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Feng S, Zhang C, Chen S, He R, Chao G, Zhang S. TLR5 Signaling in the Regulation of Intestinal Mucosal Immunity. J Inflamm Res 2023; 16:2491-2501. [PMID: 37337514 PMCID: PMC10276996 DOI: 10.2147/jir.s407521] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2023] [Accepted: 05/23/2023] [Indexed: 06/21/2023] Open
Abstract
Toll-like receptor 5 (TLR5) is a pattern recognition receptor that specifically recognizes flagellin and consequently plays a crucial role in the control of intestinal homeostasis by activating innate and adaptive immune responses. TLR5 overexpression, on the other hand, might disrupt the intestinal mucosal barrier, which serves as the first line of defense against harmful microbes. The intestine symbiotic bacteria, mucous layer, intestinal epithelial cells (IECs), adherens junctions (such as tight junctions and peripheral membrane proteins), the intestinal mucosal immune system, and cytokines make up the intestinal mucosal barrier. Impaired barrier function has been linked to intestinal illnesses such as inflammatory bowel disease (IBD). IBD is a persistent non-specific inflammatory illness of the digestive system with an unknown cause. It is now thought to be linked to infection, environment, genes, immune system, and the gut microbiota. The significance of immunological dysfunction in IBD has received more attention in recent years. The purpose of this paper is to explore TLR5's position in the intestinal mucosal barrier and its relevance to IBD.
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Affiliation(s)
- Shuyan Feng
- Zhejiang Chinese Medical University, Hangzhou, 310053, People’s Republic of China
| | - Chi Zhang
- Zhejiang Chinese Medical University, Hangzhou, 310053, People’s Republic of China
| | - Shanshan Chen
- The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, 310006, People’s Republic of China
| | - Ruonan He
- Zhejiang Chinese Medical University, Hangzhou, 310053, People’s Republic of China
| | - Guanqun Chao
- Sir Run Run Shaw Hospital of Zhejiang University, Hangzhou, 310018, People’s Republic of China
| | - Shuo Zhang
- The Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, 310005, People’s Republic of China
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Chen X, Elson CO, Dunkin D. Epicutaneous Immunotherapy with CBir1 Alleviates Intestinal Inflammation. Inflamm Bowel Dis 2023; 29:798-807. [PMID: 36651798 PMCID: PMC10152294 DOI: 10.1093/ibd/izac261] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Indexed: 01/19/2023]
Abstract
BACKGROUND Inflammatory bowel disease may be due to failed tolerance to normal gut bacteria. We demonstrate that epicutaneous immunotherapy (ET) to ovalbumin can alleviate colitis in murine models. However, most people are tolerant to or have anergy to ovalbumin. Half of Crohn's disease (CD) patients have CBir1 antibodies that can be elevated years before CD development. We determined whether ET with a CBir1 multi-epitope peptide (MEP1) could alleviate colitis. METHODS Wild type mice (C57BL/6) were transferred with CBir1 T cell receptor (TCR) T cells followed by epicutaneous application of MEP1. Proliferating Foxp3+ T cells were measured in mesenteric lymph nodes (LNs), spleen, small intestine, and colon by flow cytometry. Lymphocytes from MEP1 epicutaneously exposed and immunized C57BL/6 mice were cultured with MEP1. Interferon (IFN)-γ production was measured. Colitis was induced by transferring CD4+CD45Rbhi T cells from CBIR1 TCR or C57BL/6 mice into RAG1-/- mice. Mice were treated with ET. Body weight, colon length, colonic cytokine production, histological inflammation, inflammatory genes, and regulatory T cells (Tregs) from lamina propria were measured. RESULTS ET with 10 μg of MEP1 induced CBir1-specific Tregs that migrated to the small intestine and colon and suppressed MEP1-specific IFN-γ production. ET alleviated colitis when the model utilized CBir1 TCR T cells in mice colonized with CBir1 or A4Fla2 positive bacteria. Treated mice had improved colon length and histological inflammation and reduced colonic IFN-γ production. CONCLUSION Epicutaneous immunotherapy with MEP1 induced Tregs that migrate to intestines and suppress inflammation in mice with CBir1 or A4Fla2-positive bacterial colonization. This could be a potential strategy to treat CD and warrants further study.
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Affiliation(s)
- Xin Chen
- Division of Pediatric Gastroenterology and the Mindich Child Health and Development Institute (MCHDI), The Icahn School of Medicine at Mount Sinai (ISMMS), New York, NY, USA
| | - Charles O Elson
- Department of Medicine, Division of Gastroenterology and Hepatology, Heersink School of Medicine, The University of Alabama at Birmingham, Birmingham, AL, USA
| | - David Dunkin
- Division of Pediatric Gastroenterology and the Mindich Child Health and Development Institute (MCHDI), The Icahn School of Medicine at Mount Sinai (ISMMS), New York, NY, USA
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3
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Vent-Schmidt J, Goldsmith LJ, Steiner TS. Patients' Willingness and Perspectives Toward Chimeric Antigen Receptor T-Regulatory Cell Therapy for Inflammatory Bowel Diseases. CROHN'S & COLITIS 360 2020; 2:otaa085. [PMID: 36777762 PMCID: PMC9802168 DOI: 10.1093/crocol/otaa085] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/22/2020] [Indexed: 11/14/2022] Open
Abstract
Background Inflammatory bowel disease is a life-changing disease resulting from recurrent intestinal inflammation. Current therapies (eg, steroids and biologics) are associated with mild to severe side effects, and none provide a cure. Recent research has focused on genetically engineering gut-specific anti-inflammatory T-regulatory cells (CAR-Tregs) to control intestinal inflammation, a logistically and conceptually complex approach. The purpose of our study was to understand patients' willingness to try CAR-Treg given 2 hypothetical scenarios-in a clinical trial or as a new treatment. Methods We surveyed people living with inflammatory bowel disease about their willingness to try CAR-Treg. The online survey was developed using patient focus groups and associated literature. We recruited participants through email and social media. We used descriptive and inferential statistics to analyze closed-ended questions and inductive thematic analysis to analyze open-ended follow-up questions. Results Survey participants indicated high willingness to try CAR-Treg therapy in both a clinical trial and as a new treatment. Willingness to try was not correlated with disease state or medication history. Women were less likely than men to indicate willingness to participate in a clinical trial. Participants' reasons for being willing to try CAR-Treg therapy included the wish to change their current treatment and the calling to participate in research. Participants that were not willing to try CAR-Treg mentioned the lack of long-term data and the success of their current therapy. Conclusions This is the first study to our knowledge to investigate patient willingness to try CAR-Treg therapy. Our results demonstrate the promise of moving this therapy into clinical practice as most patients indicated willingness to try.
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Affiliation(s)
- Jens Vent-Schmidt
- Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Laurie J Goldsmith
- GoldQual Consulting, Richmond Hill, Ontario, Canada,Faculty of Health Sciences, Simon Fraser University, Burnaby, British Columbia, Canada
| | - Theodore S Steiner
- Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada,BC Children’s Hospital Research Institute, Vancouver, British Columbia, Canada,Address correspondence to: Theodore S. Steiner, Dipl-MolMed, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada ()
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Yoo JH, Donowitz M. Intestinal enteroids/organoids: A novel platform for drug discovery in inflammatory bowel diseases. World J Gastroenterol 2019; 25:4125-4147. [PMID: 31435168 PMCID: PMC6700704 DOI: 10.3748/wjg.v25.i30.4125] [Citation(s) in RCA: 52] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Revised: 06/14/2019] [Accepted: 07/19/2019] [Indexed: 02/06/2023] Open
Abstract
The introduction of biologics such as anti-tumor necrosis factor (TNF) monoclonal antibodies followed by anti-integrins has dramatically changed the therapeutic paradigm of inflammatory bowel diseases (IBD). Furthermore, a newly developed anti-p40 subunit of interleukin (IL)-12 and IL-23 (ustekinumab) has been recently approved in the United States for patients with moderate to severe Crohn’s disease who have failed treatment with anti-TNFs. However, these immunosuppressive therapeutics which focus on anti-inflammatory mechanisms or immune cells still fail to achieve long-term remission in a significant percentage of patients. This strongly underlines the need to identify novel treatment targets beyond immune suppression to treat IBD. Recent studies have revealed the critical role of intestinal epithelial cells (IECs) in the pathogenesis of IBD. Physical, biochemical and immunologic driven barrier dysfunctions of epithelial cells contribute to the development of IBD. In addition, the recent establishment of adult stem cell-derived intestinal enteroid/organoid culture technology has allowed an exciting opportunity to study human IECs comprising all normal epithelial cells. This long-term epithelial culture model can be generated from endoscopic biopsies or surgical resections and recapitulates the tissue of origin, representing a promising platform for novel drug discovery in IBD. This review describes the advantages of intestinal enteroids/organoids as a research tool for intestinal diseases, introduces studies with these models in IBD, and gives a description of the current status of therapeutic approaches in IBD. Finally, we provide an overview of the current endeavors to identify a novel drug target for IBD therapy based on studies with human enteroids/organoids and describe the challenges in using enteroids/organoids as an IBD model.
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Affiliation(s)
- Jun-Hwan Yoo
- Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States
- Digestive Disease Center, CHA Bundang Medical Center, CHA University, Seongnam 13496, South Korea
| | - Mark Donowitz
- Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States
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Ivičak-Kocjan K, Forstnerič V, Panter G, Jerala R, Benčina M. Extension and refinement of the recognition motif for Toll-like receptor 5 activation by flagellin. J Leukoc Biol 2018; 104:767-776. [PMID: 29920759 DOI: 10.1002/jlb.3vma0118-035r] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2018] [Revised: 04/24/2018] [Accepted: 05/16/2018] [Indexed: 11/07/2022] Open
Abstract
TLRs sense conserved and essential molecular components of microbes that invade multicellular organisms. The wide range of TLR agonists, differing in size and shape, is recognized either through a single or a pair of binding sites on the ectodomains of TLRs. TLR5 recognizes bacterial flagellin through two distinct binding sites on the ectodomain, the first facilitating primary binding of flagellin and the second guiding receptor dimerization necessary for signaling. The regions of flagellin recognized by TLR5 encompass key functional regions within the D1 domain of flagellin, which is also required for the assembly of functional flagella. In addition to previously identified binding sites at the N-terminal and central segment of the TLR5 ectodomain, we extended the TLR5'-D1 interaction interface on TLR5 and showed a species-specific recognition relevance of this extended region. In addition, we showed that the loop and following β-hairpin region of flagellin, previously proposed to participate in the TLR5-flagellin dimerization interface, is not accountable for these species-specific differences. We further identified residues that contribute to the interaction between two TLR5 ectodomains in an active signaling complex. Our work demonstrates that flagellin is recognized by TLR5 through a more extensive interaction surface than previously characterized.
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Affiliation(s)
- Karolina Ivičak-Kocjan
- Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia
| | - Vida Forstnerič
- Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia
| | - Gabriela Panter
- Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia
| | - Roman Jerala
- Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia
- Centre of Excellence EN-FIST, Ljubljana, Slovenia
| | - Mojca Benčina
- Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia
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Xiong X, Bales ES, Ir D, Robertson CE, McManaman JL, Frank DN, Parkinson J. Perilipin-2 modulates dietary fat-induced microbial global gene expression profiles in the mouse intestine. MICROBIOME 2017; 5:117. [PMID: 28877764 PMCID: PMC5588750 DOI: 10.1186/s40168-017-0327-x] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/22/2017] [Accepted: 08/20/2017] [Indexed: 05/06/2023]
Abstract
BACKGROUND Intestinal microbiota are critical determinants of obesity and metabolic disease risk. In previous work, we showed that deletion of the cytoplasmic lipid droplet (CLD) protein perilipin-2 (Plin2) modulates gut microbial community structure and abrogates long-term deleterious effects of a high-fat (HF) diet in mice. However, the impact of Plin2 on microbiome function is unknown. RESULTS Here, we used metatranscriptomics to identify differences in microbiome transcript expression in WT and Plin2-null mice following acute exposure to high-fat/low-carbohydrate (HF) or low-fat/high-carbohydrate (LF) diets. Consistent with previous studies, dietary changes resulted in significant taxonomic shifts. Unexpectedly, when fed a HF diet, the microbiota of Plin2-null and WT mice exhibited dramatic shifts in transcript expression despite no discernible shift in community structure. For Plin2-null mice, these changes included the coordinated upregulation of metabolic enzymes directing flux towards the production of growth metabolites such as fatty acids, nucleotides, and amino acids. In contrast, the LF diet did not appear to induce the same dramatic changes in transcript or pathway expression between the two genotypes. CONCLUSIONS Our data shows that a host genotype can modulate microbiome function without impacting community structure and identify Plin2 as a specific host determinant of diet effects on microbial function. Along with uncovering potential mechanisms for integrating how diet modulates host and microbial metabolism, our findings demonstrate the limits of 16S rRNA surveys to inform on community functional activities and the need to prioritize metatranscriptomic studies to gain more meaningful insights into microbiome function.
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Affiliation(s)
- Xuejian Xiong
- Molecular Medicine, Hospital for Sick Children, 686 Bay Street, Toronto, M5G 0A4 ON Canada
| | - Elise S. Bales
- Division of Reproductive Sciences, University of Colorado, 12700 E. 19th Avenue, Aurora, 80045 CO USA
| | - Diana Ir
- Division of Infectious Diseases, University of Colorado, 12700 E. 19th Avenue, Aurora, 80045 CO USA
| | - Charles E. Robertson
- Division of Infectious Diseases, University of Colorado, 12700 E. 19th Avenue, Aurora, 80045 CO USA
- Microbiome Research Consortium, University of Colorado, 12700 E. 19th Avenue, Aurora, 80045 CO USA
| | - James L. McManaman
- Division of Reproductive Sciences, University of Colorado, 12700 E. 19th Avenue, Aurora, 80045 CO USA
- The Center for Human Nutrition, University of Colorado, 12700 E. 19th Avenue, Aurora, 80045 CO USA
| | - Daniel N. Frank
- Division of Infectious Diseases, University of Colorado, 12700 E. 19th Avenue, Aurora, 80045 CO USA
- Microbiome Research Consortium, University of Colorado, 12700 E. 19th Avenue, Aurora, 80045 CO USA
| | - John Parkinson
- Molecular Medicine, Hospital for Sick Children, 686 Bay Street, Toronto, M5G 0A4 ON Canada
- Department of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, M5S 1A8 ON Canada
- Department of Biochemistry, University of Toronto, 1 King’s College Circle, Toronto, M5S 1A8 ON Canada
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Biotechnology approaches to produce potent, self-adjuvanting antigen-adjuvant fusion protein subunit vaccines. Biotechnol Adv 2017; 35:375-389. [PMID: 28288861 DOI: 10.1016/j.biotechadv.2017.03.005] [Citation(s) in RCA: 70] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2017] [Revised: 03/08/2017] [Accepted: 03/08/2017] [Indexed: 01/07/2023]
Abstract
Traditional vaccination approaches (e.g. live attenuated or killed microorganisms) are among the most effective means to prevent the spread of infectious diseases. These approaches, nevertheless, have failed to yield successful vaccines against many important pathogens. To overcome this problem, methods have been developed to identify microbial components, against which protective immune responses can be elicited. Subunit antigens identified by these approaches enable the production of defined vaccines, with improved safety profiles. However, they are generally poorly immunogenic, necessitating their administration with potent immunostimulatory adjuvants. Since few safe and effective adjuvants are currently used in vaccines approved for human use, with those available displaying poor potency, or an inability to stimulate the types of immune responses required for vaccines against specific diseases (e.g. cytotoxic lymphocytes (CTLs) to treat cancers), the development of new vaccines will be aided by the availability of characterized platforms of new adjuvants, improving our capacity to rationally select adjuvants for different applications. One such approach, involves the addition of microbial components (pathogen-associated molecular patterns; PAMPs), that can stimulate strong immune responses, into subunit vaccine formulations. The conjugation of PAMPs to subunit antigens provides a means to greatly increase vaccine potency, by targeting immunostimulation and antigen to the same antigen presenting cell. Thus, methods that enable the efficient, and inexpensive production of antigen-adjuvant fusions represent an exciting mean to improve immunity towards subunit antigens. Herein we review four protein-based adjuvants (flagellin, bacterial lipoproteins, the extra domain A of fibronectin (EDA), and heat shock proteins (Hsps)), which can be genetically fused to antigens to enable recombinant production of antigen-adjuvant fusion proteins, with a focus on their mechanisms of action, structural or sequence requirements for activity, sequence modifications to enhance their activity or simplify production, adverse effects, and examples of vaccines in preclinical or human clinical trials.
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8
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Li W, Ge C, Yang L, Wang R, Lu Y, Gao Y, Li Z, Wu Y, Zheng X, Wang Z, Zhang C. CBLB502, an agonist of Toll-like receptor 5, has antioxidant and scavenging free radicals activities in vitro. Int J Biol Macromol 2016; 82:97-103. [DOI: 10.1016/j.ijbiomac.2015.10.033] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2015] [Accepted: 10/11/2015] [Indexed: 10/22/2022]
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9
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Dlugosz A, Nowak P, D'Amato M, Mohammadian Kermani G, Nyström J, Abdurahman S, Lindberg G. Increased serum levels of lipopolysaccharide and antiflagellin antibodies in patients with diarrhea-predominant irritable bowel syndrome. Neurogastroenterol Motil 2015; 27:1747-54. [PMID: 26387872 DOI: 10.1111/nmo.12670] [Citation(s) in RCA: 75] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/01/2015] [Accepted: 08/11/2015] [Indexed: 12/11/2022]
Abstract
BACKGROUND Innate immune responses to conserved microbial products such as lipopolysaccharide (LPS) and flagellin are likely important in microbial-host interactions and intestinal homeostasis. We hypothesized that bacterial translocation and activation of mucosal immunity against common microbial antigens might be involved in the development of irritable bowel syndrome (IBS). We therefore compared serum levels of LPS, soluble CD14 (sCD14), and flagellin antibodies between patients with different subtypes of IBS and healthy controls. METHODS We analyzed serum obtained from 88 patients (74 females) aged 19(43)-73 years and 106 healthy volunteers (77 females) aged 19(38)-62 years. Diarrhea-predominant IBS (D-IBS) was present in 32 patients (36%), 23 patients (26%) had constipation-predominant IBS (C-IBS), and 33 patients (38%) had A-IBS. We used ELISA for sCD14 and antiflagellin immunoglobulin G and limulus amebocyte assay for LPS. Abdominal symptoms and psychiatric comorbidities were assessed using validated questionnaires. KEY RESULTS We found a significantly higher serum level of LPS in patients with D-IBS compared to controls (p = 0.0155). The level of antibodies to flagellin was higher in patients with IBS than in controls (mainly driven by higher levels in D-IBS, p = 0.0018). The levels of sCD14 were lower in D-IBS patients compared to controls (p = 0.0498). We found a weak, but significant correlation between the levels of antiflagellin antibodies and anxiety among IBS patients (ρ = 0.38; p = 0.0045). CONCLUSIONS & INFERENCES Our results support the concept that immune reactivity to luminal antigens may have a role in the development of D-IBS. The serum level of antiflagellin antibodies was found to correlate with patients' self-reported anxiety score.
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Affiliation(s)
- A Dlugosz
- Karolinska Institutet, Department of Medicine and Center for Digestive Diseases, Karolinska University Hospital, Huddinge, Stockholm, Sweden
| | - P Nowak
- Karolinska Institutet, Department of Medicine, Unit of Infectious Diseases, Karolinska University Hospital, Huddinge, Stockholm, Sweden
| | - M D'Amato
- Karolinska Institutet, Department of Biosciences and Nutrition, Karolinska University Hospital, Huddinge, Stockholm, Sweden
| | - G Mohammadian Kermani
- Karolinska Institutet, Department of Medicine and Center for Digestive Diseases, Karolinska University Hospital, Huddinge, Stockholm, Sweden
| | - J Nyström
- Karolinska Institutet, Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska University Hospital, Huddinge, Stockholm, Sweden
| | - S Abdurahman
- Department of Science and Technology, Örebro Life Science Center, Örebro University, Örebro, Sweden
| | - G Lindberg
- Karolinska Institutet, Department of Medicine and Center for Digestive Diseases, Karolinska University Hospital, Huddinge, Stockholm, Sweden
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10
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Abstract
: The human intestinal microbiome plays a critical role in human health and disease, including the pathogenesis of inflammatory bowel disease (IBD). Numerous studies have identified altered bacterial diversity and abundance at varying taxonomic levels through biopsies and fecal samples of patients with IBD and diseased model animals. However, inconsistent observations regarding the microbial compositions of such patients have hindered the efforts in assessing the etiological role of specific bacterial species in the pathophysiology of IBD. These observations highlight the importance of minimizing the confounding factors associated with IBD and the need for a standardized methodology to analyze well-defined microbial sampling sources in early IBD diagnosis. Furthermore, establishing the linkage between microbiota compositions with their function within the host system can provide new insights on the pathogenesis of IBD. Such research has been greatly facilitated by technological advances that include functional metagenomics coupled with proteomic and metabolomic profiling. This review provides updates on the composition of the microbiome in IBD and emphasizes microbiota dysbiosis-involved mechanisms. We highlight functional roles of specific bacterial groups in the development and management of IBD. Functional analyses of the microbiome may be the key to understanding the role of microbiota in the development and chronicity of IBD and reveal new strategies for therapeutic intervention.
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11
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Atif SM, Lee SJ, Li LX, Uematsu S, Akira S, Gorjestani S, Lin X, Schweighoffer E, Tybulewicz VLJ, McSorley SJ. Rapid CD4+ T-cell responses to bacterial flagellin require dendritic cell expression of Syk and CARD9. Eur J Immunol 2014; 45:513-24. [PMID: 25430631 PMCID: PMC4324162 DOI: 10.1002/eji.201444744] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2014] [Revised: 10/13/2014] [Accepted: 10/31/2014] [Indexed: 01/07/2023]
Abstract
Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR-signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin-specific CD4(+) T-cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin-specific T-cell responses.
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Affiliation(s)
- Shaikh M Atif
- Center for Comparative Medicine, Department of Anatomy, Physiology, and Cell Biology, University of California Davis, Davis, CA, USA
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Wick EC, Rabizadeh S, Albesiano E, Wu X, Wu S, Chan J, Rhee KJ, Ortega G, Huso DL, Pardoll D, Housseau F, Sears CL. Stat3 activation in murine colitis induced by enterotoxigenic Bacteroides fragilis. Inflamm Bowel Dis 2014; 20:821-34. [PMID: 24704822 PMCID: PMC4121853 DOI: 10.1097/mib.0000000000000019] [Citation(s) in RCA: 86] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND Enterotoxigenic Bacteroides fragilis (ETBF), a molecular subclass of the common human commensal, B. fragilis, has been associated with inflammatory bowel disease. ETBF colitis is characterized by the activation of Stat3 and a Th17 immune response in the colonic mucosa. This study was designed to investigate the time course and cellular distribution of Stat3 activation in ETBF-colonized mice. METHODS C57BL/6 wild-type, C57BL/6, or Rag-1 mice were inoculated with saline, nontoxigenic B. fragilis or ETBF. Histologic diagnosis and mucosal Stat activation (immunohistochemistry, Western blot, and/or electrophorectic mobility shift assay) were evaluated over time (6-24 h, 1-7 d, and 1-18 mo after inoculation). Mucosal permeability was evaluated at 16 hours, 1 day, and 3 days. Mucosal immune responses were evaluated at 1 week, and 12 and 18 months. RESULTS ETBF induced rapid-onset colitis that persisted for up to 1 year. Stat3 activation (pStat3) was noted in the mucosal immune cells within 16 hours, with colonic epithelial cell activation evident at 24 hours after inoculation. ETBF-induced increased mucosal permeability was first observed at 24 hours after inoculation, after which the initial immune cell pStat3 activation was noted. Immune cell pStat3 was present in the absence of epithelial pStat3 (C57BL/6). Epithelial pStat3 was present in the absence of T and B cells (Rag-1 mice). pStat3 persisted in the epithelial and immune cells for 1 year, characterized by isolated pStat3-positive cell clusters, with varying intensity distributed through the proximal and distal colon. Similarly, mucosal Th17 immune responses persisted for up to 1 year. Loss of fecal ETBF colonization was associated with the loss of mucosal pStat3 and Th17 immune responses. CONCLUSIONS ETBF rapidly induces immune cell pStat3, which is independent of epithelial pStat3. This occurs before ETBF-induced mucosal permeability, suggesting that ETBF, likely through B. fragilis toxin and its action on the colonic epithelial cell, triggers mucosal immune cell Stat3 activation. Peak mucosal Stat3 activation (immune and epithelial cells) occurs subsequently when other colonic bacteria may contribute to the ETBF-initiated immune response due to barrier dysfunction. ETBF induces long-lived, focal colonic Stat3 activation and Th17 immune responses dependent on the ongoing ETBF colonization. Further study is needed to evaluate the early mucosal signaling events, resulting in epithelial Stat3 activation and the sequelae of long-term colonic Stat3 activation.
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Affiliation(s)
- Elizabeth C. Wick
- Department of Surgery, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
- Department of Oncology, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
| | - Shervin Rabizadeh
- Department of Pediatrics, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
- Department of Pediatrics, Cedars-Sinai Medical Center, Los Angeles, California
| | - Emilia Albesiano
- Department of Oncology, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
| | - XinQun Wu
- Department of Medicine, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
| | - Shaoguang Wu
- Department of Medicine, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
| | - June Chan
- Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland
| | - Ki-Jong Rhee
- Department of Medicine, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
- Department of Biomedical Laboratory Science, Yonsei University, Wonju, Republic of Korea
| | - Guillermo Ortega
- Department of Medicine, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
| | - David L. Huso
- Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
| | - Drew Pardoll
- Department of Oncology, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
- Department of Medicine, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
| | - Franck Housseau
- Department of Oncology, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
| | - Cynthia L. Sears
- Department of Oncology, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
- Department of Medicine, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland
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Trained immunity or tolerance: opposing functional programs induced in human monocytes after engagement of various pattern recognition receptors. CLINICAL AND VACCINE IMMUNOLOGY : CVI 2014; 21:534-45. [PMID: 24521784 PMCID: PMC3993125 DOI: 10.1128/cvi.00688-13] [Citation(s) in RCA: 242] [Impact Index Per Article: 22.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Upon priming with Candida albicans or with the fungal cell wall component β-glucan, monocytes respond with an increased cytokine production upon restimulation, a phenomenon termed "trained immunity." In contrast, the prestimulation of monocytes with lipopolysaccharide has long been known to induce tolerance. Because the vast majority of commensal microorganisms belong to bacterial or viral phyla, we sought to systematically investigate the functional reprogramming of monocytes induced by the stimulation of pattern recognition receptors (PRRs) with various bacterial or viral ligands. Monocytes were functionally programmed for either enhanced (training) or decreased (tolerance) cytokine production, depending on the type and concentration of ligand they encountered. The functional reprogramming of monocytes was also associated with cell shape, granulocity, and cell surface marker modifications. The training effect required p38- and Jun N-terminal protein kinase (JNK)-mediated mitogen-activated protein kinase (MAPK) signaling, with specific signaling patterns directing the functional fate of the cell. The long-term effects on the function of monocytes were mediated by epigenetic events, with both histone methylation and acetylation inhibitors blocking the training effects. In conclusion, our experiments identify the ability of monocytes to acquire adaptive characteristics after prior activation with a wide variety of ligands. Trained immunity and tolerance are two distinct and opposing functional programs induced by the specific microbial ligands engaging the monocytes.
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Vinjé S, Stroes E, Nieuwdorp M, Hazen SL. The gut microbiome as novel cardio-metabolic target: the time has come! Eur Heart J 2013; 35:883-7. [PMID: 24216389 DOI: 10.1093/eurheartj/eht467] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Recent studies reveal a potential contribution of intestinal microbes in the expression of certain human cardio-metabolic diseases. The mechanisms through which intestinal microbiota and/or their metabolic products alter systemic homoeostasis and cardio-metabolic disease risks are just beginning to be dissected. Intervention studies in humans aiming to either selectively alter the composition of the intestinal microbiota or to pharmacologically manipulate the microbiota to influence production of their metabolites are crucial next steps. The intestinal microbiome represents a new potential therapeutic target for the treatment of cardio-metabolic diseases.
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Affiliation(s)
- Sarah Vinjé
- Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, AMC-UvA, Meibergdreef 9, room F4-159.2, 1105 AZ Amsterdam, The Netherlands
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15
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Knaapen M, Kootte R, Zoetendal E, de Vos W, Dallinga-Thie G, Levi M, Stroes E, Nieuwdorp M. Obesity, non-alcoholic fatty liver disease, and atherothrombosis: a role for the intestinal microbiota? Clin Microbiol Infect 2013; 19:331-7. [DOI: 10.1111/1469-0691.12170] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2012] [Revised: 01/14/2013] [Accepted: 01/17/2013] [Indexed: 12/13/2022]
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16
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Impact of HMGB1/TLR Ligand Complexes on HIV-1 Replication: Possible Role for Flagellin during HIV-1 Infection. Int J Microbiol 2012; 2012:263836. [PMID: 22719767 PMCID: PMC3375154 DOI: 10.1155/2012/263836] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2012] [Accepted: 04/16/2012] [Indexed: 12/03/2022] Open
Abstract
Objective. We hypothesized that HMGB1 in complex with bacterial components, such as flagellin, CpG-ODN, and LPS, promotes HIV-1 replication. Furthermore, we studied the levels of antiflagellin antibodies during HIV-1-infection. Methods. Chronically HIV-1-infected U1 cells were stimulated with necrotic extract/recombinant HMGB1 in complex with TLR ligands or alone. HIV-1 replication was estimated by p24 antigen in culture supernatants 48–72 hours after stimulation. The presence of systemic anti-flagellin IgG was determined in 51 HIV-1-infected patients and 19 controls by immunoblotting or in-house ELISA. Results. Flagellin, LPS, and CpG-ODN induced stronger HIV-1 replication when incubated together with necrotic extract or recombinant HMGB1 than activation by any of the compounds alone. Moreover, the stimulatory effect of necrotic extract was inhibited by depletion of HMGB1. Elevated levels of anti-flagellin antibodies were present in plasma from HIV-1-infected patients and significantly decreased during 2 years of antiretroviral therapy. Conclusions. Our findings implicate a possible role of HGMB1-bacterial complexes, as a consequence of microbial translocation and cell necrosis, for immune activation in HIV-1 pathogenesis. We propose that flagellin is an important microbial product, that modulates viral replication and induces adaptive immune responses in vivo.
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Liu Z, Chen X, Wang X, Chen X, Song CH, Du Y, Su J, Yaseen SA, Yang PC. Chemokine CXCL11 links microbial stimuli to intestinal inflammation. Clin Exp Immunol 2011; 164:396-406. [PMID: 21438871 DOI: 10.1111/j.1365-2249.2011.04382.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Interleukin (IL)-17 plays an important role in the pathogenesis in a number of immune inflammatory disorders. This study aims to investigate the mechanism by which microbial product flagellin is involved in the development of T helper type (Th)17 cells. Serum levels of IL-17 and CXCL9-11 in patients with ulcerative colitis (UC) were evaluated. The source and mechanism of CXC11 release in intestinal mucosa were examined with colonic biopsies from UC patients and a colitis mouse model. The role of flagellin in the development of Th17 cells was studied with a cell co-culture system. High serum levels of CXCL11 and IL-17 were observed in UC. Flagellin could induce the production of CXCL11 in CD14(+) cells that facilitated the development of Th17 cells. In a skewed Th1 response environment flagellin induces intestinal inflammation, with IL-17 expression predominant. CXCR3/CXCL11 pathway is involved in microbial product flagellin-induced intestinal inflammation in which the Th17 response plays an important role.
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Affiliation(s)
- Z Liu
- Department of Gastroenterology, Tenth People's Hospital of Shanghai, Tongji University, Shanghai, China
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Sánchez B, López P, González-Rodríguez I, Suárez A, Margolles A, Urdaci MC. A flagellin-producing Lactococcus strain: interactions with mucin and enteropathogens. FEMS Microbiol Lett 2011; 318:101-7. [PMID: 21323981 DOI: 10.1111/j.1574-6968.2011.02244.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Bacillus cereus CH is a probiotic strain used in human nutrition whose adhesion to mucin is dependent on its surface-associated flagellin. Flagellins from the surface of several probiotic Bacillus strains were efficiently extracted with 5 M LiCl and identified by peptide fingerprinting. Based on the proteomic analysis, cloning of the gene coding for the flagellin of B. cereus CH was performed in the lactococcal vector pNZ8110 under the control of a nisin-inducible promoter. The resulting strain, Lactococcus lactis CH, produced a surface-associated flagellin after 6 h of induction with nisin. The recombinant Lactococcus strain adhered strongly to mucin-coated polystyrene plates, whilst inhibiting competitively the adhesion of the pathogens Escherichia coli LMG2092 and Salmonella enterica ssp. enterica LMG15860 to the same molecule. Strain CH could be used in further experimentation for the characterization of the molecular mechanism of action of this probiotic B. cereus CH flagellin.
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Affiliation(s)
- Borja Sánchez
- Departamento de Microbiología y Bioquímica de Productos Lácteos, Consejo Superior de Investigaciones Científicas (IPLA-CSIC), Villaviciosa, Asturias, Spain.
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Titanji K, Velu V, Chennareddi L, Vijay-Kumar M, Gewirtz AT, Freeman GJ, Amara RR. Acute depletion of activated memory B cells involves the PD-1 pathway in rapidly progressing SIV-infected macaques. J Clin Invest 2010; 120:3878-90. [PMID: 20972331 DOI: 10.1172/jci43271] [Citation(s) in RCA: 106] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2010] [Accepted: 08/04/2010] [Indexed: 11/17/2022] Open
Abstract
Rapid progression to AIDS is a significant problem, especially in developing countries, where the majority of HIV-infected individuals reside. As rapid disease progression is also frequently observed in SIV-infected macaques, they represent a valuable tool to investigate the pathogenesis of this condition in humans. Here, we have shown that pathogenic SIV infection in rhesus macaques resulted in a rapid depletion (as early as week 2) of activated memory B (CD21-CD27+; mBAct) cells that was strongly associated with rapid disease progression. This depletion was progressive and sustained in rapid progressors, but less severe and transient in typical progressors. Because of the rapid and sustained depletion of mBAct cells, rapid progressors failed to develop SIV-specific Ab responses, showed a decline in non-SIV-specific Ab titers, and succumbed faster to intestinal bacterial infections. Depletion of mBAct cells was strongly associated with preferential depletion of mBAct cells expressing programmed death-1 (PD-1), and in vitro blockade of PD-1 improved their survival. Furthermore, in vivo PD-1 blockade in SIV-infected macaques enhanced Ab responses to non-SIV as well as SIV Ags. Our results identify depletion of mBAct cells as a very early predictor of rapid disease progression in pathogenic SIV infection and suggest an important role for the PD-1 pathway in depletion of mBAct cells and impaired humoral immune responses in SIV-infected macaques.
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Affiliation(s)
- Kehmia Titanji
- Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia, USA
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Ivison SM, Himmel ME, Hardenberg G, Wark PAJ, Kifayet A, Levings MK, Steiner TS. TLR5 is not required for flagellin-mediated exacerbation of DSS colitis. Inflamm Bowel Dis 2010; 16:401-9. [PMID: 19774646 DOI: 10.1002/ibd.21097] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND The two forms of human inflammatory bowel disease, Crohn's disease (CD) and ulcerative colitis (UC), are both associated with loss of tolerance to gut microbial antigens. The dominant antigen recognized by antibody and T-cell responses in patients with CD is bacterial flagellin. Flagellin is also the only known ligand for Toll-like receptor 5 (TLR5), a key protein in innate immunity. Although flagellin activates TLR5 to produce inflammatory responses in many cell types in the gut, there is conflicting evidence as to whether TLR5 is harmful or protective in CD and murine colitis models. A recent study found that administration of flagellin enemas to mice along with dextran sodium sulfate (DSS) made their colitis worse. METHODS We sought to determine whether this exacerbation was due to TLR5 ligation, or to TLR5-independent adaptive immune responses to flagellin as an antigen, by using a transposon insertional mutant of the Escherichia coli H18 flagellin, 2H3, which lacks TLR5 stimulatory activity. RESULTS We found that flagellin enemas produced only a mild exacerbation of DSS colitis, and that 2H3 was equivalent to or worse than wildtype flagellin. Moreover, we found that DSS colitis was more severe in TLR5(-/-) mice than wildtype C57BL/6 mice. CONCLUSIONS Together, these results suggest that flagellin-mediated exacerbation of colitis is independent of TLR5.
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Affiliation(s)
- Sabine M Ivison
- Division of Infectious Diseases, Department of Medicine, University of British Columbia, Vancouver, BC, Canada
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