Type III interferons (IFNλ) are innate immune cytokines that limit viral replication and coordinate tissue repair through the induction of interferon stimulated genes (ISGs). This response must be tightly regulated to avoid excessive responses that result in the disruption of tissue barrier integrity or inefficient responses that allow for pathogen escape. Here we examine the contribution of Mitogen Activated Protein Kinase (MAPK) signaling on IFNλ-mediated antiviral activity. We find that extracellular-signal-regulated kinase 5 (ERK5), a poorly characterized member of the conventional MAPK family, potentiates the antiviral efficacy of IFNλ. Chemical inhibition and genetic targeting of ERK5 during IFNλ treatment of cells results in a decrease in ISG induction and impaired control of viral infections. This decrease in IFNλ antiviral efficacy in the absence of ERK5 kinase activity corresponded to lowered STAT1 phosphorylation, revealing a noncanonical role for ERK5 in STAT1 activation downstream of IFNλ. In contrast, type I IFN antiviral signaling is largely resistant to ERK5 modulation. Altogether, we identify ERK5 as a potentiator of STAT1 activation, ISG expression, and antiviral activity following type III IFN stimulation.SIGNIFICANCE Regulation of type III interferons (IFNλ) at mucosal barriers in response infection to mitigate viral replication and support barrier integrity. The specific mechanistic requirements for MAPK signaling to sustain IFNλ-mediated gene expression have remained elusive. Amongt the least characterized members of the MAPK family, the role of ERK5 in regulating host inflammatory responses has been hampered by off-target effects of kinase inhibitors. Here, we combine pharmacological and genetic approaches to specifically demonstrate that ERK5 promotes antiviral immunity in epithelial cells. Mechanistically, ERK5 enhances the activation of STAT1 in response to IFN stimulation to augment the transcription of IFN-stimulated genes. Our work demonstrates that therapeutic modulation of MAPK and IFN signaling pathway co-integration could distinguish between the protective and deleterious outcomes of IFN expression.One-sentence summary ERK5 potentiates IFN lambda responses.### Competing Interest StatementThe authors have declared no competing interest.

Influenza virus activates cellular inflammasome pathways, which can be either beneficial or detrimental to infection outcomes. Here, we investigated the role of the inflammasome-activated pore-forming protein gasdermin D (GSDMD) during infection. Ablation of GSDMD in knockout (KO) mice significantly attenuated virus-induced weight loss, lung dysfunction, lung histopathology, and mortality compared with wild type (WT) mice, despite similar viral loads. Infected GSDMD KO mice exhibited decreased inflammatory gene signatures revealed by lung transcriptomics, which also implicated a diminished neutrophil response. Importantly, neutrophil depletion in infected WT mice recapitulated the reduced mortality and lung inflammation observed in GSDMD KO animals, while having no additional protective effects in GSDMD KOs. These findings reveal a new function for GSDMD in promoting lung neutrophil responses that amplify influenza virus-induced inflammation and pathogenesis. Targeting the GSDMD/neutrophil axis may provide a new therapeutic avenue for treating severe influenza.

Abstract Hypoxia, a state of insufficient oxygen availability, promotes cellular lactate production. Lactate levels are increased in lungs from patients with idiopathic pulmonary fibrosis (IPF), a disease characterized by excessive scar formation, and lactate is implicated in the pathobiology of lung fibrosis. However, the mechanisms underlying the effects of hypoxia and lactate on fibroblast phenotype are poorly understood. We exposed normal and IPF lung fibroblasts to persistent hypoxia and found that increased lactate generation by IPF fibroblasts was driven by the FoxM1‐dependent increase of lactate dehydrogenase A (LDHA) coupled with decreased LDHB that was not observed in normal lung fibroblasts. Importantly, hypoxia reduced α‐smooth muscle actin (α‐SMA) expression in normal fibroblasts but had no significant impact on this marker of differentiation in IPF fibroblasts. Treatment of control and IPF fibroblasts with TGF‐β under hypoxic conditions did not significantly change LDHA or LDHB expression. Surprisingly, lactate directly induced the differentiation of normal, but not IPF fibroblasts under hypoxic conditions. Moreover, while expression of GPR‐81, a G‐protein‐coupled receptor that binds extracellular lactate, was increased by hypoxia in both normal and IPF fibroblasts, its inhibition or silencing only suppressed lactate‐mediated differentiation in normal fibroblasts. These studies show that hypoxia differentially affects normal and fibrotic fibroblasts, promoting increased lactate generation by IPF fibroblasts through regulation of the LDHA/LDHB ratio and promoting normal lung fibroblast responsiveness to lactate through GPR‐81. This supports a novel paradigm in which lactate may serve as a paracrine intercellular signal in oxygen‐deficient microenvironments.

Fibrosis is characterized by inappropriately persistent myofibroblast accumulation and excessive extracellular matrix deposition with the disruption of tissue architecture and organ dysfunction. Regulated death of reparative mesenchymal cells is critical for normal wound repair, but profibrotic signaling promotes myofibroblast resistance to apoptotic stimuli. A complex interplay between immune cells and structural cells underlies lung fibrogenesis. However, there is a paucity of knowledge on how these cell populations interact to orchestrate physiologic and pathologic repair of the injured lung. In this context, gasdermin-D (GsdmD) is a cytoplasmic protein that is activated following cleavage by inflammatory caspases and induces regulated cell death by forming pores in cell membranes. This study was undertaken to evaluate the impact of human (Thp-1) monocyte-derived extracellular vesicles and GsdmD on human lung fibroblast death. Our data show that active GsdmD delivered by monocyte-derived extracellular vesicles induces caspase-independent fibroblast and myofibroblast death. This cell death was partly mediated by GsdmD-independent induction of cellular inhibitor of apoptosis 2 (cIAP-2) in the recipient fibroblast population. Our findings, to our knowledge, define a novel paradigm by which inflammatory monocytes may orchestrate the death of mesenchymal cells in physiologic wound healing, illustrating the potential to leverage this mechanism to eliminate mesenchymal cells and facilitate the resolution of fibrotic repair.

SESSION TITLE: Diagnosis and Characterization of ILD: From Bench to BedsideSESSION TYPE: Original InvestigationsPRESENTED ON: 10/17/2022 01:30 pm - 02:30 pmPURPOSE: (Myo)fibroblasts are the primary effector cells implicated in fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). While myofibroblast apoptosis is crucial for the resolution of wound repair, fibrotic lung fibroblasts show resistance to apoptotic stimuli. X-linked Inhibitor of Apoptosis (XIAP) is increased in IPF lung fibroblasts and regulates fibroblast resistance to apoptosis. Our goal was to investigate the expression and stability of XIAP and its structural homologue, cIAP-1, in normal and fibrotic lung fibroblasts.METHODS: The normal fibroblast cell line IMR-90 was used along with primary fibroblasts from normal (NHLFs) and IPF lungs (IPFFs). XIAP and cIAP-1 expression was assessed with quantitative real-time rtPCR and Western blotting. siRNA transfection was used to knock-down each target. Cycloheximide (CHX) and MG-132 were used to inhibit protein synthesis and proteasomal degradation, respectively.RESULTS: Increased XIAP expression in IPF fibroblasts was confirmed, and XIAP induction by TGF-β was shown in NHLF and IPFF. In contrast, cIAP-1 expression did not differ between NHLF and IPFF, and induction by TGF-β was not evident. In IMR-90 fibroblasts, CHX led to a reduction of XIAP protein within 4-6 hours while MG-132 treatment resulted in a 2-fold increase, suggesting that homeostatic expression of XIAP is tightly regulated by transcription and proteasomal degradation. This pattern was recapitulated in NHLFs. However, in IPFFs, MG-132 had no impact on XIAP protein suggesting that increased XIAP in these cells may be the result of impaired proteasomal processing. In contrast to XIAP, cIAP-1 was not clearly affected by CHX or MG-132, supporting a high degree of protein stability. Consistently, siRNA targeting XIAP was highly efficient in reduction of transcript and protein, while siRNA targeting of cIAP-1 effectively reduced transcript levels but had no impact on protein.CONCLUSIONS: XIAP and cIAP-1 expression are differentially and independently regulated in fibroblasts. XIAP undergoes rapid turnover in normal fibroblasts while cIAP-1 protein expression remains stable, suggesting distinct functions of these structurally homologous IAPs. XIAP protein has increased stability in IPFFs compared to NHLFs, suggesting defective proteasomal processing in the disease fibroblasts.CLINICAL IMPLICATIONS: Myofibroblast accumulation is a hallmark of fibrosis while myofibroblast apoptosis heralds the resolution of wound repair. XIAP is implicated in fibroblast resistance to apoptosis, and inhibition of XIAP and its structural homologues, cIAP-1 and cIAP-2, reduced lung fibrosis in vivo. The roles of individual IAPs in fibrosis have not been determined. These studies support a potential role for impaired proteasomal degradation of XIAP in the pathobiology of IPF.DISCLOSURES: No relevant relationships by Hannah BoneNo relevant relationships by Jeffrey HorowitzNo relevant relationships by Sankalp MalhotraNo relevant relationships by jayendra prasad SESSION TITLE: Diagnosis and Characterization of ILD: From Bench to Bedside SESSION TYPE: Original Investigations PRESENTED ON: 10/17/2022 01:30 pm - 02:30 pm PURPOSE: (Myo)fibroblasts are the primary effector cells implicated in fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). While myofibroblast apoptosis is crucial for the resolution of wound repair, fibrotic lung fibroblasts show resistance to apoptotic stimuli. X-linked Inhibitor of Apoptosis (XIAP) is increased in IPF lung fibroblasts and regulates fibroblast resistance to apoptosis. Our goal was to investigate the expression and stability of XIAP and its structural homologue, cIAP-1, in normal and fibrotic lung fibroblasts. METHODS: The normal fibroblast cell line IMR-90 was used along with primary fibroblasts from normal (NHLFs) and IPF lungs (IPFFs). XIAP and cIAP-1 expression was assessed with quantitative real-time rtPCR and Western blotting. siRNA transfection was used to knock-down each target. Cycloheximide (CHX) and MG-132 were used to inhibit protein synthesis and proteasomal degradation, respectively. RESULTS: Increased XIAP expression in IPF fibroblasts was confirmed, and XIAP induction by TGF-β was shown in NHLF and IPFF. In contrast, cIAP-1 expression did not differ between NHLF and IPFF, and induction by TGF-β was not evident. In IMR-90 fibroblasts, CHX led to a reduction of XIAP protein within 4-6 hours while MG-132 treatment resulted in a 2-fold increase, suggesting that homeostatic expression of XIAP is tightly regulated by transcription and proteasomal degradation. This pattern was recapitulated in NHLFs. However, in IPFFs, MG-132 had no impact on XIAP protein suggesting that increased XIAP in these cells may be the result of impaired proteasomal processing. In contrast to XIAP, cIAP-1 was not clearly affected by CHX or MG-132, supporting a high degree of protein stability. Consistently, siRNA targeting XIAP was highly efficient in reduction of transcript and protein, while siRNA targeting of cIAP-1 effectively reduced transcript levels but had no impact on protein. CONCLUSIONS: XIAP and cIAP-1 expression are differentially and independently regulated in fibroblasts. XIAP undergoes rapid turnover in normal fibroblasts while cIAP-1 protein expression remains stable, suggesting distinct functions of these structurally homologous IAPs. XIAP protein has increased stability in IPFFs compared to NHLFs, suggesting defective proteasomal processing in the disease fibroblasts. CLINICAL IMPLICATIONS: Myofibroblast accumulation is a hallmark of fibrosis while myofibroblast apoptosis heralds the resolution of wound repair. XIAP is implicated in fibroblast resistance to apoptosis, and inhibition of XIAP and its structural homologues, cIAP-1 and cIAP-2, reduced lung fibrosis in vivo. The roles of individual IAPs in fibrosis have not been determined. These studies support a potential role for impaired proteasomal degradation of XIAP in the pathobiology of IPF. DISCLOSURES: No relevant relationships by Hannah Bone No relevant relationships by Jeffrey Horowitz No relevant relationships by Sankalp Malhotra No relevant relationships by jayendra prasad