Microbial regulation of hexokinase 2 links mitochondrial metabolism and cell death in colitis

SummaryHexokinases (HK) catalyze the first step of glycolysis and thereby limit its pace. HK2 is highly expressed in the gut epithelium, plays a role in immune responses and is upregulated in inflammation and ulcerative colitis 1–3. Here, we examined the microbial regulation of HK2 and its impact on intestinal inflammation by generating mice lacking HK2 specifically in intestinal epithelial cells (Hk2ΔIEC). Hk2ΔIEC mice were less susceptible to acute intestinal inflammation upon challenge with dextran sodium sulfate (DSS). Analyzing the epithelial transcriptome from Hk2ΔIEC mice during acute colitis revealed downregulation of cell death signaling and mitochondrial dysfunction dependent on loss of HK2. Using intestinal organoids derived from Hk2ΔIEC mice and Caco-2 cells lacking HK2, we identified peptidyl-prolyl cis-trans isomerase (PPIF) as a key target of HK2-mediated regulation of mitochondrial permeability and repression of cell-death during intestinal inflammation. The microbiota strongly regulated HK2 expression and activity. The microbially-derived short-chain fatty acid (SCFA) butyrate repressed HK2 expression and oral supplementation protected wildtype but not Hk2ΔIEC mice from DSS colitis. Our findings define a novel mechanism how butyrate may act as a protective factor for intestinal barrier homeostasis and suggest targeted HK2 inhibition as a promising therapeutic avenue in intestinal inflammation.

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Matrix metalloproteinase 9-induced increase in intestinal epithelial tight junction permeability contributes to the severity of experimental DSS colitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00256.2015 ◽

2015 ◽

Vol 309 (12) ◽

pp. G988-G997 ◽

Cited By ~ 48

Author(s):

Prashant Nighot ◽

Rana Al-Sadi ◽

Manmeet Rawat ◽

Shuhong Guo ◽

D. Martin Watterson ◽

...

Keyword(s):

Epithelial Cell ◽

Protein Expression ◽

Barrier Function ◽

Intestinal Inflammation ◽

Intestinal Barrier ◽

Intestinal Barrier Function ◽

Intestinal Epithelial ◽

Dss Colitis

Recent studies have implicated a pathogenic role for matrix metalloproteinases 9 (MMP-9) in inflammatory bowel disease. Although loss of epithelial barrier function has been shown to be a key pathogenic factor for the development of intestinal inflammation, the role of MMP-9 in intestinal barrier function remains unclear. The aim of this study was to investigate the role of MMP-9 in intestinal barrier function and intestinal inflammation. Wild-type (WT) and MMP-9−/−mice were subjected to experimental dextran sodium sulfate (DSS) colitis by administration of 3% DSS in drinking water for 7 days. The mouse colonic permeability was measured in vivo by recycling perfusion of the entire colon using fluorescently labeled dextran. The DSS-induced increase in the colonic permeability was accompanied by an increase in intestinal epithelial cell MMP-9 expression in WT mice. The DSS-induced increase in intestinal permeability and the severity of DSS colitis was found to be attenuated in MMP-9−/−mice. The colonic protein expression of myosin light chain kinase (MLCK) and phospho-MLC was found to be significantly increased after DSS administration in WT mice but not in MMP-9−/−mice. The DSS-induced increase in colonic permeability and colonic inflammation was attenuated in MLCK−/−mice and MLCK inhibitor ML-7-treated WT mice. The DSS-induced increase in colonic surface epithelial cell MLCK mRNA was abolished in MMP-9−/−mice. Lastly, increased MMP-9 protein expression was detected within the colonic surface epithelial cells in ulcerative colitis cases. These data suggest a role of MMP-9 in modulation of colonic epithelial permeability and inflammation via MLCK.

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Synbindin restrains proinflammatory macrophage activation against microbiota and mucosal inflammation during colitis

Gut ◽

10.1136/gutjnl-2020-321094 ◽

2021 ◽

pp. gutjnl-2020-321094

Author(s):

Luoyan Ai ◽

Yimeng Ren ◽

Mingming Zhu ◽

Shiyuan Lu ◽

Yun Qian ◽

...

Keyword(s):

Macrophage Activation ◽

Intestinal Inflammation ◽

Fusobacterium Nucleatum ◽

Degradation Pathway ◽

Mucosal Inflammation ◽

Intestinal Epithelial ◽

Heterozygous Deletion ◽

Macrophage Response ◽

Dss Colitis ◽

Membrane Tethering

ObjectiveAs a canonical membrane tethering factor, the function of synbindin has been expanding and indicated in immune response. Here, we investigated the role of synbindin in the regulation of toll-like receptor 4 (TLR4) signalling and macrophage response to microbiota during colitis.DesignThree distinct mouse models allowing global, myeloid-specific or intestinal epithelial cell-specific synbindin heterozygous deletion were constructed and applied to reveal the function of synbindin during dextran sodium sulfate (DSS) colitis. Effects of synbindin on TLR4 signalling and macrophage activation in response to bacterial lipopolysaccharide (LPS) or Fusobacterium nucleatum were evaluated. The colocalisation and interaction between synbindin and Rab7b were determined by immunofluorescence and coimmunoprecipitation. Synbindin expression in circulating monocytes and intestinal mucosal macrophages of patients with active IBD was detected.ResultsGlobal synbindin haploinsufficiency greatly exacerbated DSS-induced intestinal inflammation. The increased susceptibility to DSS was abolished by gut microbiota depletion, while phenocopied by specific synbindin heterozygous deletion in myeloid cells rather than intestinal epithelial cells. Profoundly aberrant proinflammatory gene signatures and excessive TLR4 signalling were observed in macrophages with synbindin interference in response to bacterial LPS or Fusobacterium nucleatum. Synbindin was significantly increased in intestinal mucosal macrophages and circulating monocytes from both mice with DSS colitis and patients with active IBD. Interleukin 23 and granulocyte-macrophage colony-stimulating factor were identified to induce synbindin expression. Mechanistic characterisation indicated that synbindin colocalised and directly interacted with Rab7b, which coordinated the endosomal degradation pathway of TLR4 for signalling termination.ConclusionSynbindin was a key regulator of TLR4 signalling and restrained the proinflammatory macrophage activation against microbiota during colitis.

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Regnase-1 controls colon epithelial regeneration via regulation of mTOR and purine metabolism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809575115 ◽

2018 ◽

Vol 115 (43) ◽

pp. 11036-11041 ◽

Cited By ~ 12

Author(s):

Yasuharu Nagahama ◽

Mayuko Shimoda ◽

Guoliang Mao ◽

Shailendra Kumar Singh ◽

Yuuki Kozakai ◽

...

Keyword(s):

Acute Inflammation ◽

Intestinal Inflammation ◽

Intestinal Barrier ◽

Body Weight Loss ◽

Purine Metabolism ◽

Intestinal Epithelial ◽

Beneficial Effects ◽

Epithelial Regeneration ◽

Mtorc1 Signaling ◽

Inflammatory Bowel

Damage to intestinal epithelial cell (IEC) layers during intestinal inflammation is associated with inflammatory bowel disease. Here we show that the endoribonuclease Regnase-1 controls colon epithelial regeneration by regulating protein kinase mTOR (the mechanistic target of rapamycin kinase) and purine metabolism. During dextran sulfate sodium-induced intestinal epithelial injury and acute colitis, Regnase-1∆IEC mice, which lack Regnase-1 specifically in the intestinal epithelium, were resistant to body weight loss, maintained an intact intestinal barrier, and showed increased cell proliferation and decreased epithelial apoptosis. Chronic colitis and tumor progression were also attenuated in Regnase-1∆IEC mice. Regnase-1 predominantly regulates mTORC1 signaling. Metabolic analysis revealed that Regnase-1 participates in purine metabolism and energy metabolism during inflammation. Furthermore, increased expression of ectonucleotidases contributed to the resolution of acute inflammation in Regnase-1∆IEC mice. These findings provide evidence that Regnase-1 deficiency has beneficial effects on the prevention and/or blocking of intestinal inflammatory disorders.

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Red wine extract preserves tight junctions in intestinal epithelial cells under inflammatory conditions: implications for intestinal inflammation

Food & Function ◽

10.1039/c8fo02469c ◽

2019 ◽

Vol 10 (3) ◽

pp. 1364-1374 ◽

Cited By ~ 21

Author(s):

Carla Nunes ◽

Víctor Freitas ◽

Leonor Almeida ◽

João Laranjinha

Keyword(s):

Red Wine ◽

Intestinal Inflammation ◽

Intestinal Barrier ◽

Intestinal Epithelial ◽

Tight Junction Proteins ◽

Inflammatory Conditions ◽

Inflammatory Stimuli ◽

Wine Polyphenols ◽

Red Wine Polyphenols ◽

Junction Proteins

Red wine polyphenols protect the intestinal barrier against inflammatory stimuli by modulating the gene expression of key tight junction proteins.

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Human RIPK1 deficiency causes combined immunodeficiency and inflammatory bowel diseases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813582116 ◽

2018 ◽

Vol 116 (3) ◽

pp. 970-975 ◽

Cited By ~ 41

Author(s):

Yue Li ◽

Marita Führer ◽

Ehsan Bahrami ◽

Piotr Socha ◽

Maja Klaudel-Dreszler ◽

...

Keyword(s):

Cell Death ◽

Intestinal Inflammation ◽

Intestinal Epithelial ◽

Loss Of Function ◽

Monogenic Disorders ◽

Bowel Diseases ◽

Inflammatory Bowel ◽

Human Immune

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a critical regulator of cell death and inflammation, but its relevance for human disease pathogenesis remains elusive. Studies of monogenic disorders might provide critical insights into disease mechanisms and therapeutic targeting of RIPK1 for common diseases. Here, we report on eight patients from six unrelated pedigrees with biallelic loss-of-function mutations in RIPK1 presenting with primary immunodeficiency and/or intestinal inflammation. Mutations in RIPK1 were associated with reduced NF-κB activity, defective differentiation of T and B cells, increased inflammasome activity, and impaired response to TNFR1-mediated cell death in intestinal epithelial cells. The characterization of RIPK1-deficient patients highlights the essential role of RIPK1 in controlling human immune and intestinal homeostasis, and might have critical implications for therapies targeting RIPK1.

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Epithelial-intrinsic IKKα expression regulates group 3 innate lymphoid cell responses and antibacterial immunity

Journal of Experimental Medicine ◽

10.1084/jem.20141831 ◽

2015 ◽

Vol 212 (10) ◽

pp. 1513-1528 ◽

Cited By ~ 50

Author(s):

Paul R. Giacomin ◽

Ryan H. Moy ◽

Mario Noti ◽

Lisa C. Osborne ◽

Mark C. Siracusa ◽

...

Keyword(s):

Innate Immunity ◽

Epithelial Cell ◽

Intestinal Inflammation ◽

Intestinal Barrier ◽

Lymphoid Cells ◽

Intestinal Damage ◽

Intestinal Epithelial ◽

Mucosal Surfaces ◽

Host Mortality ◽

Group 3

Innate lymphoid cells (ILCs) are critical for maintaining epithelial barrier integrity at mucosal surfaces; however, the tissue-specific factors that regulate ILC responses remain poorly characterized. Using mice with intestinal epithelial cell (IEC)–specific deletions in either inhibitor of κB kinase (IKK)α or IKKβ, two critical regulators of NFκB activation, we demonstrate that IEC-intrinsic IKKα expression selectively regulates group 3 ILC (ILC3)–dependent antibacterial immunity in the intestine. Although IKKβΔIEC mice efficiently controlled Citrobacter rodentium infection, IKKαΔIEC mice exhibited severe intestinal inflammation, increased bacterial dissemination to peripheral organs, and increased host mortality. Consistent with weakened innate immunity to C. rodentium, IKKαΔIEC mice displayed impaired IL-22 production by RORγt+ ILC3s, and therapeutic delivery of rIL-22 or transfer of sort-purified IL-22–competent ILCs from control mice could protect IKKαΔIEC mice from C. rodentium–induced morbidity. Defective ILC3 responses in IKKαΔIEC mice were associated with overproduction of thymic stromal lymphopoietin (TSLP) by IECs, which negatively regulated IL-22 production by ILC3s and impaired innate immunity to C. rodentium. IEC-intrinsic IKKα expression was similarly critical for regulation of intestinal inflammation after chemically induced intestinal damage and colitis. Collectively, these data identify a previously unrecognized role for epithelial cell–intrinsic IKKα expression and TSLP in regulating ILC3 responses required to maintain intestinal barrier immunity.

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Apoptosis, Necrosis, and Necroptosis in the Gut and Intestinal Homeostasis

Mediators of Inflammation ◽

10.1155/2015/250762 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 53

Author(s):

Anna Negroni ◽

Salvatore Cucchiara ◽

Laura Stronati

Keyword(s):

Cell Death ◽

Immune Responses ◽

Intestinal Epithelial Cells ◽

Inflammatory Diseases ◽

Intestinal Barrier ◽

Intestinal Lumen ◽

Intestinal Epithelial ◽

Commensal Microbiota ◽

Adaptive Immune ◽

Epithelial Cell Death

Intestinal epithelial cells (IECs) form a physiochemical barrier that separates the intestinal lumen from the host’s internal milieu and is critical for electrolyte passage, nutrient absorption, and interaction with commensal microbiota. Moreover, IECs are strongly involved in the intestinal mucosal inflammatory response as well as in mucosal innate and adaptive immune responses. Cell death in the intestinal barrier is finely controlled, since alterations may lead to severe disorders, including inflammatory diseases. The emerging picture indicates that intestinal epithelial cell death is strictly related to the maintenance of tissue homeostasis. This review is focused on previous reports on different forms of cell death in intestinal epithelium.

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Neutrophil extracellular traps impair intestinal barrier functions in sepsis by regulating TLR9-mediated endoplasmic reticulum stress pathway

Cell Death and Disease ◽

10.1038/s41419-021-03896-1 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Shilong Sun ◽

Zehua Duan ◽

Xinyu Wang ◽

Chengnan Chu ◽

Chao Yang ◽

...

Keyword(s):

Er Stress ◽

Intestinal Epithelial Cell ◽

Intestinal Inflammation ◽

Intestinal Barrier ◽

Neutrophil Extracellular Traps ◽

Intestinal Epithelial ◽

Barrier Dysfunction ◽

Extracellular Traps ◽

Epithelial Cell Death ◽

Stress Activation

AbstractIncreased neutrophil extracellular traps (NETs) formation has been found to be associated with intestinal inflammation, and it has been reported that NETs may drive the progression of gut dysregulation in sepsis. However, the biological function and regulation of NETs in sepsis-induced intestinal barrier dysfunction are not yet fully understood. First, we found that both circulating biomarkers of NETs and local NETs infiltration in the intestine were significantly increased and had positive correlations with markers of enterocyte injury in abdominal sepsis patients. Moreover, the levels of local citrullinated histone 3 (Cit H3) expression were associated with the levels of BIP expression. To further confirm the role of NETs in sepsis-induced intestinal injury, we compared peptidylarginine deiminase 4 (PAD4)-deficient mice and wild-type (WT) mice in a lethal septic shock model. In WT mice, the Cit H3-DNA complex was markedly increased, and elevated intestinal inflammation and endoplasmic reticulum (ER) stress activation were also found. Furthermore, PAD4 deficiency alleviated intestinal barrier disruption and decreased ER stress activation. Notably, NETs treatment induced intestinal epithelial monolayer barrier disruption and ER stress activation in a dose-dependent manner in vitro, and ER stress inhibition markedly attenuated intestinal apoptosis and tight junction injury. Finally, TLR9 antagonist administration significantly abrogated NETs-induced intestinal epithelial cell death through ER stress inhibition. Our results indicated that NETs could contribute to sepsis-induced intestinal barrier dysfunction by promoting inflammation and apoptosis. Suppression of the TLR9–ER stress signaling pathway can ameliorate NETs-induced intestinal epithelial cell death.

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Catestatin Regulates Epithelial Cell Dynamics to Improve Intestinal Inflammation

Vaccines ◽

10.3390/vaccines6040067 ◽

2018 ◽

Vol 6 (4) ◽

pp. 67 ◽

Cited By ~ 7

Author(s):

Nour Eissa ◽

Hayam Hussein ◽

Ruth Mesgna ◽

Sandra Bonin ◽

Geoffrey Hendy ◽

...

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Intestinal Inflammation ◽

Mrna Level ◽

Intestinal Barrier ◽

Experimental Colitis ◽

Rectal Administration ◽

Intestinal Epithelial ◽

Strong Negative Correlation ◽

Promising Therapeutic Approach

Ulcerative colitis (UC) is characterized by aberrant regulation of tight junctions (TJ), signal transducer and activator of transcription 3 (STAT3), and interleukin (IL)-8/18, which lead to intestinal barrier defects. Catestatin (CST), an enterochromaffin-derived peptide, regulates immune communication and STAT-3 in the inflamed intestine. Here, we investigated the effects of CST during the development of inflammation using human biopsies from patients with active UC, human colonic epithelial cells (Caco2), and an experimental model of UC (dextran sulfate sodium [DSS]-colitis). In UC patients, the protein and mRNA level of CST was significantly decreased. Colonic expression of CST showed a strong positive linear relationship with TJ proteins and STAT3, and a strong negative correlation with IL-8 and IL-18. Intra-rectal administration of CST reduced the severity of experimental colitis, IL-18 colonic levels, maintained TJ proteins and enhanced the phosphorylation of STAT3. CST administration increased proliferation, viability, migration, TJ proteins, and p-STAT3 levels, and reduced IL-8 & IL-18 in LPS- & DSS-induced Caco2 cell epithelial injury, and the presence of STAT-3 inhibitor abolished the beneficial effect of CST. In inflammatory conditions, we conclude that CST could regulate intestinal mucosal dynamic via a potential STAT3-dependent pathway that needs to be further defined. Targeting CST in intestinal epithelial cells (IECs) should be a promising therapeutic approach such as when intestinal epithelial cell homeostasis is compromised in UC patients.

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P033 Combination of vedolizumab with tacrolimus is more efficient in reducing intestinal inflammation than vedolizumab alone in the DSS-induced acute colitis mouse model

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjz203.162 ◽

2020 ◽

Vol 14 (Supplement_1) ◽

pp. S148-S148

Author(s):

R Manzini ◽

K Atrott ◽

M Schwarzfischer ◽

A Laimbacher ◽

S Lang ◽

...

Keyword(s):

T Cells ◽

Mouse Model ◽

Molecular Mechanisms ◽

Immune Cell ◽

Intestinal Inflammation ◽

Sodium Sulphate ◽

Intestinal Epithelial ◽

Acute Colitis ◽

Human Cd34 ◽

Epithelial Cell Apoptosis

Abstract Background The humanised monoclonal antibody vedolizumab is used in the treatment of ulcerative colitis (UC) and Crohn’s disease (CD). Vedolizumab reduces intestinal inflammation through inhibition of the integrin heterodimer α 4β 7, responsible for the homing of T cells to the intestinal mucosa. Recent studies have also shown a possible involvement of vedolizumab in the regulation of the innate immune system. Particularly in CD, only a fraction of patients respond to vedolizumab treatment, and combination therapy with immunosuppressant drugs, such as the calcineurin-inhibitor tacrolimus, might prove beneficial. The aim of this study was to assess if co-treatment of vedolizumab and tacrolimus is more efficient in reducing intestinal inflammation in an acute colitis mouse model and to unravel the underlying molecular mechanisms. Methods NOD-SCID-SGM3 mice were reconstituted with human CD34+ cells and treated with 3% dextrane sodium sulphate (DSS) in drinking water to induce acute colitis. Mice were treated with vedolizumab alone (30mg/kg, inject 3 days prior to DSS-start and 50mg/kg at day 0 and at day 4 of DSS-treatment), tacrolimus alone (1mg/kg/day intraperitoneally), or a combination of tacrolimus and vedolizumab during colitis induction. Results As expected, DSS-treatment induced colitis in mice as observed by weight loss, diarrhoea, colon shortening, and endoscopic signs of inflammation categorised by the MEICS score. This translated histologically to an increased immune cell infiltration and epithelial erosion. Vedolizumab and tacrolimus treatment alone did not significantly reduce colitis severity, although endoscopy showed slightly less severe inflammation in vedolizumab-treated mice. Combination of vedolizumab and tacrolimus, however, clearly reduced colonoscopy and histology scores. DSS-treatment increased the number of CD3 T cells and CD68 macrophages in the intestine, an effect counteracted by vedolizumab or tacrolimus alone and further pronounced by combination treatment. Particularly vedolizumab treatment, either alone or in combination, caused clear reduction of pro-inflammatory M1 macrophages. Additionally, vedolizumab alone or combined decreased the levels of intestinal epithelial cell apoptosis as indicated by staining for cleaved caspase-3. Conclusion Our data demonstrate that the anti-inflammatory effect of vedolizumab is potentiated by co-treatment with tacrolimus. Notably, the combination of both drugs was more efficient in reducing T-cell and macrophage infiltration into the intestine. This indicates that the combination of vedolizumab with immunosuppressant drugs might prove beneficial for patients that do not respond to vedolizumab-only therapy.

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