Epithelial Endoplasmic Reticulum Stress Enhances the Risk of Muc5b-associated Lung Fibrosis.

The gain-of-function minor allele of the MUC5B (mucin 5B, oligomeric mucus/gel-forming) promoter (rs35705950) is the strongest risk factor for idiopathic pulmonary fibrosis (IPF), a devastating fibrotic lung disease that leads to progressive respiratory failure in adults. We have previously demonstrated that Muc5b overexpression in mice worsens lung fibrosis after bleomycin exposure and have hypothesized that excess Muc5b promotes endoplasmic reticulum (ER) stress and apoptosis, stimulating fibrotic lung injury. Here, we report that ER stress pathway members ATF4 (activating transcription factor 4) and ATF6 coexpress with MUC5B in epithelia of the distal IPF airway and honeycomb cyst and that this is more pronounced in carriers of the gain-of-function MUC5B promoter variant. Similarly, in mice exposed to bleomycin, Muc5b expression is temporally associated with markers of ER stress. Using bulk and single-cell RNA sequencing in bleomycin-exposed mice, we found that pathologic ER stress-associated transcripts Atf4 and Ddit3 (DNA damage inducible transcript 3) were elevated in alveolar epithelia of SFTPC-Muc5b transgenic (SFTPC-Muc5bTg) mice relative to wild-type (WT) mice. Activation of the ER stress response inhibits protein translation for most genes by phosphorylation of Eif2α (eukaryotic translation initiation factor 2 alpha), which prevents guanine exchange by Eif2B and facilitates translation of Atf4. The integrated stress response inhibitor (ISRIB) facilitates interaction of phosphorylated Eif2α with Eif2B, overcoming translation inhibition associated with ER stress and reducing Atf4. We found that a single dose of ISRIB diminished Atf4 translation in SFTPC-Muc5bTg mice after bleomycin injury. Moreover, ISRIB resolved the exaggerated fibrotic response of SFTPC-Muc5bTg mice to bleomycin. In summary, we demonstrate that MUC5B and Muc5b expression is associated with pathologic ER stress and that restoration of normal translation with a single dose of ISRIB promotes lung repair in bleomycin-injured Muc5b-overexpressing mice.

Idiopathic Pulmonary Fibrosis: A Genetic Disease That Involves Mucociliary Dysfunction of the Peripheral Airways.

Idiopathic pulmonary fibrosis (IPF) is an incurable complex genetic disorder that is associated with sequence changes in 7 genes (MUC5B, TERT, TERC, RTEL1, PARN, SFTPC, and SFTPA2) and with variants in at least 11 novel loci. We have previously found that 1) a common gain-of-function promoter variant in MUC5B rs35705950 is the strongest risk factor (genetic and otherwise), accounting for 30-35% of the risk of developing IPF, a disease that was previously considered idiopathic; 2) the MUC5B promoter variant can potentially be used to identify individuals with preclinical pulmonary fibrosis and is predictive of radiologic progression of preclinical pulmonary fibrosis; and 3) MUC5B may be involved in the pathogenesis of pulmonary fibrosis with MUC5B message and protein expressed in bronchiolo-alveolar epithelia of IPF and the characteristic IPF honeycomb cysts. Based on these considerations, we hypothesize that excessive production of MUC5B either enhances injury due to reduced mucociliary clearance or impedes repair consequent to disruption of normal regenerative mechanisms in the distal lung. In aggregate, these novel considerations should have broad impact, resulting in specific etiologic targets, early detection of disease, and novel biologic pathways for use in the design of future intervention, prevention, and mechanistic studies of IPF.

Spatially distinct molecular patterns of gene expression in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a heterogeneous disease that is pathologically characterized by areas of normal-appearing lung parenchyma, active fibrosis (transition zones including fibroblastic foci) and dense fibrosis. Defining transcriptional differences between these pathologically heterogeneous regions of the IPF lung is critical to understanding the distribution and extent of fibrotic lung disease and identifying potential therapeutic targets. Application of a spatial transcriptomics platform would provide more detailed spatial resolution of transcriptional signals compared to previous single cell or bulk RNA-Seq studies. METHODS: We performed spatial transcriptomics using GeoMx Nanostring Digital Spatial Profiling on formalin-fixed paraffin-embedded (FFPE) tissue from 32 IPF and 12 control subjects and identified 231 regions of interest (ROIs). We compared normal-appearing lung parenchyma and airways between IPF and controls with histologically normal lung tissue, as well as histologically distinct regions within IPF (normal-appearing lung parenchyma, transition zones containing fibroblastic foci, areas of dense fibrosis, and honeycomb epithelium metaplasia). RESULTS: We identified 254 differentially expressed genes (DEGs) between IPF and controls in histologically normal-appearing regions of lung parenchyma; pathway analysis identified disease processes such as EIF2 signaling (important for cap-dependent mRNA translation), epithelial adherens junction signaling, HIF1α signaling, and integrin signaling. Within IPF, we identified 173 DEGs between transition and normal-appearing lung parenchyma and 198 DEGs between dense fibrosis and normal lung parenchyma; pathways dysregulated in both transition and dense fibrotic areas include EIF2 signaling pathway activation (upstream of endoplasmic reticulum (ER) stress proteins ATF4 and CHOP) and wound healing signaling pathway deactivation. Through cell deconvolution of transcriptome data and immunofluorescence staining, we confirmed loss of alveolar parenchymal signals (AGER, SFTPB, SFTPC), gain of secretory cell markers (SCGB3A2, MUC5B) as well as dysregulation of the upstream regulator ATF4, in histologically normal-appearing tissue in IPF. CONCLUSIONS: Our findings demonstrate that histologically normal-appearing regions from the IPF lung are transcriptionally distinct when compared to similar lung tissue from controls with histologically normal lung tissue, and that transition zones and areas of dense fibrosis within the IPF lung demonstrate activation of ER stress and deactivation of wound healing pathways.

Global and regional burden of interstitial lung disease and pulmonary sarcoidosis from 1990 to 2019: results from the Global Burden of Disease study 2019.

BACKGROUND: Interstitial lung disease (ILD) and pulmonary sarcoidosis are common respiratory diseases with a heterogeneous distribution worldwide. The global burden and temporal trends of ILD and sarcoidosis are rarely explored. METHODS: Using the classification 'ILD and pulmonary sarcoidosis' from the Global Burden of Disease 2019 dataset, we described the age-standardised rates of incidence, mortality, disability-adjusted life-years (DALYs), and their average annual percentage change from 1990 to 2019 by sex, Sociodemographic Index (SDI) and region. RESULTS: In 2019, the global incidence and mortality of ILD and pulmonary sarcoidosis were 24.2 million and 169 833 cases, respectively. From 1990 to 2019, the global incidence, deaths and DALYs due to ILD and pulmonary sarcoidosis increased by 118.6%, 166.63% and 122.87% respectively. The global incidence of ILD and pulmonary sarcoidosis was higher in men and was mainly concentrated among persons aged 70‒79 of both sexes. Significant regional differences could be seen in the disease burden of ILD and pulmonary sarcoidosis: since 2006, high-SDI regions had higher age-standardised incidence rates but lower age-standardised death rates compared with the low-SDI regions. CONCLUSIONS: Our study suggests the incidence, mortality and DALYs from ILD and pulmonary sarcoidosis are increasing globally. To reduce the ongoing burden of this condition, early diagnosis and treatment are vital, and more targeted and specific strategies should be established in high-burden regions. Differences in incidence and mortality across regions may reflect the influence of genetic, environmental, diagnostic, pharmacotherapeutic, and health system factors.

Genes, other than Muc5b, play a role in bleomycin-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an incurable genetic disease that affects 5 million people worldwide. The gain-of-function MUC5B promoter variant rs35705950 is the dominant genetic risk factor for IPF, yet has a low penetrance. This raises the possibility that other genes and transcripts affect the penetrance of MUC5B. Previously, we have shown that the concentration of Muc5b in bronchoalveolar epithelia is directly associated with the extent and persistence of bleomycin-induced lung fibrosis in mice. In this study, we investigated whether bleomycin-induced lung injury is Muc5b dependent in genetically divergent strains of mice. Specifically, mice from the eight Diversity Outbred (DO) founders were phenotyped for Muc5b expression and lung fibrosis 3 wk after intratracheal bleomycin administration. Although we identified strains with low Muc5b expression and minimal lung fibrosis (CAST/EiJ and PWK/PhJ) and strains with high Muc5b expression and extensive lung fibrosis (NZO/H1LtJ and WSB/EiJ), there also were strains that did not demonstrate a clear relationship between Muc5b expression and lung fibrosis (129S1/SvlmJ, NOD/ShiLtJ, and C57BL/6J, A/J). Hierarchical clustering suggests that other factors may work in concert with or potentially independent of Muc5b to promote bleomycin-induced lung injury and fibrosis. This study suggests that these strains and their recombinant inbred crosses may prove helpful in identifying the genes and transcripts that interact with Muc5b and cause lung fibrosis.

Chronic Hypersensitivity Pneumonitis, an Interstitial Lung Disease with Distinct Molecular Signatures.

Rationale: Chronic hypersensitivity pneumonitis (CHP) is caused by an immune response to antigen inhalation and is characterized by variable histopathological and clinical features. A subset of subjects with CHP have usual interstitial pneumonia and appear to be clinically similar to subjects with idiopathic pulmonary fibrosis (IPF).Objectives: To determine the common and unique molecular features of CHP and IPF.Methods: Transcriptome analysis of lung samples from CHP (n = 82), IPF (n = 103), and unaffected controls (n = 103) was conducted. Differential gene expression was determined adjusting for sex, race, age, and smoking history and using false discovery rate to control for multiple comparisons.Measurements and Main Results: When compared with controls, we identified 413 upregulated and 317 downregulated genes in CHP and 861 upregulated and 322 downregulated genes in IPF. Concordantly upregulated or downregulated genes in CHP and IPF were related to collagen catabolic processes and epithelial development, whereas genes specific to CHP (differentially expressed in CHP when compared with control and not differentially expressed in IPF) were related to chemokine-mediated signaling and immune responsiveness. Using weighted gene coexpression network analysis, we found that among subjects with CHP, genes involved in adaptive immunity or epithelial cell development were associated with improved or reduced lung function, respectively, and that MUC5B expression was associated with epithelial cell development. MUC5B expression was also associated with lung fibrosis and honeycombing.Conclusions: Gene expression analysis of CHP and IPF identified signatures common to CHP and IPF, as well as genes uniquely expressed in CHP. Select modules of gene expression are characterized by distinct clinical and pathological features of CHP.

Use of a Cholestyramine Washout in a Patient With Septic Shock on Leflunomide Therapy: A Case Report and Review of the Literature.

Patients presenting with infections while receiving disease-modifying antirheumatic agents (DMARD) may be predisposed to a higher degree illness due to immunosuppression. This can be particularly problematic in patients who are receiving DMARDs with prolonged pharmacokinetic profiles. Leflunomide is a DMARD that has a prolonged half-life due to enterohepatic recirculation. We report a case of a patient with severe septic shock secondary to a prosthetic joint infection in which therapeutic levels of leflunomide were discovered, despite the patient ceasing therapy several weeks prior to admission. An orogastric cholestyramine washout was given to the patient to expedite the removal of the drug. Serum levels rapidly declined over the next several days, corresponding with resolution of her sepsis. A review of the literature relevant to the incidence of DMARD-related infections was conducted as well as discussion regarding the role of leflunomide drug monitoring and cholestyramine-facilitated removal of the drug in episodes of acute infectious syndromes.

Type I IFN-dependent T cell activation is mediated by IFN-dependent dendritic cell OX40 ligand expression and is independent of T cell IFNR expression.

Type I IFNs are important for direct control of viral infection and generation of adaptive immune responses. Recently, direct stimulation of CD4(+) T cells via type I IFNR has been shown to be necessary for the formation of functional CD4(+) T cell responses. In contrast, we find that CD4(+) T cells do not require intrinsic type I IFN signals in response to combined TLR/anti-CD40 vaccination. Rather, the CD4 response is dependent on the expression of type I IFNR (IFNαR) on innate cells. Further, we find that dendritic cell (DC) expression of the TNF superfamily member OX40 ligand was dependent on type I IFN signaling in the DC, resulting in a reduced CD4(+) T cell response that could be substantially rescued by an agonistic Ab to the receptor OX40. Taken together, we show that the IFNαR dependence of the CD4(+) T cell response is accounted for exclusively by defects in DC activation.

TLR7 enables cross-presentation by multiple dendritic cell subsets through a type I IFN-dependent pathway.

Conjugation of TLR agonists to protein or peptide antigens has been demonstrated in many studies to be an effective vaccine formula in inducing cellular immunity. However, the molecular and cellular mediators involved in TLR-induced immune responses have not been carefully examined. In this study, we identify Type I IFN and IL-12 as critical mediators of cross-priming induced by a TLR7 agonist-antigen conjugate. We demonstrate that TLR7-driven cross-priming requires both Type I IFN and IL-12. Signaling through the IFN-αßR was required for the timely recruitment and accumulation of activated dendritic cells in the draining lymph nodes. Although IL-12 was indispensable during cross-priming, it did not regulate DC function. Therefore, the codependency for these 2 cytokines during TLR7-induced cross-priming is the result of their divergent effects on different cell-types. Furthermore, although dermal and CD8α(+) DCs were able to cross-prime CD8(+) T cells, Langerhans cells were unexpectedly found to potently cross-present antigen and support CD8(+) T-cell expansion, both in vitro and in vivo. Collectively, the data show that a TLR7 agonist-antigen conjugate elicits CD8(+) T-cell responses by the coordinated recruitment and activation of both tissue-derived and lymphoid organ-resident DC subsets through a Type I IFN and IL-12 codependent mechanism.

Comparison of OX40 ligand and CD70 in the promotion of CD4+ T cell responses.

The TNF superfamily members CD70 and OX40 ligand (OX40L) were reported to be important for CD4(+) T cell expansion and differentiation. However, the relative contribution of these costimulatory signals in driving CD4(+) T cell responses has not been addressed. In this study, we found that OX40L is a more important determinant than CD70 of the primary CD4(+) T cell response to multiple immunization regimens. Despite the ability of a combined TLR and CD40 agonist (TLR/CD40) stimulus to provoke appreciable expression of CD70 and OX40L on CD8(+) dendritic cells, resulting CD4(+) T cell responses were substantially reduced by Ab blockade of OX40L and, to a lesser degree, CD70. In contrast, the CD8(+) T cell responses to combined TLR/CD40 immunization were exclusively dependent on CD70. These requirements for CD4(+) and CD8(+) T cell activation were not limited to the use of combined TLR/CD40 immunization, because vaccinia virus challenge elicited primarily OX40L-dependent CD4 responses and exclusively CD70-dependent CD8(+) T cell responses. Attenuation of CD4(+) T cell priming induced by OX40L blockade was independent of signaling through the IL-12R, but it was reduced further by coblockade of CD70. Thus, costimulation by CD70 or OX40L seems to be necessary for primary CD4(+) T cell responses to multiple forms of immunization, and each may make independent contributions to CD4(+) T cell priming.

From ARDS to pulmonary fibrosis: the next phase of the COVID-19 pandemic?

While the coronavirus disease 19 (COVID-19) pandemic has transformed the medical and scientific communites since it was first reported in late 2019, we are only beginning to understand the chronic health burdens associated with this disease. Although COVID-19 is a multi-systemic disease, the lungs are the primary source of infection and injury, resulting in pneumonia and, in severe cases, acute respiratory distress syndrome (ARDS). Given that pulmonary fibrosis is a well-recognized sequela of ARDS, many have questioned whether COVID-19 survivors will face long-term pulmonary consequences. This review is aimed at integrating our understanding of the pathophysiologic mechanisms underlying fibroproliferative ARDS with our current knowledge of the pulmonary consequences of COVID-19 disease.

Dysregulated Cell-Cell Communication Characterizes Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of older adults characterized by fibrotic replacement of functional gas exchange units in the lung. The strongest risk factor for IPF is a genetic variantin the promoter region of the gel-forming mucin, MUC5B. To better understand how the MUC5B variant influences development of fibrosis, we used the NicheNet R package and leveraged publicly available single-cell RNA sequencing data to identify and evaluate how epithelia participating in gas exchange are influenced by ligands expressed in control, MUC5B variant, and fibrotic environments. We observed that loss of type-I alveolar epithelia (AECI) characterizes the single-cell RNA transcriptome in fibrotic lung and validated the pattern of AECI loss using single nuclear RNA sequencing. Examining AECI transcriptomes, we found enrichment of transcriptional signatures for IL6 and AREG, which we have previously shown to mediate aberrant epithelial fluidization in IPF and murine bleomycin models. Moreover, we found that the protease ADAM17, which is upstream of IL6 trans-signaling, was enriched in control MUC5B variant donors. We used immunofluorescence to validate a role for enhanced expression of ADAM17 among MUC5B variants, suggesting involvement in IPF pathogenesis and maintenance.

Interleukin-6-dependent epithelial fluidization initiates fibrotic lung remodeling.

Chronic disease results from the failure of tissues to maintain homeostasis. In the lung, coordinated repair of the epithelium is essential for preserving homeostasis. In animal models and human lung disease, airway epithelial cells mobilize in response to lung injury, resulting in the formation of airway-like cysts with persistent loss of functional cell types and parenchymal architecture. Using live-cell imaging of human lung epithelial cultures and mouse precision-cut lung slices, we demonstrated that distal airway epithelia are aberrantly fluidized both after injury and in fibrotic lung disease. Through transcriptomic profiling and pharmacologic stimulation of epithelial cultures, we identified interleukin-6 (IL-6) signaling as a driver of tissue fluidization. This signaling cascade occurred independently of canonical Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling but instead was dependent on a downstream SRC family kinase (SFK)-yes-associated protein (YAP) axis. Airway epithelial-fibroblast cocultures revealed that the fibrotic mesenchyme acts as a source of IL-6 family cytokines, which drive airway fluidization. Inhibition of the IL-6-SFK-YAP cascade was sufficient to prevent fluidization in both in vitro and ex vivo models. Last, we demonstrated a reduction in fibrotic lung remodeling in mice through genetic or pharmacologic targeting of IL-6-related signaling. Together, our findings illustrate the critical role of airway epithelial fluidization in coordinating the balance between homeostatic lung repair and fibrotic airspace remodeling.

The MUC5B-associated variant rs35705950 resides within an enhancer subject to lineage- and disease-dependent epigenetic remodeling.

The G/T transversion rs35705950, located approximately 3 kb upstream of the MUC5B start site, is the cardinal risk factor for idiopathic pulmonary fibrosis (IPF). Here, we investigate the function and chromatin structure of this -3 kb region and provide evidence that it functions as a classically defined enhancer subject to epigenetic programming. We use nascent transcript analysis to show that RNA polymerase II loads within 10 bp of the G/T transversion site, definitively establishing enhancer function for the region. By integrating Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis of fresh and cultured human airway epithelial cells with nuclease sensitivity data, we demonstrate that this region is in accessible chromatin that affects the expression of MUC5B. Through applying paired single-nucleus RNA- and ATAC-seq to frozen tissue from IPF lungs, we extend these findings directly to disease, with results indicating that epigenetic programming of the -3 kb enhancer in IPF occurs in both MUC5B-expressing and nonexpressing lineages. In aggregate, our results indicate that the MUC5B-associated variant rs35705950 resides within an enhancer that is subject to epigenetic remodeling and contributes to pathologic misexpression in IPF.

Spontaneous allograft tolerance in B7-deficient mice independent of preexisting endogenous CD4+CD25+ regulatory T-cells.

BACKGROUND: Acute cardiac allograft rejection requires host, but not donor, expression of B7-1/B7-2 costimulatory molecules. However, acute cardiac rejection requires direct antigen presentation by donor-derived antigen presenting cells to CD4 T-cells and does not require indirect antigen presentation to CD4 T-cells. Given this discrepancy in the literature and that the consequence of allograft exposure in B7-deficient mice is unknown; the goal of the study was to examine the antidonor status of allografted B7-1/B7-2-deficient hosts. METHODS: C57Bl/6 B7-1/B7-2-/- mice were grafted with heterotopic BALB/c hearts. Recipients bearing long-term surviving allografts were used to examine the status of antidonor reactivity in vitro and in vivo. Tolerance was examined in vivo through adoptive transfer of splenocytes from graft-bearing animals to secondary immune-deficient Rag-1-/- hosts bearing donor-type or third-party cardiac allografts and by regulatory T-cell depletion with anti-CD25 antibody. RESULTS: When transferred to B7-replete Rag-1-/- recipients, cells from naïve B7-1/B7-2-/- mice readily initiated cardiac allograft rejection. However, splenocytes transferred from long-term allograft acceptor B7-1/B7-2-/- hosts failed to reject donor-type hearts but acutely rejected third-party allografts. In addition, such cells did not reject (donorxthird-party) F1 allografts. Finally, in vivo depletion of regulatory T-cells did not prevent long-term acceptance. CONCLUSIONS: Results demonstrate that B7-deficient T-cells are capable of acute cardiac allograft rejection in a B7-replete environment. Importantly, results also show that B7-deficient hosts do not simply ignore cardiac allografts, but rather spontaneously develop transferable, donor-specific tolerance and linked suppression in vivo. Interestingly, this tolerant state does not require endogenous CD4+CD25+ regulatory T-cells.

T cell vaccinology: exploring the known unknowns.

The objective of modern vaccine development is the safe generation of protective long-term immune memory, both prophylactic and therapeutic. Live attenuated vaccines generate potent cellular and humoral immunity [1-3], but numerous problems exist with these vaccines, ranging from production and storage issues to adverse reactions and reversion to virulence. Subunit vaccines are safer, more stable, and more amenable to mass production. However the protection they produce is frequently inferior to live attenuated vaccines and is typically confined to humoral, and not cellular immunity. Unfortunately, there are presently no subunit vaccines available clinically that are effective at eliciting cellular responses let alone cellular memory [4]. This article will provide and overview of areas of investigation that we see as important for the development of vaccines with the capacity to induce robust and enduring cellular immune responses.