Lyn deficiency leads to increased microbiota-dependent intestinal inflammation and susceptibility to enteric pathogens.

The Lyn tyrosine kinase governs the development and function of various immune cells, and its dysregulation has been linked to malignancy and autoimmunity. Using models of chemically induced colitis and enteric infection, we show that Lyn plays a critical role in regulating the intestinal microbiota and inflammatory responses as well as protection from enteric pathogens. Lyn(-/-) mice were highly susceptible to dextran sulfate sodium (DSS) colitis, characterized by significant wasting, rectal bleeding, colonic pathology, and enhanced barrier permeability. Increased DSS susceptibility in Lyn(-/-) mice required the presence of T but not B cells and correlated with dysbiosis and increased IFN-γ(+) and/or IL-17(+) colonic T cells. This dysbiosis was characterized by an expansion of segmented filamentous bacteria, associated with altered intestinal production of IL-22 and IgA, and was transmissible to wild-type mice, resulting in increased susceptibility to DSS. Lyn deficiency also resulted in an inability to control infection by the enteric pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium. Lyn(-/-) mice exhibited profound cecal inflammation, bacterial dissemination, and morbidity following S. Typhimurium challenge and greater colonic inflammation throughout the course of C. rodentium infection. These results identify Lyn as a key regulator of the mucosal immune system, governing pathophysiology in multiple models of intestinal disease.

Protective role of Akt2 in Salmonella enterica serovar typhimurium-induced gastroenterocolitis.

The Salmonella effector protein SopB has previously been shown to induce activation of Akt and protect epithelial cells from apoptosis in vitro. To characterize the role of Akt2 in host defense against Salmonella enterica serovar Typhimurium infection, wild-type (WT) mice and mice lacking Akt2 (Akt2 knockout [KO] mice) were infected using a Salmonella acute gastroenteritis model. Infected Akt2 KO mice showed a more pronounced morbidity and mortality associated with higher bacterial loads in the intestines and elevated levels of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and MCP-1, in the colons at 1 day postinfection compared to those shown in WT mice. Histopathological assessment and immunohistochemical analysis of cecal sections at 1 day postinfection revealed more severe inflammation and higher levels of neutrophil infiltration in the ceca of Akt2 KO mice. Flow cytometry analysis further confirmed an increase in the recruitment of Gr-1(+) CD11b(+) neutrophils and F4/80(+) CD11b(+) macrophages in the intestines of infected Akt2 KO mice. Additionally, enhanced levels of annexin V(+) and terminal transferase dUTP nick end labeling-positive (TUNEL(+)) apoptotic cells in the intestines of infected Akt2 KO mice were also observed, indicating that Akt2 plays an essential role in protection against apoptosis. Finally, the differences in bacterial loads and cecal inflammation in WT and Akt2 KO mice infected with WT Salmonella were abolished when these mice were infected with the sopB deletion mutant, indicating that SopB may play a role in protecting the mice from Salmonella infection through the activation of Akt2. These data demonstrate a definitive phenotypic abnormality in the innate response in mice lacking Akt2, underscoring the important protective role of Akt2 in Salmonella infection.

Impaired innate immune response and enhanced pathology during Citrobacter rodentium infection in mice lacking functional P-selectin.

The selectin family of adhesion molecules mediates recruitment of immune cells to sites of inflammation which is critical for host resistance against infection. To characterize the role of selectins in host defence against Citrobacter rodentium infection, wild-type (WT) mice and mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), P-, E- and L-selectin were infected using a Citrobacter-induced colitis model. Infected mice lacking PSGL-1 or P-selectin showed a more pronounced morbidity associated with higher bacterial load, elevated IL-12 p70, TNF-alpha, IFN-gamma, MCP-1 and IL-6 production, more severe inflammation and surprisingly higher leucocyte infiltration in the guts than WT control. Recruitment of neutrophils and macrophages and caecal inflammation were drastically reduced in infected P-selectin knockout mice receiving blocking monoclonal antibodies to ICAM-1 or LFA-1, indicating that these adhesion molecules may compensate for the loss of selectins in leucocyte recruitment. Furthermore, the adaptive immune response in mice lacking PSGL-1 or P-selectin remained functional since these infected mice were capable of eradicating the bacteria and being protected upon re-challenge with C. rodentium. These data demonstrate a definitive phenotypic impairment of innate response in mice lacking PSGL-1 or P-selectin, and suggest that these adhesion molecules are important in host innate immune response against Citrobacter infection.

Lack of functional P-selectin ligand exacerbates Salmonella serovar typhimurium infection.

The selectin family of adhesion molecules mediates the recruitment of immune cells to the site of inflammation, which is critical for host survival of infection. To characterize the role of selectins in host defense against Salmonella Typhimurium infection, wild-type (WT) mice and mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), P-, E-, or L-selectin, or the glycosyltransferase C2GlcNAcT-I (core 2) were infected using a Salmonella acute gastroenteritis model. Mice were monitored for survival and assessed for intestinal inflammation at 1 and 4 days postinfection. Infected mice lacking core 2, PSGL-1, or P-selectin showed a more pronounced morbidity and a significantly higher mortality rate associated with higher bacterial load and proinflammatory cytokine production, including that of TNF-alpha, MCP-1, and IL-6, from the colons at 4 days postinfection as compared with WT control. Surprisingly, at 1 day postinfection, more severe inflammation and higher neutrophil infiltration were observed in the ceca of mice lacking core 2, PSGL-1, or P-selectin compared with WT control. Enhanced levels of alpha(4)beta(7)(+) and MAdCAM-1(+) cells were observed in the ceca of infected mice lacking core 2, PSGL-1, or P-selectin. Neutrophil recruitment, cecal inflammation, and mortality rates were dramatically reduced in infected P-selectin knockout mice receiving blocking mAb to alpha(4)beta(7) integrin, indicating that this alternative adhesion molecule may attempt to compensate for the loss of selectins in neutrophil recruitment. These results demonstrate a definitive phenotypic abnormality in mice lacking core 2, PSGL-1, or P-selectin, suggesting that the interaction of functional PSGL-1 with P-selectin is an important process in host defense against Salmonella infection.

Temporal sequence and functional implications of V beta-specific T cell receptor down-regulation and costimulatory molecule expression following in vitro stimulation with the staphylococcal superantigen Toxic shock syndrome toxin-1.

The superantigen toxic shock syndrome toxin-1 (TSST-1) is implicated as the major cause of staphylococcal toxic shock syndrome. The temporal sequence of early signaling events in human peripheral blood mononuclear cells following TSST-1 stimulation was examined. TSST-1 induced rapid and complete down-regulation of V beta 2-specific T cell receptor (TCR), followed by transient CD154 expression on CD4(+) lymphocytes. This was sequentially followed by the up-regulation of CD86, CD80, CD40, and human leukocyte antigen-DR expression on CD14(+) monocytes. In contrast, S14N, a TSST-1 mutant toxin with a single amino acid substitution that is known to be impaired in interleukin (IL)--2, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha secretion, was deficient in both V beta 2-TCR down-regulation and CD154 and CD80/CD86 expression. Furthermore, pretreatment with monoclonal antibodies against V beta 2-TCR, CD80/CD86, and CD154 significantly inhibited TSST-1-induced IL-2, IFN-gamma, and TNF-alpha secretion. Taken together, these results indicate that early V beta-specific TCR activation, along with CD80/CD86 and CD154 costimulation, are key determinants of the TSST-1-induced proinflammatory cytokine response.