Interleukin-27 is antiviral against Zika virus at the maternal-fetal interface

Domain: neuroscience
Published: 2025-12-19
Editorial outcome: major_revision

Abstract
Congenital viral infections can have severe consequences for pregnancy and fetal outcomes. Remarkably, the fetal-derived placenta serves as a robust barrier to infection through meticulous regulation by immune effectors and cytokines. Yet, the regulatory roles of many cytokines remain undefined at the maternal-fetal interface, including Interleukin-27 (IL-27). Here, we show that trophoblast organoids derived from human placentas constitutively express both IL-27 and its receptor, and restrict Zika virus infection through IL-27 signaling. Through bulk RNA-sequencing of trophoblast organoids in the absence and presence of IL-27 signaling, we demonstrate IL-27-mediated upregulation of antiviral genes. Finally, we show that IL-27 signaling is critical to restricting placental viral burdens and protecting against pathologic fetal outcomes during murine congenital Zika virus infection. In this work, we demonstrate a novel role for IL-27 in the placenta and establish IL-27 as an innate antiviral defense at the maternal-fetal interface during congenital viral infection.

Introduction
During the critical developmental period of pregnancy, there are substantial consequences to infection. Despite complex immuneregulation at the maternal-fetal interface, a subset of pathogens can evade host immune responses, cross the placental barrier, and establish congenital infection in the fetus1. Zika virus (ZIKV), a mosquito-borne, positive-sense RNA flavivirus, is one example of a congenital viral pathogen2. Depending on the time of infection, vertical transmission of ZIKV can cause adverse pregnancy outcomes such as microcephaly, preterm birth, and even fetal demise3–5. As congenital ZIKV can have a detrimental impact on fetal health, there is a critical need to identify antiviral effectors at the maternal-fetal interface.

The fetal-derived placenta serves as an effective barrier to congenital viral infections through its constitutive expression of antimicrobial cytokines6. For example, Type I interferons (IFNs) are a family of innate antiviral cytokines that are known to restrict viral replication through the rapid induction of IFN-stimulated genes (ISGs)7. However, while type I IFN (IFNα, IFNβ) signaling is essential for healthy gestation, the classical antiviral state driven by type I IFNs can also disrupt placental development and mediate fetal pathology during congenital ZIKV infection8,9. Yet, the closely related type III IFNs (IFNλ) are constitutively produced by the placenta and have been shown to restrict ZIKV infection without causing fetal pathology10–12. While the role of type III IFNs in placental immunity is well established, it is unknown whether other cytokines perform similar antiviral functions at the maternal-fetal interface.

Interleukin 27 (IL-27) is a heterodimeric cytokine that is produced by extravillous trophoblasts and syncytiotrophoblasts in the placenta throughout gestation; yet, the functional consequence of IL-27 signaling during pregnancy is unknown13. In other contexts, IL-27 is a potent modulator of both innate and adaptive immune responses and has direct antiviral activities14,15. Interestingly, a study by Kwock et al. found that IL-27 induces antiviral gene expression in a STAT1-dependent manner to confer protection against ZIKV infection in human keratinocytes16. Together, these findings led us to ask whether IL-27 contributes to antiviral immunity at the maternal-fetal interface.

In this work, we reveal that IL-27 signaling is antiviral in fetal cells of the placenta using a primary human trophoblast organoid model. We then define the transcriptional profile of IL-27-stimulated trophoblast organoids through bulk RNA-sequencing and demonstrate IL-27-mediated antiviral gene expression in fetal trophoblasts, thus uncovering possible mechanisms of viral restriction by IL-27 at the maternal-fetal interface. We compare IL-27 to known IFNλ responses in the placenta, and identify a joint role of IL-27 and IFNλ signaling in regulating ZIKV infection of trophoblasts. Finally, we show that IL-27 signaling is critical within the context of congenital murine ZIKV infection, as IL-27 restricts placental ZIKV burdens and is protective against pathologic fetal outcomes. Together, these findings establish IL-27 as an innate antiviral defense at the maternal-fetal interface and highlight its potential for combating congenital ZIKV infections and supporting healthy fetal outcomes.

Results
Human trophoblast organoids constitutively express IL-27 and IL27RA
Prior studies indicate that IL-27 and its receptor, IL27RA, are expressed in the placenta throughout gestation13. We generated primary trophoblast (TO) and decidual (DO) organoid lines from first trimester (6-12 post-conception weeks) human placental tissues according to previously established methods and initially sought to confirm IL-27 and IL27RA expression in these models (Supplementary Table 1)17–19. Prior to experimentation, TOs and DOs were grown in Matrigel to support self-organization of 3D structures and were routinely passaged every 6-8 days, with media changes every 2-3 days. We observed that our first trimester TO and DO cultures recapitulated the morphology of previously established TO and DO lines and exhibited distinct TO and DO structures that could be visualized via bright field microscopy (Fig. 1A; Supplementary Fig. 1A)17–19. Additionally, purity of TO and DO cultures was validated prior to experimentation via over-the-counter pregnancy test strips, which detected a presence or absence of secreted trophoblast-specific hormone human chorionic gonadotropin (hCG) in TO- and DO-conditioned media, respectively (Supplementary Fig. 1, AB).

We first assessed the expression of IL-27 subunits (p28, EBI3) and IL27RA subunits (WSX1, gp130) in unstimulated TO and DO cultures via quantitative RT-PCR. We found that both TOs and DOs expressed high levels of transcripts for the shared cytokine and receptor subunits EBI3 and gp130; however, mRNA expression of unique IL-27 and IL27RA subunits p28 and WSX1 was only observed in TOs (Fig. 1B). To corroborate this finding, we measured secreted IL-27 protein in conditioned media from unstimulated TO cultures at various timepoints via enzyme linked immunosorbent assay (ELISA). We show that IL-27 is constitutively produced by TO cultures beginning as early as 24 h post-passage, with increasing production as TOs grew in culture (Fig. 1C).

To determine which cells within the multicellular TO are capable of responding to IL-27, we performed immunofluorescence microscopy for specific trophoblast markers and IL27RA. Ki67 staining identified highly proliferative cytotrophoblasts (CTB) at the periphery of TOs, while SDC1 staining identified multinuclear syncytiotrophoblasts (STB) at the center of TOs, thus demonstrating the inward-facing apical surface and “inside-out” orientation that is characteristic of TOs and other three-dimensional organoid models (Fig. 1D)18,20. We observed strong IL27RA expression within CTB regions of TOs, with only some STB regions expressing IL27RA (Fig. 1D; magenta). Together these findings indicate that IL-27 and IL27RA signaling machinery is intact in the TO model, thus validating TOs as an appropriate model to study IL-27 and IL27RA signaling in the human placenta.

We next sought to determine whether IL-27 signaling regulates congenital ZIKV infection in trophoblasts. Briefly, TO cultures derived from two independent donors were pretreated with either isotype control or IL-27-neutralizing antibodies prior to infection with Cambodian ZIKV. At 1-, 24-, or 48-h post-infection, TOs and culture supernatants were then collected for viral burden analyses (Supplementary Fig. 2A). By measuring viral RNA via quantitative RT-PCR, we showed that TO cultures were susceptible to ZIKV infection, and that neutralization of IL-27 led to significantly increased TO viral RNA levels relative to control organoids at both 24- and 48 h post-infection (Fig. 1E). Additionally, we quantified virus production in TO culture supernatants via plaque assays and found a modest increase in infectious viral particles for IL-27-neutralized TOs relative to control organoids at 24 and 48-h post-infection (Supplementary Fig. 2B). Together these data indicate that IL-27 signaling restricts ZIKV infection of fetal trophoblasts and thus is an important contributor to antiviral immunity in the placenta.

IL-27 induces antiviral gene expression in trophoblast organoids
Given the functional IL-27 signaling circuit in TOs and its known capacity to restrict viral infection in other contexts, we next sought to define IL-27-mediated gene expression changes in TOs. Although we show in Fig. 1B-C that TO cultures are constitutively producing and responding to IL-27, TOs have also been shown to constitutively produce and respond to other antiviral cytokines such as IFNλ21. Due to this robust antiviral cytokine production, we first aimed to quantify basal antiviral gene expression levels in TO cultures. To capture the basal TO state, TO cultures were treated with vehicle (PBS) and isotype control antibody (Fig. 2A; “Isotype”). To broadly mitigate signaling downstream of cytokine receptors and capture an “unstimulated” TO state, additional TO cultures were treated with Janus-kinase inhibitor Ruxolitinib (Rux) and isotype control antibody (Fig. 2A; “Rux”). We then measured the expression of common antiviral genes previously shown to be associated with IL-27 signaling (MX1, OAS2 and IFIT1) in Isotype- and Rux-treated TOs via quantitative RT-PCR16,22. As expected, we observed a strong basal expression of MX1, OAS2, and IFIT1 in Isotype-treated TOs, with antiviral gene expression largely inhibited following Rux treatment (Fig. 2B). Importantly, the antiviral gene expression observed in Isotype-treated TOs is reflective of all cytokines expressed by TOs in their basal state, including IL-27 and IFNλ. We next sought to delineate IFNλ-specific and IL-27-specific changes in antiviral gene expression in TOs. To corroborate that IFNλ can induce antiviral gene expression independently of IL-27, TO cultures were pretreated with IL-27 neutralizing antibody followed by stimulation with recombinant human IFNλ-1 and 3 (Fig. 2A; “IFNλ”). Conversely, to demonstrate that IL-27 can induce antiviral gene expression independently of IFNλ, TO cultures were pretreated with IFNλ-1 and IFNλ-3 neutralizing antibodies followed by stimulation with recombinant human IL-27 (Fig. 2A; “IL-27”). Isotype- and Rux-treated TO cultures were also included as positive and negative controls, respectively (Fig. 2A). We found that IL-27 stimulation of TOs in the absence of IFNλ significantly upregulated the expression of antiviral genes MX1, OAS2 and IFIT1, relative to Rux-treated TOs (Fig. 2C). These findings were corroborated using TOs from a second biological donor, providing support that IL-27-mediated antiviral gene expression is a representative response of human trophoblasts (Supplementary Fig. 3-B). In both donors, though we observed no significant difference in the relative antiviral gene expression levels of IL-27-stimulated TOs and Isotype-treated TOs, this was expected given that Isotype-treated TOs reflect gene expression changes downstream of all constitutively expressed cytokines. These results suggest that IL-27 has the capacity to induce antiviral gene expression in TOs independently of IFNλ, and that constitutive IL-27 signaling contributes to the robust basal antiviral state of TOs.

We next aimed to further define the transcriptional impact of IL-27 signaling in TOs and identify possible mechanisms of viral control in an unbiased manner. TO cultures were pretreated with either IL-27-neutralizing, IFNλ-neutralizing, or isotype control antibodies for 72 h prior to collection. Simultaneously, additional TO cultures were stimulated with either recombinant human IL-27, recombinant human IFNλ, or vehicle control (PBS) for 24 h prior to collection. RNA was then isolated from all TOs and bulk RNA-sequencing was performed. We confirmed that TO cultures transcriptionally recapitulate the placental tissue of origin and express various placental markers (ERVW-1, HSD3B1, XAGE2, GATA3), including those specifically associated with STBs (CGA) and CTBs (TP63) (Supplementary Fig. 4)18. As expected, we observed little-to-no expression of decidual markers MUC1, MUC5A/B, and SOX17 in the TO cultures17. Importantly, the bulk RNA-sequencing analysis recapitulated our initial findings that the IL-27 cytokine (p28, EBI3) and receptor (IL27RA, WSX-1) subunits are constitutively expressed by TOs (Supplementary Fig. 4). Our bulk RNA-sequencing analysis also revealed distinct transcriptional signatures of TOs cultured in the absence/presence of IL-27 and IFNλ (Supplementary Fig. 5A, B, Supplementary Data 1). Given the IL-27-mediated changes in gene expression observed in the basal state for vehicle and isotype controls, we instead sought to compare the transcriptional profile of IL-27-stimulated TOs to IL-27-neutralized TOs. This analysis revealed 10 genes that were significantly upregulated in IL-27-stimulated TOs relative to IL-27-neutralized TOs (Fig. 2D). Of these 10 genes, we identified 5 common antiviral genes that were also significantly upregulated in IFNλ-stimulated TOs relative to IFNλ-neutralized TOs (IFIT1, DTX3L, PARP9, PARP14, PSME1; Supplementary Table 2)23–26. Finally, gene set enrichment analysis (GSEA) revealed an upregulation of viral regulatory pathways in both IL-27- and IFNλ-stimulated TOs (Fig. 2E), with similar antiviral genes driving the enriched gene set networks (Supplementary Fig. 5C, D, Supplementary Data 2, 3).

In previous studies, IFNλ has been shown to regulate ZIKV infection of fetal trophoblasts through autocrine signaling10–12. Given that IL-27 and IFNλ modulate similar antiviral genes in TOs, we next wanted to explore the possibility of IL-27 and IFNλ signaling interplay in regulating ZIKV infection of TOs. Briefly, TO cultures were pretreated with either isotype control, α-IL-27, α-IFNλ, or a combination of both α-IL-27 and α-IFNλ antibodies. TO cultures were then infected with ZIKV-CAM for 24 h and collected for viral burden analysis via quantitative RT-PCR (Supplementary Fig. 2A). Interestingly, we demonstrate in 2 TO donors that neutralization of both IFNλ and IL-27 resulted in increased ZIKV RNA levels relative to neutralization of IFNλ or IL-27 alone (Fig. 2F, Supplementary Fig. 3C). This suggests that while IL-27 and IFNλ have independent antiviral activities at the maternal-fetal interface, together they play a joint role in regulating ZIKV infection of trophoblasts.

We next sought to determine whether the shared DEGs identified through bulk RNA-sequencing of IL-27 and IFNλ-stimulated TOs were critical effectors in the restriction of ZIKV infection in TOs. We measured the expression of our top 2 shared DEGs (IFIT1, PARP9), as well as antiviral gene MX1, in ZIKV-infected TO samples via quantitative RT-PCR. Here we observed that in infected, IL-27-neutralized TOs had significantly lower expression of IFIT1 relative to infected, Isotype-treated TOs, thus identifying IFIT1 as a possible candidate for IL-27-mediated restriction of ZIKV in TOs (Fig. 2G). Additionally, we observed the lowest expression of IFIT1, PARP9, and MX1 in IFNλ+IL-27-neutralized TOs, which exhibited the greatest ZIKV burdens (Fig. 2F, G). These analyses support that the antiviral genes identified through RNA-sequencing are likely important effectors in restricting ZIKV infection of trophoblasts.

Taken together, these data suggest that while IL-27 and IFNλ have independent antiviral activities at the maternal-fetal interface, and together they play a joint role in maintaining the basal fetal-derived antiviral state of the placenta and regulating ZIKV infection of trophoblasts.

IL-27 is primarily produced within the decidua in the murine placenta
Previous studies have demonstrated constitutive production of IL-27 subunit p28 in human placental tissues throughout gestation, therefore we sought to characterize IL-27 production and kinetics in healthy murine placental tissues13. First, we obtained lysates from whole murine placentas including maternal decidua at various gestational timepoints and measured IL-27 protein levels via ELISA. We observed that IL-27 is constitutively produced in the murine placenta throughout gestation (Fig. 3A). To visualize cellular production of IL-27 by maternal and fetal cells in the murine placenta, we next utilized p28-GFP reporter mice27. We mated p28-GFP−/− dams and p28-GFP+/− sires to generate a litter of fetuses that either lacked (p28-GFPNull) or expressed p28-GFP (p28-GFPF). In parallel, we mated p28-GFP+/− dams and p28-GFP−/− sires to generate litters of fetuses that retained maternal p28-GFP expression only (p28-GFPM) or expressed both maternal- and fetal-derived p28-GFP (p28-GFPFM) (Fig. 3B). Placentas were collected at embryonic day 13.5 (E13.5) for immunofluorescence microscopy, and the maternal decidua (Dc) and fetal labyrinth (Lb) regions were defined according to endothelial cell (CD31) architecture (Fig. 3C). We observed robust p28-GFP signal within the decidua of p28-GFPF, p28-GFPM, and p28-GFPFM mice (Fig. 3D). Interestingly, we observed significantly more fetal-derived p28-GFP than maternal-derived p28-GFP within the decidua (Fig. 3D, E). Although we hypothesized that p28 would be expressed within the fetal labyrinth, as this region contain murine trophoblast lineages analogous to the human trophoblasts shown to constitutively express IL-27, we observed minimal p28-GFP signal within the labyrinth region of p28-GFPF, p28-GFPM, and p28-GFPFM mice (Fig. 3E, Supplementary Fig. 6A, B)6,28. Together these findings indicate that IL-27 is primarily produced at the murine maternal-fetal interface within the decidua, largely by cells of fetal origin.

IL-27 limits pathologic fetal outcomes during congenital infection
We observed that IL-27 regulates ZIKV infection in human trophoblasts in vitro, therefore we next sought to determine how placental IL-27 signaling influences pregnancy outcomes during broad inflammation and viral infection in vivo. To test this, we utilized immunocompetent human STAT2 knock-in (hSTAT2 KI) mice, and hSTAT2 KI*IL27RA−/− (IL27RA−/−) mice as a genetic disruption of IL-27 signaling29. First, to test how IL-27 signaling influences outcomes to broad maternal inflammation, we challenged pregnant dams with high molecular weight polyinosinic-polycytidylic acid (HMW poly(I:C)), a synthetic double-stranded RNA and viral mimetic to trigger nonspecific antiviral immune responses in the absence of active viral replication30. We administered 20 mg/kg HMW poly(I:C) or PBS to pregnant hSTAT2 KI and IL27RA−/− dams via intraperitoneal injection at E6.5. All dams were euthanized at E13.5, a time point equivalent to the second trimester in humans, and fetal and placental tissues were collected for gross morphological analysis (Fig. 4A). Importantly, we detected increased serum IL-6 levels in the HMW poly(I:C)-treated dams at 6 h post-injection, indicating robust maternal immune activation as expected (Fig. 4B). We then assessed fetal outcomes at the time of harvest, determining “healthy”, “early resorption”, or “total resorption” phenotypes for each fetus based on gross morphology and integrity of tissues, and quantified the proportion of fetal outcomes for each condition (Fig. 4C-D). We observed that HWM poly(I:C)-stimulation of pregnant hSTAT2 KI mice resulted in some pathologic fetal outcomes (10.7% total resorption), however, this incidence was not significantly different in the absence of IL-27 signaling (3.6% early resorption, 10.7% total resorption), suggesting that IL-27 signaling does not protect against fetal pathology during broad maternal immune activation in pregnancy (Fig. 4C).

We next aimed to define how placental IL-27 signaling influences congenital viral infection. We obtained IL-27-deficient mice via antibody neutralization or genetic disruption of IL-27 signaling. For antibody neutralization, pregnant hSTAT2 KI dams were administered α-IL-27 or isotype control antibody via intraperitoneal injection beginning early in gestation and infected with mouse-adapted DAKAR ZIKV at E6.5 via footpad injection29. As a genetic disruption of IL-27 signaling, pregnant IL27RA−/− dams were also infected with mouse-adapted ZIKV at E6.5. Dams were euthanized at E13.5 and maternal serum, placentas, and fetuses were collected for further analysis (Fig. 4E). In the absence of infection, the fetuses of IL-27-neutralized and IL27RA−/− dams exhibited healthy outcomes and no observable pathology at E13.5 (Fig. 4F). During congenital ZIKV infection, a small proportion of early resorption phenotypes were observed in the fetuses of isotype control-treated dams at E13.5 (7.7%; Fig. 4F). However, there was a significantly greater incidence of resorption in the fetuses of IL-27-neutralized (28% total resorption; 8% early resorption) and IL27RA−/− (23.1% total resorption; 5.1% early resorption) dams during congenital ZIKV infection (Fig. 4F, Supplementary Fig. 7, Supplementary Table 3).

We next measured ZIKV RNA levels in the fetuses, placentas, and maternal serum via quantitative RT-PCR. To include all fetuses in our analysis of viral burden, including those exhibiting the “total resorption” phenotype that prevents accurate separation of the placenta and fetus, we first evaluated the combined fetal and placental ZIKV burdens for each conceptus. We observed significantly higher ZIKV burdens in the combined fetal/placental tissues from IL27RA−/− dams, suggesting that IL-27 may play a role in limiting ZIKV infection at the maternal-fetal interface (Fig. 4G). When considering the intact fetuses alone, we found no significant difference in ZIKV burdens in the fetuses of control, IL-27-neutralized, and IL27RA−/− dams, suggesting that fetal pathology is independent of ZIKV burden (Fig. 4H). Interestingly, we observed significantly higher ZIKV burdens in the placentas of IL-27-neutralized and IL27RA−/− dams relative to control placentas (Fig. 4H). Finally, we found that the fetal pathology phenotypes were not driven by maternal ZIKV burdens, as the viral RNA was effectively cleared in the maternal serum as expected (Fig. 4I). Collectively, these data indicate that IL-27 signaling is required to prevent fetal pathology and can restrict viral RNA replication in placental tissues during congenital ZIKV infection.

Methods
Human placental tissue samples
Placental villi and decidua were obtained from first trimester (6-12 post-conception weeks) pregnancy terminations performed at the University of Pennsylvania Family and Pregnancy Loss Center (IRB#827072). All subjects were counseled appropriately and provided written informed consent. Patients with preexisting medical conditions, pregnancy complications, or maternal infection were excluded from the study. Collected tissues were stored on ice and organoid derivation was carried out within 1 h of procurement. Information about donor tissues is detailed in Supplementary Table 1.

Derivation and culture of trophoblast and decidual organoids
Primary trophoblast organoid (TO) and decidual organoid (DO) cultures were derived from first trimester placental tissues following methods previously established by Turco et al.17–19. Briefly, placental villi and decidual tissues were separated and washed in Ham’s/F12 media (Corning, MT10-080-CV) supplemented with 1% Penicillin/Streptomycin (ThermoFisher, 15140122) to remove blood. Villous tissues were scraped with a scalpel and enzymatically digested to isolate villous trophoblast stem/progenitor cells, while decidual tissues were minced with a scalpel and enzymatically digested to isolate endometrial gland stem/progenitor cells. TO progenitor cells were seeded in 25 µl Matrigel domes, and plated with 250 µl trophoblast organoid media (TOM) consisting of Advanced DMEM/F12 (ThermoFisher, 12634-010) supplemented with 1x B27 (ThermoFisher, 17504-044), 1x N2 (ThermoFisher, 17502-048), 2mM GlutaMAX (ThermoFisher, 35050-061), 100 µg/mL Primocin (InvivoGen, ant-pm-1), 1.25mM N-acetyl-L-cysteine (Sigma-Aldrich, A9165), 5 µM Y-27632 (Sigma-Aldrich, Y0503), 50 ng/mL recombinant human EGF (Peprotech, AF-100-15), 100 ng/mL recombinant human FGF2 (Peprotech, 100-18B), 80 ng/mL recombinant human R-spondin 1 (R&D Systems, 4645-RS-01M/CF), 50 ng/mL recombinant human HGF (Peprotech, 100-39), 1.5 µM CHIR99021 (Tocris, 4423), 500 nM A83-01 (Tocris, 2939), and 2.5 µM Prostaglandin E2 (Tocris, 2296). DO progenitor cells were seeded in 25 µl Matrigel domes, and plated with 250 µl expansion media (ExM) consisting of Advanced DMEM/F12 (ThermoFisher, 12634-010) supplemented with 1x B27 (ThermoFisher, 17504-044), 1x N2 (ThermoFisher, 17502-048), 2mM GlutaMAX (ThermoFisher, 35050-061), 100 µg/mL Primocin (InvivoGen, ant-pm-1), 1.25mM N-acetyl-L-cysteine (Sigma-Aldrich, A9165), 50 ng/mL recombinant human EGF (Peprotech, AF-100-15), 100 ng/mL recombinant human FGF2 (Peprotech, 100-18B), 80 ng/mL recombinant human R-spondin 1 (R&D Systems, 4645-RS-01M/CF), 50 ng/mL recombinant human HGF (Peprotech, 100-39), 500 nM A83-01 (Tocris, 2939), 10 mM Nicotinamide (Sigma-Aldrich, N0636), and recombinant human NOGGIN (Peprotech, 120-10C). TO and DO cultures were routinely passaged every 6-8 days, with full media changes every 2-3 days, and were incubated at 37°C with 5% CO2. TOs and DOs used in this study were derived from 4 independent placentas (Supplementary Table 1). Sex of placentas and resulting TO lines were determined via quantitative RT-PCR using primers for RPS4Y1 (Taqman, Hs00606158_m1; male placentas) and Xist (Taqman, Hs01079824_m1; female placentas).

Enzyme Linked Immunosorbent Assays (ELISA)
Conditioned media (CM) was collected from TO and DO cultures at 1-, 24-, 48-, and 72-h post-passage. Secreted hCG was detected in organoid CM using One Step Pregnancy Test strips (Accumed, HCG25-PP). CM hCG levels were also measured via hCG ELISA (R&D Systems, DY9034) to validate TO and DO lines prior to experimentation. IL-27 ELISA (R&D Systems, DY2526) was performed according to manufacturer’s protocol using CM from 3-5 replicate TO wells for 3 independent TO donors, and a 2-fold serial dilution standard curve of recombinant human IL-27 plated in technical duplicate. For mouse IL-27 ELISAs, pregnant hSTAT2 KI mice were euthanized at gestational days E9.5, E13.5, and E17.5. Whole placental tissues, including maternal decidua, were collected and homogenized, and a mouse IL-27 p28 ELISA (R&D Systems, M2728) was then performed according to manufacturer’s protocol. Individual placental lysates and a 2-fold serial dilution standard curve of recombinant mouse IL-27 p28 were plated in technical duplicate. To validate maternal immune activation by HMW poly (I:C), an in-house IL-6 ELISA (Biolegend, 504501/504601) was performed on mouse serum samples. Blood was collected from HMW poly(I:C)- and control-treated dams via submandibular (cheek) bleed at 6 h post-treatment, centrifuged to remove cells, and the remaining serum was collected (Sarstedt, 41.1378.005). IL-6 ELISAs were performed using serum from 3-4 dams per group, plated in technical duplicate. A standard curve of recombinant mouse IL-6 (Biolegend, 575702) was prepared according to manufacturer’s instructions and plated in duplicate. All TO and mouse ELISA absorbance readings were determined via plate reader at 450 nm and converted to concentration using Microsoft Excel software.

Immunofluorescence microscopy
Murine placentas and TOs were fixed in 4% paraformaldehyde overnight at 4°C prior to embedding in OCT (Sakura, 4583) and sectioning (8-10 µm) with a Leica Cryostat. For all immunofluorescence experiments, slides were washed three times with 1x PBS prior to membrane disruption with PBS supplemented with 0.4% Triton X-100 (PBST; Sigma-Aldrich, T8787). All slides were blocked with 2% normal goat serum (Sigma-Aldrich, G9023-5ML) in PBST for one hour at room temperature prior to overnight incubation with primary antibodies diluted in blocking solution at 4°C. Mouse placenta sections were incubated in primary antibodies against GFP (1:200, Invitrogen, A-21311) and PECAM-1/CD31 (1:10, Developmental Studies Hybridoma Bank, 2H8s), while TO sections were incubated in primary antibodies against IL27RA (1:100, Bioss Antibodies, bs-2711R), SDC1 (1:200, Abcam, ab34164, clone B-A38), and Ki67 (1:50, Invitrogen, 50-5698-82, clone SolA15). Slides were subsequently washed three times with PBST. After washing, slides were incubated with secondary antibodies (Jackson ImmunoResearch) and 1x DAPI (ThermoFisher, AC20271010) diluted in PBST for 2 h at room temperature. Following incubation, mouse placenta slides were incubated in True Black Lipofuscin Autofluorescence Quencher (Biotium, 23007) for three minutes at room temperature to reduce background signal intensity. All slides were washed with PBST and covered with mounting media (ThermoFisher, P36961) and a coverslip prior to imaging (Eclipse Ti2, Nikon). Immunofluorescence images were processed using NIS-Elements and ImageJ software. Total area of p28-GFP signal in murine placenta images was quantified using the ImageJ particle count application.

Virus
For TO infections, ZIKV Cambodian FSS13025 strain (ZIKV-CAM) was obtained from the World Reference Center for Emerging Viruses and Arboviruses at University of Texas Medical Branch, Galveston. For in vivo infections, mouse-adapted ZIKV DAKAR was obtained from Michael Diamond’s laboratory at Washington University in St. Louis. ZIKV-CAM stocks were propagated in C6/36 mosquito cells (Aedes albopictus) (ATCC, CRL-1660), while ZIKV DAKAR stocks were grown in Vero cells (ATCC, CCL-81). C6/36 cells were maintained in Dulbecco’s Modified Eagle’s Medium (ThermoFisher, 11965092) supplemented with 10% heat-inactivated fetal bovine serum (ThermoFisher, A5670701), 1% penicillin/streptomycin (ThermoFisher, 15140122) and 1% tryptose phosphate broth (Sigma-Aldrich, T8159) at 30°C. Vero cells were maintained at 37°C in Dulbecco’s Modified Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin. Cell lines were authenticated by morphology and were routinely tested for mycoplasma contamination. For high-titer doses, ZIKV was concentrated by ultrafiltration (Centricon Plus-70; MWCO: 30,000).

Trophoblast organoid infection and stimulation
Prior to all TO experiments, TOs were routinely passaged and plated in Matrigel. For TO infections, TOs were treated with either isotype control (10 ug/mL AB-108-C, R&D; 8 ug/mL MAB003, clone 20102, R&D), 10 µg/mL α-IL-27 (R&D, AF2526) or 4 µg/mL α-IFNλ1 (R&D, MAB15981, clone 247801) and 4 µg/mL α-IFNλ3 (R&D, MAB5259, clone 567143) antibodies in fresh TOM at days 3 and 5 post-passage. At day 5 post-passage, TOs were removed from Matrigel domes and infected with 105 PFU ZIKV-CAM or PBS in fresh TOM at 37°C and 5% CO2 for 2 h. After 2 h, virus was washed from TO cultures and replaced with fresh TOM and antibodies. At 24 h post-infection, RNA was isolated from organoids and ZIKV burdens were determined via quantitative RT-PCR. Schematic of TO infection timeline included in Supplementary Fig. 2.

For Fig. 2B, C, Isotype-treated TOs were administered PBS and isotype control antibody (10 ug/mL AB-108-C, R&D; 8 ug/mL MAB003, clone 20102, R&D) in fresh TOM at days 3 and 5 post-passage. Rux-treated TOs were administered 20 µM Ruxolitinib (Stemcell, 73404) and isotype control antibody in fresh TOM at days 3 and 5 post-passage. IFNλ and IL-27-treated TOs were treated with 10 µg/mL α-IL-27 (R&D, AF2526) or 4 µg/mL α-IFNλ1 (R&D, MAB15981, clone 247801) and 4 µg/mL α-IFNλ3 (R&D, MAB5259, clone 567143) antibodies in fresh TOM at days 3 and 5 post-passage. At day 5 post-passage, IFNλ and IL-27-treated TO cultures were then stimulated with 100 ng/mL each of recombinant human IFNλ1 (R&D, 1598-IL-025/CF) and recombinant human IFNλ3 (R&D, 5259-IL-025/CF), or 500 ng/mL recombinant human IL-27 (Peprotech, 200-38) in fresh TOM, respectively. At 24 h post-stimulation, all organoids were collected for RNA isolation and quantitative RT-PCR. For bulk RNA sequencing experiments, TOs were treated with either isotype control 10 µg/mL α-IL-27 or 4 µg/mL α-IFNλ1 and 4 µg/mL α-IFNλ3 antibodies in fresh TOM at days 3 and 5 post-passage. At day 5 post-passage, additional TO cultures were stimulated with either vehicle control (PBS), 100 ng/mL each of recombinant human IFNλ1 and IFNλ3, or 500 ng/mL recombinant human IL-27 in fresh TOM. At day 6 post-passage, all organoids were collected for RNA isolation and sequencing.

RNA extraction, cDNA generation, and quantitative RT-PCR
TOs and DOs were collected at 6-8 days post-passage for RNA analysis, depending on their size and density. To ensure RNA yield, 2-4 Matrigel domes containing organoids from a single donor were pooled in 400 µl TRIzol reagent (Invitrogen, 15596026) for RNA analysis. Organoid RNA was purified via phenol-chloroform extraction and ethanol precipitation, using Phase Lock Gel Heavy tubes (QuantaBio, 2302830) according to manufacturer’s instructions. RNA quality and concentration were determined using a ThermoFisher Scientific Nanodrop One spectrophotometer. Total RNA was reverse transcribed using iScript cDNA synthesis kit (Bio-Rad). Quantitative RT-PCR was performed using SYBR Green Master Mix reagent (Thermo Fisher Scientific, A25742) and human gene-specific primers (Supplementary Table 4). All samples were run in technical duplicate on a QuantStudio 3 Real-Time PCR System. Gene expression of p28, EBI3, WSX1, and gp130 were determined based relative to housekeeping gene HPRT1 expression (ΔCT method). For TO infections, ZIKV E expression was normalized to the isotype internal reference via the ΔΔCT method.

For RNA extraction and quantification of murine viral loads, tissues were collected in 600 µl TRIzol reagent and homogenized (MP Biomedical, 116004500). Homogenates were then centrifuged for 5 min at 12,000 x g, and 100 µl of the clarified homogenate was then diluted with 300 µl TRIzol. An equal volume of 100% ethanol was then added to the diluted sample for RNA extraction. Viral RNA was extracted using a Direct-zol RNA Miniprep kit (Zymo Research, R2050) according to manufacturer’s instructions. Reverse transcription and quantitative RT-PCR were performed as described above using mouse gene-specific primers (Supplementary Table 4). ZIKV genome equivalents were determined via a 100-fold serially diluted standard curve of ZIKV RNA extracted from viral stock. Total ZIKV genome equivalents per tissue were then standardized to the tissue weight in mg.

Plaque assay
Supernatant was collected from infected TO cultures at 1-, 24-, and 48-h post-infection (hpi), followed by serial dilution in DMEM (Corning, 10-013-CV) supplemented with 2% heat-inactivated Fetal Bovine Serum (Sigma-Aldrich, F2442) and 1% Penicillin/Streptomycin (ThermoFisher, 15140122). For plaque assays, Vero cells were seeded at a density of 5 x 105 cells/well in a 6-well plate overnight. After 24 h, seeded Vero cells were treated with 250 µL of diluted TO supernatants and incubated for 1-h, with rotations every 15 min. Following incubation, inoculum was aspirated and replaced with MEM (Sigma-Aldrich, 11430030) supplemented with 5% FBS, 1% GlutaMAX, 1% Non-essential amino acids, and 0.65% agarose (Lonza, 50111). ZIKV-infected cells were fixed at 6 days post-infection with 2 mL 10% NBF (ThermoFisher, 22050105) and visualized using 0.1% crystal violet (ThermoFisher, C581-25). Plaques were manually counted, and virus titer was calculated.

Bulk RNA-sequencing
RNA was isolated from TOs for bulk RNA-sequencing using the Direct-zol RNA Miniprep kit (Zymo Research, R2050) according to manufacturer’s instructions. RNA was then processed for bulk RNA-sequencing at the Children’s Hospital of Philadelphia High Throughput Sequencing Core. Sequencing libraries were prepared using Illumina’s stranded total RNA Prep kit with Ribo-Zero and the NovaSeq 6000 SP Reagent Kit v1.5 (200 cycles) (Illumina, #20040719). All data were analyzed using an adapted form of the open-source DIY transcriptomics lecture materials50. Gene set enrichment analysis (GSEA) of bulk RNA sequencing data was performed for IL-27- and IFNλ-stimulated TOs. Log fold-change values were calculated by comparing IL-27-stimulated vs. α-IL-27 TOs and IFNλ-stimulated vs. α-IFNλ TOs. All GSEA analyses were then evaluated using a log fold-change cutoff value > 0.5. GSEA significance was determined via permutation testing with two-sided p-values, and Benjamini-Hochberg False Discovery Rate (FDR) correction. GSEA analysis described in Supplementary Data.

Mice
Wild-type (WT) C57BL/6J mice were purchased from The Jackson Laboratory (Strain #000664). Human STAT2 knock-in (hSTAT2 KI) mice (Gorman et. al. 2018) were obtained from Michael Diamond’s laboratory at Washington University in St. Louis. p28-GFP mice were obtained from Ross Kedl’s laboratory at the University of Colorado-Anschutz Medical Campus. All mice were maintained at the University of Pennsylvania under specific pathogen free conditions, on a 12-h light/dark cycle, at 21°C (±1°C) and 50% (±10%) humidity. All experiments were performed in adherence to the University of Pennsylvania’s approved IACUC protocol (#806749). All timed mating experiments utilized adult (8–15 week) male and female mice. Mouse genotypes were determined using DNA extracted from maternal tail samples or fetal yolksacs and previously described primers (Supplementary Table 4)27,29.

In vivo infections
Beginning at embryonic day 3.5 (E3.5), pregnant hSTAT2 KI dams were administered 500 µg of α-IL-27 (BioXCell, BE0326, clone MM27.7B1) or isotype control (BioXCell, BE0085, clone C1.18.4) antibody via intraperitoneal injection, with subsequent treatments every three days (E6.5, E9.5, and E12.5). At E6.5, the control and IL-27-neutralized dams were then infected with 6.75 x 105 PFUs mouse-adapted DAKAR ZIKV diluted in PBS via footpad injection. To mimic immune activation during viral infection, High Molecular Weight (HMW) Vaccigrade Poly(I:C) (Invivogen, vac-pic) was delivered to pregnant mice at E6.5 via intraperitoneal injection at 20 mg/kg. All infected and HMW poly(I:C)-stimulated dams were euthanized at E13.5 and fetal, placental, and maternal tissues were collected for further analyses. Gross morphology of fetal and placental tissues were assess upon collection. Intact embryos lacking any visible signs of pathology were characterized as “healthy.” Embryos with a distinguishable fetus and placenta but some loss in tissue integrity were characterized as “early resorption.” Visibly smaller embryos that lacked a clearly defined fetus and placenta due to hemorrhage and tissue necrosis were characterized as “total resorption.” All mouse infection and stimulation data are representative of 3-4 litters per treatment group.

Statistical analysis
Bulk RNA-sequencing data were analyzed in R studio using DIY transcriptomics lecture materials50. All graphs were plotted and statistical analyses were performed using Microsoft Excel and GraphPad Prism software. Horizontal dashed lines are used on graphs to denote limits of detection (LOD) for the given assay. All data are expressed as mean +/- standard deviation (SD) or error of mean (SEM). Exact statistical test and number of biological samples (n) are detailed in the figure legends. A one-way ANOVA was used to determine significance when determining significance between multiple groups (>3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Jurado and colleagues report the antiviral role of IL-27 at the maternal-fetal interface. While IL-27 has been studied in other immunological contexts, its function in placental immunity during congenital infections remains poorly characterized. The authors profile IL-27 and IL27RA expression in trophoblast (TO) and decidual (DO) organoids, and use TOs and in vivo models to show that IL-27 signaling restricts placental ZIKV burdens and fetal pathology during murine congenital infection. Although the study provides interesting data using established organoid and mouse models, significant revisions are needed to address mechanistic gaps and enhance novelty. The reviewer has the following comments:\n1) The conclusions that TO cultures were susceptible to ZIKV infection and IL-27 signaling restricts ZIKV infection was not supported by present data. ZIKV replication kinetics with or without IL-27 antibodies in TO and DO should be determined by PFU and RT-qPCR at different time points. Viral antigen co-staining with specific cell type markers should be performed. Additionally, scRNA-seq are suggested to identify the target cells and IL-27 producing cells in TO.\n3) The study sought to demonstrate that IL-27 restricts ZIKV infection in TOs and murine placentas, but the mechanistic underpinnings remain poorly defined. While RNA-seq data show upregulation of antiviral genes (e.g., MX1, IFIT1), the study fails to establish a causal link between IL-27 signaling and these effectors. Temporal and spatial analyses of IL-27 signaling during ZIKV infection in presence/absent of neutralization is essential. Additionally, the cross-talk and comparison of IL-27-mediated responses to the known IFNλ should be determined at the mechanistic level.\n3) Lines 179-180. The authors claimed that \"This study is the first to describe transcriptional responses to IL-27 signaling in the human trophoblast organoid model.\" Actually, many prior studies have reported transcriptomic data of ZIKV-infected TSCs, and these data should be well compared and discussed. Moreover, whether the transcriptional responses to IL-27 signaling have direct impact on ZIKV infection are fully not detected in the present study.\n4) Figure 3: Mouse experiments indicate that IL-27 is primarily produced by fetal cells in the decidual zone (Figure 3D-E), but the specific cell types (trophoblast cells? immune cells?) are not identified. Also, virological characterization supporting ZIKV infection is quite preliminary.\n5) Figure 4: Mouse placental development matures after E10.5 days, and the authors also identified IL-27RA via IFA at E13.5 days. Why authors chose E6.5 as the time point for viral challenge? In additional, fetal and placental pathology as well as viral antigen staining are essential to support the role of IL-27RA deficiency on the host’s antiviral effects.\n6) The RNA-seq analysis identifies a panel of potent antiviral genes in TOs, but none of them are not validated in mice models.", "reviewer_2": "Merlino et al describe their study of IL-27 actions in human placental trophoblast cultures, placental organoids, and mice in response to Zika virus (ZIKV) infection. This is an important study. There is concern over the use and featuring of only single donor data sets, the need for deeper interrogation of the RNAseq data analyses, and how IFN-L and IL-27 function together and separately to impart antiviral defense against ZIKV and protection form fetal disease.\nSpecific comments:\nFigure 1. E Only a single donor is shown, biological replicates from additional donors are needed. In addition the authors should include an additional treatment of both anti-IFN-lambda and anti-IL-27 abs in order to delineate which cytokines is impacting ZIKV restriction, since both clearly have similar antiviral actions.\n2C: Here it is not clear if we are looking at ab-treated cells or cytokine treated cells, and the text does not do a good job to relay what we are looking at, so this data set is unable to be interpreted. It looks like the bars show IFN-L and IL-27 treatment- is this correct? Or is it showing anti-IFNL and anti-IL-27 treatment response after adding converse cytokines? This is not described in the legend and is confusing in the text. It looks like IFN-L is inducing the genes better than IL-27. Also, that only a single donor is used here makes it difficult to determine if this response shown actually occurs in other donors and is a true representation of the trophoblast responses.\nSupplemental figure 3. This data set shows Z score comparisons where it would be best to show log2 fold change for identifying DEGs. Also, DEG networks need to be shown for functional modules that are described in the text, thus allowing the needed resolution to assess IFN-L vs. IL-27 DEG networks and gene function modules driven by each cytokine or by both cytokines. A big question here is what is the contribution of both cytokines to the gene and functional module signature? Also, how many different donors is the RNAseq data generated from? This is important to know in order to interpret the data set.\nLine 180: Claiming novelty “This study is the first to describe transcriptional responses to IL-27 signaling in the human trophoblast organoid model” ; do the authors really need to do this?\nFigure 3. These analyses are well-done, impressive, and are highly informative.\nFigure 4A-C. Dosing mice with high mw pIC will trigger TLR3, TLR7, and MDA5 signaling as well as activate PKR and OAS/RNAseL. Was the goal here to mimic an inflammatory response driven by ZIKV or to initiate a general inflammatory response that is triggered by virus infection overall? This is not clearly described for justification. In consideration of this, previous studies have shown that ZIKV triggers RLR signaling rather than TLR signaling, so the inflammatory response would be partitioned to cytoplasmic RLR actions, driving a specific and different (but some overlapping) response from high my pIC. The goal of this experiment should be better described in the text. Overall the outcome shows no impact by IL-27.\nFigure 4 D-F: These results are clear; it would be a great addition to include bulk or single cell RNAseq here but the data clearly show a role for IL-27 in restricting ZIKV fetal disease.\nDiscussion: This is concise and clear. However, inclusion of a discussion of the role of IFN-L vs IL-27 and their role together is warranted. This study is important to define a role of IL-27 in fetal protection against ZIKV but it does not really tell us how IL-27 compares to IFN-L in these actions nor what the contribution of both are to infection outcomes. These two cytokines have redundant and unique actions but the authors do not really define these beyond Venn diagrams.\nPrimary RNA-sequencing data are correctly deposited as GSE298278. All code is clear and is on GitHub", "reviewer_3": "The manuscript by Merlino et al. explores the role of IL-27 and its receptor IL27RA as modulators of antiviral gene expression, particularly MX1, IFIT1, and OAS2, using both in vitro trophoblast organoid (TO) models and in vivo approaches. To investigate this signaling axis, the authors challenged the system with the Cambodian strain of Zika virus (ZIKV), demonstrating that IL-27 signaling restricts ZIKV infection and associated pathogenesis.\nWhile IL-27 expression in human trophoblast cells and its role in immune regulation and angiogenesis during pregnancy have been previously described (doi: 10.1016/j.placenta.2007.06.004), this manuscript further explores the pathways involved in the antiviral response, and identifies shared gene expression programs regulated by IL-27 and IFN-γ in placental tissue.\nThis is a promising and timely study that adds valuable insights into the maternal–fetal immune interface, especially regarding innate antiviral defenses in the placenta. However, as the findings are derived from experiments using only one ZIKV strain, further validation is needed to support the broader conclusion that IL-27 functions as a general antiviral factor at the maternal-fetal interface.\nPoints to be addressed:\n• Cytokine profile in TO culture:\nDid the authors quantify any other cytokines secreted into the TO culture supernatant besides IL-27?\n• Biological replicates:\nAlthough technical replicates were performed, several key experiments (e.g., Figures 1E, 2B, and 2C) appear to have been conducted using TOs from a single donor. It would be important to confirm these findings using biological replicates (i.e., TOs from different placental donors), especially given known variability in susceptibility to ZIKV. For instance, twin discordance in ZIKV infection has been reported (doi: 10.1038/s41467-018-03497-1), as well as in murine models (doi: 10.1038/nature18296).\n• Fetal viability and variability in resorption rates:\nIn the fetal viability assays following IL-27 or IL27RA blockade and ZIKV infection, the authors report an increase in fetal resorption of just over 23%. While statistically significant, this effect also shows considerable variability. What factors might contribute to this variation? Could it be related to the genetic background of the TO cultures or the mice?\n• Quantification of ZIKV levels in tissues (Figure 4H):\nThe Y-axis is labeled as “PFU equivalents/mg tissue”, yet the method used is qRT-PCR, which detects ZIKV RNA, not infectious viral particles. Even though a standard curve may have been used, RNA detection alone does not confirm the presence of mature or infectious virions. To accurately assess viral infectivity, a plaque-forming unit (PFU) assay or in vitro titration would be required. The authors should consider referring to the data as Ct values or viral RNA copies, rather than PFU equivalents.\n• Figure labeling error:\nThe legend of Figure 4 contains two panels labeled as “H”. Please revise to correct this duplication."}, "author_response": "We would like to thank the reviewers for their time in carefully reviewing our manuscript and for their\nthoughtful and constructive comments, which have helped us improve this paper. We have included a\npoint-by-point response in how we addressed each reviewers’ comments:\nResponse to Reviewer #1:\nIn this manuscript, Jurado and colleagues report the antiviral role of IL-27 at the maternal-fetal interface.\nWhile IL-27 has been studied in other immunological contexts, its function in placental immunity during\ncongenital infections remains poorly characterized. The authors profile IL-27 and IL27RA expression in\ntrophoblast (TO) and decidual (DO) organoids, and use TOs and in vivo models to show that IL-27\nsignaling restricts placental ZIKV burdens and fetal pathology during murine congenital infection.\nAlthough the study provides interesting data using established organoid and mouse models, significant\nrevisions are needed to address mechanistic gaps and enhance novelty. The reviewer has the following\ncomments:\n1) The conclusions that TO cultures were susceptible to ZIKV infection and IL-27 signaling restricts ZIKV\ninfection was not supported by present data. ZIKV replication kinetics with or without IL-27 antibodies in\nTO and DO should be determined by PFU and RT-qPCR at different time points. Viral antigen co-staining\nwith specific cell type markers should be performed. Additionally, scRNA-seq are suggested to identify the\ntarget cells and IL-27 producing cells in TO.\nResponse: We thank the reviewer for this suggestion. We agree that a temporal analysis would better\nreinforce our conclusion that IL-27 signaling restricts ZIKV infection in TOs. As suggested by the reviewer,\nin our revised manuscript we now include data that assesses ZIKV replication kinetics with or without IL-\n27-neutralization in TOs from two independent donors via RT-qPCR (Figure 1E; text lines 117-124).\nThese data support our initial conclusion that IL-27 signaling restricts ZIKV infection in TOs at 24-hours\npost-infection, with an even more robust phenotype observed at 48-hours post-infection.\nFigure 1E. TOs were treated with either isotype control or -IL-27 antibody at days 3 and 5 post-\npassage. At day 5 post-passage, TOs were infected with 105 plaque forming units (PFU) of\nCambodian ZIKV (ZIKV-CAM) or PBS (Mock). TO cultures were then collected at 1-, 24-, or 48-\nhours post-infection (hpi) for RNA analysis. Graph displays ZIKV viral RNA expression in TOs as\ndetermined by quantitative RT-PCR relative to housekeeping gene HPRT1, normalized to isotype\ncontrol-treated organoids. Graph represents 3-4 independent infections performed in 2 biological\nTO donors (circles and diamonds).\nFurther, in response to the reviewer’s suggestion, we have now included plaque assays as an additional\nvirological readout of infection. These data show a modest increase in infectious viral particles for IL-27-\nneutralized TOs relative to isotype control-treated TOs at all timepoints, thus supporting our initial\nconclusions. This data has been added to the paper in Supplementary Figure 2B and text lines 124-127.\nSupplementary Figure 2B. TOs were treated with either isotype control or -IL-27 antibody at\ndays 3 and 5 post-passage. At day 5 post-passage, TOs were infected with 105 PFU Cambodian\nZIKV (ZIKV-CAM) or PBS (Mock). TO culture supernatants were then collected at 1-, 24-, or 48-\nhours post-infection (hpi) and viral plaque assays were performed to quantify infectious virus\nproduction over time. Graph displays calculated virus titers normalized to isotype control-treated\nTOs for each independent infection. Graph represent 3-4 independent infections performed in\nTOs from 2 biological donors (circles, diamonds).\nLastly, while we appreciate the reviewer’s suggestion to characterize the IL-27-responsive and target cells\nin TOs using single-cell RNA-sequencing, we consider this suggestion to be out of scope and not needed\nto support the conclusions in the manuscript.\n3) The study sought to demonstrate that IL-27 restricts ZIKV infection in TOs and murine placentas, but\nthe mechanistic underpinnings remain poorly defined. While RNA-seq data show upregulation of antiviral\ngenes (e.g., MX1, IFIT1), the study fails to establish a causal link between IL-27 signaling and these\neffectors. Temporal and spatial analyses of IL-27 signaling during ZIKV infection in presence/absent of\nneutralization is essential. Additionally, the cross-talk and comparison of IL-27-mediated responses to the\nknown IFNλ should be determined at the mechanistic level.\nResponse: In attempt to establish a link between IL-27 signaling and the antiviral effectors we identified\nthrough RNA-sequencing (Figure 2D), we evaluated the expression of shared antiviral genes (IFIT1,\nMX1, PARP9) in ZIKV-infected TO samples. In revised Figure 2G, we show that IL-27-neutralized TOs\nexhibit significantly lower expression of IFIT1 relative to Isotype-treated TOs during ZIKV infection, thus\nidentifying IFIT1 as a possible candidate for IL-27-mediated restriction of ZIKV. Finally, as suggested by\nthe reviewer, in our revised manuscript we sought to more thoroughly compare IL-27 responses to known\nIFN responses and explore crosstalk of IL-27 and IFN. We provide additional data (Figure 2F) exploring\nthe joint role of IL-27 and IFN during ZIKV infection in TOs. In the absence of both cytokines, we\nobserve higher viral burdens (Figure 2F) and lower expression of antiviral genes compared to\nneutralization of IFN or IL-27 alone (Figure 2G). These data collectively support a joint role for IL-27 and\nIFN in regulating ZIKV infection of TOs, and that the shared genes identified in our RNA-seq analysis\nare likely important antiviral effectors for restricting ZIKV in trophoblasts, and are now described in text\nlines 189-212.\nF. G.\nFigure 2F. TOs were treated with either isotype control, -IL-27, -IFN, or -IL-27 and -IFN\nantibodies at days 3 and 5 post-passage. At day 5 post-passage, TOs then were infected with 105\nPFU Cambodian ZIKV (ZIKV-CAM) or PBS (Mock) for 24 hours. TO cultures were then collected\nat day 6 post-passage for RNA analysis. ZIKV viral RNA expression in TOs as determined by\nquantitative RT-PCR relative to housekeeping gene HPRT1, normalized to isotype control treated\norganoids. Graph represents 6 independent infections from single TO donor (Donor 1). Statistical\nanalysis performed with Kruskal-Wallis ANOVA, *p<0.05, ***p<0.001.\nFigure 2G. Expression of antiviral genes IFIT1, MX1, and PARP9 by ZIKV-infected TOs as\ndetermined by quantitative RT-PCR, relative to Isotype. Graphs display 5 experimental replicates\nfrom 1 TO donor. Statistical analysis performed with Kruskal-Wallis ANOVA *p<0.05, **p<0.01.\nFurther, as suggest by the reviewer and described in the above response, in our revised manuscript we\nprovide a temporal analysis of IL-27 signaling during ZIKV infection and demonstrate that IL-27 restricts\nZIKV infection of 2 independent TO donors at both 24- and 48-hours post-infection (Text lines 117-127;\nFigure 1E, Supplementary Figure 2B). Finally, we have included citations that support effector function as\nwell as a discussion of cytokine crosstalk in our revised manuscript (text lines: 185, 305-312).\n3) Lines 179-180. The authors claimed that \"This study is the first to describe transcriptional responses to\nIL-27 signaling in the human trophoblast organoid model.\" Actually, many prior studies have reported\ntranscriptomic data of ZIKV-infected TSCs, and these data should be well compared and discussed.\nMoreover, whether the transcriptional responses to IL-27 signaling have direct impact on ZIKV infection\nare fully not detected in the present study.\nResponse: We appreciate the opportunity to specify that our transcriptional analysis is in uninfected\nsamples, not ZIKV-infected samples as suggested by reviewer’s comment. Regardless, as suggested by\nthe reviewer, we have removed this statement in the revised manuscript. Furthermore, we agree with the\nreviewer that there are prior studies that have reported transcriptomic data of ZIKV-infected TSCs.\nInterestingly, these studies identified upregulated antiviral genes in ZIKV-infected TSCs, some of which\nwere identified within our dataset, including PARP14 (PMID:35513694).\nSecond, as described in the previous comment, we have added new data supporting transcriptional\nresponses being important in ZIKV infection. In our revised manuscript we attempt to link our\ntranscriptional analysis to ZIKV infection by evaluating expression of our identified DEGs in ZIKV-infected\nTO samples (Figure 2G). Here, we observe that infected IL-27-neutralized TOs have significantly lower\nexpression of IFIT1 relative to infected, Isotype-treated TOs, thus identifying IFIT1 as a possible\ncandidate for IL-27-mediated restriction of ZIKV. Additionally, we observed the lowest expression of\nantiviral genes IFIT1, MX1 and PARP9 in IFN+IL-27-neutralized TOs, which exhibited the greatest ZIKV\nburdens. Together these data support that the identified antiviral effectors in our RNA-seq data are likely\nimportant antiviral genes for restricting ZIKV in trophoblasts.\nFigure 2G. Expression of antiviral genes IFIT1, MX1, and PARP9 by ZIKV-infected TOs as\ndetermined by quantitative RT-PCR, relative to Isotype. Graphs display 5 experimental replicates\nfrom 1 TO donor. Statistical analysis performed with Kruskal-Wallis ANOVA *p<0.05, **p<0.01.\n4) Figure 3: Mouse experiments indicate that IL-27 is primarily produced by fetal cells in the decidual\nzone (Figure 3D-E), but the specific cell types (trophoblast cells? immune cells?) are not identified. Also,\nvirological characterization supporting ZIKV infection is quite preliminary.\nResponse: We thank the reviewer for their question. Here, we provide immunofluorescence microscopy\ndata of uninfected, E13.5 mouse placentas co-stained for CD45 (magenta) to identify immune cells in the\ndecidual zone. We did not observe colocalization of CD45 with p28-GFP (green), suggesting that the IL-\n27 producer cell is not an immune cell and is instead of trophoblast cell lineage. These findings are\nconsistent with fetal trophoblast production of IL-27 observed in our TO model (Figure 1C). We further\nappreciate the opportunity to clarify that ZIKV infection was not utilized in Figure 3 and have made\nclarifying edits in text and figure legends to underscore this point (text lines: 218, and 716-739).\nRepresentative immunofluorescent images of p28-GFP and CD45 within uninfected murine\ndeciduas at E13.5. 10m placental cross-sections were obtained for p28-GFPNull and p28-GFPF\nconditions and stained for anti-GFP (green), anti-CD45 (magenta) and DAPI (blue). White box\nindicates zoom inset. Images representative of 4 placentas per condition. Scale bars 100m.\n5) Figure 4: Mouse placental development matures after E10.5 days, and the authors also identified IL-\n27RA via IFA at E13.5 days. Why authors chose E6.5 as the time point for viral challenge? In additional,\nfetal and placental pathology as well as viral antigen staining are essential to support the role of IL-27RA\ndeficiency on the host’s antiviral effects.\nResponse: In our manuscript we utilized an established model for ZIKV infection of immunocompetent\nmice in which dams are inoculated with ZIKV via subcutaneous route on day 6.5, followed by maternal\nand fetal tissue collection on day 13.5 (PMID: 29746837). This model of viral challenge at E6.5 allows for\nplacental transmission of ZIKV, thus allowing us to test how the absence or presence of IL-27 signaling\ninfluences congenital viral infection in mice. We agree with the reviewer that pathology is an important\nreadout in determining the effects of IL-27 signaling during viral infection. In our manuscript, we assessed\ngross fetal pathology upon tissue collection at E13.5 and observed a significant increase in fetal\npathology in IL-27-neutralized and IL27RA-/- mice. Of these fetuses with observable pathology, the\nmajority were classified as total resorptions, thus lacking clearly defined fetal and placental tissues. Given\nthis, we did not assess placental pathology beyond the initial gross morphological assessment upon\ncollection.\n6) The RNA-seq analysis identifies a panel of potent antiviral genes in TOs, but none of them are not\nvalidated in mice models.\nResponse: In attempt to address this comment, we evaluated the expression of the panel of 5 shared\nantiviral genes (IFIT1, PARP9, PARP14, DTX3L, PSME1) that we identified in human TOs within whole,\nuninfected hSTAT2 KI and IL27RA-/- mouse placentas. Here, we did not see differences in gene\nexpression, but we attribute this to mouse-human differences in antiviral gene repertoire and use. As\nexample, one of our identified antiviral genes, IFIT1, is a gene that has been shown to exhibit species-\nspecific differences in antiviral function between humans and mice (PMID: 27240734).\nLegend: Pregnant, uninfected hSTAT2 KI or IL27RA-/- dams were euthanized at E13.5 and\nplacental tissues were collected for quantitative RT-PCR analysis. Graphs display gene\nexpression relative to housekeeping gene Hprt1. Each data point on graph represents individual\nplacenta collected from one litter per condition.\nResponse to Reviewer #2:\nMerlino et al describe their study of IL-27 actions in human placental trophoblast cultures, placental\norganoids, and mice in response to Zika virus (ZIKV) infection. This is an important study. There is\nconcern over the use and featuring of only single donor data sets, the need for deeper interrogation of the\nRNAseq data analyses, and how IFN-L and IL-27 function together and separately to impart antiviral\ndefense against ZIKV and protection form fetal disease.\nSpecific comments:\nFigure 1. E Only a single donor is shown, biological replicates from additional donors are needed. In\naddition the authors should include an additional treatment of both anti-IFN-lambda and anti-IL-27 abs in\norder to delineate which cytokines is impacting ZIKV restriction, since both clearly have similar antiviral\nactions.\nResponse: We thank the reviewer for this helpful suggestion. To address this comment and bolster our\nconclusion that IL-27 restricts ZIKV infection of TOs, in our revised manuscript we provide an additional\nTO donor as a biological replicate for all ZIKV infection experiments (Revised Figures 1E, 2F and\nSupplementary figure 3C).\nAdditionally, we agree with the reviewer regarding the similar antiviral actions of IFN and IL-27,\nand\nsuspect interplay of the two cytokines. In our previous Figure 1E, now Figure 2F, we include an additional\ncondition where TOs were pre-treated with both anti-IFN1/3 and anti-IL-27 neutralizing antibodies (“-\nIFN + -IL-27”) in order to define the combined role of IFN and IL-27 in restricting ZIKV infection.\nInterestingly, we found that neutralization of both IFN and IL-27 resulted in higher ZIKV burdens\ncompared to IFN- or IL-27-neutralization alone. Although we observed smaller magnitudes of change in\nZIKV infection following cytokine neutralization in our second biological TO donor, we did detect\nsignificantly greater levels of viral RNA in -IFN + -IL-27- treated TOs (Supplementary Figure 3C).\nThis\nsuggests that while IL-27 and IFN have independent antiviral activities at the maternal-fetal interface,\ntogether they play a joint role in regulating ZIKV infection of trophoblasts. Given this additional data, a\ndiscussion of cytokine crosstalk has been added to lines 305-312 of the revised manuscript.\n2F. S. Fig. 3C.\nLegend: TOs from 2 biological donors were treated with either isotype control, -IL-27, -IFN,\nor -IL-27 and -IFN antibodies at days 3 and 5 post-passage. At day 5 post-passage, TOs then\nwere infected with 105 PFU Cambodian ZIKV (ZIKV-CAM) or PBS (Mock) for 24 hours. TO\ncultures were then collected at day 6 post-passage for RNA analysis. ZIKV viral RNA expression\nin TOs as determined by quantitative RT-PCR relative to housekeeping gene HPRT1, normalized\nto isotype control-treated organoids. Figure 2F; Left. Graph represents 6 independent infections\nfrom single TO donor (donor 1). Statistical analysis performed with Kruskal-Wallis ANOVA,\n*p<0.05, ***p<0.001. Supplementary Figure 3C; Right. Graph represents 3 independent\ninfections from single TO donor (donor 2). Statistical analysis performed with Kruskal-Wallis\nANOVA, **p<0.01.\n2C: Here it is not clear if we are looking at ab-treated cells or cytokine treated cells, and the text does not\ndo a good job to relay what we are looking at, so this data set is unable to be interpreted. It looks like the\nbars show IFN-L and IL-27 treatment- is this correct? Or is it showing anti-IFNL and anti-IL-27 treatment\nresponse after adding converse cytokines? This is not described in the legend and is confusing in the\ntext. It looks like IFN-L is inducing the genes better than IL-27. Also, that only a single donor is used here\nmakes it difficult to determine if this response shown actually occurs in other donors and is a true\nrepresentation of the trophoblast responses.\nResponse: We appreciate the opportunity to improve the description of data in Figures 2A-C through\nchanges to the text (Lines 134-136; 147-153) and Figure 2 legend. In Figures 2A-C, we sought to\ndemonstrate both IL-27- and IFN-mediated changes in antiviral gene expression in TOs. To demonstrate\nthat both cytokines can independently induce antiviral gene expression, we first pre-treated TO cultures\nwith either -IFN or -IL-27-neutralizing antibodies and then stimulated cultures with the converse\ncytokine. We agree with the reviewer that IL-27 is not inducing the genes as strongly as IFN; however,\nthis data supports our conclusion that IL-27 induces the expression of antiviral genes MX1, IFIT1, and\nOAS2 relative to “unstimulated”, Ruxolitinib-treated TOs, independently of IFN.\nTo further address this comment, we repeated the experiments performed in Figures 2A-C using\ntrophoblast organoids from an additional donor (Supplementary Figure 4). With this additional data, we\ndid observe some donor variability in the basal antiviral gene expression of donor 2 TOs (Supplementary\nFigure 4A), as well as differences in the magnitude of antiviral gene expression following stimulation with\neither IL-27 or IFN (Supplementary Figure 4B). We speculate that this variability could be due to deriving\nthe TOs from two separate donor placentas with inherent differences in the basal placental immune\nresponses, and a discussion of this observed donor variability has been added to text lines 318-329.\nNevertheless, with this additional data we hope to strengthen our conclusions that robust basal antiviral\ngene (MX1, IFIT1, OAS2) expression is characteristic of trophoblasts, and that IL-27-stimulation induces\nantiviral gene expression in trophoblasts in the absence of IFN.\n3A. 3B.\nSupplementary Figure 3A and 3B. Expression of antiviral genes MX1, OAS2, and IFIT1 by donor\ndisplay 4 experimental replicates from single TO donor. Statistical analysis performed with multiple\nunpaired t-tests (Mann-Whitney), *p<0.05. B. Expression of antiviral genes MX1, OAS2, and IFIT1\nby TOs as determined by quantitative RT-PCR, relative to Rux. Graphs display 4 experimental\nreplicates from single TO donor. Statistical analysis performed with Kruskal-Wallis ANOVA *p<0.05,\n**p<0.01, ***p<0.001, ****p<0.0001.\nSupplementary figure 3. This data set shows Z score comparisons where it would be best to show log2\nfold change for identifying DEGs. Also, DEG networks need to be shown for functional modules that are\ndescribed in the text, thus allowing the needed resolution to assess IFN-L vs. IL-27 DEG networks and\ngene function modules driven by each cytokine or by both cytokines. A big question here is what is the\ncontribution of both cytokines to the gene and functional module signature? Also, how many different\ndonors is the RNAseq data generated from? This is important to know in order to interpret the data set.\nLine 180: Claiming novelty “This study is the first to describe transcriptional responses to IL-27 signaling\nin the human trophoblast organoid model” ; do the authors really need to do this?\nResponse: We thank the reviewer for their comment. In our previous Supplementary figure 3, now\nSupplementary figure 5A, our primary goal was to compare IFN-stimulated vs. IFN-neutralized TOs and\nIL-27-stimulated vs. IL-27-neutralized TOs. We originally used row z-scores to evaluate gene expression\nacross all samples, as opposed to just vehicle control TOs. We hope to clarify that our bulk RNA-\nsequencing analyses utilized both log 2 fold-change (±0.5) and p-value (<0.05) cutoffs in order to obtain a\ncurated list of 66 significant DEGs that are represented within the heatmap. Additionally, the log2 fold-\nchange and p-values for specific DEGs are already included in Figure 2D volcano plots, but we provide\nthe heatmap below displaying the log 2 fold-change values for our identified DEGs when comparing IFN-\nstimulated vs. IFN-neutralized TOs and IL-27-stimulated vs. IL-27-neutralized TOs for the reviewer.\nLegend: Heat map displays average log 2 fold-change\n(L2FC) gene expression values calculated for IFN-\nstimulated TOs relative to IFN-neutralized TOs and IL-\n27-stimulated TOs relative to IL-27-neutralized TOs.\nHeatmap columns represent list of 66 significant DEGs\nidentified using log 2 fold-change (±0.5) and p-value\n(<0.05) cutoffs, clustered using Pearson’s correlation.\nAverage L2FC value is representative of 3 independent\nsamples per condition from one TO donor.\nTo further address this comment, in revised Supplementary Figure 5B we have provided an additional\nheatmap analysis in which the log 2 fold-change gene expression values were calculated for all samples\nrelative to vehicle-treated TOs for the list of 66 significant DEGs. This heatmap supports our conclusion\nthat although IL-27 and IFN-mediated changes in gene expression can be observed in TOs in the basal\n(vehicle-treated) state, stimulation with the two individual cytokines significantly upregulates antiviral gene\nexpression.\nSupplementary Figure 5B. Heat map depicts log 2 fold-change\n(L2FC) gene expression for all bulk RNA-sequencing samples\nrelative to vehicle-treated TOs. Columns represent samples\nclustered using Spearman’s correlation, and rows represent\ndifferentially expressed genes that were identified by comparing\nIL-27-stimulated to IL-27-neutralized TOs and IFN-stimulated to\nIFN-neutralized TOs, then clustered using Pearson’s correlation.\n66 genes in the heatmap met cutoffs of p-value=0.05 and log fold-\nchange=±0.5 and are listed in Supplementary Table 2. L2FC\nvalues for 66 genes were then calculated relative to vehicle-\ntreated TOs. Data are representative of 3 independent samples\nper condition from one TO donor.\nIn Figure 2E, we identify gene sets or functional modules that are enriched in IFN and IL-27-stimulated\nTOs via gene set enrichment analysis (GSEA). In our revised manuscript, we edited this figure through\nthe addition of red asterisks to better highlight the gene sets that were enriched in both groups (i.e.\nresponse to virus, innate immune response, viral genome replication, and negative regulation of viral\ngenome replication). As suggested by the reviewer, to support our initial GSEA analysis and better\ndemonstrate specific DEGs driven by IFN, IL-27, or both cytokines, we now include a network analysis\nfor IFN and IL-27-stimulated TOs in Supplementary Figures 5C-D, respectively. For each cytokine, the\nnetwork analysis highlights specific genes and their contributions to the top 8 enriched gene sets\ndescribed in Figure 5E, with the most highly connected genes positioned at the center of the network\nbeing essential to multiple gene sets. This analysis supports our conclusion that both IL-27 and IFN\ndrive viral regulatory pathways in TOs, through some overlapping and some distinct antiviral genes.\nC. D.\nSupplementary Figures 5C-D. Networks of select upregulated pathways and DEGs from gene\nset enrichment analysis (GSEA) of bulk RNA sequencing data for (C.) IFN-stimulated and (D.)\nIL-27-stimulated TOs. All GSEA analyses were performed using a log fold-change cutoff value\n<0.5. Network depicts top 8 upregulated pathways by normalized enrichment score (NES) for\nIFN-stimulated and IL-27-stimulated TOs, as shown in Figure 2E. Each pathway node is\nconnected to its modulated genes, with genes oriented near the center of the network being\nhighly connected and therefore essential to multiple upregulated pathways. Color of gene nodes\nindicates log fold-change expression; size of pathway nodes indicates number of modulated\ngenes.\nFinally, in our revised manuscript all figure legends related to bulk RNA-sequencing and GSEA analysis\nhave been edited to clarify that the data is representative of one TO donor, and line 180 has been\nremoved (revised text lines 212-213).\nFigure 3. These analyses are well-done, impressive, and are highly informative.\nResponse: We thank the reviewer for this comment.\nFigure 4A-C. Dosing mice with high mw pIC will trigger TLR3, TLR7, and MDA5 signaling as well as\nactivate PKR and OAS/RNAseL. Was the goal here to mimic an inflammatory response driven by ZIKV or\nto initiate a general inflammatory response that is triggered by virus infection overall? This is not clearly\ndescribed for justification. In consideration of this, previous studies have shown that ZIKV triggers RLR\nsignaling rather than TLR signaling, so the inflammatory response would be partitioned to cytoplasmic\nRLR actions, driving a specific and different (but some overlapping) response from high my pIC. The goal\nof this experiment should be better described in the text. Overall the outcome shows no impact by IL-27.\nResponse: We appreciate the opportunity to clarify that our goal of this experiment was to induce a\ngeneral inflammatory response that is triggered by virus infection overall. We have edited the text to\nbetter describe our goal of the experiment (text lines: 244-247).\nFigure 4 D-F: These results are clear; it would be a great addition to include bulk or single cell RNAseq\nhere but the data clearly show a role for IL-27 in restricting ZIKV fetal disease.\nResponse: We appreciate the comment that our results clearly demonstrate a role for IL-27 in restricting\nZIKV fetal disease. While we acknowledge the suggestion to include bulk or single cell RNA sequencing\ndata for our mouse model, we consider this suggestion not needed to support our conclusions and\ntherefore out of scope.\nDiscussion: This is concise and clear. However, inclusion of a discussion of the role of IFN-L vs IL-27 and\ntheir role together is warranted. This study is important to define a role of IL-27 in fetal protection against\nZIKV but it does not really tell us how IL-27 compares to IFN-L in these actions nor what the contribution\nof both are to infection outcomes. These two cytokines have redundant and unique actions but the\nauthors do not really define these beyond Venn diagrams.\nResponse: We appreciate the reviewer’s comments regarding our manuscript’s discussion and agree that\nthe joint role of IL-27 and IFN during congenital infection is worth following up on. In our revised\nmanuscript, we provide additional data (Figures 2F-G) exploring the combined role of IL-27 and IFN\nduring ZIKV infection in TOs. In the absence of both cytokines, we observe higher viral burdens (Figure\n2F) and lower expression of antiviral genes compared to neutralization of IFN or IL-27 alone (Figure 2G),\nthus suggesting a joint role during infection. This new data is further supported by our bulk RNA-seq data,\nwhich shows that IL-27 and IFNinduce similar, but distinct gene signatures in TOs, therefore we would\nexpect to see the greatest changes in viral regulation in the absence of both cytokines. Given this\nadditional data, we have expanded on this conclusion in the discussion of our revised manuscript (Text\nlines: 189-212; 305-312).\nF. G.\nFigure 2F. TOs were treated with either isotype control, -IL-27, -IFN, or -IL-27 and -IFN\nantibodies at days 3 and 5 post-passage. At day 5 post-passage, TOs then were infected with 105\nPFU Cambodian ZIKV (ZIKV-CAM) or PBS (Mock) for 24 hours. TO cultures were then collected\nat day 6 post-passage for RNA analysis. ZIKV viral RNA expression in TOs as determined by\nquantitative RT-PCR relative to housekeeping gene HPRT1, normalized to isotype control treated\norganoids. Graph represents 6 independent infections from single TO donor (Donor 1). Statistical\nanalysis performed with Kruskal-Wallis ANOVA, *p<0.05, ***p<0.001.\nFigure 2G. Expression of antiviral genes IFIT1, MX1, and PARP9 by ZIKV-infected TOs as\ndetermined by quantitative RT-PCR, relative to Isotype. Graphs display 5 experimental replicates\nfrom 1 TO donor. Statistical analysis performed with Kruskal-Wallis ANOVA *p<0.05, **p<0.01.\n(Remarks on code availability)\nPrimary RNA-sequencing data are correctly deposited as GSE298278. All code is clear and is on GitHub\nResponse: We thank the reviewer for reviewing our code.\nResponse to Reviewer #3:\nThe manuscript by Merlino et al. explores the role of IL-27 and its receptor IL27RA as modulators of\nantiviral gene expression, particularly MX1, IFIT1, and OAS2, using both in vitro trophoblast organoid\n(TO) models and in vivo approaches. To investigate this signaling axis, the authors challenged the system\nwith the Cambodian strain of Zika virus (ZIKV), demonstrating that IL-27 signaling restricts ZIKV infection\nand associated pathogenesis.\nWhile IL-27 expression in human trophoblast cells and its role in immune regulation and angiogenesis\nduring pregnancy have been previously described (doi: 10.1016/j.placenta.2007.06.004), this manuscript\nfurther explores the pathways involved in the antiviral response, and identifies shared gene expression\nprograms regulated by IL-27 and IFN-γ in placental tissue.\nThis is a promising and timely study that adds valuable insights into the maternal–fetal immune interface,\nespecially regarding innate antiviral defenses in the placenta. However, as the findings are derived from\nexperiments using only one ZIKV strain, further validation is needed to support the broader conclusion\nthat IL-27 functions as a general antiviral factor at the maternal-fetal interface.\nPoints to be addressed:\n• Cytokine profile in TO culture:\nDid the authors quantify any other cytokines secreted into the TO culture supernatant besides IL-27?\nResponse: With aim of addressing this thoughtful reviewer question, we profiled our TO bulk RNA-\nsequencing data for the expression of common cytokines, chemokines, and cytokine receptors. In a figure\nbelow, we demonstrate that in addition to IL-27, other cytokines are expressed by unstimulated TO\ncultures at the transcriptional level. Of note, we observed robust expression of several IL-6 family\ncytokines, proinflammatory cytokines, and cytokines with important functions at mucosal/barrier tissues,\nsuch as the placenta. Further, other groups have assessed cytokine/chemokine secretion in TO culture\nsupernatant via Luminex and demonstrate the constitutive release of IFNs by TO cultures (PMID:\n35975985).\nGraphs displays raw, log2 RNA counts per million (CPM) for cytokine- and chemokine- (left) or\ncytokine receptor-(right) specific genes in vehicle-treated TOs. Data are representative of 3\nindependent sequencing replicates from one TO donor.\nBiological replicates:\nAlthough technical replicates were performed, several key experiments (e.g., Figures 1E, 2B, and 2C)\nappear to have been conducted using TOs from a single donor. It would be important to confirm these\nfindings using biological replicates (i.e., TOs from different placental donors), especially given known\nvariability in susceptibility to ZIKV. For instance, twin discordance in ZIKV infection has been reported\n(doi: 10.1038/s41467-018-03497-1), as well as in murine models (doi: 10.1038/nature18296).\nResponse: We thank the reviewer for this helpful suggestion. For our revised manuscript, we now provide\nan additional TO donor as a biological replicate for several key ZIKV infection and cytokine stimulation\nexperiments. Using TOs from 2 biological donors, we show that IL-27 signaling restricts ZIKV infection of\nTOs at multiple timepoints via quantitative RT-PCR (Figure 1E) and plaque assay (Supplementary Figure\n2B), as requested by reviewer 1. In Supplementary Figures 3A-B, we repeat the experiments performed\nwithin Figures 2B and 2C and demonstrate basal antiviral gene expression and IL-27-mediated\nstimulation of antiviral genes in our second biological TO donor. Further, using TOs from 2 biological\ndonors we demonstrate a joint role for IL-27 and IFN in regulating ZIKV infection of trophoblasts, as\nrequested by reviewers 1 and 2 (Figure 2F and Supplementary Figure 3C).\nFinally, we agree with the reviewer on the possibility of donor variability and did observe this through our\nexperiments. As with twin discordance, one contributing factor to the differences we observe in viral\ninfection of trophoblasts from two separate donor placentas could be inherent differences in the basal\nplacental immune responses. We can begin to observe these differences in Figures 2B and\nSupplementary Figure 3A, which show that donor 2 TOs may exhibit more robust basal expression of\nantiviral genes OAS2 and IFIT1. A discussion of this observed donor variability has been added to our\nrevised discussion in lines 318-329.\n• Fetal viability and variability in resorption rates:\nIn the fetal viability assays following IL-27 or IL27RA blockade and ZIKV infection, the authors report an\nincrease in fetal resorption of just over 23%. While statistically significant, this effect also shows\nconsiderable variability. What factors might contribute to this variation? Could it be related to the genetic\nbackground of the TO cultures or the mice?\nResponse: We thank the reviewer for this thoughtful question. We agree that our phenotype of 23% fetal\nresorption suggests individual variation in the ultimate fetal disease outcome of lacking IL-27 signaling\nduring congenital Zika virus infection. In murine models of pregnancy/congenital infection, each individual\nimplantation site (placenta/pup unit) represents an independent virus transmission event, thereby each\nimplantation site can have varied infection kinetics/dynamics. In addition, the variability of disease\noutcome could be explained by differences of individual host response (differential basal antiviral state or\nthresholds for fetal resorption). The differences of individual host response can be seen in our TO\nexperiments (Figure 1E, Figure 2B-C/Supplementary Figure 3A-B, Figure 2F/Supplementary Figure 3C)\nwhere we observed donor variability in TO infection and antiviral gene expression. Overall, we\nhypothesize that the variability observed in our TO and murine models is related to genetic and epigenetic\nbackground differences that influence basal host responses in the placenta. We have now included\ndiscussion of this variability in the discussion (text lines: 318-329).\n• Quantification of ZIKV levels in tissues (Figure 4H):\nThe Y-axis is labeled as “PFU equivalents/mg tissue”, yet the method used is qRT-PCR, which detects\nZIKV RNA, not infectious viral particles. Even though a standard curve may have been used, RNA\ndetection alone does not confirm the presence of mature or infectious virions. To accurately assess viral\ninfectivity, a plaque-forming unit (PFU) assay or in vitro titration would be required. The authors should\nconsider referring to the data as Ct values or viral RNA copies, rather than PFU equivalents.\nResponse: We agree with the reviewer and have modified the Y-axis to “Viral RNA (PFU equivalents/mg\ntissue)”, to clarify that the readout of the assay is viral RNA copies as determined by RT-qPCR, not\ninfectious viral particles. To further specify, all values were calculated using a standard curve of RNA\nisolated from known ZIKV PFUs, thus allowing for calculation of PFU-equivalent units based on raw\nsample Ct values. After calculating the total sample PFU-equivalents based on our known standard curve,\nwe standardized according to tissue mass (mg), thus allowing us and readers to compare viral copies\nacross different tissues (fetus, placenta, serum) and across studies. This information has been added\nwithin the legend to further clarify. Additionally, our revised manuscript and discussion of Figure 4 has\nbeen edited to specify “viral RNA” (Text line: 289).\n• Figure labeling error:\nThe legend of Figure 4 contains two panels labeled as “H”. Please revise to correct this duplication.\nResponse: This error has been corrected in the revised manuscript (revised text lines 781-782)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have clarified all my previous concerns, and the MS is now acceptable.", "reviewer_2": "The authors have fully and adequately addressed my previous comments via inclusion of new data sets and text revisions.\nThis is a solid study that brings a new area of focus into maternal-fetal virus infection and immunity, defining IL-27 as a key\ncytokine in controlling ZIKV infection at this key interface."}, "author_response": ""}]

Decision letter