Pulmonary fibroblast activation during Aspergillus fumigatus infection enhances lung defense via immunomodulation and tissue remodeling

Domain: biology
Published: 2026-03-31
Editorial outcome: major_revision

Abstract
Aspergillus fumigatus is the etiologic agent of invasive aspergillosis, a life-threatening fungal pneumonia initiated by the inhalation of conidia into the lung. If the conidia are not cleared, they secrete large quantities of hydrolytic enzymes and toxins as they grow, causing extensive pulmonary damage. Fibroblasts are central mediators of tissue repair in many organs, but their functional response to pulmonary damage caused by A. fumigatus remains unexplored. Here, we employ cell lineage tracing, targeted cell ablation, and single-cell RNA sequencing to monitor fibroblast dynamics upon exposure to A. fumigatus in immunocompetent and immunosuppressed hosts. We find that a subset of pulmonary fibroblasts becomes activated in an immunocompetent host following a challenge with A. fumigatus conidia, initiating a gene expression program with acquired immunomodulatory properties and enhanced extracellular matrix-secreting capacity. Remarkably, targeted ablation of fibroblasts expressing the profibrotic activation marker periostin shows that invasive A. fumigatus infection accelerates in the absence of periostin lineage cells, accompanied by aberrant immune infiltration, tissue damage and severe alveolar hemorrhage. These findings uncover a protective immunomodulatory role for fibroblasts in limiting A. fumigatus-induced pulmonary injury and emphasize the importance of the pulmonary stroma in host defense against this invasive fungal infection.

Introduction
Environmental molds such as Aspergillus fumigatus commonly disseminate through the release of conidia (spores) into the air, inevitably exposing the lung to potential infectious threats1. Among these fungi, A. fumigatus stands out as one of the major invasive fungal infections (IFI) of global concern2. The outcome of the interaction between A. fumigatus and the lung hinges on the host's immune status. While healthy immunocompetent lungs can successfully eradicate the inhaled conidia before they can initiate infection3, individuals with mild immunosuppression or those with pre-existing lung structural defects are vulnerable to pulmonary colonization by the fungus. These colonizing infections are generally not invasive, but the constitutive release of fungal hydrolytic enzymes and toxins triggers a cycle of repeated tissue injury and inflammation that may evolve into a long-term condition known as chronic pulmonary aspergillosis4,5. In situations of more severe immunosuppression that may arise in transplant patients, the conidia germinate into the invasive hyphal form of the fungus, resulting in a life-threatening infection known as invasive pulmonary aspergillosis (IPA). IPA is characterized by the polarized growth of hyphae across anatomic boundaries, resulting in extensive tissue damage and dissemination to other tissues4. Infections caused by A. fumigatus are associated with a high rate of morbidity and mortality, even when treated, and the recent emergence of antifungal drug resistance6 underscores the need for more understanding of mechanisms involved in pulmonary protection.

The frontline of host defense against inhaled A. fumigatus spores involves the integrated activity of lung-resident macrophages and dendritic cells, along with recruited inflammatory cells7–9. However, little is known about the contribution of pulmonary stromal cells, defined here as support cells that lack hematopoietic, endothelial and epithelial markers, most of which are fibroblasts. Fibroblasts, which are best known for their role in providing structural support to a tissue through the synthesis and remodeling of the extracellular matrix (ECM), are increasingly recognized as a heterogeneous collection of matrix-secreting cells that can shift under pathologic conditions into distinct populations with diverse functionalities10–12. However, the dynamics of fibroblast behavior during the Aspergillus-host interaction is currently unexplored.

In this study, we genetically traced fibroblast activation using the previously identified activation marker periostin (Postn)13–16 and investigated the impact of these Postn-lineage (PostnLin) fibroblasts on the pathogenesis of IPA. The findings reveal that pulmonary inoculation of immunocompetent mice with A. fumigatus conidia induces Postn expression. These PostnLin cells were identified as members of the stromal fibroblast population by both immunohistochemistry and bulk RNA-sequencing (RNA-seq) and were localized to anatomic sites expected for pulmonary fibroblasts. Although these PostnLin fibroblasts represented a subset of the stromal cell population, their depletion in an inducible cell ablation model resulted in exaggerated immune infiltration, increased tissue damage, and accelerating mortality during IPA. Singe-cell RNA sequencing (scRNA-seq) of lung stromal cell populations demonstrated that exposure to A. fumigatus induced the transient appearance of two clusters of cells characterized by the expression of distinct matrix-preserving genes, as well as pattern-recognition receptors and proinflammatory cytokines and chemokines with well-established roles in host defense against IFI’s. Together, these findings reveal that fibroblasts become activated in the presence of A. fumigatus and undergo significant transcriptional rewiring toward an inflammatory profile that modulates the immune response, a phenomenon not previously reported in vivo in the context of a fungal challenge. This highlights a dual role for fibroblasts in pulmonary protection against A. fumigatus, involving both immunomodulation and ECM preservation.

Results
Collagen deposition is localized at sites of inflammation induced by A. fumigatus.
To assess the pulmonary response to A. fumigatus, we inoculated immunocompetent mice with a non-lethal dose of conidia (strain CEA10) via the oropharyngeal route (OP)17. As expected for immunocompetent animals18, colony-forming unit (CFU) analysis revealed sterilization of most conidia within 72 h (Fig. S1A). Histologic evaluation of lung tissue on day 1 post-inoculation revealed multiple inflammatory foci, which persisted for at least 7 days (Fig. 1A). However, the inflammatory foci were reduced in size on day 7 relative to day 3, suggesting the beginning of a resolution of the inflammatory response concomitant with fungal clearance. GMS-staining on day 1 post-inoculation demonstrated the presence of both conidia and small germlings within the inflammatory foci (Fig. 1B and S1C), which decreased by day 3 and only fungal debris was detected by days 5 and 7 (Fig. S1C). Trichrome staining revealed collagen fibers interspersed between the inflammatory cells on days 1, 3, 5, and 7 post-inoculation (Fig. 1C). Control sections from saline-treated mice are shown in Fig. S1B. We conclude that the accumulation of collagen within inflammatory foci is an initial response to a pulmonary challenge with A. fumigatus conidia in immunocompetent animals.

PostnLin fibroblasts emerge in response to A. fumigatus.
Detecting increased collagen deposition at sites of the fungus-host interaction implies that A. fumigatus stimulates fibroblasts to enhance ECM secretion. Changes in fibroblast activity in the context of tissue injury has been reported in many tissues and is associated with the induction of a gene expression program that modifies the collagen-producing activity of fibroblasts to maintain tissue integrity19. The periostin (Postn) gene, which encodes a matricellular protein involved in ECM organization, tissue remodeling, and the regulation of inflammatory cell migration13–16, is a well-established fibroblast activation marker. Most resident fibroblasts in adult tissues lack Postn expression, but they will activate the gene in response to injury16. In this study, we used a genetic cell lineage tracing approach to identify fibroblasts in which the Postn promoter has been induced in response to A. fumigatus.

To monitor Postn promoter activity, one Postn allele was replaced with a tamoxifen (TAM)-inducible Cre recombinase knock-in Postn-MerCreMer allele (PostnMCM) and crossed with a strain that permanently expresses the tdTomato fluorescent reporter allele upon Cre recombination (R26R-tdTomato) (Fig. 2A). These PostnLin-tracing mice were given an initial oral gavage of TAM on day -1 and then inoculated OP with a non-lethal dose of 3 x 107 conidia in saline on day 0 (Fig. 2B). Only sporadic tdTomato+ fluorescence was observed in lung tissue from PostnLin mice treated with either TAM alone or TAM plus an OP inoculation of saline (Fig. 2C, top two panels and Fig. S2A, top 3 panels; arrows highlight tdTomato+ cells), representing background levels. No sporadic tdTomato expression was detected in the PostnLin mice in the absence of TAM treatment (Fig. S2A, lower left panel), indicating tight control of the reporter gene without Cre recombination. By contrast, robust tdTomato expression within a subset of fibroblasts was apparent in lungs inoculated with conidia from the CEA10 strain of A. fumigatus (Fig. 2C, lower left panel and Fig. S2A, lower middle panel). Interestingly, heat-inactivation of the CEA10 conidia induced a similar level of activation to viable conidia, indicating that metabolic activity of the fungus is not required for this response (Fig. S2A, lower right panel). Comparable levels of expression were induced by two other A. fumigatus clinical isolates (Af293 and H237), demonstrating that the response is not A. fumigatus strain-specific (Fig S2B). No expression was seen in mice inoculated with A. fumigatus without TAM treatment, demonstrating robust control of the reporter even with a strong inducer of fibroblast activation (Fig 2C, lower right panel). TdTomato-expressing activated fibroblasts were detectable histologically on day 3 post-inoculation, with the number of labeled cells peaking on day 7 (Fig. 2D, quantified in 2E), reaching levels that were 100-fold higher, on average, than the sporadic activation observed following OP saline inoculation (Fig. 2F). The optimum dose range required to elicit a detectable activation response was 2-4 x 107 conidia administered as a single OP inoculation (Fig. S3). We conclude that the A. fumigatus-host interaction in the immunocompetent lung triggers the emergence of a population of fibroblasts characterized by expression of the Postn gene.

PostnLin fibroblasts induced by A. fumigatus are predominantly alveolar.
Characterization of pulmonary fibroblast heterogeneity by scRNA-seq has revealed unique molecular profiles for populations of fibroblasts that reside in distinct anatomic sites: directly beneath the epithelial lining of large and small conducting airways (peribronchial fibroblasts), in the connective tissue surrounding bronchovascular bundles (adventitial fibroblasts), or within the alveolar interstitial space between epithelial walls (alveolar fibroblasts)19. To determine where PostnLin fibroblasts localize, we quantified the number of tdTomato+ cells at each site by immunohistochemistry, confirming localization by co-staining with antibodies against epithelial (EpCAM), endothelial (CD31), and smooth muscle (Myh11) markers. The majority of the PostnLin cells arising 7 days post-inoculation with A. fumigatus conidia were found within the alveolar interstitium (Fig. 3B, arrows). A smaller proportion was sandwiched between the bronchiolar epithelium and the underlying smooth muscle (Fig. 3C, enlarged in Fig. 3D), and the fewest PostnLin cells showed an adventitial localization surrounding perivascular smooth muscle (Fig. 3E, enlarged in Fig. 3F). Quantification of TdTomato+ cells at the three major anatomic sites is shown in Fig. 3A.

PostnLin cells induced by A. fumigatus belong to the fibroblast population.
Platelet-derived growth factor α (Pdgfrα) is expressed by most tissue resident fibroblasts and is widely used as a marker for that cell type, including for those found in the lung11,20. We therefore used a Pdgfrα lineage (PdgfrαLin) tracing mouse to compare the morphology of quiescent PdgfrαLin resident pulmonary fibroblasts to that of A. fumigatus-induced PostnLin fibroblasts. Quiescent resident PdgfrαLin fibroblasts in untreated mice displayed a thin, elongated morphology and were widely distributed throughout the lung in the same alveolar, peribronchial and adventitial locations as A. fumigatus induced PostnLin cells (Fig. S4A-D). PostnLin fibroblasts that localized to peribronchial, and adventitial locations show a similar morphology, but those that were found clustered among regions of inflammation in the alveoli displayed a more hypertrophic and dendritic morphology with numerous branched extensions (Fig. S4E-H). To determine whether PostnLin cells express the Pdgfrα fibroblast marker, the PostnLin mice were crossed with a Pdgfrα-GFP mouse in which the endogenous Pdgfrα promoter constitutively drives expression of a histone H2B-eGFP fusion protein21,22. The resulting PostnLin/Pdgfrα-GFP strain was inoculated with 3 x 107 conidia and examination of lung sections on day 7 post-inoculation revealed a subset of PostnLin fibroblasts that also expressed Pdgfrα (Fig. 4A and B, green arrows). Quantification by flow cytometry showed that approximately 60% of the tdTomato+ cells co-expressed Pdgfrα (Fig. 4C), similar to what has been previously reported for PostnLin cells in the heart 11,16,22.

We also found that approximately 25% of the tdTomato+ cells co-stained with smooth muscle myosin heavy chain 11 (Myh11), a marker that is expressed by smooth muscle cells as well as a subset of alveolar fibroblasts that have differentiated to a contractile myofibroblast phenotype11,23 (Fig. 4D and E, colocalization shown by orange arrows). Most of the observed Myh11+/tdTomato+ colocalization was in alveolar regions where smooth muscle is absent, suggesting that the majority of the double-labeled cells represent contractile myofibroblasts. By contrast, tdTomato+ cells did not co-stain with markers for endothelial cells (CD31, Fig. 4F, G) or epithelial cells (EpCAM, Fig. 4H, I) by immunohistochemistry. Likewise, no co-labeling was observed with the hematopoietic marker CD45 (Fig. 4J–L), although CD45+ cells were frequently found in close proximity to tdTomato+ cells (Fig. 4L, inset). Flow cytometry further confirmed the absence of non-stromal labeling (EpCAM and CD31), showing only minimal tdTomato+/CD45+ populations (Fig. 4O and Fig. S5).

As some studies indicate that pericytes may express periostin, particularly under fibrotic conditions24 we investigated whether tdTomato+ cells co-express Neuron-Glial Antigen 2 (NG2, encoded by Cspg4), a canonical pericyte marker25. Both Flow cytometry and confocal immunohistochemistry analyses of A. fumigatus-induced PostnLin cells revealed no evidence of co-localization between tdTomato+ cells with NG2+ cells (Fig. 4M-O and Fig. S5H). Taken together, these findings indicate that PostnLin cells induced by A. fumigatus in the lung belong to the pulmonary fibroblast population and exhibit an activated state and morphology distinct from resident PdgfrαLin fibroblasts.

PostnLin cells express a unique transcriptional profile of matrix modification compared to resident pulmonary fibroblasts
To assess how the A. fumigatus-host interaction impacts gene expression in the stromal population, we performed bulk RNA-seq on A. fumigatus-induced PostnLin fibroblasts, using resident Pdgfrα-GFP fibroblasts as the comparator. Immunocompetent PostnLin mice were administered a single inoculum of A. fumigatus conidia to induce fibroblast activation and Pdgfrα-GFP mice were given a mock inoculation with saline. The induced PostnLin cells and the resident Pdgfrα-GFP fibroblasts were then isolated on day 7 post-inoculation by sorting tdTomato+ or GFP+ cells that were negative for vascular (CD31), epithelial (EpCAM), and hematopoietic (CD45) markers (see Fig. S6 for sorting strategy). Total RNA was extracted from the two populations and bulk RNA-Seq was performed to compare the transcriptional profile of A. fumigatus-induced PostnLin cells to that of Pdgfrα+ resident fibroblasts.

A total of 1037 differentially expressed genes (DEGs) were identified using a statistical cutoff of a Bonferroni-adjusted P-value ≤ 0.05 and a fold change greater than + 1.5 (Fig. 5A; Data S1). Among these DEGs, 552 were upregulated in the PostnLin population while 485 were downregulated, which are highlighted in the volcano plot shown in Fig. 5A. Genes involved in ECM organization, cell adhesion, and angiogenesis were among the top 10 enriched Gene Ontology (GO) terms in both the upregulated and downregulated categories (Fig. 5B and C; Data S2 and S3), demonstrating that the tissue remodeling activity of PostnLin fibroblasts is distinct from that of resident pulmonary fibroblasts. Downregulated genes were enriched in GO categories associated with signaling pathways, metabolism, gene expression, cell differentiation and proliferation (Fig. 5C; Data SI3) reflecting the comprehensive transcriptional rewiring that is necessary for resting fibroblasts to transition to an activated phenotype. Several of the genes that were downregulated in response to A. fumigatus represent markers of resident pulmonary fibroblasts, including Pdgfrα, Tcf21, and Scube226 (Fig. 5D), suggesting that pathogen exposure induces a shift away from the gene signature of resident fibroblasts.

Among the top upregulated DEGs were those involved in fibroblast activation and fibrosis (selected genes are highlighted in Fig. 5D). These include: Grem1 (Gremlin1), a bone morphogenic protein agonist involved in ECM regulation and fibrosis27,28; Cthrc1 (collagen triple helix repeat containing 1), a secreted ECM component implicated as the major effector of fibrotic processes in the injured lung26; Actg2 (actin gamma 2), linked to fibroblast activation in the lung29; IBSP (integrin binding sialoprotein (IBSP), identified in fibrotic lungs30; and Lrrc15 (leucine-rich repeat containing 15), associated with myofibroblast populations31–33. Additional DEGs previously reported to be associated with fibrotic fibroblast activity included Myh11, Tagln, Tpm2, Acta2, Des, Col1a1, Col1a2, Col5a1, Spp1, Ltbp2, Tnc and Igfbp226,29 (Fig. 5D). These results suggest that rewiring of fibroblast gene expression to modify the ECM is central to confronting an infectious threat from A. fumigatus, consistent with our observation of collagen accumulation within inflammatory foci in the lung induced by A. fumigatus (Fig. 1C).

Targeted ablation of the PostnLin lineage exacerbates the severity of invasive pulmonary aspergillosis.
To investigate the impact of PostnLin cells on IPA pathogenesis, we employed a conditional cell ablation model in which a TAM-inducible PostnMCM mouse was crossed to a recombinase-responsive diphtheria toxin subunit A (DTA) 34 expressing mouse line (Fig. 6A). In this approach, fibroblasts activating the Postn promoter express DTA, thereby selectively killing the cells and preventing fibroblast activity and downstream responses. IPA was induced using a modified triamcinolone-based immunosuppression protocol35. After inoculating mice with 1 x 106 conidia the host-pathogen interaction was allowed to occur for 5 hours without immunosuppression, after which a single dose of triamcinolone acetonide was administered for immunosuppression, and the progression of IPA was monitored over time (Fig 6B). Control mice received saline as a mock inoculation.

Infected DTA mice exhibited accelerated mortality relative to controls (Fig. 6C), accompanied by hemoptysis and evidence of gross pulmonary hemorrhage in dissected lungs (Fig. 6D). Histopathology revealed focal alveolar hemorrhage with extensive fungal angioinvasion (Fig. 6E). Quantitative scoring of histologic sections demonstrated higher levels of alveolar hemorrhage and angioinvasion in DTA mice compared to controls (Fig. 6F–G; Fig. S7). To determine whether a major loss in ECM could have predisposed to alveolar hemorrhage and angioinvasion, we assessed collagen deposition and organization by Picrosirius Red staining. As expected, collagen was present within the inflammatory foci, but overall content was not reduced following PostnLin ablation, suggesting that vascular rupture was not due to a gross structural loss of collagen support (Fig. 6H).

To exclude the possibility that the differences in mortality observed in the DTA/Cre model were independent of IPA, we challenged immunocompetent mice with a high dose inoculum of 3 x 107 A. fumigatus conidia. PostnMCM/+; R26-DTA and control mice were administered tamoxifen one day prior to OP inoculation with A. fumigatus conidia and were maintained on tamoxifen chow until day 14 post-infection for effective recombination36–38 (Fig. S8A). In contrast to the IPA model, no significant difference was observed between the two groups (Fig. S8B), indicating that IPA is necessary to elicit accelerated mortality in the DTA background. To determine whether fibroblast ablation impacts vascular integrity in the absence of IPA, fluorescently labeled dextrans of various sizes were administered into the lung at day 4 post-infection, and their subsequent appearance in the circulation was quantified. Fibroblast ablation had no detectable impact on the appearance of dextrans in the circulation (Fig. S8C), indicating that vascular permeability in the immunocompetent lung was unaffected by conditional ablation of PostnLin cells.

The two major drivers of tissue injury and mortality during IPA are fungal burden and immune-mediated tissue damage39,40. Fungal burden was assessed by qPCR analysis of lung tissue prior to the first observed deaths on day 4 (Fig. 6I). Flow cytometry confirmed effective fibroblast depletion, with ~30% fewer viable Pdgfrα⁺ cells in PostnMCM/+; R26-DTA lungs relative to controls (Fig. 6J). Fungal burden was equivalent between groups, ruling out pathogen overload as the cause of worsened outcomes and accelerated mortality (Fig. 6K).

In this transient immune suppression model of IPA, immune reconstitution by day 4 post-inoculation is known to contribute to pulmonary damage and mortality41. To determine whether fibroblast ablation impacts the extent of immune cell migration into lung tissue, distinct cellular subsets were quantified by flow cytometry. As anticipated, myeloid recruitment into the lung was evident in both control and DTA-ablated mice; however, DTA mice exhibited a striking increase in macrophages, along with elevated numbers of neutrophils, dendritic cells, eosinophils, and NK cells, while basophils, B and T cell numbers remained unchanged (Fig. 6L). We conclude that the loss of PostnLin cells in the context of IPA is associated with accelerated mortality and a dysregulated innate immune response to A. fumigatus, accompanied by pathological features consistent with vascular injury and fungal invasion.

Pulmonary fibroblast sub-populations with enhanced matrix stabilizing and immunomodulatory characteristics emerge following a challenge with A. fumigatus.
Although immune cell infiltration was increased following Postn⁺ cell ablation, immunological functions for fibroblasts were not enriched among the top GO terms in the bulk RNA-seq analysis. This RNAseq analysis was performed at day 7 post-inoculation, coinciding with the peak accumulation of tdTomato⁺ fibroblasts (Fig. 2D–E), and revealed a predominant gene expression signature of matrix-modifying activity. Importantly, the PostnLin population sequenced at this time point includes all cells that activated the Postn promoter during the preceding week, leading to permanent tdTomato labeling of both the initially activated fibroblasts and their progeny. Thus, the dataset reflects an averaged transcriptomic profile, encompassing newly activated fibroblasts as well as cells transitioning toward alternative functional states or returning to a quiescent phenotype. To determine whether fibroblasts with immune characteristics emerge at earlier stages of the host-fungal interaction and to delineate other potentially important subsets of fibroblasts that could be masked by averaging major populations, we used single-cell RNA-sequencing (scRNA-seq). Immunocompetent mice were exposed to either a single inoculum of conidia or a mock inoculation with saline. The stromal cell population was then isolated from total lung cells on days 1 and 7 post-inoculation by flow cytometry, excluding the non-stromal population based on expression of CD31, CD45 and EpCAM, consistent with the bulk RNA-seq analysis (Fig 7, see Fig. S6 for sorting strategy). The resulting stromal cell population was then subjected to a single-cell transcriptome analysis.

Cell typing was performed using unsupervised clustering and a reference scRNA-seq dataset of pulmonary fibroblast populations19. This approach identified 9 distinct stromal populations (Fig. 7B and Fig. S9), with clusters 1 and 2 classified as alveolar fibroblasts, cluster 3 as peribronchial fibroblasts, cluster 4 as pericytes, cluster 5 as adventitial fibroblasts, clusters 6-8 as A. fumigatus-induce clusters (highlighted by the asterisks), and cluster 9 as an indistinct population of fibroblasts with mixed characteristics. Representative genes used for cell typing are shown in Fig. 7C. As expected, Pdgfrα+ cells were distributed across multiple clusters, consistent with the experimental enrichment of a stromal fibroblast population. Scube2+ cells, representing an alveolar fibroblast phenotype, were distributed across 4 clusters (Fig. 7B: clusters 1, 2, 6 and 7 ); Hhip+/Cd9+ cells, indicative of peribronchial fibroblasts, were present in 2 clusters (Fig. 7B: clusters 3 and 8); Ly6a+/Cd9+ cells, indicating adventitial fibroblasts were located in cluster 5 (Fig.7B); and cluster 4 was marked by the pericyte marker Cox4i2 (Fig.7B). Cells in the alveolar and peribronchial fibroblast clusters in the saline-inoculated mice (Fig. 7D and E, left panel) reduced in number in response to A. fumigatus on day 1 post-infection (Fig. 7D and E, middle panel), coinciding with the appearance of an activated alveolar fibroblast cluster (ActivatedAlv) and an activated peribronchial fibroblast cluster (ActivatedPbr, purple and red clusters, respectively).

Analysis of the differentially expressed genes (DEGs) contained within the various activated fibroblasts identified unique and overlapping genes that fell into two notable categories: ECM synthesis and immune modulation (Data S4-5). In the ECM category, both activated clusters were enriched for genes involved in the formation of matrix structural fibers, encoding 9 different types of collagen, as well as elastin, fibrillin, and three secreted lysyl oxidase enzymes with roles in the formation of collagen and elastin crosslinks that stabilize the ECM42–44 (Data S4). In addition, several genes encoding integrins that represent the main cellular receptors used to interact with proteins in the ECM were shared between both clusters. In many organs, the TGFβ regulatory system plays a central role in driving de novo collagen synthesis42. This fibrogenic function of TGFβ is transduced by the cytosolic signaling protein Smad3 and is tightly regulated by latent TGF-β binding protein 1 (ltbp1), which sequesters TGFβ in the ECM. The observed enrichment of TGFβ, ltbp1, and Smad3 in the activated alveolar fibroblast cluster is therefore consistent with a predominant fibrogenic role for these cells (Data S4). This cluster was also remarkable for the presence of 16 DEGs encoding matrix-modifying proteins, indicating that its component cells were actively engaged in remodeling the ECM45. The expression of α-smooth muscle actin (Acta2) was notably upregulated in the activated peribronchial cluster, suggesting that some of these cells had differentiated to a contractile myofibroblast phenotype46.

The second group of DEGs in the activated clusters were associated with immune modulatory activity (Data S4), particularly in the activated alveolar fibroblast population. These included the cytokines IL-6, IL-10, and IL-11, as well as multiple cytokines that are members of the TNF, TGF-β, and BMP superfamilies (Data S4). The presence of TGF-β is particularly significant because of its dual role in regulating immune responses as well as fibrogenesis42. Although first-line defense against A. fumigatus relies heavily on tissue-resident alveolar macrophages and dendritic cells, additional inflammatory cells such as neutrophils, inflammatory monocytes, and monocyte-derived dendritic cells are recruited to the site of infection as needed47. The signal that brings these cells into the lung involves the secretion of chemokines47. Interestingly, both activated clusters were enriched for 11 of these chemokines, many of which have established roles in host defense against A. fumigatus and other IFIs748–54. Upon arrival in the lung, the functional activity of recruited myeloid cells is regulated by cytokines such CSF1 (macrophage colony-stimulating factor) and CSF3 (granulocyte colony stimulating factor), both of which were enriched in the activated fibroblasts clusters. Immune cell activation is also accomplished by lipid mediators such as the prostaglandins55. DEGs involved in prostaglandin synthesis were present in both activated clusters, indicating that activated fibroblasts share cytokine- and eicosanoid-mediated signaling to modulate the immune system. In addition to cytokines, these activated alveolar fibroblasts were enriched for receptors for 25 different cytokine and prostaglandin ligands, indicating that the communication between fibroblasts and the immune system is bidirectional.

Pattern recognition receptors (PRRs) are a class of receptors on cells of the innate immune system that are on the front lines of innate immune cell recognition, binding to pathogen-associated molecular patterns (PAMPs) on microbial surfaces and triggering downstream immune activity47. Both activated fibroblasts clusters were enriched for PRRs, suggesting that fibroblast activation would enhance the ability of these cells to recognize microbial pathogens (Data S4). PRRs identified in the clusters included the cell surface Toll-like receptors TLR-2 and TLR-4, the intracellular sensors NOD1 and NLRC3, and the secreted PRRs that include the galectin family of lectins and pentraxin-3 (PTX3)56–60.

By day 7 post-inoculation, the number of cells in the activated alveolar fibroblast cluster was dramatically reduced, coinciding with the appearance of another A. fumigatus-specific cluster that included DEGs encoding broad resident fibroblast markers Tcf21 and Pdgfrα, as well as the resident alveolar fibroblast markers Scube2, Npnt, Ces1d and Inmt (Fig. 7D and E, right panel, green cluster and Table 1).

This suggests that activated alveolar fibroblasts are beginning to deactivate by day 7, indicating a progressive return to a resident alveolar fibroblast state (Fig. 7E, arrows highlight the proposed trajectory). Similarly, by day 7 the activated peribronchial fibroblast population was no longer evident (Fig. 7D and E), while the resident peribronchial fibroblast cluster was reconstituting (Fig. 7E, arrows highlight the proposed trajectory). A representative set of inflammatory genes previously upregulated in the activated alveolar and activated peribronchiolar clusters was markedly downregulated in the deactivated alveolar fibroblast cluster (Fig. 7F), indicating progressive transition to a quiescent state. The return of activated fibroblast clusters to a resident phenotype is consistent with the observed histologic evidence of resolving inflammatory foci by day 7 post-inoculation with A. fumigatus (Fig. 1).

We conclude that exposure to an infectious threat with A. fumigatus triggers pulmonary stromal cells to differentiate into emergent populations of ECM-producing and immunomodulatory fibroblasts, which progressively return to a resident state as the infection resolves.

To determine whether human pulmonary fibroblasts can also adopt an inflammatory phenotype in response to A. fumigatus, we exposed isolated healthy primary fibroblasts to the fungus in vitro. To test their ability to directly sense fungal challenge, cells were incubated with one of four treatments: no conidia control, heat-killed conidia, heat-killed hyphae, or live conidia at a 1:50 conidia-to-fibroblast ratio (Fig. 7G). We first characterized fungal germination during co-culture, observing conidial swelling at 6 hours, germling emergence by 8 hours, and hyphal development at 12 hours (Fig. S10A).

Temporal gene expression analysis by qPCR revealed time-dependent activation of human fibroblasts during the host-pathogen interaction. At 6 hours, inflammatory gene expression was minimal (Fig. S10B). By 8 hours, coinciding with early germination, fibroblasts upregulated TLR4, CXCL1, and CXCL2. This response intensified at 12 hours and peaked at 24 hours, with marked induction of IL6 (Fig. 7G). Heat-killed conidia failed to trigger activation, while heat-killed hyphae induced a modest response relative to control. The strongest activation occurred with live conidia at 24 hours, highlighting the importance of fungal viability and morphological recognition in driving the immune response of human fibroblasts to A. fumigatus.

Together, these results demonstrate that human pulmonary fibroblasts can mount a dynamic inflammatory response to A. fumigatus, producing cytokines and chemokines capable of modulating immune cell recruitment and activation.

Methods
Animal procedures
All mouse studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council. The animal use protocol (20-09-09-02) was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Cincinnati.

Adult mice (12-16 weeks of age) were sex- and age-matched within experiments, cohoused, and subjected to identical treatment and infection protocols. No sex-specific differences were detected. A stock solution of Tamoxifen at 20 mg/mL (Chemodex, T0200) was prepared in a solution of 10% absolute ethanol and 90% olive oil. A dose of 5 mg per 30 g body weight was administered via gavage one day prior to A. fumigatus inoculation. TAM citrate-infused chow (400 mg/Kg; Inotiv TD130860) was subsequently provided to the mice for the duration of the experiment. For inoculation of conidia, 0.04 mL of a saline suspension containing 3 x 107 A. fumigatus conidia (unless otherwise stated) was administered by the oropharyngeal route, as previously described77. Control mice were inoculated with saline. To induce invasive aspergillosis, mice were immunosuppressed by subcutaneous injection of a single dose of triamcinolone acetonide (40 mg/kg body weight)78. Survival was monitored for 14 days until mice met pre-specified IACUC-approved early removal criteria.

Mouse lines
Rosa26-tdTomato (B6.Cg-Gt(rosa)26Sortm14(CAG-tdTomato)Hze/J; JAX Strain #:007914), Rosa26-DTA (B6.129S6(Cg)-Gt(ROSA))26Sortm1(DTAJpmb/J; JAX Strain #:032087), Pdgfrα-CreERT2 (B6.129S-Pdgfratm1.1(cre/ERT2)Blh/J; JAX Strain #:032770), and Pdgfrα-eGFP (B6.129S4-Pdgfratm11(EGFP)Sor/J; JAX Strain #:007669) mice were purchased from The Jackson Laboratory. PostnMerCreMer (PostnMCM) (B6.129S-Postntm2.1(cre/Esr1*)Jmol/J; JAX Strain #:029645) mice were generated as previously described16. PostnMCM and Pdgfrα-Cre mice were crossed with a Rosa26-tdTomato mouse line to enable reporter expression upon TAM-induced Cre recombination in Postn- or Pdgfra-expressing cells. Similarly, PostnMCM mice were crossed with Rosa26-DTA mice to induce specific ablation of Postn-expressing cells in the presence of TAM. Heterozygous PostnMCM and Pdgfrα-Cre mice were utilized for experiments to retain expression of the native protein, whereas homozygous Rosa26-tdTomato and Rosa26-DTA mice were used to enhance recombination efficiency. Both lineage-tracing (PostnMCM/+; R26-tdTomato) and Cre-negative (R26-DTA) littermate mice were used as controls throughout the study. For fungal dose finding studies, we used C57BL/6J mice in addition to our genetic models. All strains were backcrossed for more than 10 generations onto the C57BL/6J background or purchased from JAX as already backcrossed.

Fungal strains and growth conditions
The A. fumigatus clinical isolate CEA1074,75 was used throughout the study, while the clinical isolates Af293 and H23776 were used where indicated. Conidia were harvested before each experiment from mycelia grown for 3 days at 37⁰C on solid Aspergillus minimal medium (AMM) (1% [wt/vol] D-glucose, 1% [vol/vol] NH4 tartrate, 2% [vol/vol] salt solution [2.6% {wt/vol} KCl, 2.6% {wt/vol} MgSO4 heptahydrate, 7.6% {wt/vol} KH2PO4, and 5% {vol/vol} trace element solution]), then washed and resuspended in saline. When required, conidia or hyphae were inactivated by heat shock at 65⁰C for 30 min. The effectiveness of heat inactivation was confirmed by plating onto the center of a plate of YG medium (0.5 yeast extract, 2% glucose) plates and confirming no surviving CFUs after 4 days of incubation at 37⁰C. Mice were inoculated as described in Animal Procedures. Colony formation units (CFUs) were quantified by spreading 100 μL of serially diluted lung homogenates onto Becton Dickinson (BD) inhibitory mold agar (IMA). The plates were incubated for 16-24 h at 37⁰C. At least 3 biological replicates were analyzed per condition.

Histology and immunohistochemistry
Harvested lungs were inflated with 4% paraformaldehyde (PFA) through the trachea using gravitational flow, fixed in PFA for 1 h at room temperature, and immersed in 30% sucrose (w/v) overnight at 4°C. The lungs were then embedded in OCT (Fisher, 23730571) and stored at -80°C prior to cryosectioning. For immunohistochemistry, cryosections of 10 μm were soaked in blocking solution (PBS containing 0.1% TritonX-100, 1.5% goat serum, and 1% bovine serum albumin) for 30 min, followed by overnight incubation at 4⁰C with the primary antibody diluted 1:100 in blocking solution. The primary antibodies used in this study were Myh11(Abcam, AB224804), EpCAM/CD326 (ThermoFisher, 14579185), CD31 (BD Biosciences, 553370), CD45 (BD Biosciences, 550539) and NG2 (Abcam, AB5320). After incubation, sections were washed with PBS and incubated in blocking solution containing a 1:500 dilution of the secondary antibody for 1h at room temperature. The secondary antibodies used were Alexa Fluor 488-conjugated goat anti-rat (ThermoFisher, A11006) or anti-rabbit (ThermoFisher, A11008), and Alexa Fluor 647 goat anti-rat (ThermoFisher, A21247) or anti-rabbit (ThermoFisher, A21244). Sections were then washed, stained with DAPI (Sigma, D9542) for 5 min to label nuclei, and mounted on slides in mounting media (Vector Laboratories, H1700). Epifluorescence images were captured using an Olympus BX43 microscope equipped with an Olympus DP80 camera and connected to an X-Cite 120 fluorescence lamp illuminator. Confocal images were taken with a Leica Stellaris 8 Confocal Microscope. Brightness and contrast adjustments were made using FIJI for epifluorescence images or LAS X software for confocal images. Quantification studies were performed by counting the indicated number of cells in a full longitudinal section of the right lung lobe. At least three biological replicates were quantified per experiment, using two sections separated by 200 µm.

For histopathology, lungs were inflated with PFA, fixed, processed, sectioned, and imaged as described elsewhere79. The sections were stained with Gomori’s methenamine silver (GMS), hematoxylin and eosin (HE), Masson’s trichrome, or Picrosirius red. Alveolar hemorrhage was assessed using a modification of a semiquantitative histologic scoring system80. Each animal was scored in a blinded manner by two independent investigators, with no differences observed in scoring ratios between them. Angioinvasion was quantified by counting the number of large intrapulmonary arteries (≥100 µm)81,82 in GMS-stained sections of the left lung lobe and calculating the percentage of vessels containing fungal elements. Alveolar hemorrhage and angioinvasion were assessed in at least eight biological replicates, using two sections per replicate spaced 200 µm apart. Collagen deposition in the left lung lobe was evaluated via picrosirius red staining. Multiple pictures were taken at 100-fold of magnification and quantified by ImageJ v1.54q (National Institute of Health, USA). Briefly, measure the total area of sirius red positive area by color deconvolution and normalized to the total tissue area

FACS analysis and sorting
Harvested lungs were rinsed in cold PBS and placed in a C-tube (Miltenyi Biotec, 130093237) containing 5 mL of digestion buffer consisting of Dulbecco’s PBS with 0.9mM CaCl2, 600 U/mL Collagenase IV (Worthington Biochemistry, LS004189), 1.2 U/mL Dispase II (Gibco, 17105041) and 30 U/mL DNase I (Sigma, D4527-10KU). The samples were incubated in a water bath at 37°C for 35 min and processed using a MACS™ Dissociator (Miltenyi Biotec, 130-093-235). The homogenate was filtered through a 40 µm cell strainer. Blood cells were eliminated by incubating the samples with Red Cell Lysis Buffer (eBiosciences, 00430054) following the manufacturer’s instructions. The remaining cells were resuspended in FACS buffer (Dulbecco’s PBS with 2x Fetal Bovine Serum) and stained with the following antibodies conjugated with APC (1:100 dilution) for 30 min at room temperature: CD45 (BioLegend, 103112), CD31 (BioLegend, 102409) and EpCAM/CD326 (BioLegend, 118214). Pericytes were labeled with Alexa Fluor 647 anti-NG2 antibody (Abcam, AB283639; 1:100 dilution). Cells were washed with FACS buffer, followed by staining with the viability dye Sytox Blue (Invitrogen, S34857) or Sytox Green (Invitrogen, S34860) at a 1:500 dilution. Flow cytometry analysis was conducted using a BD LSRFortessa and sorting performed on a BD FACSAria II SFusion. Both instruments operated using the FACSDiva software. Details of the gating strategies can be found in Fig. S4 and S6.

For immune characterization, samples were washed three times in PBS before applying fixable Live/Dead Aqua stain (ThermoFisher, L34965, 1μL/mL) at room temperature for 20 min protected from light. Samples were then washed three times in fluorescence-activated cell sorting (FACS) buffer containing DPBS (Gibco, 14190-136) with 0.09% Sodium Azide and 1% FBS. Cells were stained for CD326 RB780 (BD Biosciences, 568737, 1:400), CD31 BV711 (BioLegend, 102449, 1:300), CD140a PE-Cy7 (ThermoFisher, 25-1401-82, 1:200), CD45 AF532 (ThermoFisher, 58-0451-80, 1:1000), F4/80 eFlour 450 (ThermoFisher, 48-4801-82, 1:200), CD11b BUV661 (BD Biosciences, 612977, 1:400), Ly6G PE-Fire 810 (BioLegend, 127673, 1:200), CD170 eFlour 660 (ThermoFisher, 50-1702-82, 1:200), FceR1a APC-eFlour 780 (ThermoFisher, 47-5898-82, 1:200), I-A/I-E PerCP (BioLegend, 107624, 1:2000), CD3e RY586 (BD Biosciences, 568158, 1:800), CD45R BV570 (BioLegend, 103237, 1:3000), CD19 BUV737 (BD Biosciences, 612782, 1:400), and NK1.1 BB700 (BD Biosciences, 566502, 1:200). After incubating for 30 min at 4°C protected from light, cells were washed and spectral flow cytometry analysis was performed using a Cytek Bioscience Aurora Spectral Analyzer with the following five laser configurations: 355, 405, 488, 561, 640 nm, and 64 detection channels. The spectral signature of unstained, live/dead aqua stained cells, and each fluorophore was calibrated for spectral unmixing with autofluorescence extraction using single color-stained compensation beads (Invitrogen, 01-3333-42), and unstained cells for autofluorescence. Single color live/dead controls were made by heat shocking lung cell suspension at 65°C for 5min, once cooled, dead cells mixed with a live cell suspension on ice at a 1:1 ratio. Samples were gated for debris, doublet, and dead discrimination before analysis of target populations. Flow cytometry data was generated using FlowJo v.10.10 software (BD Biosciences).

Fungal burden quantification
To quantify fungal burden, fungal DNA from mouse lung lobes was isolated using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, D6005) according to the manufacturer’s instructions. DNA concentration was determined by TaqMan PCR targeting the fungal 18S ribosomal DNA using previously reported sequences 83: forward primer, 5′-GGCCCTTAAATAGCCCGGT-3′; reverse primer, 5′-TGAGCCGATAGTCCCCCTAA-3′; and TaqMan probe, 5′-6-FAM™-AGCCAGCGGCCCGCAAATG-TAMRA™-3′. Primers and probe were synthesized by Integrated DNA Technologies (IDT). Serial 10-fold dilutions of known concentrations of fungal DNA were prepared in parallel to generate a standard curve. Each reaction consisted of 5 µL of DNA template and 15 µL of PrimeTime Gene Expression Master Mix (IDT, 1055770) containing 250 nM probe and 500 nM of each primer, for a total volume of 20 µL. All reactions were performed in triplicate on a QuantStudio™ 3 System with the following cycling conditions: 95 °C for 3 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. The resulting Ct values were used to calculate fungal genomic equivalents (GE) based on the standard curve. Total GE in each DNA extract was then normalized to lung lobe weight and expressed as GE/mg tissue.

Vascular permeability assay
To evaluate vascular leakage, mice were administered a mixture of fluorescently labeled dextrans (3 kDa, 10 kDa, 40 kDa, and 70 kDa; Thermo Fisher Scientific) via oropharyngeal instillation. Each dextran was dissolved in PBS at 2 mg/mL. Following instillation, mice were maintained for 1h to allow systemic distribution. Blood was collected by cardiac puncture, and plasma was isolated by centrifugation. Plasma samples and 10-fold serial dilutions of the dextran cocktail (0.015–150µg/mL) prepared in naïve mouse serum were loaded into black 96-well plates. Fluorescence was measured with a multimode plate reader using the following excitation/emission settings: 3kDa, 400/420nm; 10kDa, 650/670nm; 40kDa, 490/520nm; and 70kDa, 590/620nm. Blank serum from untreated mice was included to correct for autofluorescence. Dextran concentrations in plasma were interpolated from standard curves using linear regression.

Gene expression analysis of inflammatory mediators in pulmonary fibroblasts
A primary human pulmonary fibroblast line (PromoCell, C12360) was cultured in Fibroblast Growth Medium 2 (PromoCell, C23020) according to the manufacturer’s instructions. Cells were passaged 5–6 times and treated with Aspergillus fumigatus once they reached about 70% confluency. Cells were inoculated at an MOI of 10 (10 conidia per cell), except when analyzing the effect of active hyphal growth, where a lower MOI of 0.02 (1 conidium per 50 cells) was used to maintain monolayer integrity over 24h. The RNA was isolated using a NucleoSpin RNA mini kit (Macherey-Nagel, 740955.250) according to the manufacturer’s instructions, and treated with DNase I (Milipore Sigma, D7291). cDNA was synthesized using the iScript Reverse Transcription Supermix (Bio-Rad, 1708841). Gene expression was quantified with iTaq Universal SYBR Green Supermix (Bio-Rad, 1725124) on a QuantStudio™ 3 System using the following primers (5′–3′):: GAPDH-F, GAAGGTGAAGGTCGGAGT; GAPDH-R, GAAGATGGTGATGGGATTTC; IL-6-F, GGTACATCCTCGACGGCATCT; IL-6-R, GTGCCTCTTTGCTGCTTTCAC; CXCL2-F, GCTTGTCTCAACCCCGCATC; CXCL2-R, TGGATTTGCCATTTTTCAGCATCTT; CXCL1-F, GCGCCCAAACCGAAGTCATA; CXCL1-R, ATGGGGGATGCAGGATTGAG; TLR4-F, CAGAGTTTCCTGCAATGGATCA; TLR4-R, TGCTTATCTGAAGGTGTTGCACAT. All reactions were performed at least three times with the following cycling conditions: 95°C for 20s, followed by 40 cycles of 95°C for 3s and 60°C for 30s. Gene expression was normalized to the GAPDH expression and presented as fold-change in transcript levels relative to non–A. fumigatus-treated controls.

RNA sequencing and analysis
For bulk RNAseq analysis, PostnLin or Pdgfrα-GFP cells were sorted, and the RNA isolated using an RNeasy Plus Micro Kit (Qiagen, 74034) according to the manufacturer’s instructions. RNA integrity was confirmed using the RNA 6000 Pico Kit on an Agilent 2100 Bioanalyzer before sequencing. The cDNA was amplified with the Ovation RNA-Seq System v2 (Tecan Genomics), and libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina). Sequencing was performed on a NovaSeq 6000 SP v1.5 (200 cycles), yielding 100 bp paired-end reads with a sequencing depth of 30 million reads per sample. Fastq files from each sample were merged, and quality control of the sequencing reads was conducted. Adapters sequences were trimmed before further analysis. Gene expression analysis was carried out using the Mus musculus reference genome GRCm39 (mm39) in CLC Genomics Workbench (Qiagen). Differential expression was assessed between PostnLin cells treated with A. fumigatus and Pdgfrα-GFP cells treated with saline with a statistical cutoff of a Bonferroni-adjusted P-value ≤ 0.05. Gene ontology (GO) enrichment analysis was performed using the NIH DAVID Bioinformatics Functional Annotation Tool (https://david.ncifcrf.gov) applying a statistical threshold of FDR P-value  0.01 and  5 total genes per GO term. GO enrichment and volcano plots were created using RStudio (version 2024.04.2+764) on macOS.

For single-cell RNAseq analysis, sorted pulmonary stromal cells (negative for CD45, CD31 and EpCAM) were collected in a tube containing DMEM (Corning, 10013CV) with 10% FBS at a concentration of 100 cells/μL. The single-cell RNA-Seq assay was performed according to the manufacturer’s instructions (Chromium Next GSEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index), 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate a gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every GEM was reverse transcribed into cDNA, adding an Illumina TruSeq R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The cell barcoded molecules were then cleaned up with Silane DynaBeads and amplified using 14 PCR cycles. Next, full-length, barcoded cDNA was then enzymatically fragmented, sized-selected, adapter-ligated, and amplified for library construction. During the library construction, P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 were added. Samples were pooled and run on the NovaSeq 6000 sequencer with a S4 flow cell using the following sequencing parameters: R1: 28 cycles, i7: 10 cycles, i5: 10 cycles, R2: 90 cycles.

The 10x scRNA-seq data were preprocessed using Cell Ranger software (7.0.0). We used the ‘mkfastq’, ‘count’ and ‘aggr’ commands to process the 10x scRNA-seq output into one cell by gene expression count matrix using default parameters. scRNA-seq data analysis was performed with the Scanpy (1.9.0) package in Python (https://genomebiology.biomedcentral.com/articles/10.1186/s13059-017-1382-0). Genes expressed in fewer than three cells were removed from further analysis. Cells expressing less than 100 and more than 7,000 genes were also removed from further analysis. In addition, cells with a high (≥ 10%) mitochondrial genome transcript ratio were removed. For downstream analysis, we used count per million (CPM) normalization to control for library size differences in cells and transformed those into log (CPM + 1) values. After normalization, the data were then z-score normalized for each gene across all cells. We then used the following commands, tl.pca’, ‘pp.neighbors’ and ‘tl.leiden, in Scanpy to partition the single cells into clusters. Differential expression analysis was done using the Wilcoxon rank sum test at the single cell level.

Statistics & Reproducibility.
All experiments were repeated at least three times with similar results. Independent biological replicates are specified in the corresponding figure legends. No statistical method was used to predetermine sample size. Sample sizes were selected based on prior experience with the experimental models employed and on standards commonly used in the field to ensure reproducibility and feasibility. Outliers in gene expression and flow cytometry analyses were excluded based on variability. Repeated experiments confirmed the reproducibility and reliability of the data. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Statistical data analysis was performed using Microsoft Excel 365 and GraphPad Prism v10.3.1.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors investigate the role of pulmonary fibroblasts in response to Aspergillus fumigatus infection, demonstrating that fibroblast activation contributes to lung defense via immunomodulatory and extracellular matrix (ECM) remodeling functions. The authors employ novel cell lineage tracing, targeted cell ablation, single-cell RNA sequencing (scRNA-seq), and histological analysis to delineate the response of fibroblasts to fungal infection. Their findings highlight that periostin-expressing (PostnLin) fibroblasts become activated in response to A. fumigatus and contribute to infection resolution. The absence of these cells exacerbates disease progression, indicating a protective role of fibroblasts in host defense. The manuscript is very well written, the results are original, clearly communicated, reproducible, and support the conclusions of the authors. However, the significance of the findings is not clear, and the results are generally expected and unsurprising.\nMajor issues:\n1) The study is very descriptive, and not mechanistic. The finding that DTA-deletion of Postn+ fibroblasts in aspergillosis increases disease is a significant one. However, a potential mechanism is only discussed (lines 401-403), not investigated. The rest of the study characterizes fibroblast populations in the lung, which is important, but does not increase the potential impact of the study.\n2) There is no supporting data in human fibroblasts.\n3) It is unclear why bulk sequencing was done, followed by scRNAseq, when only single cell was necessary. Bulk sequencing might have some issues, especially since there appeared to be multiple variables in the cell populations that were sorted (based on both Pdgfra+/- and infection/non-infection). Also, spatial sequencing would be very appropriate for this project.\nMinor issues:\n1) It is confusing why PostnLin cells are concluded to be distinct from resident PdgfraLin fibroblasts when 60% of PostLin cells are Pdgfra+ (lines 200-202).\n2) Why was the triaminiclone model of immune suppression used? Why was it necessary to infect 5 hours prior to treatment? More information would be useful for the reader.\n3) Bulk sequencing results should probably be supplemental, as scRNAseq results are more informative.\n4) Lines 526-527. \"At least three biological replicates were quantified per replicate\" is confusing.\n5) Figure 5. Why is TAM administered one day before fungal inoculation? Is it possible that the damage from DTA-mediated killing is more important than the loss of PostnLin cells? What if TAM was administered several days before infection and mice were allowed recover DTA-mediated damage?", "reviewer_2": "In this manuscript entitled “Pulmonary fibroblast activation during Aspergillus fumigatus infection enhances lung defense via immunomodulation and tissue remodeling”, Guirao-Abad and colleagues investigated the role of fibroblasts during fungal pneumonia. The authors first showed that the lungs became fibrotic after A. fumigatus inoculation. The authors used Postn-MerCreMer mice to show Postn+ fibroblasts markedly increased after the infection. The authors showed that those de novo Postn+ cells were mostly fibroblasts in the alveolar regions, and the ablation of Postn+ cells caused increased mortality after A. fumigatus inoculation in immunosuppressed mice. By bulk RNA-seq and single cell RNA sequencing, the authors showed that alveolar fibroblasts underwent pro-fibrotic and pro-inflammatory changes after A. fumigatus inoculation. Although this manuscript demonstrated the protective function of Postn+ fibroblasts in fungal pneumonia, the authors do not adequately explore the mechanism(s) by which loss of periostin lineage cells increases mortality in immunosuppressed mice inoculated with A. fumigatus. Although the authors describe areas of hemorrhage and angio-invasion after deletion of periostin+ cells, the manuscript currently lacks quantitative data regarding the observed phenotypes and detailed information on the method to evaluate the results. There are also no experimental data explaining how the deleted cells might prevent these adverse events.\nSince periostin+ cells emerge after most of the organisms were cleared in wild type mice it is not clear how/if these cells would have contributed to immune cell recruitment. At the very least it would be important to perform quantitative immunostaining and/or flow cytometry to determine whether loss of periostin+ cells modulated the recruitment and/or activation of adaptive and innate immune cells. It would also have been useful to see whether differences in mortality were due to effects on vascular permeability.\nAdditionally, the possibility of Postn-CreER targeting pericytes rather than fibroblasts should be experimentally excluded to prove the authors’ claims.\nMajor concerns\n1. The genetic background of the mice used for the experiments should be described in detail. The Postn-MerCreMer mouse distributed in the Jackson Laboratory has been only backcrossed 5 times. How many times did the authors backcross the Postn-MerCreMer mouse? In Figure 5, what mice did the authors use for control? This information is critical since the different genetic backgrounds can show different sensitivity to infection. Additionally, did the control group in Figure 5 also receive tamoxifen treatment? Tamoxifen itself can cause various effects on the lungs. Tamoxifen food can also cause body weight loss sometimes since mice are reluctant to eat it. If the control groups in Figure 5 were not treated by tamoxifen, how do the authors exclude the possibility that the phenotype is caused by tamoxifen?\n2. The authors stated that the Postn+ cell ablation resulted in pulmonary hemorrhage and fungal angioinvasion in Figure 5. These phenotypes should be quantified and statistically evaluated.\n3. As the authors stated in the discussion, publicly available scRNA-seq data show Postn expression in pericytes at baseline. What portion of Postn-lineage cells are pericytes? Authors should be able to quantify this by Mcam (CD146) staining on flow cytometry. Although authors showed minimal tdTomato+ cells without infection by histology, this should be confirmed by flow cytometry of digested lungs for a more unbiased assessment of whole lung distribution. If there are minimal tdTomato+ cells without infection, can the authors explain why Postn-MerCreMer specifically labels fibroblasts but not pericytes, which highly express Postn at baseline?\n4. Since Postn is also highly expressed in pericytes, the authors should experimentally exclude the possibility of pericytes contributing to the phenotypes in Figure 5. The authors should be able to do this by treating the mice with fungus after treating and washing out tamoxifen or using Rosa26-DTR mice.\n5. Figure 7E is difficult to understand. Figure 1A shows that there are normal alveoli left on day 1. However, Figure 7E seems to show that all alveolar fibroblasts homogeneously become activated alveolar fibroblasts. Similar results are shown for peribronchiolar fibroblasts. If the clusters are completely driven by treatment and time points, the authors might want to try integration methods such as Harmony or Mutual Nearest Neighbors (MNN), which may allow them to identify newly emerged populations while keeping some normal populations.\n6. In Figure 7E, the authors showed only inflammatory genes for the alveolar fibroblast lineage. Differentially expressed genes for each cluster should be shown by heatmap or dot plots for readers to know the specific genes of each cluster.\n7. The authors need to include data demonstrating the efficiency of the deletion method used to delete periostin-lineage cells. It would also be good to overlay periostin expression on the uMAP plots and to have included the lineage label in the single cell sequencing experiments so the authors could determine which of the emergent stromal populations they describe were derived from periostin-expressing cells and thus which populations were likely to have been deleted.\nMinor comments\n8. It would be nice to quantify fibrosis after fungus infection by hydroxyproline assay\n9. Figure 7D, colors for activated alv and deactivated alv were swapped.\n10. Figure 4 would benefit from addition of staining for specific pericyte markers.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "This study combines robust in-host assays with newly generated reporter animal lines and scRNAseq to uncover a previously unappreciated role for pulmonary fibroblasts in limiting the severity of Aspergillus-induced pulmonary injury. The authors provide strong evidence that collagen is deposited at sites of Aspergillus-induced inflammation and that periostin-positive fibroblasts, localized primarily in the alveoli, are the major contributors. Use of a novel mouse line that can be specifically depleted for this cell type, causes exacerbation of the severity of invasive aspergillosis. In-depth bulk and scRNAseq analyses revealed that infection with A. fumigatus triggers pulmonary stromal cells to differentiate into an emergent population of fibroblasts with ECM-producing and immunomodulatory roles. Importantly, the authors also provide data showing that these activated fibroblasts return to their resident state as infection resolves.\nMajor strengths:\n• This manuscript is very well written, and the findings will have broad impact.\n• The study uncovers a novel cellular response to A. fumigatus infection that promotes appropriate host response to limit tissue damage.\n• The study demonstrates that even heat-killed conidia, that are coated in a protective barrier of hydrophobic proteins, can elicit a fibroblast response. This is critical as the hydrophobin layer has always been considered as rendering resting conidia immunologically inert, leaving the initial signals driving host response vague.\n• The study confirms that activation of fibroblast subsets also occurs in response to two additional isolates of A. fumigatus.\nPoints to address:\n1. Do the authors have any data suggesting that fungal burden is (or is not) impacted by loss of the postn lineage fibroblasts during invasive disease (Figure 5)? Maybe this is just a point for the Discussion, but is it possible that after invasive growth begins, these fibroblasts play a role in limiting fungal growth? Or, is the increased mortality of the DTA mice simply due to heightened levels of tissue damage in response to the same amount of fungal growth?\n2. In the same line of thought, the immune competent DTA mice did not experience death after infection (Line 223), but did they have prolonged time to clearance/sterilization? Even negative data may be very informative here and could be included as a Supplemental Figure, if available."}, "author_response": "Response to Reviewers for NCOMMS-25-02679\nWe are grateful to the reviewers for their thoughtful and constructive evaluations of our manuscript. We have addressed all comments, performed additional experiments, and added more than 12 new figures to strengthen the study. To improve the clarity, we have repositioned the section titled (“PostnLin cells express a unique transcriptional profile of matrix modification compared to resident pulmonary fibroblasts”) earlier in the Results.\n\nA central theme across the reviews was the mechanism by which fibroblast ablation increases mortality during A. fumigatus infection. To address this, we quantified fungal burden, immune cell recruitment, alveolar hemorrhage, and vascular invasion in both immunosuppressed and immunocompetent mice. Our results in this revised manuscript demonstrate that fungal burden remains unchanged following PostnLin ablation, whereas myeloid cell infiltration, alveolar hemorrhage, and angioinvasion are significantly increased. These findings establish that PostnLin fibroblasts protect the host by modulating inflammation and preserving vascular integrity, rather than by directly limiting fungal growth. We believe these new results directly resolve the major mechanistic concerns raised by Reviewers #1, #2, and #4.\n\nReviewer #1\n…However, the significance of the findings is not clear, and the results are generally expected and unsurprising.\nWe appreciate this perspective and have revised the text (lines 92–97, 559-565) to highlight the novelty of our findings. While fibroblasts are expected to contribute to ECM remodeling, our new data reveal that their response extends beyond matrix deposition to include a profound immunomodulatory role. For the first time, we demonstrate that ablation of activated fibroblasts accelerates mortality in IPA, with new data showing this is accompanied by exacerbated inflammation rather than increased fungal proliferation. We have revised and expanded the data presented in Fig. 6; Fig.7; Fig. S7;Fig. S8).\n\n1) The study is very descriptive, and not mechanistic. The finding that DTA-deletion of Postn+ fibroblasts in aspergillosis increases disease is a significant one. However, a potential mechanism is only discussed (lines 401-403), not investigated. The rest of the study characterizes fibroblast populations in the lung, which is important, but does not increase the potential impact of the study.\nWe performed additional experiments to provide mechanistic insights into how fibroblast ablation influences the two major determinants of disease outcome in IPA: fungal burden and the associated inflammatory response. Our results demonstrate that ablation of activated fibroblasts following A. fumigatus exposure is associated with an exacerbated inflammatory response, with no detectable alteration in fungal burden. New experiments quantifying fungal burden and immune cell recruitment have been included. Revised results are included in the manuscript at lines 268–307, 470–480; and figures Fig. 6F–L, Fig. S7).\n\n2) There is no supporting data in human fibroblasts.\nWe thank the reviewer for this comment. To address this point, we have now included new data demonstrating that primary human pulmonary fibroblasts can directly sense and respond to A. fumigatus exposure. Specifically, we performed in vitro stimulation experiments using isolated primary fibroblasts and assessed their ability to detect and respond to fungal challenge. The results revealed a strong immunomodulatory response within 24 hours, particularly in response to actively growing hyphae, whereas heat-killed conidia induced only modest activation. These findings provide the first evidence that human pulmonary fibroblasts can directly respond to A. fumigatus.\nAccordingly, we have added new Figure 7G and Figure S10 (described in lines 422–438) and expanded the Discussion section (lines 532–555) to include recent studies identifying POSTN fibroblast subsets and inflammatory fibroblast states in human lung disease, including COVID-19 and idiopathic pulmonary fibrosis. While in vivo validation through human IPA samples remains a future direction, these new data substantially strengthen the translational relevance of our findings.\n\n3) It is unclear why bulk sequencing was done, followed by scRNAseq, when only single cell was necessary. Bulk sequencing might have some issues, especially since there appeared to be multiple variables in the cell populations that were sorted (based on both Pdgfrα+/- and infection/non-infection).\nWe apologize for the lack of a clear rationale for doing both bulk RNAseq and scRNA-seq. Our bulk RNA-seq analysis specifically focused on PostnLin cells, which represent those cells that had activated the Postn promoter during the initial 1-week window, rendering that cell and its progeny permanently tagged with tdTomato. This allows us to characterize their distinct pro-fibrotic profile in response to fungal challenges. In parallel, we performed scRNA-seq at early post-challenge stages (Day 1), revealing an immunomodulatory profile that is distinct from the one observed in PostnLin cells. It is important to indicate that an isolated Postn+ expression fibroblast cluster was not detected by scRNA-seq either at Day 1 or Day 7 and bulk RNA-seq proved to be the most effective approach for analysing this specific population. This can be explained by the fact that fibrotic clusters are predominantly found in conditions that induce acute fibrosis, such as the bleomycin-induced IPF model, whereas the moderate response observed with A. fumigatus results in transient Postn expression distributed across all clusters.\nTo improve the clarity, we have repositioned the section titled (“PostnLin cells express a unique transcriptional profile of matrix modification compared to resident pulmonary fibroblasts”) earlier in the Results. The text has been revised to clarify these aspects (lines 311-324 and lines 516–531).\n\n4)…. spatial sequencing would be very appropriate for this project.\nWe appreciate this thoughtful suggestion and agree that spatial RNA sequencing would provide valuable insight into the spatial organization and stage-specific responses of fibroblast subsets during A. fumigatus infection. We have already initiated plans to incorporate these approaches; however, we encountered a resolution limitation in generating the necessary data. Because of the close proximity of myeloid cells and fibroblasts observed in Fig. 4L, we require single-cell–level spatial sequencing to distinguish these populations.\nThe 10x Genomics Visium Spatial Gene Expression platform, while capable of unbiased whole-transcriptome profiling, lacks sufficient spatial resolution for this purpose. Therefore, we are pursuing the 10x Genomics Xenium In Situ platform, which allows custom targeted gene panel design and enables single-cell and subcellular spatial mapping of fibroblast–immune–epithelial interactions. This next-generation spatial strategy will allow us to define how the tissue microenvironment at the host–pathogen interface shapes both host and fungal responses, complementing and extending the findings presented here. These experiments are currently underway and will form the basis of future studies beyond the scope of the present manuscript.\n\nMinor Comments:\n1) It is confusing why PostnLin cells are concluded to be distinct from resident PdgfrαLin fibroblasts when 60% of PostLin cells are Pdgfrα+ (lines 200-202).\nWe apologize for this confusion. Resident fibroblasts are Pdgfrα+, but do not express Postn. While all quiescent resident fibroblasts are Pdgfrα+ (represented in Fig. S4A-D), PostnLin cells represent an activated and differentiated subset that emerges in response to A. fumigatus, exhibiting a distinct morphology (Fig. S4E-H) and functional response (Fig. 5). It is important to note that, although PostnLin cells can express Pdgfrα, their expression of this receptor is reduced compared to resident fibroblasts (Fig. 5D) which is a finding that is supported by our previous study (Kanisicak et al., 2016).\nWe made the following modifications to clarify that the Pdgfrα+ lineage (PdgfrαLin) cells depicted in Fig. S4A-D are quiescent:\n-Line 175: “…the morphology of quiescent PdgfrαLin resident pulmonary fibroblasts to that of A. fumigatus-induced…”\n-Line 176: “… Quiescent resident PdgfrαLin fibroblasts displayed a thin, elongated morphology…”\n-Also added clarity to the figure tittles in Fig.4\n\n2) Why was the triaminiclone model of immune suppression used? Why was it necessary to infect 5 hours prior to treatment? More information would be useful for the reader.\nAll mouse models of IPA require some level of immunosuppression to develop IPA (similar to the situation in humans). This is generally accomplished using a corticosteroid such as triamcinolone, administered as a single dose to create transient immunosuppression, or multiple doses to maintain constant levels of immunosuppression over time. More severe immunosuppression is accomplished by combining the corticosteroid with multiple doses of the chemotherapeutic agent cyclophosphamide. We chose a single dose of triamcinolone to minimize the potential for variability that could arise with the use of multiple doses of two different immunosuppressants. Moreover, our use of a mild and transient immunosuppression protocol allowed us to analyze the immune response to infection by flow cytometry, which would be masked by the use of more severe or chronic immunosuppression protocols. We chose to infect 5 hours prior to immunosuppression to allow the initial host-pathogen interaction to occur in the absence of immunosuppression, which could potentially impact fibroblast activation.\n- Expanded rationale for triamcinolone model and infection timing (lines 466–470).\n\n3) Bulk sequencing results should probably be supplemental, as scRNAseq results are more informative.\nSince bulk RNA-seq provides key information about a cell type not captured at the time-points represented in the scRNA-seq data, we strongly suggest keeping this information in the main text. To improve clarity, we have repositioned this section titled as stated above lines 214-252. The text has been revised to clarify the rationale of this complementary and significant aspects. However, we are happy to move it to the supplementary section if collectively reviewers and editors concur that this is the best way to present the data.\nWe have revised rationale in the single cell seq data section as well in lines 311-324 and the discussion section.\n\n4) Lines 526-527. \"At least three biological replicates were quantified per replicate\" is confusing.\nThe sentence has been corrected: \"At least three biological replicates were quantified per experiment\" (Line 640-641)\n\n5) Figure 5. (Revised Figure 6) Why is TAM administered one day before fungal inoculation? Is it possible that the damage from DTA-mediated killing is more important than the loss of PostnLin cells? What if TAM was administered several days before infection and mice were allowed recover DTA-mediated damage?\nWe thank the reviewer for raising this important point. Tamoxifen (TAM) was administered one day before fungal inoculation to ensure that Cre recombination occurred concurrently with the induction of Postn expression, which is only activated in fibroblasts upon tissue injury or infection. Administering TAM several days prior to fungal challenge would not result in recombination, as Postn-MerCreMer activity requires de novo promoter activation, and quiescent fibroblasts do not express Postn. Consistent with this, “tamoxifen only” controls exhibited minimal to no tdTomato labeling, even after extended exposure (Fig. 2C, 2F).\nPrevious kinetic studies have shown that TAM-induced recombinase activation reaches 50% within 12 h and plateaus by 24 h (Feil et al., 1997), and serum levels peak within 6–7 h before declining over 24–48 h (Vanhecke et al., 2024). Thus, TAM administration one day before infection ensures efficient recombination precisely during Postn promoter activation.\nIn our hands, we determined that the PostnLin ablation achieves ~30% depletion of Pdgfrα positive fibroblasts (new Fig. 6J). Under immunocompetent conditions even with high dose of fungal challenge, PostnLin fibroblast loss did not elicit increased lethality, inflammation or vascular damage, demonstrating that the pathological phenotype observed during A. fumigatus infection in an immunocompromised setting is driven by the loss of activated Postn fibroblasts, rather than nonspecific DTA toxicity (new Figure S8 B).\nFinally, to further address the reviewer’s concern regarding potential off-target effects of fibroblast ablation, we designed a new complementary DTA ablation study in in which a modified TAM dose was used to ablate pulmonary fibroblasts utilizing the PdgfrαCreER;R26-DTA mouse line (“Reviewer’s eyes only” figure A-B) to achieve fibroblast depletion (Reviewer Figure C) similar to the depletion reached in Postn-DTA mouse (new Figure 6J), since Pdgfrα is broadly expressed by resident fibroblasts. This design allows us to compare the inflammatory outcomes of partial fibroblast depletion in the absence of fungal infection.\nConsistent with prior findings that extensive Pdgfrα fibroblast ablation does not cause tissue injury even after prolonged depletion (Kuwabara et al., 2022), our model confirmed that fibroblast loss alone did not elicit an inflammatory response. Instead, ablation of Pdgfrα fibroblasts resulted in decreased macrophage and neutrophil numbers (Reviewer Figure D), opposite to the elevated inflammation observed in the Postn-DTA model during A. fumigatus challenge, indicating that the proinflammatory phenotype in the Postn model is specifically driven by the fungal infection.\nAs this broader fibroblast–immune interaction represents a separate mechanistic focus currently under investigation, these additional data are provided for reviewer reference only and are not included in the revised manuscript to maintain its targeted scope. The text has been updated accordingly (lines 284, 452–472; Fig. 2C, 2F; Fig. 6J) and the aforementioned references were added as follows.\nFeil, R., Wagner, J., Metzger, D., & Chambon, P. (1997). Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochemical and Biophysical Research Communications, 237(3), 752–757. https://doi.org/10.1006/bbrc.1997.7124\nVanhecke, D., Bugada, V., Steiner, R. et al. Refined tamoxifen administration in mice by encouraging voluntary consumption of palatable formulations. Lab Anim 53, 205–214 (2024). https://doi.org/10.1038/s41684-024-01409-z\nKuwabara, J. T., Hara, A., Bhutada, S., Gojanovich, G. S., Chen, J., Hokutan, K., Shettigar, V., Lee, A. Y., DeAngelo, L. P., Heckl, J. R., Jahansooz, J. R., Tacdol, D. K., Ziolo, M. T., Apte, S. S., & Tallquist, M. D. (2022). Consequences of PDGFRα+ fibroblast reduction in adult murine hearts. eLife, 11, e69854. https://doi.org/10.7554/eLife.69854\n\n[REDACTED]\n[REDACTED]\n\nReviewer #2\n1) Although this manuscript demonstrated the protective function of Postn+ fibroblasts in fungal pneumonia, the authors do not adequately explore the mechanism(s) by which loss of periostin lineage cells increases mortality in immunosuppressed mice inoculated with A. fumigatus.\nTo address this point, we performed flow cytometric analysis of pulmonary immune cell populations and quantified fungal burden, representing two key determinants of disease outcome in A. fumigatus infection. Postn lineage (PostnLin) cell ablation resulted in a marked increase in myeloid cell infiltration without a significant change in fungal burden. These results indicate that accelerated mortality in the PostnLin-ablated mice arises from excessive immune activity, rather than from uncontrolled fungal proliferation. The resulting hyperinflammatory response promotes immune-mediated tissue injury, fungal angioinvasion, and alveolar hemorrhage, contributing to rapid disease progression.\nGiven that myeloid cells are the principal mediators of antifungal defense, their overrepresentation may also account for the modest reduction in fungal load observed in PostnLin-ablated mice. Thus, our findings support a model in which Postn fibroblasts restrain excessive inflammatory activation, and their loss amplifies tissue-destructive immunity.\nThese results are now incorporated into the revised manuscript and discussed in the indicated sections (lines 85–87, 290–307, 461–480; Fig. 6F–L; Fig. S7).\n\n2) Although the authors describe areas of hemorrhage and angio-invasion after deletion of periostin+ cells, the manuscript currently lacks quantitative data regarding the observed phenotypes and detailed information on the method to evaluate the results.\nWe thank the reviewer for this helpful suggestion. We have now included quantitative analyses of hemorrhagic areas and angioinvasion following Postn cell ablation, together with expanded methodological details describing how these parameters were assessed. These quantitative results and methodological details have been incorporated into the Results (lines 268–275) and Methods (lines 643–656) sections, and summarized in Figure 6F–G.\n\n3) “…no experimental data explaining how the deleted cells might prevent these adverse events.”\nAs noted above, we have performed additional experiments demonstrating that PostnLin cells play a key role in mitigating the inflammatory response to A. fumigatus. The absence of these cells results in a hyperinflammatory phenotype, consistent with increased immune-mediated injury (Fig. 6F). Importantly, these findings are supported by single-cell RNA sequencing (scRNA-seq) data obtained at early stages of infection (Day 1), which reveal the emergence of fibroblast populations with immunomodulatory transcriptional profiles characterized by elevated expression of IL6, CXCL1, and CXCL2. These gene programs are associated with myeloid cell recruitment and activation, suggesting that fibroblast-derived inflammatory cues are critical for maintaining balanced immune responses during fungal infection. Together, these data indicate that loss of PostnLin fibroblasts disrupts this regulatory network, leading to excessive inflammation and tissue injury.\n\n4) “…it would be important to perform quantitative immunostaining and/or flow cytometry to determine whether loss of periostin+ cells modulated the recruitment and/or activation of adaptive and innate immune cells.”\nWe thank the reviewer for the opportunity to address this important point. As described above, we performed flow cytometric analysis of pulmonary immune cell populations and observed a marked increase in the infiltration of macrophages, neutrophils, dendritic cells, and NK cells following PostnLin cell ablation. These innate immune populations represent the first line of defense against A. fumigatus, and their expansion indicates that loss of PostnLin fibroblasts trigger a highly specific yet dysregulated inflammatory response.\nImportantly, these findings support our proposed mechanism that Postn fibroblasts normally act to regulate innate immune activation, and that their ablation leads to uncontrolled inflammation and tissue injury during fungal infection.\nThese new data have been incorporated into the revised manuscript (lines 85–87, 290–307, 461–480; Fig. 6L).\n\n5) It would also have been useful to see whether differences in mortality were due to effects on vascular permeability.\nWe appreciate this thoughtful comment. Assessing vascular permeability in the immunosuppressed model is technically challenging, as the extensive tissue damage caused by invasive fungal growth in both control and DTA mice obscures accurate quantification of vascular integrity. To overcome this limitation, we performed permeability assays in immunocompetent mice using fluorophore-labeled dextrans of varying molecular sizes to evaluate microvascular leakage following A. fumigatus infection. Under these conditions, no significant differences in vascular permeability were detected between A. fumigatus–treated control and PostnLin ablated (PostnMCM/+;R26-DTA) mice.\nThese results indicate that loss of Postn fibroblasts alone does not compromise microvascular stability, and that the vascular injury observed in the immunosuppressed infection model likely arises secondarily from excessive inflammation and tissue damage.\nThis new dataset is presented in Figure S8, with corresponding methodological details added (lines 717-728) and text revisions included (lines 276–289. 498-501).\n\n6) Additionally, the possibility of Postn-CreER targeting pericytes rather than fibroblasts should be experimentally excluded to prove the authors’ claims.\nWe thank the reviewer for highlighting this important consideration.\nFirst, to determine whether PostnLin cells include pericytes, we performed flow cytometric and immunohistochemical analyses using antibodies against Neuron-Glial Antigen 2 (NG2, encoded by Cspg4), a canonical pericyte marker. These analyses revealed no co-labeling between tdTomato PostnLin cells and NG2 pericytes, which was further confirmed by confocal microscopy (Fig. 4M–O; Fig. S5H).\nSecond, while single-cell RNA-seq identified low-level Postn expression in a small subset of pericyte-like cells under baseline conditions, this expression was insufficient to elicit tamoxifen-induced recombination. Consistent with this, mice treated with tamoxifen alone displayed minimal-to-no tdTomato labeling, even after prolonged exposure (Fig. 2C, 2F), demonstrating that recombination occurs only upon robust Postn induction during fibroblast activation.\nFinally, if pericytes were significantly targeted or ablated, one would expect loss of vascular stability. However, vascular permeability assays using fluorophore-labeled dextrans of multiple molecular sizes revealed no increase in microvascular leakage following PostnLin cell ablation (Fig. S8).\nTogether, these findings confirm that Postn-CreER recombination selectively targets activated fibroblasts, not pericytes, and that pericyte integrity and vascular stability remain intact. This information has been incorporated into the revised manuscript (lines 204–209, 486–501; Fig. 4M–O; Fig. S5H; Fig. S8).\n\n7) How many times did the authors backcross the Postn-MerCreMer mouse? In Figure 5, what mice did the authors use for control? This information is critical since the different genetic backgrounds can show different sensitivity to infection.\nThe Postn-MerCreMer mouse line used in this study was backcrossed for more than 10 generations onto the C57BL/6J background over the past 9 years and additional mice lines purchased from JAX were already backcrossed and pure C57BL/6J background, ensuring genetic uniformity and consistency with the other transgenic strains employed.\nFor all experiments, including those shown in Figure 5 (Revised Figure 6), littermate controls were used. Specifically, Postn-MerCreMer; R26-DTA (PostnMCM/+;DTA) mice were compared to Cre-negative littermates (R26-DTA) generated from the same breeding pairs. We also utilized lineage tracing mice as controls when appropriate. All experimental and control animals were cohoused, sex- and age-matched, and subjected to identical immunosuppressive and infection protocols to control for environmental and genetic variability.\nThese details have been clarified in the Methods section of the revised manuscript (lines 595-601).\n\n8) Additionally, …did the control group in Figure 5 also receive tamoxifen treatment? Tamoxifen itself can cause various effects on the lungs. Tamoxifen food can also cause body weight loss sometimes since mice are reluctant to eat it. If the control groups in Figure 5 were not treated by tamoxifen, how do the authors exclude the possibility that the phenotype is caused by tamoxifen?\nAll groups in original Figure 5 (Revised Figure 6) received tamoxifen, ensuring that any observed phenotypic differences are not attributable to TAM treatment. Moreover, no differences in weight loss were detected between immunosuppressed control mice with or without TAM administration (Reviewers only Fig 2). Histological analysis of lung tissues from TAM-only treated mice showed no evidence of inflammation, fibrosis, or structural abnormalities, indicating that the TAM regimen used in our study does not introduce gross phenotypic alterations to tissue architecture.\nFigure 2. Body weight changes over time in immunocompetent mice challenged with A. fumigatus. Mice were treated with TAM via gavage and TAM-infused chow diet for the duration of the experiment. Each color denotes a unique mouse, with weights recorded at the indicated time points until death.\n\n9) The authors stated that the Postn+ cell ablation resulted in pulmonary hemorrhage and fungal angioinvasion in Figure 5. These phenotypes should be quantified and statistically evaluated.\nQuantitative data has been added as previously indicated. Lines 268-275, lines 643-656; Fig 6F-G.\n\n10) What portion of Postn-lineage cells are pericytes? Authors should be able to quantify this by Mcam (CD146) staining on flow cytometry. Although authors showed minimal tdTomato+ cells without infection by histology, this should be confirmed by flow cytometry of digested lungs for a more unbiased assessment of whole lung distribution. If there are minimal tdTomato+ cells without infection, can the authors explain why Postn-MerCreMer specifically labels fibroblasts but not pericytes, which highly express Postn at baseline?\nWe appreciate this comment. As outlined above, we used anti-NG2 (Cspg4) antibodies to identify pericytes, as NG2 is a widely accepted and specific pericyte marker in the lung. We avoided Mcam (CD146) because it is also expressed by smooth muscle and endothelial cells, which could confound flow cytometric analyses.\nFlow cytometry and confocal microscopy showed no overlap between tdTomato PostnLin cells and NG2 pericytes (Fig. 4M–O; Fig. S5H), and tamoxifen-only controls displayed minimal-to-no tdTomato labeling by both histology and flow cytometry (Fig. 2C, 2F). Although low baseline Postn expression was detected in pericyte-like clusters by scRNA-seq, this level was insufficient to drive Cre recombination, which occurs only after Postn promoter activation in stimulated fibroblasts.\nThese data confirm that Postn-MerCreMer recombination selectively targets activated fibroblasts, not pericytes.\n\n11) Since Postn is also highly expressed in pericytes, the authors should experimentally exclude the possibility of pericytes contributing to the phenotypes in Figure 5. The authors should be able to do this by treating the mice with fungus after treating and washing out tamoxifen or using Rosa26-DTR mice.\nAs described above, our results indicate that Postn-CreER recombination selectively targets fibroblasts rather than pericytes. To further confirm that pericytes do not contribute to the phenotypes shown in Figure 5, we quantified NG2 pericytes by flow cytometry in control and Postn-DTA mice following infection with A. fumigatus (3 × 10⁷ conidia). Mice received tamoxifen as outlined in Fig. S8A, and lungs were harvested 4 days post-inoculation. The results demonstrate that NG2 pericyte numbers were unchanged between DTA-treated and control mice, indicating that PostnLin ablation did not affect pericyte survival.\nIn addition, tamoxifen-only controls showed minimal-to-no tdTomato labeling by both histology and flow cytometry (Fig. 2C, 2F), confirming that baseline Postn expression in pericytes is insufficient to induce Cre-mediated recombination. Consistent with this, vascular permeability assays using fluorophore-labeled dextrans revealed no microvascular leakage or endothelial instability after PostnLin ablation (Fig. S8).\nTogether, these findings confirm that Postn-CreER–mediated recombination and DTA ablation specifically target activated fibroblasts, not pericytes, and that the phenotypes observed in Figure 5 (Revised Figure 6) are not attributable to pericyte loss.\nA reviewer’s-eyes-only figure is provided to illustrate that NG2 pericyte populations remain intact following PostnLin ablation.\n\n12) Figure 7E is difficult to understand. Figure 1A shows that there are normal alveoli left on day 1. However, Figure 7E seems to show that all alveolar fibroblasts homogeneously become activated alveolar fibroblasts. Similar results are shown for peribronchiolar fibroblasts. If the clusters are completely driven by treatment and time points, the authors might want to try integration methods such as Harmony or Mutual Nearest Neighbors (MNN), which may allow them to identify newly emerged populations while keeping some normal populations.\nWe appreciate the reviewer’s insightful comment and agree that batch effect correction is a critical step in single-cell data analysis. Methods such as Harmony or Mutual Nearest Neighbors (MNN) can mitigate batch-associated variability but may also over-correct, potentially masking genuine biological differences, particularly when samples represent distinct activation states.\nIn this study, we carefully evaluated batch effects prior to integration. No significant batch-driven segregation was observed between the two biologically similar saline-treated control samples (day 1 and day 7), which were processed in separate batches. Their UMAP embeddings overlapped extensively, confirming the absence of technical bias. Moreover, applying Harmony-based correction yielded a nearly identical cluster distribution, further indicating that batch effects were minimal.\nGiven these findings, we chose not to apply batch correction for the infection dataset to preserve true biological variation among fibroblast activation states.\n\n13) In Figure 7E, the authors showed only inflammatory genes for the alveolar fibroblast lineage. Differentially expressed genes for each cluster should be shown by heatmap or dot plots for readers to know the specific genes of each cluster.\nIn the original version, Figure 7E focused on inflammatory and activation-associated genes to highlight the dynamic transcriptional response of fibroblast subsets following A. fumigatus infection. To provide a more comprehensive view of fibroblast heterogeneity, we have now included differential gene expression analyses for all stromal clusters.\nSpecifically, we generated a new supplementary figure showing the top five marker genes (DEGs) defining each stromal subpopulation in dot plot (Fig. S9) and added the complete DEG list as a supplementary data table (Tables S4 and S5). These additions allow readers to evaluate the molecular identity and functional characteristics of each cluster.\nCorresponding details have been added to the Results section (lines 331–397).\n\n14) The authors need to include data demonstrating the efficiency of the deletion method used to delete periostin-lineage cells. It would also be good to overlay periostin expression on the uMAP plots and to have included the lineage label in the single cell sequencing experiments so the authors could determine which of the emergent stromal populations they describe were derived from periostin-expressing cells and thus which populations were likely to have been deleted.\nWe appreciate this important point.\nFirst, we quantified the efficacy of periostin-lineage ablation by flow cytometry as described above via labeling all fibroblast with Pdgfrα. We determined that the PostnLin ablation achieves ~30% depletion of Pdgfrα positive fibroblasts (new Fig. 6J).\nSecond, in our droplet scRNA-seq dataset at Day 1, Postn transcripts exhibit pronounced sparsity/dropout and map predominantly to a pericyte-like cluster that shows low baseline Postn expression whereas the fibroblast clusters that become activated after infection display low detectable Postn mRNA at this early time point. This is consistent with (i) the known behavior of matricellular/ECM transcripts (low capture efficiency and high dropout in 3′ droplet data), and (ii) our kinetic data indicating that periostin protein peaks later (past 24 hrs), while the Day-1 scRNA profile captures the onset of activation. As a result, a simple Postn-UMAP overlay would be misleading in our dataset. To this end we have expanded our data set and determined that the low expression of Postn in pericytes have not resulted in recombination in pericyte (Fig.4 M-O), nor resulted in ablation (Reviewer figure 3 above). Therefore, our collective data suggests the consequence of PostnLin ablation is derived from the fibroblasts.\nThird (linking to fibroblast activation states). Instead, we clarified the relationship between periostin-lineage biology and the single-cell map by providing a new supplementary figure (Fig. S9) showing the top five marker genes defining each fibroblast subpopulation and a complete DEG table (Table S4 and S5). These analyses align the emergent Day-1 inflammatory fibroblast states with the populations reduced by PostnLin ablation.\nFinally (future lineage-resolved single-cell). Our present scRNA-seq libraries did not include the fluorescent lineage reporter. In ongoing work, we are implementing feature barcoding/CITE-seq for anti-RFP and targeted spatial panels (10x Xenium) to directly map PostnLin derivatives at single-cell spatial resolution in future studies.\nWe have added these clarifications to the Results/Methods and included the new DEG visualizations (lines 311–397; Fig. S9; Table S4-5).\n\nMinor comments\n1. It would be nice to quantify fibrosis after fungus infection by hydroxyproline assay\nCollagen deposition was evaluated by Picrosirius Red staining on lung sections from immunosuppressed control and Postn-DTA mice infected with A. fumigatus. Both groups showed comparable levels and distribution of collagen, localized to areas of inflammation, indicating that Postn cell ablation did not alter total collagen content. These results further imply that pulmonary fibroblast immune modulation to be the central mechanism of action in response to A. fumigatus challenge.\nThese data are shown in Figure 6H and discussed in lines 270–275.\nBecause this histological and quantitative approach captures both the extent and spatial context of collagen deposition, a hydroxyproline assay was not employed.\n\n2. Figure 7D, colors for activated alv and deactivated alv were swapped.\nThank you! The legend has been corrected.\n\n3. Figure 4 would benefit from addition of staining for specific pericyte markers.\nFlow cytometry and confocal immunohistochemistry images using the pericyte marker NG2 have been added, showing a lack of colabeling of TdT+ cells with NG2 (Fig.4 M-O and Fig S5H).\n\nReviewer #4\n1) Do the authors have any data suggesting that fungal burden is (or is not) impacted by loss of the postn lineage fibroblasts during invasive disease (Figure 5)? Maybe this is just a point for the Discussion, but is it possible that after invasive growth begins, these fibroblasts play a role in limiting fungal growth? Or, is the increased mortality of the DTA mice simply due to heightened levels of tissue damage in response to the same amount of fungal growth?\nFungal burden was quantified in immunosuppressed Postn-DTA and control mice during invasive A. fumigatus infection. The results show that fungal load remained unchanged following ablation of PostnLin fibroblasts (Fig. 6K). However, angioinvasion of pulmonary arteries was markedly increased in the DTA group (Fig. 6G), suggesting that Postn fibroblasts contribute to maintaining vascular integrity and limiting fungal dissemination.\nCollagen quantification by Picrosirius Red staining revealed no difference in total collagen deposition between groups (Fig. 6H), indicating that the observed vascular compromise is not due to reduced ECM deposition. Instead, DTA mice displayed excessive infiltration of macrophages, neutrophils, dendritic cells, eosinophils, and NK cells, along with alveolar hemorrhage (Fig. 6D–F, 6L; Fig. S7).\nTogether, these findings indicate that fibroblast ablation disrupts immune homeostasis, leading to hyperinflammation and vascular injury, rather than impaired fungal clearance. The increased angioinvasion and hemorrhage, in the context of stable fungal burden, likely account for the accelerated mortality in the DTA model.\nThis information has been incorporated into the revised manuscript (lines 85–87, lines 267–275, 290-307, 461–480, 643-656, 700-716).\n\n2. In the same line of thought, the immune competent DTA mice did not experience death after infection (Line 223), but did they have prolonged time to clearance/sterilization? Even negative data may be very informative here and could be included as a Supplemental Figure, if available.\nTo address this point, we quantified fungal burden in immunocompetent Postn-DTA and control mice following A. fumigatus infection. At day 4 post-inoculation, which corresponds to the earliest time point at which phenotypic differences emerge in the immunosuppressed model, fungal growth was already minimal in both groups. The number of colony-forming units (CFUs) recovered from lung homogenates was consistently below 20 CFUs per plate, indicating that fungal clearance was essentially complete by this time.\nGiven this very low baseline burden, no meaningful difference in fungal sterilization kinetics could be determined between groups. These data are summarized in the accompanying supplementary table below for reviewer reference.\nTogether, these findings indicate that in the immunocompetent setting, the absence of PostnLin fibroblasts does not impair fungal clearance, consistent with their role in modulating inflammation rather than direct antifungal defense."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The manuscript presents interesting findings that highlight a potentially important role for fibroblasts in shaping immune responses and maintaining lung integrity during fungal infection. The study’s use of fibroblast lineage tracing and ablation in a model of invasive aspergillosis is important and novel and contributes to our understanding of fibroblast responses to A. fumigatus. However, while the data convincingly show altered immune cell recruitment and gene expression in the absence of periostin-lineage fibroblasts, the mechanistic conclusions advanced in the text—particularly those concerning alveolar hemorrhage, angioinvasion, and vascular compromise—are not directly supported by the experimental data. The authors reference new data additions and state that over 12 figures have been added in the revision, but these appear to primarily represent expanded or reformatted analyses of existing datasets. No new in vivo mechanistic experiments were introduced, nor were fibroblast-specific effector functions or endothelial outcomes directly tested. Thus, mechanistic claims remain speculative. Additionally, while the revised manuscript includes clearer figure labeling and additional cytokine profiling, the requested functional studies to demonstrate causality—particularly those needed to link fibroblast ablation to vascular injury or fungal dissemination—are still lacking. Based on these results, TGF-beta may be a candidate for PostLn-specific ablation. The methods are generally sound and reproducible, and the work is likely to be of interest to those studying fungal pathogenesis and pulmonary immunology. However, the authors need to provide additional experimental support for the proposed mechanism.", "reviewer_2": "The authors have adequately responded to all of the issues raised in my initial review", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors have completed an impressive amount of additional work to directly address all concerns of the previous review. The authors have included 12 new figures and altered existing figures, as well as included major edits to the presentation and discussion of results in the text. Together, these new data and revisions have greatly improved the manuscript and increased the rigor and significance of the work. I have no other critiques for the authors to consider."}, "author_response": "Response to Reviewers for NCOMMS-25-02679A\nReviewer #1:\n… However, while the data convincingly show altered immune cell recruitment and gene expression in the absence of periostin-lineage fibroblasts, the mechanistic conclusions advanced in the text—particularly those concerning alveolar hemorrhage, angioinvasion, and vascular compromise—are not directly supported by the experimental data.\nWe appreciate the reviewer’s thoughtful critique and agree that our data do not directly establish mechanistic causality for alveolar hemorrhage, angioinvasion, or vascular compromise. In response, we have revised the manuscript to more carefully constrain our conclusions to associations supported by the experimental data.\nSpecifically, we have modified the Conclusions section (Lines 303–306) to state that loss of PostnLin cells in the context of IPA is associated with accelerated mortality and altered innate immune responses to A. fumigatus, without implying direct mechanistic causation. We have also revised the Discussion (Line 472) to clarify that the observed hyperinflammatory response is consistent with immune-mediated tissue damage contributing to increased mortality, rather than asserting a direct causal mechanism.\nThese changes ensure that all interpretations regarding vascular injury and fungal invasion remain appropriately bound by the data presented.\nList of changes made:\n-Line 303-306: “…We conclude that loss of PostnLin cells in the context of IPA is associated with accelerated mortality and a dysregulated innate immune response to A. fumigatus, accompanied by pathological features consistent with vascular injury and fungal invasion.”\n-Line 472: “…This was associated with a hyperinflammatory response, consistent with immune-mediated tissue damage as a potential contributor to the observed increase in mortality (Fig. 6).”\n…The authors reference new data additions and state that over 12 figures have been added in the revision, but these appear to primarily represent expanded or reformatted analyses of existing datasets. No new in vivo mechanistic experiments were introduced, nor were fibroblast-specific effector functions or endothelial outcomes directly tested. Thus, mechanistic claims remain speculative. Additionally, while the revised manuscript includes clearer figure labeling and additional cytokine profiling, the requested functional studies to demonstrate causality—particularly those needed to link fibroblast ablation to vascular injury or fungal dissemination—are still lacking. Based on these results, TGF-beta may be a candidate for PostLn-specific ablation\nWe appreciate the reviewer’s assessment and agree that the additional figures primarily extend and refine in vivo analyses rather than introducing endothelial-targeted or single-pathway effector experiments. However, we respectfully note that the revised manuscript does include new in vivo mechanistic data at the level of immune regulation.\nSpecifically, we incorporated flow cytometric analyses demonstrating heightened activation and expansion of innate myeloid populations following PostnLin fibroblast ablation, providing direct in vivo evidence that these cells restrain inflammatory immune responses during IPA (Fig. 6I–L). Importantly, these changes occur independently of increased fungal burden, supporting a mechanistic role for PostnLin cells in regulating host immune responses rather than pathogen control.\nIn addition, experiments performed in an immunocompetent background demonstrate that the pathological features observed upon Postn ablation are infection-specific and not attributable to baseline alterations in vascular permeability (Fig. S8). Together with new in vitro data showing that human pulmonary fibroblasts directly sense A. fumigatus and acquire immunomodulatory properties (Fig. 7G and Fig. S10), these findings provide mechanistic insight into how fibroblasts shape local immune responses during fungal infection.\nWhile we do not claim identification of a single downstream effector pathway or direct endothelial injury mechanism, we have revised the manuscript to clearly frame our conclusions at the level of immune modulation and tissue homeostasis, which are directly supported by the data presented."}]

Decision letter