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Cellular senescence is crucial in the progression of idiopathic pulmonary fibrosis (IPF), but it is not evident whether the standard-of-care (SOC) drugs, nintedanib and pirfenidone, have senolytic properties. To address this question, we performed colorimetric and fluorimetric assays, qRT-PCR, and western blotting to evaluate the effect of SOC drugs and D + Q on senescent normal and IPF lung fibroblasts. In this study, we found that SOC drugs did not provoke apoptosis in the absence of death ligand in normal or IPF senescent lung fibroblasts. Nintedanib increased caspase-3 activity in the presence of Fas Ligand in normal but not in IPF senescent fibroblasts. Conversely, nintedanib enhanced B cell lymphoma 2 expression in senescent IPF lung fibroblasts. Moreover, in senescent IPF cells, pirfenidone induced mixed lineage kinase domain-like pseudokinase phosphorylation, provoking necroptosis. Furthermore, pirfenidone increased transcript levels of FN1 and COL1A1 in senescent IPF fibroblasts. Lastly, D + Q augmented growth differentiation factor 15 (GDF15) transcript and protein levels in both normal and IPF senescent fibroblasts. Taken together, these results establish that SOC drugs failed to trigger apoptosis in senescent primary human lung fibroblasts, possibly due to enhanced Bcl-2 levels by nintedanib and the activation of the necroptosis pathway by pirfenidone. Together, these data revealed the inefficacy of SOC drugs to target senescent cells in IPF.

Epithelial plasticity has been suggested in lungs of mice following genetic depletion of stem cells but is of unknown physiological relevance. Viral infection and chronic lung disease share similar pathological features of stem cell loss in alveoli, basal cell (BC) hyperplasia in small airways, and innate immune activation, that contribute to epithelial remodeling and loss of lung function. We show that a novel subset of distal airway secretory cells, intralobar serous (IS) cells, are activated to assume BC fates following influenza virus infection. Injury-induced hyperplastic BC (hBC) differ from pre-existing BC by high expression of IL-22Ra1 and undergo IL-22-dependent expansion for colonization of injured alveoli. Resolution of virus-elicited inflammation resulted in BC>IS re-differentiation in repopulated alveoli, and increased local expression of antimicrobial factors, but failed to replace normal alveolar epithelium. Epithelial plasticity therefore protects against mortality from acute respiratory viral infection but results in distal lung remodeling and loss of lung function.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease of unmet medical need. It is characterized by formation of scar tissue leading to a progressive and irreversible decline in lung function. IPF is associated with repeated injury, which may alter the composition of the extracellular matrix (ECM). Here, we demonstrate that IPF patient-derived pulmonary ECM drives profibrotic response in normal human lung fibroblasts (NHLF) in a 3D spheroid assay. Next, we reveal distinct alterations in composition of the diseased ECM, identifying potentially novel associations with IPF. Growth differentiation factor 15 (GDF15) was identified among the most significantly upregulated proteins in the IPF lung-derived ECM. In vivo, GDF15 neutralization in a bleomycin-induced lung fibrosis model led to significantly less fibrosis. In vitro, recombinant GDF15 (rGDF15) stimulated α smooth muscle actin (αSMA) expression in NHLF, and this was mediated by the activin receptor-like kinase 5 (ALK5) receptor. Furthermore, in the presence of rGDF15, the migration of NHLF in collagen gel was reduced. In addition, we observed a cell type-dependent effect of GDF15 on the expression of cell senescence markers. Our data suggest that GDF15 mediates lung fibrosis through fibroblast activation and differentiation, implicating a potential direct role of this matrix-associated cytokine in promoting aberrant cell responses in disease.

Rationale: Aberrant lung remodeling in idiopathic pulmonary fibrosis (IPF) is characterized by elevated MMP9 (matrix metalloproteinase 9) expression, but the precise role of this matrix metalloproteinase in this disease has yet to be fully elucidated. Objectives: To evaluate antifibrotic effects of MMP9 inhibition on IPF. Methods: Quantitative genomic, proteomic, and functional analyses both in vitro and in vivo were used to determine MMP9 expression in IPF cells and the effects of MMP9 inhibition on profibrotic mechanisms. Measurements and Main Results: In the present study, we demonstrate that MMP9 expression was increased in airway basal cell (ABC)-like cells from IPF lungs compared with ABC cells from normal lungs. The inhibition of MMP9 activity with an anti-MMP9 antibody, andecaliximab, blocked TGF-β1 (transforming growth factor β1)-induced Smad2 phosphorylation. However, in a subset of cells from patients with IPF, TGF-β1 activation in their ABC-like cells was unaffected or enhanced by MMP9 blockade (i.e., nonresponders). Further analysis of nonresponder ABC-like cells treated with andecaliximab revealed an association with type 1 IFN expression, and the addition of IFNα to these cells modulated both MMP9 expression and TGF-β1 activation. Finally, the inhibition of MMP9 ameliorated pulmonary fibrosis induced by responder lung cells but not a nonresponder in a humanized immunodeficient mouse model of IPF. Conclusions: Together, these data demonstrate that MMP9 regulates the activation of ABC-like cells in IPF and that targeting this MMP might be beneficial to a subset of patients with IPF who show sufficient expression of type 1 IFNs.

Rationale: Declining lung function in patients with interstitial lung disease is accompanied by epithelial remodeling and progressive scarring of the gas-exchange region. There is a need to better understand the contribution of basal cell hyperplasia and associated mucosecretory dysfunction to the development of idiopathic pulmonary fibrosis (IPF). Objectives: We sought to decipher the transcriptome of freshly isolated epithelial cells from normal and IPF lungs to discern disease-dependent changes within basal stem cells. Methods: Single-cell RNA sequencing was used to map epithelial cell types of the normal and IPF human airways. Organoid and air–liquid interface cultures were used to investigate functional properties of basal cell subtypes. Measurements and Main Results: We found that basal cells included multipotent and secretory primed subsets in control adult lung tissue. Secretory primed basal cells include an overlapping molecular signature with basal cells obtained from the distal lung tissue of IPF lungs. We confirmed that NOTCH2 maintains undifferentiated basal cells and restricts basal-to-ciliated differentiation, and we present evidence that NOTCH3 functions to restrain secretory differentiation. Conclusions: Basal cells are dynamically regulated in disease and are specifically biased toward the expansion of the secretory primed basal cell subset in IPF. Modulation of basal cell plasticity may represent a relevant target for therapeutic intervention in IPF.

RationaleCellular senescence is crucial in the progression of idiopathic pulmonary fibrosis (IPF), but it is yet unclear whether the standard-of-care (SOC) drugs nintedanib and pirfenidone have senolytic properties.ObjectivesWe attempted to illuminate the effects of SOC drugs on senescent normal and IPF lung fibroblasts in vitro.MethodsColorimetric/fluorimetric assays, qRT-PCR, and western blotting were used to evaluate the effect of SOC drugs on senescent normal and IPF lung fibroblasts.ResultsSOC drugs did not induce apoptosis in the absence of death ligands in either normal or IPF senescent cells. Nintedanib increased caspase-3 activity in the presence of Fas Ligand (FasL) in normal but not in IPF senescent fibroblasts. Conversely, nintedanib enhanced B cell lymphoma (Bcl)-2 expression in senescent IPF lung fibroblasts. Moreover, in senescent IPF cells, pirfenidone alone induced mixed lineage kinase domain-like pseudokinase (MLKL) phosphorylation, provoking necroptosis. However, fragmented gasdermin D, indicating pyroptosis, was not detected under any condition. In addition, SOC drugs increased transcript levels of fibrotic and senescence markers in senescent IPF fibroblasts, whereas D+Q inhibited all these markers. Finally, D+Q enhanced growth differentiation factor 15 (GDF15) transcript and protein levels in both normal and IPF senescent fibroblasts.ConclusionsIn the presence and absence of the extrinsic pro-apoptotic ligands, SOC drugs failed to trigger apoptosis in senescent fibroblasts, possibly due to enhanced Bcl-2 levels and the activation of the necroptosis pathway. SOC drugs elevated fibrotic and senescence markers in IPF lung fibroblasts. Together, these data demonstrated the inefficacy of SOC in targeting senescent cells. Further investigation is required to fully elucidate the therapeutic implications of SOC drugs on other senescent cell types in IPF.

Stem cell factor (SCF) and its receptor c-kit have been implicated in inflammation, tissue remodeling, and fibrosis. Ingenuity Integrated Pathway Analysis of gene expression array data sets showed an upregulation of SCF transcripts in idiopathic pulmonary fibrosis (IPF) lung biopsies compared with tissue from nonfibrotic lungs that are further increased in rapid progressive disease. SCF248, a cleavable isoform of SCF, was abundantly and preferentially expressed in human lung fibroblasts and fibrotic mouse lungs relative to the SCF220 isoform. In fibroblast-mast cell coculture studies, blockade of SCF248 using a novel isoform-specific anti-SCF248 monoclonal antibody (anti-SCF248), attenuated the expression of COL1A1, COL3A1, and FN1 transcripts in cocultured IPF but not normal lung fibroblasts. Administration of anti-SCF248 on days 8 and 12 after bleomycin instillation in mice significantly reduced fibrotic lung remodeling and col1al, fn1, acta2, tgfb, and ccl2 transcript expression. In addition, bleomycin increased numbers of c-kit+ mast cells, eosinophils, and ILC2 in lungs of mice, whereas they were not significantly increased in anti-SCF248-treated animals. Finally, mesenchymal cell-specific deletion of SCF significantly attenuated bleomycin-mediated lung fibrosis and associated fibrotic gene expression. Collectively, these data demonstrate that SCF is upregulated in diseased IPF lungs and blocking SCF248 isoform significantly ameliorates fibrotic lung remodeling in vivo suggesting that it may be a therapeutic target for fibrotic lung diseases.

Progressive tissue fibrosis, including idiopathic pulmonary fibrosis (IPF), is characterized by excessive recruitment of fibroblasts to sites of tissue injury and unremitting extracellular matrix deposition associated with severe morbidity and mortality. However, the molecular mechanisms that control progressive IPF have yet to be fully determined. Previous studies suggested that invasive fibroblasts drive disease progression in IPF. Here, we report profiling of invasive and noninvasive fibroblasts from IPF patients and healthy donors. Pathway analysis revealed that the activated signatures of the invasive fibroblasts, the top of which was ERBB2 (HER2), showed great similarities to those of metastatic lung adenocarcinoma cancer cells. Activation of HER2 in normal lung fibroblasts led to a more invasive genetic program and worsened fibroblast invasion and lung fibrosis, while antagonizing HER2 signaling blunted fibroblast invasion and ameliorated lung fibrosis. These findings suggest that HER2 signaling may be a key driver of fibroblast invasion and serve as an attractive target for therapeutic intervention in IPF.

Rationale: The Toll-like receptor 3 Leu412Phe (TLR3 L412F) polymorphism attenuates cellular anti-viral responses and is associated with accelerated disease progression in IPF. The role of TLR3 L412F in bacterial infection in IPF or in acute exacerbations (AE) has not been reported. Objective: To characterize the association between TLR3 L412F and AE-related death in IPF. To determine the effect of TLR3 L412F on the lung microbiome and on anti-bacterial TLR-responses of primary lung fibroblasts from IPF patients. Methods: TLR-mediated anti-bacterial and anti-viral responses were quantitated in L412F-wild-type and 412F-heterozygous primary lung fibroblasts from IPF patients using ELISA, western blot analysis and qPCR. Hierarchical heatmap analysis was employed to establish bacterial and viral clustering in nasopharyngeal lavage (NPL) samples from AE-IPF patients. 16S rRNA qPCR and pyrosequencing were used to determine the effect of TLR3 L412F on the IPF lung microbiome. Measurements and Main Results: A significant increase in AE-related death in 412F-variant IPF patients was reported. We established that 412F-heterozygous IPF lung fibroblasts have reduced anti-bacterial TLR responses to LPS (TLR4), Pam3CYSK4 (TLR1/2), flagellin (TLR5) and FSL-1 (TLR6/1) and have reduced responses to live Pseudomonas aeruginosa infection. Using 16S rRNA sequencing, we demonstrated that 412F-heterozygous IPF patients have a dysregulated lung microbiome with increased frequencies of Streptococcus and Staphylococcus spp. Conclusions: This study reveals that TLR3 L412F dysregulates the IPF lung microbiome and reduces the responses of IPF lung fibroblasts to bacterial TLR-agonists and live bacterial infection. These findings identify a candidate role for TLR3 L412F in viral- and bacterial-mediated AE-death.

Idiopathic pulmonary fibrosis (IPF) is the most frequently diagnosed idiopathic interstitial pneumonia and hence the most studied of the interstitial lung diseases (ILDs). Dating back to the 1970s, the consensus was that the unrelenting fibrosis in IPF/ILD was triggered by an “alveolitis” that involved key innate immune or inflammatory mechanisms. With the observation that classic immunosuppressive therapies failed to alter the disease course in IPF, focus shifted toward the pathogenesis paradigm in which alveolar/bronchiolar epithelial cells were the key drivers of fibrosis in the absence of innate immune activation in this disease. However, with the advent of more sophisticated research tools (i.e., single-cell RNA sequencing) and a more complete understanding of the complexity of the innate immune system, active investigation of strategies that modulate the activity of keys cells in the innate immune system including monocytes, macrophages, neutrophils, myeloid-derived suppressor cells (MDSC), innate lymphoid cells (ILCs), and dendritic cells (DCs) in IPF is now widespread. Thus, while the precise role of the innate immune system in IPF still remains unknown, a growing consensus from both basic and clinical research studies highlights the fact that this arm of the immune system is not an innocent bystander in IPF.

RationaleContribution of central lung tissues to pathogenesis of idiopathic pulmonary fibrosis (IPF) remains unknown.ObjectiveTo ascertain the relationship between cell types of IPF-central and IPF-peripheral lung explants using RNA sequencing (RNA-seq) transcriptome.MethodsBiopsies of paired IPF-central and IPF-peripheral along with non-IPF lungs were selected by reviewing H&E data. Criteria for differentially expressed genes (DEG) were set at false discovery rate 2. Computational cell composition deconvolution was performed. Signature scores were computed for each cell type.FindingsComparison of central IPF versus non-IPF identified 1723 DEG (1522 upregulated and 201 downregulated). Sixty-two per cent (938/1522) of the mutually upregulated genes in central IPF genes were also upregulated in peripheral IPF versus non-IPF. Moreover, 85 IPF central-associated genes (CAG) were upregulated in central IPF versus both peripheral IPF and central non-IPF. IPF single-cell RNA-seq analysis revealed the highest CAG signature score in myofibroblasts and significantly correlated with a previously published activated fibroblasts signature (r=0.88, p=1.6×10−4). CAG signature scores were significantly higher in IPF than in non-IPF myofibroblasts (p=0.013). Network analysis of central-IPF genes identified a module significantly correlated with the deconvoluted proportion of myofibroblasts in central IPF and anti-correlated with inflammation foci trait in peripheral IPF. The module genes were over-represented in idiopathic pulmonary fibrosis signalling pathways.InterpretationGene expression in central IPF lung regions demonstrates active myofibroblast features that contributes to disease progression. Further elucidation of pathological transcriptomic state of cells in the central regions of the IPF lung that are relatively spared from morphological rearrangements may provide insights into molecular changes in the IPF progression.

Idiopathic Pulmonary Fibrosis (IPF) is a disease with a devastating prognosis characterized by unrelenting lung scarring. Aberrant activation of lung fibroblasts is a key feature of this disease, yet the key pathways responsible for this are poorly understood. Mitogen-activated protein kinase, kinase, kinase- 19 (MAP3K19) was recently shown to be upregulated in IPF and this MAPK has a key role in target gene transcription in the TGF-β pathway. Herein, we further investigate the role of MAP3K19 in cultured normal and IPF fibroblasts and in a humanized SCID mouse model of IPF employing both short interfering (si) RNA and novel small-molecule inhibitors directed at this kinase. Targeting MAP3K19 had significant inhibitory effects on the expression of both alpha smooth muscle actin and extracellular matrix in cultured human IPF fibroblasts. Quantitative protein and biochemical assays, as well as histological analysis, showed that MAP3K19 was required for the development of lung fibrosis in SCID mice humanized with IPF lung fibroblasts. MAP3K19 was required for IPF myofibroblast differentiation, and targeting its activity attenuated the profibrotic activity of these cells both in vitro and in an adoptive transfer SCID model of pulmonary fibrosis.

The unfolded protein response (UPR) is a direct consequence of cellular endoplasmic reticulum (ER) stress and a key disease driving mechanism in IPF. The resolution of the UPR is directed by PPP1R15A (GADD34) and leads to the restoration of normal ribosomal activity. While the role of PPP1R15A has been explored in lung epithelial cells, the role of this UPR resolving factor has yet to be explored in lung mesenchymal cells. The objective of the current study was to determine the expression and role of PPP1R15A in IPF fibroblasts and in a bleomycin-induced lung fibrosis model. A survey of IPF lung tissue revealed that PPP1R15A expression was markedly reduced. Targeting PPP1R15A in primary fibroblasts modulated TGF-β-induced fibroblast to myofibroblast differentiation and exacerbated pulmonary fibrosis in bleomycin-challenged mice. Interestingly, the loss of PPP1R15A appeared to promote lung fibroblast senescence. Taken together, our findings demonstrate the major role of PPP1R15A in the regulation of lung mesenchymal cells, and regulation of PPP1R15A may represent a novel therapeutic strategy in IPF.

Mesenchymal vesicular GHR supports alveolar epithelial progenitor cell renewal and limits lung fibrosis., Recent studies have identified impaired type 2 alveolar epithelial cell (ATII) renewal in idiopathic pulmonary fibrosis (IPF) human organoids and severe fibrosis when ATII is defective in mice. ATIIs function as progenitor cells and require supportive signals from the surrounding mesenchymal cells. The mechanisms by which mesenchymal cells promote ATII progenitor functions in lung fibrosis are incompletely understood. We identified growth hormone receptor (GHR) is mainly expressed in mesenchymal cells, and its expression is substantially decreased in IPF lungs. Higher levels of GHR expression correlated with better lung function in patients with IPF. Profibrotic mesenchymal cells retarded ATII growth and were associated with suppressed vesicular GHR expression. Vesicles enriched with Ghr promote ATII proliferation and diminished pulmonary fibrosis in mesenchymal Ghr-deficient mice. Our findings demonstrate a previously unidentified mesenchymal paracrine signaling coordinated by GHR that is capable of supporting ATII progenitor cell renewal and limiting the severity of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant repair that diminishes lung function via mechanisms that remain poorly understood. CC chemokine receptor (CCR10) and its ligand CCL28 were both elevated in IPF compared with normal donors. CCR10 was highly expressed by various cells from IPF lungs, most notably stage-specific embryonic antigen-4-positive mesenchymal progenitor cells (MPCs). In vitro, CCL28 promoted the proliferation of CCR10+ MPCs while CRISPR/Cas9-mediated targeting of CCR10 resulted in the death of MPCs. Following the intravenous injection of various cells from IPF lungs into immunodeficient (NOD/SCID-γ, NSG) mice, human CCR10+ cells initiated and maintained fibrosis in NSG mice. Eph receptor A3 (EphA3) was among the highest expressed receptor tyrosine kinases detected on IPF CCR10+ cells. Ifabotuzumab-targeted killing of EphA3+ cells significantly reduced the numbers of CCR10+ cells and ameliorated pulmonary fibrosis in humanized NSG mice. Thus, human CCR10+ cells promote pulmonary fibrosis, and EphA3 mAb-directed elimination of these cells inhibits lung fibrosis.

Summary Pulmonary mesenchymal cells are critical players in both the mouse and human during lung development and disease states. They are increasingly recognized as highly heterogeneous, but there is no consensus on subpopulations or discriminative markers for each subtype. We completed scRNA-seq analysis of mesenchymal cells from the embryonic, postnatal, adult and aged fibrotic lungs of mice and humans. We consistently identified and delineated the transcriptome of lipofibroblasts, myofibroblasts, smooth muscle cells, pericytes, mesothelial cells, and a novel population characterized by Ebf1 expression. Subtype selective transcription factors and putative divergence of the clusters during development were described. Comparative analysis revealed orthologous subpopulations with conserved transcriptomic signatures in murine and human lung mesenchymal cells. All mesenchymal subpopulations contributed to matrix gene expression in fibrosis. This analysis would enhance our understanding of mesenchymal cell heterogeneity in lung development, homeostasis and fibrotic disease conditions., Graphical abstract, Highlights • Comprehensively profiling of lung mesenchymal subtypes in development and fibrosis. • Identification of novel lung mesenchymal cell subtypes in development and fibrosis. • Delineation of novel discriminative and consistent mesenchymal subtype markers. • Redefinition of the cell contributors of ECM in pulmonary fibrosis., Cell biology; Organizational aspects of cell biology; Pathophysiology

Aging is a critical risk factor in progressive lung fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). Loss of integrity of type 2 alveolar epithelial cells (AEC2s) is the main causal event in the pathogenesis of IPF. To systematically examine the genomic program changes of AEC2s with aging and lung injury, we performed unbiased single cell RNA-seq analyses of lung epithelial cells from either uninjured or bleomycin-injured young and old mice. Major lung epithelial cell types were readily identified with canonical cell markers in our dataset. Heterogenecity of AEC2s was apparent, and AEC2s were then classified into three subsets according to their gene signatures. Genes related to lipid metabolism and glycolysis were significantly altered within these three clusters of AEC2s, and also affected by aging and lung injury. Importantly, IPF AEC2s showed similar genomic programming and metabolic changes as that of AEC2s from bleomycin injured old mouse lungs relative to controls. Furthermore, perturbation of both lipid metabolism and glycolysis significantly changed progenitor renewal capacity in 3-Demensional organoid culture of AEC2s. Taken togather, this work identified metabolic defects of AEC2s in aging and during lung injury. Strategies to rectify these altered programs would promote AEC2 renewal which in turn improves lung repair.One sentence summaryMetabolic defects of alveolar progenitors in aging and during lung injury impair their renewal.

The heterogeneity of fibroblasts in the murine and human lung during homeostasis and disease is increasingly recognized. It remains unclear if the different phenotypes identified to date are characteristic of unique subpopulations with unique progenitors or whether they arise by a process of differentiation from common precursors. Our understanding of this ubiquitous cell type is limited by an absence of well validated, specific, markers with which to identify each cell type and a clear consensus on the distinct populations present in the lung. Here we describe single cell RNA sequencing (scRNA-seq) analysis on mesenchymal cells from the murine lung throughout embryonic (E) development (E9.5 – 17.5), at post-natal day (P1 – 15), as well as in the adult and the aged murine lungs before and after bleomycin-induced fibrosis. We carried out complementary scRNA-seq on human lung tissue from a P1 lung, a month 21 lung and lung tissue from healthy donors and patients with idiopathic pulmonary fibrosis (IPF). The murine and human data were supplemented with publicly available scRNA-seq datasets. We consistently identified lipofibroblasts, myofibroblasts, pericytes, mesothelial cells and smooth muscle cells. In addition, we identified a novel population delineated by Ebf1 (early B-cell factor 1) expression and an intermediate subtype. Comparative analysis with human mesenchymal cells revealed homologous mesenchymal subpopulations with remarkably conserved transcriptomic signatures. Comparative analysis of changes in gene expression in the fibroblast subpopulations from age matched non-fibrotic and fibrotic lungs in the mouse and human demonstrates that many of these subsets contribute to matrix gene expression in fibrotic conditions. Subtype selective transcription factors were identified and putative divergence of the clusters during development were delineated. Prospective isolation of these fibroblast subpopulations, localization of signature gene markers, and lineage-tracing each cluster are under way in the laboratory. This analysis will enhance our understanding of fibroblast heterogeneity in homeostasis and fibrotic disease conditions.

RationaleDeclining lung function in patients with interstitial lung disease is accompanied by epithelial remodeling and progressive scarring of the gas-exchange region. There is a need to better understand the contribution of basal cell hyperplasia and associated mucosecretory dysfunction to the development of idiopathic pulmonary fibrosis (IPF).ObjectivesWe sought to decipher the transcriptome of freshly isolated epithelial cells from normal and IPF lung to discern disease-dependent changes within basal stem cells.MethodsSingle cell RNA sequencing was used to map epithelial cell types of the normal and IPF human airway. Organoid and ALI cultures were used to investigate functional properties of basal cell subtypes.Measurements and Main ResultsWe found that basal cells included multipotent and secretory primed subsets in control adult lung tissue. Secretory primed basal cells include an overlapping molecular signature with basal cells obtained from distal lung tissue of IPF lungs. We confirmed that NOTCH2 maintains undifferentiated basal cells and restrict basal-to-ciliated differentiation, and present evidence that NOTCH3 functions to restrain secretory differentiation.ConclusionsBasal cells are dynamically regulated in disease and are specifically biased towards expansion of the secretory primed basal cell subset in idiopathic pulmonary fibrosis. Modulation of basal cell plasticity may represent a relevant target for therapeutic intervention in IPF.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease, with unknown etiopathogenesis and suboptimal therapeutic options. Previous reports have shown that increased T-cell numbers and CD28null phenotype is predictive of prognosis in IPF, suggesting that these cells might have a role in this disease. Flow cytometric analysis of explanted lung cellular suspensions showed a significant increase in CD8+ CD28null T cells in IPF relative to normal lung explants. Transcriptomic analysis of CD3+ T cells isolated from IPF lung explants revealed a loss of CD28-transcript expression and elevation of pro-inflammatory cytokine expression in IPF relative to normal T cells. IPF lung explant-derived T cells (enriched with CD28null T cells), but not normal donor lung CD28+ T cells induced dexamethasone-resistant lung remodeling in humanized NSG mice. Finally, CD28null T cells expressed similar CTLA4 and significantly higher levels of PD-1 proteins relative to CD28+ T cells and blockade of either proteins in humanized NSG mice, using anti-CTLA4, or anti-PD1, mAb treatment-accelerated lung fibrosis. Together, these results demonstrate that IPF CD28null T cells may promote lung fibrosis but the immune checkpoint proteins, CTLA-4 and PD-1, appears to limit this effect.

Increasing evidence highlights that senescence plays an important role in idiopathic pulmonary fibrosis (IPF). This study delineates the specific contribution of mitochondria and the superoxide they form to the senescent phenotype of lung fibroblasts from IPF patients (IPF‐LFs). Primary cultures of IPF‐LFs exhibited an intensified DNA damage response (DDR) and were more senescent than age‐matched fibroblasts from control donors (Ctrl‐LFs). Furthermore, IPF‐LFs exhibited mitochondrial dysfunction, exemplified by increases in mitochondrial superoxide, DNA, stress and activation of mTORC1. The DNA damaging agent etoposide elicited a DDR and augmented senescence in Ctrl‐LFs, which were accompanied by disturbances in mitochondrial homoeostasis including heightened superoxide production. However, etoposide had no effect on IPF‐LFs. Mitochondrial perturbation by rotenone involving sharp increases in superoxide production also evoked a DDR and senescence in Ctrl‐LFs, but not IPF‐LFs. Inhibition of mTORC1, antioxidant treatment and a mitochondrial targeting antioxidant decelerated IPF‐LF senescence and/or attenuated pharmacologically induced Ctrl‐LF senescence. In conclusion, increased superoxide production by dysfunctional mitochondria reinforces lung fibroblast senescence via prolongation of the DDR. As part of an auto‐amplifying loop, mTORC1 is activated, altering mitochondrial homoeostasis and increasing superoxide production. Deeper understanding the mechanisms by which mitochondria contribute to fibroblast senescence in IPF has potentially important therapeutic implications.

Background Recruited myeloid cells are known to promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. We tested the hypothesis that circulating blood monocytes from advanced prostate cancer (PCa) patients exhibit a protumor phenotype and directly influence the tumor microenvironment in response to tumor‐derived signals. Methods Blood monocytes from advanced and stable PCa patients were cultured, and the conditioned media (CM) were collected and analyzed using standard invasion and wound closure assays to measure effects on invasion and motility of PCa tumor cells. We then identified the proteome profile of these monocytes using proteome array and ELISA. Results Conditioned media from circulating monocytes in patients with metastatic prostate cancer (PCa‐M) increased invasion of epithelial PCa cells in vitro. Proteome Profiler Analysis revealed that monocyte‐derived CM from metastatic castration‐resistant (mCRPC) patients presented high levels of chitinase‐3‐like 1 (CHI3L1, YKL‐40) when compared to patients with stable disease (PCa‐N) and healthy control individuals (HC). The only described receptor for CHI3L1, interleukin‐13 receptor α2 (IL‐13Rα2), was significantly up‐regulated in the human metastatic PCa cell line, ARCaPM. Accordingly, we observed that the activation of IL‐13Rα2 from PCa‐M CM increased the invasiveness of ARCaPM cells while siRNA directed against this receptor significantly reduced invasiveness of these cells in the presence of CM from PCa‐M patients. Conclusions Thus, we show that circulating monocytes from metastatic PCa patients exert a tumor‐promoting role via the secretion of CHI3L1, and CHI3L1 demands further exploration as a possible therapeutic target in advanced PCa.

Made available in DSpace on 2022-04-29T08:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-07-20 Squamous cell carcinoma (SCC) is the second most common form of skin cancer and the mechanism(s) involved in the progression of this tumor are unknown. Increases in the expression of IL-33/ST2 axis components have been demonstrated to contribute to neoplastic transformation in several tumor models and interleukin-33 is correlated with poor prognosis of patients with squamous cell carcinoma of the tongue. Based on these observations, we sought to determine the role of the IL-33/ST2 pathway during the development of SCC. Our findings show that ST2- deficiency led to a marked decrease in the severity of skin lesions, suggesting that ST2 signaling contributed to tumor development. An analysis of tumor lesions in wild-type and ST2KO mice revealed that a lack of ST2 was associated with specific and significant reductions in the numbers of CD4+ T cells, CD8+ T cells, dendritic cells, and macrophages. In addition, NK cells that were isolated from ST2KO mice exhibited higher cytotoxic activity than cells isolated from wild-type mice. Notably, ST2 deficiency resulted in lower IFN-γ, TNF-α, IL-10, and IL-17 production in tumor samples. Our findings indicate that the IL-33/ST2 pathway contributes to the development of SCC by affecting leukocyte migration to tumor microenvironment and impairing NK cytotoxic activity. Department of Biological Sciences Bauru School of Dentistry University of São Paulo, Al. Dr. Octávio Pinheiro Brisolla Department of Microbiology and Immunology Institute of Biosciences of Botucatu São Paulo State University, R. Prof. Dr. Antônio Celso Wagner Zanin Department of Stomatology - Oral Pathology Bauru School of Dentistry University of São Paulo, Al. Dr. Octávio Pinheiro Brisolla Department of Biochemistry and Immunology School of Medicine of Ribeirão Preto University of São Paulo Department of Biomedical Sciences (Research Division of Immunology) and Medicine F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute Cedars-Sinai Medical Center Department of Medicine Division of Pulmonary and Critical Care Medicine Cedars-Sinai Medical Center Samuel Oschin Comprehensive Cancer Institute Cedars-Sinai Medical Center Department of Microbiology and Immunology Institute of Biosciences of Botucatu São Paulo State University, R. Prof. Dr. Antônio Celso Wagner Zanin

Recent evidence indicates that nonprofessional immune cells such as epithelial cells, endothelial cells, and fibroblasts also contribute to innate immunity via secretion of cytokines. Fibroblasts are the principal type of cell found in the periodontal connective tissues and they are involved in the immune response during periodontal disease. The role of fibroblasts in the recognition of pathogens via Toll-like receptors (TLRs) has been established; however, few studies have been conducted concerning the involvement of innate immune receptors in the recognition of Candida albicans by gingival fibroblast. In the current study, we investigate the functional activity of TLR2, cluster of differentiation 14 (CD14), and myeloid differentiation primary response gene 88 (MyD88) molecules in the recognition of C. albicans by gingival fibroblast. First, we identified that gingival fibroblasts expressed TLR2, TLR3, and TLR4. Our results showed that TLR agonists had no effect on these receptors’ expression by TLR2, MyD88, and CD14-deficient cells. Notably, C. albicans and a synthetic triacylated lipoprotein (Pam3CSK4) induced a remarkable increase of TLR3 expression on MyD88-deficient gingival fibroblasts. TLR4 expression levels were lower than TLR2 and TLR3 levels and remained unchanged after TLR agonist stimulation. Gingival fibroblasts presented morphological similarities; however, TLR2 deficiency on these cells leads to a lower proliferative response, whereas the deficiency on CD14 expression resulted in lower levels of type I collagen by these cells. In addition, the recognition of C. albicans by gingival fibroblasts had an effect on the secretion of cytokines and it was dependent on a specific recognition molecule. Specifically, tumor necrosis factor-α (TNF-α ) production after the recognition of C. albicans was dependent on MyD88, CD14, and TLR2 molecules, whereas the production of interleukin-1β (IL-1β ) and IL-13 was dependent on TLR2. These findings are the first to describe a role of gingival fibroblast in the recognition of C. albicans and the pathways involved in this process. An understanding of these pathways may lead to alternative treatments for patients with periodontal disease.

Rationale: Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung remodeling, which progressively abolishes lung function in an RTK (receptor tyrosine kinase)–dependent manner. Gas6 ...

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease of unknown etiopathogenesis with limited therapeutic options. IPF is characterized by an abundance of fibroblasts and loss of epithelial progenitors, which cumulates in unrelenting fibrotic lung remodeling and loss of normal oxygenation. IPF has been challenging to model in rodents; nonetheless, mouse models of lung fibrosis provide clues as to the natural progression of lung injury and remodeling, but many have not been useful in predicting efficacy of therapeutics in clinical IPF. We provide a detailed methodologic description of various iterations of humanized mouse models, initiated by the i.v. injection of cells from IPF lung biopsy or explants specimens into severe combined immunodeficiency (SCID)/beige or nonobese diabetic SCID γ mice. Unlike cells from normal lung samples, IPF cells promote persistent, nonresolving lung remodeling in SCID mice. Finally, we provide examples and discuss potential advantages and pitfalls of human-specific targeting approaches in a humanized SCID model of pulmonary fibrosis.

Syndecan-1 is a transmembrane proteoglycan expressed prominently by lung epithelium and has pleiotropic functions such as regulating cell migration, proliferation, and survival. Loss of syndecan-1 expression by lung cancer cells is associated with higher-grade cancers and worse clinical prognosis. We evaluated the effects of syndecan-1 in various cell-based and animal models of lung cancer and found that lung tumorigenesis was moderated by syndecan-1. We also demonstrate that syndecan-1 (or lack thereof) alters the miRNA cargo carried within exosomes exported from lung cancer cells. Analysis of the changes in miRNA expression identified a distinct shift toward augmented procancer signaling consistent with the changes found in lung adenocarcinoma. Collectively, our work identifies syndecan-1 as an important factor in lung cancer cells that shapes the tumor microenvironment through alterations in miRNA packaging within exosomes.