In the intestine, interaction between epithelial cells and macrophages (MΦs) create a unique immunoregulatory microenvironment necessary to maintain local immune and tissue homeostasis. Human intestinal epithelial cells (IECs) have been shown to express interleukin (IL)-10, which keeps epithelial integrity. We have demonstrated that bacterial signaling through Toll-like receptor (TLR) 4 induces 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) synthesis in intestinal MΦs by cyclooxygenase (Cox)-2 expression. Here, we show that TLR4 signaling generates crosstalk between IECs and MΦs that enhances IL-10 expression in IECs. Direct stimulation of TLR4 leads to the expression of IL-10 in IECs, while the presence of MΦs in a Transwell system induces another peak in IL-10 expression in IECs at a later time point. The second peak of the IL-10 expression is two times greater than the first peak. This late induction of IL-10 depends on the nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ that is accumulated in IECs by TLR4-mediated inhibition of the ubiquitin-proteasomal pathway. TLR4 signaling in MΦs in turn synthesizes 15d-PGJ2 through p38 and ERK activation and Cox-2 induction, which activates PPARγ in IECs. These results suggest that TLR4 signaling maintains IL-10 production in IECs by generating epithelial-MΦs crosstalk, which is an important mechanism in the maintenance of intestinal homeostasis mediated through host-bacterial interactions.

Recognition of Gram-negative bacteria by toll-like receptor (TLR)4 induces MyD88 and TRIF mediated responses. We have shown that TRIF-dependent responses play an important role in intestinal defense against Gram-negative enteropathogens. In the current study, we examined underlying mechanisms of how systemic TRIF activation enhances intestinal immune defense against Gram-negative bacteria. First we confirmed that the protective effect of poly I:C against enteric infection of mice with Yersinia enterocolitica was dependent on TLR3-mediated TRIF signaling by using TLR3-deficient mice. This protection was unique in TRIF-dependent TLR signaling because systemic stimulation of mice with agonists for TLR2 (Pam3CSK4) or TLR5 (flagellin) did not reduce mortality on Y. enterocolitica infection. Systemic administration of poly I:C mobilized CD11c+, F4/80+, and Gr−1hi cells from lamina propria and activated NK cells in the mesenteric lymph nodes (MLN) within 24 h. This innate immune cell rearrangement was type I IFN dependent and mediated through upregulation of TLR4 followed by CCR7 expression in these innate immune cells found in the intestinal mucosa. Poly I:C induced IFN-γ expression by NK cells in the MLN, which was mediated through type I IFNs and IL-12p40 from antigen presenting cells and consequent activation of STAT1 and STAT4 in NK cells. This formation of innate immunity significantly contributed to the elimination of bacteria in the MLN. Our results demonstrated an innate immune network in the intestine that can be established by systemic stimulation of TRIF, which provides a strong host defense against Gram-negative pathogens. The mechanism underlying TRIF-mediated protective immunity may be useful to develop novel therapies for enteric bacterial infection.

H ost defense against bacterial pathogens utilizes innate phagocytes and CD4 ϩ T cells, and successful interaction between innate and adaptive immunity establishes immunological memory. A fine interplay between innate and adaptive immune responses is necessary to eliminate pathogenic bacteria from the gastrointestinal tract without destruction of normal flora, mucosal barrier function, and gut homeostasis. However, the mechanisms regulating the interactions between innate and adaptive immunity during enteric bacterial infections have yet to be fully determined.Innate immunity covers immediate host defense against pathogens in a non-antigen-specific manner while the body is conducting initiation and calibration of adaptive immunity. In this system, pathogen-experienced antigen-presenting cells (APCs) induce differentiation of cytotoxic and helper T (Th) cells that form pathogen-specific acquired immunity. Multiple types of Th cells are generated in local lymphoid tissues during infection, while Th17 cell generation is dominant in the intestine (1). The antibacterial properties of Th17 cells have been observed in lung infections with Gram-negative extracellular bacteria (2, 3). In the intestine, however, the role of Th17 cells in host resistance to bacterial infection seems to be more complicated, as they may work as innate immune cells (4, 5). Although the importance of memory CD4 ϩ T cells in host defense against bacterial infection has been well established, the exact extent of coverage by memory Th17 cells has yet to be determined.TIR domain-containing adapter-inducing beta interferon (TRIF) is an adapter molecule that transduces intracellular signaling upon recognition of Gram-negative bacteria by Toll-like receptor 4 (TLR4) or double-stranded-RNA (dsRNA) viruses by TLR3 (6). Our previous findings regarding the unique role of innate TRIF signaling in intestinal defense against Gram-negative bacteria along with the evidence that TRIF is required for induction of costimulatory molecules and major histocompatibility complex (MHC) class II antigens suggest that TRIF may play an important role at the innate and adaptive interface (7-9).In this study, we sought to determine the role of TRIF signaling in establishing immunological memory as well as in conferring protective immunity against Gram-negative bacterial infection. We show that TRIF-deficient (Trif LPS2 ) mice failed to demonstrate increased resistance to secondary infection. TRIF deficiency resulted in the enhanced generation and maintenance of CD4 ϩ central memory T (T CM ) cells that expressed interleukin 17 (IL-17) in an antigen-specific manner. These IL-17 ϩ CD4 ϩ T cells facilitated neutrophil influx to the primary infection site and conferred on macrophages (Ms) full bactericidal function to eliminate Gram-negative pathogens only when TRIF signaling was present in innate immune cells. Therefore, our results highlight the importance of TRIF in regulating the balance between innate and adaptive immune responses to develop immune resistance to reinfecti...

Gastrointestinal mucosa reserves abundant Th17 cells where host response to commensal bacteria maintains Th17-cell generation. Although functional heterogeneity and dynamic plasticity of Th17 cells appear to be involved in chronic inflammatory disorders, how their plasticity is regulated in intestinal mucosa is unknown. Here we show that innate TRIF signaling regulates intestinal Th17-cell generation and plasticity during colitis. Absence of TRIF in mice resulted in increased severity of experimental colitis, which was associated with aberrant generation of Th17 cells especially of interferon (IFN)-γ-expressing Th17 cells in the lamina propria. The abnormal generation and plasticity of Th17 cells involved impaired expression of interleukin (IL)-27p28 by lamina propria macrophages but not dendritic cells. Treatment of TRIF-deficient mice with IL-27p28 during colitis reduced the number and IFN-γ expression of Th17 cells in the intestine. In vitro, TRIF-deficient macrophages induced more Th17 cells than wild-type (WT) macrophages during co-culture with WT naive T cells in response to cecal bacterial antigen. Many of Th17 cells induced by TRIF-deficient macrophages expressed IFN-γ due to impaired expression of IL-27p28 by macrophages and defective activation of STAT1 in T cells. These results outline TRIF-dependent regulatory mechanism by which host response to intestinal bacteria maintains Th17-cell-mediated pathology during colitis.

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