Objective Little is known about the pathogenesis of rheumatoid arthritis-related interstitial lung disease (RA-ILD). This study aimed to clarify the cellular and transcriptomic landscape of epithelial and immune cells in RA-ILD.Methods We performed single-cell RNA sequencing on fluorescence-activated cell sorted epithelial cells and immune cells from lung explants of four controls, three patients with non-RA connective tissue disease (CTD)-ILD, and five patients with RA-ILD. For T cell subclusters, we performed an integrative analysis with publicly available synovial T cell data. We performed immunofluorescence staining on lung sections from four controls, nine patients with RA-ILD, eight patients with non-RA CTD-ILD, and six patients with idiopathic pulmonary fibrosis.Results We profiled 184,814 cells in total and identified 18 distinct cell clusters. We found fewer alveolar type II cells with reciprocally higher frequencies of other epithelial cell types (basal cells and ciliated cells) and fewer FCN1+ CD14+ monocytes in RA-ILD lungs. In T cell subset analysis, peripheral helper T (Tph) cells were exclusively observed in RA-ILD lungs. Compared with synovial Tph cells, lung Tph cells had elevated expression profiles of activation and lower cytotoxic and exhausted signatures. From gene ontology analysis, genes associated with the small GTPase-mediated signal transduction were enriched in lung Tph cells. On confirmatory immunofluorescence staining, Tph cells were specifically present in RA-ILD lungs.Conclusion We report a detailed transcriptomic analysis of the epithelial and immune cells in RA-ILD lungs and include a cross-tissue comparison that demonstrates organ-specific variations in the characteristics of Tph cells.

Idiopathic pulmonary fibrosis (IPF) is an age-related, progressive, and fatal interstitial lung disease for which effective therapies remain limited. Alveolar type 2 (AT2) epithelial cells serve as facultative stem cells essential for alveolar repair; however, AT2 cell senescence disrupts epithelial regeneration and contributes to fibrotic remodeling in IPF. Syndecan-1 is a transmembrane heparan sulfate proteoglycan predominantly expressed by lung epithelial cells, but its role in AT2 dysfunction during fibrosis is poorly defined. Here, we demonstrate that syndecan-1 is robustly upregulated in AT2 cells in IPF and other fibrotic lung diseases, as well as in murine bleomycin-induced lung fibrosis. Syndecan-1 expression was further enhanced with aging and associated with increased fibrotic burden in aged mice. Using integrated human transcriptomic analyses, mouse genetic models, and epithelial cell-based systems, we show that excess syndecan-1 promotes cell-autonomous epithelial senescence and impairs AT2 progenitor function. Elevated syndecan-1 reduced AT2 renewal capacity, disrupted differentiation, and diminished surfactant protein C level, whereas genetic loss of syndecan-1 attenuated senescence and preserved epithelial function following injury. Together, these findings identify syndecan-1 as a critical epithelial regulator of AT2 senescence and maladaptive repair in pulmonary fibrosis and support targeting syndecan-1-driven epithelial dysfunction as a potential therapeutic strategy.

Alveolar type 2 (AT2) progenitor cell exhaustion and impaired regenerative capacity are key pathogenic hallmarks in idiopathic pulmonary fibrosis (IPF). Nicotinamide adenine dinucleotide (NAD + ) functions as a central regulator of cellular energy metabolism. We have previously reported that downregulation of NAD + -dependent sirtuin signaling contributes to the impaired progenitor cell function of IPF AT2 cells. In this study, we found that a key NAD + biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT), was significantly downregulated in IPF AT2 cells. NAMPT deficiency impaired AT2 renewal and enhanced lung fibrosis through downregulation of SIRT7 and SOD2, which resulted in increased oxidative stress, mitochondrial dysfunction, accumulated aberrant transitional cells, and impaired differentiation from AT2 to alveolar type 1 (AT1) cells. A mouse model with AT2-specific deletion of Nampt showed severely impaired AT2 renewal capacity and increased susceptibility to bleomycin lung injury. Activation of NAMPT by small-molecule activators promoted IPF AT2 renewal and reversed lung fibrosis in WT mice. NAMPT activation is a potentially promising therapeutic strategy for restoring AT2 progenitor cell function and halting or reversing progressive pulmonary fibrosis.

Tissue fibrosis is a common pathological outcome of chronic disease that markedly impairs organ function leading to morbidity and mortality. In the lung, idiopathic pulmonary fibrosis (IPF) is an insidious and fatal interstitial lung disease associated with declining pulmonary function. Here, we show that alveolar type 2 (AT2) cells isolated from IPF lung tissue exhibit characteristic transcriptomic features of cellular senescence. We used conditional loss of Sin3a in adult mouse AT2 cells to initiate a program of p53-dependent cellular senescence, AT2 cell depletion, and spontaneous, progressive pulmonary fibrosis. We show that p53 signaling Tgfβ signaling forms a positive feedback loop promotes AT2 to Krt8high transitional cell differentiation, AT2 senescence and progressive pulmonary fibrosis. We establish that senescence rather than loss of epithelial stem cells serves as a proximal driver for progressive fibrosis and show that either genetic or pharmacologic interventions targeting p53 activation, senescence, or Tgfβ activation, block AT2 to Krt8high transitional cell differentiation and fibrogenesis.

The commensal microbiota that colonizes mucosal surfaces in mammals influence host physiology including immune function, but the mechanisms underlying the microbiota’s influence on systemic immunity remain incompletely understood. The intestinal microbiota has been implicated in the induction of adaptive immune responses in mice and in humans, but it is unknown if long-term maintenance of memory T cells can be influenced by the commensal microbiota. We tested if antigen (ag)-specific CD4+memory Tcells generated in SPF mice could be maintained in SPF mice treated with antibiotics (AB) or in Germ-Free (GF) mice. Using TCR transgenic OT-II cells primed in vivo with their cognate ag, we found that both AB-treated and GF mice failed to maintain transferred memory CD4+T cells when compared to untreated SPF mice. Similarly, CD4+Tcells from SPF mice infected with Influenza/PR8 and adoptively transferred into secondary recipients were maintained in SPF but not in GF or Altered Schaedler’s Flora (ASF) mice. We also found that AB-treated mice failed to maintain lung tissue-resident CD4+ and CD8+ memory T cells generated by immunization with a live-attenuated influenza vaccine (FluMist). Collectively, these finds support the requirement of an intact commensal microbiota for the maintenance of CD4+ memory T cells generated upon infection and vaccination. The mechanisms mediating this effect of the commensal microbiota in memory T cell maintenance are currently under active investigation. NIH- NIAID/NHLBI Grant R01-AI103542 (to G.A.M.), NIH-NIAID Grants R21-AI142306 (To. G.A.M), R01-AI127406 (to D.U., G.A.M. and G.L.), F. Widjaja Foundation IBIRI funds (to G.A.M.), and by the CSMC Dept. of Biomedical Science “Leon Fine Translational Science Award” (to G.A. M.). AAI Intersect Fellowship for Computational Scientists and Immunologists – 2023/2024 Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Tissue-resident alveolar macrophages (AMs) rely on intrinsic stem-like programs for self-renewal and maintenance, yet the transcriptional networks that support these functions and their relevance to post-viral lung disease remain largely unknown. Here, we identify TCF4 (Tcf7l2) as a critical transcription factor that governs AM maturation and stemness. Loss of TCF4 impaired AM proliferation, shifted their identity toward a pro-inflammatory phenotype, and exacerbated host morbidity following influenza or SARS-CoV-2 infection. Conversely, enforced TCF4 expression promoted the expansion of mature AMs, and supported lung recovery, thereby protecting against severe acute viral disease. Mechanistically, TCF4 antagonized β-catenin-driven inflammatory transcription while preserving oxidative phosphorylation, defining a reciprocal regulatory axis essential for AM function. Notably, respiratory viral infections and exuberant interferon signaling suppressed TCF4 expression, which remains chronically reduced in murine and human lungs with post-COVID fibrosis. This downregulation is associated with persistent KRT8hi dysplastic epithelium and collagen deposition. Moreover, aging diminished TCF4 levels and enforced TCF4 expression dampened age-associated decline of AM self-renewal. Furthermore, in vivo TCF4 overexpression after viral clearance enhanced mature AM accumulation, promoted lung epithelium regeneration, attenuated chronic tissue fibrosis and restored pulmonary physiologial function in aged lungs in a model of persistent pulmonary fibrosis post-acute viral infection. These findings have established TCF4 as a key regulator of AM stemness and identified a promising therapeutic target for long COVID and related chronic lung diseases through the modulation of embryonic-derived macrophage regenerative capacity by targeting TCF4.

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease marked by alveolar type 2 (AT2) stem cell dysfunction and excessive matrix deposition, with no effective treatments. Recent advances have recognized that AT2 cells act as stem cells, in addition to their role in the production of pulmonary surfactants in the distal alveolar space. We and others have reported a failure of AT2 regeneration and a loss of AT2 cells in IPF. We recently further reported that there is a defect in lipid metabolism in IPF AT2 cells and we discovered a selective loss of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in AT2 cells from IPF, as well as in AT2 cells from bleomycin-injured mice. Pharmacological and genetic experiments confirm that LPCAT1 is required for AT2 cell renewal in 3D organoid assays. AT2 cell-specific Lpcat1 deletion resulted in reduced AT2 renewal, spontaneous lung fibrosis, and heightened susceptibility to bleomycin-induced fibrosis in mice in vivo. Expression-based high-content drug screening with an LPCAT1 knock-in cell line identified several drug families that upregulated LPCAT1 expression. We further confirmed that anti-malarial artesunate and PLA2 inhibitor ONO-RS-082 increased LPCAT1 mRNA expression, promoted AT2 renewal, and attenuated bleomycin-induced lung fibrosis in mice in vivo. Our findings establish LPCAT1 as a critical regulator of AT2 renewal and lipid metabolism in IPF, suggesting that reactivation of LPCAT1 could offer a novel therapeutic strategy for restoring alveolar progenitor function and mitigating lung fibrosis.

RATIONALE: Alveolar type 2 (AT2) cells play critical roles as lung stem cells in initiating lung repair and alveologenesis following injury via self-renewal and differentiation into alveolar type 1 (AT1) cells. Hence, effective AT2-to-AT1 differentiation is essential for lung repair. The over-expression of syndecan-1 in AT2 cells in lung fibrosis impacts their proliferative and reparative roles by promoting AT2 senescence. We proposed to examine the pathological roles of excess syndecan-1 in AT2 differentiation and its roles in unresolved fibrosis. METHODS: We analyzed human and mouse scRNAseq of Idiopathic pulmonary fibrosis (IPF) and bleomycin-injured WT and Sdc1-/-mice. IPF and control lungs were immunostained for transitional (KRT5, KRT8, and KRT17), AT2 (proSP-C), and AT1 (Pdpn) cell markers. Aged-WT and Sdc1-/- mice were given a single-dose bleomycin intratracheally. At day 21, the lungs were immunostained with all epithelial cell markers. The bleomycin-injured WT and Sdc1-/-AT2 3D organoid cell cultures were assessed for colony-forming efficiency and AT2 differentiation by serial imaging and immunostaining for AT1, AT2, and transitional cell markers. RESULTS: Human scRNAseq data showed that syndecan-1 was over-expressed in IPF AT2 and transitional epithelial cells compared to controls. The transcriptomic composite SenMayo Senescence score was higher in IPF AT2 cells. Spatial transcriptomic data of IPF indicated that syndecan-1 was co-expressed with KRT17 and CDKN1A [p21]. Immunostaining corroborated that syndecan-1 was co-expressed with KRT8+ KRT17+ cells in IPF. In aged mice, syndecan-1 was profibrotic, and excess syndecan-1 in the aged – WT mice was associated with the accumulation of Krt8+ cells. These findings were validated in AT2 3D organoids, where we found a significant Krt8+ and Krt17+ cell expansion in bleomycin-injured WT. Additionally, Krt17+ cells were significantly less in Sdc1-/- AT2 organoids. Further, we found normal morphology and AT1 differentiation in Sdc1-/- organoids, whereas WT AT2 organoids developed atypical morphology and maldistribution of AT1 cells (Figure 1). CONCLUSIONS: We concluded that syndecan-1 was upregulated in aberrant transitional epithelial cells of human and mouse lung fibrosis. An excess of syndecan-1 promoted an accumulation of aberrant transitional epithelial cells, thereby preventing normal AT2 differentiation and leading to ineffective lung repair in lung fibrosis. We proposed that syndecan-1 mediated an abnormal accumulation of aberrant transitional cells by maintaining the senescence state of these cells. Our future work includes the identification of regulatory molecules that syndecan-1 regulates to promote the persistent senescence state of aberrant transitional epithelial cells.

Rationale: The role of the innate immune system in idiopathic pulmonary fibrosis (IPF) remains poorly understood. However, a functional myeloid compartment is required to remove dying cells and cellular debris, as well as to mediate innate immune responses against pathogens. Aberrant macrophage activity has been described in patients with post-acute sequelae of COVID fibrosis (PASC-F), and caveolin scaffolding domain (CSD) peptides have been found to attenuate inflammation and fibrosis in mouse lung injury models. Therefore, we examined, for the first time, the effects of CSD peptide LTI-2355 on the functional and synthetic properties of human myeloid cells isolated from lung explant tissue of donor lungs as well as IPF and PASC-F lung explant tissue. Methods and Results: CD45+ myeloid cells isolated from lung explant tissue from IPF and PASC-F patients exhibited an impaired capacity to clear autologous dead cells and cellular debris. The uptake of pathogen-coated bioparticles was impaired in myeloid cells from both fibrotic patient groups independent of the type of pathogen, highlighting an intrinsic functional cell impairment. LTI-2355 improved the phagocytic activity of both IPF and PASC-F myeloid cells, and this improvement was paired with decreased proinflammatory and pro-fibrotic synthetic activity. LTI-2355 was also shown to primarily target CD206-expressing IPF and PASC-F myeloid cells. Conclusions: Primary myeloid cells from IPF and PASC-F patients exhibit dysfunctional phagocytic and synthetic properties that are modulated by LTI-2355. LTI-2355 treatment of IPF myeloid cells resulted in significantly reduced sCD163, IFN-α2, IFN-γ, IL-2, IL-10, IL-12p40, and MMP-1 in the cell supernatant. This study highlights an additional mechanism of action of the CSD peptide in the treatment of IPF and progressive fibrotic lung disease.

Peroxisomes are vital but often overlooked metabolic organelles. We found that excessive interferon signaling remodeled macrophage peroxisomes. This loss of peroxisomes impaired inflammation resolution and lung repair during severe respiratory viral infections. Peroxisomes were found to modulate lipid metabolism and mitochondrial health in a macrophage type-specific manner and enhanced alveolar macrophage-mediated tissue repair and alveolar regeneration after viral infection. Peroxisomes also prevented excessive macrophage inflammasome activation and IL-1β release, limiting accumulation of KRT8high dysplastic epithelial progenitors following viral injury. Pharmacologically enhancing peroxisome biogenesis mitigated both acute symptoms and post-acute sequelae of COVID-19 (PASC) in animal models. Thus, macrophage peroxisome dysfunction contributes to chronic lung pathology and fibrosis after severe acute respiratory syndrome coronavirus 2 infection.

Alveolar type II (AT2) progenitor cell exhaustion and impaired regenerative capacity are key pathogenic hallmarks in idiopathic pulmonary fibrosis (IPF). Nicotinamide adenine dinucleotide (NAD + ) functions as a central regulator of cellular energy metabolism. We have reported that downregulation of NAD + -dependent sirtuin signaling contributes to the impaired progenitor function of IPF AT2s. In this study, we identified that a key NAD + biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT), is significantly downregulated in IPF AT2s. NAMPT deficiency impairs AT2 renewal and enhances lung fibrosis through downregulation of SIRT7 and SOD2, which results in increased oxidative stress, mitochondrial dysfunction, induction of pathological transitional gene expression and impaired regenerative capacity to generate alveolar type I (AT1) cell required for gas exchange. Mice with deletion of Nampt in AT2s showed severely impaired AT2 renewal and increased susceptibility to bleomycin lung injury and spontaneous fibrois. Activation of NAMPT by small molecule activators promoted AT2 renewal, restored homeostasis, and reversed lung fibrosis. NAMPT activation could be a therapeutic strategy for restoring AT2 progenitor function and halting or reversing progressive pulmonary fibrosis.

A leading cause of mortality after influenza infection is the development of a secondary bacterial pneumonia. In the absence of a bacterial superinfection, prescribing antibacterial therapies is not indicated but has become a common clinical practice for those presenting with a respiratory viral illness. In a murine model, we found that antibiotic use during influenza infection impaired the lung innate immunologic defenses toward a secondary challenge with methicillin-resistant Staphylococcus aureus (MRSA). Antibiotics augment lung eosinophils, which have inhibitory effects on macrophage function through the release of major basic protein. Moreover, we demonstrated that antibiotic treatment during influenza infection caused a fungal dysbiosis that drove lung eosinophilia and impaired MRSA clearance. Finally, we evaluated 3 cohorts of hospitalized patients and found that eosinophils positively correlated with antibiotic use, systemic inflammation, and worsened outcomes. Altogether, our work demonstrates a detrimental effect of antibiotic treatment during influenza infection that has harmful immunologic consequences via recruitment of eosinophils to the lungs, thereby increasing the risk of developing a secondary bacterial infection.

Efferocytosis is a process whereby apoptotic cells are cleared to maintain tissue homeostasis. In the lungs, efferocytosis has been implicated in several acute and chronic inflammatory diseases. A long-standing method to study efferocytosis in vivo is to instill apoptotic cells into the lungs to evaluate macrophage uptake. However, this approach provides nonphysiologic levels of cells to the airspaces, where there is preferential access to the alveolar macrophages. To circumvent this limitation, we developed a new method to study efferocytosis of damaged alveolar type 2 (AT2) epithelial cells in vivo. A reporter mouse that expresses TdTomato in AT2 epithelial cells was injured with influenza (strain PR8) to induce apoptosis of AT2 cells. We were able to identify macrophages that acquire red fluorescence after influenza injury, indicating efferocytosis of AT2 cells. Furthermore, evaluation of macrophage populations led to the surprising finding that lung interstitial macrophages were the primary efferocyte in vivo. In summary, we present a novel finding that the interstitial macrophage, not the alveolar macrophage, primarily mediates clearance of AT2 cells in the lungs after influenza infection. Our method of studying efferocytosis provides a more physiologic approach in evaluating the spatiotemporal dynamics of apoptotic cell clearance in vivo and opens new avenues to study the mechanisms by which efferocytosis regulates inflammation.

Idiopathic pulmonary fibrosis (IPF) is a lethal chronic lung disease characterized by aberrant intercellular communication, extracellular matrix deposition, and destruction of functional lung tissue. While extracellular vesicles (EVs) accumulate in the IPF lung, their cargo and biological effects remain unclear. We interrogated the proteome of EV and non-EV fractions during pulmonary fibrosis and characterized their contribution to fibrosis. EVs accumulated 14 days after bleomycin challenge, correlating with decreased lung function and initiated fibrogenesis in healthy precision-cut lung slices. Label-free proteomics of bronchoalveolar lavage fluid EVs (BALF-EVs) collected from mice challenged with bleomycin or control identified 107 proteins enriched in fibrotic vesicles. Multiomic analysis revealed fibroblasts as a major cellular source of BALF-EV cargo, which was enriched in secreted frizzled related protein 1 (SFRP1). Sfrp1 deficiency inhibited the activity of fibroblast-derived EVs to potentiate lung fibrosis in vivo. SFRP1 led to increased transitional cell markers, such as keratin 8, and WNT/β-catenin signaling in primary alveolar type 2 cells. SFRP1 was expressed within the IPF lung and localized at the surface of EVs from patient-derived fibroblasts and BALF. Our work reveals altered EV protein cargo in fibrotic EVs promoting fibrogenesis and identifies fibroblast-derived vesicular SFRP1 as a fibrotic mediator and potential therapeutic target for IPF.

The long-term physiological consequences of respiratory viral infections, particularly in the aftermath of the COVID-19 pandemic-termed post-acute sequelae of SARS-CoV-2 (PASC)-are rapidly evolving into a major public health concern1-3. While the cellular and molecular aetiologies of these sequelae are poorly defined, increasing evidence implicates abnormal immune responses3-6 and/or impaired organ recovery7-9 after infection. However, the precise mechanisms that link these processes in the context of PASC remain unclear. Here, with insights from three cohorts of patients with respiratory PASC, we established a mouse model of post-viral lung disease and identified an aberrant immune-epithelial progenitor niche unique to fibroproliferation in respiratory PASC. Using spatial transcriptomics and imaging, we found a central role for lung-resident CD8+ T cell-macrophage interactions in impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. Specifically, IFNγ and TNF derived from CD8+ T cells stimulated local macrophages to chronically release IL-1β, resulting in the long-term maintenance of dysplastic epithelial progenitors and lung fibrosis. Notably, therapeutic neutralization of IFNγ + TNF or IL-1β markedly improved alveolar regeneration and pulmonary function. In contrast to other approaches, which require early intervention10, we highlight therapeutic strategies to rescue fibrotic disease after the resolution of acute disease, addressing a current unmet need in the clinical management of PASC and post-viral disease.

The composition of extracellular matrix (ECM) is altered during pathologic scarring in damaged organs including the lung. One major change in the ECM involves the cross-linking of collagen, which promotes fibroblast to myofibroblast differentiation. We examined the role of lysyl oxidase (LOX)-like 2 in lung progenitors and fibroblasts cultured from normal or IPF lung samples and in a humanized mouse model of IPF using a monoclonal antibody (Simtuzumab). Primary lung fibroblasts from normal donor lungs and IPF lung explants were examined for expression of LOXL2. Targeting LOXL2 with Simtuzumab on normal and IPF fibroblasts was examined both in vitro and in vivo for synthetic, functional, and profibrotic properties. LOXL2 was increased at transcript and protein level in IPF compared with normal lung samples. In a dose-dependent manner, Simtuzumab enhanced differentiation of fibroblasts into myofibroblasts. Inhibition of LOXL2 also enhanced fibroblast invasion and accelerated the outgrowth of fibroblasts from dissociated human lung cell preparations. Finally, preventative or delayed delivery of Simtuzumab enhanced lung fibrosis in a humanized mouse model of pulmonary fibrosis. Consistent with its failure in a Phase 2 clinical trial, Simtuzumab exhibited no therapeutic efficacy in translational in vitro and in vivo assays.

Pulmonary fibrosis is a sequelae of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection that currently lacks effective preventative or therapeutic measures. Post-viral lung fibrosis due to SARS-CoV-2 has been shown to be progressive on selected patients using imaging studies. Persistent infiltration of macrophages and monocytes, a main feature of SARS-CoV-2 pulmonary fibrosis, and long-lived circulating inflammatory monocytes might be driving factors promoting the profibrotic milieu in the lung. The upstream signal(s) that regulates the presence of these immune cells (despite complete viral clearance) remains to be explored. Current data indicate that much of the stimulating signals are localized in the lungs. However, an ongoing low-grade systemic inflammation in long Coronavirus Disease 2019 (COVID-19) symptoms suggests that certain non-pulmonary regulators such as epigenetic changes in hematopoietic stem cells might be critical to the chronic inflammatory response. Since nearly one-third of the world population have been infected, a timely understanding of the underlying pathogenesis leading to tissue remodeling is required. Herein, we review the potential pathogenic mechanisms driving lung fibrosis following SARS-CoV-2 infection based upon available studies and our preliminary findings (Graphical abstract).