Dipeptidylpeptidase 4 inhibition attenuates gestational pathologies via immune homeostasis restoration in the pulmonary-uterine axis

Domain: chemistry
Published: 2026-02-17
Editorial outcome: major_revision

Abstract
Respiratory viral infections during pregnancy threaten maternal pulmonary health and fetal development, however the mechanisms linking lung infection to distant uterine immune disruption remain unclear. Here, we demonstrate that respiratory viral infection attenuates uterine immune activation, impairing vascular remodeling and trophoblast invasion, which compromises embryonic growth. Treatment with the dipeptidylpeptidase 4 (DPP4) inhibitor sitagliptin restores immune homeostasis in both the lung and uterus, markedly reducing pregnancy complications. Mechanistically, while pulmonary infection expands interleukin-1 receptor type 2–expressing (IL1R2⁺) CD11b⁺ myeloid cells in the lung, this expansion is attenuated by DPP4 inhibition. These cells migrate to the decidua and disrupt pregnancy-maintaining immune signaling. Single-cell RNA sequencing confirms accumulation of IL1R2⁺ regulatory macrophages at both sites. Genetic Il1r2 ablation similarly reduces uterine IL1R2⁺ cells and restores gestation. This study reveals a lung-uterus immune axis and identifies DPP4 inhibition as a dual-organ therapeutic strategy against viral-induced pregnancy pathology.

Introduction
Respiratory viral infections during pregnancy are associated not only with maternal pulmonary complications but also with severe pregnancy-related outcomes, including intrauterine growth restriction, preterm birth, and neonatal mortality1–3. Moreover, such infections are linked to long-term health conditions in offspring, including cardiovascular disease and neurodevelopmental disorders such as schizophrenia4. In addition to seasonal and pandemic influenza strains5, other respiratory pathogens—such as severe acute respiratory syndrome (SARS) coronavirus6, Middle East Respiratory Syndrome (MERS) coronavirus7, novel coronavirus (SARS-CoV-2)8, and respiratory syncytial virus (RSV)9—have also been implicated in gestational complications. These associations underscore an urgent clinical need for effective interventions to mitigate pregnancy abnormalities secondary to maternal respiratory viral infections.

Current research on respiratory viral infections during pregnancy has primarily focused on hormonal fluctuations and viral transmission dynamics10,11. However, growing evidence suggests that these infections also disrupt immune function at distal mucosal sites. For instance, influenza A virus and RSV can induce significant alterations in gut microbiota, thereby impairing mucosal immunity12. Additionally, they can interfere with intestinal metabolic outputs, modulating the biosynthesis of ligands for innate-like T cells, such as natural killer T (NKT) cells13, and mucosa-associated invariant T (MAIT) cells14. Lung-derived type I interferons produced during influenza infection have also been shown to suppress intestinal immune responses against bacterial pathogens such as Salmonella, even in the absence of direct intestinal infection15. Despite the uterus being a key mucosal immune organ during pregnancy, whether it is affected by respiratory viral infections has not been elucidated.

The maternal-fetal interface is densely populated by immune cells, primarily of the innate lineage. Decidual natural killer (NK) cells constitute approximately 70% of the immune cell population, followed by macrophages at around 20%16,17. Contrary to the long-standing notion that sustained immunosuppression is essential for a successful pregnancy, recent findings highlight the importance of temporally regulated immune activation18,19. Embryo implantation requires a proinflammatory environment, characterized by the upregulation of cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1), and leukemia inhibitory factor (LIF), which are evolutionarily conserved among placental mammals20,21. Post-implantation, trophoblast cells secrete chemokines to recruit NK cells and monocytes to the maternal-fetal interface, facilitating extracellular matrix remodeling and uterine spiral artery transformation22,23. This finely tuned immune–trophoblast crosstalk, mediated through receptor–ligand interactions, ensures successful trophoblast invasion, vascular remodeling, fetal development, and maintenance of decidual integrity24,25. Whether respiratory viral infections disrupt this equilibrium in the uterus remains a critical and unexplored question.

In this study, we investigate how respiratory viral infections disrupt uterine immunity and contribute to gestational pathologies. We demonstrate that the DPP4 inhibitor sitagliptin restores fetal development by modulating lung-derived IL1R2+ myeloid cell migration and decidual immune function. These findings reveal a previously unrecognized lung-uterus immune axis and establish DPP4 inhibition as a potential dual-organ therapeutic strategy for virus-associated pregnancy complications.

Results
Respiratory influenza virus infection suppresses immune activation at the maternal-fetal interface
To evaluate immune alterations at the maternal-fetal interface following respiratory influenza virus infection, we established an H1N1 influenza A infection model in pregnant mice at embryonic day 6.5 (E6.5) and monitored pregnancy progression (Supplementary Fig. 1a). Consistent with previous reports26, infected dams exhibited reduced gestational weight gain (Supplementary Fig. 1b). Macroscopic examination of the lungs revealed dark red lesions and tissue swelling (Supplementary Fig. 1c), and histological analyses showed exacerbated inflammation, fibrosis, and heightened immune cell infiltration (Supplementary Fig. 1d–f). Influenza virus infection is known to damage alveolar epithelial cells, trigger robust immune responses, and induce proinflammatory cytokine release27. Accordingly, expression levels of Il6, Tnf, and Il1b were significantly elevated in lung tissues following infection (Supplementary Fig. 1g). Fetal assessments at E12.5 and E16.5 revealed marked intrauterine growth restriction, evidenced by significantly reduced fetal body length and weight in infected pregnancies (Supplementary Fig. 1h–k). These findings confirm that respiratory influenza infection during early gestation causes severe pulmonary pathology and fetal growth impairment, validating the robustness of our infection model.

Respiratory viruses rarely traverse the placental barrier28. Consistent with this, viral RNA was detected exclusively in lung tissues, with no detectable viral gene expression in decidual or placental tissues (Supplementary Fig. 1l), aligning with previous findings. Additionally, type I interferons were significantly upregulated in the lungs but not in the decidua or placenta (Supplementary Fig. 1m, n), indicating that fetal growth restriction is not due to direct viral infection at the maternal-fetal interface.

Transcriptomic analysis of decidual tissues revealed significant alterations in immune signaling pathways. Immune-inhibitory gene pathways—including negative regulation of innate immune responses and cytokine production—were upregulated, while immune-activating pathways were suppressed post-infection (Fig. 1a, b). Notably, several cytokines upregulated in the lung—IL-28, MIP-3α (Ccl20), IL-6, and IL-21—were downregulated in the decidua following infection (Fig. 1c, d).

We also observed significant upregulation of the type I interferon signaling inhibitor Usp18 in decidual tissues (Fig. 1e). Usp18 attenuates immune responses by deconjugating ISG15 from its substrates, thereby suppressing inflammatory cytokine production29. These findings suggest that respiratory influenza infection induces a systemic immunosuppressive state at the maternal-fetal interface.

Successful pregnancy requires localized immune activation to enable extracellular matrix remodeling and trophoblast invasion—key processes in spiral artery remodeling. VEGF and MMP9 are essential mediators of angiogenesis and tissue remodeling30. Following infection, both Vegf and Mmp9 expression were significantly reduced in decidual tissues (Fig. 1f, g), indicating compromised angiogenesis. Histological analysis further revealed impaired vascular remodeling, with increased vascular wall thickness and decreased cavity area in infected deciduas (Fig. 1h, i). Cytokeratin staining showed a reduction in trophoblast cell invasion (Fig. 1j), a critical process for adequate spiral artery remodeling and placental perfusion30. These data collectively demonstrate that respiratory influenza infection leads to suppressed immune activation at the maternal-fetal interface, impairs vascular remodeling and trophoblast invasion, and ultimately restricts fetal growth.

DPP4 inhibition rescues pregnancy outcomes following respiratory virus infection
We next investigated the mechanisms linking pulmonary infection to uterine immune dysregulation and evaluated potential therapeutic strategies. Given that the respiratory and reproductive systems are interconnected through systemic circulation, we hypothesized that blood-borne factors may mediate communication between the lungs and uterus. Proteomic analysis of serum collected at 12.5 days of gestation (6 days post-infection) revealed a significant increase in DPP4 protein levels, which was validated by ELISA (Fig. 2a, b). DPP4, a dipeptidylpeptidase expressed in both membrane-bound and soluble forms, is implicated in immune modulation, inflammation, and metabolic regulation31,32.

To assess therapeutic potential, we administered the DPP4 inhibitor sitagliptin to infected dams by daily oral gavage from E7.5 to E11.5, while controls received vehicle (Fig. 2c). Sitagliptin treatment significantly mitigated virus-induced maternal weight loss (Fig. 2d) and reduced pulmonary injury. Macroscopic examination of the lungs revealed dark red lesions and tissue swelling (Fig. 2e) and histopathological analyses demonstrated reduced pulmonary inflammation in the H1N1+Sitagliptin group (Fig. 2f, g).

Treated embryos displayed increased weight and body length, along with enlarged placental diameter and weight compared to untreated infected controls (Fig. 2h, i). Histological analysis showed that sitagliptin restored angiogenesis at the maternal-fetal interface. Vascular wall thickness and cavity area returned to levels comparable to uninfected controls, and trophoblast invasion was reinstated (Fig. 2j–l). Expression of Vegfa also increased following treatment, approximating the levels observed in uninfected pregnancies (Fig. 2m). Taken together, these results demonstrate that DPP4 inhibition via sitagliptin ameliorates the adverse effects of respiratory influenza virus infection on pregnancy by restoring immune and vascular homeostasis at the maternal-fetal interface.

To investigate whether the protective effect of sitagliptin was mediated through immunomodulation rather than via suppression of viral replication, we performed immunohistochemical staining for influenza nucleoprotein, viral titer assays, and quantification of viral gene expression in both lung and decidual tissues following influenza virus infection. These analyses consistently demonstrated that DPP4 inhibitor did not significantly affect viral replication or propagation (Supplementary Fig. 2a–e). Given the role of IL-6 as a key marker of virus-induced lung inflammation33, we assessed Il6 expression and found its significantly reduced with sitagliptin treatment compared to the H1N1 group (Supplementary Fig. 2d). These results suggest that while sitagliptin tempers hyperactive immune responses in the lung and restores immune homeostasis in the decidua, it does not impair antiviral defense. Importantly, separate administration of the inhibitor to healthy pregnant mice revealed no detectable adverse impacts on maternal well-being or prenatal development (Fig. 2d–m and Supplementary Fig. 2a–e), thus establishing its safety profile. These findings confirm its potential as a safe therapeutic candidate for respiratory virus infection in pregnancy. Furthermore, compared with uninfected controls, inhibitor treatment alone did not significantly change lung neutrophil or macrophage levels, indicating that the DPP4 inhibitor itself does not perturb the baseline pulmonary immune landscape. H1N1 infection, as expected, induced a pronounced increase in both neutrophils and macrophages, which was markedly attenuated by sitagliptin treatment (Supplementary Fig. 2f–i). These results reinforce the conclusion that sitagliptin exerts protection primarily by modulating the pulmonary immune environment.

DPP4 inhibition has a persistent protective effect towards pregnancy outcomes
To evaluate the long-term effects of sitagliptin treatment, pregnant mice were monitored until successful delivery following a 5-day therapeutic regimen. Maternal body weight was measured at two time points: immediately following treatment completion (embryonic day 11.5) and one day before expected delivery (embryonic day 18.5). Our findings demonstrate that sitagliptin treatment significantly alleviated maternal weight loss induced by the infection (Supplementary Fig. 3a). Post-delivery monitoring revealed that infected mothers exhibited lower body weight at postpartum week 1 and 2, a phenotype mitigated by sitagliptin. By the third week postpartum, maternal weight gain became comparable across all groups (Supplementary Fig. 3b). Neither infection nor treatment affected the timing of parturition (Supplementary Fig. 3c). Importantly, influenza virus infection markedly reduced the number of successfully delivered offspring. The litter size from healthy pregnancies was generally above 6 pups, whereas infection without treatment resulted in fewer than 5 surviving pups. Sitagliptin treatment substantially reversed this impairment, restoring litter sizes to levels comparable to those of uninfected dams (Supplementary Fig. 3d). These results further suggest that in untreated infections, some fetuses sustained severe damage during late gestation, preventing their successful birth. Therefore, the surviving pups from infected but untreated mothers did not show significant differences in health status compared to pups from either uninfected or treated mothers (Supplementary Fig. 3e). Collectively, these findings demonstrate that sitagliptin confers sustained benefits in both maternal recovery and offspring development, supporting its long-term protective efficacy.

Additionally, to assess the diversity of effective treatment windows for the inhibitor, mice were intranasally infected with the influenza virus after the establishment of the choriovitelline placenta at embryonic day 12.5 (E12.5)34. This was followed by a 5-day treatment regimen with sitagliptin administered daily by oral gavage from E13.5 to E17.5, while control groups received the vehicle only (Supplementary Fig. 4a). Sitagliptin treatment significantly mitigated maternal weight loss induced by the virus (Supplementary Fig. 4b) and reduced pulmonary injury. Macroscopic examination of the lungs showed dark red lesions and tissue swelling in infected animals, which were markedly improved in the sitagliptin-treated group (Supplementary Fig. 4c, d). At the embryonic level, compared with untreated infected controls, dams treated with sitagliptin exhibited increased fetal weight and body length (Supplementary Fig. 4e, f). Collectively, these findings demonstrate that sitagliptin administration after placental establishment effectively mitigates the detrimental effects of influenza infection on both maternal health and embryonic development.

DPP4 inhibition ameliorates immune dysregulation in the uterus and lung
We next examined whether DPP4 inhibition could alleviate the immune dysregulation at the maternal-fetal interface induced by respiratory viral infection. Decidual tissues were collected at 6 days post-infection (embryonic day 12.5) from Saline, H1N1, and H1N1+Sitagliptin groups for transcriptome profiling. Volcano plot analysis revealed that genes significantly downregulated by H1N1 infection, such as Lrrn4, Upk3b, Upk1b, and Car3, were restored in expression in the H1N1+Sitagliptin group (Supplementary Fig. 5a, b). Conversely, genes upregulated by H1N1 infection, including Prl7a1, Ctsm, and Gsdma, were suppressed by sitagliptin treatment (Supplementary Fig. 5a, b).

Gene ontology (GO) enrichment analysis showed that pathways related to type I interferon signaling and immunosuppressive responses, which were activated by H1N1 infection, were downregulated following sitagliptin treatment (Supplementary Fig. 5c, d). Heatmap analysis further demonstrated that the expression profiles of genes associated with interferon-β response, angiogenesis, antigenic stimulation, and extracellular matrix organization in the H1N1+Sitagliptin group resembled those of the uninfected Saline group, in contrast to the H1N1-infected group (Supplementary Fig. 5e). Notably, expression of Usp18, a negative regulator of type I interferon signaling, was significantly reduced with sitagliptin treatment compared to H1N1 infection alone (Supplementary Fig. 5f). Collectively, these findings suggest that DPP4 inhibition restores immune homeostasis at the maternal-fetal interface, supporting normal angiogenesis and promoting a favorable pregnancy environment under conditions of maternal respiratory viral infection.

To determine whether DPP4 inhibition also modulates immune responses in the lung, we performed transcriptomic analysis on lung tissues collected at the same time point. KEGG pathway analysis revealed that several immune-activating pathways—such as PI3K-Akt signaling, ECM-receptor interaction, and leukocyte transendothelial migration—were significantly upregulated in response to H1N1 infection but were suppressed by sitagliptin treatment (Supplementary Fig. 5g, h). These data indicate that sitagliptin mitigates aberrant immune activation in the lung.

DPP4 inhibition reduces IL1R2 accumulation induced by respiratory viral infection
To elucidate the mechanism by which sitagliptin modulates immune responses in both the uterus and lung, we hypothesized the involvement of a secreted factor produced in the inflamed lung that accumulates at the maternal-fetal interface. GO analysis of lung transcriptomes following H1N1 infection revealed significant enrichment of cytokine production and secretion pathways, particularly those involving the IL-1 family (Fig. 3a). The IL-1 family consists of 11 ligands (including seven active agonists), three receptor antagonists, and one anti-inflammatory cytokine, signaling through a network of six receptor chains, four signaling receptor complexes, two decoy receptors, and two negative regulators35. Within this pathway, the decoy receptor Il1r2, the antagonist Il1rn, the active ligand Il1b, and the accessory protein Il1rap were identified as key components upregulated post-infection (Fig. 3b). Expression analysis confirmed a significant upregulation of Il1r2, Il1b, and Il1rn in the lung, while Il1rap was undetectable (Fig. 3c and Supplementary Fig. 1g).

We next investigated whether Il1r2, Il1b, or Il1rn accumulated in the decidua. Proteomic profiling of decidual tissue identified IL1R2 as one of the most significantly upregulated proteins with known roles in both immune modulation and embryonic development. Notably, IL1R2 exists in both membrane-bound and soluble forms36 (Fig. 3d). Elevated IL1R2 levels in H1N1-infected decidua were further validated by ELISA (Fig. 3e). As the first-described decoy receptor37, IL1R2 structurally mimics IL-1R1, competitively inhibiting IL-1 signaling. Immunostaining showed that IL1R2-positive cells were distributed across both the decidua basalis (DB) and mesometrial lymphoid aggregate of pregnancy (MLAp) layers16,38 (Fig. 3f).

We then assessed whether sitagliptin could attenuate IL1R2 accumulation. Lung tissues from all three groups were analyzed, revealing a marked reduction in Il1r2 expression in the H1N1+Sitagliptin group (Fig. 3g). Immunofluorescence staining further confirmed reduced IL1R2 protein levels in the lungs of sitagliptin-treated animals (Fig. 3h). Together, these findings indicate that IL1R2 is induced in the lung during viral infection and subsequently accumulates at the maternal-fetal interface. DPP4 inhibition suppresses IL1R2 production in the lung, implicating this decoy receptor as a key mediator linking pulmonary inflammation to uterine immune dysregulation during respiratory viral infection.

IL1R2+ myeloid cells systemically expand in the lung, blood, and uterus following respiratory viral infection
Given the critical role of IL1R2 in mediating adverse pregnancy outcomes during respiratory influenza virus infection, we hypothesized that IL1R2 is not locally induced in the uterus but instead originates in the lung and traffics to the decidua via the circulatory system. IL1R2 is constitutively expressed in myeloid cells, including neutrophils, monocytes, and macrophages39. To investigate its systemic dynamics, we collected lung, peripheral blood, and decidual samples and analyzed both immune cell composition and IL1R2 expression by flow cytometry (Supplementary Fig. 6a).

At 4 days post-infection (E10.5), we observed a significant expansion of CD11b⁺ myeloid cells—including monocytes, neutrophils, and granulocytes—in the peripheral blood (Fig. 4a). These CD11b⁺ cells displayed markedly elevated IL1R2 expression as early as 2 days post-infection (E8.5), with sustained upregulation at 6 days post-infection (E12.5) (Fig. 4b, c).

To further assess tissue-specific IL1R2 expression, we examined immune cell populations in the lung, peripheral blood and decidua at E12.5. Infection induced a robust expansion of CD11b+ myeloid cells (macrophages, neutrophils and monocytes) in the lung, accompanied by a similar increase percentage in circulating neutrophils and monocytes (Supplementary Fig. 6b–e). By contrary, we did not observe expansion of CD11b+ myeloid cells in the decidua (Supplementary Fig. 6f, g). Moreover, IL1R2 expression on CD11b+ myeloid cells in the lung was robustly upregulated following H1N1 infection, with expression levels significantly exceeding those observed in the decidua. Nonetheless, in the decidua, IL1R2 was also elevated on monocytes, neutrophils and granulocytes (Fig. 4d, e). Co-immunofluorescence for CD11b and IL1R2 confirmed the presence of double-positive cells in both lung and decidual tissues post-infection (Fig. 4f, g). Together, these findings establish that respiratory influenza infection induces a systemic expansion of IL1R2-positive myeloid cells, with significant enrichment at the maternal-fetal interface.

Single-cell RNA-seq identifies tissue-homing IL1R2+ macrophages that link pulmonary infection to maternal–fetal interface
To trace the origin of IL1R2-expressing cells at the maternal-fetal interface, we performed single-cell RNA sequencing on CD45⁺ immune cells isolated from the lungs and decidua of pregnant mice at E12.5 following treatment with saline, H1N1 infection alone, or H1N1 infection plus sitagliptin. After quality control and doublet removal, unsupervised clustering of lung immune cells identified nine distinct subpopulations (Supplementary Fig. 7a, b). Macrophages constituted the most abundant subset and were significantly expanded following H1N1 infection, an effect that was substantially reversed by sitagliptin treatment (Supplementary Fig. 7c). Given their tissue residency and specific expression of the macrophage markers Ms4a6c (lung) and Ms4a4c (decidua), we annotated this Lyz2-sharing population as macrophages, while not excluding the possibility of some monocyte presence. Further analysis revealed a marked H1N1-induced upregulation of Il1r2 in lung macrophages, which was also significantly suppressed by sitagliptin (Supplementary Fig. 7d). In the decidua, we identified ten immune cell clusters (Supplementary Fig. 7e, f). Analysis of the major macrophage populations showed a congruent pattern: H1N1 infection robustly induced Il1r2 expression, and this induction was attenuated by sitagliptin (Supplementary Fig. 7g, h). These findings underscore macrophages as a key Il1r2-expressing population during influenza infection in pregnancy.

We next re-clustered macrophages and searched for the most relevant macrophage subpopulations. Based on macrophage types summarized previously40, lung macrophages were categorized into six distinct subsets (Fig. 5a). Among these, a subpopulation of immunoregulatory macrophages highly expressed Il1r2 (Fig. 5b, c). This subset was rare under homeostatic conditions but expanded significantly after H1N1 infection, with its proportion subsequently normalized by sitagliptin (Fig. 5d). Visualization of Il1r2 expression confirmed its specific enrichment in this immunoregulatory subset after infection, which was abrogated by treatment, suggesting these cells may serve as a key target of sitagliptin and represent a primary source of IL1R2 (Fig. 5e, f). An analogous immunoregulatory macrophage subset, also exhibiting high Il1r2 expression, was identified in the decidua (Fig. 5g–i). Its abundance increased after infection and decreased with sitagliptin treatment, and its Il1r2 expression mirrored this trend, closely recapitulating the pulmonary response (Fig. 5j–l). This suggests that these cells may serve as a critical link between the lung and maternal-fetal interface, and constitute a local source of IL1R2.

Integrated analysis of genes commonly upregulated in this macrophage subset across tissues revealed a distinct molecular signature: beyond Il1r2, these cells specifically expressed notably key mediators of myeloid cell migration and chemotaxis (Retnlg, Cxcr2, Mmp9) and other immunomodulatory genes (Chil1, Steap4, Nlrp12), implying enhanced migratory potential and immunosuppressive ability41,42 (Fig. 5m, n). In summary, our results indicate that respiratory viral infection induces a specialized subset of immunoregulatory macrophages characterized by high Il1r2 expression and a migratory gene signature, positioning them as a likely systemic source for the increased IL1R2 observed at the maternal-fetal interface.

Deletion of Il1r2 ameliorates virus-induced adverse pregnancy outcomes
Given the significant accumulation of IL1R2 at the maternal-fetal interface following H1N1 infection, we next tested whether IL1R2 mediates virus-induced pregnancy complications. IL1R2 acts as a decoy receptor that sequesters IL-1β, thereby preventing its interaction with IL-1R1 and attenuating downstream signaling39. To assess its causal role, we generated Il1r2-knockout (KO) mice and mated Il1r2-deficient females with wild-type males. Pregnant females were infected with H1N1 at E6.5 and evaluated 6 days later (Fig. 6a). Both Il1r2-WT and Il1r2-KO mice exhibited severe lung pathology post-infection, characterized by extensive hyperemia and pulmonary enlargement (Fig. 6b–e), indicating that Il1r2 deletion does not impair the host’s susceptibility to lung damage.

We then assessed pregnancy outcomes. In WT mice, influenza infection significantly impaired fetal development, as evidenced by reduced embryo length and placental diameter. Strikingly, Il1r2-KO mice maintained normal fetal and placental growth post-infection, with no signs of intrauterine growth restriction (Fig. 6f, g). These data suggest that IL1R2 plays a key role in mediating virus-induced fetal growth impairment. Importantly, Il1r2 deletion under uninfected conditions did not adversely affect embryonic development, underscoring its pathogenic role in the context of infection (Fig. 6f, g).

Histological analysis of the maternal-fetal interface further revealed that Il1r2 deletion alleviated the vascular remodeling defects induced by infection, as shown by reduced vascular wall thickening (Fig. 6h, i). Additionally, cytokeratin staining demonstrated improved trophoblast invasion in Il1r2-KO mice, reversing the impairment seen in infected WT mice (Fig. 6j). We next investigated whether Il1r2 knockout modulates viral replication. Immunohistochemical staining for influenza nucleoprotein and quantitative analysis of viral gene expression in lung and decidual tissues showed that viral replication and propagation were not significantly affected by Il1r2 deletion (Supplementary Fig. 8a–c). Collectively, these findings establish IL1R2 as a critical mediator linking respiratory viral infection to adverse pregnancy outcomes and highlight it as a potential therapeutic target for protecting pregnancy during maternal respiratory infections.

Increase of IL1R2 and fetal growth restriction in the uterus following pulmonary coronavirus infection modeled by intranasal MHV inoculation
To determine whether IL1R2 upregulation is a broader feature of other virus-induced fetal growth restriction, we extended our investigation to a model of coronavirus infection. COVID-19, caused by SARS-CoV-2, presents a major threat to maternal and fetal health during pregnancy43. Mouse hepatitis virus (MHV), a member of the same genus as SARS-CoV-2, induces clinical symptoms in mice that closely resemble those observed in COVID-19 patients, including weight loss, multi-organ pathology, and irreversible neurological damage. Therefore, MHV serves as a well-established surrogate model for studying SARS-CoV-2 pathogenesis44. Pregnant mice were intranasally infected with MHV at embryonic day 6.5 (E6.5) and monitored through E12.5 (Supplementary Fig. 9a). Infected mice exhibited impaired gestational weight gain (Supplementary Fig. 9b), as well as significant lung inflammation and enlargement (Supplementary Fig. 9c–e), in line with previous reports44 and confirming successful model establishment.

Assessment of pregnancy outcomes revealed significant fetal growth restriction following MHV infection, evidenced by reduced embryo weight and length, as well as diminished placental diameter and weight (Supplementary Fig. 9f, g). Histological analysis of the maternal-fetal interface showed impaired angiogenesis, marked by thickened vascular walls (Supplementary Fig. 9h, i). As observed in H1N1 infection (Fig. 1f, g), qPCR analysis revealed reduced expression of key angiogenic factors Vegfa and Mmp9 (Supplementary Fig. 9j), implicating disrupted angiogenesis as a contributor to impaired fetal nutrient transport and subsequent growth restriction.

Interestingly, unlike typical antiviral responses, MHV infection did not elicit a type I interferon response in the lung but instead elevated levels of IFN-γ and pro-inflammatory cytokines such as TNF-α45. Viral replication was confined to the lungs, as evidenced by localized upregulation of interferon response and Tnf, with no signs of vertical transmission (Supplementary Fig. 9k, l). Notably, expression of inflammatory marker Tnf was significantly reduced in the decidua, suggesting attenuated immune activation at the maternal-fetal interface (Supplementary Fig. 9l). These findings parallel the immunological alterations observed in influenza infection and support the notion that nasal inoculation of viruses to simulate pulmonary infection can impair placental function and fetal development via indirect, non-viral mechanisms.

We next examined whether IL1R2 expression is also elevated in this model. Transcriptomic profiling of lung tissue 6 days post-infection revealed significant upregulation of gene pathways related to immune activation, cytokine production, and type II interferon responses—consistent with observed lung pathology. In contrast, pathways involved in ciliary organization and motility were downregulated, indicating disruption of lung epithelial integrity, as seen in influenza models46 (Supplementary Fig. 10a). Notably, Gene Ontology and Sankey analyses identified upregulation of pathways regulating IL-1 family cytokines, including the immunosuppressive receptor IL1R2, which participates in feedback regulation of inflammatory responses (Supplementary Fig. 10b). FPKM and gene quantification showed markedly increased Il1r2 expression in lung tissue following infection (Supplementary Fig. 10c). Meanwhile IL1R2 protein levels increased in the decidua (Supplementary Fig. 10d), mirroring our findings in influenza-infected models (Fig. 3c, e). Immunohistochemistry further confirmed the presence of CD11b⁺ IL1R2⁺ double-positive cells in both lung and decidual tissues (Supplementary Fig. 10e, f). These results suggest that IL1R2 elevation in the decidua and the associated pregnancy complications may be a common feature across pulmonary infection modeled by intranasal H1N1 or MHV inoculation.

DPP4 inhibition alleviates pregnancy complications induced by coronavirus infection
To explore potential therapeutic strategies, we investigated whether DPP4 inhibition could mitigate coronavirus-induced pregnancy complications. Transcriptomic data revealed a significant increase in Dpp4 expression following MHV infection, suggesting a possible pathogenic role and therapeutic target (Fig. 7a, b). Administration of the DPP4 inhibitor sitagliptin significantly attenuated maternal weight loss and reversed fetal growth restriction. Treated embryos exhibited restored body length, weight, and placental size, comparable to uninfected controls (Fig. 7c–e).

To assess whether sitagliptin alleviated immunopathology in maternal tissues, we conducted transcriptomic analyses of lung and decidual tissues post-treatment. In the lung, sitagliptin reversed the downregulation of genes suppressed by infection and significantly decreased expression of pro-inflammatory markers, including Il1b, Nfkbiz, and Lyve1 (Supplementary Fig. 10g). GO enrichment analysis demonstrated recovery of pathways involved in intercellular communication, blood circulation, and tissue homeostasis (Supplementary Fig. 10h), indicating that sitagliptin attenuates excessive lung inflammation.

Histological analysis of the maternal-fetal interface revealed that sitagliptin treatment significantly reduced vascular wall thickening (Fig. 7f, g). Immunohistochemical staining further demonstrated enhanced trophoblast invasion following treatment (Fig. 7h), indicating a restoration of normal placental development. Notably, sitagliptin also significantly reduced IL1R2 protein levels in the decidua (Fig. 7i), further supporting its therapeutic potential.

To determine whether sitagliptin also restored immune balance at the maternal-fetal interface, we conducted transcriptome sequencing of decidual tissue. Volcano plot analysis revealed that sitagliptin downregulated multiple genes upregulated by intranasal MHV inoculation such as Cd40lg, Krt8, and Nme8, while upregulating genes suppressed by infection, including Prl2c5, Galnt9, and Mmp10 (Fig. 7j). GO analysis indicated that MHV infection triggered immunosuppressive pathways involving cytokines like IL-4, IL-13, and IL-10, while downregulating key signaling pathways such as JAK-STAT, extracellular matrix organization, and angiogenesis—thereby impairing maternal-fetal communication and pregnancy maintenance. Importantly, sitagliptin treatment reversed many of these effects, enhancing activation of the JAK-STAT pathway and restoring pathways related to immune activity and pregnancy support (Fig. 7k).

To further directly evaluate whether sitagliptin exerts its beneficial effects via antiviral activity in the MHV infection model, we measured viral gene replication in lung tissues after treatment. Critically, sitagliptin did not suppress MHV replication in the lungs, and MHV remained restricted to the lung without spreading to the maternal-fetal interface (Supplementary Fig. 10i), suggesting that DPP4 may not be involved in MHV amplification and that the receptor for MHV remains CEACAM1, as previously reported47. These data clearly demonstrate that the protective effect of sitagliptin on pregnancy outcomes is not mediated by direct antiviral mechanisms, but rather through modulation of the host’s immunopathological response.

Collectively, these findings demonstrate that DPP4 inhibition not only mitigates excessive lung inflammation and placental dysfunction caused by coronavirus infection but also counteracts IL1R2 elevation and immune suppression at the maternal-fetal interface. Thus, DPP4 inhibitors such as sitagliptin may represent a promising therapeutic strategy for managing adverse pregnancy outcomes associated with pulmonary infection modeled by intranasal H1N1 or MHV inoculation.

Methods
Mice
Seven-week-old female and eight-week-old male C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Company (stock number: scb0105). Il1r2+/− mice were obtained from GemPharmatech and bred to generate Il1r2+/+ and Il1r2−/− genotypes (stock number: T005564). Genotyping was performed via standard PCR using primers listed in Supplementary Table 1. All mice were maintained on a C57BL/6 background. Littermate and cage-mate controls were used throughout the study. Mice were housed under specific pathogen-free (SPF) conditions, with a 12-h light/dark cycle, ambient temperature of 20–26°C, and relative humidity of 50–70%. All animals including experimental and control mice were co-house throughout the study. All animals were fed an irradiation-sterilized mouse diet (WQJX BIOTECHNOLOGY). The established humane endpoints included: (1) weight loss exceeding 25% of initial body weight; (2) severe lethargy, hunched posture, or difficulty moving; (3) inability to access food or water independently; and (4) signs of significant distress such as dyspnea. Upon observation of any of these criteria, mice were immediately euthanized by CO₂ inhalation using a gradual fill method, followed by cervical dislocation for confirmation.

Virus infections
The mouse-adapted influenza A/PR/8/34 (H1N1) strain was propagated in chicken embryonated eggs68. Murine hepatitis virus (MHV) was generously provided by X. Zhou (Wuhan Institute of Virology, University of Chinese Academy of Sciences)69. Female and male mice were paired in the late afternoon, and vaginal plug detection was performed the following morning; plug-positive mice were designated as embryonic day 0.5 (E0.5). At E6.5, pregnant mice were anesthetized and intranasally inoculated with a sublethal dose of 16 HA of PR8 in 50 μL sterile saline or MHV in 50 μL DMEM. Mice were monitored and weighed daily and euthanized at E12.5. At E12.5 after placentation, pregnant mice were anesthetized and intranasally inoculated with a sublethal dose of PR8 in 50 μL sterile saline. Mice were monitored and weighed daily and euthanized at E18.5. Lungs and uterine tissues were harvested, photographed, weighed, and either snap-frozen or fixed for further analyses. To assess the long-term impact of prenatal infection, pregnant mice were inoculated intranasally with PR8 at E6.5 and then permitted to deliver naturally. Maternal and offspring growth was tracked for 3 weeks postpartum, including weekly body weight assessments. All infection experiments were conducted under biosafety level 2 (BSL-2) conditions at the University of Science and Technology of China.

qPCR
Total RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen, 15596018). Reverse transcription was performed using a commercial kit (Accurate Biology, AG11705), and the resulting cDNA was subjected to quantitative PCR (qPCR) using SYBR Green (Accurate Biology, AG11702). Primer sequences are listed in Supplementary Table 2. All primers were designed using Primer-BLAST and synthesized by Tsingke. Gene expression levels were quantified using the ΔCT method.

Plaque assay
Viral titers in lung and decidual tissues harvested at E12.5 were determined by plaque assay on MDCK cells. Tissues were homogenized in ice-cold serum-free DMEM, and clarified supernatants were collected after centrifugation. MDCK monolayers in 24-well plates were inoculated with serial 10-fold dilutions of tissue supernatant for 2 h at 37°C with intermittent agitation. Following adsorption, cells were overlaid with a semi-solid medium consisting of a 1:1 mixture of 2× DMEM (Procell system, PM150221) and 2% methylcellulose, supplemented with TPCK-trypsin (MedChemExpress, HY-129047C). Plates were then incubated for 2–4 days until plaque formation. Plaques were visualized by formalin fixation and crystal violet staining. Viral titers were calculated from plaques counted in appropriated dilutions and expressed as PFU per gram of tissue.

Leukocyte isolation
For lung leukocytes, lung tissue was minced and digested with 1 mg/mL collagenase type I (Sigma-Aldrich, C0130) in RPMI 1640 medium (VivaCell, Shanghai, C3018) at 37°C for 1 h. Digests were filtered through nylon mesh, followed by density gradient centrifugation in 40% Percoll at 750 × g for 20 min. Red blood cells were lysed, and leukocytes were collected. Mouse peripheral blood mononuclear cells were isolated after red blood cell lysis. For decidual leukocytes, tissues were harvested at E12.5 after embryos were carefully removed. The decidua was minced and digested in 2 mg/mL collagenase type IV (Sigma-Aldrich, C5138) in RPMI 1640 at 37°C for 45 min. The resulting suspensions were filtered, and leukocytes were isolated using the same Percoll-based protocol.

Flow cytometry analysis
Isolated cells were suspended in phosphate-buffered saline (PBS) containing 1% fetal bovine serum, blocked with Fc receptor antibodies, and stained with fluorophore-conjugated antibodies for 30 min at 4°C in the dark. Cells were washed twice with PBS and analyzed using a BD Celesta flow cytometer. Data were processed with FlowJo V10. Gating strategies for lymphocyte and myeloid populations are shown in Supplementary Fig. 5. Antibodies used are listed in Supplementary Table 3.

Histological and immunostaining analyses
Mouse lungs and decidual tissues were fixed overnight in 10% neutral-buffered formalin and embedded in paraffin following standard protocols. Sections were dewaxed with xylene, rehydrated through graded ethanol, and stained with hematoxylin and eosin (H&E). Masson’s trichrome staining was performed by XL Pathology Tissue Services (Anhui, China). For immunohistochemistry (IHC) and immunofluorescence (IF), antigen retrieval was conducted by boiling sections for 15 min. Sections were incubated with primary antibodies and visualized using either a Biotin-Streptavidin Complex System (Zhongshan Golden Bridge, PV-9001 and PV-9002) or an Alexa Fluor Tyramide SuperBoost Kit (Thermo Fisher Scientific, B40922 and B40926), following manufacturers’ protocols. Images were acquired using 3DHISTECH digital slide scanners and analyzed with CaseViewer software. Quantitative analysis of immunohistochemistry images was conducted using ImageJ software. Lung injury scoring was performed by examining ten randomly selected fields per sample. The scoring criteria were as follows: 0, normal lung with no inflammatory cells; 1, mild inflammation with occasional inflammatory cells surrounding perivascular and peribronchial areas; 2, moderate inflammation with increased numbers of inflammatory cells in perivascular and peribronchial regions, along with some infiltration into the lung parenchyma; 3, severe inflammation with extensive inflammatory cell accumulation in perivascular and peribronchial areas and the formation of large inflammatory foci within the lung parenchyma. Antibodies used for IHC and IF are listed in Supplementary Table 4.

In vivo drug administration
The DPP4 inhibitor sitagliptin (Selleck, S5079) was dissolved in DMSO and diluted in corn oil to a final concentration of 5.0 mg/mL, corresponding to a dose of 35–50 mg/kg/day. Pregnant mice received daily oral gavage of sitagliptin (200 μL) or vehicle control on E7.5, E8.5, E9.5, E10.5, and E11.5 when infected with virus at E6.5. And pregnant mice received daily oral gavage of sitagliptin (200 μL) or vehicle control on E13.5, E14.5, E15.5, E16.5, and E17.5 when infected with virus at E12.5.

Cytokine antibody array
Lung and decidual tissues were collected from pregnant mice at E12.5 after intranasal administration of saline or H1N1 at E6.5. Cytokine profiles were assessed using the Mouse Cytokine Antibody Array (RayBiotech, QAM-TH17-1) according to the manufacturer’s instructions. In brief: (i) 100 μL of sample diluent was added to each well to block the array surface for 30 min at room temperature; (ii) 100 μL of lysate (500 μg/mL) was added to each well and incubated overnight; (iii) biotinylated antibody cocktail was applied; (iv) Cy3-equivalent dye-conjugated streptavidin was used for detection. Fluorescence signals were scanned and analyzed using GenePix software.

ELISA
Cytokine concentrations in tissue supernatants or serum were measured using enzyme-linked immunosorbent assay (ELISA) kits, following manufacturers’ protocols. Kits used included IL1R2 (RayBiotech, ELM-IL1R2-1), VEGF (Multisciences, EK283), and DPP4 (Elabscience, E-EL-M3081).

RNA-Seq and data analysis
Total RNA was extracted from mouse lung and decidual tissues using TRIzol reagent (Thermo Fisher Scientific). RNA quantity and integrity were evaluated with the RNA Nano 6000 Assay Kit on a Bioanalyzer 5400 system (Agilent Technologies, CA). Library preparation and sequencing were performed on the NovaSeq 6000 platform (Novogene, Beijing, China). After quality control, libraries were pooled based on effective concentrations and expected data output and subjected to paired-end sequencing. Raw image files were processed with CASAVA software to generate sequence reads. Gene-level read counts were obtained using FeatureCounts (v2.0.6). Fragments per kilobase of exon per million mapped reads (FPKM) were calculated for each gene, normalized by transcript length and total mapped reads. Differential gene expression analysis among three experimental groups (each with three biological replicates) was conducted using DESeq2 (v1.42.0). Functional enrichment of differentially expressed genes (DEGs) was assessed using clusterProfiler (v4.8.1) for Gene Ontology (GO) terms. Enrichment analysis was also performed using the Reactome pathway database, with pathways considered significantly enriched at adjusted p values < 0.05.

Proteomic analysis
At embryonic day 12.5 (E12.5), pregnant mice were euthanized for the collection of serum and decidual tissues. Protein extraction, enzymatic digestion, LC–MS/MS analysis, and data processing were carried out by iProteome Biotechnology (Shanghai, China). Briefly, tissue and serum samples were homogenized in 100 μL of 50 mM ammonium bicarbonate (ABC) buffer, and proteins were denatured at 95°C for 5 min. After cooling, proteins were digested overnight at 37°C with trypsin at a 1:25 enzyme-to-protein ratio. Samples were analyzed using an EASY-nLC 1200 system coupled to a Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) via a nano-electrospray ion source. Mass spectrometry was performed in data-independent acquisition (DIA) mode. An initial full MS1 scan (m/z 300–1400) was acquired at a resolution of 30,000, with an AGC target of 3E+06 and maximum injection time of 20 ms. This was followed by 30 DIA segments at a resolution of 15,000, with AGC target set to 1E+06 and maximum injection time of 22 ms. Peptide identification and protein quantification were performed using the Firmiana platform (https://phenomics.fudan.edu.cn/firmiana/). Raw files were searched against the UniProt mouse proteome using FragPipe (v12.1) integrated with MSFragger (v2.2). DIA data were further analyzed using DIA-NN (v1.7.0), with peptide quantification based on the mean chromatographic fragment ion peak areas across all reference spectral libraries. Label-free quantification was applied to determine protein abundance.

Cell sorting and single-cell RNA sequencing
Mononuclear cells were isolated from lung and decidual tissues at E12.5 and stained with an anti-CD45 antibody. CD45-positive cells were sorted using a Thermo Fisher Scientific Bigfoot Full-Spectrum Flow Cytometer and processed for single-cell RNA sequencing. Cell viability was assessed microscopically using Trypan Blue staining. Single-cell suspensions were prepared at a concentration of 2×105 cells/mL in PBS (HyClone) and loaded onto a microwell chip using the Singleron Matrix NEO™ Single Cell Processing System. Barcoding Beads were collected from the chip, followed by reverse transcription of captured mRNA and cDNA synthesis via PCR amplification. The amplified cDNA was fragmented and ligated with sequencing adapters. scRNA-seq libraries were constructed using the GEXSCOPE® Single Cell RNA Library Kits (Singleron). Individual libraries were diluted to 4 nM, pooled, and sequenced on an Illumina NovaSeq 6000 system with 150 bp paired-end reads.

Raw scRNA-seq reads were processed into gene expression matrices using the CeleScope pipeline (v1.9.0; https://github.com/singleron-RD/CeleScope). Briefly, low-quality reads were removed, and poly-A tails as well as adapter sequences were trimmed using Cutadapt (v1.17). Cell barcodes and UMIs were extracted, and reads were aligned to the GRCm38 reference genome (Ensembl release 92) using STAR (v2.6.1a). UMI and gene counts per cell were quantified with featureCounts (v2.0.1), and expression matrices were generated for downstream analysis.

scRNA-seq data analysis
Count matrices were imported and analyzed in Python (v3.9) using the Scanpy package (v1.10.0). Cells with fewer than 200 detected genes or genes expressed in fewer than three cells were excluded. Cells exhibiting high mitochondrial gene content (>15%) were also removed. Potential doublets were identified and filtered out using Scrublet, applied per sample. The filtered data were normalized to 10,000 counts per cell and log1p-transformed. Highly variable genes were selected with batch key specification, and batch effects were corrected using Harmony integration within the principal component analysis (PCA) space. Corrected embeddings were used to construct a neighborhood graph, followed by UMAP visualization and clustering via the Leiden algorithm at an appropriate resolution.

For subpopulation analysis, cells previously annotated as macrophages were subsetted from the integrated dataset. These cells were then re-embedded, re-clustered, and re-analyzed independently to characterize distinct subtypes and functional states.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). No statistical method was used to predetermine sample size. In vivo experiments were randomized across treatment groups, and no data were excluded from analysis. Comparisons between two groups were evaluated using unpaired two-tailed Student’s t-tests, while comparisons among multiple groups were analyzed by one-way ANOVA. A P value < 0.05 was considered statistically significant. All data are expressed as mean ± standard error of the mean (s.e.m). Details of statistical methods are provided in the respective figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is an exciting series of studies highlighting a mechanism by which respiratory viruses can impact placenta vasculature and intrauterine growth restriction of fetuses. Identification of DPP4 and IL-1 family genes as mediators is novel and exciting because it offers therapeutic potential. My specific comments are below:\n1. Virus titers: the authors must measure virus titers in respiratory tissues. While are ample studies illustrating that neither influenza viruses nor coronaviruses replicate in reproductive tissues in mice, whether viral loads are reduced with the DPP4 or IL1R2 manipulations must be shown and cannot just be inferred from transcriptional profiling showing changes in type I IFN signaling.\n2. Rational for infecting prior to placentation: the authors should provide a rationale for only testing infection during a single first trimester human equivalent time. Would these treatments work equally well if infections occurred after embryonic day 10.5, after the placenta is formed?\n3. Immunophenotype: with a distinct focus on mucosal immunity (e.g., that is the focus of the Discussion), it was surprising that immune cell frequencies were only measured in peripheral blood and not in the lungs and placenta. This seems to be a missing link in showing that the mucosal immune profile is altered.\n4. Coronavirus data are confusing: because virus titers are not measured, it is difficult to interpret data when previous work with SARS-Cov-2 infection in dams, cited by the authors, showed that reduced control of virus replication and dampened type I IFN responses in the lungs, more than placental inflammation, caused intrauterine growth restriction, which was reversed by antiviral treatment. With MHV, the authors claim that the opposite is true but show no virus replication data. It also is concerning that MERS uses DPP4 as a virus entry receptor and it is not clear of MHV does as well. Thus, DPP4 could be acting differently with MHV than influenza infection. This is never evaluated or discussed. The authors assume that DPP4 effects are identical for both viruses, which is confusing when in Fig 7e it is apparent that sitagliptin has no effect on MHV induced intrauterine growth restriction, yet the authors conclude that sitagliptin reverses the effects of MHV on offspring. Thus, this suggests that DPP4 is not the mechanism of action for MHV.\n5. Minor: Fig 3f is not mentioned in text\n6. Minor: there is no discussion of the limitations of this study.", "reviewer_2": "The manuscript by Shi et al. attempts to unravel how lung infections might disrupt immune responses necessary for vascular remodeling and trophoblast invasion and ultimately the health of the embryos. They nicely show that respiratory infection with flu or MHV-1 enhances Cd11b+ IL1R2 expressing cells that accumulate in the uterus and the embyros show evidence of growth restriction. Importantly, a DPP4 inhibitor could reverse the detrimental effects of pulmonary infection on embryo health. The paper is very interesting and well done, but there is still confusion over how the IL1R2 is accumulating in the uterus as noted below.\nMajor\n1) Line 76 and again line 211 asserts that the IL1R2 accumulation is via soluble protein (but this is not proven in this manuscript). In reality, is the accumulation of IL1R2 in the uterus due to soluble protein spilling from the lung or Cd11b+ cell recruitment to the uterus which express IL1R2? Figure 3j seems to show all of the IL-1R2 as cell-associated. Fig 4f and g show CD11b+ cells that are IL1R2+ in the uterus, so it appears that the increase is associated with cellular influx in the uterus and this is how the discussion is framed as well. However, if this is true, it is hard to explain why the mRNA expression of IL1R2 in the decidua is not changed (Fig 3f), but the protein is higher (Fig 3g). If the increased myeloid cells bring the IL-1R2 to the uterus, shouldn’t you see evidence of increased mRNA by virtue of increased cell number? This is even more confusing when looking at the MHV data in Fig 6 m where MHV upregulates IL1R2 mRNA in the lung but not protein (so there is likely no increased IL1R2 protein to leak from lung in this case), and in the decidua, MHV elevates IL1R2 protein again without showing evidence of increased mRNA. The authors need to clarify how the elevated IL1R2 protein levels are happening. Perhaps the post-transcriptional regulation of this protein is important.\n2) The authors seem to imply that the increased percentages of myeloid cells may be responsible for the IL1R2 levels in the uterus. The data are presented only as percentages which implies that if the CD11b+ percentage is up, something should be lower, but that is not shown. Also, how do the actual numbers of cells change over time post-infection? Knowing this may help to clarify the source of the IL1R2 that accumulates in the uterus.\n3) Do you see the same impacts on the uterus if myeloid cell migration is blocked post lung infection (e.g. use of CCR2 KO mice, i.v. clodronate depletion of monocytes)? This could really help clarify whether it is cellular trafficking that is essential.\n4) Does the growth restriction of the fetus carry forward to smaller pups when born? If you let the infected mice recover and give birth, would the neonates show defects in growth or health?\nMinor:\n1) What is the cellular source of the DPP4 production?\n2) Supplemental Fig 3g proports to measure viral H1N1 protein, but in reality this is mRNA for a viral protein measured by RT-PCR it seems. The methods and text do not state what protein is measured, and for influenza, viral RNA is a poor indicator of infectious virus. The authors should determine infectious titer of flu in lung with and without the DPP4 inhibitor.\n3) What does the red font indicate in Figures 1c and d?\n4) What was the sublethal dose of PR8 used?\n5) The mechanistic link between how DPP4 inhibition limits IL1R2 accumulation in the uterus or even the lung is not defined, or speculated upon. The authors admit this in the discussion, but a hypothesis would be interesting to speculate upon.", "reviewer_3": "The authors show that gestational influenza leads to immune activation in the uterus defined by activation of inflammatory cytokines leading to intrauterine growth restriction in mice. Using transcriptomic analysis the authors show that systemic DPP4 upregulation (lung, decidua and blood) is associated with adverse outcome. Inhibition of DPP4 with Sitagliptin improves adverse outcome as defined by reduced inflammation and reduced growth restriction. This is an important study elucidating key mechanisms involved in lung infection mediated complications during pregnancy. The methodology used is sound and the conclusions drawn mostly supported by the data. However, there are major concerns that dampen my enthusiasm.\nMajor comments\n#1: DPP4 is a known protein associated with signal transduction, immune regulation and glucose metabolism (including gestational diabetes). In all the animal experiment that were involving Sitaglipin treatments, the authors did not include the control with the treatment alone. Since Sitaglipin has per se an effect on the immune function would have been important to compare the H1N1+ Sitaglipin group vs Sitaglipin, instead of only not infected. This is a major drawback of the study. These experiments are essential to support the major conclusion of the manuscript.\n#2: The authors state (lines 240-243): “Il1r2 mRNA was strongly induced in the lung but not in the uterus (Fig. 3c, f), these results suggest that respiratory virus infection induces IL1R2 expression in lung-resident myeloid cells, which then disseminate through the bloodstream and accumulate systemically, including at the maternal-fetal interface.”\nIn order to evaluate and distinguish the different subsets of myeloid cells, it would be important to include some key markers that better characterize resident and infiltrating cells. The macrophages subsets in the decidua are characterized not only by the expression of CD11b and F4/80 (classical macrophages markers), but also by CD11c, CX3CR1, TREM2 and a low expression of Ly6C, while infiltrating macrophages travelling via the bloodstream express CCR2.\n#3: Line 296, the authors state: “These findings parallel the immunological alterations observed in influenza infection and support the notion that respiratory viral infections impair placental function and fetal development via indirect, non-viral mechanisms.”\nAlthough MHV can be used as surrogate model for SARS-CoV-2 pathogenesis, it is not a respiratory borne virus, spreading mainly via fecal-oral way or blood transmission. I would suggest to completely rephrase the section on MHV, including the title, since MHV can not be compared to all respiratory viral infection, although most clinical symptoms are similar. The same counts for the abstract. Most respiratory viruses, including SARS-CoV, have a marked tropism for ciliated cells and they are mainly localized in the respiratory tract, while the infection with MHV is multi-organ. Thus, the statements made on respiratory viruses by including MHV data need to be revised throughout the manuscript accordingly.\nMinor comments:\n#1: In Fig. 2d the significance regarding the H1N1 vs H1N1-Sigtaglipin group is missing (as mentioned in the text line 134- 135). Please amend.\n#2: A minor comment regarding the gating strategy in Suppl. Fig. 4 A: the gate on CD45+ cells is set with living cells. There was a pre-gating strategy not shown with a marker for living cells (e.g. live/dead) otherwise looks like a gate towards a physical parameter. Please clarify.\n#3: The authors use the mouse-adapted influenza A/PR/8/34 (H1N1). Would have been interesting to see the effect of the treatment on a more clinically relevant seasonal strain. Please discuss.\n#4: In order to compare the percentages during steady state control and infection the absolute number of cells should have been recorded. During infection, normally, a high amount of cells are recruited and activated and there is an increase in the total amount of CD45+ cells that influence the relative percentage number. Both representative gating strategy for saline and H1N1 condition should be shown.\n#5: In il1r2-KO model would have been interesting to see how and if the immune cell composition change in relation to the migration from the lung to the placenta. Please discuss.\n#6: the authors should address why they have not used the allogenic mouse model that was shown by various groups to mimic best infection-immunity aspects during human pregnancy (Engels et al., Cell Host and Microbe 2017). Please discuss and cite accordingly.\n#7: in the methods section, the humane endpoint used is not mentioned. Please amend.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We sincerely thank the Reviewers and the Editor for their thoughtful and constructive comments. We have carefully revised the manuscript accordingly and performed additional experiments as requested. Below, we provide a point-by-point response to all the reviewers’ comments. Reviewer comments are shown in Blue color, and our responses follow each comment.\n~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis is an exciting series of studies highlighting a mechanism by which respiratory viruses can impact placenta vasculature and intrauterine growth restriction of fetuses. Identification of DPP4 and IL-1 family genes as mediators is novel and exciting because it offers therapeutic potential. My specific comments are below:\nReply: We really appreciate your comments.\n1. Virus titers: the authors must measure virus titers in respiratory tissues. While are ample studies illustrating that neither influenza viruses nor coronaviruses replicate in reproductive tissues in mice, whether viral loads are reduced with the DPP4 or IL1R2 manipulations must be shown and cannot just be inferred from transcriptional profiling showing changes in type I IFN signaling.\nReply: Thank you for pointing out the virus titers shortage in the 1st edition of our manuscript. To address your concern, we have conducted additional experiments to assess virus titers in both the DPP4 or IL1R2 treatment.\nIn the influenza infection model, we systematically evaluated viral replication and distribution using multiple approaches: (1) Immunohistochemistry for influenza nucleoprotein revealed positive signals exclusively in infected lungs, with no detection in the decidua. (2) Plaque assay quantification confirmed that infectious virus was only detectable in lung homogenates, and DPP4 inhibitor treatment did not reduce pulmonary viral titers. (3) qPCR for viral gene replication yielded consistent results, showing viral replication confined to the lungs, unaffected by sitagliptin.\nRevised Supplementary Figure 2. Influenza viral replication in the lung and decidua following sitagliptin treatment.\nIn the Il1r2 knockout model, immunohistochemistry and qPCR similarly confirmed that viral infection was strictly restricted to the lungs, and Il1r2 knockout did not alter viral replication or tissue distribution.\nRevised Supplementary Figure 8. Influenza viral replication in the lung and decidua following knocking out Il1r2.\nIn the coronavirus (MHV) infection model, qPCR analysis demonstrated that viral replication was likewise confined to the lungs and was not reduced by DPP4 inhibitor treatment.\nRevised Supplementary Figure 10i. Intranasal MHV inoculation results in the accumulation of IL1R2 and inhibition of DPP4 alleviates lung inflammation.\nThese comprehensive virological data have now been included as supplementary figures. They collectively provide strong evidence supporting our central conclusion that respiratory viral infections are strictly confined to the maternal lungs without dissemination to the maternal-fetal interface, and the protective effects of DPP4 inhibition or Il1r2 deficiency are mediated by modulating the host's immunopathological response, not by direct antiviral activity. We thank the reviewer for helping us strengthen the evidence in our paper.\n2. Rational for infecting prior to placentation: the authors should provide a rationale for only testing infection during a single first trimester human equivalent time. Would these treatments work equally well if infections occurred after embryonic day 10.5, after the placenta is formed?\nReply: Thank you for bring this important point out. According to your suggestions, we infected pregnant mice were intranasally infected with influenza virus after the establishment of the choriovitelline placenta (at gestational day 12.5, E12.5)1. This was followed by a 5-day treatment regimen with sitagliptin administered daily by oral gavage from E13.5 to E17.5, while control groups received the vehicle only. Our data showed that sitagliptin treatment still significantly mitigated virus-induced maternal weight loss and improved pregnancy outcomes. Treated embryos displayed increased weight and body length, compared to untreated infected controls (revised 2nd edition Supplementary Figure 4). These data suggest that DPP4 inhibitor sitagliptin treatments work equally well even infections occurred after embryonic day 12.5, after the placenta is formed.\nRevised Supplementary Figure 4. Inhibition of DPP4 also alleviates intrauterine growth restriction caused by respiratory influenza virus infection after placentation.\n3. Immunophenotype: with a distinct focus on mucosal immunity (e.g., that is the focus of the Discussion), it was surprising that immune cell frequencies were only measured in peripheral blood and not in the lungs and placenta. This seems to be a missing link in showing that the mucosal immune profile is altered.\nReply: Thank you for this suggestion. In the revised 2nd edition, we repeated the H1N1 infection model and analyzed immune cell frequencies in the lungs, peripheral blood, and decidua. Our data showed that Infection induced a robust expansion of CD11b⁺ myeloid cells (macrophages, neutrophils and monocytes) in the lung, accompanied by a similar increase in circulating percentage of neutrophils and monocytes. By contrary, we did not observe expansion of CD11b⁺ myeloid cells in the decidua (revised 2nd edition Supplementary Figure 6), suggesting that IL1R2+ myeloid cells subset rather than the whole myeloid cells increased in decidua.\nRevised Supplementary Figure 6. Differential alterations in myeloid cell populations in maternal lungs, peripheral blood and deciduas following influenza infection.\n4. Coronavirus data are confusing: because virus titers are not measured, it is difficult to interpret data when previous work with SARS-Cov-2 infection in dams, cited by the authors, showed that reduced control of virus replication and dampened type I IFN responses in the lungs, more than placental inflammation, caused intrauterine growth restriction, which was reversed by antiviral treatment. With MHV, the authors claim that the opposite is true but show no virus replication data. It also is concerning that MERS uses DPP4 as a virus entry receptor and it is not clear of MHV does as well. Thus, DPP4 could be acting differently with MHV than influenza infection. This is never evaluated or discussed. The authors assume that DPP4 effects are identical for both viruses, which is confusing when in Fig 7e it is apparent that sitagliptin has no effect on MHV induced intrauterine growth restriction, yet the authors conclude that sitagliptin reverses the effects of MHV on offspring. Thus, this suggests that DPP4 is not the mechanism of action for MHV.\nReply: We apologize for this confusion. We have substantially revised the text to address these concerns:\n1). Viral Replication and Mechanism: We acknowledge that the absence of viral replication data in the initial submission was an oversight. In the revised version, we clearly state that MHV replication is confined to the lungs with no vertical transmission and have included the critical new experimental data showing that sitagliptin treatment does not affect MHV replication in the lungs. This supports our conclusion that the benefits of sitagliptin stem from its modulation of host immunopathology, not a direct antiviral effect.\nRevised Supplementary Figure 10i. Intranasal MHV inoculation results in the accumulation of IL1R2 and inhibition of DPP4 alleviates lung inflammation.\n2). Thank you for mention the previous work with SARS-Cov-2 infection in dams2. They showed that reduced control of virus replication and dampened type I IFN responses in the lungs, more than placental inflammation, caused intrauterine growth restriction, which was reversed by antiviral treatment. Importantly, they indicated that infection at E16 (third trimester equivalent) rather than infection at either E6 (first trimester equivalent) or E10 (second trimester equivalent) has the above phenomena. Our study demonstrates that in an early- gestation model with a virus causing localized severe pneumonia, the magnitude of the maternal pulmonary inflammatory response is the primary driver. The differences in this mechanism observed at different stages of pregnancy might be due to the distinct roles of macrophages during the early and late stages of pregnancy.\nDuring early pregnancy, decidual macrophages are primarily tasked with immune tolerance and vascular remodeling (M2-like phenotype), essential for supporting placentation. A severe maternal inflammatory response can remotely disrupt this constructive program, leading to impaired placental development and fetal growth restriction. In late pregnancy, the dominant physiological role of these macrophages shifts toward orchestrating the pro-inflammatory cascade required for parturition. A systemic high-viral-load infection (e.g., the maSCV2 model in the JCI study) delivers a powerful inflammatory signal that prematurely activates or exacerbates this intrinsic parturition program, resulting in placental dysfunction and adverse outcomes.\nWe have added these important discussions in the revised manuscript (Page 12-13 and Line 502-512): “Our findings, demonstrating that severe maternal pulmonary inflammation—rather than direct viral burden—drives adverse pregnancy outcomes in early gestation, may initially appear to contradict a recent study, which identified uncontrolled lung viral replication as the primary cause of fetal growth restriction in late-gestation maSCV2-infected mice2. The differences in this mechanism observed at different stages of pregnancy might be due to the distinct roles of macrophages during the early and late stages of pregnancy. During early pregnancy, decidual macrophages are primarily tasked with immune tolerance and vascular remodeling (M2-like phenotype), essential for supporting placentation. In late pregnancy, the dominant physiological role of these macrophages shifts toward orchestrating the pro- inflammatory cascade required for parturition3,4. A systemic high-viral-load infection delivers a powerful inflammatory signal that prematurely activates or exacerbates this intrinsic parturition program, resulting in placental dysfunction and adverse outcomes.”\n3). Mechanism of DPP4 Action: We appreciate the reviewer’s insightful question regarding viral entry receptors. It is well established that MERS-CoV uniquely utilizes DPP4 as its primary entry receptor, which is a key determinant of its distinct pathogenesis. In contrast, the MHV strain employed in our study utilizes CEACAM15,6 as its canonical and necessary receptor for infection in mice, not DPP4. Therefore, our data indicate that in both influenza and MHV models, DPP4 inhibition alleviates fetal growth restriction by modulating the immune response, reducing pulmonary inflammation, and improving placental function, independently of suppressing viral replication. This suggests a key role for DPP4 in a common immunopathological pathway triggered by respiratory viral infections. We added these as follows in the Result (Page 10 and Line 416-423): “To further directly evaluate whether sitagliptin exerts its beneficial effects via antiviral activity in the MHV infection model, we measured viral gene replication in lung tissues after treatment. Critically, sitagliptin did not suppress MHV replication in the lungs and and MHV remained restricted to the lung without spreading to the maternal-fetal interface (Supplementary Fig. 10i), suggesting that DPP4 may not involved in MHV amplification and that the receptor for MHV remains CEACAM1, as previously reported6. These data clearly demonstrate that the protective effect of sitagliptin on pregnancy outcomes is not mediated by direct antiviral mechanisms, but rather through modulation of the host’s immunopathological response.”\n4). Interpretation of Fig 7e Data: We have re-examined the data in Fig 7e that sitagliptin treatment significantly reversed MHV-induced fetal growth restriction (reflected in embryo weight, length, and placental size), with conclusions consistent with the data presented.\nRevised Figure 7. Inhibition of DPP4 alleviates intrauterine growth restriction caused by respiratory coronavirus infection.\n5. Minor: Fig 3f is not mentioned in text.\nReply: Sorry for this problem. It has been well taken in the revised 2nd edition of manuscript.\n6. Minor: there is no discussion of the limitations of this study.\nReply: Thank you for this suggestion. We have added limitation discussion as follows (Page 12-13 and Line 513-528): “Despite the compelling evidence presented, this study has several limitations. First, in this study, we employed a syngeneic mating model to clearly delineate the core pathway through which respiratory viral infection leads to fetal growth restriction, specifically the lung inflammation-systemic immune dysregulation-placental dysfunction axis. We fully acknowledge, as demonstrated by Engels et al., that allogeneic pregnancy models, which incorporate immune tolerance mechanisms toward the semi-allogeneic fetus and better recapitulate the immunological landscape of human pregnancy and hold significant translational value7. Consequently, we will employ allogeneic models in future studies to further validate the protective effects of DPP4 inhibition. Second, while the efficacy of the inhibitor has been demonstrated using the mouse-adapted influenza A/PR/8/34 (H1N1) strain, future studies should evaluate its therapeutic effect against more clinically relevant seasonal strains. Third, it is also important to note that while the natural transmission routes for MHV include fecal-oral and blood-borne pathways and not limited to the respiratory tract, differing from the primary respiratory transmission of SARS-CoV-2, intranasal inoculation of MHV establishes a robust infection in the lungs and elicits strong pulmonary immunopathology8,9. Therefore, the animal models used may not fully recapitulate the complex immunological and physiological interplay of human pregnancy and respiratory infection, underscoring the need for validation in clinical cohorts.”\nReviewer #2 (Remarks to the Author):\nThe manuscript by Shi et al. attempts to unravel how lung infections might disrupt immune responses necessary for vascular remodeling and trophoblast invasion and ultimately the health of the embryos. They nicely show that respiratory infection with flu or MHV-1 enhances Cd11b+ IL1R2 expressing cells that accumulate in the uterus and the embryos show evidence of growth restriction. Importantly, a DPP4 inhibitor could reverse the detrimental effects of pulmonary infection on embryo health. The paper is very interesting and well done, but there is still confusion over how the IL1R2 is accumulating in the uterus as noted below.\nReply: We really appreciate your comments.\nMajor:\n1) Line 76 and again line 211 asserts that the IL1R2 accumulation is via soluble protein (but this is not proven in this manuscript). In reality, is the accumulation of IL1R2 in the uterus due to soluble protein spilling from the lung or Cd11b+ cell recruitment to the uterus which express IL1R2? Figure 3j seems to show all of the IL-1R2 as cell-associated. Fig 4f and g show CD11b+ cells that are IL1R2+ in the uterus, so it appears that the increase is associated with cellular influx in the uterus and this is how the discussion is framed as well. However, if this is true, it is hard to explain why the mRNA expression of IL1R2 in the decidua is not changed (Fig 3f), but the protein is higher (Fig 3g). If the increased myeloid cells bring the IL-1R2 to the uterus, shouldn’t you see evidence of increased mRNA by virtue of increased cell number? This is even more confusing when looking at the MHV data in Fig 6 m where MHV upregulates IL1R2 mRNA in the lung but not protein (so there is likely no increased IL1R2 protein to leak from lung in this case), and in the decidua, MHV elevates IL1R2 protein again without showing evidence of increased mRNA. The authors need to clarify how the elevated IL1R2 protein levels are happening. Perhaps the post-transcriptional regulation of this protein is important.\nReply: Thank you very much for pointing out this important point. As you have mentioned, we observed evidence that the IL1R2 is cell-associated, especially myeloid cells. However, we previously did not find mRNA level of IL1R2 significantly increased in decidua. According to your suggestions, we have reflected on our experimental methods and speculate that it might because the samples we used for the PCR tests were the whole decidua tissues, and the IL1R2+ immune cells accounted for a very limited small proportion in the entire decidua tissues. If we sort the CD45+immune cells from the lung and decidua tissues for the experiment, it is possible to observe the transcriptional changes of IL1R2+ immune cells in the lungs and uterus (revised 2nd edition Supplementary Figure 7).\nTherefore, in the revised manuscript, we analyzed single-cell RNA sequencing of CD45+ cells in the lung and uterus from dams at E12.5 following treatment with saline, H1N1 infection alone, or H1N1 infection plus sitagliptin. Our data showed that macrophages constituted the most abundant subset and were significantly expanded following H1N1 infection, an effect that was substantially reversed by sitagliptin treatment. Further analysis revealed a marked H1N1-induced upregulation of Il1r2 in lung macrophages, which was also significantly suppressed by sitagliptin. In the decidua, we identified ten immune cell clusters. Analysis of the major macrophage population showed a congruent pattern: H1N1 infection robustly induced Il1r2 expression, and this induction was attenuated by sitagliptin. These findings underscore macrophages as a key Il1r2-expressing population during influenza infection in pregnancy.\nRevised Supplementary Figure 7. A single-cell atlas of the maternal immune response to influenza infection in the lung and decidua.\nWe next re-clustered macrophages and searched for the most relevant macrophage subpopulations. A subpopulation of immunoregulatory macrophages highly expressed Il1r2 was rare under homeostatic conditions but expanded significantly after H1N1 infection, with its proportion subsequently normalized by sitagliptin. Visualization of Il1r2 expression confirmed its specific enrichment in this immunoregulatory subset after infection, which was abrogated by treatment, suggesting these cells may serve as a key target of sitagliptin and represent a primary source of IL1R2. An analogous immunoregulatory macrophage subset, also exhibiting high Il1r2 expression, was identified in the decidua. Its abundance increased after infection and decreased with sitagliptin treatment, and its Il1r2 expression mirrored this trend, closely recapitulating the pulmonary response. This suggests that these cells may serve as a critical link between the lung and maternal-fetal interface, and constitute a local source of IL1R2 (revised 2nd edition Figure 5).\nRevised Figure 5. Inhibition of DPP4 reduces migratory and Il1r2-high macrophages following influenza virus infection.\nWe also discuss this important point in the discussion part as follows (Page 12 and Line 481-492): “Notably, in both influenza and coronavirus infection models, uterine IL1R2 protein levels increased without corresponding detection in virus titers or IFNβ connected genes expression. Conversely, in lung tissue, viral infection led to elevated Il1r2 mRNA expression and significantly increased of IL1R2+ myeloid cells, implying that IL1R2 accumulates in the uterus via systemic circulation rather than in situ production. Flow cytometric analyses further confirmed increased IL1R2 expression on myeloid cells within the lung, peripheral blood, and decidua following infection. The gradient—highest in the lung, intermediate in blood, and lowest in the decidua—suggests a migration trajectory of IL1R2-high cells from the lung to the uterus. Our scRNA data further identifies tissue-homing IL1R2+ macrophages that link pulmonary infection to maternal-fetal interface. Together, these findings support a model in which IL1R2 functions as a molecular conduit linking the immune landscapes of the respiratory and reproductive tracts, reinforcing and refining the concept of the common mucosal immune system.”\n2) The authors seem to imply that the increased percentages of myeloid cells may be responsible for the IL1R2 levels in the uterus. The data are presented only as percentages which implies that if the CD11b+ percentage is up, something should be lower, but that is not shown. Also, how do the actual numbers of cells change over time post-infection? Knowing this may help to clarify the source of the IL1R2 that accumulates in the uterus.\nReply: Thank you for bring this important point up. First, our single-cell data show that pulmonary macrophages are the cell type with the greatest increase in proportion after infection, while the proportions of NK cells and T cells have decreased accordingly. The proportion of macrophages in the uterus did not increase as significantly as that in the lungs. The main immune cells that decreased were T cells (revised 2nd edition Supplementary Figure 7a, c, e and g).\nRevised Supplementary Figure 7 (a, c, e and g). A single-cell atlas of the maternal immune response to influenza infection in the lung and decidua.\nSecond, in the revised 2nd edition, we re-analyzed immune cell frequencies in the lungs, peripheral blood, and decidua in the H1N1 post-infection model. Our data showed that Infection induced a robust expansion of CD11b⁺ myeloid cells (macrophages, neutrophils and monocytes) in the lung. By contrary, we did not observe expansion of CD11b⁺ myeloid cells in the decidua (revised 2nd edition Supplementary Figure 6), suggesting that IL1R2+myeloid cells subset rather than the whole myeloid cells increased in decidua.\nRevised Supplementary Figure 6. Differential alterations in myeloid cell populations in maternal lungs, peripheral blood and deciduas following influenza infection.\nThird, to specially identify the most relevant macrophage subpopulations in decidua, decidual macrophages were categorized into distinct subsets. An analogous immunoregulatory macrophage subset, exhibiting high Il1r2 expression, was identified in the decidua. Its abundance increased after infection and decreased with sitagliptin treatment, and its Il1r2 expression mirrored this trend, closely recapitulating the pulmonary response. This suggests that these cells may serve as a critical link between the lung and maternal-fetal interface, and constitute a local source of IL1R2 (revised 2nd edition Figure 5).\nRevised Figure 5 (g-l). Inhibition of DPP4 reduces migratory and Il1r2-high macrophages following influenza virus infection.\n3) Do you see the same impacts on the uterus if myeloid cell migration is blocked post lung infection (e.g. use of CCR2 KO mice, i.v. clodronate depletion of monocytes)? This could really help clarify whether it is cellular trafficking that is essential.\nReply: Thank you for your kind suggestion. According to your suggestions, we have tried to use clodronate liposomes to effectively depleting monocytes and macrophages. However, clodronate liposomes injection to pregnant mice cause severe fetal loss, suggesting this macrophage depletion agent is not suitable for pregnant mice.\nThen, we further used anti-CSF1R antibody to depleted macrophages. Specifically, pregnant mice received an intraperitoneal (i.p.) injection of 300 μg αCSF1R at embryonic day 5.5 (E5.5). At E6.5, the mice were intranasally infected with either H1N1 PR8 influenza virus or sterile saline. To maintain macrophage depletion throughout the experimental period, additional i.p. injections of 300 μg αCSF1R were administered once daily at E7.5, E9.5, and E11.5. All mice were euthanized at E12.5 for tissue collection and evaluation of experimental endpoints. Our data indicated that H1N1+anti-CSF1R antibody group showed more severe lung pathology, indicating higher weight and gross morphological lesions. Meanwhile, we found more severe fetal growth restriction in H1N1+anti-CSF1R antibody group (Response Figure 1 as follows). This might be because macrophages are crucial for the lungs in clearing viruses, and it has been previously reported that macrophages are also very important for embryo implantation and development. Therefore, the group that had all the macrophages removed showed more severe lung lesions, and the loss of macrophages also affected embryonic development. This might indicate that eliminating all the macrophage populations is not an appropriate approach. Thank you very much for your outstanding insights.\nResponse Figure 1. Removing macrophages aggravate lung lesions and restricted embryonic growth.\n4) Does the growth restriction of the fetus carry forward to smaller pups when born? If you let the infected mice recover and give birth, would the neonates show defects in growth or health?\nReply: Thank you for this suggestion. We kept on observing the dams until successful delivery following a 5-day therapeutic regimen and their offspring until postnatal day 21. Our findings demonstrate that sitagliptin treatment significantly alleviated infection-induced maternal weight loss. Postpartum monitoring revealed that infected mothers exhibited lower body weight on days 7 and 14 after delivery, while sitagliptin administration mitigated this decline. Notably, influenza virus infection markedly reduced the number of successfully delivered offspring. The litter size from healthy pregnancies was generally above 6 pups, whereas infection without treatment resulted in fewer than 5 surviving pups. Sitagliptin treatment substantially reversed this impairment, restoring litter sizes to levels comparable to those of uninfected dams. These results further suggest that in untreated infections, some fetuses sustained severe damage during late gestation, preventing their successful birth. Importantly, the surviving pups from infected but untreated mothers did not show significant differences in health status compared to pups from either uninfected or treated mothers, indicating that sitagliptin not only confers protective effects on the dams but also supports successful delivery and early survival of offspring.\nRevised Supplementary Figure 3. Inhibition of DPP4 meliorates the compromised litter size caused by respiratory influenza virus infection.\nCollectively, these findings demonstrate that the sitagliptin confers sustained benefits in both maternal recovery and offspring development, supporting its long-term protective efficacy.\nMinor:\n1) What is the cellular source of the DPP4 production?\nReply: Thank you for raising this important question regarding the cellular sources of DPP4. According to established literature, DPP4 (CD26) is expressed on various cell types relevant to our infection model, including: (1) immune cells, particularly activated T lymphocytes, dendritic cells, and macrophage subsets; and (2) structural cells, such as lung epithelial and endothelial cells, which can upregulate DPP4 in response to inflammatory stimuli10,11.\n2) Supplemental Fig 3g proports to measure viral H1N1 protein, but in reality this is mRNA for a viral protein measured by RT-PCR it seems. The methods and text do not state what protein is measured, and for influenza, viral RNA is a poor indicator of infectious virus. The authors should determine infectious titer of flu in lung with and without the DPP4 inhibitor.\nReply: Thank you for pointing out the virus titers shortage in the 1st edition of our manuscript. To address your concern, we have performed the following key supplementary experiments, the results of which are now included in the revised manuscript (revised 2nd edition Supplementary Figure 2): (1) We performed IHC staining for influenza nucleoprotein (NP) to visually confirm the presence of viral protein and its cellular distribution. This analysis demonstrated NP-positive signals exclusively within the infected lungs, with no detection in the decidua. (2) We quantitatively assessed infectious influenza virus titers in lung homogenates from infected mice, with and without DPP4 inhibitor treatment. This assay confirms that infectious virus is present in the lungs and, critically, that sitagliptin treatment does not reduce the pulmonary titer of infectious H1N1. We have corrected the manuscript text and figure legend (formerly Supplementary Fig 3g) to accurately state that the data derived from qPCR measurement of the influenza matrix gene (viral RNA), not viral protein. The Methods section has been updated with detailed protocols for the plaque assays.\nRevised Supplementary Figure 2. Influenza viral replication in the lung and decidua following sitagliptin treatment.\nThese comprehensive virological data—encompassing infectious titer (plaque assay), viral protein localization (IHC), and genomic presence (qPCR)—collectively provide unambiguous evidence that the protective effects of DPP4 inhibition are mediated by modulating the host's immunopathological response, not by direct antiviral activity. We are grateful to you for prompting these essential clarifications and for significantly strengthening the rigor of our study.\n3) What does the red font indicate in Figures 1c and d?\nReply: Thank you for this question. The red font in Figure 1c highlights four cytokines (IL-28, MIP-3α, IL-6, and IL-21) that were significantly upregulated in lungs following respiratory influenza virus infection. In contrast, these same four cytokines were downregulated in the decidua after infection. This indicates that the lung and decidua exhibit opposite immune states following infection: the lung is in an immune-activated state, while the decidua is in an immune-suppressed state. We have added the description in the figure legend as follows: “The red font denotes four cytokines showing opposite expression between lungs and deciduas.”\n4) What was the sublethal dose of PR8 used?\nReply: Thank you for raising this important point regarding experimental details. The sublethal dose of influenza A/Puerto Rico/8/34 (PR8) virus used for intranasal infection in pregnant mice was 16 HA in 50 µL of PBS. This dose was carefully titrated in preliminary studies to consistently induce significant clinical symptoms (e.g., weight loss, lung inflammation) and robust immune activation without causing mortality in pregnant mice, thereby fulfilling the criteria of a \"sublethal\" challenge. As suggested, we have now explicitly included this information in the revised \"Methods\" section (Viral Infections) as follows: “At E6.5, pregnant mice were anesthetized and intranasally inoculated with a sublethal dose of 16 HA of PR8 in 50 μL sterile saline”.\n5) The mechanistic link between how DPP4 inhibition limits IL1R2 accumulation in the uterus or even the lung is not defined, or speculated upon. The authors admit this in the discussion, but a hypothesis would be interesting to speculate upon.\nReply: We sincerely thank you for this profound and insightful question. We fully agree that elucidating the mechanistic link between DPP4 inhibition and the reduction of IL1R2 represents a crucial direction for future research. While existing data have clearly established this association, the underlying mechanisms require further in-depth exploration. We propose the following two complementary hypotheses, which have been incorporated into the revised discussion section to stimulate broader research interest.\nDPP4 (CD26) cleaves various chemokines and cytokines, thereby modulating their biological activity. Notably, the enzymatic action of DPP4 does not always lead to complete inactivation of chemokines; in some cases, truncated forms of chemokines generated by DPP4 cleavage acquire new functional properties10,12. For example, while truncated forms of CXCL10 and CXCL11 lose their ability to mediate T cell chemotaxis via CXCR3, they may instead recruit myeloid populations such as neutrophils or monocytes by interacting with other receptors13,14. We speculate that the upregulation of DPP4 following viral infection may alter the composition and function of local chemokines, promoting the abnormal accumulation of IL1R2⁺ myeloid precursor cells at inflammatory sites such as the lungs. Conversely, DPP4 inhibition may reverse this process by restoring chemokine homeostasis, thereby limiting the infiltration of immunosuppressive cells. Additionally, since DPP4 is involved in T cell activation15, its inhibition may help shift the overall immune environment toward an anti-inflammatory state, further influencing tissue-resident macrophages and other myeloid cells to differentiate away from an IL1R2-high immunosuppressive phenotype.\nIL1R2 functions as a critical \"decoy receptor\" that negatively regulates IL-1β signaling by sequestering the cytokine16. Multiple studies suggest a potential link between DPP4 activity and IL-1β levels: exogenous DPP4 has been shown to reduce IL-1β in rheumatoid arthritis models, whereas long-term DPP4 inhibition in non-obese diabetic mice downregulates circulating IL-1β concentrations17,18. These effects may stem from DPP4's indirect regulation of immune cell infiltration and the overall inflammatory milieu. We further hypothesize that DPP4 inhibitors, by broadly mitigating tissue inflammation and cytokine release, attenuate the drive for compensatory upregulation of the decoy receptor IL1R2. From this perspective, the DPP4 inhibition-induced reduction in IL1R2 can be viewed as a consequence of systemically quelling the cytokine storm, leading to a corresponding downregulation of endogenous immunosuppressive feedback mechanisms.\nIn summary, we propose that DPP4 inhibitors do not act on a single target but rather reshape the systemic and local immunopathological landscape, ultimately normalizing the aberrant accumulation of IL1R2-high immunosuppressive cells. We have added discussion as follows (Page 11-12 and Line 471-479): “We propose that DPP4 inhibition may reduce IL1R2 accumulation through two non-exclusive mechanisms. First, by preventing the cleavage of chemokines such as CXCL10 and CXCL11, DPP4 inhibition may preserve their canonical functions and prevent the aberrant recruitment of IL1R2⁺ myeloid precursors to inflammatory sites13,14. Second, DPP4 inhibition broadly attenuates the inflammatory milieu, thereby reducing the compensatory drive for IL1R2 upregulation as a feedback brake17. Thus, DPP4 inhibition likely acts by resetting the dysregulated immunopathological environment, rather than through a single target, ultimately normalizing this immunosuppressive axis. However, the precise pathways through which DPP4 inhibition modulates Il1r2 transcription remain to be elucidated.”\nReviewer #3 (Remarks to the Author):\nThe authors show that gestational influenza leads to immune activation in the uterus defined by activation of inflammatory cytokines leading to intrauterine growth restriction in mice. Using transcriptomic analysis the authors show that systemic DPP4 upregulation (lung, decidua and blood) is associated with adverse outcome. Inhibition of DPP4 with Sitagliptin improves adverse outcome as defined by reduced inflammation and reduced growth restriction. This is an important study elucidating key mechanisms involved in lung infection mediated complications during pregnancy. The methodology used is sound and the conclusions drawn mostly supported by the data. However, there are major concerns that dampen my enthusiasm.\nReply: We really appreciate your comments.\nMajor comments:\n#1: DPP4 is a known protein associated with signal transduction, immune regulation and glucose metabolism (including gestational diabetes). In all the animal experiment that were involving Sitaglipin treatments, the authors did not include the control with the treatment alone. Since Sitaglipin has per se an effect on the immune function would have been important to compare the H1N1+ Sitaglipin group vs Sitaglipin, instead of only not infected. This is a major drawback of the study. These experiments are essential to support the major conclusion of the manuscript.\nReply：We apologize for this oversight. According to your suggestions, we have repeated the influenza infection therapeutic experiment with a complete set of four groups: Saline control, Sitagliptin alone, H1N1 infection alone, and H1N1 infection + Sitagliptin treatment. The results demonstrate that compared to the Saline control, sitagliptin treatment alone in pregnant mice induced no significant alterations in maternal weight gain, lung weight, or pulmonary pathology. Furthermore, assessment of the fetuses and the maternal-fetal interface confirmed that sitagliptin alone did not cause fetal growth restriction, impaired angiogenesis, or disrupted trophoblast invasion.\nRevised Figure 2. Inhibition of DPP4 alleviates intrauterine growth restriction caused by respiratory influenza virus infection.\nThese new data establish two critical points: First, the DPP4 inhibitor sitagliptin exhibits a favorable safety profile for both the dam and fetal development under physiological conditions. Second, the baseline immunomodulatory effect of sitagliptin during normal pregnancy is minimal, suggesting its primary role in our model is to “reset” the infection-triggered aberrant immune state rather than to profoundly perturb the normal immune environment of pregnancy.\nWe have incorporated these complete experimental results into Figure 2 in the revised manuscript. We are profoundly grateful to you for this insightful suggestion, which has significantly enhanced the rigor and persuasiveness of our study.\n#2: The authors state (lines 240-243): “Il1r2 mRNA was strongly induced in the lung but not in the uterus (Fig. 3c, f), these results suggest that respiratory virus infection induces IL1R2 expression in lung-resident myeloid cells, which then disseminate through the bloodstream and accumulate systemically, including at the maternal-fetal interface.”\nIn order to evaluate and distinguish the different subsets of myeloid cells, it would be important to include some key markers that better characterize resident and infiltrating cells. The macrophages subsets in the decidua are characterized not only by the expression of CD11b and F4/80 (classical macrophages markers), but also by CD11c, CX3CR1, TREM2 and a low expression of Ly6C, while infiltrating macrophages travelling via the bloodstream express CCR2.\nReply: Thank you for this important suggestion. In order to evaluate and distinguish the different subsets of myeloid cells, we using single-cell RNA sequencing of CD45+ cells in the lung and uterus from dams at E12.5 following treatment with saline, H1N1 infection alone, or H1N1 infection plus sitagliptin. We identified that macrophages constituted the most abundant subset and were significantly expanded following H1N1 infection, an effect that was substantially reversed by sitagliptin treatment. Further analysis revealed a marked H1N1-induced upregulation of Il1r2 in lung macrophages, which was also significantly suppressed by sitagliptin. In the decidua, we identified ten immune cell clusters. Analysis of the major macrophage population showed a congruent pattern: H1N1 infection robustly induced Il1r2 expression, and this induction was attenuated by sitagliptin. These findings underscore macrophages as a key Il1r2-expressing population during influenza infection in pregnancy.\nRevised Supplementary Figure 7. A single-cell atlas of the maternal immune response to influenza infection in the lung and decidua.\nWe next re-clustered macrophages and searched for the most relevant macrophage subpopulations. A subpopulation of immunoregulatory macrophages highly expressed Il1r2 was rare under homeostatic conditions but expanded significantly after H1N1 infection, with its proportion subsequently normalized by sitagliptin. Visualization of Il1r2 expression confirmed its specific enrichment in this immunoregulatory subset after infection, which was abrogated by treatment, suggesting these cells may serve as a key target of sitagliptin and represent a primary source of IL1R2. An analogous immunoregulatory macrophage subset, also exhibiting high Il1r2 expression, was identified in the decidua. Its abundance increased after infection and decreased with sitagliptin treatment, and its Il1r2 expression mirrored this trend, closely recapitulating the pulmonary response. This suggests that these cells may serve as a critical link between the lung and maternal-fetal interface, and constitute a local source of IL1R2 (revised 2nd edition Figure 5).\nRevised Figure 5. Inhibition of DPP4 reduces migratory and Il1r2-high macrophages following influenza virus infection.\nAccording to your suggestions, we further analyzed distinct molecular signature among these macrophages: beyond Il1r2, this macrophage subpopulation specifically expressed notably key mediators of myeloid cell migration and chemotaxis (Retnlg, Cxcr2, Mmp9) and other immunomodulatory genes (Chil1, Steap4, Nlrp12), implying enhanced migratory potential and immunosuppressive ability19-21 (revised 2nd edition Figure 5).We also observed that most macrophages in lung high expressing CCR2 while low expressing Ly6C, indicating these cells are monocyte- derived macrophages (Response Figure 2). These scRNA data together identifies tissue-homing IL1R2+ macrophages that link pulmonary infection to maternal-fetal interface.\nRevised Figure 5 (m and n). Inhibition of DPP4 reduces migratory and Il1r2-high macrophages following influenza virus infection.\nResponse Figure 2. The phenotype of pulmonary macrophages.\n#3: Line 296, the authors state: “These findings parallel the immunological alterations observed in influenza infection and support the notion that respiratory viral infections impair placental function and fetal development via indirect, non-viral mechanisms.”\nAlthough MHV can be used as surrogate model for SARS-CoV-2 pathogenesis, it is not a respiratory borne virus, spreading mainly via fecal-oral way or blood transmission. I would suggest to completely rephrase the section on MHV, including the title, since MHV can not be compared to all respiratory viral infection, although most clinical symptoms are similar. The same counts for the abstract. Most respiratory viruses, including SARS-CoV, have a marked tropism for ciliated cells and they are mainly localized in the respiratory tract, while the infection with MHV is multi-organ. Thus, the statements made on respiratory viruses by including MHV data need to be revised throughout the manuscript accordingly.\nReply: Thank you for pointing out the difference in the natural transmission route of MHV compared to typical respiratory viruses. We have rephrased the relevant sections accordingly. When introducing the MHV model, we now explicitly state its natural transmission routes (fecal-oral/blood-borne) and how they differ from primary respiratory viruses, while explaining why the intranasally inoculated MHV model remains useful for studying the distal effects of lung inflammation. And we have modified the title and relevant text throughout the manuscript. We have refined the broad term “respiratory coronavirus infection” to more precisely refer to the pulmonary coronavirus infection modeled by intranasal MHV inoculation and its systemic consequences. We avoid directly equating MHV with all naturally respiratory-transmitted viruses, instead emphasizing its utility in modeling pulmonary immunopathology and the subsequent indirect impact on pregnancy. We have correspondingly adjusted the wording in the abstract and conclusion to ensure that inferences based on MHV data are rigorous and accurate.\nMinor comments:\n#1: In Fig. 2d the significance regarding the H1N1 vs H1N1-Sigtaglipin group is missing (as mentioned in the text line 134- 135). Please amend.\nReply：Sorry for this problem. It has been well taken in the revised 2nd edition of manuscript.\n#2: A minor comment regarding the gating strategy in Suppl. Fig. 4 A: the gate on CD45+ cells is set with living cells. There was a pre-gating strategy not shown with a marker for living cells (e.g. live/dead) otherwise looks like a gate towards a physical parameter. Please clarify.\nReply：We apologize for insufficiently explaining this point in the previous version. In the revised manuscript, we showed in the gating strategy (revised 2nd edition Supplementary Figure 6a).\nRevised Supplementary Figure 6a. Differential alterations in myeloid cell populations in maternal lungs, peripheral blood and deciduas following influenza infection.\n#3: The authors use the mouse-adapted influenza A/PR/8/34 (H1N1). Would have been interesting to see the effect of the treatment on a more clinically relevant seasonal strain. Please discuss.\nReply: Thank you for this insightful comment regarding the use of seasonal influenza strains. We agree that this is a critical point for assessing the broader relevance of our findings. While our study utilized the mouse-adapted A/PR/8/34 (H1N1) strain for its well-established and reproducible pathogenesis in mice, substantial clinical and experimental evidence supports that the core immunopathological features driving fetal growth restriction are highly conserved across different influenza strains, including seasonal H1N1 viruses. This conservation underpins our hypothesis that DPP4 inhibition represents a universally effective strategy against virus-induced pregnancy complications.\nClinical cohort studies have consistently shown that pregnant women infected with seasonal influenza viruses, including H1N1, are at significantly higher risk of developing severe respiratory complications, such as pneumonia and acute respiratory distress syndrome (ARDS), compared to the general population22,23. These conditions are characterized by intense pulmonary inflammation, which is a direct result of the host's immune response to the virus. Furthermore, infection with these seasonal strains is strongly associated with adverse pregnancy outcomes, including spontaneous abortion, preterm birth, and fetal growth restriction24. The shared clinical severity and poor obstetric outcomes between the PR8 model and human infections with seasonal viruses suggest an overlap in the underlying immunopathological pathways.\nThe key mechanism we identified that severe lung inflammation leading to a systemic immunopathology that impairs placental function is not strain-specific. Research indicates that respiratory viral infections, including seasonal influenza, trigger a robust innate immune response characterized by a significant influx of myeloid cells into the lungs25-27. This inflammatory cascade and the associated cytokine spillover are central to the distal effect on the placenta. Our data demonstrate that DPP4 inhibition further counteracts the accumulation of immunosuppressive molecule IL1R2 at the maternal-fetal interface by attenuating the overall inflammatory milieu and weakening the immune negative feedback regulation. This mechanism targets the host's shared response to viral threat rather than the virus itself. Therefore, the efficacy of DPP4 inhibition in our model is contingent upon the host's stereotyped inflammatory reaction to a respiratory viral challenge—a reaction that is consistently observed in severe cases of seasonal influenza infection in pregnant women. Since the therapeutic target is the common immunopathological pathway (lung inflammation → systemic immune dysregulation → placental dysfunction) and not a virus-specific component, we have a compelling rationale to hypothesize that DPP4 inhibition would be equally effective in mitigating pregnancy complications caused by a wide range of influenza viruses, including seasonal strains. We have added discussion as follows (Page 113 and Line 519-521): “Second, while the efficacy of the inhibitor has been demonstrated using the mouse-adapted influenza A/PR/8/34 (H1N1) strain, future studies should evaluate its therapeutic effect against more clinically relevant seasonal strains”.\n#4: In order to compare the percentages during steady state control and infection the absolute number of cells should have been recorded. During infection, normally, a high amount of cells are recruited and activated and there is an increase in the total amount of CD45+ cells that influence the relative percentage number. Both representative gating strategy for saline and H1N1 condition should be shown.\nReply: Thank you for bring this important point up. In the revised 2nd edition, we re-analyzed immune cell frequencies in the lungs, peripheral blood, and decidua in the H1N1 post-infection model. Our data showed that Infection induced a robust expansion of CD11b⁺ myeloid cells (macrophages, neutrophils and monocytes) in the lung. These data confirmed that a high amount of cells are recruited into the lung. By contrary, we did not observe expansion of CD11b⁺ myeloid cells in the decidua (revised 2nd edition Supplementary Figure 6), suggesting that IL1R2+myeloid cells subset rather than the whole myeloid cells increased in decidua.\nRevised Supplementary Figure 6. Differential alterations in myeloid cell populations in maternal lungs, peripheral blood and deciduas following influenza infection.\n#5: In Il1r2-KO model would have been interesting to see how and if the immune cell composition change in relation to the migration from the lung to the placenta. Please discuss.\nReply: This is an insightful point. We fully agree that investigating potential changes in immune cell trafficking from the lung to the placenta is important.\n1) According to your suggestions, we have tried to use clodronate liposomes to effectively depleting monocytes and macrophages. However, clodronate liposomes injection to pregnant mice cause severe fetal loss, suggesting this macrophage depletion agent is not suitable for pregnant mice.\n2) Then, we further used anti-CSF1R antibody to depleted macrophages. Specifically, pregnant mice received an intraperitoneal (i.p.) injection of 300 μg αCSF1R at embryonic day 5.5 (E5.5). At E6.5, the mice were intranasally infected with either H1N1 PR8 influenza virus or sterile saline. Our data indicated that H1N1+anti-CSF1R antibody group showed more severe lung pathology, indicating higher weight and gross morphological lesions. Meanwhile, we found more severe fetal growth restriction in H1N1+anti-CSF1R antibody group (Response Figure 1 as follows). This might be because macrophages are crucial for the lungs in clearing viruses, and it has been previously reported that macrophages are also very important for embryo implantation and development. Therefore, the group that had all the macrophages removed showed more severe lung lesions, and the loss of macrophages also affected embryonic development. This might indicate that eliminating all the macrophage populations is not an appropriate approach.\nResponse Figure 1. Removing macrophages aggravate lung lesions and restricted embryonic growth.\n3) In Il1r2-KO model, by contrast, both Il1r2-WT and Il1r2-KO mice exhibited severe lung pathology post-infection, characterized by extensive hyperemia and pulmonary enlargement (revised 2nd edition Figure 6b-e). Immunohistochemical staining for influenza nucleoprotein and quantitative analysis of viral gene expression in lung and decidual tissues showed that viral replication and propagation were not significantly affected by Il1r2 deletion (revised 2nd edition Supplementary Figure 8). These results indicated that the macrophages in Il1r2 KO mice have not undergone significant changes, and this model does not cause an increase in lung inflammation as compared to the anti-CSF1R model. The Il1r2-KO model may not affect cell migration, but it might have changed the phenomenon of restricted uterine embryonic growth due to the absence of the IL1R2 protein. Thank you very much for your outstanding insights.\nRevised Supplementary Figure 8. Influenza viral replication in the lung and decidua following knocking out Il1r2.\n#6: the authors should address why they have not used the allogenic mouse model that was shown by various groups to mimic best infection-immunity aspects during human pregnancy (Engels et al., Cell Host and Microbe 2017). Please discuss and cite accordingly.\nReply: Thank you for raising this important point regarding the use of an allogeneic pregnancy model. We appreciate the opportunity to discuss our model choice in light of the seminal work by Engels et al., which elegantly demonstrates that allogeneically pregnant mice better recapitulate the severe influenza pathogenesis observed in pregnant humans7.\nIn our study, we initially employed a syngeneic mating model to first establish and dissect the core immunopathological pathway by which a distal respiratory infection impairs placental development and fetal growth. This reductionist approach allowed us to clearly delineate the causal sequence of lung inflammation, systemic immune dysregulation, placental dysfunction and fetal growth restriction, without the additional complexity of strong anti-paternal immune responses present in an allogeneic setting.\nWe fully acknowledge, in line with Engels et al., that the allogeneic model—where the maternal immune system adapts to tolerate a semi-allogeneic fetus—more closely mirrors the immunological context of human pregnancy. The cited study clearly shows that allogeneically pregnant mice exhibit more severe influenza infection, characterized by a restricted anti-viral immune response (e.g., impaired CD8⁺ T cell migration to the lung and reduced type I interferon response) compared to syngenically pregnant or non-pregnant mice. This underscores its superior translational value for studying pregnancy-specific vulnerabilities.\nTherefore, in direct response to this valuable comment, we have now included a discussion as follows (Page 12-13 and Line 516-519): “We fully acknowledge, as demonstrated by Engels et al., that allogeneic pregnancy models, which incorporate immune tolerance mechanisms toward the semi-allogeneic fetus and better recapitulate the immunological landscape of human pregnancy and hold significant translational value7.” We are convinced that our findings in the syngeneic model provide a robust mechanistic framework, and we thank the reviewer for guiding us to strengthen the translational perspective of our work by incorporating this critical discussion.\n#7: in the methods section, the humane endpoint used is not mentioned. Please amend.\nReply: Thank you for this important suggestion. We have now amended the “Methods” section to include both the humane endpoint criteria and the specific euthanasia method. The added text states: “The established humane endpoints included: (1) weight loss exceeding 25% of initial body weight; (2) severe lethargy, hunched posture, or difficulty moving; (3) inability to access food or water independently; and (4) signs of significant distress such as dyspnea. Upon observation of any of these criteria, mice were immediately euthanized by CO₂ inhalation using a gradual fill method, followed by cervical dislocation for confirmation. All animal experiments were performed according to the guidelines of animal protection law and the approved protocols by the Ethics Committee of the University of Science and Technology of China (approval number: USTCACUC27120122083) and adhered to the National Animal Research Guidelines (China).” We apologize for this oversight and appreciate your guidance in enhancing the rigor of our manuscript.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReply: We really appreciate your comments and contribution.\nReferences in this response:\nWe sincerely thank the Reviewers and the Editor for their thoughtful and constructive comments. We have carefully revised the manuscript accordingly and performed additional experiments as requested. Below, we provide a point-by-point response to all the reviewers’ comments. Reviewer comments are shown in Blue color, and our responses follow each comment.\n~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\nREVIEWER COMMENTS\nReviewer #3 and Reviewer #4 (Remarks to the Author):\nInhibition of DPP4 alleviates intrauterine growth restriction caused by respiratory influenza virus infection The authors have adequately addressed all major and minor issues. The addition of requested internal controls as well as the analysis of the different cell subsets strengthened the conclusions drawn an the manuscript has significantly improved. However, one minor issue remains. The analysis of the cell composition in the lung after Sitagliptin administration should have included internal controls as well for better comparability and validation of the data. It would be great if the authors could include this analysis as well to further strengthen the effectivity of their drug candidate.\nReply: Thank you for your positive feedback on the revised manuscript and for your valuable suggestions to further improve the quality of our work. Regarding your comment on “including internal controls for the lung cell composition analysis after Sitagliptin administration,” we have performed immunostaining analysis on lung paraffin-embedded sections from the following four groups: uninfected control, inhibitor-only treatment group, infected untreated group, and infected treated group (Revised Supplementary Figure 2f-i as follows). The results showed that compared with the uninfected control group, inhibitor treatment alone did not cause significant changes in lung neutrophils and macrophages, indicating that the DPP4 inhibitor itself does not alter the baseline pulmonary immune landscape. Furthermore, H1N1 infection indeed triggered a significant increase in neutrophils and macrophages, which was markedly alleviated by Sitagliptin treatment. These findings reinforce that Sitagliptin exerts its protective role by modulating the pulmonary immune environment. Once again, we sincerely appreciate your thorough and professional review, which has helped strengthen the rigor and completeness of our conclusions.\nRevised Supplementary Figure 2f-i. Influenza viral replication in the lung and decidua and pulmonary immune cells infiltration following sitagliptin treatment."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors did a very thorough job responding to our concerns and provide substantial evidence to support claims in the manuscript. We have no additional concerns or comments.", "reviewer_2": "The revisions have strengthened the manuscript and conclusions and addressed my concerns.", "reviewer_3": "Inhibition of DPP4 alleviates intrauterine growth restriction caused by respiratory influenza virus infection The authors have adequately addressed all major and minor issues. The addition of requested internal controls as well as the analysis of the different cell subsets strengthened the conclusions drawn an the manuscript has significantly improved. However, one minor issue remains. The analysis of the cell composition in the lung after Sigagliptin administration should have included internal controls as well for better comparability and validation of the data. It would be great if the authors could include this analysis as well to further strengthen the effectivity of their drug candidate.", "reviewer_4": "Inhibition of DPP4 alleviates intrauterine growth restriction caused by respiratory influenza virus infection The authors have adequately addressed all major and minor issues. The addition of requested internal controls as well as the analysis of the different cell subsets strengthened the conclusions drawn an the manuscript has significantly improved. However, one minor issue remains. The analysis of the cell composition in the lung after Sigagliptin administration should have included internal controls as well for better comparability and validation of the data. It would be great if the authors could include this analysis as well to further strengthen the effectivity of their drug candidate."}, "author_response": ""}]

Decision letter