Atypical pericapillary Ly6G⁺Nur77⁺ macrophages initiate type-2 immune responses to allergens in the mouse lung

Domain: neuroscience
Published: 2026-01-22
Editorial outcome: major_revision

Abstract
The mechanisms of airway allergen sensing and type 2 immune response initiation remain poorly understood. Using a mouse house dust mite (HDM)-induced allergic airway model, we identify a population of lung macrophages located close to alveolar capillaries that express Ly6G and the nuclear receptor Nr4a1/Nur77. These atypical Ly6G+Nur77+ macrophages preferentially capture airway-delivered allergens and play an important role in initiating HDM-driven T helper type 2 (Th2) responses. They sense the major HDM allergen, the cysteine protease Der p 1, via protease-activated receptor 2 (PAR2), and their activation and accumulation require both PAR2 and Nr4a1/Nur77. These Ly6G+Nur77+ macrophages regulate the migration of conventional migratory dendritic cells (mDCs) to draining mediastinal lymph nodes (mLNs) through cysteinyl leukotriene (CysLT) production, which enhances mDC migration toward CCL21 for T cell priming. Inhibiting CysLT biosynthesis reduces mDC migration and dampens Th2 allergic responses, highlighting possible therapeutic avenues in type 2 immunity.

Introduction
The mechanisms by which the immune system detects pathogens are well understood, involving pathogen-associated molecular patterns (PAMPs) recognized by pattern recognition receptors (PRRs) that trigger innate immune responses1. However, the recognition of common environmental allergens is less clear, as allergens often lack distinct PAMPs. Therefore, understanding how allergens are detected and initiate allergic inflammation remains a critical unanswered question.

House dust mites (HDM) are the leading cause of indoor allergic diseases, including asthma and allergic rhinitis, affecting more than 50% of allergic patients2. Despite their clinical significance, the mechanisms by which HDM components are recognized by the immune system and trigger allergic inflammation remain controversial and poorly understood. Lipopolysaccharide (LPS) content in HDM and Toll-like receptor 4 (TLR4) activation have been implicated in immune activation3,4. However, other studies have demonstrated that neither LPS nor TLR4 is required and, in fact, suppress rather than promote type 2 allergic inflammation induced by HDM5–9. Other research has focused on the protease activity of HDM allergens2, particularly cysteine protease activity, which has been shown to promote type 2 skin inflammation10. In this context, Der p 1, the major allergen in HDM, exhibits potent cysteine protease activity and has been compared to papain, a well-characterized model allergen known to activate type 2 immune responses11–13. However, the specific receptors that recognize protease activities in allergens and the precise mechanisms that initiate type 2 allergic inflammation, particularly in the airways, remain unclear.

We demonstrate that detecting cysteine protease activity in HDM is crucial for initiating T helper type 2 (Th2) responses and type 2 immunity in the airways. This protease activity is recognized by protease-activated receptor 2 (PAR2) on a population of perivascular lung macrophages expressing Ly6G and the nuclear receptor Nr4a1/Nur77. These macrophages regulate the migration of conventional-migratory dendritic cells (mDC) to the mediastinal lymph nodes (mLN) by producing cysteinyl leukotrienes (CysLTs) for efficient T cell priming and expansion. Furthermore, inhibiting CysLT production reduces mDC migration and specifically weakens Th2 allergic responses without affecting other types of immune responses.

Results
PAR2 and allergen protease sensing are required for Th2 cell responses to HDM

HDM is the most common cause of type 2 persistent allergic airway disease. In mice, intranasal (i.n.) HDM sensitization followed by i.n. HDM challenge (Fig. 1a) induced a robust accumulation of CD44hiCD4+ T cells (Fig. 1c), IL-13/IL-5-producing Th2 cells (Fig. 1b, d), and eosinophils (Supplementary Fig. 1a) in the lung. To determine the role of different protease activities within HDM in promoting sensitization, HDM were either heat-inactivated (HI) or pretreated with irreversible inhibitors of cysteine (E-64) or serine (4-(2-aminoethyl)-benzene-sulfonyl fluoride [AEBSF]) proteases to eliminate all or specific protease activity. These modified HDM preparations were then used to sensitize mice, followed by HDM challenge. Sensitization with HDMHI or HDME64 failed to induce accumulation of Th2 cells (Fig. 1b–d) and eosinophils (Supplementary Fig. 1a) in the lungs of challenged mice, whereas HDMAEBSF sensitization followed by HDM challenge induced similar Th2-driven inflammation as control HDM-sensitized and challenged mice (Fig. 1b–d, Supplementary Fig. 1a). These findings indicate that cysteine protease activity in HDM is the primary driver of the Th2 immune response. The papain-like cysteine protease Der p 1 is the most abundant and major allergen in HDM14. Sensitization and challenge with Der p 1 induced a robust accumulation of CD44hiCD4+ T cells (Fig. 1f) and Th2 cells (Fig. 1e, g) in the lungs of mice, similar to HDM. This response depended on the cysteine protease activity of Der p 1, as E-64 pretreatment during Der p 1 sensitization prevented Th2 cell accumulation (Fig. 1e–g). Likewise, papain elicited a similar response (Fig. 1h–j), as expected from their shared enzymatic mechanism15. Overall, HDM, Der p 1, and papain induce Th2 responses through a common pathway that relies on cysteine protease activity.

Previous studies found that HDM can promote allergic inflammation due to LPS contamination4. Our HDM extract contained low endotoxin/LPS levels (<30 endotoxin units (EU)/mg). We compared its response to two additional standardized dried HDM extracts: one with high endotoxin levels (B82: ~8×10³ EU/mg) and another with low endotoxin levels (B91: 20 EU/mg). We found that our HDM extract and HDM B91 potently induced Th2 responses (Supplementary Fig. 1b, c) and eosinophil accumulation (Supplementary Fig. 1f) in the lung, whereas the high-endotoxin HDM B82 did not induce Th2-driven inflammation but instead promoted Th1 and Th17 cell accumulation in the lung (Supplementary Fig. b–f). These data suggest that LPS contamination suppresses type 2 immunity, as we have previously published5–7. To confirm this, we used Tlr4−/− mice, which lack the receptor for LPS. After sensitization and challenge with HDM, these mice generated robust Th2-driven responses, similar to WT controls (Supplementary Fig. 1g–k), demonstrating that our HDM does not rely on LPS or TLR4 activation to induce Th2 immune responses. Instead, cysteine protease activity in HDM is the primary driver of Th2 responses.

HDM sensitization induces the priming of allergen-specific CD4+ T cells in the lung-draining mediastinal lymph node (mLN), which subsequently migrate to the lung after challenge16. To analyze differences in T-cell priming to HDM with or without cysteine protease activity, we transferred IL-4-GFP reporter (4get) OTII TCR-transgenic CD4+ T cells, followed by sensitization with HDM or HDME-64 in the presence of OVA (Fig. 1k). HDME-64 was ineffective at inducing OTII cell expansion and GFP-IL-4 expression compared to HDM. Consequently, HDME-64 failed to induce the accumulation of IL-4+ OTII cells in mLNs (Fig. 1l, m). Similar results were observed with HDMHI (Supplementary Fig. 1l, m). Likewise, sensitization with Der p 1 or papain in the presence of OVA induced robust OTII cell expansion and GFP-IL-4 expression, which was abrogated when allergens were pretreated with E-64 (Fig. 1n–p). These data suggest that cysteine protease activity in HDM, Der p 1, and papain is necessary for the expansion and Th2 lineage commitment of allergen-specific CD4+ T cells.

PAR2 is a G protein-coupled receptor that becomes activated when its extracellular domain is cleaved by proteases, including cysteine proteases2. This activation has been implicated in allergic inflammation2. To investigate whether PAR2 is required to mediate responses to protease allergens, WT and Par2−/− mice were sensitized and challenged with papain (Fig. 1q). Th2 cells failed to accumulate in the lungs of Par2−/− mice compared to WT mice (Fig. 1r, s). We observed a similar response to HDM (Supplementary Fig. 1n, o). To test whether PAR2 expression was intrinsically needed in allergen-responsive CD4+ T cells, we sensitized and challenged WT:Par2−/− mixed bone marrow (BM) chimeras with HDM (Supplementary Fig. 1p). We found no difference in the accumulation of naïve, activated CD44hiCD4+ T cells, Th2 cells, or Der p 1:I-Ab-specific T cells in the lung or mLNs of the Par2−/− compartment compared to the WT compartment (Supplementary Fig. 1q). These results suggest that PAR2 essentially contributes to Th2 responses to HDM and other protease allergens but is not intrinsically required in responding T cells.

To analyze differences in T-cell priming in a PAR2-deficient environment, we transferred 4get.OT-II cells into WT and Par2−/− mice, sensitized the recipients, and analyzed the donor OT-II cells (Fig. 1t). OT-II cells did not accumulate in mLNs of Par2−/− mice compared with WT mice, although they still upregulated IL-4-GFP expression (Fig. 1u, v). The impaired expansion of OT-II cells resulted in a reduced number of IL-4+ OT-II cells in the mLNs of Par2−/− mice (Fig. 1u, v). When these recipient mice were further challenged, we observed reduced accumulation of total OT-II and IL-4+ OT-II cells in the lungs of Par2−/− mice (Fig. 1w–y). Similar results were obtained when recipient mice were treated with the PAR2 antagonist AZ3451 during allergen sensitization (Supplementary Fig. 1r, s). However, donor OT-II cell responses to i.n. immunization with LPS+OVA or to i.n. Influenza A virus strain PR8 engineered to express the OT-II OVA epitope (PR8-OTII) was comparable between Par2−/− and WT mice in both the mLNs and lungs (Supplementary Fig. 1t–x). These findings suggest that PAR2 is specifically required for Th2 responses to protease allergens.

PAR2 and allergen protease sensing promote migration of mDCs

Since T cell priming was impaired in the absence of protease activity in allergens, we tested whether the capture of allergen by mDCs in the lung and their migration to the mLNs could also be defective. HDM and HDMHI were labeled with Alexa-647 prior to sensitization, and HDM-bearing, Alexa-647+ cells in the lung and mLNs were analyzed 24 h later (Fig. 2a). No differences were found in HDM+ cells in the lung, including total cells (Fig. 2b) or mDCs (both type 1 mDC1 and type 2 mDC2) (Fig. 2c), between HDM and HDMHI. These results suggest that protease inactivation of HDM does not affect the capacity of DC subsets to capture the allergen in the lung. However, we found reduced frequencies of HDM-bearing, Alexa-647+ cells in the mLNs of mice treated with HDMHI (Fig. 2d). Phenotypical analysis revealed that HDM+ cells in mLNs corresponded exclusively to single-H-Ly6C−CD11c+MHCIIhi mDCs, including both CD103+ mDC1 and CD11b+ mDC2 (Fig. 2e). Both mDC1 and mDC2 were found in reduced frequencies in the mLNs of mice treated with HDMHI (Fig. 2f). These results suggest that protease activity in HDM is essential for promoting DC migration to the mLNs.

We next sensitized WT and Par2−/− mice with Alexa-647-labeled papain (Fig. 2g). No differences were found in papain+ cells in the lung, including total cells or mDCs (mDC1 and mDC2) (Fig. 2h), between WT and Par2−/− mice 24 h after treatment. However, we observed reduced accumulation of papain+ cells in the mLNs of Par2−/− mice (Fig. 2i), which included both mDC1 and mDC2 subsets (Fig. 2j). Similar results were obtained using HDM or when mice were treated with the PAR2 antagonist AZ3451 at the time of allergen exposure instead of using Par2−/− mice (Supplementary Fig. 2a–d). Since these data indicated that PAR2 is required for mDC migration to the mLNs in response to allergens, we tested whether PAR2 expression was intrinsically required in mDCs. Thus, we sensitized WT:Par2−/− mixed BM chimeras with Alexa-647-labeled papain (Fig. 2k). We found similar accumulation of papain+ mDCs in both the Par2−/− and WT compartments. Overall, our data show that allergen protease activity and PAR2 are required for DC migration to the mLNs following allergen exposure, but PAR2 is not intrinsically required in mDCs.

Atypical Ly6G+ macrophages express and depend on PAR2 for lung accumulation following allergen exposure

To gain insight into which cells express PAR2 to regulate mDC and Th2 responses to allergens, we generated BM chimeric mice to determine whether PAR2 expression was required in the hematopoietic or structural compartment. Thus, irradiated hematopoietic-depleted WT and Par2−/− recipients were reconstituted with BM from either WT or Par2−/− mice to restore the hematopoietic compartment (Fig. 3a). Mice lacking Par2 in hematopoietic cells (Par2−/− > Par2−/− and Par2−/− > WT) exhibited diminished lung Th2 cell responses compared to mice with WT hematopoietic cells (WT > WT and WT > Par2−/−) following papain sensitization and challenge (Fig. 3b, c). Furthermore, mice with Par2−/− hematopoietic cells displayed defective migration of papain+ mDCs to the mLNs 24 h after allergen exposure (Fig. 3d). These results suggest that PAR2 expression in the hematopoietic compartment is required for optimal mDC migration and Th2 responses to protease allergens.

Next, to determine whether the required PAR2-expressing cells are host-resident or circulating, we surgically conjoined congenic WT and Par2−/− mice for 28 days to enable shared blood circulation, followed by exposure to Alexa-647-labeled papain. Par2−/− co-joined mice showed reduced migration of papain+ mDCs to the mLNs 24 h after allergen exposure compared to WT co-joined mice (Fig. 3e). Although mDCs (WT and Par2−/−) in the lungs of WT and Par2−/− mice took up papain similarly (Fig. 3f), both WT and Par2−/− mDCs in the mLNs of Par2−/− hosts contained significantly fewer papain+ cells than mDCs in the mLNs of WT co-joined mice (Fig. 3g), suggesting that host-resident PAR2-expressing cells regulate mDC migration.

We next analyzed PAR2 expression in lung cells and found it associated with a distinct population characterized by the expression of monocyte-macrophage markers, including CX3CR1, F4/80, CD64, CD115, and Ly6C (Fig. 3h, i). Notably, this PAR2+ population also expressed Ly6G, a marker typically associated with neutrophils (Fig. 3i), leading us to designate this unique population as Ly6G+ macrophages (Ly6G+ MΦ). Ly6G+ MΦ homogeneously expressed PAR2, whereas other lung cells were negative (Supplementary Fig. 3a). Importantly, PAR2 surface expression on Ly6G+ MΦ was markedly down-modulated 24 h after HDM or papain exposure compared with naïve controls, consistent with receptor activation and internalization following protease stimulation (Supplementary Fig. 3b). A most extensive phenotypic analysis of 24 h papain-treated mice showed that Ly6G+ MΦ had a unique expression profile distinct from neutrophils, monocyte subsets, or monocyte-derived DCs (moDCs). Ly6G+ MΦ were characterized by high expression of molecules involved in adhesion and migration, such as CD11a, CD49d, CD43, CD66a, and CD97. Additionally, they highly expressed molecules involved in immune and homeostatic regulation, such as Treml4, Clec4a1, CD305, CD273, CD274, CD143, and CD36 (Supplementary Fig. 3b). Ly6G+ MΦ also showed high staining for Ki-67 and EdU (5-ethynyl-2’-deoxyuridine), suggesting that these cells are highly proliferative (Supplementary Fig. 3c). To further investigate their proliferative dynamics, we performed an EdU pulse-chase experiment. We found that Ly6G+ MΦ retained EdU labeling over time, although fluorescence intensity gradually declined, consistent with active proliferation of a stable population. In contrast, monocytes rapidly lost EdU signal, indicative of high turnover and replacement by newly generated cells (Supplementary Fig. 3d). Ly6G+ MΦ exhibited high forward scatter (FSC) and side scatter (SSC) profiles, indicating that these cells are larger in size and internally granular, although less so than alveolar macrophages (Supplementary Fig. 3b). Morphology of Ly6G+ MΦ was distinct from neutrophils, monocyte subsets and alveolar macrophages (Supplementary Fig. 3e). We found that Ly6G+ MΦ in the lung uptake allergens and represented approximately 10–20% of the total cells that internalized HDM or papain (Fig. 3j–l). Other cells that uptake allergens included alveolar macrophages, classical monocytes, moDCs, neutrophils, and mDCs (Fig. 3j–l). Ly6G+ MΦ only constitute about 1% of the total cells in the lung (Fig. 3m, n), but their high representation among allergen+ cells suggests a preferential ability of Ly6G+ MΦ to take up allergens. Indeed, more than 60% of Ly6G+ MΦ internalized HDM or papain, and along with alveolar macrophages, are the cell subsets with the most preferential ability to access and take up allergens in the lung (Supplementary Fig. 3f–h). Ly6G+ MΦ remained of host origin after parabiotic surgery (Fig. 3o); however, they were radiosensitive and completely replaced by BM progenitors after irradiation depletion (Fig. 3p). In contrast, alveolar macrophages were radioresistant, with more than 70% remaining of host origin (Fig. 3p). Using Ms4a3TdT reporter mice, an established fate-mapping model for progeny of granulocyte-monocyte progenitors (GMPs)17, we found that Ly6G+ MΦ, as well as monocyte subsets, neutrophils, and eosinophils in the lung, all originate from GMPs (Fig. 3q) and were already detectable in the lung by postnatal day 7 (P7) (Supplementary Fig. 3i). In contrast, alveolar and interstitial macrophages were mostly not GMP-derived (Fig. 3q). We observed no defect in the generation of lung Ly6G+ MΦ in Ccr2−/− mice (Fig. 3r), suggesting that Ly6G+ MΦ may develop independently of CCR2+ monocytes. However, after irradiation depletion, Ccr2−/− BM cells were defective in repopulating lung Ly6G+ MΦ (Fig. 3s) and were completely unable to compete with WT BM for the generation of Ly6G+ MΦ (Fig. 3t). These data suggest that CCR2+ classical monocytes are precursors of Ly6G+ MΦ when a niche is available, although compensatory pathways might be able to substitute for the absence of CCR2 during the homeostatic development of this population.

Ly6G+ MΦ rapidly and transiently accumulated in the lungs of mice following allergen exposure (Fig. 3u). However, HDM+ Ly6G+ MΦ did not accumulate in the lungs of HDMHI-treated mice compared to those treated with HDM (Fig. 3v). HDM+ classical monocytes and moDCs were also reduced in the lungs of HDMHI-treated mice (Fig. 3v), suggesting that the accumulation of allergen-bearing Ly6G+ macrophages, classical monocytes, and moDCs depends on protease activity in HDM. Similarly, total and papain+ Ly6G+ MΦ did not accumulate in the lungs of treated Par2−/− mice compared to WT mice (Fig. 3w, Supplementary Fig. 3j). MoDCs also did not differentiate (Fig. 3w), indicating that PAR2 is required for the accumulation of allergen-bearing Ly6G+ MΦ and moDCs. To test whether PAR2 expression was intrinsically required in these cells, we sensitized WT:Par2−/− mixed BM chimeras with labeled papain (Fig. 3x). We found that papain+ Par2−/− Ly6G+ MΦ severely failed to accumulate in the lung, and only WT Ly6G+ MΦ were able to accumulate in the lung (Fig. 3y). In contrast, other papain+ lung cells showed similar accumulation in both the Par2−/− and WT compartments (Fig. 3y). Thus, Ly6G+ MΦ require PAR2 for lung accumulation after allergen exposure. Overall, Ly6G+ MΦ are a population of macrophages with a unique expression profile, high proliferative capacity, and superior ability to take up inhaled allergens. Ly6G+ MΦ are radiosensitive and derive from GMP progenitors and CCR2+ monocytes. Ly6G+ MΦ accumulate in the lung after treatment with protease allergens and require PAR2 for this process.

Ly6G+ MΦ represent a transcriptionally distinct perivascular MΦ subset in the lung

To understand Ly6G+ MΦ in relation to other cells that encounter and capture allergens in the lung—beyond population-level analysis—we transcriptionally profiled individual papain+ cells from the lung 24 h post-treatment using scRNA-seq. Papain+ cells clustered into 17 transcriptional groups, one of which (cluster 4) expressed a signature associated with Ly6G+ MΦ (Supplementary Fig. 4a, b). Other clusters were categorized based on their transcriptional signatures into alveolar macrophages (clusters 2, 14), neutrophils (clusters 0, 1, 6, 15), classical/CCR2+ monocytes (clusters 5, 7), moDCs (clusters 3, 11), and conventional DCs, including type 1 (cluster 8), type 2, (cluster 13) and migratory (cluster 10) DC phenotypes. Additionally, we identified smaller clusters representing endothelial cells, NK cells, and T cells (Supplementary Fig. 4a, b). Signature genes of Ly6G+ MΦ included markers highly expressed in these cells: Csf1r (CD115), Cx3cr1, Fcgr4 (CD16.2), Itgal (CD11a), Itga4 (CD49d), Spn (CD43), Clec4a1, Treml4, Cd36, Ace (CD143), and Lair1 (CD305) (Supplementary Fig. 4b, c). Other notable genes included Nr4a1, Bcl2, Adgre4, Pparg, Eno3, Tgfbr3, and Cd300e (Supplementary Fig. 4c). Ly6g transcripts were not detected in any cell in the scRNA-seq dataset. We isolated the Ly6G+ MΦ cluster along with other monocyte-related clusters (classical monocytes and moDCs) for further analysis. In this refined analysis, Ly6G+ MΦ segregated into two clusters (cluster 2, 3) (Fig. 4a). Since classical monocytes are precursors to both Ly6G+ MΦ (Fig. 3s, t) and moDCs6, we explored their differentiation pathways using pseudotime trajectory analysis. This revealed two distinct trajectories, one leading to cluster 3 and subsequently to cluster 2 within the Ly6G+ MΦ population, suggesting classical monocytes differentiate into Ly6G+ MΦ through two developmental states. The second trajectory led to moDCs, indicating an alternative differentiation pathway. These findings highlight the plasticity of classical monocytes, capable of giving rise to both Ly6G+ MΦ and moDCs via separate pathways (Supplementary Fig. 4d). We then performed Gene Set Enrichment Analysis (GSEA) using signatures of monocyte subsets (Supplementary Data 118.). This analysis revealed that Ly6G+ MΦ clusters were enriched for a non-classical monocyte signature, whereas classical monocyte and moDC clusters were enriched for the classical monocyte signature (Fig. 4b). These findings suggest that Ly6G+ MΦ may differentiate through a pathway involving a non-classical monocyte transition. Gene ontology (GO) analysis showed that Ly6G+ MΦ clusters were highly associated with adhesion, proliferation and DNA repair, differentiation, and activation processes (Fig. 4c, Supplementary Data 2). Notably, cluster 2 displayed more GO terms related to activation than cluster 3, suggesting it may represent a more activated state of Ly6G+ MΦ. To further characterize Ly6G+ MΦ relative to non-classical monocytes (ncMo), we performed an integrated scRNA-seq analysis of isolated populations. The integrated UMAP revealed that the two cell types clustered separately, demonstrating distinct transcriptional identities (Supplementary Fig. 5a, b). ncMo were predominantly localized to clusters 0 and 1, whereas Ly6G+ MΦ distributed across clusters 2.0, 2.1, 3, 4, and 5 (Supplementary Fig. 5c). Despite their distinct clustering, both populations shared expression of a core set of genes including Csf1r, Cx3cr1, Nr4a1, Bcl2, Pparg, Tgfbr3, Il10ra, Klf2, Klf4, Clec4a1, Cebpb, Itga4, Itgal, Spn, Cd9, Cd36, Lair1, Treml4, Fcgr4, and Ace. However, Ly6G+ MΦ uniquely expressed higher levels of genes associated with effector and inflammatory programs, such as Arg2, Dhrs9, Dusp1, Hdc, Alox5, Ptgs2, Il1r2, Il1rn, Lmnb1, and Ly6g (Supplementary Fig. 5d). To explore the relationship between ncMo and Ly6G+ MΦ, we performed pseudotime trajectory analysis. From ncMo, two branching developmental pathways were observed: Branch 1 was enriched for pathways associated with cell migration, activation, adhesion, angiogenesis, vasculature regulation, and cytokine/chemokine production, suggesting a differentiation program leading to immune activation/regulation and tissue remodeling. Branch 2 was enriched for pathways related to translation, ribosome biogenesis, oxidative phosphorylation (OXPHOS), nucleoside/nucleotide synthesis, protein folding, apoptosis, and p53 stress signaling, indicating a metabolic and stress-response program (Supplementary Fig. 5e, f, Supplementary Data 3). Cell cycle analysis revealed that differentiation of Ly6G+ MΦ along Branch 1 was accompanied by an increased proportion of G2/M-phase cells, consistent with active proliferation during this trajectory (Supplementary Fig. 5g). These data indicate that Ly6G+ MΦ represent a specialized macrophage lineage with a developmental relationship to ncMo.

We next performed RNA-seq to analyze gene expression changes during Ly6G+ MΦ development by comparing the transcriptomes of Ly6G+ MΦ and their precursors, CCR2+ classical BM monocytes. Approximately 1500 genes were differentially upregulated by at least a two-fold change. These include signature genes of Ly6G+ MΦ (Fig. 4d) that were previously identified at the transcript level in our scRNA-seq analysis (Supplementary Fig. 4b, c, Fig. 5d) and at the protein level by flow cytometry (Supplementary Fig. 3b). Ly6G+ MΦ were enriched for a non-classical monocyte signature and negatively enriched for the classical monocyte signature (Fig. 4e, Supplementary Data 1), consistent with our scRNA-seq analyses (Fig. 4b, Supplementary Fig. 5d). Ingenuity Pathway Analysis (IPA) identified biological functions in Ly6G+ MΦ that were similar to those observed in our scRNA-seq analysis, including adhesion, migration, movement, proliferation, and inflammatory response (Fig. 4f, Supplementary Data 4). IPA canonical pathways were associated with degranulation, alternative macrophage activation via IL-4/13, tissue remodeling and fibrosis, and tumor microenvironment (Fig. 4g, Supplementary Data 4). We then performed GSEA using an atlas of human macrophage subset signatures previously identified across various tissues during prenatal development19. Ly6G+ MΦ showed strong enrichment for a human perivascular angiogenic macrophage subset (Fig. 4h and Supplementary Data 5), which is predominantly found in the lung and skin19. Since these data suggested that Ly6G+ MΦ might represent a perivascular population in the lung, we conducted short-term intravascular labeling experiments. Mice were intravenously (i.v.) injected with labeled CD45 prior to analysis to stain circulating cells and those in perivascular locations accessible from the bloodstream. We found that Ly6G+ MΦ were highly stained with i.v. CD45 in both naïve and allergen-treated mice (Fig. 4i). Additionally, monocyte subsets were labeled. However, unlike monocytes, Ly6G+ MΦ were not detected in circulation (Supplementary Fig. 5h), suggesting perivascular localization in the lung. In contrast, moDCs, neutrophils, and alveolar macrophages were protected from labeling (Fig. 4i), indicating that they resided within lung tissue. Overall, our findings indicate that Ly6G+ MΦ constitute a population of perivascular macrophages characterized by a non-classical monocyte signature, with features of increased cell adhesion, migration, proliferation, alternative macrophage activation, and tissue remodeling.

Ly6G+ MΦ intrinsically depend on the transcription factor Nr4a1/Nur77 for their function in controlling the migration of mDCs and the initiation of Th2 responses

Since one of the top identified signature genes of Ly6G+ MΦ in our scRNA-seq and RNA-seq analyses was Nr4a1 (Fig. 4d, Supplementary Fig. 4b, c and Supplementary Fig. 5d), we used NR4A1-GFP reporter mice to study Nr4a1 expression upon allergen exposure. We found that Nr4a1 was not expressed in mLNs 24 h after allergen exposure but was present in the lung, where its expression was specifically restricted to Ly6G+ MΦ and ncMo (Supplementary Fig. 6a, b). Among lung cells that took up allergens, only Ly6G+ MΦ expressed Nr4a1 (Fig. 5a). Since Nr4a1 is a specific marker for Ly6G+ MΦ, we used NR4A1-GFP reporter mice to assess the location of Ly6G+ MΦ in the lung. We found that Nr4a1+ Ly6G+ CX3CR1+ MΦ were primarily localized along the CD31+ capillary network within the perivascular-interstitial compartment of the alveolar parenchyma (Supplementary Fig. 6c). We next used Nr4a1 enhancer domain E2-deficient (se_2−/−) mice, which lack Nr4a1 expression specifically in the monocyte lineage20, to analyze the role of Nr4a1 in Ly6G+ MΦ. We found that se_2−/− mice exhibited defective accumulation of Ly6G+ MΦ and ncMo in the lung 24 h following allergen exposure (Fig. 5b). To test whether Nr4a1 was intrinsically required in allergen-bearing Ly6G+ MΦ, we sensitized WT:se_2−/− mixed BM chimeras with labeled papain (Fig. 5c). We found that papain+ se_2−/− Ly6G+ MΦ failed to accumulate in the lung, with WT Ly6G+ MΦ accounting for >80% of the accumulation (Fig. 5c). In contrast, other papain+ lung cells showed similar accumulation in both the se_2−/− and WT compartments (Fig. 5c). Thus, Ly6G+ MΦ require the transcription factor Nr4a1 (Nur77) for lung accumulation following allergen exposure. Ly6G+ MΦ significantly increased Ki-67 expression in papain-treated WT mice, indicating cell proliferation following allergen-driven activation (Supplementary Fig. 6d). However, in papain-treated se_2−/− mice, Ly6G+ MΦ failed to upregulate Ki-67 (Supplementary Fig. 6d), suggesting a defect in cell cycle entry, which may contribute to the impaired accumulation of Ly6G+ MΦ in the lung in these mice.

Since se_2−/− mice have defects in Ly6G+ MΦ, we assessed Th2 responses in these mice. We found that Th2 cells failed to accumulate in the lungs of se_2−/− mice compared to WT mice after sensitization and challenge with papain (Fig. 5d). Similar results were observed with HDM (Supplementary Fig. 6e). However, we found that Nr4a1se_2 expression was not intrinsically required in allergen-responsive CD4+ T cells (Supplementary Fig. 6f). Additionally, we observed that se_2−/− mice had impaired expansion of donor OT-II cells in the mLNs following allergen sensitization (Fig. 5e) and reduced accumulation of total OT-II and IL-4+ OT-II cells in the lungs following challenge (Fig. 5f). We next evaluated mDC migration in se_2−/− mice and observed reduced accumulation of papain+ mDC1 and mDC2 cells in the mLNs of se_2−/− mice 24 h after treatment, compared to WT mice (Fig. 5g). No differences were found in papain+ cells in the lungs, including mDCs (Supplementary Fig. 6g), indicating defective migration of mDCs to the mLNs in se_2−/− mice. However, in WT:se_2−/− mixed BM chimeras treated with labeled papain, we found similar accumulation of papain+ mDCs in both the se_2−/− and WT mLN compartments (Fig. 5h), suggesting that Nr4a1se_2 expression is not intrinsically required in mDCs for migration to the mLNs. Overall, our data suggest that Nr4a1 expression is intrinsically required in Ly6G+ MΦ to control mDC migration to the mLN and drive allergen-specific Th2 responses. To confirm this idea, we generated Par2−/−:se_2−/− mixed BM chimeras (50% s/P−/−), reasoning that Ly6G+ MΦ, uniquely dependent on both genes, would be selectively impaired in these mice compared with control WT:se_2−/− and WT:Par2−/− BM chimeras (Fig. 5i). 24 h after papain treatment, we found a defect in allergen-driven lung accumulation of Ly6G+ MΦ in 50% s/P−/− chimeras compared with control chimeras, as expected (Fig. 5j). Other lung cell populations were unaffected (Fig. 5j). Accompanying the Ly6G+ MΦ defect, we observed defective migration of mDCs to the mLNs (Fig. 5k), defective expansion of donor OTII cells in the mLNs after sensitization (Fig. 5l), and defective accumulation of Th2 cells (Fig. 5m), donor OTII cells (Fig. 5m), and eosinophils (Fig. 5m) in the lung after challenge. This suggests that defective activation and accumulation of Ly6G+ MΦ after allergen exposure impair mDC migration to the mLNs and the development of Th2 responses and type 2 inflammation. Immunofluorescence of lung sections showed that in naïve lungs, CCR7+ DCs were sparsely distributed throughout the parenchyma (Supplementary Fig. 6h) and were often found in close proximity to Nr4a1+ Ly6G+ MΦ near CD31+ capillaries within the alveolar-airway parenchyma (Supplementary Fig. 6i), whereas after papain treatment, CCR7+ DCs clustered around LYVE-1+ lymphatics (Supplementary Fig. 6h). These findings raise the possibility that activation of Ly6G+ MΦ may help guide DCs toward afferent lymphatics to promote their migration to the mLNs.

Ly6G+ MΦ promote mDC migration by producing cysteinyl leukotrienes (CysLTs)

CCR7 directs mDC migration to mLNs21. To test whether CCR7 is required for this migration in response to papain, we generated WT:Ccr7−/− BM chimeras (Fig. 6a). In the lung, papain+ WT and Ccr7−/− mDCs were equivalently represented 24 h after allergen administration (Fig. 6b). However, only papain+ WT mDCs were detected in mLNs (Fig. 6b), suggesting that CCR7 expression in mDCs is essential for their migration to mLNs in response to papain. Interestingly, although mDCs upregulated CCR7 expression following common Th1/Th17 stimuli, such as LPS treatment or PR8 influenza infection, we observed a reduction in CCR7 expression in papain-treated mice compared with naïve mice (Fig. 6c). This prompted us to question how mDCs are still able to migrate increasingly to mLNs in response to papain, even when CCR7 expression is reduced. Since Ly6G+ MΦ are required for this enhanced migration, we examined the differentially expressed genes in our Ly6G+ MΦ RNA-seq database. We found specific genes related to the eicosanoid signaling network to be upregulated, particularly Alox5, Alox5ap, and Ltc4s (Supplementary Fig. 7a, b), which encode critical enzymes involved in the synthesis of CysLTs, including LTC4, LTD4, and LTE4 (Supplementary Fig. 7a). Flow cytometry confirmed that Ly6G+ MΦ express high levels of ALOX5 in naïve mice compared to other cell populations in the lung (Supplementary Fig. 7c). Additionally, we analyzed changes in eicosanoid pathway genes in Ly6G+ MΦ from naïve and papain-treated mice and found significant upregulation of Ltc4s in Ly6G+ MΦ from papain-treated mice (Fig. 6d). Overall, these results suggest that Ly6G+ MΦ have the capability to produce CysLTs, and this activity may be enhanced by exposure to allergens. To directly assess the capacity of Ly6G+ MΦ to produce CysLTs, we purified Ly6G+ MΦ from the lungs and stimulated them ex vivo with papain. WT Ly6G+ MΦ robustly produced LTC4, whereas PAR2−/− Ly6G+ MΦ displayed impaired production (Supplementary Fig. 7d), indicating that papain-induced PAR2 signaling promotes LTC4 production in these cells. In line with this, we observed that in vivo exposure to papain caused a transient increase in LTC4 levels in the lungs (Fig. 6e). However, this response was not present in Par2−/− and Nr4a1−/− mice (Fig. 6f), which have defective Ly6G+ MΦ.

To test whether CysLTs are required for mDC migration to mLNs following papain treatment, we generated BM chimeras using WT and Alox5−/− BM donor cells and sensitized them with papain. As expected, Alox5−/− BM chimeras exhibited reduced LTC4 levels in the lung after papain treatment (Fig. 6g). Although allergen capture in the lung was similar between Alox5−/− and WT mice, we observed reduced accumulation of papain+ mDCs in mLNs of Alox5−/− mice compared to WT mice (Fig. 6h), suggesting defective migration from the lung to the mLN. This defect was rescued by LTC4 inoculation at the time of allergen exposure (Fig. 6h). These findings indicate that LTC4 production is essential for mDC migration to mLNs following allergen exposure. In addition, we show that both Ly6G+ MΦ-deficient Par2−/− and se_2−/− mice, which had defective LTC4 production and impaired mDC migration to the mLN; this latter defect could be rescued by LTC4 or LTD4 inoculation at the time of allergen exposure, but not by LTB4 inoculation (Fig. 6i, Supplementary 7e–g). This suggests a link between Ly6G+ MΦ and CysLT production in the control of mDC migration to allergens. To confirm this idea, we generated se_2−/−:Alox5−/− mixed BM chimeras (s/A−/−) to selectively target Alox5 deficiency to Ly6G+ MΦ. Since se_2−/− BM cells are unable to efficiently reconstitute Ly6G+ MΦ in the lung, most Ly6G+ MΦ in these mice are expected to be derived from Alox5−/− BM cells and therefore expected to lack the capacity to produce CysLTs. We compared these mice to control WT:se_2−/− (WT/s−/−) and WT:Alox5−/− (WT/A−/−) BM chimeras (Fig. 6j). We confirmed that both experimental s/A−/− and control mice had normal numbers of Ly6G+ MΦ after papain sensitization (Fig. 6k). Additionally, no differences were observed in other lung cells (Supplementary Fig. 7h). As expected, Ly6G+ MΦ in experimental s/A−/− chimeras showed defective expression of ALOX5 compared to control chimeras. (Fig. 6l, m). No differences in ALOX5 expression were found in other lung cells (Supplementary Fig. 7i). We found s/A−/− chimeras were unable to produce detectable levels of LTC4 in the lung following papain exposure (Fig. 6n), suggesting Ly6G+ MΦ are a major source of CysLTs in the lung post-allergen exposure. We also found reduced accumulation of papain+ mDCs in mLNs of s/A−/− mice compared to control mice (Fig. 6o) despite similar allergen capture in the lung (Supplementary Fig. 7j), suggesting defective migration from the lung to the mLN. Furthermore, this migration defect was rescued by LTC4 inoculation at the time of allergen exposure (Fig. 6o, Supplementary Fig. 7j). These results indicate that Ly6G+ MΦ regulate mDC migration to mLN after allergen exposure by producing CysLTs. Finally, to address how CysLTs may regulate CCR7-dependent migration of mDCs toward their ligands, CCL19 and CCL21, we assessed mDCs from mice treated with either papain or LPS in vitro chemotaxis assays. mDCs from papain-treated mice exhibited poor migration toward CCL21 compared to those from LPS-treated mice (Fig. 6p, q). However, the addition of LTC4 or LTD4 enhanced migration to CCL21, while no effect was observed for CCL19 (Fig. 6p). These findings suggest that LTC4 and LTD4 specifically enhance CCR7-dependent mDC migration toward CCL21.

Inhibition of CysLT production blocks mDC migration and prevents Th2 immune responses to allergens

Blocking the action of CysLTs on CysLT receptor 1 (CysLT1R) using drug antagonists is a common treatment for asthma and allergic rhinitis22; however, no current drug blocks CysLT2R and CysLT3R, which have distinct but complementary roles to CysLT1R in mediating CysLT signaling23. As a potentially more effective approach, we tested whether broadly inhibiting CysLT production could prevent Th2 immunity. First, we tested whether treatment of mice with the LTC4 synthase (LTC4S) inhibitor AZD9898 at the time of allergen exposure could impact mDC migration to the mLNs. AZD9898 treatment strongly reduced mDC migration to the mLNs in papain-treated mice (Fig. 7a, b, Supplementary Fig. 7k) but had no effect in mice treated with papain+LPS (Fig. 7a, c), suggesting that LTC4S blockade specifically inhibits mDC migration to allergens. Consistent with this, we found that a single AZD9898 treatment at the onset of allergen sensitization (Fig. 7d) blocked the expansion of donor OTII cells in the mLNs of papain-sensitized mice (Fig. 7e, f) but not in mice additionally treated with LPS (Fig. 7e, g). Finally, a single AZD9898 treatment during allergen sensitization effectively suppressed Th2 responses in the lung following allergen challenge (Fig. 7h, i). These data suggest that LTC4S inhibition during allergen exposure can effectively prevent the development of Th2 cell responses. To determine whether LTC4S inhibition could also prevent the development of type 2 inflammation in sensitized mice, we administered AZD9898 at the beginning of the challenge phase rather than during sensitization (Fig. 7j, l). Strikingly, this later treatment produced similar effects, including reduced mDC migration and re-expansion of donor OTII cells in the mLNs (Fig. 7j, k), and suppressed Th2 responses (Fig. 7l, m) and eosinophilia (Fig. 7n) in the lung. These findings indicate that LTC4S inhibition is effective at both the sensitization and challenge phases, highlighting its therapeutic potential for allergic airway disease.

In conclusion, we have identified a unique perivascular/pericapillary Ly6G+ macrophage population that highly expresses PAR2 and Nr4a1/Nur77 and responds to cysteine protease activity within aeroallergens. Activation of these Ly6G+ macrophages is essential for driving a full Th2 allergic response, as they regulate mDC migration to mLNs by producing LTC4, which synergizes with CCR7 on mDCs to enhance migration toward CCL21, thereby facilitating T cell priming and expansion. Importantly, targeting the LTC4S-driven CysLT biosynthesis pathway effectively and specifically reduces mDC migration to mLNs following allergen exposure and represents a promising strategy for preventing and treating Th2 cell-driven allergic inflammation.

Methods
Animals
All animal procedures were conducted in accordance with institutional guidelines and approved by the UAB and NIAID Institutional Animal Care and Use Committee (IACUC). Mice were housed under specific pathogen-free conditions. Unless otherwise stated, mice were 6–12 weeks old, maintained on a C57BL/6J background, and both sexes were used in balanced numbers. Animals were euthanized using CO2 asphyxiation followed by cervical dislocation in accordance with approved protocols. The mice strains used in these experiments include: C57BL/6J (B6), B6.SJL-Ptprca Pepcb/BoyJ (CD45.1+ B6 congenic), C57BL/6-Tg(TcraTcrb)425Cbn/J (OTII), B6.129-Il4tm1Lky/J (B6.4get IL-4-GFP reporter), B6.Cg-F2rl1tm1Mslb/J (Par2−/−), C57BL/6-Rr39em1Ched/J (Nr4a1se_2/se_2), B6;129S2-Nr4a1tm1Jmi/J (Nur77−/−), B6.129S4-Ccr2tm1Ifc/J (Ccr2−/−), C57BL/6-Tg(Nr4a1-EGFP/cre) 820Khog/J (Nur77GFP), B6(Cg)-Tlr4tm1.2Karp/J (Tlr4−/−), B6;129S2-Alox5tm1Fun/J (Alox5−/−), C57BL/6J-Ms4a3em2(cre)Fgnx/J (Ms4a3cre), and B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Ai9). Nr4a1se_2/se_2 were developed and kindly provided by Dr. Catherine C. Hedrick. All other mice were originally obtained from Jackson Laboratory and bred onsite. To produce BM chimeras, recipient mice were irradiated with an 800 Rad split dose from a high-energy X-ray source and reconstituted with 107 total BM cells. Mice were analyzed 7–8 weeks later.

For parabiosis experiments, sex- and weight-matched mice were cohoused for 20 days before surgery. The flanks of the partnered mice were shaved 2–3 days prior to the operation. Longitudinal incisions were made from the forelimb to the hindlimb, and sutures were used to join the flanks of the mice. Mice were analyzed 4 weeks post-surgery.

Allergen exposure, adoptive transfer and treatments
Mice were i.n. administered with the following allergens: 100µg of HDM (Dermatophagoides pteronyssinus) extract (Greer Laboratories), 10µg of Papain (Sigma-Aldrich), or 10µg of Der p 1 (Prospec) for 1–3 days, followed by an i.n. challenge with 100µg of HDM, 10µg of Papain, or 10µg of Der p 1 for 3 days, 2 weeks apart. In some experiments, allergens were co-administered with 5µg endotoxin-free OVA (EndoFit OVA, InvivoGen). In certain experiments, allergens were heat-inactivated (100°C for 45 min) or treated with protease inhibitors: E-64 (100nM, 37°C for 30 min) or AEBSF (1mM, 37°C for 30 min) (Sigma-Aldrich). Some allergens were labeled with Alexa Fluor 647 (Invitrogen) before administration. Additional experimental conditions included i.n. administration of PAR2 antagonist AZ3451 (30µg, MedChem Express), LPS from Escherichia coli O111:B4 (5µg, Sigma-Aldrich), or 2nM of Leukotriene B4, C4, or D4 (Cayman Chemical Company) at the time of initial sensitization. For influenza virus infection, mice were i.n. administered 500 VFU of PR8-OTII influenza virus. In some experiments, mice received i.p. administration of 200µL of 2.5mg/mL EDU (Invitrogen) or 200µg of AZD9898 (MedChem Express). For intra- and perivascular staining, mice were i.v. administered 3µg anti-CD45.2 antibody (clone 104, BD Biosciences) 5 min before euthanasia and tissue harvest.

Flow cytometry and cell sorting
Lungs were isolated, cut into small fragments, and digested for 45 min at 37°C with 0.6 mg/mL collagenase A (Sigma) and 30 mg/mL DNase I (Sigma) in RPMI-1640 medium (GIBCO). Digested lungs or mLNs were mechanically disrupted by passage through a wire mesh. Blood was collected in Dextran-EDTA buffer. Bone marrow was collected from the femurs and tibias and filtered through a wire mesh. Red blood cells were lysed with 150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA. Fc receptors were blocked using anti-mouse CD16/32 (clone 93; BioXCell, BE0307), followed by staining with fluorochrome-conjugated antibodies against surface and intracellular markers. Antibodies included CD4 (GK1.5; BD Biosciences, 553729), CD11b (M1/70; BD Biosciences, 553311), Ly6G (1A8; BioLegend, 127603), MHC-II (M5/114.15.2; BioLegend, 107620), and Siglec-F (E50-2440; BD Biosciences, 565526), among others. Unless otherwise noted, antibodies were used at manufacturer-recommended dilutions (typically 1:100 for surface staining and 1:50–1:100 for intracellular staining). A full list of all antibodies, clones, catalog numbers, RRIDs, and dilutions is provided in the Reporting Summary. Dead cells were excluded using 7AAD (Sigma-Aldrich). For intracellular cytokine staining, cell suspensions were stimulated with PMA (20ng/mL) and calcimycin (1mg/mL) in the presence of BD GolgiPlug for 5 h, followed by surface staining, fixation, and permeabilization using the BD Cytofix/Cytoperm Plus Kit. Intranuclear staining was performed using the Mouse Regulatory T Cell Staining Kit (eBioscience). EdU incorporation was measured with the Click-iT Plus EdU Flow Cytometry Assay Kit (Invitrogen). Data were acquired on an Attune NxT or BD LSRFortessa and analyzed with FlowJo v10.10. Cell sorting was performed on a BD FACSAria II (post-sort purity >98%). In all flow cytometry dot plots, scale bars include a darker reference bar indicating the 0 baseline, with signal extending up to 10^6.

OTII cell transfer
CD4+ T cells were isolated from the spleens of naïve 4get.OTII TCR-transgenic mice using MACS (Miltenyi Biotec) or the EasySep CD4+ Positive Selection Kit (STEMCELL Technologies) (post-isolation purity >95%). Equivalent numbers (~50,000) of naïve OTII cells were transferred i.v. into naïve congenic recipients one day before OVA treatments.

Cell morphology
100µL of sorted cells (1×10^5 cells/mL) were loaded into cuvettes attached to micro slides and spun at 500–600 rpm for 5 min. The cuvette was then removed, and the slides were stained using the Hema 3 Stat Pack (Fisher Scientific). Slides were covered and sealed with Cytoseal Mounting Medium (Electron Microscopy Sciences). Images were captured using an Echo Revolve 4 microscope.

Immunofluorescence microscopy
Lungs from Nur77-GFP mice were fixed overnight in 1% paraformaldehyde (Electron Microscopy Sciences) at 4°C, washed with PBS and ammonium chloride, and cryoprotected in 30% sucrose overnight. Tissues were embedded in OCT (Fisher Healthcare), and 12µm cryosections were prepared. Sections were blocked with 5% donkey serum (Sigma-Aldrich) and stained with antibodies against CCR7 (4B12; eBioscience, 13-1971-82), CD31 (MEC13.3; BioLegend, 102516), CX3CR1 (SA011F11; BioLegend, 149005), Ly6G (1A8; BioLegend, 127603), LYVE-1 (ALY7; eBioscience, 53-0443-82), GFP (A21311; Life Technologies), and DAPI. Slides were mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were captured on a Leica SP8 confocal microscope and analyzed with LAS X software (v3.5.21594.6).

Migration assays
100ng of CCL21 or CCL19 in RPMI medium with 1% FBS was added to the bottom chamber of each 24-well Transwell plate (polycarbonate filter with a 5-μm pore; Costar). mDCs (50×10^4 cells per Transwell) were added to the upper chamber in the presence or absence of 100nM LTC4 or LTD4, followed by incubation for 90 min at 37°C. Cells that had transmigrated were collected from the lower chamber and counted. Results are presented as the number of input cells that migrated in the assay. No spontaneous migration in response to the control medium was observed.

Cell culture and stimulation
Sorted Ly6G+ macrophages were cultured in 96-well flat-bottom plates pre-coated with fibronectin (10µg/mL) in the presence of M-CSF (10ng/mL), IL-3 (5ng/mL), TGF-β (1ng/mL), and IL-10 (10ng/mL) and incubated overnight at 37°C. The following day, cells were stimulated for 1 h at 37°C with control media or papain (20–100µg/mL). Plates were then placed on ice, and supernatants were collected for LTC4 quantification by ELISA.

ELISA
LTC4 was quantified in lung homogenates and cell culture supernatants using the Leukotriene C4 ELISA Kit (Cayman Chemical Company), following the kit instructions.

RNA sequencing (RNA-seq)
Primary analysis. Library preparation and RNA-seq were conducted through Genewiz (Azenta Life Sciences). Libraries were sequenced on the HiSeq 3000 platform. The quality of raw sequence fastq-formatted files was assessed using fastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Sequences were trimmed using TrimGalore (version 0.4.4) with phred33 scores, paired-end reads, and the Nextera adapter options (https://github.com/FelixKrueger/TrimGalore). Trimmed sequences were aligned using STAR (version 2.5.2a) with mouse GRCm39 and default options49. Aligned reads were counted with HTseq-count (version 0.6.1p1) set for unstranded reads using the GRCm39 annotation file50.

Downstream analysis
The R package edgeR version 4.4.151,52 was used to assess pairwise differential expression between sample groups. Only genes with counts per million greater than 1 for at least three samples were considered for further analysis. Comparison of our data to other published datasets was accomplished using gene set enrichment analysis (GSEA, version 4.3.2)53. Pathway and function enrichment analyses were performed using Ingenuity Pathway Analysis (IPA, Qiagen Inc. (https://digitalinsights.qiagen.com/IPA)54. Differentially expressed genes (DEGs) were identified based on the criteria of FDR < 0.001 and log2 fold-change > 1 or < −1.

Single cell RNA sequencing (scRNA-seq)
Cell suspension and 10x barcoded gel beads were loaded into 10x Chromium™ Single Cell Chip G (PN 1000120) to capture single cells in Nanoliter-scale oil droplets by 10x Genomics Chromium X Controller using Chromium Next GEM Single Cell 3’ v3.1 reagents (PN-1000268). The single-cell 3’ v3 gene expression library was sequenced on a NovaSeq 6000 101 cycle + 101 cycle symmetric run. The OpenOmics/cell-seek pipeline v2.0.1 and v3.0.2 (https://doi.org/10.5281/zenodo.11106856), leveraging 10x Genomics Cell Ranger v8.0.0 and v9.0.0 generated matrices from FASTQ files using the 10x Genomics GRCm39-2024-A transcriptome reference and performed Seurat QC filtering using Seurat v5.1.0 and v5.2.155 in R v4.3.3 and v4.4.0 (https://www.R-project.org/). The Seurat filtering was performed to remove low-count cells and potential doublets by applying 3 median absolute deviations from the median log-library size filter to the gene and read counts. The same filtering was applied to the mitochondrial percentage with only an upper threshold. Cells were log10 normalized, dimension reduction performed using 30 principal components, and data visualized using Uniform Manifold Approximation and Projections (UMAP). For the allergen+ dataset, unsupervised clustering was performed at resolution 0.6, and monocyte/macrophage populations were isolated by subsetting clusters 3, 4, 5, 7, and 11. The subset was re-normalized, scaled, and re-clustered at resolution 0.4. For the comparative analysis of Ly6G+ MΦ and ncMo, datasets were first merged and clustered at a resolution of 0.2. Based on marker inspection, clusters 0–2 and 5–7 were selected and reanalyzed. Samples were maintained as separate layers and reprocessed with 30 PCs using Seurat’s joint PCA integration workflow. After integration, reclustering was performed at resolution 0.2, and cluster 2 was further subclustered at resolution 0.1. Top marker analysis of the Seurat clusters and branches was performed using Model-based Analysis of Single-cell Transcriptomics (MAST) (https://github.com/RGLab/MAST/) and the Bonferroni test for multiple test correction. Top markers with an average log2 fold-change of greater than 0.5 and adjusted p-value less than 0.05 were used as input for Over-Representation Analysis (ORA). ORA was performed using the R package clusterProfiler v4.10.1 and v4.14.656 with the GO Biological Process and KEGG databases. Gene Set Enrichment Analysis was performed using the Bioconductor package UCell v2.6.257. Trajectory analysis was performed using Monocle 3 v1.3.758 with closed loops set to false. Feature plots, UMAPs, and violin plots were visualized using the R package scCustomize v3.0.1 (https://zenodo.org/records/14529706). The dot plot and heatmap were visualized using the Bioconductor package ComplexHeatmap v2.18.059. Cell cycle scoring was performed with Seurat using mouse G2/M and S phase markers.

Statistical analysis
Data analysis was performed using GraphPad Prism (Version 10.2.0). Statistical tests used are indicated in the figure legends. P-values of less than 0.05 were considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, **P < 0.0001).

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) under accession GSE291081, and scRNA-seq data have been deposited in GEO under accessions GSE289548 and GSE305119 and are publicly available. Any additional information required to reanalyze the data reported in this paper is available from the corresponding author upon request. All other data are available in the article and its Supplementary files or from the corresponding author upon request. Source data are provided with this paper.

Code availability
The code used to analyze the scRNA-seq data can be accessed at https://doi.org/10.5281/zenodo.15149503 and https://doi.org/10.5281/zenodo.16848446.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Meloun et al. presents a compelling study uncovering a novel function of Ly6G⁺ perivascular macrophages in the initiation of type 2 immune responses in the lung. By elucidating a PAR2–NR4A1–ALOX5–LTC₄ signaling cascade that promotes dendritic cell (DC) migration and subsequent Th2 cell differentiation, this work provides mechanistic insights into how innate cells sense and respond to allergen exposure. The study is methodologically sound, conceptually novel, and highly relevant to our understanding of type 2 immunity and allergic inflammation.\nThat said, there are a few aspects where the authors could further strengthen their conclusions and enhance the impact of this already promising work.\nMajor Comments\n1. Clarification of the direct role of PAR2 activation in NR4A1 induction\nThe manuscript proposes that HDM-derived cysteine proteases cleave PAR2 in Ly6G⁺ macrophages, leading to NR4A1 induction and subsequent ALOX5 expression and LTC₄ production. While this signaling axis is supported by in vivo genetic loss-of-function experiments, the mechanistic link between PAR2 cleavage and NR4A1 activation remains indirect. To strengthen this critical aspect of the model, the authors are encouraged to:\n・Provide direct evidence of PAR2 cleavage, for example using cleavage-specific antibodies in Western blot analysis.\n・Examine whether ALOX5 expression and LTC₄ production are induced following in vitro stimulation of sorted Ly6G⁺ macrophages with HDM or papain.\n・Determine whether these responses are abrogated in PAR2-deficient cells or upon treatment with protease inhibitors.\nThese additional experiments would help establish a more direct causal relationship between PAR2 activation and the downstream transcriptional and metabolic responses.\n2. Elucidation of whether Ly6G⁺ macrophages and antigen-loaded DCs physically interact\nIt remains unclear whether the effect of Ly6G⁺ macrophages on DCs is mediated via direct contact or paracrine signaling. Including imaging data to assess spatial proximity or contact between Ly6G⁺ cells and OVA⁺ DCs would help clarify the nature of this cellular interaction.\n3. Anatomical localization of Ly6G⁺ macrophages near lymphatic vessels\nGiven that tissue DCs typically migrate to draining lymph nodes via afferent lymphatic vessels, it would be valuable to demonstrate whether Ly6G⁺ macrophages are anatomically positioned near lymphatic structures (e.g., LYVE1⁺ or Prox1⁺ vessels), particularly at sites where DCs initiate egress. Triple immunostaining for Ly6G, ALOX5, and lymphatic markers would support this point.\n4. Retention of antigen-loaded DCs in the lung upon LTC₄ or ALOX5 inhibition\nIf Ly6G⁺ macrophages promote DC migration via LTC₄, inhibition of this pathway should result in increased retention of antigen-loaded DCs in the lung. While the authors show an accumulation of total CD11c⁺ cells, it is not clear whether these include antigen-bearing DCs. Tracking OVA⁺ DCs (e.g., via OVA-AF647 labeling) under ALOX5-deficient or LTC₄-inhibited conditions would directly support the proposed mechanism.\n5. Th2 cell transfer to distinguish initiation vs. effector stages\nTo confirm that Ly6G⁺ macrophages are required specifically for initiating Th2 differentiation (rather than affecting effector cell function), the authors could assess whether adoptively transferred pre-polarized OT-II Th2 cells bypass the requirement for Ly6G⁺ macrophages or LTC₄. This would pinpoint the step at which this innate mechanism operates.\nMinor Comments\n1. Characterization of Ly6G⁺ macrophages\nThe Ly6G⁺ macrophages shown in Extended Data Fig. 2c appear to display a binucleated morphology. Similar nuclear features have been observed in certain atypical monocyte populations, such as the SatM cells described in a fibrosis model (Satoh et al., Nature, 2017; PMID: 28002407). While the relevance may be limited, briefly acknowledging this morphological similarity might help place these cells within the broader diversity of monocyte/macrophage subsets.\n2. The selectivity of PAR2 over other PARs\nWhile the authors focus on PAR2, it is worth noting that HDM-derived cysteine proteases can potentially activate multiple members of the PAR family (PAR1–4). Notably, recent studies have shown that PAR1 is highly expressed in Th2 cells and contributes to allergic inflammation by enhancing IL-5 and IL-13 production (Kumagai et al., PNAS, 2023; PMID: 38015852). Although additional experiments are not essential, a brief discussion on the potential roles or expression levels of other PARs—particularly PAR1—could help contextualize the specificity of the PAR2-dependent mechanism highlighted in this study.", "reviewer_2": "The study by Meloun et al. identifies an as-yet-unknown sensor of allergens with protease activity and an important actor of type 2 immunity to allergens, namely atypical macrophages expressing PAR2 and Ly6G. The experimental design is robust and appropriate, the experiments are very well executed, the amount of data is impressive and they support most of the authors’ conclusions. The manuscript is very well written and easy to read, and the authors should be commended for their high-quality work. Since such Ly6G+ macrophages have not been previously described in the healthy mouse lung, I think that their characterization at steady-state warrants some additional investigation, especially regarding their localization. Comments from a to e below are related to this.\nMajor comments:\na. My understanding of the data in Fig. 3 is that Ly6G+ Macs at steady-state derive from GMPs postnatally and are not rapidly replaced by circulating monocytes. The authors use the word “tissue-resident” – what is their definition of tissue-residence? This should be clarified, as the possibility remains that they are intravascular (see below).\nb. Related to this, it seems that Ly6G+ macs are relatively long-lived. EdU-pulse experiments could provide insights into their half-life.\nc. If they derived postnatally from GMPs, as shown with the Ms3a3Tdtom mice, when do they appear in the lung after birth?\nd. In Fig. 3y, an important missing control is the chimeric data in non papaïn-injected mice. The authors state that accumulation of Ly6G+ macs in the lung after treatment with protease allergens requires PAR2, but they gate on papain+ cells, not all the cells, and they only look at treated mice, not naïve mice.\ne. My main conceptual concern relates to the localization of these cells. The authors state that Ly6G+ Macs are “perivascular” based on anti-CD45 iv staining, then later they say that Nr4a1+ Ly6G+ Macs are primarily present in the alveolar interstitium, located between the alveolar epithelium and the CD31+ capillary endothelium. First, to me, “perivascular” would rather refer to their preferential localization around bigger blood vessels (arterioles, venules), not capillaries, while the alveolar interstitium is not “perivascular” per se. I think that this is particularly important to clarify since it also relates to the site of sensitization to protease allergens and DC uptake? Does it occur in the alveoli? Or rather at the level of bronchial epithelium, which is suggested in many review and original articles in the field? Second, I understand that the confocal microscopy pictures shown in Extended Data Fig. 4c are taken from a naïve mouse lung. If correct, Nr4a1+Ly6G+ “Macs” are strikingly abundant on the slide, while they are a very rare population. An alternative, more likely, scenario is that the Nr4a1+Ly6G+ lung cells are lung neutrophils, which also express Nr4a1 (unpublished observations – and this does not fit with the data shown in Extended Data Fig. 4b – are Neu from the lung, as stated? or from the blood?). Third, it is impossible to draw the conclusion, based on the pictures shown, that Ly6G+ Macs are not intravascular, especially since they stain positive for anti-CD45 iv. Hence, it is imperative to re-assess the localization of Ly6G+ Macs at steady-state using additional markers: the best would be the use of Cx3cr1 GFP mice, or alternatively, the use of CD68, with a particular focus at peribronchial/perivascular locations.\nf. All the “data not shown” should be included in the manuscript, even though I appreciate already the substantial amount of data.\nComments on figures:\n• Figure 2a–f: The authors aim to evaluate the role of HDM proteases in promoting mDC migration. Why was heat-inactivated HDM used instead of HDM pretreated with E-64, a selective inhibitor of cysteine protease activity? This alternative would more precisely address the role of cysteine proteases.\n• Extended Data Figure 2b: The gating on histograms appears to be suboptimal. Given the high autofluorescence of macrophages, it is recommended to use Fluorescence Minus One (FMO) controls to accurately define positive populations. Similarly, for EdU staining, cells from non-injected mice should be subjected to the Click reaction to serve as a proper negative control.\n• Figure 4i: Gating on density plots is preferable, as it facilitates clearer discrimination between positive and negative populations. Furthermore, cells from PBS-injected control mice should be included as a negative control for CD45 intravenous staining.\n• Extended Data Figure 3a-b: the moDC transcriptome fits perfectly with the signature of lung interstitial macrophages (IM), as they express all the macrophages and IM-related markers (Mafb, Fcgr1, C1q, Mrc1, etc), but do not express DC-specific markers. Even though the discrimination between moDCs and IMs remain contentious, I am curious why the authors chose to name these cells moDCs.\n• Extended Data Figure 3e: Instead of gating on CD11b++ cells, staining for Ly6G would provide a more accurate identification of the target population.\n• Figure 4b: The authors demonstrate that Nr4a1 is necessary for the accumulation of Ly6G+ macrophages and ncMo. However, it should be considered that ncMo may drive the differentiation of Ly6G+ macrophages upon allergen exposure. Therefore, the observed reduction in Ly6G+ macrophages in these mice may be secondary to the absence of ncMo, rather than reflecting a direct requirement of Nr4a1 in Ly6G+ macrophages themselves. This should be clarified.\n• Figures 4c–d: The authors conduct mixed bone marrow chimeras (WT/se_2-/-) to assess allergen uptake by Ly6G+ macrophages in the absence of Nr4a1, showing reduced papain capture by Ly6G+ macrophages from se_2-/- origin. To strengthen these findings, it would be important to report the chimerism levels in Ly6G+ macrophage population. If Ly6G+ macrophages require Nr4a1 for differentiation, then cells of se_2-/- origin should fail to differentiate into Ly6G+ macrophages, and this underrepresentation may account for the reduced frequency of papain+ Ly6G+ macrophages observed in Figure 4c.\nFurther suggestions for improvement can be found below.\n• Across all experiments, the figure panels are very small and should be enlarged with a focus on the most important findings (some gating strategies could be moved to supplement) for better readability.\n• All the data converge to the possibility that ncMo give rise to Ly6G+ Macs. This is particularly interesting since a previous study identified CD16.2+ ncMo in the mouse lung, also expressing Ace and Plac8, like Ly6G+ Macs. This possibility could be discussed.\n• Recent findings have shown that (likely other) recruited Ly6G+ macrophages can play a role in alveolar repair following influenza virus infection, and such Ly6G+ macs depend on both GM-CSF and type 2 cytokine signaling. Since cysteine proteases have previously been reported to induce GM-CSF production in non-classical monocytes (ncMo) (Kaur et al., Cell Reports, 2021), and since alternative activation via IL4/13 pops up in the transcriptomic data, it would be a nice mechanistic addition to investigate the extent to which Ly6G+ macrophages rely on these signaling pathways. Alternatively, this possibility could be discussed.", "reviewer_3": "Meloun and colleagues report, for the first time, that in an allergic model of pulmonary inflammation, Ly6G+ Macrophages produce CysLTs via PAR2, which facilitates dendritic cell migration to mediastinal lymph nodes (mLN). This study presents several novel findings, including the presence of Ly6g+ Macrophages, similar to those reported in an IAV model of lung inflammation (REF 30). The activation of Ly6G+ macrophages through Nr4a1 and PAR to produce CysLTs. And the effects of CysLTs on DCs migration to mLN to activate T-cells. The methodology includes the use of various chimeras and single-cell sequencing. The data presented support the main conclusions. I offered several observations and recommendations to further refine and expand the study.\nThe authors note in the discussion that lung Ly6g+ macrophages have previously been identified (Ref. 30). There are reported similarities between the Ly6G+ macrophages described in this study and those in Ref. 30 identified in a viral model of lung inflammation, including their origin from GMP precursors and their alveolar location. The authors may wish to consider investigating additional potential similarities, such as whether papain-induced Ly6g+ macrophages express Arg1. Are they short-lived, meaning are they only involved in sensitization? Additionally, how CysLTs enhance DC migration in response to chemokines?\nFigure 3w: The authors state that “papain+ Ly6G+ MΦ did not accumulate in the lungs of treated Par2-/- mice compared to WT”; however, there appears to be only a 50% reduction. Furthermore, given that moDCs were also reduced in PAR2-/-, is PAR2 necessary for cMo or non-classical Mo differentiation into Ly6G+ macrophages, or for their proliferation?\nExtended Data 3b. Although Ly6G+ macrophages and non-classical monocytes share certain genes, non-classical monocytes were not identified in the transcriptional signature clusters.\nLine 364. It would be informative to review the data on HDM, even if just as Extended Data.\nIn the BM chimera with WT and Alox5-/- BM donors, papain sensitization yields minimal lung LTC4 (∼5 ng/ml). Yet, in figure 6n (WT/A-) under the same conditions, LTC4 levels reach about 40 ng/ml. What accounts for this difference?\nFigure 6k. Ly6g+ macrophage counts are comparable in s/A-/- chimeras and controls. However, does ALOX5 deficiency alter their activation, proliferation, or recruitment?\nLine 455. The statement that the three CysLT receptors “have redundant functions” is inaccurate. CysLT1, CysLT2, and CysLT3 each preferentially bind LTD4, LTC4, and LTE4, respectively, resulting in distinct effects based on cell type and receptor expression.\nFigure 7. Asthmatic patients are already sensitized to allergens. Does AZD9898 given during the challenge phase suppress T-cell responses, and are these results relevant to real allergens like house dust mites (HDM)?\nDoes AZD9898 affect Ly6G+ macrophage numbers or activation?\nLine 513: The authors discuss Ruscitti et al. (ref 30) and indicate differences but do not highlight similarities in Ly6G+ macrophages, especially in relation to their location and development. Including this information would provide readers with a more comprehensive understanding.\nMinor\nLine 349: add “Fig. 5b”\nLine 374: add “Fig. 5h″"}, "author_response": "We sincerely thank the Editor and Reviewers for their thoughtful and constructive comments, which have greatly improved the quality and clarity of our manuscript. In this revised version, we have carefully addressed all the points raised. Specifically, we have experimentally assessed the direct function of PAR2, clarified the longevity and steady-state presence of Ly6G⁺ macrophages, and incorporated new data and discussion regarding their localization. We have also performed AZD8989 inhibitor experiments in sensitized animals, expanded the discussion of potential developmental pathways, and comprehensively revised the text to address all other reviewer comments with new data, updated figures, and clarifying explanations throughout the manuscript.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript by Meloun et al. presents a compelling study uncovering a novel function of Ly6G⁺ perivascular macrophages in the initiation of type 2 immune responses in the lung. By elucidating a PAR2–NR4A1–ALOX5–LTC₄ signaling cascade that promotes dendritic cell (DC) migration and subsequent Th2 cell differentiation, this work provides mechanistic insights into how innate cells sense and respond to allergen exposure. The study is methodologically sound, conceptually novel, and highly relevant to our understanding of type 2 immunity and allergic inflammation.\nThat said, there are a few aspects where the authors could further strengthen their conclusions and enhance the impact of this already promising work.\nMajor Comments\n1. Clarification of the direct role of PAR2 activation in NR4A1 induction\nThe manuscript proposes that HDM-derived cysteine proteases cleave PAR2 in Ly6G⁺ macrophages, leading to NR4A1 induction and subsequent ALOX5 expression and LTC₄ production. While this signaling axis is supported by in vivo genetic loss-of-function experiments, the mechanistic link between PAR2 cleavage and NR4A1 activation remains indirect. To strengthen this critical aspect of the model, the authors are encouraged to:\n・Provide direct evidence of PAR2 cleavage, for example using cleavage-specific antibodies in Western blot analysis.\n・Examine whether ALOX5 expression and LTC₄ production are induced following in vitro stimulation of sorted Ly6G⁺ macrophages with HDM or papain.\n・Determine whether these responses are abrogated in PAR2-deficient cells or upon treatment with protease inhibitors.\nThese additional experiments would help establish a more direct causal relationship between PAR2 activation and the downstream transcriptional and metabolic responses.\nWe agree that establishing a more direct connection between PAR2 activation by protease allergens and downstream LTC₄ synthesis in Ly6G⁺ macrophages would strengthen our model. We have now added new in vitro data using sorted Ly6G⁺ macrophages, as well as supporting in vivo observations:\n Papain induces LTC₄ synthesis in Ly6G⁺ macrophages in a PAR2-dependent manner. Sorted Ly6G⁺ macrophages were stimulated with papain (20-100 µg mL⁻¹) for 1 h. LTC₄ levels in the supernatant increased in WT but not in Par2⁻/⁻ cells (new Extended Data Fig. 7d; see lines 446–450). This experiment directly addresses the reviewer’s request to test in vitro LTC₄ induction by protease allergens and to demonstrate its deficiency in PAR2-deficient Ly6G⁺ macrophages. The rapid kinetics are consistent with de novo eicosanoid synthesis, supported by the constitutive expression of Alox5 and Ltc4s in Ly6G⁺ macrophages (Extended Data Fig. 7a-c). Because sorted Ly6G⁺ macrophages did not survive extended culture, we could not reliably assess whether PAR2 activation further increased Ltc4s transcription, as observed in vivo (Fig. 6d), nor could we evaluate other long-term effects of PAR2 activation in vitro.\n PAR2 surface expression is down-modulated following protease stimulation. By flow cytometry, surface PAR2 expression decreased on WT Ly6G⁺ macrophages in vivo 24 h after HDM or papain exposure compared with naïve mice (new Extended Data Fig. 3b; see lines 222–225), suggesting activation-associated receptor internalization or down-modulation. We fully agree that cleavage-specific assays (e.g., neo-epitope antibodies or Western blot) would more precisely define the biochemical step. However, such assays were not feasible with our limited primary cell yields within this revision.\nTogether with the in vivo genetic loss-of-function data, these findings provide direct functional evidence linking protease-dependent PAR2 activation to early eicosanoid output in Ly6G⁺ macrophages. As a note, Ly6G⁺ macrophages constitutively express NR4A1, PAR2, ALOX5, and LTC₄ S, suggesting that they possess a transcriptionally poised machinery for leukotriene synthesis. However, LTC₄ production is only triggered upon protease-dependent PAR2 activation and requires NR4A1 expression. Both NR4A1 and PAR2 are therefore essential for this response, although their mechanistic relationship remains to be defined. We have clarified this point in the revised discussion (see lines 555-557).\n2. Elucidation of whether Ly6G⁺ macrophages and antigen-loaded DCs physically interact\nIt remains unclear whether the effect of Ly6G⁺ macrophages on DCs is mediated via direct contact or paracrine signaling. Including imaging data to assess spatial proximity or contact between Ly6G⁺ cells and OVA⁺ DCs would help clarify the nature of this cellular interaction.\n3. Anatomical localization of Ly6G⁺ macrophages near lymphatic vessels\nGiven that tissue DCs typically migrate to draining lymph nodes via afferent lymphatic vessels, it would be valuable to demonstrate whether Ly6G⁺ macrophages are anatomically positioned near lymphatic structures (e.g., LYVE1⁺ or Prox1⁺ vessels), particularly at sites where DCs initiate egress. Triple immunostaining for Ly6G, ALOX5, and lymphatic markers would support this point.\nPoints 2 & 3: Anatomical localization of Ly6G⁺ macrophages and DCs in the lung.\nWe thank the reviewer for these insightful comments regarding the spatial relationship between Ly6G⁺ macrophages and dendritic cells (DCs) in the lung. To investigate whether these cell types are spatially associated, we performed additional immunofluorescence analyses of lung sections from naïve and papain-treated mice. In naïve lungs, CCR7⁺ DCs were sparsely distributed throughout the parenchyma and frequently located in close proximity to Nr4a1⁺ Ly6G⁺ macrophages near CD31⁺ capillaries (new Extended Data Fig. 6h,i). Following papain treatment, however, CCR7⁺ DCs formed prominent clusters around LYVE1⁺ lymphatic vessels (Extended Data Fig. 6h). Thus, Ly6G⁺ macrophages are primarily associated with the CD31⁺ capillary network within the alveolar-airway parenchyma, where DCs can also localize, particularly in naïve lungs, likely allowing them to access inhaled antigens. Together with our functional data showing that Ly6G⁺ macrophage activation upon allergen exposure promotes DC migration to the mediastinal lymph nodes through the production of cysteinyl leukotrienes (cysLTs), these histological observations suggest that activated Ly6G⁺ macrophages communicate locally with DCs in the alveolar-airway parenchyma to enhance their CCR7-dependent chemotactic migration toward lymphatic vessels. This model is consistent with our in vivo migration assays showing that cysLT signaling enhances CCR7-dependent DC chemotaxis toward CCL21 (Fig. 6p,q). Together, these findings support a model in which activated Ly6G⁺ macrophages located in the perivascular-interstitial space locally instruct DCs through the production of cysteinyl leukotrienes, thereby enhancing their CCR7-dependent responsiveness to CCL21 and guiding their migration toward CCL21-producing LYVE1⁺ lymphatic vessels for egress to the draining lymph nodes (see lines 419-425).\n4. Retention of antigen-loaded DCs in the lung upon LTC₄ or ALOX5 inhibition\nIf Ly6G⁺ macrophages promote DC migration via LTC₄ , inhibition of this pathway should result in increased retention of antigen-loaded DCs in the lung. While the authors show an accumulation of total CD11c⁺ cells, it is not clear whether these include antigen-bearing DCs. Tracking OVA⁺ DCs (e.g., via OVA-AF647 labeling) under ALOX5-deficient or LTC₄ -inhibited conditions would directly support the proposed mechanism.\nWe tracked papain⁺ DCs in the lung and mediastinal lymph node of Alox5⁻/⁻ mice, Alox5⁻/⁻ mixed bone marrow chimeras, and after pharmacological inhibition of LTC₄ synthase with AZD9898. Although we observed a marked reduction in the migration of allergen-bearing cells to the draining lymph node, this was not accompanied by a detectable increase in the proportion or relative accumulation of these cells in the lung (Fig. 6h; Extended Data Fig. 7j,k). This likely reflects the fact that the total resident DC pool in the lung is much larger than the small CCR7⁺ subset that migrates to lymph nodes; thus, even when cysLT-dependent egress is impaired, the relative change in total or antigen-bearing DCs within the lung remains below detection. In addition, DCs with delayed migration may undergo apoptosis or be cleared rather than accumulating as intact antigen⁺ cells. Therefore, the most informative measure of cysLT-dependent DC trafficking is the arrival of allergen-bearing DCs in the draining lymph node, rather than their relative retention in the lung.\n5. Th2 cell transfer to distinguish initiation vs. effector stages\nTo confirm that Ly6G⁺ macrophages are required specifically for initiating Th2 differentiation (rather than affecting effector cell function), the authors could assess whether adoptively transferred pre-polarized OT-II Th2 cells bypass the requirement for Ly6G⁺ macrophages or LTC₄ . This would pinpoint the step at which this innate mechanism operates.\nWe appreciate this thoughtful suggestion. Our data indicate that Ly6G⁺ macrophages act upstream during the initiation phase of the type-2 response by promoting CCR7-dependent dendritic cell migration required for efficient naïve CD4⁺ T-cell expansion. When Ly6G⁺ macrophage activation or the LTC₄ pathway is disrupted, naïve antigen-specific T cells fail to expand efficiently due to defective DC migration; however, the small fraction of T cells that do receive adequate stimulation still polarize to Th2, as IL-4–producing cells are preserved among the residual responders. Thus, the dominant defect lies in T-cell priming and expansion, not in Th2 effector differentiation.\nWhile adoptive transfer of pre-polarized OT-II Th2 cells could, in principle, bypass the initiation step, interpretation of such experiments would be complicated by the fact that in vitro-polarized Th2 cells exhibit altered and non-physiological homing patterns. Our data already demonstrate impaired expansion with intact per-cell Th2 polarization within the draining lymph node. We therefore focused on this aspect, recognizing that further assessment of effector Th2 homing to the lung or their local effector functions would involve distinct mechanisms beyond the scope of the present study.\nMinor Comments\n1. Characterization of Ly6G⁺ macrophages\nThe Ly6G⁺ macrophages shown in Extended Data Fig. 2c appear to display a binucleated morphology. Similar nuclear features have been observed in certain atypical monocyte populations, such as the SatM cells described in a fibrosis model (Satoh et al., Nature, 2017; PMID: 28002407). While the relevance may be limited, briefly acknowledging this morphological similarity might help place these cells within the broader diversity of monocyte/macrophage subsets.\nThank you for this insightful comment. We agree that it is important to acknowledge this study and have now incorporated this point into the Discussion (paragraph, lines 564–571, with the reference added in line 566). This will be an interesting direction for future studies to further explore potential relationships among these monocyte/macrophage subsets.\n2. The selectivity of PAR2 over other PARs\nWhile the authors focus on PAR2, it is worth noting that HDM-derived cysteine proteases can potentially activate multiple members of the PAR family (PAR1–4). Notably, recent studies have shown that PAR1 is highly expressed in Th2 cells and contributes to allergic inflammation by enhancing IL-5 and IL-13 production (Kumagai et al., PNAS, 2023; PMID: 38015852). Although additional experiments are not essential, a brief discussion on the potential roles or expression levels of other PARs—particularly PAR1—could help contextualize the specificity of the PAR2-dependent mechanism highlighted in this study.\nWe appreciate this insightful suggestion. Although cysteine proteases such as Der p 1 may have the potential to cleave multiple PARs under certain experimental conditions, our study specifically demonstrates that activation of Ly6G⁺ macrophages and the subsequent initiation of Th2 responses are mediated through PAR2. Given that our data clearly establish a dominant and specific requirement for PAR2, we believe that expanding the discussion to include other PARs would not enhance the mechanistic clarity of our study. We have therefore chosen to keep the discussion focused on the experimentally supported PAR2-dependent pathway.\nReviewer #2 (Remarks to the Author):\nThe study by Meloun et al. identifies an as-yet-unknown sensor of allergens with protease activity and an important actor of type 2 immunity to allergens, namely atypical macrophages expressing PAR2 and Ly6G. The experimental design is robust and appropriate, the experiments are very well executed, the amount of data is impressive and they support most of the authors’ conclusions. The manuscript is very well written and easy to read, and the authors should be commended for their high-quality work. Since such Ly6G+ macrophages have not been previously described in the healthy mouse lung, I think that their characterization at steady-state warrants some additional investigation, especially regarding their localization. Comments from a to e below are related to this.\nMajor comments:\na. My understanding of the data in Fig. 3 is that Ly6G+ Macs at steady-state derive from GMPs postnatally and are not rapidly replaced by circulating monocytes. The authors use the word “tissue-resident” – what is their definition of tissue-residence? This should be clarified, as the possibility remains that they are intravascular (see below).\nWe appreciate the opportunity to clarify and expand on the biology and dynamics of Ly6G⁺ macrophages. In our manuscript, we do not use the term tissue-resident but instead refer to this population as host-resident, based on our parabiosis experiments demonstrating exclusive derivation from the host partner (Fig. 3o). At steady state, our data indicate that these macrophages arise postnatally from GMP progenitors (Fig. 3q) and are not rapidly replaced by circulating monocytes (Fig. 3r); however, they can be replenished by circulating monocytes following depletion (Fig. 3s-t).\nThey display high proliferative capacity, which is further enhanced upon allergen stimulation (Extended Data Figs. 3c, 5g, 6d). We detect these cells in the lung but not in circulation (Extended Data Figs. 5h). Intravascular labeling and immunofluorescence imaging show that they occupy a perivascular niche closely associated with pulmonary capillaries, rather than being intravascular (Fig. 4i). Collectively, these findings suggest that Ly6G⁺ macrophages are maintained locally within the lung and are not readily replenished by circulating monocytes under steady-state conditions, consistent with a self-renewing, long-lived population. As suggested by the reviewer, we have now included EdU pulse–chase experiments (see below) to further illustrate their proliferative dynamics and turnover within this stable population. In addition, we have summarized these findings in the discussion to emphasize that Ly6G⁺ macrophages likely represent a long-lived, self-renewing population in the lung that is poorly replenished from the circulation (see lines 545-548).\nb. Related to this, it seems that Ly6G+ macs are relatively long-lived. EdU-pulse experiments could provide insights into their half-life.\nThank you for this valuable suggestion. As recommended, to further investigate the long-lived maintenance and proliferative dynamics of Ly6G⁺ macrophages, we performed an EdU pulse-chase experiment. We found that Ly6G⁺ MΦ retained EdU labeling over time, although fluorescence intensity gradually declined, consistent with active proliferation within a stable population. In contrast, monocytes rapidly lost EdU signal, indicating high turnover and replacement by newly generated cells (Extended Data Fig. 3d; lines 232–237). These findings support the conclusion that Ly6G⁺ macrophages represent a relatively long-lived, self-renewing population in the lung.\nc. If they derived postnatally from GMPs, as shown with the Ms3a3Tdtom mice, when do they appear in the lung after birth?\nAs suggested, we examined the early postnatal appearance of Ly6G⁺ macrophages in the lung. We found that Ly6G⁺ MΦ were already detectable by postnatal day 7 (P7), indicating that they arise early after birth (Extended Data Fig. 3i; lines 253–254).\nd. In Fig. 3y, an important missing control is the chimeric data in non papaïn-injected mice. The authors state that accumulation of Ly6G+ macs in the lung after treatment with protease allergens requires PAR2, but they gate on papain+ cells, not all the cells, and they only look at treated mice, not naïve mice.\nWe agree that it is important to distinguish allergen-induced accumulation from potential baseline differences. As shown in our data, the presence of Ly6G⁺ macrophages in the lung is comparable between Par2⁻/⁻ and wild-type mice under steady-state conditions, indicating no difference in this population at baseline (Extended Data Fig. 3j). However, following papain administration, Par2⁻/⁻ mice fail to accumulate Ly6G⁺ macrophages in the lung (Extended Data Fig. 3j). Similarly, allergen-bearing Ly6G⁺ macrophages were markedly reduced in Par2⁻/⁻ mice after papain treatment compared with wild-type controls (Fig. 3w). Together, these data indicate that accumulation of Ly6G⁺ macrophages in response to protease allergens requires PAR2. In competitive 50% WT : 50% Par2⁻/⁻ bone marrow chimeras, papain⁺ Ly6G⁺ macrophages derived from WT donors selectively accumulated after papain challenge, whereas Par2⁻/⁻-derived cells did not (Fig. 3y). These findings further confirm that PAR2 signaling is required in a cell-intrinsic manner for the allergen-induced accumulation of Ly6G⁺ macrophages in the lung.\ne. My main conceptual concern relates to the localization of these cells. The authors state that Ly6G+ Macs are “perivascular” based on anti-CD45 iv staining, then later they say that Nr4a1+ Ly6G+ Macs are primarily present in the alveolar interstitium, located between the alveolar epithelium and the CD31+ capillary endothelium. First, to me, “perivascular” would rather refer to their preferential localization around bigger blood vessels (arterioles, venules), not capillaries, while the alveolar interstitium is not “perivascular” per se. I think that this is particularly important to clarify since it also relates to the site of sensitization to protease allergens and DC uptake? Does it occur in the alveoli? Or rather at the level of bronchial epithelium, which is suggested in many review and original articles in the field? Second, I understand that the confocal microscopy pictures shown in Extended Data Fig. 4c are taken from a naïve mouse lung. If correct, Nr4a1+Ly6G+ “Macs” are strikingly abundant on the slide, while they are a very rare population. An alternative, more likely, scenario is that the Nr4a1+Ly6G+ lung cells are lung neutrophils, which also express Nr4a1 (unpublished observations – and this does not fit with the data shown in Extended Data Fig. 4b – are Neu from the lung, as stated? or from the blood?). Third, it is impossible to draw the conclusion, based on the pictures shown, that Ly6G+ Macs are not intravascular, especially since they stain positive for anti-CD45 iv. Hence, it is imperative to re-assess the localization of Ly6G+ Macs at steady-state using additional markers: the best would be the use of Cx3cr1 GFP mice, or alternatively, the use of CD68, with a particular focus at peribronchial/perivascular locations.\nOur flow cytometry data demonstrate that Nr4a1⁺Ly6G⁺ cells in the lung correspond precisely to the Ly6G⁺ macrophage population of interest (Extended Data Fig. 6a–b). Regarding the concern that these cells might represent neutrophils, our Nr4a1-GFP reporter analysis (Extended Data Fig. 6a–b) shows that lung neutrophils do not express Nr4a1, whereas Nr4a1 expression is restricted to non-classical monocytes and Ly6G⁺ macrophages in the lung. Furthermore, flow cytometric (Extended Data Fig. 6a) and newly incorporated imaging analyses (Extended Data Fig. 6c) confirm that Nr4a1⁺Ly6G⁺ cells express CX3CR1, further distinguishing them from neutrophils and supporting their macrophage identity. Thus, this population does not contain neutrophils.\nIn addition, we have now included scRNA-seq data from sorted Nr4a1⁺Ly6G⁺ macrophages, which clearly demonstrate their macrophage lineage and expression of core macrophage genes including Csf1r, Cx3cr1, Nr4a1, Bcl2, Pparg, Tgfbr3, Il10ra, Klf2, Klf4, Clec4a1, Cebpb, Itga4, Itgal, Spn, Cd9, Cd36, Lair1, Treml4, Fcgr4, and Ace, as well as Arg2, Dhrs9, Dusp1, Hdc, Alox5, Ptgs2, Il1r2, Il1rn, Lmnb1, and Ly6g, defining them as a unique Ly6G⁺ macrophage population (Extended Data Fig. 5; lines 314-335).\nOur imaging data show that Nr4a1⁺Ly6G⁺ macrophages are closely associated with the capillary network within the lung parenchyma, perivascular in the sense of pericapillary rather than around larger arterioles or venules. To prevent confusion, we have clarified this terminology throughout the manuscript and now describe these cells as residing at the alveolar-capillary interface. Confocal imaging (now shown in Extended Data Fig. 6c) was performed after allergen exposure (24 h after papain treatment), when Ly6G⁺ macrophages are more abundant and activated. Although these cells are rare at steady state, they can also be detected in naïve lungs at lower frequency and occupy the same anatomical niche.\nOur data further show that Ly6G⁺ macrophages are not intravascular. They are absent from the circulation, and anti-CD45 i.v. labeling combined with flow cytometry and confocal imaging demonstrates that they have access to blood antigens yet reside outside the vascular lumen, in close apposition to capillaries, consistent with a pericapillary, interstitial localization (see amplified inset in Extended Data Fig. 6c).\nThis localization places Ly6G⁺ macrophages strategically between the alveolar epithelium and capillary endothelium, within a region directly accessible to inhaled allergens. In our model, allergens reach nearly all alveolar macrophages (Extended Data Fig. 3f), confirming that the distal airspaces are highly exposed to inhaled material and likely represent regions where allergens can readily reach the underlying tissue, given the thinness and permeability of the alveolar barrier. While protease allergens can also access the bronchial epithelium, our data specifically define the anatomical localization of Ly6G⁺ macrophages near the alveolar region, in close association with the capillary network. This positioning places them at the alveolar-capillary interface, a site also reached by inhaled allergens in our experimental model.\nOur data support a key role for Ly6G⁺ macrophages in promoting Th2 sensitization. These cells occupy an interstitial niche where they are ideally positioned to take up soluble allergens that traverse the epithelial barrier, sense protease activity, and interact with dendritic cells (Extended Data Fig. 6h–i, lines 419–425), thereby orchestrating the initiation of Th2 immune responses.\nCollectively, these data establish that Ly6G⁺ macrophages represent a distinct perivascular/pericapillary macrophage population positioned at the alveolar–capillary interface. We have revised the manuscript to clarify this localization and included new supporting data (Extended Data Fig. 6c, inset showing Ly6G⁺ macrophages adjacent to CD31⁺ capillaries and co-localization of CX3CR1 with Nr4a1⁺Ly6G⁺ cells in the lung).\nf. All the “data not shown” should be included in the manuscript, even though I appreciate already the substantial amount of data.\nAll data previously referred to as “not shown” have now been included in the revised manuscript. Specifically:\n Extended Data Fig. 1l-m: Accumulation of IL-4⁺ OT-II cells in mLNs following HDM and HDMHI treatment.\n Extended Data Fig. 1n-o: Th2 response to HDM in Par2⁻/⁻ mice.\n Extended Data Fig. 1r-s: Effect of the PAR2 antagonist AZ3451 on the accumulation of IL-4⁺ OT-II cells in mLNs.\n Extended Data Fig. 2a-d: Effect of the PAR2 antagonist AZ3451 on mDC migration.\n Extended Data Fig. 6e: Th2 response to HDM in se_2⁻/⁻ mice.\n Extended Data Fig. 6g: Frequency of papain⁺ cells in the lungs of se_2⁻/⁻ mice 24 h after treatment.\nThe only exception is the data from global Nr4a1⁻/⁻ mice (see line 385), which were not included because they are equivalent to those from se_2⁻/⁻ mice. The se_2⁻/⁻ line represents a super-enhancer knockout that prevents high-level Nr4a1 expression, while Nr4a1⁻/⁻ mice are global knockouts exhibiting an indistinguishable phenotype in Ly6G⁺ macrophages. We believe that adding these data would not provide additional insight, as they are redundant and would only make the manuscript denser without improving clarity. However, if the reviewers or editors consider them necessary, we would be glad to include them.\nComments on figures:\n• Figure 2a–f: The authors aim to evaluate the role of HDM proteases in promoting mDC migration. Why was heat-inactivated HDM used instead of HDM pretreated with E-64, a selective inhibitor of cysteine protease activity? This alternative would more precisely address the role of cysteine proteases.\nIn our study, we used an HDM allergen model together with two complementary approaches to inactivate cysteine protease activity—heat inactivation (HDM-HI) and selective cysteine protease inhibition (HDM-E-64)—alongside well-characterized cysteine protease (C1A) allergens (HDM-derived Der p 1 and papain, which share an identical catalytic mechanism). These approaches were employed to evaluate the role of cysteine protease activity in type 2 inflammation, Th2-cell accumulation, antigen-specific T-cell priming, dendritic-cell migration, and Ly6G⁺ macrophage activation. Although not every experiment was performed with all HDM preparations or protease allergens, parallel data using HDM-E-64 and the cysteine proteases Der p 1 and papain consistently demonstrated that cysteine protease activity is the critical enzymatic function driving Th2 immunity in our model. Importantly, our results further show that PAR2 serves as the sensor of this cysteine protease activity, mediating the downstream responses that promote allergen-induced accumulation and activation of Ly6G⁺ macrophages, dendritic cell migration, and subsequent Th2-cell priming. Accordingly, we used heat-inactivated HDM in Figure 2a–f as a general control to abolish overall protease activity, while complementary experiments using HDM-E-64, Der p 1, and papain (shown in other figures) confirm that cysteine protease activity—and its sensing through PAR2—specifically mediate the observed effects. To streamline the presentation, papain was used as a model cysteine protease to mechanistically dissect this pathway.\n• Extended Data Figure 2b: The gating on histograms appears to be suboptimal. Given the high autofluorescence of macrophages, it is recommended to use Fluorescence Minus One (FMO) controls to accurately define positive populations. Similarly, for EdU staining, cells from non-injected mice should be subjected to the Click reaction to serve as a proper negative control.\nWe appreciate the reviewer’s concern; however, we would like to clarify that all flow cytometry data were acquired and analyzed using rigorous gating strategies and appropriate controls. Our laboratory has extensive experience with flow cytometric analysis of diverse immune cell populations, including lung macrophages, and all gating thresholds were established based on well-defined controls. Furthermore, the expression of the markers identified by flow cytometry in Ly6G⁺ macrophages has been independently validated by three separate experiments involving both scRNA-seq and bulk RNA-seq analyses (Extended Data Figs. 4–5 and Fig. 4), which confirm the expression of the same defining markers. Therefore, we respectfully disagree that the histograms appear “suboptimal,” as the data were generated rigorously following established cytometric standards and are consistent across multiple orthogonal platforms.\n• Figure 4i: Gating on density plots is preferable, as it facilitates clearer discrimination between positive and negative populations. Furthermore, cells from PBS-injected control mice should be included as a negative control for CD45 intravenous staining.\nWe thank the reviewer for this suggestion. All gating for CD45 intravenous staining was defined using PBS-injected control mice, which served as negative controls in each experiment. In the figure, we presented representative histograms rather than density plots to facilitate direct comparison of fluorescence intensity across conditions within the same axis range. Because discrimination is based on a single fluorescence channel, the gating outcome is identical whether displayed as histograms or density plots.\n• Extended Data Figure 3a-b: the moDC transcriptome fits perfectly with the signature of lung interstitial macrophages (IM), as they express all the macrophages and IM-related markers (Mafb, Fcgr1, C1q, Mrc1, etc), but do not express DC-specific markers. Even though the discrimination between moDCs and IMs remain contentious, I am curious why the authors chose to name these cells moDCs.\nWe thank the reviewer for this thoughtful comment. We agree that the distinction between monocyte-derived dendritic cells (moDCs) and interstitial macrophages remains an area of active discussion in the field. Our classification is based on both functional and developmental evidence. Following allergen exposure, our flow cytometry analysis detects a strong induction and allergen uptake by Cx3cr1⁺F4/80⁺Ly6C⁺CD11b⁺ monocyte-derived dendritic cells that markedly upregulate antigen-presentation molecules (Fig. 3j).\nConsistent with this, our scRNA-seq analysis of allergen-bearing cells identifies clusters annotated as moDCs that upregulate genes involved in antigen processing and presentation while co-expressing myeloid markers shared with monocytes and macrophages. Pseudotime analysis further links these clusters developmentally to Ccr2⁺ classical monocytes, supporting their derivation from recruited monocytes. We recognize that different interpretations of this population are possible; however, our evidence indicates that these cells represent monocyte-derived antigen-presenting cells induced by allergen exposure. We therefore chose the term “moDCs” to reflect both their monocyte origin and their strong antigen-presenting phenotype. This distinction is not the central focus of the manuscript, and readers can interpret the data accordingly if they prefer alternative terminology.\n• Extended Data Figure 3e: Instead of gating on CD11b++ cells, staining for Ly6G would provide a more accurate identification of the target population.\nIn the revised version, we have now included staining for Ly6G, which more precisely defines the Ly6G⁺ macrophage population (Extended Data Fig. 5h). The updated figure confirms that Ly6G⁺ macrophages are present in the lung but absent from the blood, supporting the accuracy of our previous gating strategy.\n• Figure 4b: The authors demonstrate that Nr4a1 is necessary for the accumulation of Ly6G+ macrophages and ncMo. However, it should be considered that ncMo may drive the differentiation of Ly6G+ macrophages upon allergen exposure. Therefore, the observed reduction in Ly6G+ macrophages in these mice may be secondary to the absence of ncMo, rather than reflecting a direct requirement of Nr4a1 in Ly6G+ macrophages themselves. This should be clarified.\nWe thank the reviewer for this insightful comment. To address this point, we performed additional analyses to define the relationship between Ly6G⁺ macrophages and non-classical monocytes (ncMo). Integrated scRNA-seq profiling of isolated Ly6G⁺ macrophages and ncMo revealed that these two populations cluster separately, indicating distinct transcriptional identities (Extended Data Fig. 5a–c; see lines 314-335). Although they share a core set of genes (Csf1r, Cx3cr1, Nr4a1, Bcl2, Pparg, Tgfbr3, Il10ra, Klf2, Klf4, etc.), Ly6G⁺ macrophages uniquely expressed genes associated with effector and inflammatory programs, including Arg2, Dhrs9, Dusp1, Hdc, Alox5, Ptgs2, Il1r2, Il1rn, and Ly6g (Extended Data Fig. 5d; see lines 314-335).\nPseudotime trajectory analysis linked ncMo to Ly6G⁺ macrophages through two distinct differentiation branches: one enriched for activation, adhesion, angiogenesis, and cytokine/chemokine pathways (Branch 1), and another for metabolic and stress-response pathways (Branch 2) (Extended Data Fig. 5e,f; see lines 314-335). Differentiation along Branch 1 was accompanied by increased G2/M-phase representation, consistent with active proliferation (Extended Data Fig. 5g; see lines 314-335). These data indicate that Ly6G⁺ macrophages represent a specialized macrophage lineage developmentally related to, but transcriptionally and functionally distinct from, ncMo.\nFunctionally, we have already linked the defective Ly6G⁺ macrophage phenotype to reduced Th2 immunity rather than to any secondary effect of ncMo loss. In mixed bone marrow chimeras (WT:Par2⁻/⁻, WT:se_2⁻/⁻, and Par2⁻/⁻:se_2⁻/⁻ combinations; Fig. 5i), Ly6G⁺ macrophages—uniquely dependent on both Par2 and Nr4a1se_2 expression—were selectively impaired in Par2⁻/⁻:se_2⁻/⁻ chimeras after papain challenge (Fig. 5i,j), whereas ncMo and other lung populations remained unaffected. This selective defect was accompanied by reduced mDC migration to the mLNs (Fig. 5k), impaired OT-II expansion (Fig. 5l), and decreased Th2-cell and eosinophil accumulation in the lung (Fig. 5m,n).\nTogether, these findings demonstrate that although Ly6G⁺ macrophages may be developmentally related to ncMo, they are transcriptionally and functionally distinct and uniquely require both Nr4a1 and Par2 intrinsically for their activation and accumulation after allergen exposure. The observed phenotype therefore reflects a direct requirement for Ly6G⁺ macrophages in mediating Th2 immunity to protease allergens, rather than an effect mediated by ncMo.\n• Figures 4c–d: The authors conduct mixed bone marrow chimeras (WT/se_2-/-) to assess allergen uptake by Ly6G+ macrophages in the absence of Nr4a1, showing reduced papain capture by Ly6G+ macrophages from se_2-/- origin. To strengthen these findings, it would be important to report the chimerism levels in Ly6G+ macrophage population. If Ly6G+ macrophages require Nr4a1 for differentiation, then cells of se_2-/- origin should fail to differentiate into Ly6G+ macrophages, and this underrepresentation may account for the reduced frequency of papain+ Ly6G+ macrophages observed in Figure 4c.\nWe thank the reviewer for this thoughtful comment. The degree of chimerism within the Ly6G⁺ macrophage population in WT:se_2⁻/⁻ mixed bone marrow chimeras was comparable between WT and se_2⁻/⁻ donor cells, both in the total Ly6G⁺ macrophage pool and within the allergen⁺ fraction after papain exposure. Importantly, allergen⁺ Ly6G⁺ macrophages constituted the majority (~75–80%) of the total Ly6G⁺ macrophages following papain treatment (see Extended Data Fig. 3f). Thus, the reduced frequency of se_2⁻/⁻-derived allergen⁺ Ly6G⁺ macrophages does not reflect impaired antigen uptake but rather reduced accumulation of se_2⁻/⁻ Ly6G⁺ macrophages. Consistent with this, we found that Ly6G⁺ macrophages require Nr4a1 (Nur77) for their expansion, as papain-treated WT mice showed a significant increase in Ki-67 expression within Ly6G⁺ macrophages, indicating proliferation upon allergen-driven activation (Extended Data Fig. 6d). These findings suggest that Nr4a1 controls the proliferative accumulation of Ly6G⁺ macrophages in response to protease allergens, rather than their antigen uptake capacity. This conclusion is explicitly discussed in the main text.\nFurther suggestions for improvement can be found below.\n• Across all experiments, the figure panels are very small and should be enlarged with a focus on the most important findings (some gating strategies could be moved to supplement) for better readability.\nWe thank the reviewer for this helpful suggestion. We agree that figure readability is important; however, due to space constraints, we have retained the current layout. We believe that all panels are clearly legible and that keeping the key data within the main figures allows readers to fully evaluate the primary findings without needing to consult supplementary materials.\n• All the data converge to the possibility that ncMo give rise to Ly6G+ Macs. This is particularly interesting since a previous study identified CD16.2+ ncMo in the mouse lung, also expressing Ace and Plac8, like Ly6G+ Macs. This possibility could be discussed.\nWe thank the reviewer for this thoughtful comment. We also considered this possibility and examined the relationship between Ly6G⁺ macrophages and the previously described CD16.2⁺ ncMo population expressing Ace and Plac8. To address this, we performed GSEA using the published CD16.2⁺ ncMo transcriptional signature; however, we did not detect any enrichment of this signature within our Ly6G⁺ macrophage population. In contrast, we observed strong enrichment with the canonical ncMo signature as shown in Figure 4b and 4e. As the CD16.2⁺ ncMo signature did not overlap with our Ly6G⁺ macrophage transcriptome, we prefer to refrain at this stage from directly linking these two populations.\n• Recent findings have shown that (likely other) recruited Ly6G+ macrophages can play a role in alveolar repair following influenza virus infection, and such Ly6G+ macs depend on both GM-CSF and type 2 cytokine signaling. Since cysteine proteases have previously been reported to induce GM-CSF production in non-classical monocytes (ncMo) (Kaur et al., Cell Reports, 2021), and since alternative activation via IL4/13 pops up in the transcriptomic data, it would be a nice mechanistic addition to investigate the extent to which Ly6G+ macrophages rely on these signaling pathways. Alternatively, this possibility could be discussed.\nWe thank the reviewer for this thoughtful comment. We agree that examining the contribution of GM-CSF, type 2 cytokine, or other signaling pathways to Ly6G⁺ macrophage activation is an important direction for future work. This represents a long-term interest of our group but falls beyond the scope of the present study. In our dataset, Ly6G⁺ macrophages express a broad cytokine receptor profile,\nincluding Csf1r, Csf2rb, Tgfbr1, Tgfbr2, Tgfbr3, Il10ra, Il2rg, Il4ra, Il13ra1, Il27ra, and Il31ra, suggesting potential responsiveness to M-CSF, GM-CSF, TGF-β, IL-10, IL-4/IL-13, IL-27, and IL-31 signaling pathways. This expression pattern supports the idea that Ly6G⁺ macrophages are equipped to integrate multiple regulatory and remodeling cues within the lung microenvironment. We are currently further exploring the functional relevance of these cytokine–receptor pathways, which lies beyond the scope of the present study. We have noted this point in the Discussion (lines 557–560), highlighting that Ly6G⁺ macrophages display IL-4/IL-13–associated activation and remodeling signatures that warrant further investigation.\nReviewer #3 (Remarks to the Author):\nMeloun and colleagues report, for the first time, that in an allergic model of pulmonary inflammation, Ly6G+ Macrophages produce CysLTs via PAR2, which facilitates dendritic cell migration to mediastinal lymph nodes (mLN). This study presents several novel findings, including the presence of Ly6g+ Macrophages, similar to those reported in an IAV model of lung inflammation (REF 30). The activation of Ly6G+ macrophages through Nr4a1 and PAR to produce CysLTs. And the effects of CysLTs on DCs migration to mLN to activate T-cells. The methodology includes the use of various chimeras and single-cell sequencing. The data presented support the main conclusions. I offered several observations and recommendations to further refine and expand the study.\nThe authors note in the discussion that lung Ly6g+ macrophages have previously been identified (Ref. 30). There are reported similarities between the Ly6G+ macrophages described in this study and those in Ref. 30 identified in a viral model of lung inflammation, including their origin from GMP precursors and their alveolar location. The authors may wish to consider investigating additional potential similarities, such as whether papain-induced Ly6g+ macrophages express Arg1. Are they short-lived, meaning are they only involved in sensitization? Additionally, how CysLTs enhance DC migration in response to chemokines?\nWe thank the reviewer for these insightful suggestions. We acknowledge in the Discussion that there may be similarities between the Ly6G⁺ macrophages described here and those reported in Ref. 30 (now Ref. 31 in the revised manuscript). However, when we explored potential overlaps, we did not observe enrichment of the transcriptomic signature of the previously described population in our dataset (GSEA, not shown), indicating that these cells are not transcriptionally similar. In our data, Arg1 is highly and uniquely expressed in the moDC population (cluster 3 in Extended Data Fig. 4a; cluster 0 in Fig. 4a). Although these data are not explicitly shown in the figures, they are available in the deposited GEO dataset. In contrast, Ly6G⁺ macrophages do not express Arg1 as a feature marker but instead show high and specific expression of Arg2 (new data shown in Extended Data Fig. 5d). This gene is also highly represented in our bulk RNA-seq dataset, which, although not explicitly shown in the figures, is included in the data deposited in GEO. Thus, our analyses do not reveal obvious transcriptional similarities between the Ly6G⁺ macrophages described here and those reported in Ref. 30 (now Ref. 31).\nOur results, including parabiosis experiments (Fig. 3o) and newly added EdU pulse–chase analyses (Extended Data Fig. 3d), indicate that lung Ly6G⁺ macrophages are long-lived and possess strong self-renewing and proliferative capacity (Extended Data Figs. 3c, 5g, 6d). Nonetheless, our data show that this population can be depleted by irradiation or possibly by severe tissue injury, as may occur in viral infection models, after which it is repopulated by monocytes. This may account for model-dependent differences between our findings and those reported in Ref. 30 (now Ref. 31). Overall, although we acknowledge that certain features partly resemble the previously described Ly6G⁺ macrophages, we also observe key differences—including long-term persistence, proliferative capacity, and a distinct transcriptional profile—that likely reflect either unique macrophage subsets or divergent activation states arising from specific experimental contexts. This will require further investigation, but our data indicate that the Ly6G⁺ macrophages identified here are long-lived and transcriptionally distinct from the short-lived populations described in viral infection models. Thus, while we recognize some shared features, the aim of this study was to define the identity and function of the Ly6G⁺ macrophages described here under steady-state and allergen-challenge conditions, rather than to establish equivalence with previously reported populations.\nRegarding CysLT-mediated dendritic-cell migration, our data show that CysLTs selectively enhance CCR7-dependent migration toward CCL21, but not CCL19, as demonstrated in transwell assays (Fig. 6p).\nFigure 3w: The authors state that “papain+ Ly6G+ MΦ did not accumulate in the lungs of treated Par2-/- mice compared to WT”; however, there appears to be only a 50% reduction. Furthermore, given that moDCs were also reduced in PAR2-/-, is PAR2 necessary for cMo or non-classical Mo differentiation into Ly6G+ macrophages, or for their proliferation?\nWe thank the reviewer for this important comment. We agree that it is critical to distinguish allergen-induced accumulation from potential baseline differences. As shown in our data, the presence of Ly6G⁺ macrophages in the lung is comparable between Par2⁻/⁻ and wild-type mice under steady-state conditions, indicating no difference at baseline (Extended Data Fig. 3j). However, following papain administration, Par2⁻/⁻ mice fail to accumulate Ly6G⁺ macrophages in the lung (Extended Data Fig. 3j). Likewise, allergen-bearing Ly6G⁺ macrophages are markedly reduced in Par2⁻/⁻ mice compared with wild-type controls (Fig. 3w).\nOur competitive 50% WT : 50% Par2⁻/⁻ bone marrow chimeras demonstrate that only WT-derived Ly6G⁺ macrophages accumulate after papain challenge, whereas Par2⁻/⁻-derived Ly6G⁺ macrophages do not (Fig. 3y). These findings indicate that PAR2 signaling is required in a cell-intrinsic manner for the allergen-induced accumulation of Ly6G⁺ macrophages in the lung. While moDCs are also reduced in Par2⁻/⁻ mice, the competitive chimera experiments show that this effect is not cell-intrinsic, as both WT and Par2⁻/⁻ progenitors give rise to moDCs at comparable frequencies.\nImportantly, our parabiosis experiments combined with allergen challenge show that Ly6G⁺ macrophages are almost exclusively host-derived (Fig.3o), in contrast to cMo and ncMo populations, which equilibrate between donor and host partners. This indicates that Ly6G⁺ macrophages are largely maintained locally and are not continuously replenished from circulating monocytes during allergen exposure. Thus, the loss of these cells in Par2⁻/⁻ mice reflects a failure of resident Ly6G⁺ macrophages to expand or persist in response to allergen, rather than a defect in monocyte recruitment or differentiation. Collectively, these data demonstrate that PAR2 signaling is specifically required within Ly6G⁺ macrophages for their allergen-induced accumulation.\nExtended Data 3b. Although Ly6G+ macrophages and non-classical monocytes share certain genes, non-classical monocytes were not identified in the transcriptional signature clusters.\nWe thank the reviewer for this insightful comment. We show that Ly6G⁺ macrophages and non-classical monocytes (ncMo) share transcriptional features, as both populations express a conserved core signature. However, ncMo were not detected in our allergen-induced scRNA-seq dataset because the analysis was performed on purified allergen⁺ cells, and ncMo do not take up allergen in the lung under these conditions.\nTo directly address the relationship between ncMo and Ly6G⁺ macrophages, we have now performed additional integrated scRNA-seq analyses of isolated Ly6G⁺ macrophages and ncMo. These analyses revealed that the two populations cluster separately, indicating distinct transcriptional identities (Extended Data Fig. 5a–c; see lines 314-335). While they share a core gene set (Csf1r, Cx3cr1, Nr4a1, Bcl2, Pparg, Tgfbr3, Il10ra, Klf2, Klf4, Clec4a1, Cebpb, Itga4, Itgal, Spn, Cd9, Cd36, Lair1, Treml4, Fcgr4, and Ace), Ly6G⁺ macrophages uniquely express a group of effector and inflammatory genes—including Arg2, Dhrs9, Dusp1, Hdc, Alox5, Ptgs2, Il1r2, Il1rn, and Ly6g—that distinguish them from ncMo (Extended Data Fig. 5d; see lines 314-335). Pseudotime trajectory analysis further linked ncMo to Ly6G⁺ macrophages through two developmental branches: one enriched for activation, adhesion, angiogenesis, and cytokine/chemokine pathways (Branch 1), and another enriched for metabolic and stress-response programs (Branch 2) (Extended Data Fig. 5e,f; see lines 314-335). Differentiation along Branch 1 was associated with increased representation of cells in G₂ /M phase, consistent with active proliferation (Extended Data Fig. 5g; see lines 314-335).\nMoreover, Ly6G⁺ macrophages uniquely require both Nr4a1 and Par2 in a cell-intrinsic manner. In mixed bone marrow chimeras (WT:Par2⁻/⁻, WT:se_2⁻/⁻, and Par2⁻/⁻:se_2⁻/⁻ combinations; Fig 5i), Ly6G⁺ macrophages—dependent on both Par2 and Nr4a1se_2 expression—were selectively impaired in Par2⁻/⁻:se_2⁻/⁻ chimeras after papain challenge (Fig. 5i,j), whereas ncMo and other lung populations remained unaffected (Fig. 5i,j). This selective defect was accompanied by reduced mDC migration to the mLNs (Fig. 5k), impaired OT-II expansion (Fig. 5l), and decreased Th2-cell and eosinophil accumulation in the lung (Fig. 5m,n). Thus, the defective Ly6G⁺ macrophage phenotype leads to reduced Th2 immunity, whereas ncMo remain unaffected.\nTogether, these findings demonstrate that although Ly6G⁺ macrophages may be developmentally related to ncMo, they are transcriptionally and functionally distinct and uniquely require both Nr4a1 and Par2 intrinsically for their activation and accumulation after allergen exposure. The observed phenotype therefore reflects a direct requirement for Ly6G⁺ macrophages in mediating Th2 immunity to protease allergens.\nLine 364. It would be informative to review the data on HDM, even if just as Extended Data.\nWe thank the reviewer for this suggestion. The requested data have now been incorporated as Extended Data Fig. 6e, showing that Th2 cells fail to accumulate in the lungs of se_2⁻/⁻ mice compared to WT controls following sensitization and challenge with HDM. This addition is now referenced in the main text (line 396).\nIn the BM chimera with WT and Alox5-/- BM donors, papain sensitization yields minimal lung LTC4 (∼5 ng/ml). Yet, in figure 6n (WT/A-) under the same conditions, LTC4 levels reach about 40 ng/ml. What accounts for this difference?\nWe thank the reviewer for this observation. The mean LTC₄ levels in the experiment with WT and Alox5⁻/⁻ BM donors were approximately 20 ng/ml for the WT group, whereas those in the WT/A⁻ chimeras shown in Fig. 6n averaged around 40 ng/ml. These experiments were performed independently, and the variation observed falls within the expected range of inter-experimental variability for LTC₄ quantification in lung tissue.\nFigure 6k. Ly6g+ macrophage counts are comparable in s/A-/- chimeras and controls. However, does ALOX5 deficiency alter their activation, proliferation, or recruitment?\nWe thank the reviewer for this question. As shown in Fig. 6k, Ly6G⁺ macrophage accumulation in the lungs of s/A⁻/⁻ chimeras is comparable to that of controls, indicating that ALOX5 deficiency does not affect their overall accumulation, recruitment, or expansion. Taken together, our findings suggest that ALOX5 is not required for Ly6G⁺ macrophage accumulation but rather for their effector functions, including LTC₄ production and the promotion of dendritic-cell migration.\nLine 455. The statement that the three CysLT receptors “have redundant functions” is inaccurate. CysLT1, CysLT2, and CysLT3 each preferentially bind LTD4, LTC4, and LTE4, respectively, resulting in distinct effects based on cell type and receptor expression.\nWe thank the reviewer for this accurate clarification. We have revised the sentence to reflect that CysLT1R, CysLT2R, and CysLT3R have distinct but complementary roles in mediating CysLT signaling, rather than redundant functions. The corrected text now appears in lines 496–497.\nFigure 7. Asthmatic patients are already sensitized to allergens. Does AZD9898 given during the challenge phase suppress T-cell responses, and are these results relevant to real allergens like house dust mites (HDM)?\nWe thank the reviewer for this excellent and important comment. We have now performed additional experiments to address this point. To determine whether LTC₄ S inhibition could also prevent the development of type 2 inflammation in previously sensitized mice, we administered AZD9898 at the beginning of the challenge phase rather than during sensitization (Fig. 7j and 7l). Strikingly, this later treatment produced similar effects, including reduced mDC migration and re-expansion of donor OT-II cells in the mLNs (Fig. 7j,k), and suppression of Th2 responses (Fig. 7 l,m) and eosinophilia (Fig. 7n) in the lung. These findings demonstrate that LTC₄ S inhibition remains effective when administered after sensitization, thereby modeling the therapeutic setting of allergen-experienced patients (see lines 508-515 and line 524).\nAlthough these new experiments were performed using papain as a representative cysteine protease allergen, our study directly links this mechanism to HDM-induced inflammation. As detailed in the manuscript, we used multiple HDM preparations—including heat-inactivated (HDM-HI) and cysteine-protease–inhibited (HDM-E-64) HDM—together with the purified HDM protease Der p 1 and papain, which share an identical catalytic mechanism. Across these parallel systems, inhibition or inactivation of cysteine protease activity consistently abrogated Th2-cell priming, dendritic-cell migration, and Ly6G⁺ macrophage activation. Thus, papain serves as a mechanistic model allergen that recapitulates the same PAR2-dependent, cysteine-protease–driven pathway underlying type 2 inflammation in HDM exposure. Together, these findings highlight that LTC₄S inhibition targets a conserved effector pathway relevant to both model and clinically significant allergens such as HDM.\nDoes AZD9898 affect Ly6G+ macrophage numbers or activation?\nAZD9898 treatment did not affect the number/accumulation of Ly6G⁺ macrophages in the lung.\nLine 513: The authors discuss Ruscitti et al. (ref 30) and indicate differences but do not highlight similarities in Ly6G+ macrophages, especially in relation to their location and development. Including this information would provide readers with a more comprehensive understanding.\nWe again thank the reviewer for this helpful comment regarding potential similarities with previously reported Ly6G⁺ macrophages. We agree that it is important to acknowledge potential overlap between the Ly6G⁺ macrophages described in our study and those reported by Ruscitti et al. (now Ref. 31). As discussed previously, we clarified in the Discussion that, based on currently available data, the main shared feature between these populations is the expression of Ly6G. While both populations are found near the alveoli, the Ly6G⁺ macrophages described here localize predominantly to perivascular and pericapillary regions and display a long-lived, self-renewing phenotype, whereas those reported by Ruscitti et al. appear transient arising following tissue injury. In addition, their developmental trajectories and broader transcriptional profiles are distinct. Additional studies will be required to determine whether these populations are developmentally or functionally related, or whether the observed differences reflect model-specific activation states.\nMinor\nLine 349: add “Fig. 5b”\nWe thank the reviewer for noting this. We have now added the reference to Fig. 5b at the indicated location (now in line 380) in the revised manuscript.\nLine 374: add “Fig. 5h″\nWe thank the reviewer for noting this. We have now added the reference to Fig. 5h at the indicated location (now in line 405-406) in the revised manuscript.\nREVIEWERS' COMMENTS\nReviewer #1 comments were assessed by in house editors and considered to be addressed.\nWe thank the reviewer and the in-house editors for confirming that all comments have been satisfactorily addressed.\nReviewer #2 (Remarks to the Author):\nThe authors have performed a tremendous amount of work during these revisions. In my opinion, the manuscript is now fully acceptable for publication in Nature Communications, and the authors should be commended for their impressive work.\nWe thank the reviewer for the positive assessment and greatly appreciate their supportive comments.\nReviewer #3 (Remarks to the Author):\nI would like to express my gratitude to the Authors for their thorough response to my inquiries and for expanding their dataset. I am particularly pleased to note the identification of Ly6G+ macrophages as long-lived cells and their developmental relationship with non-classical monocytes (ncMo).\nI agree with Review 2 and the authors’ decision to clarify the description of “perivascular\" macrophages, specifying their localization to the alveolar-pericapillary region. This description should be consistently incorporated throughout the manuscript, including the title, to enhance clarity and precision.\nWe thank the reviewer for this helpful suggestion. The manuscript already included the recommended clarifications specifying that these macrophages localize to the alveolar–pericapillary region. To align the title with this anatomical precision, we have updated the title accordingly. Because the micro-anatomical details are fully described in the text, this targeted modification provides the most accurate localization: pericapillary rather than the broader term perivascular, while keeping the title concise and clear.\nFigure 6. The finding that Ly6G+ macrophages produce LTC , which subsequently induces CCR7+ dendritic cells (DC) to migrate toward CCL21, represents an intriguing mechanism. Although this may extend beyond the scope of the current study, it would be interesting to investigate which receptor is specifically involved in the novel Ly6G+ macrophage-induced activation of DC.\nThank you for this thoughtful suggestion. In our study, we show that both LTC₄ and LTD₄ promote DC migration to the lymph nodes in vivo and that both equally potentiate CCR7-driven DC migration toward CCL21 in vitro. Because these mediators signal through CysLTRs and can engage both CysLTR1 and CysLTR2, it is likely that either receptor, or both, contribute to this pathway. However, given their overlapping receptor usage and the fact that our data already demonstrate functional enhancement of DC migration by both LTC₄ and LTD₄, further delineating which receptor, either one or both, is responsible would add only limited mechanistic depth within the scope of the current study. While interesting, we consider this question beyond the primary focus of our manuscript.\nRegarding Figure 3v, I appreciate the authors’ response to my comment. However, I maintain that the phrase “did not accumulate” might be an overstatement, given the comparable reduction observed in DC.\nThank you for this clarification. In our study, Ly6G⁺ macrophages increase approximately two-fold 24h after allergen exposure compared to basal levels in naïve lungs. This allergen-driven accumulation is completely absent when protease activity is removed from the allergen or when the host cannot detect protease activity. Therefore, in this specific context, we use “did not accumulate” to indicate that Ly6G⁺ macrophages fail to undergo their characteristic allergen-induced expansion under protease-deficient or protease-insensitive conditions.\nRegarding Figure 6k, while it may extend beyond the primary scope of this study, considering the potential autocrine effects of CysLTs, I am curious whether the absence of Alox5 influences the production of other molecules by these macrophages upon activation.\nThank you for this insightful comment. Our focus in this study was specifically to test the functional consequences of Ly6G⁺ macrophage–derived cysteinyl leukotrienes on dendritic cell migration and subsequent Th2 priming. We demonstrate a direct effect of these macrophage-derived CysLTs on enhancing CCR7⁺ DC migration toward CCL21. While Alox5 deficiency could theoretically alter additional activation-associated mediators, determining broader autocrine or paracrine effects falls outside the scope of this work. Our data directly address the key mechanistic link we aimed to test: the requirement for Ly6G⁺ macrophage–derived CysLTs in promoting DC migration and Th2 initiation."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have performed a tremendous amount of work during these revisions. In my opinion, the manuscript is now fully acceptable for publication in Nature Communications, and the authors should be commended for their impressive work.", "reviewer_3": "I would like to express my gratitude to the Authors for their thorough response to my inquiries and for expanding their dataset. I am particularly pleased to note the identification of Ly6G+ macrophages as long-lived cells and their developmental relationship with non-classical monocytes (ncMo).\nI agree with Review 2 and the authors’ decision to clarify the description of “perivascular\" macrophages, specifying their localization to the alveolar-pericapillary region. This description should be consistently incorporated throughout the manuscript, including the title, to enhance clarity and precision.\nFigure 6. The finding that Ly6G+ macrophages produce LTC , which subsequently induces CCR7+ dendritic cells (DC) to migrate toward CCL21, represents an intriguing mechanism. Although this may extend beyond the scope of the current study, it would be interesting to investigate which receptor is specifically involved in the novel Ly6G+ macrophage-induced activation of DC.\nRegarding Figure 3v, I appreciate the authors’ response to my comment. However, I maintain that the phrase “did not accumulate” might be an overstatement, given the comparable reduction observed in DC.\nRegarding Figure 6k, while it may extend beyond the primary scope of this study, considering the potential autocrine effects of CysLTs, I am curious whether the absence of Alox5 influences the production of other molecules by these macrophages upon activation."}, "author_response": ""}]

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