Targeting alarmin release reverses Sjogren's syndrome phenotype by revitalizing Ca2+ signalling.

BACKGROUND: Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease that is embodied by the loss of salivary gland function and immune cell infiltration, but the mechanism(s) are still unknown. The aim of this study was to understand the mechanisms and identify key factors that leads to the development and progression of pSS. METHODS: Immunohistochemistry staining, FACS analysis and cytokine levels were used to detect immune cells infiltration and activation in salivary glands. RNA sequencing was performed to identify the molecular mechanisms involved in the development of pSS. The function assays include in vivo saliva collection along with calcium imaging and electrophysiology on isolated salivary gland cells in mice models of pSS. Western blotting, real-time PCR, alarmin release, and immunohistochemistry was performed to identify the channels involved in salivary function in pSS. RESULTS: We provide evidence that loss of Ca2+ signaling precedes a decrease in saliva secretion and/or immune cell infiltration in IL14α, a mouse model for pSS. We also showed that Ca2+ homeostasis was mediated by transient receptor potential canonical-1 (TRPC1) channels and inhibition of TRPC1, resulting in the loss of salivary acinar cells, which promoted alarmin release essential for immune cell infiltration/release of pro-inflammatory cytokines. In addition, both IL14α and samples from human pSS patients showed a decrease in TRPC1 expression and increased acinar cell death. Finally, paquinimod treatment in IL14α restored Ca2+ homeostasis that inhibited alarmin release thereby reverting the pSS phenotype. CONCLUSIONS: These results indicate that loss of Ca2+ signaling is one of the initial factors, which induces loss of salivary gland function along with immune infiltration that exaggerates pSS. Importantly, restoration of Ca2+ signaling upon paquinimod treatment reversed the pSS phenotype thereby inhibiting the progressive development of pSS.

Neutrophils Facilitate Prolonged Inflammasome Response in the DAMP-Rich Inflammatory Milieu.

Aberrant inflammasome activation contributes to various chronic inflammatory diseases; however, pyroptosis of inflammasome-active cells promptly terminates local inflammasome response. Molecular mechanisms underlying prolonged inflammasome signaling thus require further elucidation. Here, we report that neutrophil-specific resistance to pyroptosis and NLRP3 desensitization can facilitate sustained inflammasome response and interleukin-1ß secretion. Unlike macrophages, inflammasome-activated neutrophils did not undergo pyroptosis, indicated by using in vitro cell-based assay and in vivo mouse model. Intriguingly, danger-associated molecular patterns (DAMP)-rich milieu in the inflammatory region significantly abrogated NLRP3-activating potential of macrophages, but not of neutrophils. This macrophage-specific NLRP3 desensitization was associated with DAMP-induced mitochondrial depolarization that was not observed in neutrophils due to a lack of SARM1 expression. Indeed, valinomycin-induced compulsory mitochondrial depolarization in neutrophils restored inflammasome-dependent cell death and ATP-induced NLRP3 desensitization in neutrophils. Alongside prolonged inflammasome-activating potential, neutrophils predominantly secreted interleukin-1ß rather than other proinflammatory cytokines upon NLRP3 stimulation. Furthermore, inflammasome-activated neutrophils did not trigger efferocytosis-mediated M2 macrophage polarization essential for the initiation of inflammation resolution. Taken together, our results indicate that neutrophils can prolong inflammasome response via mitochondria-dependent resistance to NLRP3 desensitization and function as major interleukin-1ß-secreting cells in DAMP-rich inflammatory region.

Long-chain saturated fatty acids in breast milk are associated with the pathogenesis of atopic dermatitis via induction of inflammatory ILC3s.

Breastfeeding influences the immune system development in infants and may even affect various immunological responses later in life. Breast milk provides a rich source of early nutrition for infant growth and development. However, the presence of certain compounds in breast milk, related to an unhealthy lifestyle or the diet of lactating mothers, may negatively impact infants. Based on a cohort study of atopic dermatitis (AD), we find the presence of damage-associated molecular patterns (DAMPs) activity in the mother's milk. By non-targeted metabolomic analysis, we identify the long-chain saturated fatty acids (LCSFA) as a biomarker DAMPs (+) breast milk samples. Similarly, a mouse model in which breastfed offspring are fed milk high in LCSFA show AD onset later in life. We prove that LCSFA are a type of damage-associated molecular patterns, which initiate a series of inflammatory events in the gut involving type 3 innate lymphoid cells (ILC3s). A remarkable increase in inflammatory ILC3s is observed in the gut, and the migration of these ILC3s to the skin may be potential triggers of AD. Gene expression analysis of ILC3s isolated from the gut reveal upregulation of genes that increase ILC3s and chemokines/chemokine receptors, which may play a role in ILC migration to the skin. Even in the absence of adaptive immunity, Rag1 knockout mice fed a high-LCSFA milk diet develop eczema, accompanied by increased gut ILC3s. We also present that gut microbiota of AD-prone PA milk-fed mice is different from non-AD OA/ND milk-fed mice. Here, we propose that early exposure to LCSFAs in infants may affect the balance of intestinal innate immunity, inducing a highly inflammatory environment with the proliferation of ILC3s and production of interleukin-17 and interleukin-22, these factors may be potential triggers or worsening factors of AD.

Proteomics reveals distinct mechanisms regulating the release of cytokines and alarmins during pyroptosis.

A major pathway for proinflammatory protein release by macrophages is inflammasome-mediated pyroptotic cell death. As conventional secretion, unconventional secretion, and cell death are executed simultaneously, however, the cellular mechanisms regulating this complex paracrine program remain incompletely understood. Here, we devise a quantitative proteomics strategy to define the cellular exit route for each protein by pharmacological and genetic dissection of cellular checkpoints regulating protein release. We report the release of hundreds of proteins during pyroptosis, predominantly due to cell lysis. They comprise constitutively expressed and transcriptionally induced proteins derived from the cytoplasm and specific intracellular organelles. Many low-molecular-weight proteins including the cytokine interleukin-1ß, alarmins, and lysosomal-cargo proteins exit cells in the absence of cell lysis. Cytokines and alarmins are released in an endoplasmic reticulum (ER)-Golgi-dependent manner as free proteins rather than by extracellular vesicles. Our work provides an experimental framework for the dissection of cellular exit pathways and a resource for pyroptotic protein release.

Role of Alarmins in the Pathogenesis of Systemic Sclerosis.

Systemic sclerosis (SSc) is a rare chronic autoimmune disease associated with significant morbidity and mortality. Two main subsets of SSc are recognized: (i) diffuse cutaneous SSc with rapidly progressive fibrosis of the skin, lungs, and other internal organs; and (ii) limited cutaneous SSc, which is dominated by vascular manifestations, with skin and organ fibrosis generally limited and slowly progressing. In spite of intense investigation, both etiology and pathogenesis of SSc are still unknown. Genetic and environmental factors, as well as abnormalities of immune functions, are strongly suggested for etiology, while microvascular abnormalities, immune system activation, and oxidative stress are suggested for the pathogenesis. Recently, it has been found that a multitude of mediators and cytokines are implicated in the fibrotic processes observed in SSc. Among these, a central role could be exerted by "alarmins", endogenous and constitutively expressed proteins/peptides that function as an intercellular signal defense. This review describes, in a detailed manner, the role of alarmins in the pathogenesis of scleroderma.

A longitudinal study highlights shared aspects of the transcriptomic response to cardiogenic and septic shock.

BACKGROUND: Septic shock (SS) and cardiogenic shock (CS) are two types of circulatory shock with a different etiology. Several studies have described the molecular alterations in SS patients, whereas the molecular factors involved in CS have been poorly investigated. We aimed to assess in the whole blood of CS and SS patients, using septic patients without shock (SC) as controls, transcriptomic modifications that occur over 1 week after ICU admission and are common to the two types of shock. METHODS: We performed whole blood RNA sequencing in 21 SS, 11 CS, and 5 SC. In shock patients, blood samples were collected within 16 h from ICU admission (T1), 48 h after ICU admission (T2), and at day 7 or before discharge (T3). In controls, blood samples were available at T1 and T2. Gene expression changes over time have been studied in CS, SS, and SC separately with a paired analysis. Genes with p value < 0.01 (Benjamini-Hochberg multiple test correction) were defined differentially expressed (DEGs). We used gene set enrichment analysis (GSEA) to identify the biological processes and transcriptional regulators significantly enriched in both types of shock. RESULTS: In both CS and SS patients, GO terms of inflammatory response and pattern recognition receptors (PRRs) were downregulated following ICU admission, whereas gene sets of DNA replication were upregulated. At the gene level, we observed that alarmins, interleukin receptors, PRRs, inflammasome, and DNA replication genes significantly changed their expression in CS and SS, but not in SC. Analysis of transcription factor targets showed in both CS and SS patients, an enrichment of CCAAT-enhancer-binding protein beta (CEBPB) targets in genes downregulated over time and an enrichment of E2F targets in genes with an increasing expression trend. CONCLUSIONS: This pilot study supports, within the limits of a small sample size, the role of alarmins, PRRs, DNA replication, and immunoglobulins in the pathophysiology of circulatory shock, either in the presence of infection or not. We hypothesize that these genes could be potential targets of therapeutic interventions in CS and SS. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02141607. Registered 19 May 2014.

Protective role of mesenchymal stem cells and mesenchymal stem cell-derived exosomes in cigarette smoke-induced mitochondrial dysfunction in mice.

BACKGROUND: Cigarette smoke (CS)-induced lung inflammation and Chronic Obstructive Pulmonary disease (COPD) involves mitochondrial dysfunction. Mesenchymal stem cells (MSC) and MSC-derived exosomes (EXO) are reported to show therapeutic effects in many animal models of inflammation and injury. In the present study, we determined the role of MSC and EXO intervention in CS-induced lung inflammation with a focus on mitochondrial dysfunction. METHODS: EXO were characterized using Western blot for exosomal markers, tunable resistive pulse sensing by qNano and transmission electron microscopy (TEM). Mitochondrial reporter mice (mt-Keima and mito-QC) were exposed to air or CS for 10 days. mt-Keima mice were treated with intraperitoneal injections of MSC or EXO or MSC and EXO (MSC + EXO) for 10 days. Total cell counts, differential cell counts were performed using automated cell counter and flow cytometry respectively. Further, the levels of pro-inflammatory mediators in bronchoalveolar lavage (BAL) fluid were measured using ELISA. Western blot analysis, quantitative PCR, confocal microscopy were used in the current study to determine the effects in the lungs of CS exposed mice. Seahorse flux analyzer was used to measure the oxidative-phosphorylation (OXPHOS) in the BEAS2B cells and BEAS2B - mMSC co-culture experiments. RESULTS: CS exposure increased the inflammatory cellular infiltrations in the lungs of the mt-Keima mice. MSC + EXO treatment showed protection compared to individual treatments (MSC or EXO alone). There were no changes in the mitophagy proteins like PINK1 and Parkin, which was also found using the mito-QC mice. CS exposure led to significant increase in the mitochondrial fission protein DRP1 and other DAMPs pathway mediators like S100A4 and S100A8, HMGB1, RAGE and AGE. MSC + EXO treatment increased the gene expression of (fusion genes) mfn1, mfn2 and opa1. Additionally, the rhot1 gene expression was increased in MSC + EXO treatment group compared to Air- and CS exposed groups. BEAS2B-mMSC co-cultures showed protective response against the CSE-altered mitochondrial respiration parameters, confirming the beneficial effect of MSC towards human bronchial lung epithelial cells. CONCLUSION: CS affects some of early mitochondrial genes involved in the fission/fusion process, enhancing the damage response along with altered cytokine levels. MSC + EXO combination treatment showed their protective effects. MSC + EXO combination treatment may act against these early events caused by CS exposure owing to its anti-inflammatory and other mitochondrial transfer mechanisms.

Molecules of Damage-Associated Patterns in Bronchoalveolar Lavage Fluid and Serum in Chronic Obstructive Pulmonary Disease.

Chronic exposure to detrimental environmental factors may induce immunogenic cell death of structural airway cells in chronic obstructive pulmonary disease (COPD). Damage-associated molecular patterns (DAMPs) is a family of heterogeneous molecules released from injured or dead cells, which activate innate and adaptive immune responses on binding to the pattern recognition receptors on cells. This study seeks to define the content of DAMPs in the bronchoalveolar lavage fluid (BALF) and serum of COPD patients, and the possible association of these molecules with clinical disease features. Thirty COPD in advanced disease stages were enrolled into the study. Pulmonary function tests, arterial blood gas content, 6-minute walk test, and BODE index were assessed. The content of DAMPs was estimated using the commercial sandwich-ELISA kits. We found differential alterations in the content of various DAMP molecules. In the main, BALF DAMPs positively associated with age, forced expiratory volume in one second (FEV1), and residual volume (RV); and inversely with PaO2, residual volume/total lung capacity (RV/TLC) ratio, and the disease severity staging. In serum, DAMPS positively associated with the intensity of smoking and inversely with age, PaO2, and TLC. In conclusion, DAMPs are present in both BALF and serum of COPD patients, which points to enhanced both local in the lung environment as well as systemic pro-inflammatory vein in this disease. These molecules appear involved with the lung damage and clinical variables featuring COPD. However, since the involvement of various DAMPs in COPD is variable, the exact role they play is by far unsettled and is open to further exploration.

Current knowledge on biomarkers for contact sensitization and allergic contact dermatitis.

Contact sensitization is common and affects up to 20% of the general population. The clinical manifestation of contact sensitization is allergic contact dermatitis. This is a clinical expression that is sometimes difficult to distinguish from other types of dermatitis, for example irritant and atopic dermatitis. Several studies have examined the pathogenesis and severity of allergic contact dermatitis by measuring the absence or presence of various biomarkers. In this review, we provide a non-systematic overview of biomarkers that have been studied in allergic contact dermatitis. These include genetic variations and mutations, inflammatory mediators, alarmins, proteases, immunoproteomics, lipids, natural moisturizing factors, tight junctions, and antimicrobial peptides. We conclude that, despite the enormous amount of data, convincing specific biomarkers for allergic contact dermatitis are yet to be described.