. In HEK293T cells, immunoprecipitation of transfected Flag-tagged TLR3 effectively pulled down transfected Myc-tagged HRD1, and treatment with poly(I:C) led to a higher yield of HRD1 pulldown (Fig. 1c). Under basal condition, immunoprecipitation of endogenous TLR3 or HRD1 effectively pulled down endogenous HRD1 or TLR3, respectively in macrophages (Figs. 1d and S1b). The physical interaction of endogenous TLR3 and HRD1 was increased shortly after poly(I:C) treatment (Figs. 1d and S1b). We then tested how HRD1 interacts with TLR3. HRD1 is a multi-pass transmembrane protein with a large cytosolic domain. In HEK293T cells, immunoprecipitation of truncated HRD1 proteins revealed that both the transmembrane and cytosolic RING domains were required for its interaction with TLR3 (Figs. 1e, f and S1c). The TLR3 protein is composed of a N-terminal leucine-rich repeat (LRR) ectodomain, a transmembrane (TM) domain, and a cytosolic Toll/interleukin-1 receptor (TIR) domain (Fig. 1g)31. Immunoprecipitation of truncated TLR3 proteins showed that TM domain of TLR3 was required for the effective interaction between TLR3 and HRD1 (Figs. 1h and S1d). Taken together, HRD1 interacts with TLR3, and both the protein level of HRD1 and their interactions increase upon dsRNA stimulation.

HRD1 positively regulates TLR3-mediated immune signaling
To directly test the role of HRD1 in the regulation of TLR3-mediated immune signaling, we compared the immune response of WT and Hrd1−/− cells to TLR3 agonist stimulation. While protein levels of TLR3 downstream effectors TBK1, IRF3 and P65 were comparable, these proteins, as well as IκBα, were hypo-phosphorylated in Hrd1−/− RAW 264.7 macrophages compared to WT cells upon treatment with poly(I:C) (Fig. 2a). We then tested whether HRD1’s regulation of TLR3 signaling is applicable to human cells or more physiologically relevant primary macrophages. The overexpression of HRD1 in TLR3-stable-expression HEK293T-TLR3 cells increased the phosphorylation of TBK1, P65, and IκBα upon stimulation in a dose-dependent manner, while HRD1 deficiency in HEK293T-TLR3 cells decreased their phosphorylation (Figs. 2b and S2a). Knocking down Hrd1 in primary macrophages using siRNAs (Fig. S2b, c) or treating primary macrophages with LS-102 (Fig. 2c)—a specific small-molecule HRD1 inhibitor that suppresses its E3 ubiquitin ligase catalytic activity32–34—significantly reduced the phosphorylation levels of TLR3 downstream effectors TBK1, IRF3, P65 and IκB in response to poly(I:C) treatment.

The stimulation of TLR3 consistently resulted in a notable reduction in the expression of various inflammatory cytokine genes (Ifnb1, Il1b, Tnfa, Il6, and Ccl5) in Hrd1−/− macrophages compared to WT cells, using poly(I:C) (Fig. 2d). As expected, Hrd1−/− macrophages showed a significant upregulation of the UPR gene Bip (Fig. 2d). However, the reduced inflammatory gene expression is not due to UPR activation, as shown later. Treatment with LS-102 significantly reduced the transcription of inflammatory genes (Fig. S2d) and the secretion of cytokines, including TNFα, IL6 and IFNβ (Fig. 2e), in poly(I:C)-stimulated primary macrophages. LS-102 treatment did not reduce poly(I:C)-induced transcription of inflammatory genes in Hrd1−/− macrophages, excluding an off-target effect (Fig. S2e). Consistently, Hrd1 knockdown in primary macrophages also led to reduced transcription of inflammatory genes and decreased secretion of TNFα, IL6 and IFNβ upon poly(I:C)-stimulation (Fig. S2f, g). Poly(I:C)-induced NF-κB promoter activity and interferon-stimulated response element (ISRE) activity was also reduced in Hrd1−/− HEK293T-TLR3 cells compared to WT cells (Fig. S2h). Therefore, the HRD1-TLR3 signaling regulatory axis represents a general mechanism across different cells. We next evaluated the potency of secretomes from poly(I:C)-stimulated WT and Hrd1−/− macrophages in inhibiting HSV-1 replication. Replication of the HSV-1 was significantly higher in Vero cells treated with secretome from Hrd1−/− cells when compared to the control group (Figs. 2f and S2i). Taken together, we conclude that HRD1 enhances TLR3-mediated innate immune signaling.

HRD1 and TLR3 signaling form a positive feedback loop between each other
HRD1 is a positive regulator of TLR3 signaling (Fig. 2a–f), and its protein levels and transcription are increased upon poly(I:C) stimulation (Figs. 1b, and 3a), indicating that TLR3 signaling upregulates HRD1 to form a positive feedback loop. Consistent with previous study showing HRD1 expression can be induced via extra-cellular signal-regulated kinase 1/2 (ERK1/2) activation and transcriptional activity of ETS135, poly(I:C) stimulation elevated ERK activation, as indicated by the hyper-phosphorylation of ERK1/2 (Fig. 3b), and increased the transcription of Ets1 and Hrd1 (Fig. 3c). Knocking down Ets1 in primary macrophages using siRNAs inhibited poly(I:C)-induced increase in Hrd1 transcription (Fig. S3a, b). Furthermore, both PD98059, an inhibitor of the ERK1/2 upstream mitogen-activated protein kinase kinase 1/2 (MEK1/2), and LS-102 inhibited the elevation of ERK phosphorylation and significantly reduced the transcription of Ets1 and Hrd1 following poly(I:C) stimulation (Fig. 3b, c). The suppressive effects of LS-102 on ERK phosphorylation and the transcription of Ets1 and Hrd1 were rescued by the addition of various TLR3 downstream cytokines (Fig. 3d, e, f).

Tlr3 transcription and protein levels were also increased in macrophage upon poly(I:C) stimulation (Fig. 3g, h), which was dependent on HRD1 and signal transducer and activator of transcription 1 (STAT1) activity, as both LS-102 and the STAT1 inhibitor Fludarabine suppressed poly(I:C)-induced Tlr3 upregulation (Fig. 3h–j). Previous studies showed that IFNβ and interferon regulatory factors (IRFs) promote TLR3 expression36. Adding back IFNβ directly induced STAT1 activation in the presence of LS-102 (Fig. 3k) and rescued the induction of Tlr3 transcription (Fig. 3l). Additionally, Irf1 and Irf2 induction was observed upon poly(I:C) stimulation (Fig. 3j, l), which was suppressed by LS-102 and Fludarabine (Fig. 3j, l) but rescued by IFNβ (Fig. 3l). Taken together, TLR3 signaling induces HRD1 expression through ERK1/2-ETS1 activity. In turn, HRD1 activity contributes to the increased transcription of Tlr3 in a STAT1-dependent manner, thereby amplifying the inflammatory response.

The effect of HRD1 on TLR3 signaling is uncoupled from ERAD and UPR
The classical function of HRD1 is to ubiquitinate misfolded or misfolding-prone substrate proteins and promote their proteasomal degradation17,18. For example, HRD1 deficiency leads to the accumulation and impaired function of pro-arginine vasopressin (proAVP) and pro-opiomelanocortin (POMC)23,24. Considering that TLR3 is synthesized and mostly resides in the ER of resting cells3,4, we then tested whether HRD1 promotes TLR3 signaling by regulating the abundance of TLR3 protein in the ER. Surprisingly, there was no difference in the abundance of the full-length TLR3 protein between WT and Hrd1−/− macrophages (Fig. 4a). Consistent with this, the translation shut-off assay using cycloheximide (CHX) revealed that the full-length TLR3 protein levels decreased by a comparable ratio within 9 h of treatment in both WT and Hrd1−/− macrophages (Fig. 4b). The overexpression of HRD1 in HEK293T-TLR3 cells did not change the protein levels of TLR3 at varying doses (Fig. 4c). These findings indicate that HRD1 does not directly regulate the degradation and abundance of the TLR3 protein in the ER. We also used Eeyarestatin I that targets Valosin-containing protein (VCP) to inhibit ERAD37,38, and ERAD inhibitor pretreatment induced expression of UPR genes, including Xbp1s and Bip, but did not reduce poly(I:C)-induced expression of pro-inflammatory genes in macrophages (Fig. 4d). Additionally, irrespective of Eeyarestatin I pretreatment, poly(I:C)-induced inflammatory gene expression remained consistently impaired in HRD1-deficient macrophages compared to WT macrophages (Fig. 4d).

ERAD and UPR, two key quality-control machineries, are closely interconnected to maintain ER homeostasis16, we next examined whether HRD1 effect on TLR3 is mediated by UPR. Hrd1−/− cells exhibited mild ER stress compared to WT cells under basal conditions, as indicated by Eif2α phosphorylation levels (Fig. 4e) and expression of UPR genes Bip, Xbp1s, and Chop (Fig. 4f). Treatment with thapsigargin (Tg) induced robust ER stress in both WT and Hrd1−/− cells (Fig. 4e, f). However, ER stress induction did not suppress poly(I:C)-induced TLR3 signaling in WT cells, and regardless of Tg treatment, poly(I:C)-induced TLR3 signaling and inflammatory gene expression remained consistently reduced in Hrd1−/− macrophages compared to WT macrophages (Fig. 4e, f). Taken together, compromised TLR3 signaling in Hrd1-deficient cells is not due to the inhibition of ERAD activity or the induction of ER stress.

IRE1α-XBP1s branch of the UPR is reported to potentiate the production of pro-inflammatory cytokines upon TLR2 and TLR4 stimulation in macrophage39 and IRE1α is significantly increased in Hrd1−/− macrophages (Fig. 4e)30, we next asked whether IRE1α mediates HRD1 effect on TLR3 signaling. We used an IRE1α-specific inhibitor 4μ8c40, which expectedly suppressed Xbp1 mRNA splicing in both WT and Hrd1−/− macrophages (Fig. 4g). 4μ8c pretreatment reduced the expression of pro-inflammatory genes in both genotypes but did not reverse the reduction in pro-inflammatory gene expression in Hrd1−/− RAW 264.7 cells upon poly(I:C) stimulation (Fig. 4g). Taken together, these data demonstrate that the effect of HRD1 on TLR3 pathway is ERAD-, UPR- and IRE1α-independent.

HRD1 modulates TLR3 signaling through ubiquitinating TLR3
Ubiquitination not only guides protein degradation by the proteasome but also exerts regulatory roles through diverse mechanisms41. We then tested whether HRD1 ubiquitinates TLR3 to regulate its signaling. In addition to the observed physical interaction between HRD1 and TLR3 (Fig. 1c–h), HRD1 indeed robustly ubiquitinated both exogenously overexpressed (Fig. 5a–b) and endogenous TLR3 proteins (Fig. 5c). Intriguingly, the ubiquitination of TLR3 by HRD1 was observed to increase significantly following poly(I:C) treatment, highlighting the physiological relevance of this modification (Fig. 5d). Moreover, HRD1 efficiently ubiquitinated ER-resident TLR3 when TLR3 transport from ER to Golgi was blocked by Brefeldin A (BFA) (Figs. 5e, and S3c). Similar as previously reported substrates42, the catalytic RING domain of HRD1 is critically needed to mediate the ubiquitination of TLR3, as demonstrated by the reduction of poly-ubiquitination of TLR3 in cells expressing HRD1 RING ligase-dead C291A/C294A (C2A) mutant (lanes 2 vs. 3, Figs. 5f, and S3d) or ΔRING mutant (lanes 2 vs. 4, Figs. 5f, and S3d). TM domain of TLR3 was required for the effective interaction between TLR3 and HRD1 (Fig. 1h), TM domain of HRD1 also plays a crucial role in facilitating the ubiquitination of TLR3 as ΔTM HRD1 mutant with intact RING domain failed to ubiquitinate TLR3 despite of its high expression level (lanes 2 vs. 5, Figs. 5f, and S3d). The overexpression of WT HRD1 significantly enhanced the poly(I:C)-triggered NF-κB promoter and ISRE activity, when compared to the overexpression of C2A, ΔRING or ΔTM mutants, further demonstrating the significance of HRD1 E3 ligase activity in TLR3 signaling (Fig. S3e). Consistent with the inhibition of TLR3 signaling by LS-102 (Figs. 2c, e, S2d), LS-102 also suppresses the ubiquitination of TLR3 by HRD1 (Figs. 5g, and S3f). Taken together, HRD1 directly ubiquitinates ER-located TLR3 via its RING domain, and the ubiquitin-ligation function of HRD1 is essential for its regulation of TLR3 signaling.

The type of ubiquitin linkage formed could be critical for the fate of the target protein, we next defined the polyubiquitin chain topology involved in TLR3 ubiquitination. Mutations of ubiquitin at K48 and K63 (K48R and K63R mutants) led to a significant reduction in the level of TLR3 polyubiquitination (Fig. 5h). In consistent, the K48- and K63-linked ubiquitination of TLR3 increased significantly following poly(I:C) treatment in macrophage in a HRD1-dependent way (Fig. S3g). VCP engages with ERAD substrates and is required for retrotranslocation and proteasomal degradation of misfolded proteins43. We further tested the interaction between VCP and TLR3 and found that ubiquitinated TLR3 interacted with HRD1 but not with VCP, instead reducing the interaction between HRD1 and VCP (Figs. 5i, and S3h). Taken together, HRD1-mediated ubiquitination of TLR3 primarily occurs through the K48 and K63 residues of ubiquitin, in contrast to K27-linked ubiquitination observed in other HRD1 substrates, such as STING and CREBH30,44. This conformation of ubiquitin chain, together with other unrecognized features of TLR3 might allow it escape VCP engagement and proteasomal degradation after ubiquitination by HRD1.

In addition, no interaction was observed between TLR3 with SEL1L, BIP, or OS9, which facilitate the recruitment of ERAD substrates, both in WT and Hrd1−/− cells (Fig. 5j). While the adaptor protein SEL1L normally forms a complex with HRD1 and is required for HRD1 stability20, previous studies suggest that they may have independent functions21,45.

To further test the role of SEL1L, we utilized siRNA to reduce SEL1L protein levels without bringing down HRD1 in primary macrophages (Fig. S4a, b), and this approach demonstrated that reducing SEL1L alone did not decrease the expression of various inflammatory cytokine genes upon poly(I:C) stimulation (Fig. S4c). Furthermore, SEL1L deficiency did not reduce exogenously expressed HRD1-mediated TLR3 ubiquitination levels in HEK293T cells (Fig. S4d). Thus, HRD1 could modify TLR3 and regulate its signaling independent of ERAD activities.

HRD1 promotes TLR3 Trafficking into endolysosome for cleavage
Ubiquitination can facilitate the efficient sorting and the accurate delivery of membrane proteins to their destination organelles46. TLR3 primarily resides in the ER, but it undergoes trafficking through Golgi to endolysosomes upon poly(I:C) stimulation for proteolytic cleavage and activation3,4. Next, we investigated whether intracellular trafficking of TLR3 is impaired due to deficient HRD1 ubiquitination activity in Hrd1−/− cells. We performed confocal microscopy to examine TLR3 location at different organelles in WT and Hrd1−/− macrophages treated with or without poly(I:C) for the indicated times, including the ER (KDEL), trans-Golgi network (TGN38), and lysosomes (LAMP1) (Figs. 6a-c, and S5a–c). Our confocal microscopy studies revealed that in resting-state WT and Hrd1−/− cells, TLR3 was primarily located in the ER (Figs. 6a, d and S5a). Upon treatment with poly(I:C), the localization levels of TLR3 in the Golgi did not change in Hrd1−/− cells compared to WT cells (Figs. 6b, e, and S5b). In WT cells, poly(I:C) significantly increased TLR3 localization in the endolysosome, whereas TLR3 transportation to the endolysosome was substantially decreased in Hrd1−/− cells compared to WT cells (Figs. 6c, f, and S5c). Similarly, by using TLR3-Flag expressing WT and Hrd1−/− HEK293T cells, we observed that the location of TLR3 in the ER (Calnexin) and its transportation to the Golgi (GM130) did not change between WT and Hrd1−/− cells (Figs. 6g, h, and S5d-e, h-i).

However, poly(I:C) treatment significantly increased TLR3 transport to the early endosome (Rab5) and endolysosome (Rab7) in WT cells, whereas this transport was inhibited in poly(I:C)-treated Hrd1−/− cells (Figs. 6i, j, and S5f-g, j–k).

We then used Western blot analysis to directly examine the levels of cleaved TLR3 in HEK293T cells. Remarkably, we observed a significant reduction in the level of cleaved, activable TLR3 in poly(I:C)-treated Hrd1−/− cell compared to WT cells (Fig. 6k, with quantitation shown in Fig. S6a), and in LS102-treated cells compared to control cells (Fig. 6l, with quantitation shown in Fig. S6b). The uncleaved TLR3 were primarily high-mannose endoglycosidase (Endo) H-sensitive in both WT and Hrd1−/− cells, suggesting uncleaved TLR3 molecules were largely retained within the ER in both cell types (Fig. 6m, lanes 1–2 vs. 3-4, highlighted with arrow). A fraction of uncleaved TLR3 was endoH-resistant and peptide-N-glycosidase F (PNGaseF)-sensitive (Fig. 6m, lanes 3-4 vs. 5-6, highlighted in red box), indicating that this specific fraction of TLR3 is located in post-ER compartments. And the relative protein levels of EndoH-resistant TLR3 in WT cells were higher compared to Hrd1−/− cells (Fig. 6m, lanes 3 vs. 4, with quantitation shown in Fig. S6c). In line with earlier reports suggesting that TLR3 underwent trafficking through the Golgi network before reaching endosomes for cleavage47, the cleaved form of TLR3 exhibited partial resistance to EndoH treatment (Fig. 6m, lanes 3-4 vs. 5-6), and the level of cleaved TLR3 is decreased in Hrd1−/− cells compared to WT cells (Fig. 6m, lanes 1 vs. 2, lanes 3 vs. 4, lanes 5 vs. 6). We further examined the distribution of TLR3 within the ER, Golgi and endosome following the isolation of these organelles. We observed a significant reduction in both uncleaved and cleaved TLR3 within the fraction of endosomes in Hrd1−/− cells as compared to WT cells after poly(I:C) treatment (Fig. 6n). The regulation of TLR3 cleavage and intracellular trafficking by HRD1 was also examined in mouse embryonic fibroblasts (MEFs). HRD1 deficiency and LS-102 treatment resulted in a marked decrease of cleaved TLR3 levels (Fig. S6d-e). Furthermore, the proportion of post-ER uncleaved TLR3 was significantly lower in Hrd1−/− MEFs compared to WT cells (Fig S6f). Following poly(I:C) stimulation, both uncleaved and cleaved TLR3 were notably reduced in the endosomal fraction of Hrd1−/− MEFs versus WT cells (Fig. S6g). Together, these results strongly indicate that HRD1 is essential for proper endosomal localization of TLR3.

The ESCRT (endosomal sorting complexes required for transport) machinery play a critical role in the sorting of ubiquitinated membrane proteins to endolysosome48. Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), Tumor susceptibility gene 101 (TSG101), and Vacuolar protein-sorting-associated protein 36 (VPS36), the key components of the ESCRT-0, ESCRT-I, and ESCRT-II complexes respectively, were previously reported to recognize, bind and collectively orchestra the sorting of polyubiquitinated TLR3 to endolysosome for cleavage3. Overexpression of WT HRD1, but not the HRD1 C2A mutant, significantly enhanced the binding of TLR3 with HRS, TSG101 and VPS36 (Fig. 6o–q). Furthermore, the interaction between endogenous TLR3 with HRS, TSG101 and VPS36 was significantly augmented in macrophage upon poly(I:C) treatment in a HRD1-dependent way (Fig. S7a).

E3 ligase TRIM3, localized in the Golgi apparatus and endosomes, mediates the ubiquitination of TLR3 at K831 to enhance its trafficking3. To delineate the relationship between TRIM3 and HRD1 in TLR3 trafficking, we utilized siRNA to knockdown Trim3 in Hrd1−/− RAW 264.7 cells, and found that the reduction of Trim3 further decreased the expression of inflammatory cytokine genes upon poly(I:C) stimulation in cells with Hrd1 deficiency (Fig. S7b). Additionally, the expression of HRD1 did not affect TRIM3-mediated ubiquitination of TLR3 (Fig. S7c). These findings collectively suggest that HRD1’s E3 ubiquitination activity is a prerequisite for ESCRT-mediated recognition and sorting of TLR3 to endosomes.

Ubiquitination of TLR3 at K813 by HRD1 is indispensable for TLR3 trafficking
The cytosolic domain of TLR3, rather than the N-terminal ectodomain truncate, underwent polyubiquitination by HRD1 (Figs. 7a, and S8a), suggesting that the ubiquitination of TLR3 primarily takes place within its cytosolic domain. To further map the exact ubiquitination site(s), we next replaced each of the 11 cytosolic K residues to Arg (R) individually. TLR3 K813R mutant showed mostly reduced level of ubiquitination by HRD1 (Fig. S8b, c), and polyubiquitin chains composed of WT, K48 and K63 ubiquitin moieties were consistently observed to be conjugated on TLR3 via K813 residue (Fig. 7b). Moreover, Brefeldin A treatment led to the stuck of TLR3 K813R in the ER but did not induce its ubiquitination by HRD1, indicating HRD1 ubiquitinates ER-located TLR3 at K813 (Fig. S8d).

Ubiquitination-defective TLR3 K813R mutant exhibited reduced levels of cleaved TLR3 at basal stage and upon poly(I:C) treatment in MEF cells (Fig. 7c, and quantifications shown in Fig. S8e), further supporting a crucial role of HRD1-mediated TLR3 ubiquitination in promoting its cleavage. By using EndoH and PNGaseF assays, we further observed that the level of uncleaved TLR3 K813R in the post-ER fraction was lower compared to wild-type TLR3, indicating a traffic defect of this mutant (Fig. 7d, lanes 3 vs. 4, highlighted in red box, and quantification shown in Fig. S8f). Following the isolation of cellular organelles, we observe a significant reduction in both uncleaved and cleaved TLR3 K813R within the fraction of endosomes as compared to WT TLR3 after poly(I:C) treatment (Fig. 7e). Furthermore, K813R mutation also resulted in a substantial impairment of signaling transduction following poly(I:C) treatment, as indicated by a notable decrease in the phosphorylation levels of TBK1 and P65, along with an elevated stabilization of IκBα (Fig. 7f). Thus, in contrast to HRD1-mediated ubiquitination of STING and non-secreted immunoglobulin light chain, which occurs at multiple types of amino acids to promote the degradation of these target proteins30,49, HRD1 specially mediates the ubiquitination of TLR3 at residue K813 to positively regulate its trafficking and signaling.

HRD1 promotes TLR3-mediated inflammation in vivo
To assess the physiological relevance of TLR3 regulation by HRD1, we examined whether targeting HRD1 could mitigate the systemic inflammation and tissue damage in mice following poly(I:C)-injection. Consistent with its in vitro effects (Fig. 2c, e), LS-102 treatment in vivo significantly reduced serum cytokines levels of TNFα, IL6 and IFNβ (Fig. 8a), as well as lethality rates (Fig. 8b), in C57BL/6 mice following poly(I:C)-injection. Histological analysis revealed that poly(I:C)-induced leukocyte infiltration, alveolar wall thickening, and edema in the lung were markedly alleviated in LS-102-treated mice (Fig. 8c). Additionally, hepatocyte cell death, indicated by TUNEL staining, was also significantly reduced (Fig. 8c).

To further establish the physiological relevance of HRD1-mediatd K813 ubiquitination of TLR3 in vivo, we generated knock-in (KI) mice carrying the K813R mutation on the C57BL/6J genetic background using the CRISPR/Cas9-based methods (Fig. S9a). Three genotype-confirmed F0 mice were used to generate F1 mice independently by crossing each of them with C57BL/6J WT mice. F1 heterozygous KI mice were inter-crossed to generate two independent homozygous KI mice lines, with genotype confirmed by PCR and sequencing (Fig. S9b). Each KI line was characterized independently, and similar results were obtained from each line. Bone marrow-derived macrophages (BMDMs) from TLR3K813R KI mice displayed a notably decreased response to poly(I:C) stimulation compared to those from WT littermates, including lower transcriptional levels of various inflammatory cytokines (Figs. 8d, and S9c), as well as reduced phosphorylation levels of TBK1, P65, and IκBα (Fig. 8e). Consistently, serum cytokines levels of TNFα, IL6 and IFNβ (Fig. 8f), as well as lethality rates (Fig. 8g), were significantly lower in TLR3K813R mice compared to their WT littermates after poly(I:C) treatment. Histological analysis confirmed that poly(I:C)-induced lung inflammation and hepatocyte cell death were markedly reduced in TLR3K813R cohorts (Fig. 8h). Taken together, HRD1-mediated regulation and K813 ubiquitination of TLR3 are essential for TLR3 agonist-driven inflammation in vivo.

Methods
Mice
TLR3K813R KI mice were generated at GemPharmatech Co., Ltd using the CRISPR-Cas9 technology. The single guide RNA (sgRNA) was designed to target mouse genome TLR3 exon 6 where the mutation is located: 5’-TGATAACGAAAATGATTTTT-3’. The sgRNA was transcribed in vitro, and the homologous recombination vector (donor vector) carrying Tlr3 target mutation sequence was constructed and verified through sequencing. The CRISPR/Cas9 system, donor vector, and sgRNA were mixed and microinjected into fertilized mouse eggs of C57BL/6JGpt background. The surviving injected fertilized eggs were then transferred into pseudopregnant females. Tail and toe samples were collected from 1-week-old pups born from the recipient mice to extract genomic DNA. A pair of primers was designed for PCR and sequencing: F (5’-GTTTCAGTGCATCGGATTCTTGG-3’) and R (5’-CTCAATAGCTTGCTGAACTGCGTG-3’). Genotype-positive mice carrying the desired mutations were established as F0 founders. Three independent founders were then bred separately to WT C57BL6/J mice to obtain F1 heterozygous TLR3K813R KI mice. F1 heterozygous mice were inter-crossed to generate homozygous TLR3K813R KI mice, WT and heterozygous littermates. C57BL/6J mice were purchased from the Model Animal Research Center of Nanjing University (China). All mice were housed in 20°C, 40–60% humidity room with 12 h light cycle and fed a normal-chow diet (13% fat, 57% carbohydrate, and 30% protein). All animal procedures were approved by and done in accordance with the Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University (2021-0135).

Power analysis of the animal size
Based on sample size formula of the power analysis, N=8 (CV)2 [1+(1-PC)2]/(PC)2, to reach the error=0.05, Power=0.80, percentage change in means (PC)=20%, co-efficient of variation (CV)=10–15% (varies between the experiments), 4–6 mice per group are the minimal number of mice to obtain statistical significance and to ensure adequate power. Mice in each group were randomly chosen based on the age, genotype and gender.

Cell culture and drug treatment
Peritoneal macrophages were obtained 4 d after 2 ml aged 4% brewed thioglycollate broth (VWR 90000-294) was intraperitoneal injected. Mice were euthanized and macrophages were collected by injection of 1x PBS into the peritoneal cavity. The peritoneal exudate cells were centrifuged at ~1000 rpm for 10 min, followed with red blood cell lysis buffer treatment to remove red blood cells and resuspended in culture medium in six-well plates for further analyses as previously described. HEK293T (Cat.# CRL-3216), RAW264.7 (Cat.# TIB-71), and MEF (Cat.# CRL-2991) cell lines were originally obtained from ATCC. Cells were maintained in DMEM (Cytiva) supplemented with 10% FBS (Braserum) and 1% penicillin/streptomycin at 37°C under 5% CO2. 5μM Eeyarestatin I (MedChemExpress), and 0.1mM 4μ8c (MedChemExpress) were used in cell culture. TLR3 ligand poly(I:C) (High molecular weight, InvivoGen, tlrl-pic) was dissolved in sterile endotoxin-free physiological water and directly added into the culture medium at 50 µg/ml. LS-102 (MedChemExpress) was dissolved to 10 mM in DMSO and used at 5 µM. Thapsigargin (EMD Calbiochem) was dissolved to 0.6 mM in DMSO and used at 300 nM; Cycloheximide (CHX, Sigma) was dissolved in DMSO and used at 50 µg/ml; MG132 (MedChemExpress) was dissolved in DMSO and used at 25 μM; BFA (Sellck) was dissolved in DMSO and used at 10 µg/ml. The recombinant cytokines (MedChemExpress) were dissolved in complete medium and used at a final concentration 25 ng/ml in cell culture.

CRISPR-mediated gene KO cells
Generation of CRISPR-based Hrd1−/− RAW 264.7 cells, Hrd1−/− HEK293T cells, Sel1l−/− HEK293T cells, and Hrd1−/− MEF cells was performed using the lentiCRISPRv2 vector from the Zhang laboratory at MIT. The Hrd1 and Sel1l sgRNAs were designed, synthesized and cloned into the lentiCRISPRv2 vector, which expresses the sgRNA, Cas9 protein, and pruomycin resistance gene. The mouse sgRNA sequence for Hrd1 was GAUCCAUGCGGCAUGUCGGG. The human sgRNA sequence for Hrd1 was CUUGGUCAGGUACACCACAG. The sgRNA sequences for Sel1l were sgRNA-1: GCUGACGCUGCUGCUGUGUG and sgRNA-2: GGAGGGAAGAUGGCAGACUG.

Generation of BMDMs
Bone marrow cells were isolated from tibiae and femur of mice. After filtration and centrifugation, the bone marrow cells were cultured in DMEM medium containing 10% FBS and 50 ng/ml M-CSF (PeproTech) for 3-5 days to generate BMDMs.

Western blot and image quantitation
Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 % NP-40, 0.5 % sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, 1× Protease inhibitor cocktail). Phosphatase Inhibitor (LC laboratories) was added to inhibit phosphatase activity in lysates. Protein concentration was determined by BCA assay (Beyotime). Samples were subjected to SDS-PAGE, transferred to PVDF and incubated with primary antibodies (4°C, overnight) and HRP-conjugated secondary antibodies (RT, 1 h). Signals were developed with ECL. For Endo H treatment, the lysates were treated with Endo H or PNGase F (New England BioLab) per manufacturer’s instruction. Antibodies used in this study were: Flag (Sigma F1804; 1:2000); HA (Proteintech 51064-2-AP; 1:4000), HRD1 (Proteintech 13473-1-AP; 1:1000), SEL1L (Abclonal; A12073, 1:2000), IRE1α (Cell Signaling 3294; 1:3000), PERK (Proteintech 20582-1-AP; 1:3000), Eif2α (Cell Signaling 5324; 1:3000), p-Eif2α (Cell Signaling 5324; 1:2000), β-tubulin (Proteintech 10068-1-AP; 1:3000), HSP90 (Proteintech 13171-1-AP; 1:5000), Myc (ABclonal AE010; 1:2000), V5 (ABclonal AE017; 1:2000), phospho-IκBα (ABclonal AP0707; 1:1000), HRS (ABclonal A1790; 1:3000), TSG101 (ABclonal A1692; 1:1000), VPS36 (ABclonal A15157; 1:1000), TLR3 (NOVUS NBP2-24875; 2 μg/ml),, p-TBK1 (Ser172) (Cell Signaling D52C2; 1:1000), TBK1 (Cell Signaling E9H5S; 1:2000), p-IRF3 (Ser396) (Cell Signaling D601M; 1:2000), IRF3 (Cell Signaling D83B9; 1:2000), IκBα (Cell Signaling 9242; 1:2000), β-actin (Cell Signaling 13E5; 1:5000), p65 (Cell Signaling D14E12; 1:2000), phospho-p65 (Cell Signaling 93H1; 1:1000), Ubiquitin (linkage-specific K48) (Cell Signaling D9D5; 1:2000), Ubiquitin (linkage-specific K63) (Cell Signaling D7A11; 1:2000), GOLPH2 (ABclonal A26409PM; 1:1000), EEA1 (ABclonal A5057; 1:2000), Ubiquitin (Santa Cruz P4D1; 1:200), TGN38 (Santa Cruz sc-166594; 1:200), and mouse IgG (Santa Cruz sc-2025), Rabbit IgG (Abclonal; AC005) ERK1/2 (Cell Signaling 4695; 1:2000), p-ERK1/2 (Cell Signaling 9101; 1:2000), STAT1 (Cell Signaling 14994; 1:2000), p-STAT1 (Cell Signaling 9167; 1:2000). Western blot band density was quantitated by the use of Image Lab software 4.1 on the ChemiDOC XRS+ system (Bio-Rad). Protein levels were normalized to HSP90, β-tubulin or β-actin. Phosphorylated forms of proteins were normalized to levels of total proteins. Data are presented as mean ± SEM unless otherwise specified.

Coimmunoprecipitation
Cells were incubated with 20 mM N-ethylmaleimide (NEM) in PBS for 10 min on ice, snap frozen and lysed in lysis buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl pH 7.5, protease inhibitor cocktail (Sigma), 0.5 % Nonidet P-40 (NP40), 10 mM NEM) on ice for 15 min. Cells were centrifuged at 12,000 g at 4°C for 10 min. Supernatants were collected and protein concentration was measured using the BCA assay (Beyotime). For each immunoprecipitation, the supernatant was incubated with 0.5 μg of the indicated antibody or control IgG at 4°C for overnight, followed by incubation with 20 μl of Protein A/G PLUS-Agarose (Santa Cruz) at 4°C for another 3 h. The agarose beads were washed for five times with lysis buffer before immunoblot analysis. For ubiquitination analysis, the transfected cells were lysed in lysis buffer containing 1% SDS and denatured by boiling for 5 min. The supernatant was diluted tenfold with lysis buffer before immunoprecipitation using the indicated antibodies. Immunocomplexes were eluted by boiling for 5 min in SDS sample buffer followed by SDS-PAGE and Western blot analysis.

IP-MASS
The WT and Hrd1−/− RAW 264.7 cells were lysed and immunoprecipitated using anti-HRD1 antibody and Protein A/G PLUS-Agarose (Santa Cruz). The binding proteins were eluted and denaturized from the beads after washing, and subjected to SDS-PAGE. Protein samples in gel were digested into peptides by trypsin, and then analyzed by liquid chromatography-tandem mass spectrometry using an UltiMate 3000 nanoLC system and Orbitrap Exploris 480 (Thermo Fisher Scientific). Peptides were separated on a home-made reversed-phase analytical column (30 cm, 100 μm i.d.) packed with 1.9 μm Reprosil-Pur C18 beads (Dr. Maisch, Ammerbuch, Germany) at a constant flow rate of 450 nl/min on an UltiMate 3000 nanoLC system. Solvent A was 0.1% formic acid. The gradient was comprised of an increase from 3% to 10% solvent B (0.1% formic acid in 98% acetonitrile) over 3 min, 10% to 24% in 37 min, 24% to 38% in 12 min and climbing to 80% in 4 min, then holding at 80% for the last 4 min. For data acquisition, the peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Orbitrap Exploris 480 (Thermo Fisher Scientific) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 400 to 1200 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 60,000. Peptides were then selected for MS/MS using NCE setting as 27 and the fragments were detected in the Orbitrap at a resolution of 15,000. Each precursor ion was fragmented with HCD. The collision energy of the HCD was set to 27. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 30 s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The resulting MS/MS data were processed using Proteome Discoverer software v2.5 (Thermo Fisher). Tandem mass spectra were searched against SwissProt Mouse database using the SEQUEST algorithm. Trypsin (full) was specified as cleavage enzyme allowing up to 2 missing cleavages. The minimum peptide length was 6 amino acids with a maximum of 5 modifications per peptide. The mass tolerance for precursor ions was set as 10 ppm, and the mass tolerance for fragment ions was set as 0.02 Da. Proteins and PSMs were filtered with a maximum FDR of 1%, which constituted the full list of proteins identified from the respective Hrd1+/+ and Hrd1−/− immunoprecipitates. To identify HRD1-specific binding proteins, we excluded those proteins found in both Hrd1+/+ and Hrd1−/− immunoprecipitates. Given that HRD1 localizes to the ER, we focused on selecting ER-resident proteins that interact with HRD1. Fig 1a shows the top ten specific HRD1-interacting ER proteins, ranked by sum PEP score. The sum PEP score is calculated based on the posterior error probability (PEP) values of the peptide spectrum matches (PSM), with PEP indicating the likelihood that an observed PSM is a random event. The sum PEP score is the negative logarithm of the PEP values of the connected PSMs.

Plasmid construction
Flag-tagged TLR3 and V5-tagged HRS, TSG101, VPS36 and VCP were inserted into pEGFP-N2 plasmid, with the EGFP deletion. Myc-tagged HRD1 was generated as previously reported30. Plasmids containing HA-tagged Ub, its mutant and truncation were constructed by standard molecular biology techniques. For stable expression, WT and K813R TLR3 with or without Flag tag were individually subcloned into pCDH-CMV-MCS-EF1-copGFP plasmid. All of the plasmids were confirmed by DNA sequencing.

RNA extraction, reverse transcription (RT) and quantitative PCR (Q-PCR)
Total RNA was isolated from cells using RNAiso plus kit (Takara). cDNA was generated using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time, Takara), and amplified using TB Green Premix Ex Taq (Tli RNaseH Plus, Takara) per the manufacturer’s instructions. Q-PCR data were collected using QuantStudio™ 5 (Applied Biosystems) and gene expression was normalized to ribosomal L32 gene for each sample.

The sequences of the primers used are listed below:
Tnfa (TCAGCCGATTTGCTATCTCATA; AGTACTTGGGCAGATTGACCTC),
Il6 (AGACAAAGCCAGAGTCCTTCAG; TGCCGAGTAGATCTCAAAGTGA),
Il1b (CCAAGCAACGACAAAATACC; GTTGAAGACAAACCGTTTTTCC),
Ifnb1 (AGATCAACCTCACCTACAGG; TCAGAAACACTGTCTGCTGG),
Ccl5 (GCTGCTTTGCCTACCTCTCC; TCGAGTGACAAACACGACTGC),
Bip (TGTGGTACCCACCAAGAAGTC; TTCAGCTGTCACTCGGAGAAT),
Chop (TATCTCATCCCCAGGAAACG; GGGCACTGACCACTCTGT),
Xbp1u (ACTATGTGCACCTCTGCAGC, GTCCAGAATGCCCAAAA GG),
Xbp1s (GAGTCCGCAGCAGGTG; GTGTCAGAGTCCATGGGA),
L32 (GAGCAACAAGAAAACCAAGCA; TGCACACAAGCCATCTACTCA),
Ets1 (TGGGTGATGTGGGCTGTG; GGCTCCATTAACCTGATATGGT),
Hrd1 (AGCTACTTCAGTGAACCCCACT; CTCCTCTACAATGCCCAC TGAC),
Irf1 (CGAGGGGACATTGGGATAG; CCTGAATAGAGGGCGGCA),
Irf2 (GAAAGAAACCAAAGACAGAAAAAG; TATAGCTGAAGTCAGGACCGC),
Tlr3 (AGTGTCACAGGACCCCAGA; GAACTACAGAAGCCAAAAAGC),
Sel1l (TGGGTTTTCTCTCTCTCCTCTG; CCTTTGTTCCGGTTACTTCTTG),
Trim3 (CGTCTCAGCCCTACAAAACAA; ACAGGCCTCACAGTAAAACTCC).

The Q-PCR conditions were: 94°C for 5 min, 40 cycles of (94°C for 15 s, 58°C for 15 sec and 72°C for 30 sec), followed by dissociation curve analysis.

Dual-luciferase reporter assay
Cells were transfected with the NF-κB or ISRE luciferase reporter with TK Renilla and the indicated plasmids using lipo8000 (Beyotime). 24 hours after transfection, the cells were either left untreated or subjected to treatment with poly(I:C) for the indicated durations. Subsequently, whole-cell lysates were collected, and luciferase activity was measured using a dual-luciferase reporter assay system (Promega) per the manufacturer’s instructions.

Stable cell line establishment
HEK293T cells were cotransfected with the plasmids of TLR3-containing pCDH-CMV-MCS-EF1-copGFP, psPAX2, and pMD2G. The culture medium was collected 48–72 h after transfection and filtered. WT and Hrd1−/− cells were infected with the virus-containing medium in the presence of polybrene (8 μg/ml, Beyotime). The positive cells were selected by sorting GFP-positive cells.

In vitro ubiquitination assays
The full-length and ΔTM mutant of HRD1 were constructed into the pMBP vector to generate MBP-fusion proteins. Recombinant proteins were expressed in the E. coli BL21 (DE3) Codon-Plus strain (TransGen Biotech) induced with 0.1 mM IPTG at 16°C for 20 h, and purified using Ni-NTA affinity chromatography (Beyotime). The purified proteins were then processed by MBP tag removal (Beyotime) to obtain HRD1 and HRD1-ΔTM proteins, and further identified by western blotting. Flag-tagged TLR3 was immunoprecipitated from HEK293T cells topically expressing TLR3-Flag using an anti-Flag affinity gel (MCE). For in vitro ubiquitination assays, the immunoprecipitated Flag-tagged TLR3 was incubated with HRD1, HRD1-ΔTM proteins, ubiquitin, E1 (Abcam, ab139467) and E2 (UBE2G2 from MCE) in the ubiquitination buffer (Abcam, ab139467) containing MgCl2 and ATP for 3 h at 37°C according to the instructions. The reactions were terminated by adding an equal volume of 2× non-reducing gel loading buffer and analyzed by immunoblotting.

Immunofluorescent staining
Cells were mounted on coverslips and left untreated or treated with poly(I:C) for the indicated times, then fixed with 4% paraformaldehyde for 15 min. After rinse with PBS, the cells were permeabilized and blocked in a buffer containing 0.1% saponin and 5% normal goat serum in PBS at room temperature for 1 hour. Subsequently, the cells were stained with the indicated primary antibodies at 4°C overnight, followed by secondary antibodies at room temperature for 1 h. Following extensive washes with PBS containing 0.1% saponin, slides were covered with antifade mounting medium with DAPI (Beyotime). Primary antibodies for immunofluorescent staining included: TLR3 (Proteintech 17766-1-AP, 1:200), Calnexin (Proteintech 10427-2-AP, 1:200), GM130 (Proteintech 11308-1-AP, 1:1000), Rab5 (Cell Signaling C8B1; 1: 200), Rab7 (Cell Signaling D95F2; 1: 200), KDEL (Novus Biologicals NBP1-97469, 1:200), TGN38 (Santa Cruz sc-166594, 1:200), LAMP1 (Proteintech 65050-1-Ig, 1:1000), Flag (Sigma F1804, 1: 200). Fluorescence images were captured by a Leica TCS SP8 microscope with a 100× objective at the Imaging Core Facility of the First Affiliated Hospital at Zhejiang University School of Medicine. Quantitative analysis of colocalization images was performed using Fiji (ImageJ) software.

HSV1 infection
WT or Hrd1−/− RAW 264.7 cells were cultured in six-well plates, and once reaching 100% confluency, they were stimulated by 100 μg/mL poly(I:C) for 18 h (1.5 mL per well). The supernatants were collected as secretomes and filtered to remove cell debris. Vero cells were seeded on 12-well plates with 600 μL of the indicated secretome per well, and infected with GFP-HSV1 virus at indicated concentrations. After 24 h of incubation, fluorescence images were captured using a Leica DMi8 Fluorescence microscope at the Imaging Core Facility of the First Affiliated Hospital at Zhejiang University School of Medicine. Quantitative analysis of the GFP-positive area was performed using Fiji (ImageJ) software.

Poly(I:C) challenge
Sex- and age-matched mice were injected i.p. with poly(I:C) (InvivoGen, tlrl-pic) at 0.7 mg/kg body weight plus D-galactosamine (Sigma-Aldrich, G1639) at 0.5 g/kg body weight and observed for survival every 1 h. Serum was collected at 2 h time point post injection for cytokine analysis. The tissues for HE and TUNEL were collected at 4 h post injection.

Tissues HE and TUNEL staining
The lung and liver tissues were fixed in 4% formaldehyde for 24 h immediately after dissection from the mice. Fixed tissues were then processed through a series of ethanol solutions for dehydration and cleared in xylene before being embedded in paraffin wax. Paraffin-embedded tissues are sectioned into 4 μm thick slices and mounted onto glass slides. The sections were deparaffinized in xylene and rehydrated through graded ethanol solutions. For HE staining, the lung sections staining begins with Mayer’s hematoxylin, which binds to nucleic acids, imparting a blue color to nuclei. After differentiation in running tap water, sections are counterstained with eosin Y, which stains cytoplasmic and extracellular matrix components pink. Following staining, sections are dehydrated in absolute alcohol, cleared in xylene, and mounted with a resinous medium. The final preparation is examined under a Leica DM2500 LED microscope for histological analysis. For liver TUNEL staining, the liver sections were stained using One Step TUNEL Apoptosis Assay Kit (Beyotime). Briefly, the liver sections were digested with protease K (20 μg/ml) at 37°C for 30 min and rinsed with PBS. The liver sections incubated in the TUNEL detection solution at 37°C for 60 min in the dark. After rinsing with PBS, the slides were covered with antifade mounting medium with DAPI (Beyotime). Fluorescence images were captured by a Leica TCS SP8 microscope at the Imaging Core Facility of the First Affiliated Hospital at Zhejiang University School of Medicine.

ELISA analysis
Macrophages were cultured in 12 well plate (1×106 cells/well), stimulated with poly(I:C) for indicated time, and the supernatant was collected. The levels of mouse TNFα, IL6, and IFNβ in the cell culture supernatants and mouse serum were quantified using ELISA commercial kits (BioLegend). All ELISAs were performed per suppliers’ protocols.

Isolation of ER, Golgi and endosome
ER Enrichment kit (Invent Biotechnologies), Golgi Apparatus Enrichment Kit (Invent Biotechnologies) and Endosome Isolation and Cell Fractionation kit (Invent Biotechnologies) were used to isolate ER, Golgi and endosome according to the manufacturer’s instructions, respectively. Briefly, 3×107 cells were collected and washed in cold PBS. For ER enrichment, the cells were freezed and resuspended in Buffer A, and transferred to a filter cartridge for centrifugation twice. All the supernatants were transferred to a new fresh tube and centrifuged. The supernatant was mixed with buffer B and centrifuged. The pellet was resuspended with buffer A and vortexed vigorously. After mixing with buffer C, the sample was centrifuged. Buffer D was added into the supernatant for the final centrifugation. The pellet was dissolved in SDS loading buffer. For Golgi enrichment, the cells suspended in buffer A were transferred to a filter cartridge, followed by centrifugation. The supernatant mixed with buffer B and centrifuged. After centrifugation, the pellet was resuspended in cold buffer A and centrifuged. Cold buffer C was added into the supernatant with vigorous vortex. After centrifugation, the pellet was dissolved in SDS loading buffer. For endosome isolation, the cell suspension with buffer A was transferred to a filter cartridge and centrifuged. Then, the supernatant was mixed with buffer B and incubated at 4°C for overnight. Following centrifugation, the pellet was dissolved in SDS loading buffer.

siRNA
The siRNAs used in this study were synthesized by Sangon Biotech. The sequences targeting Hrd1 were siHrd1-1: sense, GCAUGGCAGUCCUGUACAUTT and antisense, AUGUACAGGACUGCCAUGCTT; siHrd1-2: sense, GAGCCAUCCGCAACAUGAATT and antisense, UUCAUGUUGCGGAUGGCUCTT. The sequences targeting Ets1 were siEts1: sense, GCUUCAUCACAGAGUCCUATT and antisense, UAGGACUCUGUGAUGAAGCTT; siEts1-2: sense, AGGUGUUAACUCCAGACAATT and antisense, UUGUCUGGAGUUAACACCUTT. The sequences targeting Sel1l were siSel1l-1: sense, GGCUCUUGUAUAUUAUACUTT and antisense, AGUAUAAUAUACAAGAGCCTT; siSel1l-2: sense, GUAGUUGCUGGCCAGAUAUTT and antisense, AUAUCUGGCCAGCAACUACTT. The sense and antisense sequences of siTrim3 are CUGAGGUUUUGCUAGUAAGTT and CUUACUAGCAAAACCUCAGTT, respectively.

Statistical analysis
Results were expressed as mean ± SEM. Comparisons between groups were made using unpaired two-tailed Student’s t-test. Data distribution was assumed to be normal, but this was not formally tested. No sample size calculation was performed for in vitro experiments. No animals or samples were excluded from the analysis. All experiments were repeated with at least three independent biological replicates.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The ER-associated degradation (ERAD) pathway is typically involved in the degradation of misfolded or misfolding-prone proteins in the ER through the cytosolic proteasome system. HRD1, an ER-resident E3 ubiquitin ligase, plays a central role in ERAD by directly binding and ubiquitinating substrates for retrotranslocation and proteasomal degradation. In this study, Zhao et al. have identified TLR3 as a new substrate of HRD1. Specifically, HRD1 binds to and facilitates the ubiquitination of TLR3 at K813 on the ER, thereby promoting the trafficking of TLR3 via the Golgi to the endolysosome for cleavage and activation through the ESCRT machinery, demonstrating a role of HRD1 in TLR3 sorting and trafficking rather than degradation in conventional ERAD. Consistently, the authors observed that TLR3 downstream signaling e.g. the expression of inflammatory genes and the production of inflammatory cytokines was compromised upon TLR3 stimulation when HRD is deficient or inhibited. In addition, the TLR3K813R knockin mice phenocopy the Hrd1-/- mice regarding TLR3 activation. This study reveals a novel role of HRD1 in the trafficking and maturation of the innate immune sensor TLR3 and a new mechanism for TLR3 activation, which advances our understanding of innate immune regulation. Overall, the manuscript is well-written and the data presented are of high quality.\nMajor concerns:\n1. The authors show that the K813R mutation in TLR3 significantly impairs its cleavage and signaling using TLR3-stably expressed HEK293T cells and BMDMs from TLR3 K813R knock-in mice. To further confirm the physiological relevance of K813 ubiquitination, it is recommended to examine phenotypic differences between TLR3 WT and K813R KI mice after poly(I:C) challenges, such as circulating cytokines levels and tissue inflammation.\n2. The study shows that TLR3 binds to HRD1 but not to SEL1L. However, knockdown of SEL1L results in increased inflammatory gene expression without affecting HRD1 protein levels, which is unexpected. It would be beneficial to assess whether SEL1L influences TLR3 protein stability in SEL1L-deficient macrophages to better understand this effect.\n3. The discussion briefly mentions the potential implications of HRD1-mediated regulation of TLR3 and STING in cancer therapy. Since this study does not explore HRD1 or TLR3 functions in the context of cancer, it would be more appropriate to omit or reframe this section to maintain focus on the current study's findings.\nMinor comments:\n1. In Fig 6d, the Y-axis label is missing, making it difficult to interpret the correlation method and the data presented.\n2. Because TLR7/9 require similar sorting and cleavage for activation as TLR3, it would be better if the authors test (or at least discuss) whether HRD1 regulates the activation of TLR7/9.\n3. The manuscript contains several grammatical errors that need to be corrected to improve clarity.", "reviewer_2": "The manuscript by Zhao L et al., titled ‘The HRD1 quality control pathway is essential for TLR3-mediated innate immunity by regulating its trafficking,’ explores the regulatory mechanisms of TLR3 trafficking. Using co-immunoprecipitation coupled with mass spectrometry (CoIP-MS), the authors unexpectedly identified the endoplasmic reticulum (ER)-resident E3 ubiquitin ligase HMG-CoA reductase degradation protein 1 (HRD1), a key factor in ER-associated degradation (ERAD), as an interactor with TLR3, influencing its signaling. Mechanistically, the authors propose that HRD1 mediates TLR3 ubiquitination at lysine 813, and facilitates its trafficking from the Golgi to endolysosomes via the endosomal sorting complex required for transport (ESCRT) machinery, ultimately regulating TLR3-mediated immune responses. Importantly, they assert that HRD1’s role in TLR3 trafficking is independent of its conventional ERAD function. Although the manuscript introduces a novel function for HRD1 in TLR3 ubiquitination and trafficking, the presented data are insufficient to fully substantiate the authors’ claims, as detailed below:\nMajor comments:\n1. In this manuscript, the majority of the data, except for Fig. 7h-i, were generated using HEK293T or RAW264.7 cell lines. To validate their conclusions, the authors should validate their findings in more physiologically relevant systems, such as primary macrophages or mouse embryonic fibroblasts (MEFs). This includes confirming TLR3 signaling, cytokine expression, as well as TLR3 ubiquitination and cleavage in these primary cell types.\n2. In this study, the authors focused exclusively on the influence of HRD1 on TLR3. To provide a broader understanding of HRD1’s role in the innate immune response, the authors should also assess whether HRD1 is involved in regulating other TLRs, particularly endosomal TLRs such as TLR7, TLR8, and TLR9, which share similarities in trafficking and signaling pathways with TLR3.\n3. HRD1 is primarily responsible for the recognition and removal of misfolded and aberrant proteins in the ER through ubiquitination and proteasomal degradation. How does HRD1 mediate both K48- and K63-linked ubiquitination on TLR3 to promote its trafficking to endolysosomes rather than catalyzing K27-linked ubiquitination, which typically leads to ERAD-dependent degradation? The authors should clarify the mechanism by which HRD1 distinguishes between its protein substrates, directing them either toward endolysosomal trafficking or ERAD-mediated degradation.\n4. It is well known that TLR3 undergoes proteolytic processing in acidic endosomes (PMID: 22865861). If HRD1-mediated TLR3 ubiquitination is critical for its trafficking from the Golgi to endolysosomes, one would expect a significant reduction in the cleaved, mature form of TLR3 in HRD1-deficient cells. However, in Figures 6k and 6m, the cleaved TLR3 form shows only a marginal decrease in HRD1-deficient cells. Additionally, if TLR3 cleavage is impaired in HRD1-deficient cells, one would expect an accumulation of the full-length TLR3. Contrary to this, both the full-length and cleaved forms of TLR3 are decreased in HRD1 knockout cells, suggesting that HRD1's primary role might not be in trafficking but rather in TLR3 degradation after activation.\n5. The authors state that HRD1 directly ubiquitinates TLR3; however, they do not provide any supporting evidence for this claim. To strengthen their argument, the authors should perform in vitro ubiquitination assays.\n6. Poly(I:C) is a synthetic ligand for TLR3, and while the authors claim that HRD1 is involved in TLR3-mediated antiviral function, Figure 2f only indirectly assesses type I interferon levels by monitoring the anti-HSV-1 effect. To provide more direct evidence, the authors should evaluate HRD1’s role in TLR3 signaling, immune responses, and anti-viral immunity after actual viral challenge, such as with encephalomyocarditis virus (EMCV), to further substantiate HRD1’s involvement in antiviral defense both in vitro and in vivo.\n7. The authors claim that TLR3 signaling induces HRD1 expression via ERK1/2-EST1 activity. However, the data presented only demonstrate that ERK1/2 stimulates HRD1 and EST1 expression, without directly showing the essential role of EST1 in TLR3-triggered HRD1 expression. The authors need to specifically investigate and demonstrate the involvement of EST1 in the TLR3-mediated induction of HRD1 to support their claim.\n8. In Fig. 4a-c, the authors assessed whether HRD1 regulates TLR3 protein levels at steady state. However, since poly(I:C) induces the interaction between HRD1 and TLR3, the authors should also evaluate TLR3 protein levels in wild-type and HRD1-deficient cells at different time points following poly(I:C) stimulation. This time-course analysis would provide important insights into whether HRD1 regulates TLR3 protein folding and stability during inflammation.\n9. In Fig. 4a-f, the authors conclude that the compromised TLR3 signaling in Hrd1-deficient cells is not due to the inhibition of ERAD activity or the induction of ER stress. However, the authors do not provide direct comparisons of TLR3 signaling between wild-type and Hrd1-deficient cells under conditions of ERAD inhibition or ER stress induction. Without this comparison, it is unclear how the authors arrived at this conclusion. The authors should perform these experiments to substantiate their claim.\n10. In Fig. 4g, the authors should include a treatment group with only poly(I:C) to allow for a direct comparison of the effects of IRE1alpha on HRD1-mediated TLR3 signaling.\n11. A previous study reported that only cleaved endosomal TLRs are located in endosomes/phagosomes after activation (PMID: 18820679). In Fig. 6n and 7e, the authors should show the protein levels of both full-length and cleaved TLR3 in all cellular fractions, including the endosomal fraction, to fully support their conclusion about the trafficking and localization of TLR3.\n12. In Fig. S6d, the TLR3 K746R mutant, rather than the K813R mutant, showed a more pronounced reduction in ubiquitination by HRD1. The authors should perform a detailed quantification analysis of the ubiquitination levels for all mutants. Additionally, the authors should validate whether HRD1 also ubiquitinates TLR3 at K746 and consider generating a double mutant (TLR3 K746R/K813R) if necessary to fully assess HRD1's role in TLR3 ubiquitination.\n13. Since previous studies have demonstrated that TRIM3 ubiquitinates TLR3 at the same lysine residue (K813) to regulate its trafficking to endolysosomes and subsequent immune responses (PMID: 32878999), the data presented in Fig. 7 are not novel and do not adequately support the claim that HRD1 modulates TLR3 trafficking and inflammatory response through ubiquitination at the K813 residue. The authors should investigate whether the depletion of HRD1 in TLR3K813R knock-in cells results in any further reduction of TLR3-mediated signaling and cytokine production.\nMinor comments:\n1. The authors utilized LS-102 to inhibit HRD1 ligase activity in this manuscript. However, does LS-102 specifically target HRD1? It may be beneficial for the authors to generate an enzymatically inactive form of HRD1 (C291A) as previously described (PMID: 19864457) and evaluate its impact on TLR3 trafficking and signaling.\n2. In Fig. S2e, poly(I:C)-induced NF-κB promoter activity was only marginally reduced in HRD1-deficient cells compared to WT cells, which contrasts with the authors' claim of significant reduction. Additionally, since TLR3-driven type I IFN production plays a critical role in antiviral immune responses, the authors should also perform an interferon promoter-driven luciferase reporter assay in HRD1-deficient cells to more comprehensively assess the impact of HRD1 on TLR3 signaling.", "reviewer_3": "This is an interesting and comprehensive study showing through several sound approaches that ubiquitination of TLR3 by HRD1 regulates TLR3 trafficking and function and that this is unexpectedly not through the ERAD pathway. The following concerns should be addressed.\n-Numbering of lines and/or pages, which are missing, would have made the reviewer’s life easier.\n1) The title “The HRD1 quality control pathway is essential for TLR3-mediated innate immunity by regulating its trafficking“ does not match well the findings, because the authors show involvement of HRD1 ubiquitination, but not its quality control activity and targeting to ERAD. They don’t find involvement of other proteins of this pathway either. I would suggest “Ubiquitination by HRD1 is essential for…..” or similar.\n2) Fig. 1a. The immunoprecipitation seems to have a high non-specific background. Details are missing in the figure legend and/or Methods describing the analysis, what cutoff was used for the difference between Hrd1+/+ and -/-, fold differences, etc.\nAn immunoblot with anti-HRD1 comparing the two cell lines should be shown to demonstrate the knockout.\n3) Fig. 1c. Why did treatment with poly(I:C) lead to a higher yield of HRD1 pulldown when exogenously expressed myc-tagged HRD1 is expressed?\n4) Section titled “HRD1 positively regulates TLR3-mediated immune signaling”\nIt should be explained why different cell types were used.\nAs Hrd1-/- increases BiP significantly (Fig. 2d), all the results in this section could be explained by an indirect effect of the UPR. Therefore, it should be mentioned at this stage that this effect is not due to the UPR, as shown later.\n5) Fig. 4C, the TLR3 blot is overexposed, so it cannot be quantified.\n6) “Taken together, HRD1-mediated ubiquitination of TLR3 primarily occurs through the K48 and K63 residues of ubiquitin, in contrast to lysine(K) 27-linked ubiquitination that leads to HRD1 substrates degradation…” HRD1 can use K27 but it can also use K48 for targeting to ERAD.\n7) The English is in general good but I spotted a few errors of grammar and wording. The text should be edited by a native English speaker.", "reviewer_4": "In the manuscript entitled \"The HRD1 Quality Control Pathway is Essential for TLR3-Mediated Innate Immunity by Regulating Its Trafficking,\" the authors propose a hypothesis to explain the mechanisms enabling the intracellular transport and cleavage of TLR3 to regulate the innate immune response. Central to this hypothesis is HRD1, a protein residing in the endoplasmic reticulum and a member of the ERAD machinery that ubiquitinates and targets proteins for proteasomal degradation. The authors claim that in the case of TLR3, HRD1 labels TLR3 for activation rather than degradation, despite functioning as an E3 ubiquitin ligase in the context of ERAD. While the hypothesis is intriguing, the authors fail to demonstrate that the ubiquitin activation of TLR3 is attributable to HRD1:\n1. The authors assert that the impairment \"is caused by a marked reduction in TLR3 trafficking from the Golgi to endolysosomes and its subsequent proteolytic cleavage.\" However, the experiments showing a) intracellular localization by immunofluorescence, b) isolation of the ER, Golgi, and endosomes, and c) glycosylation of TLR3 are unconvincing. First, the co-localization studies do not demonstrate an increase in endosomal trafficking; the Mander’s coefficient shows only weak significance. The isolation of organelles appears to lack resolution, and TLR3 is not found diminished in endosomes. The EndoH and PNGase experiments did not reveal marked differences in TLR3 in the absence of HRD1. None of these experiments were performed with at least three biological replicates, and the blots were not quantified or shown with error bars.\n2. The next claim, \"Importantly, HRD1's role in TLR3 trafficking is decoupled from its canonical ERAD activity and the ER stress sensor inositol-requiring enzyme 1 alpha (IRE1α),\" is not demonstrated. Most of the experiments intended to show that HRD1 absence does not induce ERAD reduction or UPR activation were conducted only once, lacking replicates, quantitation, and other critical analyses. Additionally, the proteins tested were arbitrarily chosen, and IRE1α was not quantified at all.\n3. The remaining figures in the manuscript were superficially executed. Western blot experiments were performed a maximum of twice, were not shown to be reproducible, and were not quantified or represented with error bars."}, "author_response": "We sincerely thank all four reviewers for their insightful and constructive comments. We have carefully addressed each of their comments, which have been instrumental in enhancing and strengthening our manuscript.\nReviewers' Comments:\nReviewer #1 (Remarks to the Author):\nThe ER-associated degradation (ERAD) pathway is typically involved in the degradation of misfolded or misfolding-prone proteins in the ER through the cytosolic proteasome system. HRD1, an ER-resident E3 ubiquitin ligase, plays a central role in ERAD by directly binding and ubiquitinating substrates for retrotranslocation and proteasomal degradation. In this study, Zhao et al. have identified TLR3 as a new substrate of HRD1. Specifically, HRD1 binds to and facilitates the ubiquitination of TLR3 at K813 on the ER, thereby promoting the trafficking of TLR3 via the Golgi to the endolysosome for cleavage and activation through the ESCRT machinery, demonstrating a role of HRD1 in TLR3 sorting and trafficking rather than degradation in conventional ERAD. Consistently, the authors observed that TLR3 downstream signaling e.g. the expression of inflammatory genes and the production of inflammatory cytokines was compromised upon TLR3 stimulation when HRD is deficient or inhibited. In addition, the TLR3K813R knockin mice phenocopy the Hrd1-/- mice regarding TLR3 activation. This study reveals a novel role of HRD1 in the trafficking and maturation of the innate immune sensor TLR3 and a new mechanism for TLR3 activation, which advances our understanding of innate immune regulation. Overall, the manuscript is well-written and the data presented are of high quality.\nWe thank the reviewer for their insightful comments. We have addressed all the issues raised, which have significantly improved our work. In particular, our new experiments have strengthened the physiological significance of K813 ubiquitination of TLR3 mediated by HRD1 in vivo and revealed the broader involvement of HRD1 in the regulation of endosomal TLRs.\nMajor concerns:\n1. The authors show that the K813R mutation in TLR3 significantly impairs its cleavage and signaling using TLR3-stably expressed HEK293T cells and BMDMs from TLR3 K813R knock-in mice. To further confirm the physiological relevance of K813 ubiquitination, it is recommended to examine phenotypic differences between TLR3 WT and K813R KI mice after poly(I:C) challenges, such as circulating cytokines levels and tissue inflammation.\nWe thank the reviewer for this insightful comment. In response, we have examined the phenotypic differences between WT and TLR3K813R KI mice following poly(I:C) challenge, including circulating cytokine levels, tissue inflammation, and survival rates (Response Figure 1a-c). Our new data demonstrate that circulating levels of TNFα, IL-6, and IFNβ, as well as mouse lethality, were significantly lower in TLR3K813R KI mice compared to their WT littermates after poly(I:C) treatment (Response Figure 1a-b). Histological analysis revealed that poly(I:C)-treated WT mice exhibited substantial leukocyte infiltration, increased alveolar wall thickness, edema, and hemorrhage, while these inflammatory responses were markedly alleviated in TLR3K813R KI mice (Response Figure 1c). Consistently, hepatocyte cell death, as indicated by TUNEL staining, was significantly reduced in the TLR3K813R KI group following poly(I:C) treatment (Response Figure 1c). These findings further support the physiological significance of K813 ubiquitination of TLR3 by HRD1 in vivo. Response Figure 1a-c have now been incorporated into Figure 8 f-g of the revised manuscript. Together with the in vivo data using the HRD1 inhibitor LS102, these findings highlight the physiological significance of TLR3 ubiquitination by HRD1 in vivo. To improve the logical flow, the LS102 in vivo data have been relocated from Figure 2g-h to Figure 8a–c in the revised manuscript.\nResponse Figure 1. Attenuated systemic inflammation and improved survival in TLR3K813R KI mice following poly(I:C) challenge. a, ELISA analysis of serum cytokines TNFα, IL6 and IFNβ in sex- and age-matched WT and TLR3K813R KI mice 2 hours after poly(I:C) injection (n=9 mice per group). b, Survival curves of sex- and age-matched WT and TLR3K813R KI mice following poly(I:C) plus D-GalN injection (n= 9 mice per group). c, Histological analysis of lung and liver tissues from poly(I:C)-challenged mice. Sex- and age-matched WT and TLR3K813R KI mice were injected i.p. with poly(I:C) plus D-GalN for 4 hours. Lung sections were assessed by H&E staining to evaluate inflammation, and liver sections by TUNEL staining to evaluate hepatocyte cell death. Data are representative of three independent repeats. Values represent mean ± s.e.m. *P < 0.05; **P < 0.01, by unpaired two-tailed Student’s t-test.\n2. The study shows that TLR3 binds to HRD1 but not to SEL1L. However, knockdown of SEL1L results in increased inflammatory gene expression without affecting HRD1 protein levels, which is unexpected. It would be beneficial to assess whether SEL1L influences TLR3 protein stability in SEL1L-deficient macrophages to better understand this effect.\nWe thank the reviewer for this valuable comment. In response, we examined the protein levels and stability of TLR3 in Sel1l-/- primary macrophages derived from myeloid cell-specific Sel1L-deficient mice (Sel1lLyz2), compared to WT macrophages from Sel1lflox/flox (Sel1lf/f) littermate controls 1. Our analysis revealed no differences in TLR3 protein levels (Response Figure 2a-b) or stability (Response Figure 2c-d) between WT and Sel1l-/- macrophages. These findings support the conclusion that SEL1L does not bind to TLR3 or regulate its stability and protein levels in the ER.\nResponse Figure 2. SEL1L deficiency does not affect TLR3 protein levels or stability. a-b, Immunoblot analysis of the indicated proteins in WT and Sel1l-/- macrophages (a), with quantitation of full-length (FL) TLR3 shown in (b). Data are combined from five independent repeats. Arrow indicates the FL TLR3 bands. c-d, Immunoblot analysis of WT and Sel1l-/- macrophages treated with cycloheximide (CHX) for the indicated times (c), with quantitation from three independent experiments shown in (d). Arrow indicates the SEL1L bands. Values represent mean ± s.e.m. NS, not significant, by unpaired, two-tailed Student’s t-test (b, d).\n3. The discussion briefly mentions the potential implications of HRD1-mediated regulation of TLR3 and STING in cancer therapy. Since this study does not explore HRD1 or TLR3 functions in the context of cancer, it would be more appropriate to omit or reframe this section to maintain focus on the current study's findings.\nWe thank the reviewer for the comment. In response to the suggestion, we have removed the word “cancer” on line 498 and the phrase “in cancer treatment” on line 500 from this section.\nMinor comments:\n1. In Fig 6d, the Y-axis label is missing, making it difficult to interpret the correlation method and the data presented.\nWe thank the reviewer for the comment. The Y-axis label has been added accordingly.\n2. Because TLR7/9 require similar sorting and cleavage for activation as TLR3, it would be better if the authors test (or at least discuss) whether HRD1 regulates the activation of TLR7/9.\nWe thank the reviewer for this insightful comment. TLR3, TLR7, and TLR9 are synthesized in the ER and subsequently transported to endosomes for activation. Despite their similarities, these TLRs recognize distinct nucleic acid types, have different pathological implications, and depend on unique molecular pathways for trafficking and activation2, 3. Specifically, TLR3 transport requires specific chaperones and ubiquitination modifications, such as TRIM3-mediated K831 ubiquitination4-6. In contrast, TLR7 and TLR9 utilize distinct trafficking routes and regulatory mechanisms. Notably, while TLR9 is released from UNC93B1 within endosomes for ligand binding and signaling7, TLR7 uniquely remains associated with UNC93B1, recruiting syntenin-1 to regulate its signaling7-9. These specialized mechanisms enable rapid antiviral responses while preventing excessive activation.\nIn response to the reviewer’s comment, we conducted experiments to examine the inflammatory response and downstream effector protein activation in WT and Hrd1⁻/⁻ macrophages following stimulation with TLR7 and TLR9 agonists. Our findings confirm that HRD1 positively regulates TLR7/9 activation, establishing it as a master regulator of endosomal TLRs. Given the intrinsic complexity and distinct pathological relevance of TLR7/9 signaling, we consider HRD1's involvement highly significant; however, a comprehensive and dedicated investigation of this aspect is needed and is beyond the scope of the current manuscript.\nResponse Figure 3. HRD1 positively regulates the TLR7- and TLR9-mediated inflammatory response. a, qPCR analysis of Tnfa, Ifnb1, Il6, and Ccl5 in WT versus Hrd1−/− RAW 264.7 cells treated with vehicle, 2 μg/ml TLR7 agonist R837, or 2 μM TLR9 agonist ODN for 16 hours (n=4 per group). Data are representative of three independent repeats. mRNA, messenger RNA. b-c, Immunoblot analysis of indicated proteins in WT and Hrd1−/− RAW 264.7 cells treated with R837 (b) or ODN (c) for the indicated times. Relative levels of TBK1 phosphorylation are shown below the blots. Data are representative of three independent repeats.\n3. The manuscript contains several grammatical errors that need to be corrected to improve clarity.\nWe thank the reviewer for the comment. We apologize for the grammatical errors and have carefully reviewed the text to correct all of them.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Zhao L et al., titled ‘The HRD1 quality control pathway is essential for TLR3-mediated innate immunity by regulating its trafficking,’ explores the regulatory mechanisms of TLR3 trafficking. Using co-immunoprecipitation coupled with mass spectrometry (CoIP-MS), the authors unexpectedly identified the endoplasmic reticulum (ER)-resident E3 ubiquitin ligase HMG-CoA reductase degradation protein 1 (HRD1), a key factor in ER-associated degradation (ERAD), as an interactor with TLR3, influencing its signaling. Mechanistically, the authors propose that HRD1 mediates TLR3 ubiquitination at lysine 813, and facilitates its trafficking from the Golgi to endolysosomes via the endosomal sorting complex required for transport (ESCRT) machinery, ultimately regulating TLR3-mediated immune responses. Importantly, they assert that HRD1’s role in TLR3 trafficking is independent of its conventional ERAD function. Although the manuscript introduces a novel function for HRD1 in TLR3 ubiquitination and trafficking, the presented data are insufficient to fully substantiate the authors’ claims, as detailed below:\nWe thank the reviewer for their insightful comments. We have addressed the issues raised below, which have significantly improved our work and strengthened our model.\nMajor comments:\n1. In this manuscript, the majority of the data, except for Fig 7h-i, were generated using HEK293T or RAW264.7 cell lines. To validate their conclusions, the authors should validate their findings in more physiologically relevant systems, such as primary macrophages or mouse embryonic fibroblasts (MEFs). This includes confirming TLR3 signaling, cytokine expression, as well as TLR3 ubiquitination and cleavage in these primary cell types.\nWe appreciate the reviewer’s valuable comment. In response, we used primary macrophages and knocked down (KD) Hrd1 expression with siRNAs, followed by poly(I:C) stimulation. Our results showed that poly(I:C) induced weaker TLR3 downstream signaling in Hrd1 KD macrophages, as evidenced by reduced phosphorylation of signaling effectors, lower expression of inflammatory genes, and decreased cytokine secretion (Response Fig 4a-d). Together with our in vivo studies using HRD1 inhibitors and primary cell studies from TLR3K813R KI mice (Fig 8a-e in the revised manuscript), these findings highlight the physiological significance of the HRD1-TLR3 regulatory axis. Response Fig 4a-d have been incorporated into Supplementary Fig 2b, c, f, g in the revised manuscript.\nResponse Figure 4. The inflammatory response is reduced in Hrd1-knockdown primary macrophages following poly(I:C) stimulation. a, Primary macrophages were transfected with Hrd1 siRNAs for 72 hours, followed by qPCR analysis to asses Hrd1 expression (n=4 per group). NC, negative control. b, Primary macrophages were transfected with Hrd1 siRNAs for 72h and then stimulated with poly(I:C) (50 μg/ml) for 1h. Cell lysates were subjected to Immunoblot analysis of the indicated proteins. c-d, Primary macrophages were transfected with Hrd1 siRNAs for 72h, followed by poly(I:C) (50 μg/ml) stimulation for 3h. qPCR analysis was performed on cell lysates (c), and cell culture supernatants were collected for ELISA-based cytokine analysis ( n=4 per group). Data are representative of three independent repeats. Values represent mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001, by unpaired, two-tailed Student’s t-test.\n2. In this study, the authors focused exclusively on the influence of HRD1 on TLR3. To provide a broader understanding of HRD1’s role in the innate immune response, the authors should also assess whether HRD1 is involved in regulating other TLRs, particularly endosomal TLRs such as TLR7, TLR8, and TLR9, which share similarities in trafficking and signaling pathways with TLR3.\nWe thank the reviewer for this valuable comment. As the same question was raised by Reviewer 1, please refer to our earlier response and Response Figure 3.\n3. HRD1 is primarily responsible for the recognition and removal of misfolded and aberrant proteins in the ER through ubiquitination and proteasomal degradation. How does HRD1 mediate both K48- and K63-linked ubiquitination on TLR3 to promote its trafficking to endolysosomes rather than catalyzing K27-linked ubiquitination, which typically leads to ERAD-dependent degradation? The authors should clarify the mechanism by which HRD1 distinguishes between its protein substrates, directing them either toward endolysosomal trafficking or ERAD-mediated degradation.\nWe thank the reviewer for this insightful comment. We are fully aware that the canonical role of HRD1 is to recognize and eliminate misfolded and aberrant proteins in the ER through ubiquitination and proteasomal degradation. In this study, we demonstrated that HRD1 mediates both K48- and K63-linked ubiquitination of TLR3 to regulate its trafficking. This unique ubiquitin linkage pattern facilitates TLR3 transport, consistent with previous findings that TLR3 sorting from the Golgi to endolysosomes requires K63-linked ubiquitination, particularly through its interaction with the core subunit of ESCRT-0, HRS4. Additionally, HRS has been shown to preferentially associate with both K48- and K63-linked ubiquitinated substrates10. To further validate this mechanism, we conducted experiments demonstrating that HRD1-mediated K48- and K63-linked ubiquitination of TLR3 is essential for its interaction with HRS (Response Fig. 5a). These findings further strengthen our proposed working model.\nRecent mouse studies have identified a wide range of endogenous HRD1 substrates in a cell type-specific manner. While some substrates are inherently prone to misfolding (e.g. pro-AVP 11 and POMC 12), others appear to fold normally1, 13-15. Despite decades of investigation, it remains unclear what intrinsic structural features determine whether a protein is targeted by HRD1-ERAD pathway for ubiquitination and proteasomal degradation. Understanding how HRD1 distinguishes between its substrates and directs them either toward ERAD-mediated degradation or endo-lysosomal trafficking remains a fundamental but unresolved question. Based on current knowledge, we propose the following potential mechanisms: (1) ERAD substrates such as pro-AVP 11 and POMC 12 tend to form high-molecular-weight or insoluble aggregates. In contrast, TLR3 does not exhibit such aggregation in either WT or HRD1-deficient macrophages (Response Figure 5b-c). (2) HRD1-mediated K27-linked ubiquitination of substrates such like STING or non-secreted immunoglobulin light chains occurs on multiple amino acids, including lysine (K), serine (S), threonine (T), and cysteine (C)1, 16. However, our in vitro and in vivo data indicate that HRD1-mediated ubiquitination of TLR3 occurs predominantly at K813 (Figure 7a-f, 8d-h), rather than on multiple amino acid residues. (3) Previous studies have established that SEL1L is essential for HRD1-mediated ERAD function, playing a crucial role in substrate recruitment and ubiquitination 13, 17, 18. However, our results show that SEL1L does not bind to TLR3 (Fig. 5i), and that SEL1L deficiency does not affect TLR3 abundance or stability (Response Figure 2). The lack of SEL1L involvement, combined with the specific ubiquitination pattern observed in TLR3, suggests that TLR3 is directed toward an alternative pathway involving K48- and K63-linked ubiquitination rather than ERAD. Future investigation is needed to provide a more cohesive understanding of HRD1 substrate selection. We have added a discussion of this point in the manuscript at lines 485 and 488.\nResponse Figure 5. HRD1 mediates K48- and K63-linked ubiquitination of TLR3 to regulate its trafficking. a, HEK293T cells were transfected with the indicated plasmids for 24h, followed by immunoprecipitation and immunoblot analysis. Data are representative of three independent repeats. b, Immunoblot analysis of TLR3 protein levels in wild-type and HRD1-deficient RAW 264.7 cells. For non-reducing SDS-PAGE, lysates were prepared in 5×non-denaturing loading buffer (250 mM Tris-HCl pH 6.8, 1% SDS, 50% glycerol and 0.05% bromophenol blue) and were not boiled prior to gel separation. Data are representative of three independent repeats. c, Western blot analysis of TLR3 in NP-40 soluble (S) and insoluble (P) fractions of Hrd1+/+ and Hrd1-/- RAW 264.7 cells lysed in buffer containing 0.5% NP-40. HSP90 and H2A were used as markers for the soluble and insoluble fractions, respectively. Data are representative of three independent repeats.\n4. It is well known that TLR3 undergoes proteolytic processing in acidic endosomes (PMID: 22865861). If HRD1-mediated TLR3 ubiquitination is critical for its trafficking from the Golgi to endolysosomes, one would expect a significant reduction in the cleaved, mature form of TLR3 in HRD1-deficient cells. However, in Figures 6k and 6m, the cleaved TLR3 form shows only a marginal decrease in HRD1-deficient cells. Additionally, if TLR3 cleavage is impaired in HRD1-deficient cells, one would expect an accumulation of the full-length TLR3. Contrary to this, both the full-length and cleaved forms of TLR3 are decreased in HRD1 knockout cells, suggesting that HRD1's primary role might not be in trafficking but rather in TLR3 degradation after activation.\nWe thank the reviewer for this insightful comment. We have performed four biologically independent experiments to compare the levels of full-length and cleaved TLR3 in WT and Hrd1⁻/⁻ cells. Our data consistently show that full-length TLR3 levels are comparable between the two groups, whereas cleaved TLR3 bands are significantly reduced in Hrd1⁻/⁻ cells compared to WT cells (Response Figure 6a).\nThe Endo-H digestion experiments in Fig 6m and Fig 7d revealed that the majority of full-length (FL) TLR3 is Endo H-sensitive and resides in the ER (indicated by the black arrow). Only a small fraction of uncleaved TLR3 is Endo H-resistant (highlighted in the red box), indicating its localization in post-ER compartments. Notably, the amount of post-ER uncleaved TLR3 is reduced in Hrd1⁻/⁻ cells compared to WT cells, with quantitation shown in Response Figure 6b. These data have now been incorporated into Fig S5l-m of the revised manuscript.\nFurther fractionation of the post-ER uncleaved TLR3 into Golgi and endosomal compartments showed that the endosome-localized full-length TLR3—which represents only a small fraction of total FL TLR3 and is likely rapidly cleaved upon Golgi-to-endosome trafficking—was decreased in both Hrd1⁻/⁻ and TLR3 K813R cells compared to WT cells (Fig 6n, Fig 7e). Additionally, CHX chase experiments following poly(I:C) treatment showed that the half-life (degradation rate) of FL TLR3 was unchanged between WT and Hrd1⁻/⁻ cells (Response Figure 6c-d). Based on these findings, we concluded that FL TLR3 trafficking to the endosome is reduced in Hrd1⁻/⁻ and TLR3 K813R cells, leading to decreased cleaved TLR3 bands. To avoid ambiguity, we have revised the manuscript wording from \"impaired cleavage\" to \" a reduced level of cleaved TLR3 \" (line 370).\nResponse Figure 6. TLR3 trafficking to the endosome is reduced in Hrd1⁻/⁻ cells. a-b, Quantitation of data from Fig 6k and 6m in the manuscript, showing decreased levels of cleaved TLR3 (a) and Endo H-resistant FL TLR3 (b) in Hrd1⁻/⁻ cells. Data are combined from four independent experiments. Values represent mean ± s.e.m. NS, not significant, **P < 0.01, ***P < 0.001, by unpaired, two-tailed Student’s t-test. c-d, Wild-type and HRD1-deficient RAW 264.7 cells were pretreated with poly(I:C) for 3h and then treated with CHX for the indicated times, followed by Immunoblot analysis of TLR3 protein levels. Arrow indicates the bands of FL TLR3. These data are representative of three independent repeats. Values represent mean ± s.e.m. NS, not significant.\n5. The authors state that HRD1 directly ubiquitinates TLR3; however, they do not provide any supporting evidence for this claim. To strengthen their argument, the authors should perform in vitro ubiquitination assays.\nWe thank the reviewer for this valuable comment. In response, we conducted an in vitro ubiquitination assay. As prokaryotic expression of full-length TLR3 is not feasible, we ectopically expressed TLR3-Flag in HEK293T cells and purified it using an anti-Flag affinity gel. HRD1 and HRD1-ΔTM were cloned into the pMBP vector to generate MBP-fusion proteins, which were then processed by MBP tag removal to obtain purified HRD1 and HRD1-ΔTM proteins. The in vitro ubiquitination assay using these proteins demonstrated that HRD1 efficiently ubiquitinate TLR3 in vitro (Response Figure 7).\nResponse Figure 7. HRD1 ubiquitinates TLR3 in vitro. Full-length TLR3-Flag was ectopically expressed in HEK293T cells and purified using anti-Flag Affinity Gel (HY-K0217, MCE). HRD1 and HRD1-ΔTM were cloned into the pMBP vector to generate MBP-fusion proteins. Recombinant proteins were expressed in the E. coli BL21 (DE3) strain cultured in L-broth supplemented with ampicillin. Protein expression was induced with 0.1 mM IPTG at 16 ℃ for 16 h, followed by purification using Ni-NTA affinity chromatography. The resulting recombinant proteins were incubated overnight with TEV plus protease (Beyotime, P2305M) to remove MBP tag, yielding HRD1 and HRD1-ΔTM proteins. These proteins then used for an in vitro ubiquitylation assay according to the protocol of the kit (ab139467, Abcam). Arrows indicate the bands corresponding to HRD1 and HRD1-ΔTM.\n6. Poly(I:C) is a synthetic ligand for TLR3, and while the authors claim that HRD1 is involved in TLR3-mediated antiviral function, Figure 2f only indirectly assesses type I interferon levels by monitoring the anti-HSV-1 effect. To provide more direct evidence, the authors should evaluate HRD1’s role in TLR3 signaling, immune responses, and anti-viral immunity after actual viral challenge, such as with encephalomyocarditis virus (EMCV), to further substantiate HRD1’s involvement in antiviral defense both in vitro and in vivo.\nWe thank the reviewer for this insightful comment. We agree that using the EMCV infection model would be ideal to demonstrate the role of HRD1 in regulating TLR3-mediated antiviral function. To pursue this, we contacted several virology and immunology institutes in Beijing, Shanghai, and Wuhan, as well as commercial sources, in an effort to obtain EMCV viral stocks. However, due to stringent regulations on infectious viruses, EMCV stocks are currently not accessible in our country. Given these constraints, our current study utilizes poly(I:C) stimulation to investigate the role of HRD1 in regulating TLR3-mediated innate immune responses both in vitro and in vivo. To clarify this point, we have revised the term “antiviral immunity” to “inflammatory response” (line 49) or “inhibiting HSV-1 replication” (line 182).\n7. The authors claim that TLR3 signaling induces HRD1 expression via ERK1/2-EST1 activity. However, the data presented only demonstrate that ERK1/2 stimulates HRD1 and EST1 expression, without directly showing the essential role of EST1 in TLR3-triggered HRD1 expression. The authors need to specifically investigate and demonstrate the involvement of EST1 in the TLR3-mediated induction of HRD1 to support their claim.\nWe thank the reviewer for this valuable comment. To confirm the involvement of ETS1 in TLR3 signaling-mediated HRD1 expression, we performed additional experiments. Using siRNAs to knock down Ets1 in primary macrophages, we observed that Hrd1 expression increased in control cells but not in Ets1-knockdown cells upon poly(I:C) treatment. These results clearly demonstrate that ETS1 plays a role in the TLR3-mediated induction of HRD1 (Response Figure 8a-b). These data have now been incorporated into Fig S3a-b in the revised manuscript.\nResponse Figure 8. ETS1 plays a role in TLR3-mediated induction of HRD1. a, qPCR analysis of Ets1 expression in primary macrophages transfected with Ets1 siRNAs for 72h (n=4). NC, negative control. b, qPCR analysis of Hrd1 expression in primary macrophages transfected with Ets1 siRNAs for 72h and stimulated with poly(I:C) (50 μg/ml) for 3h (n=4). Data are representative of three independent repeats. Values represent mean ± s.e.m. ***P < 0.001, by unpaired, two-tailed Student’s t-test.\n8. In Fig. 4a-c, the authors assessed whether HRD1 regulates TLR3 protein levels at steady state. However, since poly(I:C) induces the interaction between HRD1 and TLR3, the authors should also evaluate TLR3 protein levels in wild-type and HRD1-deficient cells at different time points following poly(I:C) stimulation. This time-course analysis would provide important insights into whether HRD1 regulates TLR3 protein folding and stability during inflammation.\nWe thank the reviewer for this insightful comment. In response to this request, we performed an experiment to assess TLR3 protein levels in wild-type and HRD1-deficient RAW 264.7 cells at different time points following poly(I:C) stimulation. Our data showed that TLR3 protein and mRNA levels significantly increased in WT cells after 3–6 hours of poly(I:C) stimulation, whereas this upregulation was suppressed in HRD1-deficient cells (Response Figure 9a-b). This trend is consistent with the data presented in the manuscript, where the HRD1 inhibitor LS-102 suppressed TLR3 upregulation at both the mRNA and protein levels (Figure 3g, h, j).\nTaken together, these findings indicate that HRD1 deficiency does not affect the abundance or stability of full-length TLR3 protein in unstimulated macrophages. However, upon poly(I:C) stimulation, HRD1 deficiency leads to a reduction in TLR3 protein levels—not due to altered stability of full-length TLR3 (refer to Response Figure 6c-d) — but rather due to the loss of the HRD1-dependent positive feedback loop that enhances TLR3 expression upon activation.\nResponse Figure 9. The effect of HRD1 on TLR3 levels in poly(I:C) stimulated cells. a-b, Immunoblot analysis of TLR3 protein levels (a) and qPCR analysis of Tlr3 expression (b) in wild-type and HRD1-deficient RAW 264.7 cells at different time points following poly(I:C) stimulation. The image data are representative of three independent repeats. Arrow indicates the bands corresponding to full-length (FL) TLR3 ( n=3) for (b). Values are presented as mean ± s.e.m. ***P < 0.001, by unpaired, two-tailed Student’s t-test.\n9. In Fig. 4a-f, the authors conclude that the compromised TLR3 signaling in Hrd1-deficient cells is not due to the inhibition of ERAD activity or the induction of ER stress. However, the authors do not provide direct comparisons of TLR3 signaling between wild-type and Hrd1-deficient cells under conditions of ERAD inhibition or ER stress induction. Without this comparison, it is unclear how the authors arrived at this conclusion. The authors should perform these experiments to substantiate their claim.\nWe thank the reviewer for this insightful comment. In response, we conducted further experiments to demonstrate that the compromised TLR3 signaling observed in HRD1-deficient cells is not due to ERAD inhibition or ER stress induction. First, our newly added data show that IRE1α, a known substrate of HRD1-mediated ERAD19, is significantly stabilized and accumulated in HRD1-deficient macrophages (Response Figure 10a), indicating impaired ERAD activity. However, this impaired activity does not lead to the accumulation of full-length TLR3 protein. Furthermore, treatment with Eeyarestatin I and NMS-873—two ERAD inhibitors—did not stabilize full-length TLR3 protein (Response Figure 10b). These findings support the conclusion that HRD1 does not directly target full-length TLR3 for ER-associated degradation.\nTo further address the reviewer’s concern, we conducted experiments comparing TLR3-mediated inflammatory responses between WT and HRD1-deficient cells under ERAD-inhibition conditions using Eeyarestatin I. While pretreatment with the inhibitor mildly induced UPR gene expression in WT cells, it did not reduce poly(I:C)-induced inflammatory gene expression (ERAD inhibitor + poly(I:C) vs. vehicle + poly(I:C)). Moreover, regardless of Eeyarestatin I pretreatment, poly(I:C)-induced inflammatory gene expression remained consistently impaired in HRD1-deficient macrophages compared to WT macrophages (Response Figure 10c). These findings further support that the impaired TLR3-mediated inflammatory response in HRD1-deficient macrophages is not due to ERAD inhibition.\nWe also examined the potential contribution of ER stress in both WT and HRD1-deficient cells. Our new data show that HRD1-deficient cells exhibit mild ER stress compared to WT cells under basal conditions, as indicated by increased EIF2α phosphorylation (Response Figure 10d) and elevated expression of UPR genes Xbp1s, Bip, and Chop (Response Figure 10e). Treatment with thapsigargin (Tg) induced robust ER stress in both WT and HRD1-deficient cells (Response Figure 10d-e). However, ER stress induction did not suppress poly(I:C)-induced TLR3 signaling in WT cells, and poly(I:C)-induced TLR3 signaling remained consistently reduced in HRD1-deficient macrophages, regardless of Tg treatment (Response Figures 10d–e).\nThese findings indicate that reduced TLR3 signaling in HRD1-deficient cells is not due to increased ER stress. Response Figure 10c-e have now been incorporated into Figure 4d-f in the revised manuscript.\nResponse Figure 10. The effect of HRD1 on TLR3 signaling is uncoupled from ERAD and UPR. a. Immunoblot analysis of the indicated proteins in WT and HRD1-deficient RAW 264.7 cells treated with CHX for the indicated times, with relative quantitation of IRE1α from three independent repeats shown on the right. b. Immunoblot analysis of the indicated proteins in RAW 264.7 cells treated with CHX and/or MG132, NMS873, Eeyarestatin I for 9h. Data are representative of three independent repeats (a-b). c, qPCR analysis of Xbp1s, Bip, Tnfa, Ifnb1, Il6 and Il1b in WT and Hrd1−/−RAW 264.7 cells treated with Eeyarestatin I for 8 h and/or poly(I:C) for 6 h (n = 4 per group). d, Immunoblot analysis of indicated proteins in WT and Hrd1−/−RAW 264.7 cells treated with thapsigargin for 1.5 h and/or poly(I:C) for 1 h. Representative of three independent repeats. Tg, thapsigargin. p-, phosphorylated PERK; n-, non-phosphorylated PERK. Quantitation of the ratio of phosphorylated to total protein (p/t) is shown below each blot. e, qPCR analysis of Bip, Xbp1s, Tnfa, Il6, Chop, Ccl5, Ifnb1 and Il1b in WT and Hrd1−/−RAW 264.7 cells treated with thapsigargin and/or poly(I:C) for 6 h (n = 4 per group), representative of three independent repeats. Values represent mean ± s.e.m. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001, by unpaired, two-tailed Student’s t-test (c, e).\n10. In Fig. 4g, the authors should include a treatment group with only poly(I:C) to allow for a direct comparison of the effects of IRE1alpha on HRD1-mediated TLR3 signaling.\nWe thank the reviewer for this insightful comment. In response, we repeated the experiments to include a poly(I:C)-only treatment group. Our results show that IRE1α activity positively regulates TLR3-mediated inflammatory gene induction (Response Figure 11) and therefore does not contribute to the downregulation of TLR3-mediated inflammatory gene expression observed in Hrd1-/- cells. These findings have now been incorporated into Figure 4g in the revised manuscript.\nResponse Figure 11. IRE1α does not contribute to the downregulation of TLR3-mediated inflammation in Hrd1-/- cells. WT and Hrd1-/- RWA 264.7 cells were pretreated with 4μ8c (0.1 mM) for 24h, followed by stimulation with poly(I:C) (50μg/ml) for 6h before qPCR analysis of the indicated genes. Data are representative of three independent repeats. Values represent mean ± s.e.m. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001, by unpaired, two-tailed Student’s t-test.\n11. A previous study reported that only cleaved endosomal TLRs are located in endosomes/phagosomes after activation (PMID: 18820679). In Fig. 6n and 7e, the authors should show the protein levels of both full-length and cleaved TLR3 in all cellular fractions, including the endosomal fraction, to fully support their conclusion about the trafficking and localization of TLR3.\nWe sincerely appreciate the reviewer's insightful comment. Despite multiple attempts across different experimental batches, we were unable to detect cleaved TLR3 bands in isolated endosomes from macrophages. This is likely due to technical challenges, such as the susceptibility of cleaved TLR3 to degradation during the macrophage endosome isolation process. To address this challenge, we overexpressed TLR3-Flag in WT and Hrd1⁻/⁻ 293T cells. Our new data show that the protein levels of both full-length and cleaved TLR3 in endosomes are reduced in Hrd1⁻/⁻ cells (Response Fig 12). Together with our confocal colocalization imaging data (Fig 6a–j) and Endo H-treatment western blot data (Response Fig 6b, see our response to Point 4 above), we conclude that TLR3 trafficking into endosome is defective in Hrd1⁻/⁻ cells. The data from Response Fig 12 have been incorporated into Figure 6n in the revised manuscript.\nResponse Figure 12. Full-length and cleaved TLR3 in endosomes are reduced in Hrd1⁻/⁻ cells. WT and Hrd1−/− HEK293T cells were transfected with TLR3-Flag plasmid and treated with 50 μg/ml poly(I:C) for 12 h. Immunoblot analysis was performed on the indicated proteins following subcellular fractionation to isolate the ER, Golgi, and endosomes. Relative quantitation of full-length (FL) and cleaved (CL) TLR3 levels in the indicated organelle fractions is shown below the blot. Data are representative of three independent repeats.\n12. In Fig. S6d, the TLR3 K746R mutant, rather than the K813R mutant, showed a more pronounced reduction in ubiquitination by HRD1. The authors should perform a detailed quantification analysis of the ubiquitination levels for all mutants. Additionally, the authors should validate whether HRD1 also ubiquitinates TLR3 at K746 and consider generating a double mutant (TLR3 K746R/K813R) if necessary to fully assess HRD1's role in TLR3 ubiquitination.\nWe thank the reviewer for this great comment. We performed three biologically independent repeats of this experiment, and a more representative dataset is shown in Response Figure 13a. Quantification of ubiquitination levels for all mutants across the three repeats is presented in Response Figure 13b. Furthermore, we generated a TLR3 K746R/K813R double mutant, which did not show a further decrease in ubiquitination levels compared to the TLR3 K813R single mutant (Response Figure 13c). These findings further support our model that HRD1 ubiquitinates TLR3 at K813. The data from Response Fig 13a-b have now been incorporated into Fig S6b-c in the revised manuscript.\nResponse Figure 13. HRD1 ubiquitinates TLR3 at K813R. a-b, HEK293T cells were transfected with the indicated plasmids for 24h and subjected to immunoprecipitation followed by immunoblot analysis (a) and quantitation from three independent experiments (b). c, HEK293T cells were transfected with the indicated plasmids for 24h and subjected to immunoprecipitation and immunoblot analysis. Data are representative of three independent repeats. Values represent mean ± s.e.m. NS, not significant; *P < 0.05 by unpaired, two-tailed Student’s t-test (b).\n13. Since previous studies have demonstrated that TRIM3 ubiquitinates TLR3 at the same lysine residue (K813) to regulate its trafficking to endolysosomes and subsequent immune responses (PMID: 32878999), the data presented in Fig. 7 are not novel and do not adequately support the claim that HRD1 modulates TLR3 trafficking and inflammatory response through ubiquitination at the K813 residue. The authors should investigate whether the depletion of HRD1 in TLR3K813R knock-in cells results in any further reduction of TLR3-mediated signaling and cytokine production.\nWe thank the reviewer for this comment. It has been reported that TRIM3, which localizes to the Golgi apparatus and endosomes, ubiquitinates TLR3 at K8314. In contrast, our findings reveal that HRD1 targets a different site—K813—for TLR3 ubiquitination in the ER. Additionally, our data show that knockdown of Trim3 further decreases the expression of inflammatory genes upon poly(I:C) stimulation in Hrd1 deficiency cells (manuscript Fig S6b). Thus, our findings suggest a novel regulatory mechanism for TLR3 trafficking and highlight a previously unrecognized role of the ER-resident E3 ligase HRD1 in innate immune regulation.\nMinor comments:\n1. The authors utilized LS-102 to inhibit HRD1 ligase activity in this manuscript. However, does LS-102 specifically target HRD1? It may be beneficial for the authors to generate an enzymatically inactive form of HRD1 (C291A) as previously described (PMID: 19864457) and evaluate its impact on TLR3 trafficking and signaling.\nWe thank the reviewer for this comment. LS-102 has been used in previous studies as a specific inhibitor of HRD1 E3 ligase activity 20-23. To further verify the specificity of LS-102, we treated HRD1 knockout cells and found that LS-102 had no effect on TLR3-mediated inflammatory gene expression in these cells (manuscript Fig S2e), further confirming its specificity. Additionally, the enzymatically inactive HRD1 RING-finger mutant C2A (C291A/C294A)12, 19 has been used throughout our studies to evaluate its impact on TLR3. This mutant is now described in the revised manuscript (line 266).\n2. In Fig. S2e, poly(I:C)-induced NF-κB promoter activity was only marginally reduced in HRD1-deficient cells compared to WT cells, which contrasts with the authors' claim of significant reduction. Additionally, since TLR3-driven type I IFN production plays a critical role in antiviral immune responses, the authors should also perform an interferon promoter-driven luciferase reporter assay in HRD1-deficient cells to more comprehensively assess the impact of HRD1 on TLR3 signaling.\nWe thank the reviewer for this insightful comment. In response, we have performed interferon promoter-driven luciferase reporter assays. Our results show that HRD1 enhances TLR3-driven type I IFN activity (Response Figure 14a-b). These findings have been incorporated into Fig S2h and FigS 3e in the revised manuscript.\nResponse Figure 14. HRD1 enhances TLR3-driven type I IFN activity. a, Measurement of interferon-stimulated response element (ISRE) transcriptional activity by luciferase-based reporter assays in Hrd1+/+ and Hrd1-/- TLR3-stable HEK293T cells treated with or without poly(I:C) for 12 h (n=3 per group). b, Measurement of ISRE transcriptional activity by luciferase-based reporter assays in Hrd1-/- HEK293T cells stably expressing TLR3, transfected with or without the indicated HRD1 plasmids and treated with poly(I:C) for 12 h (n=3 per group).. Data are representative of three independent repeats. Values represent mean ± s.e.m. NS, not significant; ***P < 0.001; ****P < 0.0001, by unpaired, two-tailed Student’s t-test.\nReviewer #3 (Remarks to the Author):\nThis is an interesting and comprehensive study showing through several sound approaches that ubiquitination of TLR3 by HRD1 regulates TLR3 trafficking and function and that this is unexpectedly not through the ERAD pathway. The following concerns should be addressed.\nWe thank the reviewer for their insightful comments. We have now addressed all the issues raised below, which has significantly improved our work and strengthened our model.\n-Numbering of lines and/or pages, which are missing, would have made the reviewer’s life easier.\nWe thank the reviewer for this helpful comment. Line numbering has been added in the revised manuscript.\n1) The title “The HRD1 quality control pathway is essential for TLR3-mediated innate immunity by regulating its trafficking “does not match well the findings, because the authors show involvement of HRD1 ubiquitination, but not its quality control activity and targeting to ERAD. They don’t find involvement of other proteins of this pathway either. I would suggest “Ubiquitination by HRD1 is essential for…..” or similar.\nWe thank the reviewer for this valuable comment. We have revised the title to “Ubiquitination by HRD1 Is Essential for TLR3 Trafficking and Its Innate Immune Signaling”.\n2) Fig. 1a. The immunoprecipitation seems to have a high non-specific background. Details are missing in the figure legend and/or Methods describing the analysis, what cutoff was used for the difference between Hrd1+/+ and -/-, fold differences, etc.\nAn immunoblot with anti-HRD1 comparing the two cell lines should be shown to demonstrate the knockout.\nWe thank the reviewer for this great comment. We have now provided additional details regarding the analysis of IP-MS data in the Methods section titled \"IP-MASS.\" Briefly, the resulting MS/MS data were processed using Proteome Discoverer software v2.5 (Thermo Fisher). Tandem mass spectra were searched against the SwissProt Mouse database using the SEQUEST algorithm. Proteins and peptide spectrum matches (PSMs) were filtered using a maximum FDR of 1%, which constituted the complete list of proteins identified from the respective Hrd1+/+ and Hrd1-/- immunoprecipitates.\nTo identify HRD1-specific binding proteins, we excluded proteins found in both Hrd1+/+ and Hrd1-/- immunoprecipitates. Given that HRD1 localizes to the ER, we focused on selecting ER-resident proteins that interact with HRD1. Figure 1a shows the top ten specific HRD1-interacting ER proteins, ranked by their sum PEP score. The sum PEP score is calculated based on the posterior error probability (PEP) values of the PSMs, with PEP indicating the likelihood that an observed PSM is a random event. The sum PEP score is computed as the negative logarithm of the PEP values from the associated PSMs. To provide greater clarity, we have included a supplementary table detailing these top ten hits. The IP-MASS section in the supplementary materials of the current manuscript has been updated accordingly.\nProtein Exp. q-value Sum PEP Coverage Peptides PSMs\nScore [%]\nHRD1 0 180.375 23 16 45\nSEL1L 0 168.414 38 18 55\nOS9 0 133.929 31 15 43\nTLR3 0 128.951 28 21 55\nHADHB 0 87.176 32 11 18\nERLEC1 0 86.236 36 10 17\nACTR1A 0 67.255 31 8 13\nGDI2 0 54.742 35 11 15\nDNASE1L1 0 42.286 32 7 17\nALB 0 33.077 7 5 15\nIn addition, we have included immunoblot data comparing HRD1 protein levels in WT and Hrd1-/- RAW 264.7 cell lines (Response Figure 15). These data have now been incorporated into Fig S1a in the revised manuscript.\nResponse Figure 15, Immunoblot analysis of HRD1 in WT and Hrd1-/- RAW 264.7 cells, representative of three independent repeats.\n3) Fig. 1c. Why did treatment with poly(I:C) lead to a higher yield of HRD1 pulldown when exogenously expressed myc-tagged HRD1 is expressed?\nWe sincerely appreciate the reviewer's insightful comment. Our results indicate that poly(I:C) treatment enhances the interaction between HRD1 and TLR3 in RAW 264.7 cells, with a noticeable increase observed as early as 0.5 hours after treatment (manuscript Figure 1d)—prior to the upregulation of TLR3 protein levels. Similarly, in HEK293T cells transfected with TLR3, poly(I:C) stimulation activates TLR3, leading to an increased physical interaction between HRD1 and TLR3. As a result, HRD1 pull-down efficiency is higher, even though the overall HRD1 protein level remains unchanged due to the exogenous expression of Myc-tagged HRD1.\n4) Section titled “HRD1 positively regulates TLR3-mediated immune signaling”\nIt should be explained why different cell types were used.\nAs Hrd1-/- increases BiP significantly (Fig. 2d), all the results in this section could be explained by an indirect effect of the UPR. Therefore, it should be mentioned at this stage that this effect is not due to the UPR, as shown later.\nWe thank the reviewer for this insightful comment. In this section, we primarily used WT and Hrd1-/- RAW 264.7 macrophages to examine TLR3-mediated immune signaling, including inflammatory gene expression, downstream effector activation, and cytokine secretion. Primary macrophages were also included, particularly in experiments involving LS-102 treatment and HRD1 knockdown via siRNA, as these cells are more physiologically relevant. Additionally, we used 293T cells to demonstrate that HRD1’s regulation of TLR3 signaling is a general mechanism, not limited to professional innate immune cells or mouse cells, but also applicable to non-immune human cells. We have clarified this point in the revised manuscript, specifically in lines 156-158. Furthermore, we added a sentence in lines 170-172 to clarify that although Bip expression is increased in Fig 2d, the reduced inflammatory gene expression is not due to UPR activation, as demonstrated later.\n5) Fig. 4C, the TLR3 blot is overexposed, so it cannot be quantified.\nWe thank the reviewer for this insightful comment. We have repeated the experiment, and the immunoblot data have been updated with representative results using normal exposure setting (Response Figure 16). These updated data have now been incorporated into Fig 4c in the revised manuscript.\nResponse Figure 16. HRD1 does not affect TLR3 protein levels. Immunoblot analysis of indicated proteins in HEK293T cells stably expressing TLR3, transfected with increasing amounts of plasmid encoding HRD1 protein, Data are representative of at least three independent repeats.\n6) “Taken together, HRD1-mediated ubiquitination of TLR3 primarily occurs through the K48 and K63 residues of ubiquitin, in contrast to lysine(K) 27-linked ubiquitination that leads to HRD1 substrates degradation…” HRD1 can use K27 but it can also use K48 for targeting to ERAD.\nWe thank the reviewer for this insightful comment. We have revised the text to: “HRD1-mediated ubiquitination of TLR3 primarily occurs through the K48 and K63 residues of ubiquitin, in contrast to lysine(K) 27-linked ubiquitination observed in other HRD1 substrates, such as STING and CREBH” in the revised manuscript (lines 289-291).\n7) The English is in general good but I spotted a few errors of grammar and wording. The text should be edited by a native English speaker.\nWe thank the reviewer for this comment. We apologize for the grammatical and wording errors and have carefully reviewed and revised the text accordingly.\nReviewer #4 (Remarks to the Author):\nIn the manuscript entitled \"The HRD1 Quality Control Pathway is Essential for TLR3-Mediated Innate Immunity by Regulating Its Trafficking,\" the authors propose a hypothesis to explain the mechanisms enabling the intracellular transport and cleavage of TLR3 to regulate the innate immune response. Central to this hypothesis is HRD1, a protein residing in the endoplasmic reticulum and a member of the ERAD machinery that ubiquitinates and targets proteins for proteasomal degradation. The authors claim that in the case of TLR3, HRD1 labels TLR3 for activation rather than degradation, despite functioning as an E3 ubiquitin ligase in the context of ERAD. While the hypothesis is intriguing, the authors fail to demonstrate that the ubiquitin activation of TLR3 is attributable to HRD1:\nWe thank the reviewer for these comments. We have now addressed all the issues raised below, which has significantly improved our work and strengthened our model. Additionally, we carried out a new experiment to demonstrate that TLR3 can be ubiquitinated by HRD1 in vitro (please refer to response Fig 7).\n1. The authors assert that the impairment \"is caused by a marked reduction in TLR3 trafficking from the Golgi to endolysosomes and its subsequent proteolytic cleavage.\" However, the experiments showing a) intracellular localization by immunofluorescence, b) isolation of the ER, Golgi, and endosomes, and c) glycosylation of TLR3 are unconvincing. First, the co-localization studies do not demonstrate an increase in endosomal trafficking; the Mander’s coefficient shows only weak significance. The isolation of organelles appears to lack resolution, and TLR3 is not found diminished in endosomes. The EndoH and PNGase experiments did not reveal marked differences in TLR3 in the absence of HRD1. None of these experiments were performed with at least three biological replicates, and the blots were not quantified or shown with error bars.\nWe thank the reviewer for this insightful comment. In the previous manuscript, we did not include a direct comparison to demonstrate increased TLR3 colocalization with endosomes following poly(I:C) treatment. In this revision, we have conducted such an analysis, and our data clearly show that TLR3 colocalization with the endosomal marker LAMP1 significantly increases in WT cells at both 15 and 60 minutes post-treatment, whereas in Hrd1-/- cells, only a slight increase is observed at 60 minutes (Response Figure 17a, Manuscript Figure 6f). Furthermore, the colocalization of TLR3 with the endosomal marker is significantly higher in WT cells than in Hrd1-/- RAW 264.7 cells at both time points (Response Figure 17a, Manuscript Figure 6f). Additionally, reanalysis of our data revealed a significant increase in endosomal trafficking of TLR3 in WT 293T cells following poly(I:C) stimulation, along with a clear difference in endosomal trafficking between Hrd1+/+ and Hrd1-/- 293T cells (Response Figure 17b, Manuscript Figure S5j–k). We also clarified the text in the revised manuscript (lines 318 and 323).\nRegarding organelle isolation, we conducted multiple experiments to optimize quality. Our new data showed that the protein levels of both full-length and cleaved TLR3 in endosomes are reduced in Hrd1⁻/⁻ cells (please refer to Response Fig12). Additionally, we repeatedly observed a clear reduction in uncleaved TLR3 K813R within endosomesas compared to WT TLR3 after poly(I:C) treatment (Response Figure 17c, Manuscript Figure 7e).\nRegarding the EndoH and PNGase experiments, four biologically independent repeats consistently showed that the amount of post-ER uncleaved TLR3 was reduced in Hrd1⁻/⁻ macrophages compared to WT cells. The quantification of this reduction is provided above in the response to Reviewer 2, point 4 (Response Figure 6b, Manuscript Fig S5m).\nTogether, these results support the model that HRD1 regulates TLR3 trafficking to endolysosomes, thereby affecting its subsequent proteolytic cleavage. All confocal images and immunoblot data shown are representative of at least three biologically independent replicates, and key blots have been quantified in the manuscript where appropriate.\nResponse Figure 17. HRD1 regulates TLR3 trafficking to endolysosomes. a, Quantitation of TLR3 colocalization with lysosomes in WT and Hrd1−/− RAW 264.7 cells treated with or without poly(I:C) for the indicated times. Mander's overlap coefficient was used to measure colocalization. b, Quantitation of TLR3 localization in early and late endosomes in WT and Hrd1−/− HEK293T cells transfected with TLR3-Flag plasmid, with or without poly(I:C) treatment for the indicated times. c, Immunoblot analysis of the indicated proteins following subcellular fractionation of the ER, Golgi, and endosomes from HEK293T cells stably expressing WT and K813R TLR3, treated with poly(I:C) (50 μg/ml). Relative quantitation of TLR3 bands in indicated organelles shown below the gel. Values represent mean ± s.e.m. *P < 0.05, ***P < 0.001, ****P < 0.0001, by unpaired, two-tailed Student’s t-test. Data in (a-b) are pooled from three independent experiments; (C) is representative of three biologically independent repeats.\n2. The next claim, \"Importantly, HRD1's role in TLR3 trafficking is decoupled from its canonical ERAD activity and the ER stress sensor inositol-requiring enzyme 1 alpha (IRE1α),\" is not demonstrated. Most of the experiments intended to show that HRD1 absence does not induce ERAD reduction or UPR activation were conducted only once, lacking replicates, quantitation, and other critical analyses. Additionally, the proteins tested were arbitrarily chosen, and IRE1α was not quantified at all.\nWe thank the reviewer for this insightful comment. We have conducted additional experiments to further demonstrate that HRD1's role in TLR3 regulation is independent of its canonical ERAD activity, UPR, and IRE1α (please refer to response to Reviewer 2, points 9 and 10; Response Figures 10 and 11). These experiments were performed with three biological replicates and included with appropriate control groups, quantification, and critical analysis of the UPR, as shown in Response Figures 10 and 11. Most of these findings have been incorporated into the revised manuscript (Figure 4d–g), further strengthening our hypothesis.\n3. The remaining figures in the manuscript were superficially executed. Western blot experiments were performed a maximum of twice, were not shown to be reproducible, and were not quantified or represented with error bars.\nWe appreciate the reviewer’s comment. All Western blot experiments were performed at least three times, and the number of replicates is provided in the figure legends of the manuscript. The original data demonstrating reproducibility are now provided. Additionally, the essential Western blot data have been quantified."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed all my previous concerns. The manuscript is much improved and acceptable for publication in Nat Commun.", "reviewer_2": "The revised manuscript by Zhao L et al. entitled 'Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling' has addressed most of the comments from the previous review. However, there are still a few comments, as outlined below:\n1. In Response Figure 9, the authors should assess the levels of both full-length and cleaved TLR3 at various time points following poly(I:C) treatment in the presence of a protein synthesis inhibitor (CHX), in order to draw a definitive conclusion about the role of HRD1 in regulating TLR3 protein stability during inflammation.\n2. Several studies have reported that TLR3 is delivered to endosomes or endolysosomes in HEK293T cells without undergoing proteolytic cleavage (J Biol Chem. 2012 Sep 21;287(39):32617–29; EMBO J. 2006 Jul 26;25(14):3335–46; Immunity. 2006 Feb;24(2):153–63). Additionally, HEK293T cells express low levels of UNC93B1, and studying the trafficking of endosomal TLRs in this system typically requires UNC93B1 overexpression. Therefore, HEK293T cells are not an appropriate model to investigate TLR3 trafficking and cleavage. The authors should confirm the impact of HRD1 on TLR3 trafficking and cleavage using a more physiologically relevant system, such as macrophages or mouse embryonic fibroblasts (MEFs). Therefore, the authors should carry out Fig. 6g-n and 7c-e in macrophages or MEFs.\n3. In Response Figure 12, the authors state that they were unable to detect endogenous cleaved TLR3. This is likely due to the fact that most commercial TLR3 antibodies recognize epitopes within the N-terminal region, which is lost upon TLR3 maturation. To properly assess the cleaved form of TLR3, the authors should generate a knock-in (KI) model with a C-terminal tag on TLR3.\n4. In the new Figure 7c, neither the WT nor the TLR3(K813R) mutant shows an increase in the cleaved TLR3 band following poly(I:C) treatment, which again demonstrate TLR3 in HEK293T cells does not undergo properly proteolytic cleavage. Additionally, the blot appears to be overexposed; the authors should provide a quantitative analysis of the cleaved-to-full-length TLR3 ratio to clearly illustrate the impact of K813 ubiquitination on TLR3 processing. Moreover, in Figure 7d, the cleaved TLR3 bands appear similar between WT and K813R (lanes 1 and 2), which raises concerns about the functional significance of the mutation. The authors should clarify this observation. Finally, in Figure 7e, the upper blot should display both full-length and cleaved TLR3 bands, as was done in Figure 6n, to allow for proper comparison and interpretation.\n5. Does HRD1-mediated ubiquitination of TLR3 affect its interaction with UNC93B1, and consequently influence TLR3 trafficking?\n6. The authors should include detailed experimental conditions—such as incubation times and treatment durations—in each figure legend to ensure reproducibility and clarity.\n7. The in vitro ubiquitination data presented in Response Figure 7 are important for supporting the conclusions and should be incorporated into the main manuscript.", "reviewer_3": "The manuscript is significantly improved. Only one of my concerns has still not been answered satisfactorily:\n5) Fig. 4C, the TLR3 blot is overexposed, so it cannot be quantified.\nAuthor: We thank the reviewer for this insightful comment. We have repeated the experiment, and\nthe immunoblot data have been updated with representative results using normal exposure\nsetting (Response Figure 16). These updated data have now been incorporated into Fig\n4c in the revised manuscript\nReviewer: The anti-TLR3 blot is still overexposed, showing too strong saturated detection, so it cannot be stated that HRD1 does not affect TLR3 protein levels. In addition, a quantification of the blot should be shown to make that statement.", "reviewer_4": "The revised manuscript, now titled “Ubiquitination by HRD1 Is Essential for TLR3 Trafficking and Its Innate Immune Signaling,” focuses on the mechanisms regulating TLR3-mediated immune responses. Specifically, it proposes that TLR3 activation is determined at the endoplasmic reticulum (ER) by HRD1-mediated ubiquitination. According to the authors, HRD1—known to function in complex with SEL1L to mediate proteasomal degradation of ER-synthesized proteins—does not participate in the ER-associated degradation (ERAD) of TLR3. Instead, HRD1 is suggested to facilitate the trafficking of TLR3 to post-Golgi compartments, where it is cleaved into its active form.\nAlthough the revised version includes some new experiments and several repetitions of prior ones, the manuscript fails to demonstrate a consistent molecular mechanism by which HRD1 regulates TLR3 cleavage into its signalling-competent form. Rather, in the absence of HRD1, TLR3 appears to be degraded prior to cleavage, possibly via alternative degradation pathways such as autophagy or lysosomal processing.\nDue to the lack of a clear mechanistic understanding of how HRD1 deficiency impacts TLR3 signaling, this manuscript is not suitable for publication in Nature Communications.\nMore specifically:\n• The experiments assessing TLR3 expression and turnover in the absence of HRD1 or SEL1L are unconvincing.\no For SEL1L, only a single (non-replicated) experiment is shown (Response Figure 2). At 6 hours after cycloheximide (CHX) treatment, TLR3 levels are reported to be higher than at 0 and 3 hours, which is not plausible. Moreover, SEL1L’s half-life is reportedly approximately 3 hours, not 9 hours as stated by the authors. This experiment is unclear and more likely reflects degradation of SEL1L in the absence of HRD1, as previously reported.\n• Although the authors propose that HRD1 facilitates TLR3 cleavage in endosomes, their data only show that uncleaved TLR3 is degraded more rapidly in HRD1 deficient cells.\n• Figure 4b suggests that TLR3 degradation is accelerated in the absence of HRD1, with a half-life difference of at least 1 hour (7.5 h vs. >9 h), in contrast with authors conclusion. Furthermore, the three supplementary replicates for Figure 4b are inconsistent and contain unspecified background bands.\n• Regarding EndoH and PNGase experiments: not all replicates (we do not know whether they are biological replicates or just repeats) display the cleaved form of TLR3, indicating poor validation of results. More importantly, the ratio of uncleaved to cleaved TLR3 is unchanged across all conditions, suggesting no HRD1-specific effect. Furthermore, the half-life of TLR3 in HRD1 KO cells is shown in Response Figure 6c to be reduced from ~6 h to ~3 h, but the quantification in Figure 6d is inaccurate.\n• Figure 6n: only 2 out of 3 replicates show cleaved TLR3 in endosomes. In Figure 7e, the three replicates show inconsistent electrophoretic patterns.\n• The role of IRE1 is not clearly addressed. There is no quantification of phosphorylated IRE1 or PERK, which are the active forms involved in the unfolded protein response (UPR) and ERAD. A comparison of phosphorylated IRE1 in cells with and without HRD1 could reveal altered signaling, but this was not performed.\n• TLR3 folding is not demonstrated in the manuscript, despite the authors' claim: “We then tested whether HRD1 promotes TLR3 signaling by regulating the folding and abundance of TLR3 protein in the ER.”"}, "author_response": "We sincerely thank all four reviewers for their insightful and constructive comments. We have carefully addressed each of their comments, which have been instrumental in enhancing and strengthening our manuscript.\nReviewers' Comments:\nReviewer #1 (Remarks to the Author):\nThe authors have adequately addressed all my previous concerns. The manuscript is much improved and acceptable for publication in Nat Commun.\nWe sincerely thank this reviewer for his/her positive feedback and constructive comments.\nReviewer #2 (Remarks to the Author):\nThe revised manuscript by Zhao L et al. entitled 'Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling' has addressed most of the comments from the previous review. However, there are still a few comments, as outlined below:\nWe sincerely thank this reviewer for his/her positive feedback and constructive comments, which have greatly contributed to further improving our manuscript.\n1. In Response Figure 9, the authors should assess the levels of both full-length and cleaved TLR3 at various time points following poly(I:C) treatment in the presence of a protein synthesis inhibitor (CHX), in order to draw a definitive conclusion about the role of HRD1 in regulating TLR3 protein stability during inflammation.\nWe thank the reviewer for emphasizing the need to examine both the full-length and the cleaved forms of TLR3. To address this, we repeated the cycloheximide (CHX)-chase assay in wild-type and HRD1-deficient MEF cells stably expressing TLR3-Flag and monitored the decay of each species side-by-side: Cells were exposed to poly(I:C) in the presence of CHX for 0, 3, 6 h, and 9h. Whole-cell lysates were immunoblotted with anti-Flag antibody to show both the ~130 kDa full-length receptor and the ~80 kDa cleaved fragment (new Response Fig. 1).\nIn agreement with our previous data (1st-round Response Fig. 6), the full-length protein decayed at similar rates in WT and HRD1-deficient cells (new Response Fig. 1b). To avoid confusion, we wish to clarify that poly(I:C) stimulation alone increases TLR3 expression at both the mRNA and protein levels (1st-round Response Fig. 9a). However, the co-addition of CHX effectively blocks new synthesis of TLR3, resulting in decreased TLR3 protein levels at later time points when both poly(I:C) and CHX are present. Importantly, the cleaved TLR3 fragment also displayed a comparable decay pattern, as shown by normalization to its own 0 h time point in each genotype (new Response Fig. 1c). These findings demonstrate that HRD1 doesn’t regulate TLR3 signaling via regulating its turnover.\nResponse Figure 1. HRD1 does not regulate the turnover of both full-length and cleaved TLR3 during poly(I:C) activation. a, Wild-type and HRD1-deficient MEF cells stably expressing TLR3-Flag were treated with poly(I:C) (50 μg/ml) in the presence of CHX (50 μg/ml) at various time points, and the turnover of both full-length (FL) and cleaved TLR3 was analyzed using anti-Flag antibody. b-c, The quantitation of FL (b) and cleaved TLR3 (c) from three independent experiments. Values are represented as mean ± s.e.m. NS, not significant, by unpaired, two-tailed Student’s t-test (b, c).\n2. Several studies have reported that TLR3 is delivered to endosomes or endolysosomes in HEK293T cells without undergoing proteolytic cleavage (J Biol Chem. 2012 Sep 21;287(39):32617–29; EMBO J. 2006 Jul 26;25(14):3335–46; Immunity. 2006 Feb;24(2):153–63). Additionally, HEK293T cells express low levels of UNC93B1, and studying the trafficking of endosomal TLRs in this system typically requires UNC93B1 overexpression. Therefore, HEK293T cells are not an appropriate model to investigate TLR3 trafficking and cleavage. The authors should confirm the impact of HRD1 on TLR3 trafficking and cleavage using a more physiologically relevant system, such as macrophages or mouse embryonic fibroblasts (MEFs). Therefore, the authors should carry out Fig. 6g-n and 7c-e in macrophages or MEFs.\nWe thank the reviewer for this insightful comment. Although endosomal TLRs were known by imaging studies to localize to endosomes as early as 2006, the earliest reports demonstrating that endosomal TLRs undergo proteolytic cleavage for activation came later—for TLR9 in 2008(1) and for TLR3 in 2011(2) .\nTherefore, the studies published in EMBO J. 2006 and Immunity 2006 should be interpreted in the context that the proteolytic processing of TLR3 had not yet been discovered at that time, rather than as evidence that TLR3 is delivered to endosomes or endolysosomes in HEK293T cells without undergoing cleavage. Indeed, subsequent studies have shown that TLR3 in HEK293 cells can undergo proteolytic cleavage (3, 4).\nTo further establish the physiological relevance of HRD1, we repeated the key assays in wild-type and HRD1-knock-out mouse embryonic fibroblasts (MEFs) stably expressing FLAG-tagged TLR3. The new data show that HRD1 loss or inhibition impairs both TLR3 cleavage (new Response Fig. 2a-b) and trafficking (new Response Fig. 2c-d), fully recapitulating the effects originally observed in HEK293T cells (Fig. 6k–n). For Fig. 6g-j, data obtained from macrophage cells are already shown in Fig. 6a–f. For Fig. 7c-e, please refer to new response Fig.3 below.\nCollectively, published work and our new data from MEF cells demonstrate that (i) HEK293T cells are a valid model for studying TLR3 cleavage, and (ii) HRD1 is indispensable for the physiological maturation of TLR3 across cell types. We have incorporated the data of new response Fig.2 into the revised manuscript (supplementary Fig. S6d-g).\nResponse Figure 2. HRD1 promotes TLR3 Trafficking into endolysosome for cleavage in MEF cells. a, Immunoblot analysis of FL and cleaved TLR3 in WT and Hrd1−/− MEF cells stably expressing TLR3-Flag treated with 50 μg/ml poly(I:C) for 12h. Quantification of full-length and cleaved TLR3 levels, normalized to WT cells is shown on the right. b, Immunoblot analysis of FL and cleaved TLR3 in WT MEF cells stably expressing TLR3-Flag pretreated with LS-102 (10 μM) and sequentially treated with 50 μg/ml poly(I:C) for 12h. Quantification of full-length and cleaved TLR3 levels, normalized to untreated cells is shown on the right. c, Cells were treated as in (a) before preparation of cell lysates, and immunoblot analysis of TLR3-Flag in these cell lysates treated with or without Endo H or PNGase F. Arrow, Endo H-sensitive; Red box, Endo H-resistant. Quantitation of Endo H resistant TLR3, normalized to WT cells is shown on the right. d, Immunoblot analysis of indicated proteins following the isolation of the ER, Golgi and endosomes from WT and Hrd1−/− MEF cells stably expressing TLR3-Flag treatment with 50 μg/ml poly(I:C) for 60 minutes. FL: full length TLR3; CL: cleaved TLR3. All these data are representative of three independent repeats. Values represent mean ± s.e.m. NS, not significant; **P < 0.01; ***P < 0.001, by unpaired, two-tailed, Student’s t-test.\n3. In Response Figure 12, the authors state that they were unable to detect endogenous cleaved TLR3. This is likely due to the fact that most commercial TLR3 antibodies recognize epitopes within the N-terminal region, which is lost upon TLR3 maturation. To properly assess the cleaved form of TLR3, the authors should generate a knock-in (KI) model with a C-terminal tag on TLR3.\nWe thank the reviewer for this insightful comment. To assess the physiological relevance of HRD1 in regulating TLR3 trafficking, we performed experiments using wild-type and HRD1-deficient MEF cells stably expressing TLR3 with a C-terminal FLAG tag, thereby enabling detection of the cleaved C-terminal fragment in endosome. As shown in the new Response Fig. 2c–d, HRD1 deficiency markedly reduced the levels of Endo H–resistant full-length TLR3, as well as both full-length and cleaved TLR3 localized in the endosome. Together with our confocal colocalization studies of endogenous TLR3 (Fig. 6a–f), which revealed impaired trafficking of TLR3 into endosomes in HRD1-deficient cells, these findings strongly support a critical role for HRD1 in facilitating proper TLR3 endosomal localization.\n4. In the new Figure 7c, neither the WT nor the TLR3(K813R) mutant shows an increase in the cleaved TLR3 band following poly(I:C) treatment, which again demonstrate TLR3 in HEK293T cells does not undergo properly proteolytic cleavage. Additionally, the blot appears to be overexposed; the authors should provide a quantitative analysis of the cleaved-to-full-length TLR3 ratio to clearly illustrate the impact of K813 ubiquitination on TLR3 processing. Moreover, in Figure 7d, the cleaved TLR3 bands appear similar between WT and K813R (lanes 1 and 2), which raises concerns about the functional significance of the mutation. The authors should clarify this observation. Finally, in Figure 7e, the upper blot should display both full-length and cleaved TLR3 bands, as was done in Figure 6n, to allow for proper comparison and interpretation.\nWe thank the reviewer for the insightful comment on Figure 7c. We have now repeated the experiment in MEF cells stably expressing either wild-type TLR3 or the K813R mutant. The new Response Fig 3a show that cleavage of WT TLR3 increases following 30 minutes of poly(I:C) stimulation, whereas cleavage of the K813R mutant is delayed and only becomes notable after 60 minutes. These results suggest that lysine 813 is critical for efficient TLR3 processing. And the data has been incorporated into Fig. 7c and Fig. S8e of revised manuscript.\nWe thank the reviewer for the insightful comment on Figure 7d. We have repeated the experiment in MEF cells as suggested by the reviewer, densitometric quantification comparing cleaved TLR3 levels between WT and K813R (lanes 1 vs. 2) revealed a ~65 % reduction in cleavage in the K813R mutant, suggesting that lysine 813 is essential for proper TLR3 maturation (new Response Fig. 3b). Furthermore, the K813R mutant exhibited impaired trafficking compared to WT, as indicated by the reduction of Endo H–resistant full-length TLR3 in the K813R mutant (red square) (new Response Fig. 3b). And the data has been incorporated into Fig. 7d and Fig. S8f of revised manuscript.\nWe thank the reviewer for the insightful comment on Figure 7e . We have now shown both the full-length and cleaved TLR3 bands (new Response Fig. 3d). And the data has been incorporated into Fig. 7e of revised manuscript.\nResponse Figure 3. Lysine 813 is essential for TLR3 trafficking. a, Immunoblot analysis of full- length (FL) and cleaved (CL) TLR3 in MEF cells stably expressing Flag-tagged WT TLR3 or the K813R mutant (a). Cells were treated with 50 μg/ml poly(I:C) for the indicated durations. b, Quantification of the cleaved-to-full-length TLR3 ratio in a (n=4). c, MEF cells stably expressing Flag-tagged WT or K813R TLR3 were treated with poly(I:C) for 12h. Cell lysates were subsequently treated with or without Endo-H or PNGase F prior to immunoblot analysis of TLR3-Flag. Arrow, Endo H-sensitive; Red box, Endo H-resistant. d, Quantification of relative Endo-H resistant TLR3 in c is shown on the left, n=3; Quantification of relative CL TLR3 in c (lanes 1 vs 2) is shown on the right, n=3. e, Immunoblot analysis of the indicated proteins following subcellular fractionation (ER, Golgi, and endosomes) from MEF cells stably expressing WT or K813R TLR3 after 60 minutes of poly(I:C) treatment (50 μg/m). Values are presented as mean ± s.e.m. NS, not significant; **P < 0.01; ***P < 0.001 by unpaired, two-tailed, Student’s t-test.\n5. Does HRD1-mediated ubiquitination of TLR3 affect its interaction with UNC93B1, and consequently influence TLR3 trafficking?\nWe thank the reviewer for this insightful comment. To address this point, we performed Co-IP analyses in WT and HRD1 KO RAW 264.7 cells. As previously shown (Fig. 5c), HRD1 deficiency reduces TLR3 ubiquitination. In the new Response Fig. 4, we further demonstrate that HRD1 deficiency does not alter the interaction between TLR3 and UNC93B1, indicating that HRD1 does not regulate TLR3 trafficking through its association with the UNC93B1 protein.\nResponse Figure 4. HRD1 does not affect the interaction between TLR3 and UNC93B1. Co-IP of endogenous TLR3 was performed in WT and HRD1-knockout RAW 264.7 macrophages. Cell lysates were immunoprecipitated using an anti-TLR3 antibody, following by immunoblotting for the indicated proteins. IgG was used as an isotype control. Representative of three independent repeats.\n6. The authors should include detailed experimental conditions—such as incubation times and treatment durations—in each figure legend to ensure reproducibility and clarity.\nWe thank the reviewer for this insightful comment. We have revised the figure legends to include detailed experimental conditions.\n7. The in vitro ubiquitination data presented in Response Figure 7 are important for supporting the conclusions and should be incorporated into the main manuscript.\nWe thank the reviewer for this insightful comment. We have now incorporated the in vitro ubiquitination data into Fig. 5b of the revised manuscript.\nReviewer #3 (Remarks to the Author):\nThe manuscript is significantly improved. Only one of my concerns has still not been answered satisfactorily:\n5) Fig. 4C, the TLR3 blot is overexposed, so it cannot be quantified.\nAuthor: We thank the reviewer for this insightful comment. We have repeated the experiment, and the immunoblot data have been updated with representative results using normal exposure setting (Response Fig.16). These updated data have now been incorporated into Fig 4c in the revised manuscript.\nReviewer: The anti-TLR3 blot is still overexposed, showing too strong saturated detection, so it cannot be stated that HRD1 does not affect TLR3 protein levels. In addition, a quantification of the blot should be shown to make that statement.\nWe thank the reviewer for this comment. In response, we repeated the experiment with two additional replicates. The immunoblot data have been updated with representative results in revised Fig. 4c, and the accompanying quantitative analysis (new Response Fig. 5) indicates that HRD1 expression does not affect total TLR3 protein levels.\nResponse Figure 5. HRD1 expression does not alter TLR3 protein levels. a, HEK293T-TLR3 cells were transfected with increasing amounts of HRD1-expressing plasmid. Cell lysates were analyzed by Immunoblotting for the indicated proteins. Blots shown are representative of five independent experiments. b, Quantitative densitometry of TLR3 signal normalized to β-actin is plotted (mean ± s.e.m., n = 5). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. NS, not significant.\nReviewer #4 (Remarks to the Author):\nIn the manuscript entitled \"The HRD1 Quality Control Pathway is Essential for TLR3-Mediated Innate Immunity by Regulating Its Trafficking,\" the authors propose a hypothesis to explain the mechanisms enabling the intracellular transport and cleavage of TLR3 to regulate the innate immune response. Central to this hypothesis is HRD1, a protein residing in the endoplasmic reticulum and a member of the ERAD machinery that ubiquitinates and targets proteins for proteasomal degradation. The authors claim that in the case of TLR3, HRD1 labels TLR3 for activation rather than degradation, despite functioning as an E3 ubiquitin ligase in the context of ERAD. While the hypothesis is intriguing, the authors fail to demonstrate that the ubiquitin activation of TLR3 is attributable to HRD1:\nWe thank the reviewer for their thoughtful comments. Our study presents clear and conceptional advancement, as acknowledged by reviewers in the 1st round of evaluation: “this study reveals a novel role of HRD1 in the trafficking and maturation of the innate immune sensor TLR3 and a new mechanism for TLR3 activation, which advances our understanding of innate immune regulation”; “introduces a novel function for HRD1 in TLR3 ubiquitination and trafficking”; “This is an interesting and comprehensive study showing through several sound approaches that ubiquitination of TLR3 by HRD1 regulates TLR3 trafficking and function and that this is unexpectedly not through the ERAD pathway”.\nIn addition, the insightful and constructive feedback from all reviewers over two rounds of review has greatly improved the manuscript. The revised version now presents a coherent molecular mechanism and a well-supported model demonstrating that HRD1 acts as a positive regulator of TLR3 signaling. Specifically, HRD1-mediated ubiquitination of TLR3 facilitates its trafficking into endolysosomal compartments, thereby promoting its cleavage into the signaling-competent form. Notably, this modification does not significantly alter the stability or abundance of the full-length TLR3 precursor.\nMore specifically:\n• The experiments assessing TLR3 expression and turnover in the absence of HRD1 or SEL1L are unconvincing.\no For SEL1L, only a single (non-replicated) experiment is shown (Response Figure 2). At 6 hours after cycloheximide (CHX) treatment, TLR3 levels are reported to be higher than at 0 and 3 hours, which is not plausible. Moreover, SEL1L’s half-life is reportedly approximately 3 hours, not 9 hours as stated by the authors. This experiment is unclear and more likely reflects degradation of SEL1L in the absence of HRD1, as previously reported.\nWe thank the reviewer for this comment and the opportunity to clarify this point. However, we respectfully disagree with the reviewer’s interpretation for the following reasons:\nTLR3 turnover: Our data consistently show that TLR3 expression and turnover are not significantly altered by HRD1 deficiency (Fig. 4a-b), or by SEL1L deficiency (1st round Response Fig. 2). Data in 1st round Response Fig. 2b or d were from 5 or 3 biologically independent repeats respectively, as indicated. In our cycloheximide (CHX) chase experiments under SEL1L deficiency (1st round Response Fig. 2c-d), we observed a clear trend of progressive decrease in TLR3 protein levels over time in both wild-type and SEL1L-deficient cells. While individual data points show minor fluctuations—such as slightly higher levels at later time point compared to 0 h—we attribute these to inherent technical variability in Western blotting and CHX chase assays. Such fluctuations is not unusual and have been reported in high-profile studies including recent papers in Nature Communication(5-9). Importantly, these small variations do not alter the overall interpretation or the trend.\nSEL1L half-life: We agree with the reviewer that SEL1L stability is relevant in this context. However, extensive work by Dr. Ling Qi—a co-author of this study who has focused on SEL1L for over a decade—has consistently shown that SEL1L is a relatively stable protein , consistent with an estimated half-life of approximately 9 hours- rather than the 3 hours suggested (8, 10, 11).\nWe appreciate the opportunity to clarify these points and maintain that our data and interpretations are consistent with published literature and are experimentally robust.\n• Although the authors propose that HRD1 facilitates TLR3 cleavage in endosomes, their data only show that uncleaved TLR3 is degraded more rapidly in HRD1 deficient cells.\nWe thank the reviewer for this comment and appreciate the opportunity to clarify our conclusions. Our data (Fig. 6 and Fig.7) directly support a model in which HRD1 facilitates the trafficking of full-length TLR3 to endolysosomal compartments, thereby enabling its subsequent proteolytic cleavage into the signaling-competent form. In this way, HRD1 ensures a sufficient reservoir of TLR3 within endolysosomal compartments to support its efficient proteolytic maturation. We have carefully reviewed the manuscript to ensure that this mechanistic interpretation is conveyed clearly.\nRegarding the reviewer’s concern that “uncleaved TLR3 is degraded more rapidly in HRD1 deficient cells,” we acknowledge that this point is also raised in the following comment, and we will address it comprehensively in our subsequent response.\n• Figure 4b suggests that TLR3 degradation is accelerated in the absence of HRD1, with a half-life difference of at least 1 hour (7.5 h vs. >9 h), in contrast with authors conclusion. Furthermore, the three supplementary replicates for Figure 4b are inconsistent and contain unspecified background bands.\nWe acknowledge the reviewer’s comment and concern. It is important to note that cycloheximide (CHX) chase assays conducted over extended time periods can introduce variability due to cytotoxic effects from prolonged inhibition of protein synthesis, particularly when assessing the half-life of stable proteins. In our previous Fig. 4b, a modest acceleration of TLR3 degradation at the 9-hour time point was observed, which likely reflects technical limitations inherent to long-duration CHX chase assays. To address this, we performed two additional biologically independent replicates and have clarified this point in the Fig. 4b of revised manuscript.\n• Regarding EndoH and PNGase experiments: not all replicates (we do not know whether they are biological replicates or just repeats) display the cleaved form of TLR3, indicating poor validation of results.\nWe thank the reviewer for this comment. We confirm that all experiments presented are based on biological replicates. To address the concern regarding the detection of cleaved TLR3 in EndoH and PNGase F assays, we have added newly repeated biological replicates (Fig. 6m, quantification shown in Fig. S6c, in the revised manuscript), in which the cleaved form of TLR3 is clarified. Additionally, per the suggestion of reviewer 2, we repeated Endo-H and PNGase F experiments using more physiologically relevant MEF cells and observed the same change, see new response Fig. 2c (Fig. S6f in the revised manuscript) and new response Fig. 3b (Fig. 7d and Fig. S8f in the revised manuscript). These updated results further strengthen the validity and reproducibility of our findings.\nMore importantly, the ratio of uncleaved to cleaved TLR3 is unchanged across all conditions, suggesting no HRD1-specific effect. Furthermore, the half-life of TLR3 in HRD1 KO cells is shown in Response Figure 6c to be reduced from ~6 h to ~3 h, but the quantification in Figure 6d is inaccurate.\nWe thank the reviewer for the comment and the opportunity to clarify this point. With more rigorous quantification and statistical analysis, our updated data show that the levels of uncleaved TLR3 were unchanged, but cleaved TLR3 is reduced in HRD1 knockout cells (revised Fig. S6a) and in cells treated with HRD1 inhibitor (revised Fig. S6b). In addition, our updated quantitation data also show that the levels of cleaved TLR3 is reduced in cells with the K813R mutant (revised Fig. S8e). These findings are consistent with our proposed model that HRD1 facilitates TLR3 trafficking into endosome for subsequent TLR3 cleavage.\nRegarding the half-life analysis of TLR3 in HRD1-deficient cells (1st round Response Fig. 6c), we respectfully disagree with the reviewer’s interpretation. Our data indicate that TLR3 stability is not significantly altered by the absence of HRD1. Importantly, each time point in the CHX chase assay was normalized to its corresponding 0 h value for each genotype, ensuring accurate comparison of degradation kinetics. The quantification in 1st round Response Fig. 6d accurately reflects this normalization. The apparently lower TLR3 protein level at the 0 h time point in HRD1 KO cells—which may contribute to the confusion, was thoroughly addressed in our first-round response (see 1st Response Fig. 9).\n• Figure 6n: only 2 out of 3 replicates show cleaved TLR3 in endosomes. In Figure 7e, the three replicates show inconsistent electrophoretic patterns.\nWe thank the reviewer for the comment. While some variation was observed across biological replicates—likely due to technical and biological factors. As reviewer 2 also suggested to verify the data in Figure 6n and Figure 7e, particularly using physiologically relevant MEF cells, we have repeated these experiments accordingly (see new response Fig.2 and new response Fig.3). In addition, more replicates for Fig. 6n have been included. These updated data improve consistency and further support the reproducibility and clarity of our findings.\n• The role of IRE1 is not clearly addressed. There is no quantification of phosphorylated IRE1 or PERK, which are the active forms involved in the unfolded protein response (UPR) and ERAD. A comparison of phosphorylated IRE1 in cells with and without HRD1 could reveal altered signaling, but this was not performed.\nWe thank the reviewer for the insightful comment. In the Figure 4e of the manuscript, we have directly accessed the phosphorylation levels of Eif2α, the immediate downstream effector of PERK, in WT and Hrd1-/- cells treated with vehicle, thapsigargin (Tg), poly(I:C), or a combination of both. We have now used a Phos-tag gel to examine the phosphorylation status of IRE1 under these conditions (new response Fig. 6). Collectively, our data demonstrate that Hrd1-/- cells exhibit mild ER stress under basal conditions, as evidenced by a slight increase in eIF2α phosphorylation (Figure 4e) and p-IRE1 levels (new response Fig. 6) compared to WT cells. Treatment with Tg, but not poly(I:C), induced robust ER stress in both WT and Hrd1-/- cells, as indicated by a marked increase in phosphorylation of both eIF2α and IRE1 (Figure 4e and new response Fig. 6, respectively). These data further support the reproducibility and clarity of our findings.\nResponse Figure 6. HRD1 deficiency does not affect IRE1a activation. Samples were prepared as described in Fig. 4e. Phosphorylated IRE1a was detected using Phos-tag gel electrophoresis followed by immunoblotting. Representative of three independent repeats. p-, phosphorylated IRE1α (upper band in IRE1α phostag blot); t-, total IRE1α .\n• TLR3 folding is not demonstrated in the manuscript, despite the authors' claim: “We then tested whether HRD1 promotes TLR3 signaling by regulating the folding and abundance of TLR3 protein in the ER.”\nWe thank the reviewer for this insightful comment. We accessed the inter-molecular disulfide bond formation, solubility, aggregate formation of TLR3, which are directly influenced by its folding status, in our first-round response (see 1st Response Fig. 5b-c). However, we acknowledge that these analyses may not fully capture all aspects of TLR3 folding. In light of this, we have revised the wording in the manuscript to more accurately reflect our data and to avoid overinterpretation."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have addressed all comments raised in my review of the previous version of their manuscript.", "reviewer_3": "The authors have adequately addressed my concerns. The manuscript is acceptable for publication in Nat Commun.", "reviewer_4": "In the revised manuscript, the authors maintain their original claim that TLR3 activation is determined at the endoplasmic reticulum through HRD1-mediated ubiquitination. Thus, without HRD1, TLR3 would not be ubiquitinated, could not traffic to post-Golgi compartments, and would not be cleaved into its active form.\nHowever, their data merely show that before cleavage, TLR3 is degraded more rapidly in HRD1-deficient cells. Even with additional replicates, the degradation pattern remains the same as previously noted: TLR3 degradation is accelerated without HRD1, resulting in less cleaved (active) TLR3. However, the current experiments do not support these conclusions, as the observed degradation kinetics suggest that TLR3 is degraded before cleavage. This impaired processing is unrelated to altered post-ER trafficking or N-glycosylation, as the authors claim. The EndoH-resistant/EndoH-sensitive ratio is nearly identical in HRD1-deficient and control cells, indicating no HRD1-specific effect on glycosylation.\nMoreover, the authors were unable to reproduce the relevant experiments or clarify whether SEL1L is degraded in the absence of HRD1, which would in turn affect TLR3’s intracellular fate. The explanations concerning SEL1L turnover, or the toxicity of the cycloheximide (CHX) assay and lack of new experimental approaches fail to address the central issue: the absence of a mechanistic explanation for TLR3 inactivation.\nAs stated in previous review rounds, the lack of a clear mechanistic understanding of how HRD1 deficiency affects TLR3 signaling makes this manuscript unsuitable for publication in Nature Communications."}, "author_response": "We sincerely thank all the reviewers for their thoughtful comments and for the time dedicated to evaluating our manuscript. The constructive feedback provided throughout the review process has substantially enhanced the rigor and clarity of our study.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed all comments raised in my review of the previous version of their manuscript.\nWe sincerely thank this reviewer for the positive feedback and constructive comments.\nReviewer #3 (Remarks to the Author):\nThe authors have adequately addressed my concerns. The manuscript is acceptable for publication in Nat Commun.\nWe sincerely thank this reviewer for the supportive comments and for the helpful feedback across revisions.\nReviewer #4 (Remarks to the Author):\nIn the revised manuscript, the authors maintain their original claim that TLR3 activation is determined at the endoplasmic reticulum through HRD1-mediated ubiquitination. Thus, without HRD1, TLR3 would not be ubiquitinated, could not traffic to post-Golgi compartments, and would not be cleaved into its active form.\nHowever, their data merely show that before cleavage, TLR3 is degraded more rapidly in HRD1-deficient cells. Even with additional replicates, the degradation pattern remains the same as previously noted: TLR3 degradation is accelerated without HRD1, resulting in less cleaved (active) TLR3. However, the current experiments do not support these conclusions, as the observed degradation kinetics suggest that TLR3 is degraded before cleavage. This impaired processing is unrelated to altered post-ER trafficking or N-glycosylation, as the authors claim. The EndoH-resistant/EndoH-sensitive ratio is nearly identical in HRD1-deficient and control cells, indicating no HRD1-specific effect on glycosylation.\nMoreover, the authors were unable to reproduce the relevant experiments or clarify whether SEL1L is degraded in the absence of HRD1, which would in turn affect TLR3’s intracellular fate. The explanations concerning SEL1L turnover, or the toxicity of the cycloheximide (CHX) assay and lack of new experimental approaches fail to address the central issue: the absence of a mechanistic explanation for TLR3 inactivation.\nAs stated in previous review rounds, the lack of a clear mechanistic understanding of how HRD1 deficiency affects TLR3 signaling makes this manuscript unsuitable for publication in Nature Communications.\nWe thank the reviewer for the additional comments. All points raised in this round were already addressed in detail in our previous response letters. For clarity, we briefly reiterate the key evidences. Our data consistently show TLR3 stability is unchanged in HRD1-deficient cells, as shown in current manuscript Fig. 4b, 2nd round Response Figure 1 and 1st round Response Fig. 6c-d.\nThis impaired processing is unrelated to altered post-ER trafficking or N-glycosylation, as the authors claim. The EndoH-resistant/EndoH-sensitive ratio is nearly identical in HRD1-deficient and control cells, indicating no HRD1-specific effect on glycosylation.\nOur data show that the quantitation of EndoH-resistant TLR3 (post-ER form) is reduced in HRD1-deficient cells, consistent with impaired post-ER trafficking, as shown in 2nd round Response Fig. 2c-d or current manuscript Fig. S6f. Same pattern is also observed in TLR3 K813R mutant，as shown in 2nd round Response Fig. 3c-d or current manuscript Fig. 7d and Fig. S8f.\nMoreover, the authors were unable to reproduce the relevant experiments or clarify whether SEL1L is degraded in the absence of HRD1, which would in turn affect TLR3’s intracellular fate. The explanations concerning SEL1L turnover, or the toxicity of the cycloheximide (CHX) assay and lack of new experimental approaches fail to address the central issue: the absence of a mechanistic explanation for TLR3 inactivation.\nAs stated in previous review rounds, the lack of a clear mechanistic understanding of how HRD1 deficiency affects TLR3 signaling makes this manuscript unsuitable for publication in Nature Communications.\nOur previous study has shown that SEL1L protein levels are elevated in HRD1-deficient macrophages (Fig. 3c, Nat Cell Biol, 2023，PMID: 37142791), consistent with multiple published literatures which consistently shown that SEL1L is a stable protein. These points, including literature references, were already provided in detail in the prior round revision.\nCollectively, our data well support the mechanism that HRD1-mediated ubiquitination licenses TLR3 to exit the ER and reach endolysosomes for proteolytic processing. HRD1 deficiency results in TLR3 trafficking defect and reduction of the cleaved, signaling-competent TLR3 form.\nWe appreciate the reviewer’s engagement and respectfully note that the concerns raised in this round were fully addressed with the additional data and clarifications provided in the previous revision."}]

Decision letter