Search

PDF file - 2MB

It has become increasingly appreciated that autoimmune responses against neuronal components play an important role in type 1 diabetes (T1D) pathogenesis. In fact, a large proportion of islet-infiltrating B lymphocytes in the NOD mouse model of T1D produce Abs directed against the neuronal type III intermediate filament protein peripherin. NOD-PerIg mice are a previously developed BCR-transgenic model in which virtually all B lymphocytes express the H and L chain Ig molecules from the intra-islet–derived anti-peripherin–reactive hybridoma H280. NOD-PerIg mice have accelerated T1D development, and PerIg B lymphocytes actively proliferate within islets and expand cognitively interactive pathogenic T cells from a pool of naive precursors. We now report adoptively transferred T cells or whole splenocytes from NOD-PerIg mice expectedly induce T1D in NOD.scid recipients but, depending on the kinetics of disease development, can also elicit a peripheral neuritis (with secondary myositis). This neuritis was predominantly composed of CD4+ and CD8+ T cells. Ab depletion studies showed neuritis still developed in the absence of NOD-PerIg CD8+ T cells but required CD4+ T cells. Surprisingly, sciatic nerve–infiltrating CD4+ cells had an expansion of IFN-γ− and TNF-α− double-negative cells compared with those within both islets and spleen. Nerve and islet-infiltrating CD4+ T cells also differed by expression patterns of CD95, PD-1, and Tim-3. Further studies found transitory early B lymphocyte depletion delayed T1D onset in a portion of NOD-PerIg mice, allowing them to survive long enough to develop neuritis outside of the transfer setting. Together, this study presents a new model of peripherin-reactive B lymphocyte–dependent autoimmune neuritis.

Loss of alveolar type 2 cells (AEC2s) and the ectopic appearance of basal cells in the alveoli characterize severe lung injuries such as idiopathic pulmonary fibrosis (IPF). Here we demonstrate that human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, transdifferentiate into basal cells in response to fibrotic signalling in the lung mesenchyme, in vitro and in vivo. Single-cell analysis of normal hAEC2s and mesenchymal cells in organoid co-cultures revealed the emergence of pathologic fibroblasts and basaloid cells previously described in IPF. Transforming growth factor-β1 and anti-bone morphogenic protein signalling in the organoids promoted transdifferentiation. Trajectory and histologic analyses of both hAEC2-derived organoids and IPF epithelium indicated that hAEC2s transdifferentiate into basal cells through alveolar-basal intermediates that accumulate in proximity to pathologic CTHRC1hi/TGFB1hi fibroblasts. Our study indicates that hAEC2 loss and expansion of alveolar metaplastic basal cells in severe human lung injuries are causally connected through an hAEC2-basal cell lineage trajectory driven by aberrant mesenchyme.

Epithelial cells are charged with protection at barrier sites, but whether this normally beneficial response might sometimes become dysfunctional still needs definition. Here, we recognized a pattern of imbalance marked by basal epithelial cell growth and differentiation that replaced normal airspaces in a mouse model of progressive postviral lung disease due to the Sendai virus. Single-cell and lineage-tracing technologies identified a distinct subset of basal epithelial stem cells (basal ESCs) that extended into gas-exchange tissue to form long-term bronchiolar-alveolar remodeling regions. Moreover, this cell subset was selectively expanded by crossing a cell-growth and survival checkpoint linked to the nuclear-localized alarmin IL-33 that was independent of IL-33 receptor signaling and instead connected to autocrine chromatin accessibility. This mechanism creates an activated stem-progenitor cell lineage with potential for physiological or pathological function. Thus, conditional loss of Il33 gene function in basal epithelial cells disrupted the homeostasis of the epithelial barrier at skin and gut sites but also markedly attenuated postviral disease in the lung based on the downregulation of remodeling and inflammation. Thus, we define a basal ESC strategy to deploy innate immune machinery that appears to overshoot the primordial goal of self-defense. Our findings reveal new targets to stratify and correct chronic and often deadly postviral disease.

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive interstitial fibrosis, which leads to severe debilitation, respiratory failure, and death. In IPF, environmental exposures interact with genetic risk factors to engender critical patho-etiological events in lung epithelial cells, including endoplasmic reticulum (ER) stress and TGFβ signaling, but the interactions between these disparate pathways are not well understood. We previously showed that kinase inhibitors of the IRE1α bifunctional kinase/RNase—a central mediator of the unfolded protein response (UPR) to ER stress—protected mice from bleomycin-induced pulmonary fibrosis. Here we show that a nanomolar-potent, mono-selective kinase inhibitor of IRE1α (KIRA8) decreases ER-stress induced TGFβ signaling and the senescence-associated secretory phenotype (SASP) in the lung epithelium after bleomycin exposure. A recently-described subset of “damage-associated transient progenitors” (DATPs) display IRE1α-regulated pathological gene signatures that are quelled by KIRA8, in vivo. After injury, these cells uniquely express integrin αvβ6, a key activator of TGFβ in pulmonary fibrosis. KIRA8 inhibition of IRE1α decreases both DATP number and Itgb6 expression in remaining cells, with a decrease in local collagen accumulation. Single-cell RNA sequencing from IPF lungs revealed an analogous Itgb6+ cell population that may also be regulated by IRE1α. These findings suggest that lung epithelial progenitor cells sit at the center of the fibrotic niche, and IRE1α signaling locks them into a dysfunctional state that establishes and perpetuates pathological fibrosis.

Type 1 diabetes (T1D) in both humans and NOD mice is caused by T cell–mediated autoimmune destruction of pancreatic β cells. Increased frequency or activity of autoreactive T cells and failures of regulatory T cells (Tregs) to control these pathogenic effectors have both been implicated in T1D etiology. Due to the expression of MHC class I molecules on β cells, CD8 T cells represent the ultimate effector population mediating T1D. Developing autoreactive CD8 T cells normally undergo extensive thymic negative selection, but this process is impaired in NOD mice and also likely T1D patients. Previous studies identified an allelic variant of Nfkbid, a NF-κB signal modulator, as a gene strongly contributing to defective thymic deletion of autoreactive CD8 T cells in NOD mice. These previous studies found ablation of Nfkbid in NOD mice using the clustered regularly interspaced short palindromic repeats system resulted in greater thymic deletion of pathogenic CD8 AI4 and NY8.3 TCR transgenic T cells but an unexpected acceleration of T1D onset. This acceleration was associated with reductions in the frequency of peripheral Tregs. In this article, we report transgenic overexpression of Nfkbid in NOD mice also paradoxically results in enhanced thymic deletion of autoreactive CD8 AI4 T cells. However, transgenic elevation of Nfkbid expression also increased the frequency and functional capacity of peripheral Tregs, in part contributing to the induction of complete T1D resistance. Thus, future identification of a pharmaceutical means to enhance Nfkbid expression might ultimately provide an effective T1D intervention approach.

EGCG and Pulmonary Fibrosis Nine patients who were scheduled to undergo lung biopsy for pulmonary fibrosis were given epigallocatechin gallate (EGCG) for 2 weeks. Levels of fibrotic markers in lung...

Alveolar epithelium plays a pivotal role in protecting the lungs from inhaled infectious agents. Therefore, the regenerative capacity of the alveolar epithelium is critical for recovery from these insults in order to rebuild the epithelial barrier and restore pulmonary functions. Here, we show that sublethal infection of mice with Streptococcus pneumoniae, the most common pathogen of community-acquired pneumonia, led to exclusive damage in lung alveoli, followed by alveolar epithelial regeneration and resolution of lung inflammation. We show that surfactant protein C–expressing (SPC-expressing) alveolar epithelial type II cells (AECIIs) underwent proliferation and differentiation after infection, which contributed to the newly formed alveolar epithelium. This increase in AECII activities was correlated with increased nuclear expression of Yap and Taz, the mediators of the Hippo pathway. Mice that lacked Yap/Taz in AECIIs exhibited prolonged inflammatory responses in the lung and were delayed in alveolar epithelial regeneration during bacterial pneumonia. This impaired alveolar epithelial regeneration was paralleled by a failure to upregulate IκBa, the molecule that terminates NF-κB–mediated inflammatory responses. These results demonstrate that signals governing resolution of lung inflammation were altered in Yap/Taz mutant mice, which prevented the development of a proper regenerative niche, delaying repair and regeneration of alveolar epithelium during bacterial pneumonia.

Loss of alveolar type 2 cells (AEC2s) and the ectopic appearance of basal cells in the alveoli characterize severe lung injuries such as idiopathic pulmonary fibrosis (IPF). Here we demonstrate that human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, transdifferentiate into basal cells in response to fibrotic signalling in the lung mesenchyme, in vitro and in vivo. Single-cell analysis of normal hAEC2s and mesenchymal cells in organoid co-cultures revealed the emergence of pathologic fibroblasts and basaloid cells previously described in IPF. Transforming growth factor-β1 and anti-bone morphogenic protein signalling in the organoids promoted transdifferentiation. Trajectory and histologic analyses of both hAEC2-derived organoids and IPF epithelium indicated that hAEC2s transdifferentiate into basal cells through alveolar-basal intermediates that accumulate in proximity to pathologic CTHRC1

The human lung differs substantially from its mouse counterpart, resulting in a distinct distal airway architecture affected by disease pathology in chronic obstructive pulmonary disease. In humans, the distal branches of the airway interweave with the alveolar gas-exchange niche, forming an anatomical structure known as the respiratory bronchioles. Owing to the lack of a counterpartin mouse, the cellular and molecular mechanisms that governrespiratory bronchioles in the human lung remain uncharacterized. Here we show that human respiratory bronchioles contain a unique secretory cell population that is distinct from cells in larger proximal airways. Organoid modelling reveals that these respiratory airway secretory (RAS) cells act as unidirectional progenitors for alveolar type 2 cells, which are essential for maintaining and regenerating the alveolar niche. RAS cell lineage differentiation into alveolar type 2 cells is regulated by Notch and Wnt signalling. In chronic obstructive pulmonary disease, RAS cells are altered transcriptionally, corresponding to abnormal alveolar type 2 cell states, which are associated with smoking exposure in both humans and ferrets. These data identify a distinct progenitor in a region of the human lung that is not found in mouse that has a critical role in maintaining the gas-exchange compartment and is altered in chronic lung disease.

Tumor extracellular matrix has been associated with drug resistance and immune suppression. Here, proteomic and RNA profiling reveal increased collagen levels in lung tumors resistant to PD-1/PD-L1 blockade. Additionally, elevated collagen correlates with decreased total CD8+ T cells and increased exhausted CD8+ T cell subpopulations in murine and human lung tumors. Collagen-induced T cell exhaustion occurs through the receptor LAIR1, which is upregulated following CD18 interaction with collagen, and induces T cell exhaustion through SHP-1. Reduction in tumor collagen deposition through LOXL2 suppression increases T cell infiltration, diminishes exhausted T cells, and abrogates resistance to anti-PD-L1. Abrogating LAIR1 immunosuppression through LAIR2 overexpression or SHP-1 inhibition sensitizes resistant lung tumors to anti-PD-1. Clinically, increased collagen, LAIR1, and TIM-3 expression in melanoma patients treated with PD-1 blockade predict poorer survival and response. Our study identifies collagen and LAIR1 as potential markers for immunotherapy resistance and validates multiple promising therapeutic combinations., Tumor extracellular matrix has been associated with cancer progression, therapy resistance and immune suppression. Here, the authors show that collagen generates resistance to PD-1/PD-L1 immunotherapy by upregulating LAIR1 expression and downstream signaling, leading to increased CD8+ T cell exhaustion.

We recently identified epigallocatechin gallate (EGCG), a trihydroxyphenolic compound, as a dual inhibitor of lysyl oxidase-like2 and transforming growth factor-β1 (TGFβ1) receptor kinase that when given orally to patients with idiopathic pulmonary fibrosis (IPF) reversed profibrotic biomarkers in their diagnostic biopsies. Here, we extend these findings to advanced pulmonary fibrosis using cultured precision-cut lung slices from explants of patients with IPF undergoing transplantation. During these experiments, we were surprised to discover that not only did EGCG attenuate TGFβ1 signalling and new collagen accumulation but also activated matrix metalloproteinase-dependent collagen I turnover, raising the possibility of slow fibrosis resolution with continued treatment.

SUMMARYUnderstanding differential lineage potential of orthologous stem cells across species can shed light on human disease. Here, utilizing 3D organoids, single cell RNAseq, and xenotransplants, we demonstrate that human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, are multipotent and able to transdifferentiate into KRT5+ basal cells when co-cultured with primary fibroblasts in 3D organoids. Trajectory analyses and immunophenotyping of epithelial progenitors in idiopathic pulmonary fibrosis (IPF) indicate that hAEC2s transdifferentiate into metaplastic basal cells through alveolar-basal intermediate (ABI) cells that we also identify in hAEC2-derived organoids. Modulating hAEC2-intrinsic and niche factors dysregulated in IPF can attenuate metaplastic basal cell transdifferentiation and preserve hAEC2 identity. Finally, hAEC2s transplanted into fibrotic immune-deficient murine lungs engraft as either hAEC2s or differentiated KRT5+ basal cells. Our study indicates that hAEC2s-loss and expansion of alveolar metaplastic basal cells in IPF are causally connected, which would not have been revealed utilizing murine AEC2s as a model.HighlightsHuman AEC2s transdifferentiate into KRT5+ basal cells when accompanied by primary adult human lung mesenchyme in 3D organoid culture.Alterations of hAEC2-intrinsic and niche factors dysregulated in IPF can modify metaplastic hAEC2 transdifferentiation.hAEC2s engraft into fibrotic lungs of immune-deficient mice and transdifferentiate into metaplastic basal cells.Transcriptional trajectory analysis suggests that hAEC2s in IPF gives rise to metaplastic basal cells via alveolar-basal intermediate cells.

Targeting human T-cell metabolism for modulating immune function requires an understanding of macronutrient utilization. Using metabolic inhibition during activation of human naïve CD8+ T-cells, we demonstrate blocking glycolysis or mitochondrial respiration prevents T-cell proliferation. However, after activation and differentiation, the metabolic program changes. Inhibition of glycolysis abolished cytotoxic T-lymphocyte (CTL) activity, whereas mitochondrial inhibition had no effect on CTL lytic function. Studies with uniformly labeled 13C-glucose confirmed CTL convert the majority of glucose to lactate. The role of glycolysis in CTL function was assessed using NOD models of Type 1 diabetes (T1D). Treatment of NOD models with a glycolysis inhibitor resulted in reduced and delayed T1D incidence and significantly preserved β-cell mass. We conclude glycolysis and mitochondrial ATP production are essential for efficient T-cell activation, but only glycolysis is essential for CTL lytic function. These data suggest targeting glycolysis in CTLs is a promising pathway to prevent T-cell-mediated autoimmunity.

It has become increasingly appreciated that autoimmune responses against neuronal components play an important role in type 1 diabetes (T1D(4)) pathogenesis. In fact, a large proportion of islet-infiltrating B-lymphocytes in the NOD mouse model of T1D produce antibodies directed against the neuronal type III intermediate filament protein, peripherin. NOD-PerIg mice are a previously developed BCR-transgenic model in which virtually all B-lymphocytes express the heavy and light chain immunoglobulin molecules from the intra-islet derived anti-peripherin reactive hybridoma H280. NOD-PerIg mice have accelerated T1D development and PerIg B-lymphocytes actively proliferate within islets and expand cognitively interactive pathogenic T-cells from a pool of naïve precursors. We now report adoptively transferred T-cells or whole splenocytes from NOD-PerIg mice expectedly induce T1D in NOD.scid recipients, but depending on the kinetics of disease development, can also elicit a peripheral neuritis (with secondary myositis). This neuritis was predominantly composed of CD4(+) and CD8(+) T-cells. Antibody depletion studies showed neuritis still developed in the absence of NOD-PerIg CD8(+) T-cells, but required CD4(+) T-cells. Surprisingly, sciatic nerve-infiltrating CD4(+) cells had an expansion of IFN-γ(−) and TNF-α(−) double negative cells compared to those within both islets and spleen. Nerve and islet infiltrating CD4(+) T-cells also differed by expression patterns of CD95, PD-1 and Tim-3. Further studies found transitory early B-lymphocyte depletion delayed T1D onset in a portion of NOD-PerIg mice allowing them to survive long enough to develop neuritis outside of the transfer setting. Together, this study presents a new model of peripherin-reactive B-lymphocyte dependent autoimmune neuritis.

Lung injury activates specialized adult epithelial progenitors to regenerate the epithelium. Depending on the extent of injury, both remaining alveolar type II cells (AEC2s) and distal airway stem/progenitors mobilize to cover denuded alveoli and restore normal barriers. The major source of airway stem/progenitors other than basal-like cells remains uncertain. Here, we define a distinct subpopulation (∼5%) of club-like lineage-negative epithelial progenitors (LNEPs) marked by high H2-K1 expression critical for alveolar repair. Quiescent H2-K1high cells account for virtually all invitro regenerative activity of airway lineages. After bleomycin injury, H2-K1 cells expand and differentiate invivo to alveolar lineages. However,injured H2-K1 cells eventually develop impaired self-renewal with features of senescence, limiting complete repair. Normal H2-K1high cells transplanted into injured lungs differentiate into alveolar cells and rescue lung function. These findings indicate that small subpopulations of specialized stem/progenitors are required for effective lung regeneration and are a potential therapeutic adjunct after major lung injury.

Cancer cells function as primary architects of the tumor microenvironment. Yet, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblasts (CAFs) are distinguishable on the basis of gene expression signatures they acquire in co-culture with epithelial- or mesenchymal-like lung adenocarcinoma (LUAD) cells. High expression of the EMT activator ZEB1 endows LUAD cells with the capacity to activate a soluble factor exchange that leads to CAF reprogramming, to generate CAF-led invasive projections in multicellular aggregates, and to respond to pro-metastatic signals from CAFs in mice. Thus, ZEB1-expressing LUAD cells are positioned at the apex of a signaling hierarchy in the tumor microenvironment.

The lung's unique extracellular matrix (ECM), while providing structural support for cells, is critical in the regulation of developmental organogenesis, homeostasis and injury-repair responses. The ECM, via biochemical or biomechanical cues, regulates diverse cell functions, fate and phenotype. The composition and function of lung ECM become markedly deranged in pathological tissue remodeling. ECM-based therapeutics and bioengineering approaches represent promising novel strategies for regeneration/repair of the lung and treatment of chronic lung diseases. In this review, we assess the current state of lung ECM biology, including fundamental advances in ECM composition, dynamics, topography, and biomechanics; the role of the ECM in normal and aberrant lung development, adult lung diseases and autoimmunity; and ECM in the regulation of the stem cell niche. We identify opportunities to advance the field of lung ECM biology and provide a set recommendations for research priorities to advance knowledge that would inform novel approaches to the pathogenesis, diagnosis, and treatment of chronic lung diseases.

GWAS have repeatedly mapped susceptibility loci for emphysema to genes that modify hedgehog signaling, but the functional relevance of hedgehog signaling to this morbid disease remains unclear. In the current study, we identified a broad population of mesenchymal cells in the adult murine lung receptive to hedgehog signaling, characterized by higher activation of hedgehog surrounding the proximal airway relative to the distal alveoli. Single-cell RNA-sequencing showed that the hedgehog-receptive mesenchyme is composed of mostly fibroblasts with distinct proximal and distal subsets with discrete identities. Ectopic hedgehog activation in the distal fibroblasts promoted expression of proximal fibroblast markers and loss of distal alveoli and airspace enlargement of over 20% compared with controls. We found that hedgehog suppressed mesenchymal-derived mitogens enriched in distal fibroblasts that regulate alveolar stem cell regeneration and airspace size. Finally, single-cell analysis of the human lung mesenchyme showed that segregated proximal-distal identity with preferential hedgehog activation in the proximal fibroblasts was conserved between mice and humans. In conclusion, we showed that differential hedgehog activation segregates mesenchymal identities of distinct fibroblast subsets and that disruption of fibroblast identity can alter the alveolar stem cell niche, leading to emphysematous changes in the murine lung.

Type 1 diabetes (T1D) is characterized by T cell–mediated destruction of the insulin-producing β cells of the pancreatic islets. Among the loci associated with T1D risk, those most predisposing are found in the MHC region. HLA-B*39:06 is the most predisposing class I MHC allele and is associated with an early age of onset. To establish an NOD mouse model for the study of HLA-B*39:06, we expressed it in the absence of murine class I MHC. HLA-B*39:06 was able to mediate the development of CD8 T cells, support lymphocytic infiltration of the islets, and confer T1D susceptibility. Because reduced thymic insulin expression is associated with impaired immunological tolerance to insulin and increased T1D risk in patients, we incorporated this in our model as well, finding that HLA-B*39:06–transgenic NOD mice with reduced thymic insulin expression have an earlier age of disease onset and a higher overall prevalence as compared with littermates with typical thymic insulin expression. This was despite virtually indistinguishable blood insulin levels, T cell subset percentages, and TCR Vβ family usage, confirming that reduced thymic insulin expression does not impact T cell development on a global scale. Rather, it will facilitate the thymic escape of insulin-reactive HLA-B*39:06–restricted T cells, which participate in β cell destruction. We also found that in mice expressing either HLA-B*39:06 or HLA-A*02:01 in the absence of murine class I MHC, HLA transgene identity alters TCR Vβ usage by CD8 T cells, demonstrating that some TCR Vβ families have a preference for particular class I MHC alleles.

After influenza infection, lineage-negative epithelial progenitors (LNEPs) exhibit a binary response to reconstitute epithelial barriers: activating a Notch-dependent ΔNp63/cytokeratin 5 (Krt5) remodelling program or differentiating into alveolar type II cells (AEC2s). Here we show that local lung hypoxia, through hypoxia-inducible factor (HIF1α), drives Notch signallingandKrt5pos basal-like cell expansion. Single-cell transcriptional profiling of human AEC2s from fibrotic lungs revealed a hypoxic subpopulation with activated Notch, suppressed surfactant protein C (SPC), and transdifferentiation toward a Krt5pos basal-likestate. Activated murine Krt5pos LNEPs and diseased human AEC2s upregulate strikingly similar core pathways underlyingmigration and squamous metaplasia. While robust, HIF1α-driven metaplasia is ultimately inferior to AEC2 reconstitution inrestoring normal lung function. HIF1α deletion or enhanced Wnt/β-catenin activity in Sox2pos LNEPs blocks Notch andKrt5activation, instead promoting rapid AEC2 differentiation and migration and improving the quality of alveolar repair.

B lymphocytes play a key role in type 1 diabetes (T1D) development by serving as a subset of APCs preferentially supporting the expansion of autoreactive pathogenic T cells. As a result of their pathogenic importance, B lymphocyte–targeted therapies have received considerable interest as potential T1D interventions. Unfortunately, the B lymphocyte–directed T1D interventions tested to date failed to halt β cell demise. IgG autoantibodies marking humans at future risk for T1D indicate that B lymphocytes producing them have undergone the affinity-maturation processes of class switch recombination and, possibly, somatic hypermutation. This study found that CRISPR/Cas9-mediated ablation of the activation-induced cytidine deaminase gene required for class switch recombination/somatic hypermutation induction inhibits T1D development in the NOD mouse model. The activation-induced cytidine deaminase protein induces genome-wide DNA breaks that, if not repaired through RAD51-mediated homologous recombination, result in B lymphocyte death. Treatment with the RAD51 inhibitor 4,4′-diisothiocyanatostilbene-2, 2′-disulfonic acid also strongly inhibited T1D development in NOD mice. The genetic and small molecule–targeting approaches expanded CD73+ B lymphocytes that exert regulatory activity suppressing diabetogenic T cell responses. Hence, an initial CRISPR/Cas9-mediated genetic modification approach has identified the AID/RAD51 axis as a target for a potentially clinically translatable pharmacological approach that can block T1D development by converting B lymphocytes to a disease-inhibitory CD73+ regulatory state.

How lung epithelium and endothelium co-develop to maintain structural integrity of alveoli remains unclear. In this issue of Developmental Cell, Ellis etal. define how epithelial Vegfa directs development of a distinct endothelial cell population that ultimately plays a critical role in ensuring appropriate alveolar septation during alveologenesis.

Type 1 diabetes development in the NOD mouse model is widely reported to be dependent on high-level production by autoreactive CD4+ and CD8+ T cells of interferon-γ (IFN-γ), generally considered a proinflammatory cytokine. However, IFN-γ can also participate in tolerance-induction pathways, indicating it is not solely proinflammatory. This study addresses how IFN-γ can suppress activation of diabetogenic CD8+ T cells. CD8+ T cells transgenically expressing the diabetogenic AI4 T-cell receptor adoptively transferred disease to otherwise unmanipulated NOD.IFN-γnull, but not standard NOD, mice. AI4 T cells only underwent vigorous intrasplenic proliferation in NOD.IFN-γnull recipients. Disease-protective IFN-γ could be derived from any lymphocyte source and suppressed diabetogenic CD8+ T-cell responses both directly and through an intermediary nonlymphoid cell population. Suppression was not dependent on regulatory T cells, but was associated with increased inhibitory STAT1 to STAT4 expression levels in pathogenic AI4 T cells. Importantly, IFN-γ exposure during activation reduced the cytotoxicity of human-origin type 1 diabetes–relevant autoreactive CD8+ T cells. Collectively, these results indicate that rather than marking the most proinflammatory lymphocytes in diabetes development, IFN-γ production could represent an attempted limitation of pathogenic CD8+ T-cell activation. Thus, great care should be taken when designing possible diabetic intervention approaches modulating IFN-γ production.

Tumor extracellular matrix has been associated with drug resistance and immune suppression. Here, proteomic and RNA profiling reveal increased collagen levels in lung tumors resistant to PD-1/PD-L1 blockade. Additionally, elevated collagen correlates with decreased total CD8

Accumulation of senescent cells is associated with the progression of pulmonary fibrosis, but mechanisms accounting for this linkage are not well understood. To explore this issue, we investigated whether a class of biologically active profibrotic lipids, the leukotrienes (LT), is part of the senescence-associated secretory phenotype. The analysis of conditioned medium (CM), lipid extracts, and gene expression of LT biosynthesis enzymes revealed that senescent cells secreted LT, regardless of the origin of the cells or the modality of senescence induction. The synthesis of LT was biphasic and followed by antifibrotic prostaglandin (PG) secretion. The LT-rich CM of senescent lung fibroblasts (IMR-90) induced profibrotic signaling in naive fibroblasts, which were abrogated by inhibitors of ALOX5, the principal enzyme in LT biosynthesis. The bleomycin-induced expression of genes encoding LT and PG synthases, level of cysteinyl LT in the bronchoalveolar lavage, and overall fibrosis were reduced upon senescent cell removal either in a genetic mouse model or after senolytic treatment. Quantification of ALOX5(+) cells in lung explants obtained from idiopathic pulmonary fibrosis (IPF) patients indicated that half of these cells were also senescent (p16(Ink4a+)). Unlike human fibroblasts from unused donor lungs made senescent by irradiation, senescent IPF fibroblasts secreted LTs but failed to synthesize PGs. This study demonstrates for the first time to our knowledge that senescent cells secrete functional LTs, significantly contributing to the LT pool known to cause or exacerbate IPF.

SUMMARY Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis., In brief Bota-Rabassedas et al. show that EMT in lung adenocarcinoma cells activates a secretory process that governs CAF heterogeneity and, in turn, sensitizes lung adenocarcinoma cells to pro-metastatic signals from CAFs. Thus, EMT positions lung adenocarcinoma cells at the apex of a signaling hierarchy in the tumor microenvironment., Graphical Abstract

While the autoimmune destruction of pancreatic ß-cells underlying type 1 diabetes (1D) development is ultimately mediated by T-cells in NOD mice and also likely humans, B-lymphocytes play an additional key pathogenic role. It appears expression of plasma membrane bound immunoglobulin (Ig) molecules that efficiently capture ß-cell antigens allows autoreactive B-lymphocytes bypassing normal tolerance induction processes to be the subset of antigen presenting cells most efficiently activating diabetogenic T-cells. NOD mice transgenically expressing Ig molecules recognizing antigens that are (insulin) or not (hen egg lysozyme; HEL) expressed by ß-cells have proven useful in dissecting the developmental basis of diabetogenic B-lymphocytes. However, these transgenic Ig specificities were originally selected for their ability to recognize insulin or HEL as foreign, rather than autoantigens. Thus, we generated and characterized NOD mice transgenically expressing an Ig molecule representative of a large proportion of naturally occurring islet-infiltrating B-lymphocytes in NOD mice recognizing the neuronal antigen peripherin. Transgenic peripherin autoreactive B-lymphocytes infiltrate NOD pancreatic islets, acquire an activated proliferative phenotype, and potently support accelerated T1D development. These results support the concept of neuronal autoimmunity as a pathogenic feature of T1D, and targeting such responses could ultimately provide an effective disease intervention approach.

In both NOD mice and humans, the development of type 1 diabetes (T1D) is dependent in part on autoreactive CD8+ T cells recognizing pancreatic β cell peptides presented by often quite common MHC class I variants. Studies in NOD mice previously revealed that the common H2-Kd and/or H2-Db class I molecules expressed by this strain aberrantly lose the ability to mediate the thymic deletion of pathogenic CD8+ T cell responses through interactions with T1D susceptibility genes outside the MHC. A gene(s) mapping to proximal chromosome 7 was previously shown to be an important contributor to the failure of the common class I molecules expressed by NOD mice to mediate the normal thymic negative selection of diabetogenic CD8+ T cells. Using an inducible model of thymic negative selection and mRNA transcript analyses, we initially identified an elevated Nfkbid expression variant as a likely NOD-proximal chromosome 7 region gene contributing to impaired thymic deletion of diabetogenic CD8+ T cells. CRISPR/Cas9–mediated genetic attenuation of Nfkbid expression in NOD mice resulted in improved negative selection of autoreactive diabetogenic AI4 and NY8.3 CD8+ T cells. These results indicated that allelic variants of Nfkbid contribute to the efficiency of intrathymic deletion of diabetogenic CD8+ T cells. However, although enhancing thymic deletion of pathogenic CD8+ T cells, ablating Nfkbid expression surprisingly accelerated T1D onset that was associated with numeric decreases in both regulatory T and B lymphocytes in NOD mice.

Endoplasmic reticulum stress (ER stress) has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease of progressive fibrosis and respiratory failure. ER stress activates a signaling pathway called the unfolded protein response (UPR) that either restores homeostasis or promotes apoptosis. The bifunctional kinase/RNase IRE1α is a UPR sensor that promotes apoptosis if ER stress remains high (i.e., a “terminal” UPR). Using multiple small molecule inhibitors against IRE1α, we show that ER stress-induced apoptosis of murine alveolar epithelial cells can be mitigated in vitro. In vivo, we show that bleomycin exposure to murine lungs causes early ER stress to activate IRE1α and the terminal UPR prior to development of pulmonary fibrosis. Small-molecule IRE1α kinase-inhibiting RNase attenuators (KIRAs) that we developed were used to evaluate the importance of IRE1α activation in bleomycin-induced pulmonary fibrosis. One such KIRA—KIRA7—provided systemically to mice at the time of bleomycin exposure decreases terminal UPR signaling and prevents lung fibrosis. Administration of KIRA7 14 days after bleomycin exposure even promoted the reversal of established fibrosis. Finally, we show that KIRA8, a nanomolar-potent, monoselective KIRA compound derived from a completely different scaffold than KIRA7, likewise promoted reversal of established fibrosis. These results demonstrate that IRE1α may be a promising target in pulmonary fibrosis and that kinase inhibitors of IRE1α may eventually be developed into efficacious anti-fibrotic drugs.

© 2018 Cold Spring Harbor Laboratory Press; all rights reserved. Activation of TGF-β1 initiates a program of temporary collagen accumulation important to wound repair in many organs. However, the outcome of temporary extracellular matrix strengthening all too frequently morphs into progressive fibrosis, contributing to morbidity and mortality worldwide. To avoid this maladaptive outcome, TGF-β1 signaling is regulated at numerous levels and intimately connected to feedback signals that limit accumulation. Here, we examine the current understanding of the core functions of TGF-β1 in promoting collagen accumulation, parallel pathways that promote physiological repair, and pathological triggers that tip the balance toward progressive fibrosis. Implicit in better understanding of these processes is the identification of therapeutic opportunities that will need to be further advanced to limit or reverse organ fibrosis.

In both NOD mice and humans, the development of type 1 diabetes (T1D) is dependent in part on autoreactive CD8(+) T-cells recognizing pancreatic ß-cell peptides presented by often quite common MHC class I variants. Studies in NOD mice previously revealed the common H2-K(d) and/or H2-D(b) class I molecules expressed by this strain aberrantly lose the ability to mediate the thymic deletion of pathogenic CD8(+) T-cell responses through interactions with T1D susceptibility (Idd) genes outside the MHC. A gene(s) mapping to proximal Chromosome 7 was previously shown to be an important contributor to the failure of the common class I molecules expressed by NOD mice to mediate the normal thymic negative selection of diabetogenic CD8(+) T-cells. Using an inducible model of thymic negative selection and mRNA transcript analyses we initially identified an elevated Nfkbid expression variant as a likely NOD proximal Chromosome 7 region gene contributing to impaired thymic deletion of diabetogenic CD8(+) T-cells. CRISPR/Cas9-mediated genetic attenuation of Nfkbid expression in NOD mice resulted in improved negative selection of autoreactive diabetogenic AI4 and NY8.3 CD8(+) T-cells. These results indicated allelic variants of Nfkbid contribute to the efficiency of intrathymic deletion of diabetogenic CD8(+) T-cells. However, while enhancing thymic deletion of pathogenic CD8(+) T-cells, ablating Nfkbid expression surprisingly accelerated T1D onset in NOD mice that was associated with numeric decreases in both regulatory T- and B-lymphocytes (Tregs/Bregs).

Type 1 diabetes (T1D) is characterized by T cell-mediated destruction of the insulin-producing βcells of the pancreatic islets. Among the loci associated with T1D risk, those most predisposing are found in the MHC region. HLA-B*39:06 is the most predisposing class I MHC allele and is associated with an early age of onset. To establish an NOD mouse model for the study of HLA-B*39:06, we expressed it in the absence of murine class I MHC. HLA-B*39:06 was able to mediate the development of CD8 T cells, support lymphocytic infiltration of the islets, and confer T1D susceptibility. Because reduced thymic insulin expression is associated with increased T1D risk in patients, we incorporated this in our model as well, finding that HLA-B*39:06-transgenic NOD mice with reduced thymic insulin expression have an earlier age of disease onset and a higher overall prevalence as compared to littermates with typical thymic insulin expression. This was despite virtually indistinguishable blood insulin levels, T cell subset percentages, and TCR Vβ family usage, indicating that reduced thymic insulin expression does not impact T cell development on a global scale. Rather, we propose that it allows the thymic escape of insulin-reactive HLA-B*39:06-restricted T cells which participate in β cell destruction. We also found that in mice expressing either HLA-B*39:06 or HLA-A*02:01 in the absence of murine class I MHC, HLA transgene identity alters TCR Vβ usage, which may contribute to varying diabetogenic CD8 T cell repertoires in the presence of different HLA class I alleles.

In both NOD mice and humans, the development of type 1 diabetes (T1D) is dependent in part on autoreactive CD8+ T-cells recognizing pancreatic ß-cell peptides presented by often quite common MHC class I variants. Studies in NOD mice previously revealed the common H2-Kd and/or H2-Db class I molecules expressed by this strain acquire an aberrant ability to mediate pathogenic CD8+ T-cell responses through interactions with T1D susceptibility (Idd) genes outside the MHC. A gene(s) mapping to the Idd7 locus on proximal Chromosome 7 was previously shown to be an important contributor to the failure of the common class I molecules expressed by NOD mice to mediate the normal thymic negative selection of diabetogenic CD8+ T-cells. Using an inducible model of thymic negative selection and mRNA transcript analyses we initially identified an elevated Nfkbid expression variant is likely an NOD Idd7 region gene contributing to impaired thymic deletion of diabetogenic CD8+ T-cells. CRISPR/Cas9-mediated genetic attenuation of Nfkbid expression in NOD mice resulted in improved negative selection of autoreactive diabetogenic AI4 and NY8.3 CD8+ T-cells. These results indicated allelic variants of Nfkbid represent an Idd7 gene contributing to the efficiency of intrathymic deletion of diabetogenic CD8+ T-cells. However, while enhancing thymic deletion of pathogenic CD8+ T-cells, ablation of Nfkbid expression surprisingly accelerated T1D onset in NOD mice likely at least in part by numerically decreasing regulatory T- and B-lymphocytes (Tregs/Bregs), thereby reducing their peripheral immunosuppressive effects.

Broadly, tissue regeneration is achieved in two ways: by proliferation of common differentiated cells and/or by deployment of specialized stem/progenitor cells. Which of these pathways applies is both organ- and injury-specific. Current models in the lung posit that epithelial repair can be attributed to cells expressing mature lineage markers. By contrast, here we define the regenerative role of previously uncharacterized, rare lineage-negative epithelial stem/progenitor (LNEP) cells present within normal distal lung. Quiescent LNEPs activate a ΔNp63 (a p63 splice variant) and cytokeratin 5 remodelling program after influenza or bleomycin injury in mice. Activated cells proliferate and migrate widely to occupy heavily injured areas depleted of mature lineages, at which point they differentiate towards mature epithelium. Lineage tracing revealed scant contribution of pre-existing mature epithelial cells in such repair, whereas orthotopic transplantation of LNEPs, isolated by a definitive surface profile identified through single-cell sequencing, directly demonstrated the proliferative capacity and multipotency of this population. LNEPs require Notch signalling to activate the ΔNp63 and cytokeratin 5 program, and subsequent Notch blockade promotes an alveolar cell fate. Persistent Notch signalling after injury led to parenchymal 'micro-honeycombing' (alveolar cysts), indicative of failed regeneration. Lungs from patients with fibrosis show analogous honeycomb cysts with evidence of hyperactive Notch signalling. Our findings indicate that distinct stem/progenitor cell pools repopulate injured tissue depending on the extent of the injury, and the outcomes of regeneration or fibrosis may depend in part on the dynamics of LNEP Notch signalling.

© 2014 Elsevier Inc. TGF-β signaling is essential in many processes, including immune surveillance, and its dysregulation controls various diseases, including cancer, fibrosis, and inflammation. Studying the innate host defense, which functions in most cell types, we found that RLR signaling represses TGF-β responses. This regulation is mediated by activated IRF3, using a dual mechanism of IRF3-directed suppression. Activated IRF3 interacts with Smad3, thus inhibiting TGF-β-induced Smad3 activation and, in the nucleus, disrupts functional Smad3 transcription complexes by competing with coregulators. Consequently, IRF3 activation by innate antiviral signaling represses TGF-β-induced growth inhibition, gene regulation and epithelial-mesenchymal transition, and the generation of Treg effector lymphocytes from naive CD4+lymphocytes. Conversely, silencing IRF3 expression enhances epithelial-mesenchymal transition, TGF-β-induced Treg cell differentiation upon virus infection, and Treg cell generation invivo. Wepresent a mechanism of regulation of TGF-β signaling by the antiviral defense, with evidence for its role in immune tolerance and cancer cell behavior.

Segmented filamentous bacterium (SFB) a gram-positive, anaerobic, and intestinal commensal organism directly influences the development of Th17 helper cells in the small intestine of mice. In NOD mice, SFB colonization interferes with the development of type 1 diabetes (T1D), a T-cell–mediated autoimmune disease, suggesting that SFB may influence Th17 cells to inhibit Th1 populations associated with the anti-β-cell immune response. This effect is a serious concern for investigators who use NOD mice for diabetes research because the expected incidence of disease decreases markedly when they are colonized by SFB. A room housing mice for T1D studies at The Jackson Laboratory was determined by fecal PCR testing to have widespread SFB colonization of multiple NOD strains after a steady decline in the incidence of T1D was noted. Rederivation of all NOD-related mouse strains was not feasible; therefore an alternative treatment using antibiotics to eliminate SFB from colonized mice was undertaken. After antibiotic treatment, soiled bedding from NOD mouse strains housed in SFB-free high-health–status production barrier rooms was used to reintroduce the gastrointestinal microbiota. Over the past 16 mo since treating the mice and disinfecting the mouse room, regular PCR testing has shown that no additional SFB colonization of mice has occurred, and the expected incidence of T1D has been reestablished in the offspring of treated mice.

During the acute respiratory distress syndrome, epithelial cells, primarily alveolar type (AT) I cells, die and slough off, resulting in enhanced permeability. ATII cells proliferate and spread onto the denuded basement membrane to reseal the barrier. Repair of the alveolar epithelium is critical for clinical recovery; however, mechanisms underlying ATII cell proliferation and spreading are not well understood. We hypothesized that hypoxia-inducible factor (HIF)1α promotes proliferation and spreading of ATII cells during repair after lung injury. Mice were treated with lipopolysaccharide or hydrochloric acid. HIF activation in ATII cells after injury was demonstrated by increased luciferase activity in oxygen degradation domain-Luc (HIF reporter) mice and expression of the HIF1α target gene GLUT1. ATII cell proliferation during repair was attenuated in ATII cell-specific HIF1α knockout (SftpcCreERT2+/-;HIF1αf/f) mice. The HIF target vascular endothelial growth factor promoted ATII cell proliferation invitro and after lung injury invivo. In the scratch wound assay of cell spreading, HIF stabilization accelerated, whereas HIF1α shRNA delayed wound closure. SDF1 and its receptor, CXCR4, were found to be HIF1α-regulated genes in ATII cells and were up-regulated during lung injury. Stromal cell-derived factor 1/CXCR4 inhibition impaired cell spreading and delayed the resolution of permeability after lung injury. We conclude that HIF1α is activated in ATII cells after lung injury and promotes proliferation and spreading during repair.

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Type 1 diabetes (T1D) in both humans and the NOD mouse model is caused by the autoimmune mediated killing of pancreatic β-cells. Increased frequency or activity of autoreactive T cells and failures of regulatory T cells (Tregs) to control autoreactive T cells have both been implicated in T1D etiology. Due to the expression of MHC-I molecules on β-cells, autoreactive CD8+ T cells are likely the ultimate mediators of this destruction. Developing CD8+ T cells undergo selection processes in the thymus whereby most autoreactive clonotypes are eliminated during their development. Fine mapping of a genetic locus associated with defective thymic deletion of autoreactive CD8+ AI4 TCR transgenic (AI4) T cells in NOD mice led to the identification of Nfkbid, a NFκB signal modulator, as contributing to this phenotype. Ablation of Nfkbid in NOD mice by CRISPR/Cas9 results in a decreased frequency of autoreactive CD8+ AI4 T cells and an unexpected acceleration of T1D incidence, associated with reductions in the frequency and functional capacity of peripheral Tregs. Transgenic overexpression of Nfkbid in NOD mice paradoxically results in further decreased levels of circulating autoreactive CD8+ AI4 T cells. However, transgenic elevation of Nfkbid expression also increased the frequency and functional capacity of peripheral Tregs with a striking complete absence of T1D development in these mice. Studies are ongoing to define the mechanisms of T1D resistance mediated by transgenic overexpression of the transcriptional co-factor Nfkbid.