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Introduction: Primary Sjögren's syndrome (SS) is an autoimmune exocrinopathy that affects the structure and function of salivary and lachrymal glands. Labial salivary gland (LSG) acinar cells from SS patients lose cellular homeostasis and experience endoplasmic reticulum and oxidative stress. The integrated cellular stress response (ISR) is an adaptive pathway essential for restoring homeostasis against various stress-inducing factors, including pro-inflammatory cytokines, and endoplasmic reticulum and oxidative stress. ISR activation leads eIF2α phosphorylation, which transiently blocks protein synthesis while allowing the ATF4 expression, which induces a gene expression program that seeks to optimize cellular recovery. PKR, HRI, GCN2, and PERK are the four sentinel stress kinases that control eIF2α phosphorylation. Dysregulation and chronic activation of ISR signaling have pathologic consequences associated with inflammation. Methods: Here, we analyzed the activation of the ISR in LSGs of SS-patients and non-SS sicca controls, determining the mRNA, protein, and phosphorylated-protein levels of key ISR components, as well as the expression of some of ATF4 targets. Moreover, we performed a qualitative characterization of the distribution of ISR components in LSGs from both groups and evaluated if their levels correlate with clinical parameters. Results: We observed that the four ISR sensors are expressed in LSGs of both groups. However, only PKR and PERK showed increased expression and/or activation in LSGs from SS-patients. eIF2α and p-eIF2α protein levels significantly increased in SS-patients; meanwhile components of the PP1c complex responsible for eIF2α dephosphorylation decreased. ATF4 mRNA levels were decreased in LSGs from SS-patients along with hypermethylation of the ATF4 promoter. Despite low mRNA levels, SS-patients showed increased levels of ATF4 protein and ATF4-target genes involved in the antioxidant response. The acinar cells of SS-patients showed increased staining intensity for PKR, p-PKR, p-PERK, p-eIF2α, ATF4, xCT, CHOP, and NRF2. Autoantibodies, focus score, and ESSDAI were correlated with p-PERK/PERK ratio and ATF4 protein levels. Discussion: In summary, the results showed an increased ISR activation in LSGs of SS-patients. The increased protein levels of ATF4 and ATF4-target genes involved in the redox homeostasis could be part of a rescue response against the various stressful conditions to which the LSGs of SS-patients are subjected and promote cell survival.
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Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) and can be treated with glucocorticoids (GC), although some patients are unresponsive to this therapy. The transcription factor LRH-1/NR5A2 is critical to intestinal cortisol production (intestinal steroidogenesis), being reduced in UC patients. However, the relationship between LRH-1 expression and distribution with altered corticosteroid responses is unknown. To address this, we categorized UC patients by their steroid response. Here, we found that steroid-dependent and refractory patients presented reduced glucocorticoid receptor (GR)-mediated intestinal steroidogenesis compared to healthy individuals and responder patients, possibly related to increased colonic mucosa GR isoform beta (GRß) content and cytoplasmic LRH-1 levels in epithelial and lamina propria cells. Interestingly, an intestinal epithelium-specific GR-induced knockout (GRiKO) dextran sodium sulfate (DSS)-colitis mice model presented decreased epithelial LRH-1 expression, whilst it increased in the lamina propria compared to DSS-treated control mice. Mechanistically, GR directly induced NR5A2 gene expression in CCD841CoN cells and human colonic organoids. Furthermore, GR bound to two glucocorticoid-response elements within the NR5A2 promoter in dexamethasone-stimulated CCD841CoN cells. We conclude that GR contributes to intestinal steroidogenesis by inducing LRH-1 in epithelial cells, suggesting LRH-1 as a potential marker for glucocorticoid-impaired response in UC. However, further studies with a larger patient cohort will be necessary to confirm role of LRH-1 as a therapeutic biomarker.
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Colite Ulcerativa , Animais , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Humanos , Mucosa Intestinal/metabolismo , Intestinos , Camundongos , Esteroides/metabolismoRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs (sRNA), that alter gene expression by binding to target messenger RNAs (mRNAs) and repressing translation. Dysregulated miRNA expression has been implicated in the pathogenesis of autoimmune diseases such as Sjögren's syndrome (SS). The aim of this study was to characterize the global profile of sRNAs in labial salivary glands (LSG) from SS-patients and to validate potential miRNA candidates implicated in glandular inflammation. LSG from 21 SS-patients and 9 sicca controls were analyzed. A global next generation sequencing (NGS)-based sRNA profiling approach was employed to identify direct targets whereby differentially expressed miRNAs were predicted using bioinformatics tools. miRNA levels were validated by TaqMan and target mRNA levels were determined by quantitative real-time PCR. We also performed in vitro assays using recombinant TNF-α. NGS shows that ~30% of sRNAs were miRNAs. In comparison with samples from sicca controls, four miRNAs were found differentially expressed in LSG from SS-patients with low focus score (LFS) and 18 from SS-patients with high focus score (HFS). The miRNA with the most significant changes identified by NGS was hsa-miR-181d-5p and downregulation was confirmed by TaqMan analysis. Levels of TNF-α mRNA, a direct target of hsa-miR-181d-5p, were significantly increased and negatively correlated with hsa-miR-181d-5p presence. Moreover, positive correlations between TNF-α transcript levels, focus score, ESSDAI, and autoantibody levels were also detected. Furthermore, TNF-α stimulation decreased hsa-miR-181d-5p levels in vitro. Downregulation of hsa-miR-181d-5p in LSG from SS-patients could contribute to the glandular pro-inflammatory environment by deregulation of its direct target TNF-α. Further dissection of the pathophysiological mechanisms underlying the hsa-miR-181d-5p-mediated action in inflammatory conditions could be useful to evaluate the benefits of increasing hsa-miR-181d-5p levels for restoration of salivary gland epithelial cell architecture and function.
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MicroRNAs , Síndrome de Sjogren , Regulação para Baixo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , Síndrome de Sjogren/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Sjögren's syndrome (SS) is an autoimmune disease that mainly affects salivary glands (SG) and is characterized by overactivation of the type I interferon (IFN) pathway. Type I IFNs can decrease the levels of hsa-miR-145-5p, a miRNA with anti-inflammatory roles that is downregulated in SG from SS-patients. Two relevant targets of hsa-miR-145-5p, mucin 1 (MUC1) and toll-like receptor 4 (TLR4) are overexpressed in SS-patients and contribute to SG inflammation and dysfunction. This study aimed to evaluate if hsa-miR-145-5p modulates MUC1 and TLR4 overexpression in SG from SS-patients in a type I IFN dependent manner. Labial SG (LSG) biopsies from 9 SS-patients and 6 controls were analyzed. We determined hsa-miR-145-5p levels by TaqMan assays and the mRNA levels of MUC1, TLR4, IFN-α, IFN-ß, and IFN-stimulated genes (MX1, IFIT1, IFI44, and IFI44L) by real time-PCR. We also performed in vitro assays using type I IFNs and chemically synthesized hsa-miR-145-5p mimics and inhibitors. We validated the decreased hsa-miR-145-5p levels in LSG from SS-patients, which inversely correlated with the type I IFN score, mRNA levels of IFN-ß, MUC1, TLR4, and clinical parameters of SS-patients (Ro/La autoantibodies and focus score). IFN-α or IFN-ß stimulation downregulated hsa-miR-145-5p and increased MUC1 and TLR4 mRNA levels. Hsa-miR-145-5p overexpression decreased MUC1 and TLR4 mRNA levels, while transfection with a hsa-miR-145-5p inhibitor increased mRNA levels. Our findings show that type I IFNs decrease hsa-miR-145-5p expression leading to upregulation of MUC1 and TLR4. Together, this suggests that type I interferon-dependent hsa-miR-145-5p downregulation contributes to the perpetuation of inflammation in LSG from SS-patients.
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Interferon Tipo I/metabolismo , MicroRNAs/metabolismo , Mucina-1/metabolismo , Síndrome de Sjogren/metabolismo , Receptor 4 Toll-Like/metabolismo , Adulto , Regulação para Baixo , Feminino , Humanos , Inflamação/metabolismo , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Mucina-1/genética , Glândulas Salivares Menores/metabolismo , Síndrome de Sjogren/genética , Receptor 4 Toll-Like/genética , Adulto JovemRESUMO
Relevant reviews highlight the association between dysfunctional mitochondria and inflammation, but few studies address the contribution of mitochondria and mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) to cellular homeostasis and inflammatory signaling. The present review outlines the important role of mitochondria in cellular homeostasis and how dysfunctional mitochondrion can release and misplace mitochondrial components (cardiolipin, mitochondrial DNA (mtDNA), and mitochondrial formylated peptides) through multiple mechanisms. These components can act as damage-associated molecular patterns (DAMPs) and induce an inflammatory response via pattern recognition receptors (PRRs). Accumulation of damaged ROS-generating mitochondria, accompanied by the release of mitochondrial DAMPs, can activate PRRs such as the NLRP3 inflammasome, TLR9, cGAS/STING, and ZBP1. This process would explain the chronic inflammation that is observed in autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type I diabetes (T1D), and Sjögren's syndrome. This review also provides a comprehensive overview of the importance of MERCs to mitochondrial function and morphology, cellular homeostasis, and the inflammatory response. MERCs play an important role in calcium homeostasis by mediating the transfer of calcium from the ER to the mitochondria and thereby facilitating the production of ATP. They also contribute to the synthesis and transfer of phospholipids, protein folding in the ER, mitochondrial fission, mitochondrial fusion, initiation of autophagosome formation, regulation of cell death/survival signaling, and regulation of immune responses. Therefore, alterations within MERCs could increase inflammatory signaling, modulate ER stress responses, cell homeostasis, and ultimately, the cell fate. This study shows severe ultrastructural alterations of mitochondria in salivary gland cells from Sjögren's syndrome patients for the first time, which could trigger alterations in cellular bioenergetics. This finding could explain symptoms such as fatigue and malfunction of the salivary glands in Sjögren's syndrome patients, which would contribute to the chronic inflammatory pathology of the disease. However, this is only a first step in solving this complex puzzle, and several other important factors such as changes in mitochondrial morphology, functionality, and their important contacts with other organelles require further in-depth study. Future work should focus on detecting the key milestones that are related to inflammation in patients with autoimmune diseases, such as Sjögren´s syndrome.
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Síndrome de Sjogren , DNA Mitocondrial/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Inflamação/metabolismo , MitocôndriasRESUMO
OBJECTIVE: This systematic review aims to describe the value of saliva as a noninvasive sample for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in comparison with the current method for sample collection, the nasopharyngeal swab. STUDY DESIGN: We conducted a systematic review of the literature following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations. We searched in 5 databases (PubMed, Cochrane, EBSCO, Elsevier, and MEDLINE) and included articles published between December 2019 and July 2020. RESULTS: This review included 22 publications that met inclusion criteria, 17 of which were case series, 2 of which were case reports, and 3 of which were massive screenings. All articles compared saliva with nasopharyngeal swabs. The detection rate of SARS-CoV-2 in saliva was similar to that for nasopharyngeal swabs. The sensitivity ranged between 20% and 97%, and specificity ranged between 66% and 100%. CONCLUSIONS: This systematic review found that saliva might be an appropriate, fast, painless, simple, and noninvasive sample for SARS-CoV-2 detection, making it ideal for massive screening of SARS-CoV-2 infection.
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COVID-19 , SARS-CoV-2 , Humanos , Programas de Rastreamento , SalivaRESUMO
OBJECTIVE: Altered homeostasis of salivary gland (SG) epithelial cells in Sjögren's syndrome (SS) could be the initiating factor that leads to inflammation, secretory dysfunction and autoimmunity. Autophagy is an important homeostatic mechanism, whose deficiency is associated with inflammation and accumulation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) components. We aimed to evaluate whether autophagy is altered in labial SG (LSG) epithelial cells from primary SS (pSS) patients and whether this contributes to inflammation through the JAK-STAT pathway. Furthermore, we investigated the anti-inflammatory effect of the JAK inhibitor tofacitinib in autophagy-deficient (ATG5 knockdown) three-dimensional (3D)-acini. METHODS: We analysed LSG biopsies from 12 pSS patients with low focus score and 10 controls. ATG5-deficient 3D-acini were generated and incubated with IL-6 in the presence or absence of tofacitinib. Autophagy markers, pro-inflammatory cytokine expression, and JAK-STAT pathway activation were evaluated by PCR or western blot, along with correlation analyses between the evaluated markers and clinical parameters. RESULTS: LSG from pSS patients showed increased p62 and decreased ATG5 expression, correlating negatively with increased activation of JAK-STAT pathway components (pSTAT1 and pSTAT3). Increased expression of STAT1 and IL-6 correlated with EULAR Sjögren's syndrome disease activity index and the presence of anti-Ro antibodies. ATG5-deficient 3D-acini reproduced the findings observed in LSG from pSS patients, showing increased expression of pro-inflammatory markers such as IL-6, which was reversed by tofacitinib. CONCLUSION: Decreased expression of ATG5 in LSG epithelial cells from pSS patients possibly contributes to increased inflammation associated with JAK-STAT pathway activation, as evidenced in ATG5-deficient 3D-acini. Interestingly, these results suggest that tofacitinib could be used as an anti-inflammatory agent in pSS patients.
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Autofagia/efeitos dos fármacos , Interleucina-6/metabolismo , Piperidinas/farmacologia , Pirimidinas/farmacologia , Adolescente , Adulto , Idoso , Western Blotting , Estudos de Casos e Controles , Feminino , Imunofluorescência , Humanos , Interleucina-6/antagonistas & inibidores , Janus Quinases/metabolismo , Masculino , Pessoa de Meia-Idade , Piperidinas/uso terapêutico , Pirimidinas/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição STAT/metabolismo , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Síndrome de Sjogren , Adulto JovemRESUMO
OBJECTIVES: Xerostomia in SS patients has been associated with low quality and quantity of salivary mucins, which are fundamental for the hydration and protection of the oral mucosa. The aim of this study was to evaluate if cytokines induce aberrant mucin expression and whether tauroursodeoxycholic acid (TUDCA) is able to counteract such an anomaly. METHODS: Labial salivary glands from 16 SS patients and 15 control subjects, as well as 3D acini or human submandibular gland cells stimulated with TNF-α or IFN-γ and co-incubated with TUDCA, were analysed. mRNA and protein levels of Mucin 1 (MUC1) and MUC7 were determined by RT-qPCR and western blot, respectively. Co-immunoprecipitation and immunofluorescence assays for mucins and GRP78 [an endoplasmic reticulum (ER)-resident protein] were also performed. mRNA levels of RelA/p65 (nuclear factor-κB subunit), TNF-α, IL-1ß, IL-6, SEL1L and EDEM1 were determined by RT-qPCR, and RelA/p65 localization was evaluated by immunofluorescence. RESULTS: MUC1 is overexpressed and accumulated in the ER of labial salivary gland from SS patients, while MUC7 accumulates throughout the cytoplasm of acinar cells; however, MUC1, but not MUC7, co-precipitated with GRP78. TUDCA diminished the overexpression and aberrant accumulation of MUC1 induced by TNF-α and IFN-γ, as well as the nuclear translocation of RelA/p65, together with the expression of inflammatory and ER stress markers in 3D acini. CONCLUSION: Chronic inflammation alters the secretory process of MUC1, inducing ER stress and affecting the quality of saliva in SS patients. TUDCA showed anti-inflammatory properties decreasing aberrant MUC1 accumulation. Further studies are necessary to evaluate the potential therapeutic effect of TUDCA in restoring glandular homeostasis in SS patients.
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Células Acinares/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Mucina-1/efeitos dos fármacos , Glândulas Salivares Menores/efeitos dos fármacos , Síndrome de Sjogren/metabolismo , Glândula Submandibular/efeitos dos fármacos , Ácido Tauroquenodesoxicólico/farmacologia , Xerostomia/metabolismo , Células Acinares/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/genética , Feminino , Proteínas de Choque Térmico/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Imunoprecipitação , Técnicas In Vitro , Interferon gama/farmacologia , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Mucina-1/genética , Mucina-1/metabolismo , Mucinas/efeitos dos fármacos , Mucinas/genética , Mucinas/metabolismo , Proteínas/efeitos dos fármacos , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Glândulas Salivares Menores/metabolismo , Proteínas e Peptídeos Salivares/efeitos dos fármacos , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismo , Síndrome de Sjogren/genética , Glândula Submandibular/citologia , Glândula Submandibular/metabolismo , Fator de Transcrição RelA/efeitos dos fármacos , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Xerostomia/genéticaRESUMO
Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by mucosa damage associated with an uncontrolled inflammatory response. This immunological impairment leads to altered inflammatory mediators such as IL-33, which is shown to increase in the mucosa of active UC (aUC) patients. MicroRNAs present a distorted feature in inflamed colonic mucosa and are potential IL-33 regulating candidates in UC. Therefore, we studied the microRNA and mRNA profiles in inflamed colonic samples of UC patients, evaluating the effect of a microRNA (selected by in silico analysis and its expression in UC patients), on IL-33 under inflammatory conditions. We found that inflamed mucosa (n = 8) showed increased expression of 40 microRNAs and 2,120 mRNAs, while 49 microRNAs and 1,734 mRNAs were decreased, as determined by microarrays. In particular, IL-33 mRNA showed a 3.8-fold increase and eight members of a microRNA family (miR-378), which targets IL-33 mRNA in the 3'UTR, were decreased (-3.9 to -3.0 times). We selected three members of the miR-378 family (miR-378a-3p, miR-422a, and miR-378c) according to background information and interaction energy analysis, for further correlation analyses with IL-33 expression through qPCR and ELISA, respectively. We determined that aUC (n = 24) showed high IL-33 levels, and decreased expression of miR-378a-3p and miR-422a compared to inactive UC (n = 10) and controls (n = 6). Moreover, both microRNAs were inversely correlated with IL-33 expression, while miR-378c does not show a significant difference. To evaluate the effect of TNFα on the studied microRNAs, aUC patients with anti-TNF therapy were compared to aUC receiving other treatments. The levels of miR-378a-3p and miR-378c were higher in aUC patients with anti-TNF. Based on these findings, we selected miR-378a-3p to exploring the molecular mechanism involved by in vitro assays, showing that over-expression of miR-378a-3p decreased the levels of an IL-33 target sequence ß-gal-reporter gene in HEK293 cells. Stable miR-378a-3p over-expression/inhibition inversely modulated IL-33 content and altered viability of HT-29 cells. Additionally, in an inflammatory context, TNFα decreased miR-378a-3p levels in HT-29 cells enhancing IL-33 expression. Together, our results propose a regulatory mechanism of IL-33 expression exerted by miR-378a-3p in an inflammatory environment, contributing to the understanding of UC pathogenesis.
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Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Regulação da Expressão Gênica , Interleucina-33/metabolismo , MicroRNAs/genética , Adolescente , Adulto , Idoso , Alarminas/genética , Alarminas/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Biópsia , Linhagem Celular , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunossupressores/uso terapêutico , Interleucina-33/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Interferência de RNA , RNA Mensageiro/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adulto JovemRESUMO
[This corrects the article DOI: 10.3389/fimmu.2019.01394.].
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[This corrects the article DOI: 10.3389/fimmu.2019.00277.].
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In colorectal cancer (CRC), cancer-associated fibroblasts (CAFs) are the most abundant component from the tumor microenvironment (TM). CAFs facilitate tumor progression by inducing angiogenesis, immune suppression and invasion, thus altering the organization/composition of the extracellular matrix (i.e., desmoplasia) and/or activating epithelial-mesenchymal transition (EMT). Soluble factors from the TM can also contribute to cell invasion through secretion of cytokines and recently, IL-33/ST2 pathway has gained huge interest as a protumor alarmin, promoting progression to metastasis by inducing changes in TM. Hence, we analyzed IL-33 and ST2 content in tumor and healthy tissue lysates and plasma from CRC patients. Tissue localization and distribution of these molecules was evaluated by immunohistochemistry (using localization reference markers α-smooth muscle actin or α-SMA and E-cadherin), and clinical/histopathological information was obtained from CRC patients. In vitro experiments were conducted in primary cultures of CAFs and normal fibroblasts (NFs) isolated from tumor and healthy tissue taken from CRC patients. Additionally, migration and proliferation analysis were performed in HT29 and HCT116 cell lines. It was found that IL-33 content increases in left-sided CRC patients with lymphatic metastasis, with localization in tumor epithelia associated with abundant desmoplasia. Although ST2 content showed similarities between tumor and healthy tissue, a decreased immunoreactivity was observed in left-sided tumor stroma, associated to metastasis related factors (advanced stages, abundant desmoplasia, and presence of tumor budding). A principal component analysis (including stromal and epithelial IL-33/ST2 and α-SMA immunoreactivity with extent of desmoplasia) allowed us to distinguish clusters of low, intermediate and abundant desmoplasia, with potential to develop a diagnostic signature with benefits for further therapeutic targets. IL-33 transcript levels from CAFs directly correlated with CRC cell line migration induced by CAFs conditioned media, with rhIL-33 inducing a mesenchymal phenotype in HT29 cells. These results indicate a role of IL-33/ST2 in tumor microenvironment, specifically in the interaction between CAFs and epithelial tumor cells, thus contributing to invasion and metastasis in left-sided CRC, most likely by activating desmoplasia.
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Neoplasias Colorretais/patologia , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Invasividade Neoplásica/patologia , Microambiente Tumoral/fisiologia , Adulto , Idoso , Fibroblastos Associados a Câncer/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Ulcerative colitis (UC) and Crohn's disease (CD), collectively known as Inflammatory Bowel Diseases (IBD), are caused by a complex interplay between genetic, immunologic, microbial and environmental factors. Dysbiosis of the gut microbiome is increasingly considered to be causatively related to IBD and is strongly affected by components of a Western life style. Bacteria that ferment fibers and produce short chain fatty acids (SCFAs) are typically reduced in mucosa and feces of patients with IBD, as compared to healthy individuals. SCFAs, such as acetate, propionate and butyrate, are important metabolites in maintaining intestinal homeostasis. Several studies have indeed shown that fecal SCFAs levels are reduced in active IBD. SCFAs are an important fuel for intestinal epithelial cells and are known to strengthen the gut barrier function. Recent findings, however, show that SCFAs, and in particular butyrate, also have important immunomodulatory functions. Absorption of SCFAs is facilitated by substrate transporters like MCT1 and SMCT1 to promote cellular metabolism. Moreover, SCFAs may signal through cell surface G-protein coupled receptors (GPCRs), like GPR41, GPR43, and GPR109A, to activate signaling cascades that control immune functions. Transgenic mouse models support the key role of these GPCRs in controlling intestinal inflammation. Here, we present an overview of microbial SCFAs production and their effects on the intestinal mucosa with specific emphasis on their relevance for IBD. Moreover, we discuss the therapeutic potential of SCFAs for IBD, either applied directly or by stimulating SCFAs-producing bacteria through pre- or probiotic approaches.
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Ácidos Graxos Voláteis/fisiologia , Doenças Inflamatórias Intestinais/etiologia , Mucosa Intestinal/metabolismo , Animais , Bactérias/metabolismo , Proliferação de Células , Microbioma Gastrointestinal , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/microbiologia , Mucosa Intestinal/imunologia , Prebióticos , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G/fisiologiaRESUMO
Sjögren's syndrome (SS) is an autoimmune exocrinopathy associated with severe secretory alterations by disruption of the glandular architecture integrity, which is fundamental for a correct function and localization of the secretory machinery. Syt-1, PI(4,5)P2 and Ca2+ are significant factors controlling exocytosis in different secretory cells, the Ca2+ role being the most studied. Salivary acinar cells from SS-patients show a defective agonist-regulated intracellular Ca2+ release together with a decreased IP3R expression level, and this condition may explain a reduced water release. However, there are not reports where Syt-1, PI(4,5)P2 and Ca2+ in acinar cells of SS patients had been studied. In the present study, we analyzed the expression and/or localization of Syt-1 and PI(4,5)P2 in acinar cells of labial salivary gland biopsies from SS-patients and control individuals. Also, we evaluated whether the overexpression of Syt-1 and the loss of cell polarity induced by TNF-α or loss of interaction between acinar cell and basal lamina, alters directionality of the exocytosis process, Ca2+ signaling and α-amylase secretion in a 3D-acini model stimulated with cholinergic or ß-adrenergic agonists. In addition, the correlation between Syt-1 protein levels and clinical parameters was evaluated. The results showed an increase of Syt-1â¯mRNA and protein levels, and a high number of co-localization points of Syt-1/STX4 and PI(4,5)P2/Ezrin in the acinar basolateral region of LSG from SS-patients. With regard to 3D-acini, Syt-1 overexpression increased exocytosis in the apical pole compared to control acini. TNF-α stimulation increased exocytic events in the basal pole, which was further enhanced by Syt-1 overexpression. Additionally, altered acinar cell polarity affected Ca2+ signaling and amylase secretion. Overexpression of Syt-1 was associated with salivary gland alterations revealing that the secretory dysfunction in SS-patients is linked to altered expression and/or localization of secretory machinery components together with impaired epithelial cell polarity. These findings provide a novel insight on the pathological mechanism implicated in ectopic secretory products to the extracellular matrix of LSG from SS-patients, which might initiate inflammation.
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Expressão Gênica , Glândulas Salivares/metabolismo , Síndrome de Sjogren/etiologia , Síndrome de Sjogren/metabolismo , Sinaptotagmina I/genética , Adulto , Biomarcadores , Biópsia , Cálcio/metabolismo , Sinalização do Cálcio , Suscetibilidade a Doenças , Feminino , Glicosilação , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Glândulas Salivares/patologia , Transdução de Sinais , Síndrome de Sjogren/diagnóstico , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
A complex network of chemokines can influence cancer progression with the recruitment and activation of hematopoietic cells, including macrophages to the supporting tumor stroma promoting carcinogenesis and metastasis. The aim of this study was to investigate the relation between tissue and plasma chemokine levels involved in macrophage recruitment with tumor-associated macrophage profile markers and clinicopathological features such as tumor-node-metastases stage, desmoplasia, tumor necrosis factor-α, and vascular endothelial growth factor plasma content. Plasma and tumor/healthy mucosa were obtained from Chilean patients undergoing colon cancer surgery. Chemokines were evaluated from tissue lysates (CCL2, CCL3, CCL4, CCL5, and CX3CL1) by Luminex. Statistical analysis was performed using Wilcoxon match-paired test ( p < 0.05). Macrophage markers (CD68, CD163, and iNOS) were evaluated by immunohistochemistry samples derived from colorectal cancer patients. Correlation analysis between chemokines and macrophage markers and clinicopathological features were performed using Spearman's test. Plasmatic levels of chemokines and inflammatory mediators' vascular endothelial growth factor and tumor necrosis factor-α were evaluated by Luminex. Tumor levels of CCL2 (mean ± standard deviation = 530.1 ± 613.9 pg/mg), CCL3 (102.7 ± 106.0 pg/mg), and CCL4 (64.98 ± 48.09 pg/mg) were higher than those found in healthy tissue (182.1 ± 116.5, 26.79 ± 22.40, and 27.06 ± 23.69 pg/mg, respectively p < 0.05). The tumor characterization allowed us to identify a positive correlation between CCL4 and the pro-tumor macrophages marker CD163 ( p = 0.0443), and a negative correlation of iNOS with desmoplastic reaction ( p = 0.0467). Moreover, we identified that tumors with immature desmoplasia have a higher CD163 density compared to those with a mature/intermediated stromal tissue ( p = 0.0288). Plasmatic CCL4 has shown a positive correlation with inflammatory mediators (tumor necrosis factor-α and vascular endothelial growth factor) that have previously been associated with poor prognosis in patients. In conclusion High expression of CCL4 in colon cancer could induce the infiltration of tumor-associated macrophages and specifically a pro-tumor macrophage profile (CD163+ cells). Moreover, plasmatic chemokines could be considered inflammatory mediators associated to CRC progression as well as tumor necrosis factor-α and vascular endothelial growth factor. These data reinforce the idea of chemokines as potential therapeutic targets or biomarker in CRC.
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Biomarcadores Tumorais/metabolismo , Quimiocina CCL2/metabolismo , Quimiocina CCL3/metabolismo , Quimiocina CCL4/metabolismo , Neoplasias Colorretais/patologia , Macrófagos/patologia , Microambiente Tumoral , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Neoplasias Colorretais/metabolismo , Feminino , Seguimentos , Humanos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Crohn's disease (CD) is a chronic inflammatory bowel disorder characterized by deregulated inflammation triggered by environmental factors. Notably, adherent-invasive Escherichia coli (AIEC), a bacterium with the ability to survive within macrophages is believed to be one of such factors. Glucocorticoids are the first line treatment for CD and to date, it is unknown how they affect bactericidal and inflammatory properties of macrophages against AIEC. The aim of this study was to evaluate the impact of glucocorticoid treatment on AIEC infected macrophages. First, THP-1 cell-derived macrophages were infected with a CD2-a AIEC strain, in the presence or absence of the glucocorticoid dexamethasone (Dex) and mRNA microarray analysis was performed. Differentially expressed mRNAs were confirmed by TaqMan-qPCR. In addition, an amikacin protection assay was used to evaluate the phagocytic and bactericidal activity of Dex-treated macrophages infected with E. coli strains (CD2-a, HM605, NRG857c, and HB101). Finally, cytokine secretion and the inflammatory phenotype of macrophages were evaluated by ELISA and flow cytometry, respectively. The microarray analysis showed that CD2-a, Dex, and CD2-a + Dex-treated macrophages have differential inflammatory gene profiles. Also, canonical pathway analysis revealed decreased phagocytosis signaling by Dex and anti-inflammatory polarization on CD2-a + Dex macrophages. Moreover, amikacin protection assay showed reduced phagocytosis upon Dex treatment and TaqMan-qPCR confirmed Dex inhibition of three phagocytosis-associated genes. All bacteria strains induced TNF-α, IL-6, IL-23, CD40, and CD80, which was inhibited by Dex. Thus, our data demonstrate that glucocorticoids impair phagocytosis and induce anti-inflammatory polarization after AIEC infection, possibly contributing to the survival of AIEC in infected CD patients.
Assuntos
Doença de Crohn/microbiologia , Dexametasona/farmacologia , Infecções por Escherichia coli/imunologia , Glucocorticoides/farmacologia , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Animais , Aderência Bacteriana , Doença de Crohn/imunologia , Citocinas/imunologia , Escherichia coli/patogenicidade , Humanos , Inflamação , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Proteína Adaptadora de Sinalização NOD2/genética , Reação em Cadeia da Polimerase em Tempo Real , Células THP-1 , Fator de Necrose Tumoral alfa/imunologiaRESUMO
For many years, researchers in the field of autoimmunity have focused on the role of the immune components in the etiopathogenesis of autoimmune diseases. However, some studies have demonstrated the importance of target tissues in their pathogenesis and the breach of immune tolerance. The immune system as well as target tissue cells (plasmatic, ß-pancreatic, fibroblast-like synoviocytes, thyroid follicular and epithelial cells of the lachrymal glands, salivary glands, intestine, bronchioles and renal tubules) share the characteristic of secretory cells with an extended endoplasmic reticulum (ER). The function of these cells depends considerably on a normal ER function and calcium homeostasis, so they can produce and secrete their main components, which include glycoproteins involved in antigenic presentation such as major histocompatibility complex (MHC) class I and II. All these proteins are synthesized and modified in the ER, and for this reason disturbances in the normal functions of this organelle such as protein folding, protein quality control, calcium homeostasis and redox balance, promote accumulation of unfolded or misfolded proteins, a condition known as ER stress. Autoimmune diseases are characterized by inflammation, which has been associated with an ER stress condition. Interestingly, patients with these diseases contain circulating auto-antibodies against chaperone proteins (such as Calnexin and GRP94), thus affecting the folding and assembly of MHC class I and II glycoproteins and their loading with peptide. The main purpose of this article is to review the involvement of the protein quality control and unfolded protein response (UPR) in the ER protein homeostasis (proteostasis) and their alterations in autoimmune diseases. In addition, we describe the interaction between ER stress and inflammation and evidences are shown of how autoimmune diseases are associated with an ER stress condition, with a special emphasis on the second most prevalent autoimmune rheumatic disease, Sjögren's syndrome.
Assuntos
Autoimunidade , Estresse do Retículo Endoplasmático , Dobramento de Proteína , Proteínas/metabolismo , Síndrome de Sjogren/fisiopatologia , Resposta a Proteínas não Dobradas , Animais , Retículo Endoplasmático/metabolismo , Humanos , Proteínas/químicaRESUMO
Here, we determined the 5-hydroxymethylcytosine (5hmC), 5-methylcytosine (5mC), Ten Eleven Translocation (TETs), and DNA methyltransferases (DNMTs) levels in epithelial and inflammatory cells of labial salivary glands (LSG) from Sjögren's syndrome (SS)-patients and the effect of cytokines on HSG cells. LSG from SS-patients, controls and HSG cells incubated with cytokines were analysed. Levels of 5mC, 5hmC, DNMTs, TET2 and MeCP2 were assessed by immunofluorescence. In epithelial cells from SS-patients, an increase in TET2, 5hmC and a decrease in 5mC and MeCP2 were observed, additionally, high levels of 5mC and DNMTs and low levels of 5hmC were detected in inflammatory cells. Cytokines increased TET2 and 5hmC and decreased 5mC levels. Considering that the TET2 gene.promoter contains response elements for transcription factors activated by cytokines, together to in vitro results suggest that changes in DNA hydroxymethylation, resulting from altered levels of TET2 are likely to be relevant in the Sjögren's syndrome etiopathogenesis.
Assuntos
5-Metilcitosina/análogos & derivados , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/genética , Proteína 2 de Ligação a Metil-CpG/genética , Proteínas Proto-Oncogênicas/genética , Glândulas Salivares Menores/metabolismo , Síndrome de Sjogren/genética , 5-Metilcitosina/metabolismo , Adulto , Idoso , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Citocinas/imunologia , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/genética , Dioxigenases/imunologia , Dioxigenases/metabolismo , Epigênese Genética , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/metabolismo , Lábio , Masculino , Proteína 2 de Ligação a Metil-CpG/metabolismo , Pessoa de Meia-Idade , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/imunologia , Oxigenases de Função Mista/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Glândulas Salivares Menores/citologia , Glândulas Salivares Menores/imunologia , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Adulto Jovem , DNA Metiltransferase 3BRESUMO
Objectives: Labial salivary glands (LSGs) of SS patients show alterations related to endoplasmic reticulum stress. Glandular dysfunction could be partly the consequence of an altered inositol-requiring enzyme 1α (IRE1α)/X box-binding protein 1 (XBP-1) signalling pathway of the unfolded protein response, which then regulates genes involved in biogenesis of the secretory machinery. This study aimed to determine the expression, promoter methylation and localization of the IRE1α/XBP-1 pathway components in LSGs of SS patients and also their expression induced by IFN-γ in vitro. Methods: IRE1α, XBP-1 and glucose-regulated protein 78 (GRP78) mRNA and protein levels were measured by qPCR and western blot, respectively, in LSGs of SS patients (n = 47) and control subjects (n = 37). Methylation of promoters was evaluated by methylation-sensitive high resolution melting, localization was analysed by immunofluorescence and induction of the IRE1α/XBP-1 pathway components by IFN-γ was evaluated in 3D acini. Results: A significant decrease of IRE1α, XBP-1u, XBP-1s, total XBP-1 and GRP78 mRNAs was observed in LSGs of SS patients, which was correlated with increased methylation levels of their respective promoters, and consistently the protein levels for IRE1α, XBP-1s and GRP78 were observed to decrease. IFN-γ decreased the mRNA and protein levels of XBP-1s, IRE1α and GRP78, and increased methylation of their promoters. Significant correlations were also found between IRE1α/XBP-1 pathway components and clinical parameters. Conclusion: Decreased mRNA levels for IRE1α, XBP-1 and GRP78 can be partially explained by hypermethylation of their promoters and is consistent with chronic endoplasmic reticulum stress, which may explain the glandular dysfunction observed in LSGs of SS patients. Additionally, glandular stress signals, including IFN-γ, could modulate the expression of the IRE1α/XBP-1 pathway components.
Assuntos
Endorribonucleases/genética , Regulação da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Glândulas Salivares/fisiopatologia , Síndrome de Sjogren/genética , Proteína 1 de Ligação a X-Box/genética , Adulto , Idoso , Western Blotting , Metilação de DNA , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Endorribonucleases/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/biossíntese , Glândulas Salivares/metabolismo , Transdução de Sinais , Síndrome de Sjogren/metabolismo , Síndrome de Sjogren/patologia , Proteína 1 de Ligação a X-Box/biossíntese , Adulto JovemRESUMO
The ST2/IL33 signalling pathway has been associated with ulcerative colitis (UC). ST2, encoded by the IL1RL1 gene, is expressed as both a membrane-anchored receptor (ST2L) activated by IL33 and as a soluble receptor (sST2) with anti-inflammatory properties. In UC patients, sST2 is further increased by corticosteroid treatment; however, the glucocorticoid-mediated molecular regulation remains unknown. We therefore tested whether genetic variants in the IL1RL1 distal promoter are involved in UC and affect glucocorticoid-mediated ST2 expression. Serum ST2 levels and genetic variants in the IL1RL1 distal promoter were examined by ELISA and PCR sequencing in UC patients receiving corticosteroids. Glucocorticoid-mediated ST2 production was evaluated in intestinal mucosa cultures. Molecular regulation of glucocorticoid-mediated ST2 was assessed by RT-qPCR, ChIP assay and luciferase reporter assay. Dexamethasone effect on ST2 transcript expression was analyzed in leukocytes and related to IL1RL1 variants. Sequencing of a distal IL1RL1 promoter region demonstrated that SNPs rs6543115(C) and rs6543116(A) are associated with increased sST2 in UC patients on corticosteroids. Dexamethasone up-regulated sST2 transcription through interaction with the glucocorticoid-response element (GRE) carrying rs6543115(C) variant. Our data indicate that IL1RL1 SNPs rs6543115(C) confer susceptibility to UC and is contained in the GRE, which may modulate glucocorticoid-induced sST2 expression.