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Systemic sclerosis (SSc), also known as scleroderma, is an autoimmune-driven connective tissue disorder that results in fibrosis of the skin and internal organs such as the lung. Fibroblasts are known as the main effector cells involved in the progression of SSc through the induction of extracellular matrix (ECM) proteins and myofibroblast differentiation. Here, we demonstrate that 4'-(cyclopropylmethyl)-N2-4-pyridinyl-[4,5'-bipyrimidine]-2,2'-diamine (PIK-III), known as class III phosphatidylinositol 3-kinase (PIK3C3/VPS34) inhibitor, exerts potent antifibrotic effects in human dermal fibroblasts (HDFs) by attenuating transforming growth factor-beta 1 (TGF-β1)-induced ECM expression, cell contraction and myofibroblast differentiation. Unexpectedly, neither genetic silencing of PIK3C3 nor other PIK3C3 inhibitors (e.g., SAR405 and Autophinib) were able to mimic PIK-III-mediated antifibrotic effect in dermal fibroblasts, suggesting that PIK-III inhibits fibroblast activation through another signaling pathway. We identified that PIK-III effectively inhibits p38 activation in TGF-β1-stimulated dermal fibroblasts. Finally, PIK-III administration significantly attenuated dermal and lung fibrosis in bleomycin-injured mice. [ABSTRACT FROM AUTHOR]

Background: Regulation of the thermogenic response by brown adipose tissue (BAT) is an important component of energy homeostasis with implications for the treatment of obesity and diabetes. Our preliminary analyses of RNA-Seq data uncovered many nodes representing epigenetic modifiers that are altered in BAT in response to chronic thermogenic activation. Thus, we hypothesized that chronic thermogenic activation broadly alters epigenetic modifications of DNA and histones in BAT. Results: Motivated to understand how BAT function is regulated epigenetically, we developed a novel method for the first-ever unbiased top-down proteomic quantitation of histone modifications in BAT and validated our results with a multi-omic approach. To test our hypothesis, wildtype male C57BL/6J mice were housed under chronic conditions of thermoneutral temperature (TN, 28°C), mild cold/room temperature (RT, 22°C), or severe cold (SC, 8°C) and BAT was analyzed for DNA methylation and histone modifications. Methylation of promoters and intragenic regions in genomic DNA decrease in response to chronic cold exposure. Integration of DNA methylation and RNA expression datasets suggest a role for epigenetic modification of DNA in regulation of gene expression in response to cold. In response to cold housing, we observe increased bulk acetylation of histones H3.2 and H4, increased histone H3.2 proteoforms with di- and trimethylation of lysine 9 (K9me2 and K9me3), and increased histone H4 proteoforms with acetylation of lysine 16 (K16ac) in BAT. Conclusions: Our results reveal global epigenetically-regulated transcriptional "on" and "off" signals in murine BAT in response to varying degrees of chronic cold stimuli and establish a novel methodology to quantitatively study histones in BAT, allowing for direct comparisons to decipher mechanistic changes during the thermogenic response. Additionally, we make histone PTM and proteoform quantitation, RNA splicing, RRBS, and transcriptional footprint datasets available as a resource for future research. [ABSTRACT FROM AUTHOR]

Rationale: Within chronic obstructive pulmonary disease (COPD), emphysema is characterized by a significant yet partially understood B cell immune component. Objectives: To characterize the transcriptomic signatures from lymphoid follicles (LFs) in ever-smokers without COPD and patients with COPD with varying degrees of emphysema. Methods: Lung sections from 40 patients with COPD and eversmokers were used for LF proteomic and transcriptomic spatial profiling. Formalin- and O.C.T.-fixed lung samples obtained from biopsies or lung explants were assessed for LF presence. Emphysema measurements were obtained from clinical chest computed tomographic scans. High-confidence transcriptional target intersection analyses were conducted to resolve emphysema-induced transcriptional networks. Measurements and Main Results: Overall, 115 LFs from eversmokers andGlobal Initiative for ChronicObstructive LungDisease (GOLD) 1-2 andGOLD3-4 patients were analyzed. No LFs were found in never-smokers. Differential gene expression analysis revealed significantly increased expression of LF assembly and B cellmarker genes in subjects with severe emphysema. High-confidence transcriptional analysis revealed activation of an abnormal B cell activity signature in LFs (q-value= 2.56E-111). LFs frompatients withGOLD 1-2 COPDwith emphysema showed significantly increased expression of genes associated with antigen presentation, inflammation, and B cell activation and proliferation. LFs frompatients with GOLD1-2COPD without emphysema showed an antiinflammatory profile. The extent of centrilobular emphysema was significantly associated with genes involved in B cellmaturation and antibody production. Protein-RNA network analysis showed that LFs in emphysema have a unique signature skewed toward chronic B cell activation. Conclusions: An off-targeted B cell activation within LFs is associated with autoimmune-mediated emphysema pathogenesis. [ABSTRACT FROM AUTHOR]

Conventional dendritic cells (cDCs) generate protective cytotoxic T lymphocyte (CTL) responses against extracellular pathogens and tumors. This is achieved through a process known as cross-presentation (XP), and, despite its biological importance, the mechanism(s) driving XP remains unclear. Here, we show that a cDC-specific pore-forming protein called apolipoprotein L 7C (APOL7C) is up-regulated in response to innate immune stimuli and is recruited to phagosomes. Association of APOL7C with phagosomes led to phagosomal rupture and escape of engulfed antigens to the cytosol, where they could be processed via the endogenous MHC class I antigen processing pathway. Accordingly, mice deficient in APOL7C did not efficiently prime CD8 + T cells in response to immunization with bead-bound and cell-associated antigens. Together, our data indicate the presence of dedicated apolipoproteins that mediate the delivery of phagocytosed proteins to the cytosol of activated cDCs to facilitate XP.

Adaptive immunity is critical to eliminate malignant cells, while multiple tumor-intrinsic factors can alter this protective function. Melanoma antigen-A4 (MAGE-A4), a cancer-testis antigen, is expressed in several solid tumors and correlates with poor survival in non-small cell lung cancer (NSCLC), but its role in altering antitumor immunity remains unclear. We found that expression of MAGE-A4 was highly associated with the loss of PTEN , a tumor suppressor, in human NSCLC. Here we show that constitutive expression of human MAGE-A4 combined with the loss of Pten in mouse airway epithelial cells results in metastatic adenocarcinoma enriched in CD138 + CXCR4 + plasma cells, predominantly expressing IgA. Consistently, human NSCLC expressing MAGE-A4 showed increased CD138 + IgA + plasma cell density surrounding tumors. The abrogation of MAGE-A4-responsive plasma cells (MARPs) decreased tumor burden, increased T cell infiltration and activation, and reduced CD163 + CD206 + macrophages in mouse lungs. These findings suggest MAGE-A4 promotes NSCLC tumorigenesis, in part, through the recruitment and retention of IgA + MARPs in the lungs.

Analysis of lung alveolar type 2 (AT2) progenitor stem cells has highlighted fundamental mechanisms that direct their differentiation into alveolar type 1 cells (AT1s) in lung repair and disease. However, microRNA (miRNA) mediated post-transcriptional mechanisms which govern this nexus remain understudied. We show here that the let-7 miRNA family serves a homeostatic role in governance of AT2 quiescence, specifically by preventing the uncontrolled accumulation of AT2 transitional cells and by promoting AT1 differentiation to safeguard the lung from spontaneous alveolar destruction and fibrosis. Using mice and organoid models with genetic ablation of let-7a1/let-7f1/let-7d cluster ( let-7afd ) in AT2 cells, we demonstrate prevents AT1 differentiation and results in aberrant accumulation of AT2 transitional cells in progressive pulmonary fibrosis. Integration of enhanced AGO2 UV-crosslinking and immunoprecipitation sequencing (AGO2-eCLIP) with RNA-sequencing from AT2 cells uncovered the induction of direct targets of let-7 in an oncogene feed-forward regulatory network including BACH1/EZH2 which drives an aberrant fibrotic cascade. Additional analyses by CUT&RUN-sequencing revealed loss of let-7afd hampers AT1 differentiation by eliciting aberrant histone EZH2 methylation which prevents the exit of AT2 transitional cells into terminal AT1s. This study identifies let-7 as a key gatekeeper of post-transcriptional and epigenetic chromatin signals to prevent AT2-driven pulmonary fibrosis., Competing Interests: COMPETING INTEREST STATEMENT The authors declare no competing interests.