Toll-like Receptor 9 Inhibition Mitigates Fibroproliferative Responses in Translational Models of Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease for which current treatment options only slow clinical progression. Previously, we identified a subset of patients with IPF with an accelerated disease course associated with fibroblast expression of Toll-Like Receptor 9 (TLR9) mediated by interactions with its ligand mitochondrial DNA (mtDNA). OBJECTIVES: We aimed to show that TLR9 activation induces fibroproliferative responses that are abrogated by its antagonism by using two commercially-available indirect inhibitors and a proprietary, selective direct small molecule inhibitor. METHODS: We employed two independent cohorts of patients with IPF, multiple in vitro fibroblast cell culture platforms, an in vivo mouse model, and an ex vivo human precision cut lung slices system to investigate the clinical and biologic significance of TLR9 in this disease. MEASUREMENTS AND MAIN RESULTS: In two independent IPF cohorts, plasma mtDNA activates TLR9 in a manner associated with the expression of MCP-1, IL-6, TNFα, and IP-10 and worsened transplant-free survival. Our cell culture platform showed that TLR9 mediates fibroblast activation via TGFß1 and stiff substrates, and that its antagonism, particularly direct inhibition, ameliorates this process, including production of these TLR9 associated pharmacodynamic endpoints. We further demonstrated that direct TLR9 inhibition mitigates these fibroproliferative responses in our in vivo and ex vivo models of pulmonary fibrosis. CONCLUSIONS: In this novel study, we found that direct TLR9 inhibition mitigates fibroproliferative responses in preclinical models of pulmonary fibrosis. Our work demonstrates the therapeutic potential of direct TLR9 antagonism in IPF and related fibrotic lung diseases.

Plasma collagen neoepitopes are associated with multiorgan disease in the ACCESS and GRADS sarcoidosis cohorts.

INTRODUCTION: The pathogenesis of sarcoidosis involves tissue remodelling mediated by the accumulation of abnormal extracellular matrix, which is partly the result of an imbalance in collagen synthesis, cross-linking and degradation. During this process, collagen fragments or neoepitopes, are released into the circulation. The significance of these circulating collagen neoepitopes in sarcoidosis remains unknown. METHODS: We employed plasma samples from patients with sarcoidosis enrolled in A Case Control Etiologic Study of Sarcoidosis (ACCESS) and Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS), and healthy control patients recruited from the Yale community. Plasma concentrations of type III and VI collagen degradation (C3M and C6M) and formation (PRO-C3 and PRO-C6) were quantified via neoepitope-specific competitive ELISA, and statistical associations were sought with clinical phenotypes. RESULTS: Relative to healthy controls, the plasma of both sarcoidosis cohorts was enriched for C3M and C6M, irrespective of corticosteroid use and disease duration. While circulating collagen neoepitopes were independent of Scadding stage, there was a significant association between multiorgan disease and PRO-C3, PRO-C6 and C3M in the ACCESS cohort; PRO-C3 and C6M displayed this property in GRADS. These findings were unrelated to plasma levels of interleukin-4 (IL-4), IL-5, IL-6, IL-9, IL-10 and IL-13. Moreover, PRO-C3 was associated with dermatological disease in both cohorts. DISCUSSION: In two well-characterised sarcoidosis cohorts, we discovered that the plasma is enriched for neoepitopes of collagen degradation (C3M and C6M). In multiorgan disease, there was an association with circulating neoepitopes of type III formation (PRO-C3), perhaps mediated by dermatological sarcoidosis. Further investigation in this arena has the potential to foster new insights into the pathogenic mechanisms of this complex disease.

Endoscopic submucosal dissection for colorectal polyps: outcome determining factors.

BACKGROUND: Endoscopic submucosal dissection (ESD) has gained increasing popularity in the management of complicated colorectal polyps. However, clinical outcomes for ESD have remained highly inconsistent worldwide. This study investigated and analysed factors that significantly affect ESD outcomes. METHODS: We conducted a single-centred retrospective study on 220 colorectal polyps removed by ESD from 1st January 2016 to 31st December 2020. Data were collected and retrieved from clinical records. Variables studied included patient demographics, ESD technicalities and polyp characteristics. The primary outcome was completeness of resection based on en bloc and R0 resection rates. The secondary outcomes were recurrence, complications and hospital stay. Further analysis was performed for significant outcome determining factors. RESULTS: The en bloc resection and R0 resection rates were 97.3% and 65% respectively. Intraprocedural and delayed perforation rates were 3.2% and 0.5% respectively. Intraprocedural and delayed bleeding rates were both 1.8%. Post-polypectomy syndrome rate was 2.7%. The median hospital stay was 4 days. Submucosal fibrosis was a significant determining factor for lower en bloc resection (p = 0.004), lower R0 resection (p = 0.002), intraprocedural perforation (p = 0.001), intraprocedural bleeding (p = 0.025) and post-polypectomy syndrome (p = 0.039). Hybrid snaring was associated with lower en bloc resection (p < 0.001), while longer ESD time was associated with lower R0 resection (p = 0.003) and post-polypectomy syndrome (p = 0.025). Other significant factors for post-polypectomy syndrome included young age (p = 0.021) and large polyp size (p = 0.018). Secondary analysis showed that submucosal fibrosis was significantly associated with non-granular lesions (p < 0.001) and prior biopsy (p = 0.003). CONCLUSION: Submucosal fibrosis, hybrid snaring, ESD time, age and polyp size were significant outcome determining factors for ESD. By identifying these factors, strategies may be formulated to improve ESD outcomes.

Review on colorectal endoscopic submucosal dissection focusing on the technical aspect.

BACKGROUND: Endoscopic submucosal dissection (ESD) enables removal of broad-based colorectal polyps or lateral spreading tumors with a higher rate of en bloc resection and a lower risk of local recurrence. However, it is technically demanding. Over the past 20 years, various equipment and innovative techniques have been developed to reduce the difficulty of ESD. The information in the literature is scattered. Our aim is to provide a comprehensive review on the setup and technical aspects of colorectal ESD. METHODS: We searched the PubMed database and systemically reviewed all original and review articles related to colorectal ESD. Further manual search according to reference lists of identified articles were done. The selected articles were categorized and reviewed. Original figures were created to help readers understand some of the ESD techniques. RESULTS: A total of 216 articles were identified, in which 25 of them were review articles and 191 of them were original articles. They were categorized and reviewed. An in-depth appraisal of the setting, equipment, and technical aspects of colorectal ESD was performed. CONCLUSIONS: Although ESD is a technically demanding procedure that requires expert endoscopic skills, it can be mastered. With good peri-procedural preparation, sufficient knowledge of the equipment, and thorough understanding of the useful endoscopic tricks and maneuvers, colorectal ESD can be performed smoothly and safely.

A Nerve-Fibroblast Axis in Mammalian Lung Fibrosis.

Tissue fibrosis contributes to pathology in vital organs including the lung. Curative therapies are scant. Myofibroblasts, pivotal effector cells in tissue fibrosis, accumulate via incompletely understood interactions with their microenvironment. In an investigative platform grounded in experimental lung biology, we find that sympathetic innervation stimulates fibrotic remodeling via noradrenergic α1-adrenergic receptor engagement in myofibroblasts. We demonstrate the anti-fibrotic potential of targeted sympathetic denervation and pharmacological disruption of noradrenergic neurotransmitter functions mediated by α1-adrenoreceptors (α1-ARs). Using the α1-adrenoreceptor subtype D as a representative α1-AR, we discover direct noradrenergic input from sympathetic nerves to lung myofibroblasts utilizing established mouse models, genetic denervation, pharmacologic interventions, a newly invented transgenic mouse line, advanced tissue mimetics, and samples from patients with diverse forms of pulmonary fibrosis. The discovery of this previously unappreciated nerve-fibroblast axis in the lung demonstrates the crucial contribution of nerves to tissue repair and heralds a novel paradigm in fibrosis research.

cGAS Expression is Enhanced in Systemic Sclerosis Associated Interstitial Lung Disease and Stimulates Inflammatory Myofibroblast Activation.

Objective: The lungs of patients with Systemic Sclerosis Associated Interstitial Lung Disease (SSc-ILD) contain inflammatory myofibroblasts arising in association with fibrotic stimuli and perturbed innate immunity. The innate immune DNA binding receptor Cyclic GMP-AMP synthase (cGAS) is implicated in inflammation and fibrosis, but its involvement in SSc-ILD remains unknown. We examined cGAS expression, activity, and therapeutic potential in SSc-ILD using cultured fibroblasts, precision cut lung slices (PCLS), and a well-accepted animal model. Methods: Expression and localization of cGAS, cytokines, and type 1 interferons were evaluated in SSc-ILD lung tissues, bronchoalveolar lavage (BAL), and isolated lung fibroblasts. CGAS activation was assessed in a publicly available SSc-ILD single cell RNA sequencing dataset. Production of cytokines, type 1 interferons, and αSMA elicited by TGFß1 or local substrate stiffness were measured in normal human lung fibroblasts (NHLFs) via qRT-PCR, ELISA, and immunofluorescence. Small molecule cGAS inhibition was tested in cultured fibroblasts, human PCLS, and the bleomycin pulmonary fibrosis model. Results: SSc-ILD lung tissue and BAL are enriched for cGAS, cytokines, and type 1 interferons. The cGAS pathway shows constitutive activation in SSc-ILD fibroblasts and is inducible in NHLFs by TGFß1 or mechanical stimuli. In these settings, and in human PCLS, cGAS expression is paralleled by the production of cytokines, type 1 interferons, and αSMA that are mitigated by a small molecule cGAS inhibitor. These findings are recapitulated in the bleomycin mouse model. Conclusion: cGAS signaling contributes to pathogenic inflammatory myofibroblast phenotypes in SSc-ILD. Inhibiting cGAS or its downstream effectors represents a novel therapeutic approach.

Damage sensing through TLR9 Regulates Inflammatory and Antiviral Responses During Influenza Infection.

Host response aimed at eliminating the infecting pathogen, as well as the pathogen itself, can cause tissue injury. Tissue injury leads to the release of a myriad of cellular components including mitochondrial DNA, which the host senses through pattern recognition receptors. How the sensing of tissue injury by the host shapes the anti-pathogen response remains poorly understood. In this study, we utilized mice that are deficient in toll-like receptor-9 (TLR9), which binds to unmethylated CpG DNA sequences such as those present in bacterial and mitochondrial DNA. To avoid direct pathogen sensing by TLR9, we utilized the influenza virus, which lacks ligands for TLR9, to determine how damage sensing by TLR9 contributes to anti-influenza immunity. Our data show that TLR9-mediated sensing of tissue damage promotes an inflammatory response during early infection, driven by the epithelial and myeloid cells. Along with the diminished inflammatory response, the absence of TLR9 led to impaired viral clearance manifested as a higher and prolonged influenza components in myeloid cells including monocytes and macrophages rendering them highly inflammatory. The persistent inflammation driven by infected myeloid cells led to persistent lung injury and impaired recovery in influenza-infected TLR9-/- mice. Further, we show elevated TLR9 activation in the plasma samples of patients with influenza and its association with the disease severity in hospitalized patients, demonstrating its clinical relevance. Overall, we demonstrate an essential role of damage sensing through TLR9 in promoting anti-influenza immunity and inflammatory response.

Targeting the NLRP3 inflammasome and associated cytokines in scleroderma associated interstitial lung disease.

SSc-ILD (scleroderma associated interstitial lung disease) is a complex rheumatic disease characterized in part by immune dysregulation leading to the progressive fibrotic replacement of normal lung architecture. Because improved treatment options are sorely needed, additional study of the fibroproliferative mechanisms mediating this disease has the potential to accelerate development of novel therapies. The contribution of innate immunity is an emerging area of investigation in SSc-ILD as recent work has demonstrated the mechanistic and clinical significance of the NLRP3 inflammasome and its associated cytokines of TNFα (tumor necrosis factor alpha), IL-1ß (interleukin-1 beta), and IL-18 in this disease. In this review, we will highlight novel pathophysiologic insights afforded by these studies and the potential of leveraging this complex biology for clinical benefit.

Mitochondrial DNA Sensing Pathogen Recognition Receptors in Systemic Sclerosis Associated Interstitial Lung Disease: A Review.

Purpose of the review: Systemic sclerosis (SSc) is a condition of dermal and visceral scar formation characterized by immune dysregulation and inflammatory fibrosis. Approximately 90% of SSc patients develop interstitial lung disease (ILD), and it is the leading cause of morbidity and mortality. Further understanding of immune-mediated fibroproliferative mechanisms has the potential to catalyze novel treatment approaches in this difficult to treat disease. Recent findings: Recent advances have demonstrated the critical role of aberrant innate immune activation mediated by mitochondrial DNA (mtDNA) through interactions with toll-like receptor 9 (TLR9) and cytosolic cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS). Summary: In this review, we will discuss how the nature of the mtDNA, whether oxidized or mutated, and its mechanism of release, either intracellularly or extracellularly, can amplify fibrogenesis by activating TLR9 and cGAS, and the novel insights gained by interrogating these signaling pathways. Because the scope of this review is intended to generate hypotheses for future research, we conclude our discussion with several important unanswered questions.

Circulating Mitochondrial DNA Is Associated With High Levels of Fatigue in Two Independent Sarcoidosis Cohorts.

BACKGROUND: Patients with sarcoidosis who develop severe clinical phenotypes of pulmonary fibrosis or multiorgan disease experience debilitating symptoms, with fatigue being a common chief complaint. Studies that have investigated this patient-related outcome measure (PROM) have used the Fatigue Assessment Scale (FAS), a self-reported questionnaire that reflects mental and physical domains. Despite extensive work, its cause is unknown and treatment options remain limited. Previously, we showed that the plasma of patients with sarcoidosis with extrapulmonary disease endorsing fatigue was enriched for mitochondrial DNA (mtDNA), a ligand for the innate immune receptor toll-like receptor 9 (TLR9). Through our cross-disciplinary platform, we investigated a relationship between sarcoidosis-induced fatigue and circulating mtDNA. RESEARCH QUESTION: Is there a psychobiologic mechanism that connects sarcoidosis-induced fatigue and mtDNA-mediated TLR9 activation? STUDY DESIGN AND METHODS: Using a local cohort of patients at Yale (discovery cohort) and the National Institutes of Health-sponsored Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis study (validation cohort), we scored the FAS and quantified in the plasma, mtDNA concentrations, TLR9 activation, and cytokine levels. RESULTS: Although FAS scores were independent of corticosteroid use and Scadding stage, we observed a robust association between FAS scores, which included mental and physical domains, and multiorgan sarcoidosis. Subsequently, we identified a significant correlation between plasma mtDNA concentrations and all domains of fatigue. Additionally, we found that TLR9 activation is associated with all aspects of the FAS and partially mediates this PROM through mtDNA. Last, we found that TLR9-associated soluble mediators in the plasma are independent of all facets of fatigue. INTERPRETATION: Through our cross-disciplinary translational platform, we identified a previously unrecognized psychobiologic connection between sarcoidosis-induced fatigue and circulating mtDNA concentrations. Mechanistic work that investigates the contribution of mtDNA-mediated innate immune activation in this PROM and clinical studies with prospective cohorts has the potential to catalyze novel therapeutic strategies for this patient population and those with similar conditions.

Multi-modality machine learning approach for risk stratification in heart failure with left ventricular ejection fraction ≤ 45.

AIMS: Heart failure (HF) involves complex remodelling leading to electrical and mechanical dysfunction. We hypothesized that machine learning approaches incorporating data obtained from different investigative modalities including atrial and ventricular measurements from electrocardiography and echocardiography, blood inflammatory marker [neutrophil-to-lymphocyte ratio (NLR)], and prognostic nutritional index (PNI) will improve risk stratification for adverse outcomes in HF compared to logistic regression. METHODS AND RESULTS: Consecutive Chinese patients referred to our centre for transthoracic echocardiography and subsequently diagnosed with HF, between 1 January 2010 and 31 December 2016, were included in this study. Two machine learning techniques, multilayer perceptron and multi-task learning, were compared with logistic regression for their ability to predict incident atrial fibrillation (AF), transient ischaemic attack (TIA)/stroke, and all-cause mortality. This study included 312 HF patients [mean age: 64 (55-73) years, 75% male]. There were 76 cases of new-onset AF, 62 cases of incident TIA/stroke, and 117 deaths during follow-up. Univariate analysis revealed that age, left atrial reservoir strain (LARS) and contractile strain (LACS) were significant predictors of new-onset AF. Age and smoking predicted incident stroke. Age, hypertension, type 2 diabetes mellitus, chronic kidney disease, mitral or aortic regurgitation, P-wave terminal force in V1, the presence of partial inter-atrial block, left atrial diameter, ejection fraction, global longitudinal strain, serum creatinine and albumin, high NLR, low PNI, and LARS and LACS predicted all-cause mortality. Machine learning techniques achieved better prediction performance than logistic regression. CONCLUSIONS: Multi-modality assessment is important for risk stratification in HF. A machine learning approach provides additional value for improving outcome prediction.