bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of CD34+CD10+CD19−Pax5+ cells on M2b macrophage polarization
Authors: OHAMA Hideko , Asai Akira , Yasuoka Hidetaka , Tsuchimoto Yusuke , Fukunishi Shinya , Higuchi Kazuhide
Published: January 2023

M2b macrophages (Mf)/monocytes play a role in the increased susceptibility of alcoholics to infections. However, the mechanism involved in M2bMf polarization remains unclear. In this study, we investigated the role of CD34+CD10+CD19−Pax5+ cells (Pre/Pro-B cells) in the peripheral blood of alcoholics on alcohol-related M2bMf/monocyte polarization through the production of high mobility group box-1 (HMGB1). M2b monocytes were identified as few bactericidal activity, IL-10 and CCL1 productions. Pre/Pro-B cells isolated from 18 alcoholics and 11 healthy donors (HD) were cultured with 10 nM of retinoic acid for 24hrs and culture medium obtained were assayed for HMGB1 by ELISA. CD14+cells (1 × 106 cells/ml) from HD were stimulated with 10% (v/v) of each culture media of Pre/Pro-B cells for 24hrs and their producing abilities of IL-10 and CCL1 were assayed. And 1 × 106 cells/ml of these cells were infected with 2 × 106 CFU /ml Enterococcus faecalis (E. faecalis). The bactericidal activity was assayed for colony counting methods. In the results, Pre/Pro-B cells from HD did not produce HMGB1(10.5 ± 5.5 ng/mL), while these cells from alcoholics did (70.3 ± 20.2 ng/mL). When CD14+ cells from HD were stimulated with each culture media from alcoholics, these CD14+ cells produced IL-10 (102.3 ± 1.7 pg/mL) and CCL1 (3.12 ± 1.33 ng/mL) and did not have bactericidal activity against E. faecalis (17.0 ± 8.6%). However, same CD14+ cells from HD were stimulated with each culture media from HD, these cells did not produce IL-10 (10.3 ± 3.4 pg/mL) and CCL1 (0.3 ± 0.01 ng/mL) and have the bactericidal activity (62.8 ± 3.8%). These results indicate that Pre/Pro-B cells play a crucial role in M2bMf/monocyte polarization via HMGB1 production in alcoholics.
Type III interferons are expressed in tuberculosis granulomas and promote an inflammatory phenotype in macrophages that differs from type I interferon
Authors: Talukdar Priyanka , Mattila Joshua T. , Fallert Junecko Beth A
Published: January 2023

Humans and non-human primates express four subtypes of type III interferons (IFNλs; IFNλ1-IFNλ4). Unlike type I interferons, which have been thoroughly investigated in tuberculosis (TB), the role of IFNλs and their effects on immunity in TB remain unknown, especially at the disease site. To better understand the role of IFNλs in TB, we examined IFNλ1 and IFNλ4 expression in cynomolgus macaques with TB and investigated the effects of IFNα/β, IFNλ1 and IFNλ4 signaling on macaque macrophages. Using IHC, we identified differential IFNλ1 and IFNλ4 expression in macrophages and neutrophils, including IFNλ4 localization in the nuclei of epithelioid macrophages. CD206+ macrophages and CD3+ cells in the airway expressed IFNλR while CD11c+ macrophages in the lymphocyte cuff of the granuloma expressed IFNλR. To measure IFNλ1 and IFNλ4’s effect on macrophage gene expression and compare these cytokines against IFNα/β, we used NanoString transcriptional profiling and Ingenuity Pathway Analysis (IPA) on cytokine-stimulated macrophages to identify differentially regulated pathways. We found that IFNα/β upregulated the greatest number of ISGs, followed by IFNλ1, whereas IFNλ4 stimulation had minimal effect on gene expression. Interestingly, pro-inflammatory genes including IL-1β, IL-8, TLR1, BATF were upregulated by IFNλ1 while IL-1β and TLR1 were downregulated by IFNα/β. To determine the effects of IFNλs on anti-mycobacterial macrophage responses, we used a reporter Mtb strain to determine how IFNλ1 and IFNλ4 effect the viability of Mycobacterium tuberculosis. Our data suggest that IFNλs have non-redundant properties with type 1 interferons that may promote macrophage activation, inflammation, and antibacterial activity in TB.
Effect of temperature on functional activity of macrophages in three different species
Authors: Hassan Intisar A , Renfro Zachary , Blake Harrison , Rath Satyajit , Durdik Jeannine M
Published: January 2023

Temperature affects body physiological functions, including immunity in ways that influence survival. This study investigated the effect of temperature on macrophage activation and metabolism across species. Macrophages were isolated from mice, chicken or rainbow trout primary tissue or from macrophage cell lines and were activated with lipopolysaccharide (LPS) or IFN-γ. For mice, both Raw264.7 and primary cells, nitric oxide (NO) production was similar at 35°C and 37°C but dropped dramatically at temperatures below 35°C. At fever temperature (39°C), NO release increased in response to LPS. Young bone marrow derived macrophage (BMDM) and peritoneal resident macrophage (PRM) showed increased protein synthesis at 39°C compared to 37°C. Chicken splenic macrophages (CSM) showed NO responses that were similar at 37, 39, and at 41°C (normal for avian). A fever of 42°C had a large stimulatory effect on NO production compared to 41°C. A chicken liver derived macrophage cell line, HTC, showed the same pattern. They also showed higher protein synthesis at 42°C compared with (41°C) after LPS stimulation. Trout head-kidney macrophages (THM) showed the highest NO responses at 19°C when compared to more typical stream temperatures of 13, 15, and 16°C. They still showed some response at 28 and 37°C. Thus, their macrophages respond at higher temperatures than the fish can tolerate. Stimulated THM cells with LPS at 19°C showed increased protein synthesis compared to 13 and 16°C. We can conclude from our experiments that fish macrophages had a much broader range of temperatures at which they could respond by NO generation and protein synthesis compared to mice and chickens and all had increased responses at fever temperatures.
Computational approach to identifying universal macrophage biomarker
Authors: Dang Dharanidhar , Prince Lawrence S , Sahoo Debashis
Published: January 2023

Macrophages are a type of white blood cell, of the immune system, that engulfs and digests cellular debris, cancer cells, and anything else that does not have the type of proteins specific to healthy body cells on its surface. Understanding gene expression dynamics in macrophages are crucial for studying human diseases. Recent advances in high-throughput technologies have enabled the collection of immense amounts of biological data. A reliable marker of macrophage is essential to study their function. Traditional approaches use a number of markers that may have tissue specific expression patterns. To identify universal biomarker of macrophage, we used a previously published computational approach called BECC (Boolean Equivalent Correlated Clusters) that was originally used to identify universal cell cycle genes. We performed BECC analysis on a seed gene CD14, a known macrophage marker. FCER1G and TYROBP were among the top candidates which were validated as strong candidates for universal biomarkers for macrophages in human and mouse tissues. To our knowledge, such a finding is first of its kind.
Selective killing of proinflammatory human M1 macrophages by SMAC mimetics and anti-inflammatory M2 macrophages by SMAC mimetics and caspase inhibitors
Authors: Kumar Ashok , Ali Hamza
Published: January 2023

The inflammatory and anti-inflammatory macrophages have been implicated in many diseases including rheumatoid arthritis, multiple sclerosis and leprosy. Targeting macrophage function and activation may represent a potential target to treat these diseases. Herein, we investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of human pro- and anti-inflammatory macrophage subsets. We have shown previously, that human monocytes are susceptible to the cytotoxic effects of SMs, however, differentiated macrophages (M0) are resistant. Whether human macrophage subsets are also resistant to the cytotoxic effects of SM remains unknown. We show that differentiation of M0 macrophages towards M1 state rendered them highly susceptible to SM-induced cell death, whereas M2a, M2b and M2c subsets were resistant, with M2c being the most resistant. SM-induced cell death in M1 macrophages was mediated by apoptosis as well as necroptosis, and activated both extrinsic and intrinsic pathways of apoptosis. The susceptibility of M1 macrophages to SM-induced cell death was attributed to the IFNg-mediated polarization as JAK inhibitor reversed their susceptibility. In contrast, M2c and M0 macrophages experienced cell death through necroptosis pathway following simultaneous blockage of the IAPs pathways by SM and the caspase pathways by the pan-caspase inhibitors. Overall, the results suggest that survival of human macrophages is critically linked to the activation of the IAPs pathways. Moreover, agents blocking the cellular IAP1/2 and/or caspases can be exploited therapeutically to address inflammation-related diseases.
Characterization of renal macrophage (RM) fate, dynamics, and niches: embryonic RMs are more sensitive to immune challenges than bone marrow-derived RMs
Authors: Qin Xuebin , Liu Fengming , Qin Zhongnan , Rappaport Jay
Published: January 2023

Renal macrophages (RMs) participate in tissue homeostasis, inflammation, and repair. RMs consist of embryo-derived (EMRMs) and bone marrow-derived RMs (BMRMs), but the fate, dynamics, replenishment, functions, and metabolic states of the two RM populations remain unclear. Answering these questions will advance our understanding of the pathophysiological functions of tissue macrophages, in general, and RMs, in particular. To begin to address these important questions, we systematically investigated and functionally characterized RMs at different ages by conditionally labeling and ablating RM populations in several lines of transgenic mice. Here, we demonstrate: 1) RMs expand and mature in parallel with renal growth after birth; 2) RMs are mainly derived from fetal liver monocytes before birth, and self-maintain through adulthood with an approximately 40% contribution from peripheral monocytes; 3) After the RMs niche is emptied, peripheral monocytes rapidly differentiate into BMRMs, which preserve longevity; 4) The CX3CR1/CX3CL1 axis provides RMs niche signaling essential for the maintenance and regeneration of both EMRMs and BMRMs; 5) EMRMs have a higher capacity for scavenging immune complexes and are more sensitive to immune challenge than BMRMs; and 6) Differences in the glycolytic capacities of EMRMs and BMRMs correlate with distinct intrinsic functions and the response to immune challenge of the two RM populations.
Immune profiles in experimental two-Kidney-one-cuff (2K-1C) model of renovascular hypertension that predicts reversible versus irreversible renal injury
Authors: Osman Mazen , De Lorenzo Silvana , Johnson Ruth , Grande Joseph
Published: January 2023

Renal artery stenosis is common in the elderly population; biomarkers that predict response to restoration of blood flow have not been previously established.

Methods

We have previously shown in murine model that cuff removal 7 days (7D Cuff-off) after initial placement on the renal artery results in reversible injury whereas cuff removal 14 days (14D Cuff-off) after placement results in irreversible renal injury, when assessed at 28 days after initial surgery. Positive and negative controls consisted of cuff placement for 28 days (28D) and sham surgeries respectively. We performed RNASeq on whole renal cortex to identify innate immune pathways that correlate with reversible versus irreversible injury. Data were normalized to appropriate sham controls in all the groups. Induction of greater than log2 ratio > 2 and p < 0.00001 were considered significant (following adjustment for multiple comparisons).

Results

As assessed by RNASeq, progression of renal disease (14D Cuff-off & 28D mice) was associated with persistent upregulation of genes associated with T cell signaling and macrophage polarization compared to reversible injury (7D Cuff off). Upregulated genes included but not limited to: CCL5, CCR5, CCL2, CXCL1, CXCL9, CXCL10, CXCR3, TLR2, KLF6, CD74, CD4, CD14, CD44, CD83, CD84, IL1R1, IL1B, SPP1, AIF1, SLC7A2, TRAF1, S100A4, SLC9A9, CRIP1, CLEC7A, CLEC10A, EGR2, MGL2, SOCS2, CTLA2B, and FCGR1.

Conclusion

We have identified genes associated with T cell and macrophage activation that were down regulated in the 7D Cuff-off versus 14D Cuff-off and 28D mice; future studies in isolated T cells and macrophages will determine whether these genes represents biomarker(s) of renal disease progression in 2K-1C renovascular hypertension.
PRMT5 has roles in thymic/peripheral T cell development and EAE
Authors: Sengupta Shouvonik , Webb Lindsay M , NarvaezMiranda Janiret , Amici Stephanie A , Edell Claudia , Guerau-de-Arellano Mireia
Published: January 2023

Protein Arginine Methyltransferase (PRMT) 5 is a type-II methyltransferase involved in oncogenesis, embryonic and hematopoietic development. However, the role of PRMT5 T-cell biology is only now coming into focus. To evaluate PRMT5’s functions in T-cells, we have developed a conditional knock-out (KO) model that allow for a deletion of PRMT5 during thymic development, by knocking-out PRMT5 in all peripheral T cells (T-PRMT5D/D). Using this model that deletes both protein-coding isoforms of Prmt5, we investigated the role of the PRMT5 gene during T-cell development, proliferation and experimental autoimmune encephalomyelitis (EAE). We saw reduced numbers of CD4+ T-cells, Tregs and iNK T-cells in the thymus. CD4+, Treg and iNK T-cell number defects were seen in the peripheral lymphoid organs such as spleen and lymph nodes. Peripheral analyses also showed reduced CD8+ T-cells, naive, effector and central memory CD4+ T-cells. We also induced PRMT5 deletion specifically in peripheral CD4+ T cells (iCD4-PRMT5D/D). We utilize this model to induce PRMT5 deletion specifically in peripheral CD4+ T cells while ensuring that the mice develop normal numbers of peripheral T cells. Using this model, we showed that EAE was completely abolished in the mice. Total CNS infiltrating T-cell numbers were substantially reduced, and significant losses were observed in the Th1 and Th17 cytokine production from these cells. These data substantively demonstrate a driver role for PRMT5 in thymic CD4 Th cell development, peripheral CD4+, iNKT and CD8+ T-cell maintenance and Th cell-mediated EAE autoimmunity.
Enhanced N-glycan branching on CD8 T cells exacerbates autoimmune diabetes in non-obese diabetic mice
Authors: Chien MingWei , Sytwu HueyKang
Published: January 2023

β1,6N-acetylglucosaminyltransferase V (Mgat5), is responsible for tetra-antennary N-glycans in the biosynthesis of glycoprotein and the resultant N-glycan branching serves as a backbone for additional glycosylation such as poly-N-aceyllactosamine (poly-LacNAc) structure. The N-glycan branching is involved in the pathological conditions such as malignancies, but the modulatory roles of N-glycan branching on T cells in autoimmune diabetes is not completely understood. Here, we report that the levels of N-glycan branching on T cells is positively correlated with the diabetogenesis in NOD mice. Since the levels of N-glycan branching is regulated during T-cell development and highly increases in pathogenic lymphocytes in pancreas, we generated established T cell-specific Mgat5 transgenic and mutant NOD mice. Interestingly, our results demonstrated that the diabetic incidence in Mgat5 transgenic mice was significantly higher and that of Mgat5 mutant mice markedly attenuated compared to control NOD mice. In adoptive transfer experiments, NOD/SCID mice that received control CD4 T cells plus transgenic CD8 T cells developed diabetes more rapidly than the mice received control or transgenic CD4 T cells plus control CD8 T cells, suggesting that the diabetogenicity in Mgat5 transgenic mice is a CD8 T cell–autonomous manner. Moreover, we generated Mgat5/NY8.3 doubly transgenic NOD mice expressing highly pathogenic MHC class-I-restricted 8.3 T cell receptor, and observed that these mice have higher diabetic incidence than wild-type NY8.3 transgenic NOD mice. Taken together, enhanced N-glycan branching on CD8 T cells exacerbates the development of diabetes in NOD mice.
The C3-like molecule CD109 controls Th1 versus Th17 induction in CD4+ T cells
Authors: Merle Nicolas S , Kolev Martin , Rahman Jubayer , West Erin , Yan Bingyu , Kazemian Majid , Afzali Behdad , Kemper Claudia
Published: January 2023

Recent work defined an unexpected and critical role for intracellular/autocrine active complement in human Th1 biology. Specifically, autocrine stimulation of CD46 via TCR-induced C3b generation is needed for IFN-γ production by human CD4+T cells. Analysis of the CD46-driven gene signature in Th1 cells revealed CD109, a C3-like surface protein known to regulate TGF-β signaling, as a direct target of CD46. We confirmed that T cells from patients with CD46 deficiency, which suffer from recurrent infection due to reduced Th1 responses, and from healthy donors knocked-out for CD46 via CRISPR-Cas9 technology failed to upregulate CD109. CD109 is expressed on keratinocytes, endothelial and stem cells and overexpression is connected with cancer but its function on T cells remains unexplored.

In vitro activation of mouse Cd109−/− CD4+ T-cells showed a strong increase of Th1- and Th17-related cytokines compare to their WT counterpart. In line with this observation, Cd109−/− mouse CD4+ T cells showed an increase in IL-17A+ and IFN-γ+/IL-17A+ T cell numbers in vivo and caused significantly more weight loss and disease pathology in a T cell-transfer colitis model. Although CD109 is known to regulate TGF-β signaling, our RNA-sequencing analysis of activated Cd109−/− mouse T cells revealed that this molecule is restraining Th17 induction rather via impact on cell cycle and apoptosis related pathways, which we are investigating now.

Together, these data suggest that the complement-related protein CD109 serves as an important and novel molecular switch on CD4+ T-cells, where it regulates the balance between Th1 and Th17-induction pathways.
Pregnancy drives TCR-dependent and -independent changes in maternal T cells
Authors: Krupp Katharine , Xu Rong , Porrett Paige
Published: January 2023

Pregnancy is a significant immunological event, which has been shown to induce changes in maternal T cells. Studies examining maternal T cell responses in pregnancy have largely been conducted using mice that express transgenic “surrogate” fetal antigens, and have focused mainly on divided alloreactive T cells. Yet, maternal T cells may exhibit a broader range of responses during pregnancy some of which may not rely upon TCR signaling or cellular proliferation. To investigate the full spectrum of endogenous maternal T cell responses in natural pregnancy, we used the Nur77 mouse model, which allows for a graded assessment of TCR signaling using a GFP reporter. Splenocytes from female CD45.2 Nur77 mice were labeled with CellTrace Violet (CTV) to detect cellular division and transferred into female CD45.1 congenic mice. These host females were then mated with either syngeneic CD45.1 or allogeneic Balb/c males or left naive non-mated controls. Splenocytes were collected on either embryonic day 10 or 18 (E10 or E18) and analyzed using flow cytometry.

t-SNE analysis revealed few phenotypic differences between the experimental groups at E10 with significantly increased separation by E18, which was most evident in the CD4 compartment. Interestingly, this separation was driven primarily by changes in the expression of selectins (i.e. downregulation of CD62L) and cytokine receptors (i.e. increased expression of CD25) as opposed to changes in TCR signaling (GFP) or cellular division (CTV). Accordingly, we conclude that maternal T cell responses in pregnancy are significantly shaped by non-antigen driven signals. The functional impact of these non-antigen driven changes in cellular phenotype are unknown and currently under investigation.
Surfactant protein receptor SP-R210 isoforms play a role in regulating macrophage immune response in conjunction with epigenetic changes during Influenza A infection
Authors: Yau Eric , Song Chunhua , Kawasawa Yuka Imamura , Chroneos Zissis C
Published: January 2023

Alveolar macrophages (AMs) and surfactant proteins are critical for host defense and regulation of immune responses to airborne pathogens like influenza A virus (IAV). Previous findings showed that the surfactant protein A receptor SP-R210 regulates macrophage responses to LPS and phagocytic flux. Our goal is to understand how the long (L) and short (S) isoforms of SP-R210 alter macrophage responses to respiratory pathogens. Here we investigated how altering expression of the L isoform changes macrophage epigenetic landscape during IAV infection. Chromatin immunoprecipitation and sequencing were used to identify alterations in H3K4me3, H3K9me3, and H3K27me3 histone methylation and PU.1 transcription factor binding in L knockdown (KD) cells at baseline and after IAV infection. The UCSC genome browser was used to identify spatial relations of enriched binding regions, revealing reduced PU.1 binding at genes associated with macrophage differentiation and signaling between innate and adaptive immune cells, such as interleukin 18 (IL-18) and colony-stimulating factor receptors (CSF1R, CSF2RB) in KD cells. These changes were mirrored with decreased H3K4me3 marks in KD macrophages. Further analysis with ChIPseeker revealed enrichment in Toll-like receptor and interferon signaling pathways in KDmacrophages. IAV infection upregulated PU.1 binding to genes associated with IL-3, IL-5 and TLR4 signaling pathways among others. RNA sequencing confirmed increased TLR4 and TLR6 and decreased IL-18 transcripts in KDmacrophages. These findings support the idea that the L isoform of SP-R210 regulates chromatin architecture and accessibility of transcription factors at genes with roles in macrophage differentiation and activation.
Cardiolipin dynamics regulate proinflammatory cytokine production in methicillin-resistant Staphylococcus aureus--infected macrophages
Authors: Reynolds Mack B , Abuaita Basel H , O’Riordan Mary X
Published: January 2023

During the immune response, mitochondria serve as a hub for innate immune signaling molecules, such as mitochondrial antiviral signaling protein (MAVS) and Nod Like Receptor Family Pyrin Domain Containing 3 (NLRP3). Cardiolipin, a class of phospholipids found primarily in the inner mitochondrial membrane (IMM), regulates mitochondrial function and cellular stress. In lipopolysaccharide (LPS)-stimulated macrophages, CL translocates from the IMM to the outer mitochondrial membrane (OMM) and recruits NLRP3 to promote inflammasome activation. However, the role of CL in macrophage antibacterial responses remains ill defined. Here, we have made CL-deficient RAW264.7 (RAW) cells by stably knocking down Cardiolipin Synthase (Crls1). Upon infection with methicillin-resistant Staphylococcus aureus (MRSA), Crls1 KD RAW cells showed markedly decreased expression and production of cytokines IL-6, IL-1β, and IFN-β compared to control cells, while TNF-α levels remained similar. For IL-6 and IFN-β, transcript and protein levels in Crls1 KD RAW cells were comparable to unstimulated controls. Surprisingly, these cells showed normal MRSA killing and NF-kB p65 nuclear translocation. These findings suggest a novel role for CL in transcript-level regulation of cytokines during infection. To further investigate CL-dependent innate immune functions, we will use RAW cells expressing a GFP-tagged CL-binding protein, Stomatin-like Protein-2-GFP, to track CL dynamics at membrane-level resolution using 3D structured illumination microscopy. This approach supports earlier studies that describe increased CL OMM localization in response to LPS. Our data suggest an underappreciated role for CL in macrophage responses to MRSA infection.
TPL2 kinase action and control of inflammation
Authors: Xu Daqi , Kwon Youngsu , Senger Kate , Pham Victoria , Varfolomeev Eugene , Francis George , Hackney Jason , Parr Brendan , Rathore Nisha , Setiadi Francesca , Boenig Gladys , Harris Seth , Lupardus Patrick , Matsumoto Marissa , Wang Yuanyuan , Hanson Jesse , Maher Jonathan , Wu Kai , Shaw Andrey , Scherl Alexis , Wu Jiansheng , Lu Cheng , Huang Zhiyu , Zhang Juan , Park Summer , Sujatha-Bhaskar Swathi , Almazan Alice , Xu Min , Caplazi Patrick , Brightbill Hans , Ghilardi Nico , Townsend Michael , Lee Wyne , Johnson Adam , McKenzie Brent , Zarrin Ali A , Wilson Mark
Published: January 2023

Tumor progression locus 2 (TPL2; also known as MAP3K8) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that phosphorylates the MAPK kinases MEK1 and MEK2 (MEK1/2), which, in turn, activates the MAPKs extracellular signal–regulated kinase 1 (ERK1) and ERK2 (ERK1/2). We describe an additional conserved and critical role for TPL2 in mediating the effector functions of myeloid cells including neutrophils, macrophages, and microglia through the activation of the p38 MAPK signaling pathway. Gene expression profiling and functional studies of neutrophils and monocytes revealed a MEK1/2-independent branch point downstream of TPL2 in neutrophils. Genetic ablation of the catalytic activity of TPL2 or therapeutic intervention with TPL2-specific inhibitors reduced inflammatory responses in vitro and in vivo. Besides myeloid cells, TPL2 also plays critical roles in fibroblast synoviocytes, indicating a potential role in rheumatoid arthritis via stromal cells.
TLR-TRIF signaling induces GLI3 to modulate inflammation
Authors: Matissek Stephan Josef , Koeppen Katja , Elsawa Sherine F
Published: January 2023

Chronic inflammation is associated with several disorders and therefore the identification of novel regulators of inflammation is needed. We identified GLI3 as a novel component of the TLR-TRIF signaling. Stimulation of monocytes with LPS increased GLI3 expression in a hedgehog-independent mechanism. We stimulated cells with MPLA, (TLR4 ligand that signals through TRIF) and polyI:C (TLR3 ligand) and found that IRF3 directly binds to the GLI3 promoter and IRF3 increased GLI3 expression. We generated a conditional knockout (cko) mouse where GLI3 was deleted in myeloid cells using LysM-Cre mediated recombination. IFA-elicited macrophages from cko mice stimulated with LPS showed reduced IL-6, CCL2 and TNF-a secretion compared to macrophages from wild-type (wt) littermates. We also performed RNA sequencing of LPS treated or untreated macrophages from GLI3 cko or wt mice. Using a generalized linear model in edgeR, we identified 495 genes with significant interaction effects between genotype and LPS treatment. Ingenuity Pathway Analysis of the interaction genes revealed “Inflammatory Response” and “Immune Cell Trafficking” pathways as most significantly enriched. The 25 significant interaction genes on these pathways included 9 with a positive interaction (Ccl25, Cd226, Cgas, Hmgn5, Il12b, Lrrc8a, Rapgef3, Rnf122, and Tlr8) and 16 with a negative interaction (Acacb, Adipoq, Ccl1, Ccne1, Cx3cl1, Fry, Gstk1, Mertk, Nqo2, Slc6a4, Sort1, Timd4, Tnfrsf18/GITR, Tnfsf13/APRIL, Tnfsf13b/BAFF, and Trem2). Taken together, these results identify a novel role for GLI3 in mediating TLR-TRIF-induced inflammation and identify a novel set of inflammatory genes that are dependent on GLI3 in response to TLR-TRIF signaling.
Autophagy-related protein VPS34 controls the homeostasis and function of macrophages
Authors: Yang Guan , Song Wenqiang , Postoak J. Luke , Martinez Jennifer , Zhang Jianhua , Wu Lan , Van Kaer Luc
Published: January 2023

Vacuolar protein sorting 34 (VPS34; also known as PIK3C3) plays a role in both canonical and noncanonical autophagy, key processes that control immune cell responsiveness to a variety of stimuli. Our previous studies found that VPS34 is a critical regulator that controls the development, homeostasis, and function of dendritic cells and T cells. In this study, we investigated the role of VPS34 in macrophage biology using myeloid cell-specific Vps34-deficient mice. We found that Vps34-deficient macrophages express increased surface levels of MHC class I and class II molecules. Interestingly, Vps34 ablation in macrophages caused a partial impairment in the homeostatic maintenance of TIM-4+ macrophages and defective uptake of apoptotic cells. In addition, myeloid cell-specific Vps34-deficient animals showed significantly reduced severity of experimental autoimmune encephalomyelitis (EAE), a primarily CD4+ T cell-mediated mouse model of multiple sclerosis. Importantly, peritoneal macrophages from mice deficient in the VPS34-associated protein RUBICON, which is critical for a noncanonical form of autophagy called light chain (LC) 3-associated phagocytosis (LAP), showed normal MHC class I, MHC class II, and TIM-4 expression and Rubicon−/− mice developed signs of EAE similar to wild-type control mice. These results suggested that the canonical autophagy-dependent activities of VPS34 play a critical role in controlling macrophage homeostasis and function. Collectively, our studies establish VPS34 as an important regulator of macrophage functions and macrophage-mediated regulation of EAE. Our findings also have important implications for the development of small-molecule inhibitors of VPS34 for therapeutic purposes.
Fc receptor-like 6 (FCRL6) delineates innate-like B cell progenitors that undergo pre-BCR dependent and independent pathways of natural antibody selection
Authors: Davis Randall S , Honjo Kazuhito , Won Woong-Jai , King Rodney G , Ianov Lara , Crossman David K , Easlick Juliet L , Shakhmatov Mikhail , Khass Mohamed , Vale Andre M , Stephan Robert P , Li Ran
Published: January 2023

Innate-like B-1a B cells produce ‘natural’ antibodies (Abs) to neutralize pathogens and clear neo self-antigens, but the early selection mechanisms that shape their polyreactive repertoires are poorly understood. Fc receptor-like (Fcrl) genes encode surface regulatory proteins preferentially expressed by B cells. Here, we identified a B cell progenitor subset defined by FCRL6 expression, harboring emerging Ighv repertoire, migration, and differentiation properties conducive for natural Ab generation. Receptor-specific monoclonal antibodies identified subpopulations of FCRL6+ pro B cells in E18 fetal liver (FL), neonatal tissues, and adult bone marrow (BM). While it was absent on Hardy Fr. A cells, FCRL6 expression became evident in Fr. B, peaked in Fr. C, and declined in Fr. C′. By the Dorshkind scheme, FCRL6 marked subsets of B-1P and pro B cells in the FL and BM, but was not detected on B-2P cells. Compared to FCRL6− pro B cells, the repressed mitotic, DNA damage repair, and signaling activity of FCRL6+ progenitors, yielded VH repertoires with biased distal Ighv segment accessibility, constrained diversity, and hydrophobic and charged CDR-H3 sequences. Similar features were apparent among B-1a and chronic lymphocytic leukemia (CLL) Ighv sequences. Beyond nascent autoreactivity, VH11 productivity, which predominates phosphatidylcholine-specific B-1a B cell receptors (BCRs), was higher for FCRL6+ cells as was pre-BCR formation, which was required for Myc induction and VH11, but not VH12, B-1a development. Thus, FCRL6 revealed unexpected heterogeneity in the developmental origins, regulation, and selection of natural Abs at the pre-BCR checkpoint with implications for autoimmunity and lymphoproliferative disorders.
The E3 ubiquitin ligase Fbw7 controls survival pathways to prevent apoptosis in mature B cells
Authors: Ramezani-Rad Parham , Leung Charlotte R , Apgar John R , Rickert Robert C
Published: January 2023

Proteolytic processing of proteins is crucial for cellular homeostasis, but also regulates signaling pathways required for effector functions and cell fate decisions. Importantly, dysregulation of proteolysis poses the risk of malignant transformation through abnormal accumulation of oncogenes. The E3 ubiquitin ligase, Fbw7, mandates proteasomal degradation of several oncogenes involved in cellular proliferation, growth, and survival. To elucidate the role of Fbw7 in B cells, we studied the function of Fbw7 in a conditional knockout mouse model. Ablation of Fbw7 during early B cell development diminished the mature recirculating B cell pool in the bone marrow and decreased the B cell population in the spleen. Further, we found a drastic reduction of the B1 cells in the peritoneal cavity. A novel acute deletion model for Fbw7 in mature B1 and B2 cells, showed similar losses of mature B cells in the bone marrow and spleen. Analysis of B cell proteomes revealed defective NF-κB signaling in the absence of Fbw7. BCR-stimulation of Fbw7-deficient B cells ex vivo showed impaired growth, poor proliferation and upregulation of apoptosis. In addition to defective NF-κB signaling, the loss of Fbw7 decreased PI3K pathway signaling, hyperstabilized the well-characterized Fbw7 substrate Myc, and induced pro-apoptotic Bim protein, which supports the role of Fbw7 in survival signaling. Ectopic expression of BCL2 in Fbw7-deficient B cells added context-specific survival signals and largely rescued the apoptotic fate of these cells. Our results indicate for the first time that Fbw7 is a critical regulator in B cells, contributing to survival signaling and controlling cell death in mature B cells.
TFG is upregulated during plasma cell differentiation and regulates apoptosis, ER stress and autophagy in a B cell line
Authors: Steinmetz Tobit Daniel , Reimer Dorothea , Urbanczyk Sophia , Schlötzer-Schrehardt Ursula , Winkler Thomas , Jäck Hans-Martin , Mielenz Dirk
Published: January 2023

Long-term survival and antibody production of plasma cells depend on an intact endoplasmic reticulum (ER) quality control and autophagy. The cytosolic adaptor protein TRK-fused gene (TFG) has been shown to function in ER-Golgi transport, secretion and proteasome regulation. We show here that TFG becomes upregulated by LPS and CpG in primary B1 and B2 cells, and the highest expression of TFG was found in plasma cells.

To address the function of TFG we disrupted the tfg gene in B1-like CH12 lymphoma cells and analyzed mice with heterozygous TFG deletion. TFG KO CH12 cells had higher spontaneous apoptosis rates and were more sensitive to ER stress induced by Tunicamycin, MG132 or an ER-trapped μ heavy chain. Autophagy flux as determined by LC3 turnover, electron microscopy and RFP-GFP-LC3 turnover assays indicated a late suppression of the autophagy pathway in TFG KO CH12 cells. While homozygous deletion of TFG was embryonically lethal, TFG+/− mice had normal B development and germinal center B cells, but significantly less CD138+/TACI+ plasmablast/plasma cells in Peyer’s patches, concomitant with reduced serum IgA.

Based on these data we suggest that TFG is a critical factor of ER homeostasis and autophagy in B cell lines. We propose that TFG counters ER stress by enhancing autophagy and sustain cell survival. Especially plasma cells in Peyer’s patches may critically depend on these TFG-mediated mechanisms.

This work is supported in part by grants TRR130 and GRK1660 from the German Research Foundation to D.M and H.-M.J
Characterization of immune anomalies caused by the ptpn6Ala457Thr mutation associated with a specific form of pulmonary emphysema
Authors: Pineault Marie , Milad Nadia , Lechasseur Ariane , Beaulieu Marie-Josée , Routhier Joanie , Aubin Sophie , Maltais François , Morissette Mathieu
Published: January 2023

A new mutation (PTPN6Ala455Thr) was recently discovered in a family in which many members developed a severe, early and panacinar form of pulmonary emphysema. The mutation was found to be located in the PTPN6 gene, encoding a tyrosine phosphatase known as SHP-1. SHP-1 regulates immunological signaling pathways, such as those involved in B cells maturation and activation.

To characterize the impact of the ptpn6Ala457Thr mutation in a mouse model on subpopulations of B lymphocytes and on their response to immunization.

Proportions of B1a et B2 cells were analyzed in the lungs and spleens of 4-month old mice carrying the ptpn6Ala457Thr mutation by flow cytometry. Serum immunoglobulin levels were assessed. Another group of mice were immunized with a T cells-independent antigen (NP-Ficoll, 50ug i.p.). Plasma was collected at different timepoints after immunization and NP-specific antibodies were measured by ELISA (IgG1, IgG3, IgM and IgA).

Mice carrying the ptpn6Ala457Thr mutation mice had a higher proportion of B1a cells and a lower proportion of B2 cells in the spleen and in the lungs. Total IgG and IgM levels in the serum were higher in ptpn6Ala457Thr mice compared to wild-type animals. Following immunization with NP-Ficoll, ptpn6Ala457Thr mice displayed levels of NP-specific IgG3 and IgM similar to those observed in wild-type mice. However, ptpn6Ala457Thr mice had lower levels of NP-specific IgG1 antibodies compared to the wild-type group.

SHP-1 seems to be important for B cell differentiation and functions, as the mutation led to significant changes in B cell subpopulations. It seems that SHP-1 plays an important role in the immune response to immunization. The full scope of the immunological effects of this mutation requires further study.
Dietary fats directly impact viral immunity by modifying membrane flexibility and how T cells see antigen
Authors: Kolawole Elizabeth Motunrayo , Evavold Brian
Published: January 2023

Dietary supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) have are increasingly consumed for their health benefits and exhibit immunosuppressive effects on both innate and adaptive immunity. Although omega-3s can lead to the production of less inflammatory metabolites, we focused on the direct effects of a high omega 3 diet on T cell activation during viral infection. Specifically, we analyzed T cell recognition of antigen and the resulting functional responses during lymphocytic choriomeningitis (LCMV) infection. The omega-3 based diet manipulated structural lipid content in plasma membranes leading to stiffer T cells which subsequently resulted in reduced frequency and activation of the prevalence immunodominant epitopes. We further found that a high omega-3 diet decreased 2D affinity and number of tetramer positive polyclonal as well as CD8 T cell receptor transgenic cells at peak anti-viral immunity. In addition to affinity, the bond lifetime of antigen recognition was also decreased. This resulted in decreased numbers of SLEC and effector cytokines without impeding viral clearance. Our work indicates that a major effect of dietary omega-3 fatty acids on T cells is the modification of the cell membrane to dampen the initial recognition of antigen leading to decreased but effective immunity.
Characterization of the regulatory TCR repertoire in the context of infection, inflammatory disease, and tumor microenvironment
Authors: Wilson Taylor Leigh , Crawford Jeremy Chase , Samarasinghe Amali , McGargill Maureen Ann , Thomas Paul Glyndwr
Published: January 2023

Regulatory T cells (Tregs) are key negative regulators of the immune system. Conventional T cells use their T cell receptor (TCR) to direct their effector function against specific targets. While much is known about the role of Tregs in their microenvironment, whether Tregs require this TCR specificity to elicit their effector functions is not clear. If so, Treg-specific antigens are also undefined. Investigation of the TCR repertoire can help to answer these questions as it provides insight into the degree of antigen recognition T cells experienced during a response. Since Tregs have been implicated in infection, autoimmunity, allergy, and cancer, we have chosen to characterize the Treg TCR repertoire in murine models for influenza, type I diabetes, asthma, neuroblastoma, and non-small cell lung carcinoma. Using an algorithm generated in our lab, TCRdist, we analyzed features of the TCR repertoire such as clonal expansion, TCR diversity, and V and J region biases. Our preliminary data show lung Tregs from asthmatic mice display the greatest antigen specificity while Tregs in influenza and neuroblastoma have minimal specificity based on clonal expansion and enrichment of TCR motifs. Additionally, we have identified Treg receptors that occur in more than one disease type. These data suggest that Treg TCR-dependency is condition-specific and may be determined by the local tissue as a pathological antigen. Future studies include associating these differences in the repertoire with TCR signaling and Treg functional capacities. Understanding the antigen-specificity of Tregs and identifying highly effective Tregs across diverse diseases can provide opportunities to develop broadly applicable, targeted therapeutics.
Translational regulation directs fate decisions involved in memory CD8 T cell differentiation
Authors: Araki Koichi , Ando Satomi
Published: January 2023

mTOR regulates the formation of memory CD8 T cells that circulate in the blood and are present in lymphoid organs. These memory CD8 T cells are the basis for long-lived T cell immunity and efficiently control re-infection to the previously encountered pathogens. Here, we show that the fate decisions between memory precursor and terminal effector CD8 T cells are regulated by the cap-dependent translation that is the downstream pathway of mTOR. Experiments using RNA interference to inhibit expression of eIF4E, which is a cap-binding protein and a translation initiation factor, in antigen-specific CD8 T cells revealed that high eIF4E expression induced terminal effector cells and low expression promoted memory precursor cells. This eIF4E dependent regulation of memory CD8 T cell differentiation was also confirmed by the overexpression of a constitutively active form of 4E-BP that inhibits cap-dependent translation by binding to eIF4E. RNA immunoprecipitation analysis with anti-eIF4E antibody between naïve and activated CD8 T cells showed that, rather than all mRNAs, the interaction of specific groups of mRNAs (i.e., translation and oxidative phosphorylation) with eIF4E was dynamically regulated during activation of the T cells. Transcripts related to translation and oxidative phosphorylation inefficiently bound to eIF4E in naïve CD8 T cells but the level of competence of these transcripts in binding to eIF4E was significantly improved in activated CD8 T cells. Thus, our data show that eIF4E-dependent translation is actively and selectively regulated during naïve to effector CD8 T cell differentiation and has an important role in fate decisions involved in the formation of memory CD8 T cells.
Single-cell metabolic analysis by mass cytometry reveals distinct transitional states of CD8 T cell differentiation
Authors: Levine Lauren , Hiam Kamir J. , Marquez Diana M. , Tenvooren Iliana , Contreras Diana C , Rathmell Jeffrey Charles , Spitzer Matthew H
Published: January 2023

Mass cytometry permits high-dimensional analysis of diverse aspects of cellular behavior. Here, we adapted this platform to simultaneously profile metabolism, signaling, cell cycle, and effector function with single-cell resolution. Using this approach, we measured enzymes characterizing glycolysis, the TCA cycle, fatty acid oxidation, oxidative phosphorylation, and nutrient transport in conjunction with downstream targets of TCR signaling regulating translation, cell cycle progression, proliferation and cytotoxicity. By integrating these functional measurements with surface markers and transcription factors delineating CD8 T cell differentiation and fate acquisition, unsupervised high-dimensional single-cell metabolic analysis by mass cytometry permits identification of unique metabolic and differentiation states of extremely rare cell populations, such as antigen-specific T cells. We interrogated antigen-specific CD8 T cell activation in vitro in addition to the trajectory of the CD8 T cell response over the first nine days of Listeria monocytogenes infection, a well-characterized model for studies of T cell differentiation. This integrated, high-dimensional approach revealed a novel, activated, and highly metabolically active transitional T cell subset emerging at four days post-infection. These cells simultaneously exhibit glycolytic and oxidative functional programs that we propose represents a key metabolic inflection point in CD8+ T cell differentiation. This approach should be useful for mechanistic investigations of metabolic regulation of immune responses in vivo.
T cell-intrinsic expression of HIC1 links retinoic acid to tissue residency
Authors: Bramhall Michael , Rodrigues Grace , Christo Susan , Mackay Laura , Zaph Colby
Published: January 2023

The interaction between micronutrients and the immune system is emerging as a key control point in immune cell function in homeostasis and disease. For example, the vitamin A metabolite all-trans retinoic acid (atRA) has established roles in immune cell migration and effector function. Vitamin A deficiency results in a significant reduction in intestinal immune cells, which is thought to be associated with a lack of intestinal homing receptor expression and reduced migration to the intestine. However, the precise molecular pathways controlling intestinal immune cell homeostasis remains unclear.

Here, we show that the atRA-inducible BTB-POZ transcriptional repressor Hypermethylated in cancer 1 (HIC1, ZBTB29) controls intestinal T cell residency. Mice with a T cell-intrinsic deletion of HIC1 (Hic1ΔT mice) have reduced numbers of CD69+ CD103+ tissue resident memory T (TRM) cells in the intestine, demonstrating that the atRA/HIC1 axis is critical for establishing and maintaining TRM cells. Further, following influenza A virus infection, numbers of TRM cells in the lungs are significantly reduced in Hic1ΔT mice, as well as in mice treated with the pan-retinoic acid receptor inhibitor BMS493, or in mice with a T cell-specific deletion of RARa (RaraΔT mice).

Consistent with this, activation of naive CD8+ T cells from mice or humans in the presence of IL-2, IL-15, TGFβ and atRA results in the generation of CD69+ CD103+ ‘TRM-like’ cells that share the transcriptional profile of bona fide TRM cells. Taken together, these results identify atRA as a critical regulator of T cell tissue residency, acting by inducing expression of HIC1 and promoting the tissue residency gene program.
Essential respiratory chain subunits encoded by mitochondrial DNA control plasma cell development
Authors: Urbanczyk Sophia , Baris Olivier , Mougiakakos Dimitrios , Wiesner Rudolf , Jäck Hans-Martin , Mielenz Dirk
Published: January 2023

The contribution of mitochondrial metabolism to plasma cell development is largely unknown. To define the function of the mitochondrial respiratory chain during B cell activation and differentiation, we used a mouse model in which CD23-Cre drives the expression of a dominant-negative mutant of the mitochondrial helicase Twinkle (DNTwinkle) and the reporter GFP in mature B cells. DNTwinkle expression induces mitochondrial DNA (mtDNA) deletions in dividing cells and results in impaired respiratory chain activity. Flow cytometry in non-immunized DNTwinkle mice did not reveal a difference in either total cell numbers or frequencies of mature B cell subsets. However, only about 8% of the plasma cells in the bone marrow were positive for DNTwinkle/GFP, indicating that these cells have a significant disadvantage in the presence of wildtype plasma cells. T-dependent immunization showed a decreased primary immune response, whereas the secondary immune response was not affected. Strikingly, in immunized mice, all antigen-specific plasma cells in the bone marrow were GFP-negative, again highlighting the advantage of escaper cells. Comparing to in vitro stimulated wild-type B cells, DNTwinkle-positive splenic B cells showed comparable survival, but proliferation, Ig class switch, and plasmablast differentiation were impaired. Also, OxPhos was reduced, but glycolysis and lactate secretion were increased. Finally, AMPK activation was enhanced along with reduced mTOR activity, which could explain the observed impaired Blimp-1 expression and plasmablast differentiation of DNTwinkle B cells. Our results place mitochondrial DNA replication and respiratory chain activity upstream of mTOR and Blimp-1 via AMPK.