TLR2/TLR4 activation induces Tregs and suppresses intestinal inflammation caused by Fusobacterium nucleatum in vivo

PLOS ONE, Nov 2019

Toll-like receptors (TLRs) 2 and 4 play critical roles in intestinal inflammation caused by Fusobacterium nucleatum (F. nucleatum) infection, but the role of TLR2/TLR4 in regulation of proinflammatory cytokines remains unknown. In this study, through microarray analysis and qRT-PCR, we showed that TLR2/TLR4 are involved in the F. nucleatum-induced inflammatory signaling pathway in Caco-2 cells, C57BL/6 mice and human clinical specimens. In TLR2-/- and TLR4-/- mice, F. nucleatum infection resulted in increased colonization of the bacteria and production of the proinflammatory cytokines IL-8, IL-1β and TNF-α. In addition, the ratio of Foxp3+ CD4+ T cells in the total CD4+ T cells in TLR2-/- and TLR4-/- mice was less than that in wild-type mice, and the ratio in hybrid mice was more than that in knockout mice, which suggested that TLR2/TLR4 mediated the number of Tregs. Furthermore, it was observed that inflammatory cytokine levels were reduced in TLR2-/- mice after Treg transfer. Thus, these data indicate that TLR2/TLR4 regulate F. nucleatum-induced inflammatory cytokines through Tregs in vivo.

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TLR2/TLR4 activation induces Tregs and suppresses intestinal inflammation caused by Fusobacterium nucleatum in vivo

October TLR2/TLR4 activation induces Tregs and suppresses intestinal inflammation caused by Fusobacterium nucleatum in vivo Yin-ping Jia 0 1 Kun Wang 0 1 Zhu-jun Zhang 0 1 Ya-nan Tong 0 1 Dan Han 0 1 Chun-yu Hu 0 1 Qian Li 0 1 Yang Xiang 0 1 Xu-hu Mao 0 1 Bin Tang 0 1 0 Department of Clinical Microbiology and Immunology, Southwest Hospital & College of Medical Laboratory Science, Third Military Medical University , Chongqing , China , 2 Emei Sanatorium of PLA Rocket Force , Emeishan , China 1 Editor: Mathias Chamaillard, "INSERM" , FRANCE Toll-like receptors (TLRs) 2 and 4 play critical roles in intestinal inflammation caused by Fusobacterium nucleatum (F. nucleatum) infection, but the role of TLR2/TLR4 in regulation of proinflammatory cytokines remains unknown. In this study, through microarray analysis and qRT-PCR, we showed that TLR2/TLR4 are involved in the F. nucleatum-induced inflammatory signaling pathway in Caco-2 cells, C57BL/6 mice and human clinical specimens. In TLR2-/- and TLR4-/- mice, F. nucleatum infection resulted in increased colonization of the bacteria and production of the proinflammatory cytokines IL-8, IL-1β and TNF-α. In addition, the ratio of Foxp3+ CD4+ T cells in the total CD4+ T cells in TLR2-/- and TLR4-/- mice was less than that in wild-type mice, and the ratio in hybrid mice was more than that in knockout mice, which suggested that TLR2/TLR4 mediated the number of Tregs. Furthermore, it was observed that inflammatory cytokine levels were reduced in TLR2-/- mice after Treg transfer. Thus, these data indicate that TLR2/TLR4 regulate F. nucleatum-induced inflammatory cytokines through Tregs in vivo. - Funding: This work was supported by Grant Nos. 81501796 (for Q.L.) from National Natural Science Foundation of China (NSFC,), and the project of medical science and technology for training youth scholars of PLA (14QNP054) (for B.T.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Fusobacterium nucleatum (F. nucleatum), an anaerobic gram-negative bacterium, is normally prevalent in the oral cavity. This bacteria is the main cause of periodontal disease, and it is implicated in abscesses, inflammatory bowel disease (IBD), and colon cancer [ 1 ]. Intestinal inflammation is a well-known risk factor for colorectal cancer [ 2 ]. An accumulating number of investigations have found that F. nucleatum infection can lead to an inappropriate inflammatory response in intestinal epithelial cells [ 3 ], and F. nucleatum is significantly concentrated in inflammatory bowel tissue in patients with IBD [4±6]. However, the mechanism by which intestinal inflammation is induced by F. nucleatum infection remains unclear. Toll-like receptors (TLRs) play a critical role in initiating both the innate and adaptive immune system defense [7±8]. TLRs are mainly expressed in the plasma membrane and the membrane of intracellular vesicles. Plasma membrane TLRs include TLR1, TLR2, TLR4, Competing interests: The authors have declared that no competing interests exist. TLR5, and TLR6 [ 9 ]. Particularly, TLR2 is the signaling receptor for the gram-positive bacteria cell wall components peptidoglycan (PGN) and lipoteichoic acid (LTA), and TLR4 is the primary signaling receptor for the gram-negative bacterial cell wall component lipopolysaccharide. Se-Ra Park et al. reported that TLR2/TLR4 may take part in cytokine production by macrophages against F. nucleatum infection [ 10 ]. Accumulating studies have shown that F. nucleatum induces the epithelial inflammatory response through the nuclear factor-kappa B pathway [ 11 ]. These studies have focused primarily on periodontal epithelial cell models in F. nucleatum infection. However, studies of F. nucleatum infection in human intestinal epithelial cells and in vivo models are rare. Regulatory T cells (Tregs) are a subpopulation of T cells that express the biomarkers CD4, Foxp3, and CD25 and are critical for mediating autoimmune disease and immune responses to infectious microorganisms [ 12 ]. During host immune responses, Tregs can be recruited and expanded to regulate the immune response [ 12 ]. To date, the mechanism underlying the immunomodulatory effect of Tregs by F. nucleatum infection remains largely unknown. In the present study, the relationships between F. nucleatum and Toll-like receptors, Tregs and inflammation were investigated in Caco-2 cells and mice. We hypothesized that TLR2/ TLR4 activation induces Tregs and suppresses intestinal inflammation caused by Fusobacter ium nucleatum in mice. Materials and methods Ethics statement The experiments involving specimens from human subjects (adult volunteers) and all animal experiments were approved by the Ethics Review Board of the Third Military Medical University (Chongqing; permit number 2011±04), and volunteers gave their written informed consent. Animal surgery was performed under sodi (...truncated)


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Yin-ping Jia, Kun Wang, Zhu-jun Zhang, Ya-nan Tong, Dan Han, Chun-yu Hu, Qian Li, Yang Xiang, Xu-hu Mao, Bin Tang. TLR2/TLR4 activation induces Tregs and suppresses intestinal inflammation caused by Fusobacterium nucleatum in vivo, PLOS ONE, 2017, Volume 12, Issue 10, DOI: 10.1371/journal.pone.0186179