JCI Insight

Post-acute sequelae of COVID (PASC) persist in many patients for weeks and months after recovery from initial SARS-CoV-2 infection. Recent evidence suggests that pathological changes in skeletal muscle may contribute significantly to ongoing pain and fatigue, particularly post-exertional malaise. This study aimed to investigate the underlying mechanisms of PASC-related fatigue by examining skeletal muscle function and circulating factors in affected individuals. We conducted a cross-sectional case-control study of patients with fatigue-associated PASC who had experienced mild to moderate COVID-19 without hospitalization. Skeletal muscle biopsies revealed reduced mitochondrial respiration and content in PASC participants compared to healthy controls. This lower respiratory capacity was accompanied by markedly elevated circulating levels of soluble IL-2 receptor alpha subunit (sIL2R), a T cell-specific receptor. In vitro experiments demonstrated that sIL2R directly impairs mitochondrial oxygen consumption and reduces mitochondrial complex III subunit protein levels in cultured muscle cells. These findings suggest a mechanism linking systemic immune dysregulation to muscle-specific mitochondrial dysfunction in PASC. This work provides new insights into the pathophysiology of PASC identifying sIL2R as a promising therapeutic target for addressing mitochondrial deficits in PASC-related fatigue and opening avenues for developing targeted interventions.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with poorly defined pathogenic mechanism and no cure. It is characterized by chronic inflammation, myofibroblast accumulation, and aberrant extracellular matrix (ECM) remodeling. Fibrosis progression is considered to occur due to sustained aberrant fibroblast mechanotransduction: sensing "normal" soft tissue as stiff scarred tissue leading to the overproduction of ECM that then stiffens the microenvironment, thus reinforcing a progressive, stiffness-dependent fibrotic program. How chronic inflammation leads to aberrant mechanotransduction is not well understood. Thy-1 is a regulator of mechanotransduction in fibroblasts. Thy-1 expression is lost in fibroblastic foci, the active sites of fibrosis, although the mechanism of this loss is unknown. We demonstrate that in IPF tissue, the SMA+ fibroproliferative foci express the Type 1 IL-1 receptor (IL-1RI) and IL-1RI-deficient mice did not develop bleomycin-induced pulmonary fibrosis. Using ASC speck formation during inflammasome activation as a marker of mature IL-1{beta} release, we identified the immune compartment as the source of active IL-1{beta} during bleomycin-induced fibrosis. Furthermore, incubating mouse lung fibroblasts on soft (2kPa) hydrogels with IL-1{beta} was sufficient to reduce Thy-1 surface expression and induce v{beta}3 integrin activation. As expected, Thy-1 negative fibroblasts exhibited elevated v{beta}3 integrin activation but surprisingly, Thy-1 negative fibroblasts also expressed higher levels of IL-1RI, potentially linking the immuno-responsive and mechanosensitivity of this fibroblast subpopulation. Leveraging the non-resolving fibrosis that occurs in Thy-1-/- mice, we observed that crossing Thy-1-/- mice onto the IL-1RI-/- background was sufficient to reduce fibrosis. Together, these data indicate that Thy-1 negative fibroblasts are an immuno-responsive subpopulation that also display altered mechanotransduction, potentially serving as the link between the noted inflammation and aberrant mechanotransduction observed in IPF.

Pulmonary fibrosis (PF) is a chronic progressive lung disease histopathologically characterized by fibrotic remodeling and the presence of pathological epithelial and mesenchymal cell populations in the distal lung parenchyma. Within the epithelial compartment, a subset of alveolar type 2 cells (AT2s) enter and persist in an aberrant transitional state. Whether and how these aberrant transitional cells participate in lung fibrosis is not known. To address this, we exploited the SftpcC121G mouse model, where we previously demonstrated that chronic expression of a PF-associated point mutation (C121G) in the AT2-specific surfactant protein C (Sftpc) gene results in spontaneous and progressive fibrosis driven by intrinsic AT2 dysfunction. We utilized single cell RNA sequencing to demonstrate the emergence of pathologic epithelial and mesenchymal cells in the SftpcC121G murine lung fibrosis model, including aberrant transitional alveolar epithelial cells as well as transitional and fibrotic fibroblasts. Aberrant transitional alveolar epithelial cells share similar transcriptional profiles to human aberrant basaloid cells, including the upregulation of profibrotic gene markers (Fn1, Ctgf, Tgfb2, Pdgfb, Spp1), and develop a unique interactome with pathogenic lung fibroblasts. We developed a method to reliably flow sort aberrant transitional alveolar epithelial cells, and we highlight their ability to cause fibrotic activation of fibroblasts in ex vivo organoid assays and using conditioned supernatant, suggesting a profibrotic secretome. We conclude that aberrant transitional alveolar epithelial cells actively contribute to fibrotic lung remodeling through pathogenic activation of alveolar fibroblasts.

The distal bronchioles in Idiopathic Pulmonary Fibrosis (IPF) exhibit histopathological abnormalities such as bronchiolization, peribronchiolar fibrosis and honeycomb cysts that contribute to the overall architectural remodeling of lung tissue seen in the disease. Here we describe an additional histopathologic finding of epithelial desquamation in patients with IPF, wherein epithelial cells detach from the basement membrane of the distal bronchioles. To understand the mechanism driving this pathology, we performed spatial transcriptomics of the epithelial cells and spatial proteomics of the basement membrane of the distal bronchioles from IPF patients and patients with no prior history of lung disease. Our findings reveal a downregulation of cell junctional components, upregulation of epithelial-mesenchymal transition signatures and dysregulated basement membrane matrix in IPF distal bronchioles, facilitating epithelial desquamation. Further, functional assays identified regulation between Collagen IV in the matrix, and the junctional genes JUP and PLEC, that is crucial for maintaining distal bronchiolar homeostasis. In IPF, this balanced regulation between matrix and cell-junctions is disrupted, leading to loss of epithelial adhesion, peribronchiolar fibrosis and epithelial desquamation. Overall, our study suggests that in IPF the interplay between the loss of cell junctions and a dysregulated matrix results in desquamation of distal bronchiolar epithelium and lung remodeling, exacerbating the disease. One Sentence SummaryTwo-way regulation of cell junctional proteins and matrix proteins drives cellular desquamation and fibrosis in the distal bronchioles of patients with Idiopathic Pulmonary Fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two FDA approved anti-fibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Using an in-silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor, originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. We investigated the anti-fibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: (i) in vitro in normal human lung fibroblasts (NHLFs); (ii) in vivo in bleomycin and recombinant adenovirus transforming growth factor-beta (Ad-TGF-{beta}) murine models of pulmonary fibrosis; and (iii) ex vivo in precision cut lung slices from these mouse models. In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone.

A hallmark of idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases is dysregulated repair of the alveolar epithelium. The Hippo pathway effector transcription factors YAP and TAZ have been implicated as essential for type 1 and type 2 alveolar epithelial cell (AT1 and AT2) differentiation in the developing lung, yet aberrant activation of YAP/TAZ is a prominent feature of the dysregulated alveolar epithelium in IPF. In these studies, we sought to define the functional role of YAP/TAZ activity during alveolar regeneration. We demonstrate that Yap and Taz are normally activated in AT2 cells shortly after injury, and deletion of Yap/Taz in AT2 cells led to pathologic alveolar remodeling, failure of AT2 to AT1 cell differentiation, increased collagen deposition, exaggerated neutrophilic inflammation, and increased mortality following injury induced by a single dose of bleomycin. Loss of Yap/Taz activity prior to a LPS injury prevented AT1 cell regeneration, led to intra-alveolar collagen deposition, and resulted in persistent innate inflammation. Together these findings establish that AT2 cell Yap/Taz activity is essential for functional alveolar epithelial repair and prevention of fibrotic remodeling.

Signaling via G protein-coupled receptors (GPCRs) can modulate levels of cyclic adenosine monophosphate (cAMP) and shape the functions of fibroblasts in idiopathic pulmonary fibrosis (IPF). We have identified Chemokine (C-X-C) Motif Ligand 6 (CXCL6) as a potential pro-fibrotic GPCR ligand. We tested the function of CXCL6 in ex vivo human donor and fibrotic lung fibroblasts and in an animal model of pulmonary fibrosis. We also measured levels of CXCL6 in the blood and bronchoalveolar lavage (BAL) of patients with IPF. CXCL6 decreased cAMP levels in a dose-dependent manner in Donor and IPF Fibroblasts. CXCL6 mRNA and protein were localized to epithelial cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased Collagen I and -SMA levels in Donor and IPF Fibroblasts. Silencing of CXCR1/2 as well as Reparixin, a CXCR1/2 inhibitor, blocked effects of CXCL6. Treprostinil blocked effects of CXCL6 only on levels of -SMA but not on Collagen I. CXCL6 levels in the BAL of two separate cohorts of patients with IPF was associated with poor survival. We conclude that high CXCL6 drives fibroblast function and correlates with poor outcomes in IPF.

RationaleMechanical ventilation rapidly induces slow and fast fiber contractile dysfunction in the human diaphragm, which could be attenuated by phrenic nerve stimulation. Here, we present data from a controlled trial of intraoperative phrenic stimulation to offset slow and fast fiber contractile dysfunction and myofilament protein derangements. ObjectivesIn this study, we tested the hypothesis that intraoperative hemidiaphragm stimulation would mitigate slow and fast fiber loss of contractile function in the human diaphragm. MethodsNineteen adults (9 females, age 59 {+/-}12 years) consented to participate. Unilateral phrenic twitch stimulation was applied for one minute, every 30 minutes during cardiothoracic surgery. Thirty minutes following the last stimulation bout, biopsies were obtained from the hemidiaphragms for single fiber force mechanics and quantitation of thin filament protein abundance. Effects of stimulation and fiber type on force mechanics were evaluated with linear mixed models with the subject treated as a random intercept effect. Measurements and Main ResultsSubjects underwent 6 {+/-}2 hemidiaphragm stimulations at 17 {+/-}6 mA, during 278 {+/-}68 minutes of mechanical ventilation. In slow-twitch fibers, cross-sectional area (p<0.0001) and specific force (p<0.0005) were significantly greater on the stimulated side. Longer-duration surgeries were associated with lower slow-twitch specific force (p<0.001). Stimulation did not alter contractile function of fast-twitch fibers or calcium-sensitivity in either fiber type. There were no differences in abundance or phosphorylation of myofilament proteins. ConclusionUnilateral phrenic stimulation during open chest surgery preserved contractile function of slow-twitch diaphragm fibers, but had no effect on relative abundance of sarcomeric proteins.

Myofibroblast differentiation, essential for driving extracellular matrix synthesis in pulmonary fibrosis, requires increased glycolysis. While glycolytic cells must export lactate, the contributions of lactate transporters to myofibroblast differentiation are unknown. In this study, we investigated how MCT1 and MCT4, key lactate transporters, influence myofibroblast differentiation and experimental pulmonary fibrosis. Our findings reveal that inhibiting MCT1 or MCT4 reduces TGF{beta}-stimulated pulmonary myofibroblast differentiation in vitro and decreases bleomycin-induced pulmonary fibrosis in vivo. Through comprehensive metabolic analyses, including bioenergetics, stable isotope tracing, metabolomics, and imaging mass spectrometry in both cells and mice, we demonstrate that inhibiting lactate transport enhances oxidative phosphorylation, reduces reactive oxygen species production, and diminishes glucose metabolite incorporation into fibrotic lung regions. Furthermore, we introduce VB253, a novel MCT4 inhibitor, which ameliorates pulmonary fibrosis in both young and aged mice, with comparable efficacy to established antifibrotic therapies. These results underscore the necessity of lactate transport for myofibroblast differentiation, identify MCT1 and MCT4 as promising pharmacologic targets in pulmonary fibrosis, and support further evaluation of lactate transport inhibitors for patients for whom limited therapeutic options currently exist. SUMMARYSmall molecule inhibitors of lactate transporters, including the novel MCT4 inhibitor VB253, reprogram fibroblast metabolism to prevent myofibroblast differentiation and decrease bleomycin-induced pulmonary fibrosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by progressive multiorgan fibrosis. While the cause of SSc remains unknown, a perturbed vasculature is considered a critical early step in the pathogenesis. Using fibrinogen as a marker of vascular leakage, we found extensive extravascular fibrinogen deposition in the dermis of both limited and diffuse systemic sclerosis disease, and it was present in both early and late-stage patients. Based on a timed series of excision wounds, retention on the fibrin deposit of the splice variant domain, fibrinogen EC, indicated a recent event, while fibrin networks lacking the EC domain were older. Application of this timing tool to SSc revealed considerable heterogeneity in EC domain distribution providing unique insight into disease activity. Intriguingly, the fibrinogen-EC domain also accumulated in macrophages. These observations indicate that systemic sclerosis is characterized by ongoing vascular leakage resulting in extensive interstitial fibrin deposition that is either continually replenished and/or there is impaired fibrin clearance. Unresolved fibrin deposition might then incite chronic tissue remodeling.

The tendon healing process is regulated by the coordinated interaction of multiple cell types and molecular processes. However, these processes are not well-defined leading to a paucity of therapeutic approaches to enhance tendon healing. Scleraxis-lineage (ScxLin) cells are the major cellular component of adult tendon and make time-dependent contributions to the healing process. Prior work from our lab and others suggests heterogeneity within the broader ScxLin population over the course of tendon healing; therefore delineating the temporal and spatial contributions of these cells is critical to understanding and improving the healing process. In the present study we utilize lineage tracing of the adult aScxLin population to determine whether these cells undergo cellular activation and subsequent myofibroblast differentiation, which is associated with both proper healing and fibrotic progression in many tissues. We show that adult aScxLin cells undergo transient activation in the organized cellular bridge at the tendon repair site, contribute to the formation of an organized neotendon, and contribute to a persistent myofibroblast population in the native tendon stubs. The mechanisms dictating this highly specialized spatial response are unknown. We therefore utilized spatial transcriptomics to better define the spatio-molecular program of tendon healing. Integrated transcriptomic analyses across the healing time-course identifies five distinct molecular regions, including key interactions between the inflammatory bridging tissue and highly reactive tendon tissue at the repair site, with adult ScxLin cells being a central player in the transition from native tendon to reactive, remodeling tendon. Collectively, these data provide important insights into both the role of adult ScxLin cells during healing as well as the molecular mechanisms that underpin and coordinate the temporal and spatial healing phenotype, which can be leveraged to enhance the healing process.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease marked by progressive scarring with unknown causes and limited treatments. Myofibroblasts drive fibrosis by depositing excess matrix, however the mechanisms driving fibroblast-to-myofibroblast transformation and how myofibroblast-secreted factors disrupt the alveolar niche, undermining lung repair and regeneration remain poorly understood. Here we show that YAP and TAZ are activated in lung fibroblasts from pulmonary fibrosis patients and bleomycin-treated mice. Targeted deletion of Yap/Taz in fibroblasts significantly dampened the fibroinflammatory response, decreased myofibroblast activation, and ultimately resulted in attenuated fibrosis and enhanced regeneration of alveolar epithelial cells after bleomycin-induced injury. Conversely, fibroblast-specific overexpression of constitutively active YAP (YAP5SA) aggravated fibrosis by amplifying fibroinflammatory responses and simultaneously suppressing alveolar epithelial regeneration. Pharmacological inhibition of YAP/TAZ using verteporfin halted the development of bleomycin-induced fibrosis and even reversed established fibrosis in mice. Verteporfin effectively prevented fibroblast-to-myofibroblast transition and promoted collagen I degradation by lowering TIMP levels and enhancing activation of MMP1 and MMP9, demonstrating the significance of the YAP-TIMP-MMP1/9 axis in facilitating ECM breakdown. Furthermore, YAP activation in fibroblasts disrupted alveolar type II (AT2) cell homeostasis by inducing senescence through IGF1-IGF1R-mediated paracrine signaling. Blocking IGF1 signaling with a neutralizing antibody reduced the number of senescent AT2 cells, demonstrating the significance of the YAP-IGF1-IGF1R axis in maintaining alveolar epithelial cell homeostasis. Targeting YAP/TAZ may offer therapeutic strategies to mitigate pulmonary fibrosis by simultaneously mitigating pathological fibroblast activation, fibroinflammatory response, reducing AT2 cell senescence, and promoting alveolar epithelial regeneration.

Sarcoidosis is an idiopathic inflammatory disorder that is commonly treated with glucocorticoids and there are no approved steroid-sparing medications. There is emerging evidence that Janus kinase (JAK) inhibitors, which inhibit JAK-dependent cytokine activity, may hold promise in sarcoidosis. In this open-label trial, 10 patients with recalcitrant sarcoidosis with cutaneous involvement were treated with tofacitinib 5 mg twice daily. There was no washout period and patients were permitted to continue, taper, or discontinue other treatments. The primary outcome was the change in the Cutaneous Sarcoidosis Activity and Morphology Instrument (CSAMI) activity score after 6 months. Change in internal organ disease activity was also assessed using total lesion glycolysis (TLG) determined by full-body positron emission tomography. A mean reduction in the CSAMI activity score of 82.7% was observed, with 6 patients showing a complete response. Internal organ response data was available in 8 patients; a decrease in TLG of [&ge;]50% was noted in 5 patients, with complete or near complete resolution in 3 (>98% reduction in TLG). Patients were generally able to significantly taper or discontinue their baseline immunosuppressive regimen, which included prednisone in 5 patients. Single cell RNA-sequencing, bulk RNA-sequencing, and high-throughput proteomic analyses were performed on skin and blood as a function of treatment in order to delineate changes in immunologic signals with therapy. We identified CD4+ T cell derived IFN-{gamma} as a central cytokine driver of sarcoidosis and inhibition of its activity was achieved with tofacitinib and correlated closely with clinical improvement. Tofacitinib appears to have impressive activity in treatment of sarcoidosis and likely acts by inhibiting IFN-{gamma}, larger, controlled studies are warranted.

TGF-{beta}-activating integrins promote solid-organ fibrosis, suggesting their use as a molecular marker of disease. Chronic lung allograft dysfunction (CLAD), a progressive fibrotic complication that limits lung transplant survival, is driven by intragraft TGF-{beta} activation. However, the expression patterns of TGF-{beta}-activating integrins remain undefined in lung transplants. Single-cell RNA sequencing in a mouse CLAD model revealed high levels of the TGF-{beta}-activating integrin v{beta}6, which was mainly localized to fibrosis-associated Krt8+ transitional alveolar cells (AT1/2), while tolerant transplants lacked both v{beta}6 expression and Krt8+AT1/2 cells. Molecular imaging with a newly developed positron emission tomography radiotracer specific for v{beta}6, [64Cu]Cu-DOTA-A20-K16R, showed significantly higher uptake in CLAD versus tolerant transplants. In contrast, [64Cu]Cu-DOTA-A20-K16R allograft uptake was reduced by treatments that lowered v{beta}6 expression and CLAD severity. Finally, [64Cu]Cu-DOTA-A20-K16R autoradiographic analysis on human explanted lungs with CLAD showed elevated activity that correlated with v{beta}6 expression. Collectively, these findings demonstrate the potential utility of v{beta}6 molecular imaging to detect CLAD pathogenesis.

Emerging evidence suggests that SARS-CoV-2 infections are characterized by systemic immune responses that appear to be dysregulated with more severe CoViD-19 disease. Lymphopenia and delayed antibody responses are commonly identified in CoViD-19 subjects, and recent reports have demonstrated abrogation of germinal centers in severe CoViD-19. This work assessed a potential mechanistic basis for impaired humoral responses, focusing on the T follicular helper (Tfh) and B cell interface that is critical for germinal center reactions. Here we demonstrated that Tfh activity is impaired in hospitalized relative to ambulatory CoViD-19 subjects, potentially due to decreased expression of the costimulatory molecule ICOS-L on B cells. Functional impairment manifested as a diminished ability to stimulated Tfh derived IFN{gamma} and IL-21, the latter of which is critical for B cell proliferation and differentiation. Activation of Tfh cells by agonism of the ICOS receptor ex vivo by an agonistic antibody stimulated the generation of IFN{gamma}/IL-21 double positive cells from hospitalized CoViD-19 subjects. This report establishes an immunological defect that differentiates ambulatory from hospitalized CoViD and suggests that agents that could restore impaired mechanisms at the Tfh-B cell interface may be of therapeutic value.

The alveolus, the lungs primary gas exchange unit, relies on tightly coordinated interactions between epithelial and endothelial layers. In idiopathic pulmonary fibrosis (IPF), a progressive interstitial lung disease, this architecture is profoundly disrupted. While epithelial and mesenchymal compartments have been extensively studied, the role of pulmonary microvascular endothelial cells (PMVECs) in IPF pathogenesis remains underexplored. Here, we characterize PMVEC alterations in IPF using single-cell RNA sequencing and spatial transcriptomics, identifying subtype-specific markers and demonstrating their progressive loss in fibrotic lungs. To model endothelial dysfunction, we established robust protocols for isolating and culturing primary human ECs and applied a pharmacologically relevant cytokine cocktail (IPF-RC) that mimics the IPF microenvironment. IPF-RC exposure induced hallmark features of endothelial injury, including VE-cadherin loss, increased ICAM1/VCAM1 signaling, impaired barrier integrity, and reduced wound healing and angiogenic capacity. To address the need for translational tools in drug discovery, we optimized and validated a suite of functional, scalable test systems and their endpoints using both primary and commercial endothelial cells. These mechanistic assays reliably recapitulate fibrotic endothelial injury and enable quantitative assessment of therapeutic interventions. Notably, treatment with a cAMP analog partially restored endothelial function, supporting the utility of these models for regenerative and pharmacological screening. Our findings position PMVECs as active participants in IPF progression and present novel, scalable test systems that bridge mechanistic insight with translational application. These models offer a valuable platform for identifying endothelial-targeted therapies aimed at restoring alveolar capillary integrity in fibrotic lung disease.

AbstractFibrosis is driven by the emergence of myofibroblasts, which are the primary producers of the extracellular matrix proteins that form fibrotic lesions. Despite this critical role, detecting myofibroblasts remains challenging due to the paucity of selective markers. We therefore screened for novel myofibroblast-specific markers, discovering that the expression of the close homolog of L1 (ChL-1) and embigin (Emb) distinguish activated myofibroblasts from their quiescent precursors. We report that ChL-1+/Emb+ fibroblasts: (1) emerge during cardiac inflammation, (2) have elevated expression of collagens and inflammatory factors and (3) localise to fibrotic zones - consistent with activated myofibroblasts. Mechanistically, Chl1+/Emb+ myofibroblasts differentiate from resident fibroblasts which upregulate these markers in response to proinflammatory cytokines, such as IL-1 and IL-17. Moreover, we show that embigin could be exploited to target antibody-based therapies to myofibroblasts and confirm this protein as a conserved marker of activated fibroblasts in multiple tissues and settings. Collectively, these findings identify ChL-1 and embigin as novel myofibroblast surface-markers that could be used to identify, enumerate and target this pathogenic population.

Capillary regression destabilizes tissue homeostasis and contributes to chronic organ dysfunction, yet the inflammatory pathways that drive pathological vessel loss remain incompletely defined. We previously identified the inflammatory cytokines TNF and IL-1 as conserved mediators of physiological vessel regression in the neonatal mouse eye, but whether these cytokines contribute to pathological capillary regression in adult mice is unknown. In this study, we investigated the capillary regression that occurs along with inflammation in murine kidneys following irreversible unilateral ureteral obstruction (UUO) surgical challenge. Mice lacking genes encoding global TNF, the endothelial IL-1 receptor IL-1R1, or both (double knockout, DKO) were examined at 10 days after UUO surgery. While loss of the individual genes did not affect peritubular capillary (PTC) regression, PTC regression was significantly reduced in DKO mice. This reduction in PTC regression correlated with less expression of the tubular epithelial injury marker KIM-1. DKO kidneys also displayed less fibrosis by Picrosirius Red and Massons trichrome staining. These findings demonstrate that TNF and endothelial IL-1R1 cooperatively drive pathological capillary regression in the irreversible UUO model of chronic kidney injury and that preservation of PTCs correlates with less renal tubular injury and fibrosis at 10 days after injury. NEW & NOTEWORTHYPathological PTC regression drives progressive kidney injury, but its inflammatory triggers are unclear. Using the UUO model, this study identifies cooperative TNF and endothelial IL-1R1 signaling as key drivers of capillary loss. While deletion of either pathway alone was insufficient, combined loss preserved PTCs, reduced tubular injury, and attenuated fibrosis. These findings highlight synergistic inflammatory signaling in microvascular loss and suggest dual targeting may help preserve PTC integrity in chronic kidney disease.

The renin-angiotensin system is a well-characterized regulator of tissue homeostasis whose clinical relevance has expanded to include lung disorders such as chronic obstructive pulmonary disease (COPD)-associated emphysema, idiopathic pulmonary fibrosis, and COVID-19. Despite this interest, the cell-specific localization of angiotensin receptors in the human lung has remained poorly defined, in part due to limitations of available antibody reagents. Here, we define the expression patterns of the two predominant angiotensin receptors, AGTR1 and AGTR2, using complementary bulk and single-nucleus transcriptomic datasets from human lung tissue. We demonstrate that these receptors exhibit mutually exclusive, compartment-specific localization, with AGTR1 expressed in lung pericytes and AGTR2 expressed in alveolar epithelial type 2 cells. AGTR1 is detectable in isolated lung pericytes, and spatial colocalization with pericyte markers confirmed within the airspace microvasculature compartment by RNAscope. Airspace pericyte abundance was reduced in an experimental emphysema model but restored by pharmacologic attenuation of AGTR1 signaling commensurate with airspace repair. In COPD lungs, AGTR1 expression showed heterogeneous, disease-associated dysregulation across stromal populations, including upregulation in alveolar fibroblasts. Bulk transcriptomics also revealed aging-associated redistribution of AGTR1 expression into stromal compartments. Angiotensin II and cigarette smoke impaired pericyte migration toward endothelial cells, while combined exposure suppressed pericyte proliferation. Together, these findings identify AGTR1 as a new highly selective marker of lung pericytes and a regulator of pericyte behaviors within the airspace microvasculature. These findings provide a cell-resolved framework for angiotensin signaling with direct relevance to airspace resilience and therapeutic targeting.

Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion-inhibited pig airway CF model revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Utilizing proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, proteomics analyses of CF sputum revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and within SMGs of the CF lung.