Autophagy, a process of degradation and recycling of macromolecules and organelles to maintain cellular homeostasis, has also been shown to help eliminate invading pathogens. Conversely, various pathogens including parasites have been shown to modulate/exploit host autophagy facilitating their intracellular infectious cycle. In this regard, Cryptosporidium parvum (CP), a protozoan parasite of small intestine is emerging as a major global health challenge. However, the pathophysiology of cryptosporidiosis is mostly unknown. We have recently demonstrated CP-induced epithelial barrier disruption via decreasing the expression of specific tight junction (TJ) and adherens junction (AJ) proteins such as occludin, claudin-4 and E-cadherin. Therefore, we utilised confluent Caco-2 cell monolayers as in vitro model of intestinal epithelial cells (IECs) to investigate the potential role of autophagy in the pathophysiology of cryptosporidiosis. Autophagy was assessed by increase in the ratio of LC3II (microtubule associated protein 1 light chain 3) to LC3I protein and decrease in p62/SQSTM1 protein levels. CP treatment of Caco-2 cells for 24 hr induced autophagy with a maximum effect observed with 0.5 × 106 oocyst/well. CP decreased mTOR (mammalian target of rapamycin, a suppressor of autophagy) phosphorylation, suggesting autophagy induction via mTOR inactivation. Measurement of autophagic flux utilizing the lysosomal inhibitor chloroquine (CQ) showed more pronounced increase in LC3II level in cells co-treated with CP + CQ as compared to CP or CQ alone, suggesting that CP-induced increase in LC3II was due to enhanced autophagosome formation rather than impaired lysosomal clearance. CP infection did not alter ATG7, a key autophagy protein. However, the decrease in occludin, claudin-4 and E-cadherin by CP was partially blocked following siRNA silencing of ATG7, suggesting the role of autophagy in CP-induced decrease in these TJ/AJ proteins. Our results provide novel evidence of autophagy induction by CP in host IECs that could alter important host cell processes contributing to the pathophysiology of cryptosporidiosis.
Abstract Commensal bacteria are critical regulators of both tissue homeostasis and the development and exacerbation of autoimmunity. However, it remains unclear how the intestinal microbiota contributes to inflammation in tissues such as the central nervous system (CNS) where these microbes are typically absent and whether T cell receptor (TCR) specificity for commensal-derived antigens is important to the development of tissue inflammation-related outcomes. Here, we found that ileum- and cecum-colonizing segmented filamentous bacteria (SFB)-specific T cells (clone TCR 7B8 ) can infiltrate the CNS wherein they can be reactivated and produce high levels of inflammatory cytokines including IFNγ, IL-17A, TNFα, and GM-CSF in the absence of regulatory T cells. In contrast, other SFB-specific T cells (clone TCR 1A2 ) recognizing an epitope in which 8/9 amino acids overlap with those recognized by TCR 7B8 failed to induce such neuroinflammation. Despite their similar SFB-derived peptide antigen targets, TCR 7B8 was found to recognize peptides derived from host proteins including receptor tyrosine-protein kinase ErbB2, trophinin 1, and anaphase-promoting complex subunit 2 in vitro , whereas TCR 1A2 did not, indicating that TCR 7B8 induces CNS inflammation via molecular mimicry. Immune checkpoint blockade accelerated TCR 7B8 -mediated CNS inflammation, suggesting a potential cause of immune-related adverse events induced in cancer patients undergoing such treatment. Together, our findings reveal a potential mechanism whereby gut commensal-specific T cells are dysregulated and contribute to extraintestinal inflammation.
I nflammatory B owel D isease (IBD) is a global health burden currently affecting around 3 million people in the United States with increasing incidence worldwide. The pathogenesis of IBD is still unclear due to the multifactorial nature of the disease. Compromised intestinal barrier is one of the most critical early events linked to the onset of intestinal inflammation. In this regard, SLC26A3 or DRA ( D own R egulated in A denoma) is an essential transporter for chloride absorption in the mammalian intestine and has been identified as an IBD susceptibility gene in certain cohorts. Further, DRA levels are markedly decreased in colonic mucosa of IBD patients and in mouse models of gut inflammation. Also, DRA KO mice are more susceptible to DSS colitis. However, the mechanisms underlying increased susceptibility to inflammation by loss of DRA are not fully understood. Aims To examine the impact of loss of DRA (in DRA KO mice) on epithelial barrier function and expression of key tight junction and adherens junction proteins and elucidate underlying mechanisms. Methods Wild type and DRA KO mice (8–10 weeks old, M/F) were used. Protein expression was measured by immunoblotting and immunofluorescence. FITC‐dextran flux was examined utilizing Ussing chamber. Results DRA KO mice exhibited an increase in colonic paracellular permeability (~ 5 fold, p<0.05) indicating impaired barrier function. This was associated with a decrease in levels of TJ protein ZO‐1 and occludin (~ 65%, p<0.05) and major AJ protein, E‐cadherin (~ 60%, p<0.05). Also, the expression of pore forming, Claudin 2 was significantly upregulated in DRA KO mouse colon (~ 3 fold, p<0.05). No significant changes were observed in claudin 1 or 3. Since mouse intestine is a complex physiological system, we next examined if DRA silencing in a model epithelial Caco‐2 cell line, affects barrier function and tight junction protein expression. shRNA Knock‐down of DRA in Caco‐2 cells resulted in a marked decrease in TEER (~ 40%, p<0.05) and occludin expression (~ 60%, p<0.05). Similarly, enteroids derived from DRA KO mice exhibited decreased expression of occludin and E‐cadherin proteins (~ 50%, p<0.05). There was no alteration in mRNA levels of TJ/AJ proteins in DRA KO mice indicating role of post‐transcriptional mechanisms. To understand the mechanisms underlying these alterations, we next examined the expression of RNA‐binding proteins that are known to regulate the translation of occludin and E‐cadherin. Our results showed a marked increase in expression of CUGBP1 (a negative regulator of TJ/AJ proteins) and a marked decrease in expression of intact HuR (a positive regulator). Conclusion Our studies suggest a novel role of a membrane chloride transporter DRA in maintenance of intestinal barrier function and thus a down regulation of this protein may play a key role in pathogenesis of IBD. Support or Funding Information Supported by NIDDK and Department of Veterans Affairs This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Serotonin transporter (SERT) deficiency has been implicated in metabolic syndrome, intestinal inflammation, and microbial dysbiosis. Interestingly, changes in microbiome metabolic capacity and several alterations in host gene expression, including lipid metabolism, were previously observed in SERT
Down-regulation of chloride transporter SLC26A3 or down-regulated in adenoma (DRA) in colonocytes has recently been linked to the pathogenesis of ulcerative colitis (UC). Because exaggerated immune responses are one of the hallmarks of UC, these current studies were undertaken to define the mechanisms by which loss of DRA relays signals to immune cells to increase susceptibility to inflammation.
Background: Serotonin transporter (SLC6A4; SERT) plays a key role in regulating the availability of 5-HT (5-hydroxytryptamine, a neurotransmitter and hormone) in the brain and the gut mucosa. A decrease in intestinal SERT expression, associated with increased availability of 5-HT, is implicated in the pathogenesis of infectious and inflammatory disorders of the gut. Therefore, it is critical to understand the regulation of SERT, as a novel target for the treatment of gastrointestinal disorders. In this regard, long noncoding RNAs (LncRNAs, small transcripts >200 nucleotides) are known to modulate gene expression via chromatin remodeling, transcriptional and posttranscriptional mechanisms. Notably, elevated levels of LncRNA H19 are observed in IBD patients and mouse models of inflammation and are known to promote mucosal regeneration and wound healing and restore the intestinal homeostasis. Whether LncRNA H19 regulates SERT expression remains unknown and was the focus of the current study. Methods: Caco-2 cells were transiently transfected with either H19 mammalian expression vector or H19 siRNA using Lipofectamine 2000. Protein and mRNA expression was measured by immunoblotting and real-time PCR, respectively. Age-matched H19 lncRNA- deficient mice and wild-type (WT) littermates were used to determine the role of genetic deletion of H19 on SERT expression in ileum. Results: H19 overexpression in Caco-2 cells increased SERT mRNA levels (~13-fold; p<0.05) at 48h post-transfection compared to an empty vector. Consistent with the mRNA levels, H19 also increased SERT protein expression (~2-fold, p<0.05). Furthermore, siRNA-mediated knockdown of H19 in Caco-2 cells resulted in a significant decrease in SERT mRNA levels (~80%, p<0.05). Parallel to the in vitro results, H19 deficient mice also showed a significant decrease in SERT mRNA expression (~69 %, p<0.05) in the ileum compared to the WT mice. Interestingly, in comparison to WT, the expression of other key transporters involved in intestinal NaCl absorption (SLC9A3, SLC26A3, and SLC26A6) remained unaltered in H19 deficient mice. Conclusion: These data demonstrate a novel role of LncRNA H19 in regulating the expression of SERT in the intestine under physiological conditions. We speculate that H19 mediated increase in SERT expression may provide valuable insights into the mechanisms restoring decreased SERT expression in inflammatory or infectious diseases of the gut. (Supported by VA/NIH). VA and NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
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