Stromal‐immune cell cross talk fundamentally alters the lung microenvironment following tissue insult

Communication between stromal and immune cells is essential to maintain tissue homeostasis, mount an effective immune response, and promote tissue repair. This ‘crosstalk’ occurs in both the steady state and following a variety of insults, for example, in response to local injury, at sites of infection, or cancer. What do we mean by crosstalk between cells? Reciprocal activation and/or regulation occurs between immune and stromal cells, by direct cell contact and indirect mechanisms, including the release of soluble cytokines. Moving beyond cell to cell contact, and this review investigates the complexity of ‘cross‐space’ cellular communication. We highlight different examples of cellular communication by a variety of lung stromal and immune cells following tissue insults. This review examines how the ‘geography of the lung microenvironment’ is altered in various disease states, more specifically we investigate how this influences lung epithelial cells and fibroblasts via their communication with immune cells and each other

Antigen presenting cells: professionals, amateurs, and spectators in the 'long game' of lung immunity

The lung is frequently and repeatedly exposed to invading pathogens and thus requires constant immunosurveillance. Professional antigen presenting cells (APCs), including dendritic cells, engulf invading pathogens and present their peptides via major histocompatibility complexes (MHC) I and II, to CD8 or CD4 T cells. Epithelial cells and stromal cells (including fibroblasts) provide more than structural support, they are increasingly recognised as key players in the immune response, acting as non-professional APCs through interactions with antigen experienced T cells that migrate to the lung. The importance of the contributions of non-professional and professional APCs to T cell function in vivo, is currently unclear. This review summarises the roles of professional and non-professional APCs in lung immunity, at the steady state and following viral insult, with particular emphasis on their ability to interact with and influence T cells

HDAC1 interacts with the p50 NF-κB subunit via its nuclear localization sequence to constrain inflammatory gene expression

The NF-κB p50 subunit is an important regulator of inflammation, with recent experimental evidence to support it also having a tumor suppressor role. Classically, p50 functions in heterodimeric form with the RelA (p65) NF-κB subunit to activate inflammatory genes. However, p50 also forms homodimers which actively repress NF-κB-dependent inflammatory gene expression and exert an important brake on the inflammatory process. This repressive activity of p50:p50 is thought to be in part mediated by an interaction with the epigenetic repressor protein Histone Deacetylase 1 (HDAC1). However, neither the interaction of p50 with HDAC1 nor the requirement of HDAC1 for the repressive activities of p50 has been well defined. Here we employed in silico prediction with in vitro assays to map sites of interaction of HDAC1 on the p50 protein. Directed mutagenesis of one such region resulted in almost complete loss of HDAC1 binding to p50. Transfected mutant p50 protein lacking the putative HDAC1 docking motif resulted in enhanced cytokine and chemokine expression when compared with cells expressing a transfected wild type p50. In addition, expression of this mutant p50 was associated with enhanced chemoattraction of neutrophils and acetylation of known inflammatory genes demonstrating the likely importance of the p50:HDAC1 interaction for controlling inflammation. These new insights provide an advance on current knowledge of the mechanisms by which NF-κB-dependent gene transcription are regulated and highlight the potential for manipulation of p50:HDAC1 interactions to bring about experimental modulation of chronic inflammation and pathologies associated with dysregulated neutrophil accumulation and activation

CXCR3A promotes the secretion of the anti-fibrotic decoy receptor sIL-13Rα2 by pulmonary fibroblasts

CXCR3A and its IFN-inducible ligands CXCL9 and CXCL10 regulate vascular remodelling and fibroblast motility. IL‑13 is a pro‑fibrotic cytokine implicated in the pathogenies of inflammatory and fibro-proliferative conditions. Previous work from our lab has shown that CXCR3A is negatively regulated by IL-13 and is necessary for the basal regulation of the IL-13 receptor subunit IL-13Rα2. This study investigates the regulation of fibroblast phenotype, function and downstream IL-13 signalling by CXCR3A in vitro. CXCR3A was overexpressed via transient transfection. CXCR3A-/- lung fibroblastswere isolated for functional analysis. Additionally, the contribution of CXCR3A to tissue remodelling following acute lung injury was assessed in vivo using wild type (WT) and CXCR3-/- mice challenged with IL-13. CXCR3 and IL‑13Rα2 displayed a reciprocal relationship following stimulation with either IL-13 or CXCR3 ligands. CXCR3A reduced expression of fibroblast activation makers, soluble collagen production and proliferation. CXCR3A enhanced the basal expression of pERK1/2 while inducing IL-13 mediated down‑regulation of NFκB‑p65. CXCR3A-/- pulmonary fibroblasts were increasingly proliferative and displayed reduced contractility and α‑smooth muscle actin expression. IL-13 challenge regulated expression of the CXCR3 ligands and soluble IL-13Rα2 levels in lungs and broncho‑alveolar lavage fluid (BALF) of WT mice, this response was absent in CXCR3-/- mice. Alveolar macrophage accumulation and expression of genes involved in lung remodelling was increased in CXCR3-/- mice. We conclude that CXCR3A is a central anti-fibrotic factor in pulmonary fibroblasts, limiting fibroblast activation and reducing ECM production. Therefore targeting of CXCR3A may be a novel approach to regulate fibroblast activity in lung fibrosis and remodelling

Triphasic production of IFNγ by innate and adaptive lymphocytes following influenza A virus infection

Interferon gamma (IFNγ) is a potent antiviral cytokine that can be produced by many innate and adaptive immune cells during infection. Currently, our understanding of which cells produce IFNγ and where they are located at different stages of an infection is limited. We have used reporter mice to investigate in vivo expression of Ifnγ mRNA in the lung and secondary lymphoid organs during and following influenza A virus (IAV) infection. We observed a triphasic production of Ifnγ expression. Unconventional T cells and innate lymphoid cells, particularly NK cells, were the dominant producers of early Ifnγ, while CD4 and CD8 T cells were the main producers by day 10 post-infection. Following viral clearance, some memory CD4 and CD8 T cells continued to express Ifnγ in the lungs and draining lymph node. Interestingly, Ifnγ production by lymph node Natural Killer (NK), NKT and innate lymphoid type 1 cells also continued to be above naïve levels, suggesting memory-like phenotypes for these cells. Analysis of the localisation of Ifnγ+ memory CD4 and CD8 T cells demonstrated that cytokine+ T cells were located near airways and in the lung parenchyma. Following a second IAV challenge, lung IAV specific CD8 T cells rapidly increased their expression of Ifnγ while CD4 T cells in the draining lymph node increased their Ifnγ response. Together, these data suggest that Ifnγ production fluctuates based on cellular source and location, both of which could impact subsequent immune responses

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment

cRel expression regulates distinct transcriptional and functional profiles driving fibroblast matrix production in systemic sclerosis

Objectives: NF-κB regulates genes that control inflammation, cell proliferation, differentiation and survival. Dysregulated NF-κB signalling alters normal skin physiology and deletion of cRel limits bleomycin-induced skin fibrosis. This study investigates the role of cRel in modulating fibroblast phenotype in the context of SSc. Methods: Fibrosis was assessed histologically in mice challenged with bleomycin to induce lung or skin fibrosis. RNA sequencing and pathway analysis was performed on wild type and Rel-/- murine lung and dermal fibroblasts. Functional assays examined fibroblast proliferation, migration and matrix production. cRel overexpression was investigated in human dermal fibroblasts. cRel immunostaining was performed on lung and skin tissue sections from SSc patients and non-fibrotic controls. Results: cRel expression was elevated in murine lung and skin fibrosis models. Rel-/- mice were protected from developing pulmonary fibrosis. Soluble collagen production was significantly decreased in fibroblasts lacking cRel while proliferation and migration of these cells was significantly increased. cRel regulates genes involved in extracellular structure and matrix organization. Positive cRel staining was observed in fibroblasts in human SSc skin and lung tissue. Overexpression of constitutively active cRel in human dermal fibroblasts increased expression of matrix genes. An NF-κB gene signature was identified in diffuse SSc skin and nuclear cRel expression was elevated in SSc skin fibroblasts. Conclusion: cRel regulates a pro-fibrogenic transcriptional programme in fibroblasts that may contribute to disease pathology. Targeting cRel signalling in fibroblasts of SSc patients could provide a novel therapeutic avenue to limit scar formation in this disease