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1.
Immun Inflamm Dis ; 12(2): e1144, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38363052

RESUMO

Interleukin-22 (IL-22) is an important cytokine in the intestinal environment. IL-22 is mainly produced by immune cells and targeted at nonimmune cells such as epithelial and stromal cells in a broad array of tissues such as -but not restricted to- the liver and adipose tissue. IL-22 therefore connects immune functions with metabolic functions of the host, and since it is induced by the microbiota, connects host functioning to the outside environment. IL-22 induces epithelial cell proliferation aiding in rapid epithelium regeneration and wound healing. Additionally, IL-22 activates antiapoptotic genes and DNA damage response pathways, enhancing epithelial cell survival. Recently, it has also been shown that IL-22 induces Paneth cell differentiation in humans. However, IL-22 can also contribute to intestinal epithelium damage and reduces microbial diversity in the intestine directly or indirectly by inducing excessive antimicrobial peptide production by epithelial cells. Moreover, IL-22 enhances angiogenesis and may therefore support tumorigenesis in the intestine. In conclusion, it appears that whether IL-22 has a beneficial or harmful effect in the mammalian intestine largely depends on its regulation. This review aims to provide a comprehensive overview of the current literature and emphasizes that IL-22 signaling outcome depends on the timing and duration of IL-22 production, the presence of it regulators such as IL-22BP, and the specific location of the cytokine production in the gastrointestinal tract.


Assuntos
Intestinos , Animais , Humanos , Citocinas/metabolismo , Mucosa Intestinal , Homeostase , Mamíferos
2.
J Clin Immunol ; 44(3): 63, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38363399

RESUMO

Inflammatory bowel disease (IBD) occurring following allogeneic stem cell transplantation (aSCT) is a very rare condition. The underlying pathogenesis needs to be better defined. There is currently no systematic effort to exclude loss- or gain-of-function mutations in immune-related genes in stem cell donors. This is despite the fact that more than 100 inborn errors of immunity may cause or contribute to IBD. We have molecularly characterized a patient who developed fulminant inflammatory bowel disease following aSCT with stable 100% donor-derived hematopoiesis. A pathogenic c.A291G; p.I97M HAVCR2 mutation encoding the immune checkpoint protein TIM-3 was identified in the patient's blood-derived DNA, while being absent in DNA derived from the skin. TIM-3 expression was much decreased in the patient's serum, and in vitro-activated patient-derived T cells expressed reduced TIM-3 levels. In contrast, T cell-intrinsic CD25 expression and production of inflammatory cytokines were preserved. TIM-3 expression was barely detectable in the immune cells of the patient's intestinal mucosa, while being detected unambiguously in the inflamed and non-inflamed colon from unrelated individuals. In conclusion, we report the first case of acquired, "transplanted" insufficiency of the regulatory TIM-3 checkpoint linked to post-aSCT IBD.


Assuntos
Receptor Celular 2 do Vírus da Hepatite A , Doenças Inflamatórias Intestinais , Humanos , Receptor Celular 2 do Vírus da Hepatite A/genética , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/etiologia , Citocinas/metabolismo , Mucosa Intestinal , DNA
3.
Nat Commun ; 15(1): 1470, 2024 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-38368394

RESUMO

Disrupted host-microbe interactions at the mucosal level are key to the pathophysiology of IBD. This study aimed to comprehensively examine crosstalk between mucosal gene expression and microbiota in patients with IBD. To study tissue-specific interactions, we perform transcriptomic (RNA-seq) and microbial (16S-rRNA-seq) profiling of 697 intestinal biopsies (645 derived from 335 patients with IBD and 52 from 16 non-IBD controls). Mucosal gene expression patterns in IBD are mainly determined by tissue location and inflammation, whereas the mucosal microbiota composition shows a high degree of individual specificity. Analysis of transcript-bacteria interactions identifies six distinct groups of inflammation-related pathways that are associated with intestinal microbiota (adjusted P < 0.05). An increased abundance of Bifidobacterium is associated with higher expression of genes involved in fatty acid metabolism, while Bacteroides correlates with increased metallothionein signaling. In patients with fibrostenosis, a transcriptional network dominated by immunoregulatory genes is associated with Lachnoclostridium bacteria in non-stenotic tissue (adjusted P < 0.05), while being absent in CD without fibrostenosis. In patients using TNF-α-antagonists, a transcriptional network dominated by fatty acid metabolism genes is linked to Ruminococcaceae (adjusted P < 0.05). Mucosal microbiota composition correlates with enrichment of intestinal epithelial cells, macrophages, and NK-cells. Overall, these data demonstrate the presence of context-specific mucosal host-microbe interactions in IBD, revealing significantly altered inflammation-associated gene-taxa modules, particularly in patients with fibrostenotic CD and patients using TNF-α-antagonists. This study provides compelling insights into host-microbe interactions that may guide microbiota-directed precision medicine and fuels the rationale for microbiota-targeted therapeutics as a strategy to alter disease course in IBD.


Assuntos
Interações entre Hospedeiro e Microrganismos , Doenças Inflamatórias Intestinais , Humanos , Interações entre Hospedeiro e Microrganismos/genética , Fator de Necrose Tumoral alfa/genética , Doenças Inflamatórias Intestinais/patologia , Fenótipo , Inflamação/genética , Inflamação/patologia , Ácidos Graxos , Mucosa Intestinal/patologia
4.
Nat Commun ; 15(1): 1282, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38346956

RESUMO

TNF acts as one pathogenic driver for inducing intestinal epithelial cell (IEC) death and substantial intestinal inflammation. How the IEC death is regulated to physiologically prevent intestinal inflammation needs further investigation. Here, we report that EF-hand domain-containing protein D2 (EFHD2), highly expressed in normal intestine tissues but decreased in intestinal biopsy samples of ulcerative colitis patients, protects intestinal epithelium from TNF-induced IEC apoptosis. EFHD2 inhibits TNF-induced apoptosis in primary IECs and intestinal organoids (enteroids). Mice deficient of Efhd2 in IECs exhibit excessive IEC death and exacerbated experimental colitis. Mechanistically, EFHD2 interacts with Cofilin and suppresses Cofilin phosphorylation, thus blocking TNF receptor I (TNFR1) internalization to inhibit IEC apoptosis and consequently protecting intestine from inflammation. Our findings deepen the understanding of EFHD2 as the key regulator of membrane receptor trafficking, providing insight into death receptor signals and autoinflammatory diseases.


Assuntos
Colite , Receptores Tipo I de Fatores de Necrose Tumoral , Humanos , Camundongos , Animais , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Intestinos/patologia , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Apoptose , Colite/patologia , Inflamação/patologia , Fatores de Despolimerização de Actina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo
5.
Sci Rep ; 14(1): 3479, 2024 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-38347087

RESUMO

Reduced butyrate-production capacity has been reported in fecal microbial communities in patients with active ulcerative colitis. However, the butyrate-production capacity of the mucosal microbiome from active vs quiescent mucosa in ulcerative colitis has been unexplored. We sought to determine the diversity and relative abundance of mucosal bacterial and fungal communities from endoscopically active vs quiescent mucosa in patients with UC, and aimed to predict contributions of mucosal microbial communities to butyrate synthesis. Systematic, segmental right- and left-sided biopsies were obtained from endoscopically active (n = 13) or quiescent (n = 17) colonic mucosa, among 15 patients with pan-colonic ulcerative colitis. Dietary fiber intake of patients was performed using the validated five-item FiberScreen questionnaire. Amplicon sequencing of mucosal bacteria and fungi was performed. The diversity and relative abundance of mucosal bacterial and fungal taxa were quantified, and predicted contributions to butyrate synthesis were ascertained. Bacterial alpha and beta diversity were similar between active vs quiescent mucosa. Butyrogenic taxa were significantly increased in quiescence, including Butyricimonas, Subdoligranulum, and Alistipes. Predicted butyrate kinase activity was significantly and concomitantly increased in quiescent mucosa. Fiber intake was positively correlated with butyrogenic microbes. Compared to mucosal bacterial prevalence, mucosal fungi were detected in low prevalence. Butyrogenic microbes are relatively increased in quiescent mucosa in ulcerative colitis, and may be related to increased fiber intake during quiescence. Manipulation of the mucosal microbiome towards butyrate-producing bacteria may be associated with endoscopic quiescence.


Assuntos
Colite Ulcerativa , Humanos , Colite Ulcerativa/patologia , Butiratos , Colo/patologia , Biópsia , Mucosa Intestinal/patologia , Bactérias/genética
6.
Immunity ; 57(2): 195-197, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38354697

RESUMO

How commensals influence intestinal immunity is incompletely understood. In this issue of Immunity, Eshleman et al. demonstrate that microbiota-derived butyrate restrains tuft cell development via HDAC3 modulation in intestinal epithelial cells, showing how microbial metabolites impact intestinal type 2 immunity.


Assuntos
Mucosa Intestinal , Microbiota , Amor , Intestinos , Células Epiteliais
7.
Int J Mol Sci ; 25(3)2024 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-38338944

RESUMO

We aimed to test how the postbiotic butyrate impacts select gut bacteria, small intestinal epithelial integrity, and microvascular endothelial activation during acute ethanol exposure in mice and primary human intestinal microvascular endothelial cells (HIMECs). Supplementation during an acute ethanol challenge with or without tributyrin, a butyrate prodrug, was delivered to C57BL/6 mice. A separate group of mice received 3 days of clindamycin prior to the acute ethanol challenge. Upon euthanasia, blood endotoxin, cecal bacteria, jejunal barrier integrity, and small intestinal lamina propria dendritic cells were assessed. HIMECs were tested for activation following exposure to ethanol ± lipopolysaccharide (LPS) and sodium butyrate. Tributyrin supplementation protected a butyrate-generating microbe during ethanol and antibiotic exposure. Tributyrin rescued ethanol-induced disruption in jejunal epithelial barrier, elevated plasma endotoxin, and increased mucosal vascular addressin cell-adhesion molecule-1 (MAdCAM-1) expression in intestinal microvascular endothelium. These protective effects of tributyrin coincided with a tolerogenic dendritic response in the intestinal lamina propria. Lastly, sodium butyrate pre- and co-treatment attenuated the direct effects of ethanol and LPS on MAdCAM-1 induction in the HIMECs from a patient with ulcerative colitis. Tributyrin supplementation protects small intestinal epithelial and microvascular barrier integrity and modulates microvascular endothelial activation and dendritic tolerizing function during a state of gut dysbiosis and acute ethanol challenge.


Assuntos
Células Endoteliais , Etanol , Camundongos , Humanos , Animais , Etanol/farmacologia , Ácido Butírico/farmacologia , Ácido Butírico/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Mucosa Intestinal/metabolismo
8.
Int J Mol Sci ; 25(3)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38339023

RESUMO

The gastrointestinal (GI) tract of multicellular organisms, especially mammals, harbors a symbiotic commensal microbiota with diverse microorganisms including bacteria, fungi, viruses, and other microbial and eukaryotic species. This microbiota exerts an important role on intestinal function and contributes to host health. The microbiota, while benefiting from a nourishing environment, is involved in the development, metabolism and immunity of the host, contributing to the maintenance of homeostasis in the GI tract. The immune system orchestrates the maintenance of key features of host-microbe symbiosis via a unique immunological network that populates the intestinal wall with different immune cell populations. Intestinal epithelium contains lymphocytes in the intraepithelial (IEL) space between the tight junctions and the basal membrane of the gut epithelium. IELs are mostly CD8+ T cells, with the great majority of them expressing the CD8αα homodimer, and the γδ T cell receptor (TCR) instead of the αß TCR expressed on conventional T cells. γδ T cells play a significant role in immune surveillance and tissue maintenance. This review provides an overview of how the microbiota regulates γδ T cells and the influence of microbiota-derived metabolites on γδ T cell responses, highlighting their impact on immune homeostasis. It also discusses intestinal neuro-immune regulation and how γδ T cells possess the ability to interact with both the microbiota and brain.


Assuntos
Linfócitos T CD8-Positivos , Microbiota , Animais , Linfócitos T CD8-Positivos/metabolismo , Neuroimunomodulação , Mucosa Intestinal/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta , Homeostase , Mamíferos/metabolismo
9.
Parasite ; 31: 6, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38334686

RESUMO

Previous studies have shown that recombinant Trichinella spiralis galectin (rTsgal) is characterized by a carbohydrate recognition domain sequence motif binding to beta-galactoside, and that rTsgal promotes larval invasion of intestinal epithelial cells. Galactomannan is an immunostimulatory polysaccharide composed of a mannan backbone with galactose residues. The aim of this study was to investigate whether galactomannan inhibits larval intrusion of intestinal epithelial cells and enhances antibody-dependent cellular cytotoxicity (ADCC), killing newborn larvae by polarizing macrophages to the M1 phenotype. The results showed that galactomannan specially binds to rTsgal, and abrogated rTsgal facilitation of larval invasion of intestinal epithelial cells. The results of qPCR, Western blotting, and flow cytometry showed that galactomannan and rTsgal activated macrophage M1 polarization, as demonstrated by high expression of iNOS (M1 marker) and M1 related genes (IL-1ß, IL-6, and TNF-α), and increased CD86+ macrophages. Galactomannan and rTsgal also increased NO production. The killing ability of macrophage-mediated ADCC on larvae was also significantly enhanced in galactomannan- and rTsgal-treated macrophages. The results demonstrated that Tsgal may be considered a potential vaccine target molecule against T. spiralis invasion, and galactomannan may be a novel adjuvant therapeutic agent and potential vaccine adjuvant against T. spiralis infection.


Title: Le galactomannane inhibe l'invasion par Trichinella spiralis des cellules de l'épithélium intestinal et améliore la cytotoxicité cellulaire dépendante des anticorps tuant les larves en activant la polarisation des macrophages. Abstract: Des études antérieures ont montré que la galectine recombinante de Trichinella spiralis (rTsgal) est caractérisée par un motif de séquence de domaines de reconnaissance des glucides se liant au bêta-galactoside, et que la rTsgal favorise l'invasion larvaire des cellules épithéliales intestinales. Le galactomannane est un polysaccharide immunostimulateur composé d'un squelette mannane avec des résidus galactose. Le but de cette étude était de déterminer si le galactomannane inhibe l'intrusion larvaire des cellules épithéliales intestinales et améliore la cytotoxicité cellulaire dépendante des anticorps (CCDA) tuant les larves nouvelles-nées en polarisant les macrophages au phénotype M1. Les résultats ont montré que le galactomannane se liait spécialement au rTsgal et supprimait la facilitation du rTsgal sur l'invasion larvaire des cellules épithéliales intestinales. Les résultats de la qPCR, du Western blot et de la cytométrie en flux ont montré que le galactomannane et le rTsgal activaient la polarisation des macrophages M1, comme le démontre la forte expression de l'iNOS (marqueur de M1) et des gènes liés à M1 (IL-1ß, IL-6 et TNF-α), et l'augmentation des macrophages CD86+. Le galactomannane et le rTsgal ont également augmenté la production de NO. La capacité de destruction de la CCDA médiée par les macrophages sur les larves était également significativement améliorée dans les macrophages traités au galactomannane et au rTsgal. Les résultats ont démontré que Tsgal pourrait être considéré comme une molécule cible potentielle d'un vaccin contre l'invasion par T. spiralis, et que le galactomannane pourrait être un nouvel agent thérapeutique adjuvant et un adjuvant vaccinal potentiel contre l'infection à T. spiralis.


Assuntos
Galactose/análogos & derivados , Doenças dos Roedores , Trichinella spiralis , Triquinelose , Animais , Camundongos , Mananas/farmacologia , Mananas/metabolismo , Larva/genética , Mucosa Intestinal , Citotoxicidade Celular Dependente de Anticorpos , Camundongos Endogâmicos BALB C
10.
Sci Immunol ; 9(92): eadk4348, 2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-38335269

RESUMO

TCRαß+CD8αα+ intraepithelial lymphocytes (CD8αα+ αß IELs) are a specialized subset of T cells in the gut epithelium that develop from thymic agonist selected IEL precursors (IELps). The molecular mechanisms underlying the selection and differentiation of this T cell type in the thymus are largely unknown. Here, we found that Bcl6 deficiency in αß T cells resulted in the near absence of CD8αα+ αß IELs. BCL6 was expressed by approximately 50% of CD8αα+ αß IELs and by the majority of thymic PD1+ IELps after agonist selection. Bcl6 deficiency blocked early IELp generation in the thymus, and its expression in IELps was induced by thymic TCR signaling in an ERK-dependent manner. As a result of Bcl6 deficiency, the precursors of IELps among CD4+CD8+ double-positive thymocytes exhibited increased apoptosis during agonist selection and impaired IELp differentiation and maturation. Together, our results elucidate BCL6 as a crucial transcription factor during the thymic development of CD8αα+ αß IELs.


Assuntos
Linfócitos Intraepiteliais , Receptores de Antígenos de Linfócitos T alfa-beta , Camundongos , Animais , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linfócitos Intraepiteliais/metabolismo , Mucosa Intestinal , Camundongos Knockout
11.
Sci Adv ; 10(6): eadi2671, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38335286

RESUMO

The adult intestine is a regionalized organ, whose size and cellular composition are adjusted in response to nutrient status. This involves dynamic regulation of intestinal stem cell (ISC) proliferation and differentiation. How nutrient signaling controls cell fate decisions to drive regional changes in cell-type composition remains unclear. Here, we show that intestinal nutrient adaptation involves region-specific control of cell size, cell number, and differentiation. We uncovered that activation of mTOR complex 1 (mTORC1) increases ISC size in a region-specific manner. mTORC1 activity promotes Delta expression to direct cell fate toward the absorptive enteroblast lineage while inhibiting secretory enteroendocrine cell differentiation. In aged flies, the ISC mTORC1 signaling is deregulated, being constitutively high and unresponsive to diet, which can be mitigated through lifelong intermittent fasting. In conclusion, mTORC1 signaling contributes to the ISC fate decision, enabling regional control of intestinal cell differentiation in response to nutrition.


Assuntos
Mucosa Intestinal , Intestinos , Mucosa Intestinal/metabolismo , Linhagem da Célula , Diferenciação Celular , Células-Tronco/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Nutrientes , Proliferação de Células
12.
BMC Immunol ; 25(1): 15, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38336646

RESUMO

BACKGROUND AND AIMS: We aimed to investigate the immune characteristics of intestinal CD8+ gamma delta T (CD8+ γδ T) cells in Crohn's disease (CD) and their correlation with disease activity. METHODS: The study cohorts included 21 CD patients and 21 healthy individuals. CD8+ γδ T cells were isolated from human ileal mucosa for detection by flow cytometry. The activation or inhibition status of cells was detected by detecting the expression of activation marker HLA-DR and the immunosuppressive molecule PD-1 on cells. The cytotoxicity of cells was assessed by detecting the expression of cytotoxic molecules (Perforin, Granzyme B, and TRAIL) in cells. Ratios of investigated cells were calculated as prediction factors by receiver operating characteristic curve (ROC) analysis. RESULTS: The study revealed a reduction in intestinal CD8+ γδT cells among active CD patients, with a more pronounced reduction observed in moderately active patients compared to mildly active patients. Moreover, active CD patients exhibited heightened activation levels in their intestinal CD8+ γδT cells, whereas the activation was comparatively weakened in moderately active patients compared with mildly active patients. Additionally, the cytotoxicity of intestinal CD8+ γδT cells was enhanced solely in mildly active patients, while it was impaired in moderately active patients compared with mildly active patients. Furthermore, HLA-DR+ CD8+ γδT cell ratio, CD8+ γδT ratio, and CD8+ γδT count were identified as indicators in the diagnosis of active CD. Meanwhile, the ratios of Granzyme B+ CD8+ γδT cell and Perforin+ CD8+ γδT cell were identified as indicators that distinguish mildly moderately active CD cases. CONCLUSIONS: Intestinal CD8+ γδT was reduced in active CD patients, but their activation and cytotoxicity were enhanced. However, with increased disease activity, intestinal CD8+ γδ T cells became dysfunctional. CD-specific perturbations observed in various phenotypic markers in CD8+ γδ T cells can be used as indicators to assist in diagnosing CD patients.


Assuntos
Doença de Crohn , Linfócitos Intraepiteliais , Humanos , Granzimas , Linfócitos Intraepiteliais/metabolismo , Perforina , Linfócitos T Citotóxicos , Mucosa Intestinal , Antígenos HLA-DR , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo
13.
Methods Mol Biol ; 2763: 71-78, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38347401

RESUMO

In the intestine, mucus covering the mucosa plays a critical role in maintaining gut homeostasis by protecting the mucosa from invasion by commensal bacteria. The gut mucus is composed primarily of MUC2 mucin secreted by goblet cells. MUC2 is highly O-glycosylated, and O-glycans are necessary for the function and polymer structure of MUC2. In addition, recent evidence revealed that several glycan modifications, such as sialylation and sulfation, confer resistance of mucins to proteolysis and affect the viscosity and lubricity of mucus. Therefore, characterizing glycan structures of mucins is required to understand their functions fully. In this chapter, we describe how to purify secreted mucins from the mammalian intestine for analysis of their glycan structures. This description includes the extraction of MUC2 mucin from the mucosal surface of the mouse colon and colon explants.


Assuntos
Mucosa Intestinal , Mucinas , Animais , Camundongos , Mucinas/química , Mucosa Intestinal/microbiologia , Mucina-2 , Células Caliciformes , Polissacarídeos , Mamíferos
14.
Clin Immunol ; 260: 109923, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38316201

RESUMO

Celiac Disease (CD) is a T-cell mediated disorder caused by immune response to gluten, although the mechanisms underlying CD progression are still elusive. We analyzed immune cell composition, plasma cytokines, and gliadin-specific T-cell responses in patients with positive serology and normal intestinal mucosa (potential-CD) or villous atrophy (acute-CD), and after gluten-free diet (GFD). We found: an inflammatory signature and the presence of circulating gliadin-specific IFN-γ+ T cells in CD patients regardless of mucosal damage; an increased frequency of IL-10-secreting dendritic cells (DC-10) in the gut and of circulating gliadin-specific IL-10-secreting T cells in potential-CD; IL-10 inhibition increased IFN-γ secretion by gliadin-specific intestinal T cells from acute- and potential-CD. On GFD, inflammatory cytokines normalized, while IL-10-producing T cells accumulated in the gut. We show that IL-10-producing cells are fundamental in controlling pathological T-cell responses to gluten: DC-10 protect the intestinal mucosa from damage and represent a marker of potential-CD.


Assuntos
Doença Celíaca , Humanos , Gliadina , Interleucina-10 , Glutens , Citocinas , Mucosa Intestinal
16.
Mol Med ; 30(1): 17, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38302880

RESUMO

BACKGROUND: In sepsis, intestinal barrier dysfunction is often caused by the uncontrolled death of intestinal epithelial cells (IECs). CD4CD8αα intraepithelial lymphocytes (IELs), a subtype of CD4+ T cells residing within the intestinal epithelium, exert cytotoxicity by producing granzyme B (GrB) and perforin (Prf). Extracellular cold-inducible RNA-binding protein (eCIRP) is a recently identified alarmin which stimulates TLR4 on immune cells to induce proinflammatory responses. Here, we hypothesized that eCIRP enhances CD4CD8αα IEL cytotoxicity and induces IEC death in sepsis. METHODS: We subjected wild-type (WT) and CIRP-/- mice to sepsis by cecal ligation and puncture (CLP) and collected the small intestines to isolate IELs. The expression of GrB and Prf in CD4CD8αα IELs was assessed by flow cytometry. IELs isolated from WT and TLR4-/- mice were challenged with recombinant mouse CIRP (eCIRP) and assessed the expression of GrB and Prf in CD4CD8αα by flow cytometry. Organoid-derived IECs were co-cultured with eCIRP-treated CD4CD8αα cells in the presence/absence of GrB and Prf inhibitors and assessed IEC death by flow cytometry. RESULTS: We found a significant increase in the expression of GrB and Prf in CD4CD8αα IELs of septic mice compared to sham mice. We found that GrB and Prf levels in CD4CD8αα IELs were increased in the small intestines of WT septic mice, while CD4CD8αα IELs of CIRP-/- mice did not show an increase in those cytotoxic granules after sepsis. We found that eCIRP upregulated GrB and Prf in CD4CD8αα IELs isolated from WT mice but not from TLR4-/- mice. Furthermore, we also revealed that eCIRP-treated CD4CD8αα cells induced organoid-derived IEC death, which was mitigated by GrB and Prf inhibitors. Finally, histological analysis of septic mice revealed that CIRP-/- mice were protected from tissue injury and cell death in the small intestines compared to WT mice. CONCLUSION: In sepsis, the cytotoxicity initiated by the eCIRP/TLR4 axis in CD4CD8αα IELs is associated with intestinal epithelial cell (IEC) death, which could lead to gut injury.


Assuntos
Linfócitos Intraepiteliais , Sepse , Animais , Camundongos , Mucosa Intestinal/metabolismo , Intestinos , Camundongos Endogâmicos C57BL , Sepse/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
17.
J Vis Exp ; (203)2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38314824

RESUMO

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.


Assuntos
Cálcio , Intestinos , Humanos , Cálcio/análise , Mucosa Intestinal/química , Organoides/química , Microscopia de Fluorescência/métodos
18.
Front Immunol ; 15: 1351427, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38318169

RESUMO

One of the leading causes of infectious diarrhea in newborn calves is the apicomplexan protozoan Cryptosporidium parvum (C. parvum). However, little is known about its immunopathogenesis. Using next generation sequencing, this study investigated the immune transcriptional response to C. parvum infection in neonatal calves. Neonatal male Holstein-Friesian calves were either orally infected (N = 5) or not (CTRL group, N = 5) with C. parvum oocysts (gp60 subtype IIaA15G2R1) at day 1 of life and slaughtered on day 7 after infection. Total RNA was extracted from the jejunal mucosa for short read. Differentially expressed genes (DEGs) between infected and CTRL groups were assessed using DESeq2 at a false discovery rate < 0.05. Infection did not affect plasma immunohematological parameters, including neutrophil, lymphocyte, monocyte, leucocyte, thrombocyte, and erythrocyte counts as well as hematocrit and hemoglobin concentration on day 7 post infection. The immune-related DEGs were selected according to the UniProt immune system process database and were used for gene ontology (GO) and pathway enrichment analysis using Cytoscape (v3.9.1). Based on GO analysis, DEGs annotated to mucosal immunity, recognizing and presenting antigens, chemotaxis of neutrophils, eosinophils, natural killer cells, B and T cells mediated by signaling pathways including toll like receptors, interleukins, tumor necrosis factor, T cell receptor, and NF-KB were upregulated, while markers of macrophages chemotaxis and cytosolic pattern recognition were downregulated. This study provides a holistic snapshot of immune-related pathways induced by C. parvum in calves, including novel and detailed feedback and feedforward regulatory mechanisms establishing the crosstalk between innate and adaptive immune response in neonate calves, which could be utilized further to develop new therapeutic strategies.


Assuntos
Doenças dos Bovinos , Criptosporidiose , Cryptosporidium parvum , Cryptosporidium , Fenômenos do Sistema Imunitário , Animais , Bovinos , Masculino , Humanos , Cryptosporidium parvum/genética , Cryptosporidium/genética , Transcriptoma , Doenças dos Bovinos/genética , Mucosa Intestinal , Fator de Necrose Tumoral alfa/genética , Imunidade Adaptativa
19.
J Invest Surg ; 37(1): 2308809, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38323630

RESUMO

The human intestinal epithelium has an impressive ability to respond to insults and its homeostasis is maintained by well-regulated mechanisms under various pathophysiological conditions. Nonetheless, acute injury and inhibited regeneration of the intestinal epithelium occur commonly in critically ill surgical patients, leading to the translocation of luminal toxic substances and bacteria to the bloodstream. Effective therapies for the preservation of intestinal epithelial integrity and for the prevention of mucosal hemorrhage and gut barrier dysfunction are limited, primarily because of a poor understanding of the mechanisms underlying mucosal disruption. Noncoding RNAs (ncRNAs), which include microRNAs (miRNAs), long ncRNAs (lncRNAs), circular RNAs (circRNAs), and small vault RNAs (vtRNAs), modulate a wide array of biological functions and have been identified as orchestrators of intestinal epithelial homeostasis. Here, we feature the roles of many important ncRNAs in controlling intestinal mucosal growth, barrier function, and repair after injury-particularly in the context of postoperative recovery from bowel surgery. We review recent literature surrounding the relationships between lncRNAs, microRNAs, and RNA-binding proteins and how their interactions impact cell survival, proliferation, migration, and cell-to-cell interactions in the intestinal epithelium. With advancing knowledge of ncRNA biology and growing recognition of the importance of ncRNAs in maintaining the intestinal epithelial integrity, ncRNAs provide novel therapeutic targets for treatments to preserve the gut epithelium in individuals suffering from critical surgical disorders.


Assuntos
MicroRNAs , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , MicroRNAs/genética , Mucosa Intestinal
20.
Crit Rev Eukaryot Gene Expr ; 34(3): 83-99, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38305291

RESUMO

In Crohn's disease (CD), gut dysbiosis is marked by the prevalence of pathogenic bacterial species. Although several microbes have been reported as risk factors or causative agents of CD, it is not yet clear which is the real trigger of the disease. Thirty years ago, a new pathovar of Escherichia coli strain was isolated in the ileal mucosa of CD patients. This strain, called adherent invasive E. coli (AIEC), for its ability to invade the intestinal mucosa, could represent the causative agent of the disease. Several authors studied the mechanisms by which the AIEC penetrate and replicate within macrophages, and release inflammatory cytokines sustaining inflammation. In this review we will discuss about the role of AIEC in the pathogenesis of CD, the virulence factors mediating adhesion and invasion of AIEC in mucosal tissue, the environmental conditions improving AIEC survival and replication within macrophages. Finally, we will also give an overview of the new strategies developed to limit AIEC overgrowth.


Assuntos
Doença de Crohn , Infecções por Escherichia coli , Humanos , Doença de Crohn/epidemiologia , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Escherichia coli , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Aderência Bacteriana , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia
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