Publication Date

2009-09-22

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Microbiology and Immunology (Medicine)

Date of Defense

2009-09-03

First Committee Member

Rebecca D. Adkins - Committee Chair

Second Committee Member

Kenneth A. Fields - Committee Member

Third Committee Member

Kurt Schesser - Committee Member

Fourth Committee Member

Bonnie Blomberg - Committee Member

Fifth Committee Member

Matthias Salathe - Outside Committee Member

Sixth Committee Member

Susan C. Straley - Outside Committee Member

Abstract

Neonates are generally thought to be more likely to suffer from gastrointestinal disease, owing in part to diminished immune cell function. To gain insight into the development of mucosal immune responses during early life, we developed a model of orogastric infection with the Gram-negative bacterium Yersinia enterocolitica using murine neonates. Remarkably, neonatal mice of either the BALB/c or C57BL/6 mouse strains showed markedly enhanced survival after infection compared to adult mice. Both innate and adaptive immune components appear to contribute to this phenomenon. First, the increased resistance of neonates coincided with containment of the bacteria in the intestinal tissue with low dissemination into the spleen and liver. In contrast, the bacteria readily disseminated to the peripheral tissues in adult mice. Flow cytometric and histological studies revealed increased levels of neutrophils and macrophages in the neonatal mesenteric lymph nodes (MLN) compared to adult mice. Similar results were obtained using two different high virulence Y. enterocolitica strains. The rapid mobilization of innate cells sequestered the bacteria to the intestinal tissue, since in vivo neutrophil depletion led to efficient dissemination of Y. enterocolitica to the spleen and liver of neonates. Together, these results support the hypothesis that the neonatal intestinal immune system is competent to mount a strong antibacterial response by rapidly mobilizing innate phagocytes and thereby confining the bacterial infection to the gut, resulting in a high level of resistance. Second, we have also demonstrated that the adaptive immune system was mobilized during primary and secondary infection with this pathogen and that some of these factors may contribute to the enhanced resistance of neonatal mice to infection. Primary infection in neonates led to increased levels of antigen presenting cells, B and T cells with an activated phenotype in the MLN. MLN CD4+ Th cells from infected neonates were found to produce greater levels of IFN-gamma and IL-17A, compared to CD4+ Th cells from adult mice. These Th responses are likely to be functionally significant because neonatal mice deficient in CD4+ T cells were found to be more susceptible than adult mice to primary infection. CD4+ T cells adoptively transferred into CD4 deficient mice rescued the majority of mice from lethal infection and led to the production of IFN-gamma and IL-17A by MLN cells. In addition, primary T cell-dependent IgG1 and IgG2a serum antibodies specific for the Yersinia immunogen LcrV were increased compared to adult mice, and the absence of B cells partially increased the susceptibility of neonatal mice to primary infection. During secondary infection, however, neonatal and adult mice mounted quantitatively and qualitatively similar Yersinia-specific memory antibody responses, demonstrating that infection with Y. enterocolitica promotes mature B cell responses in neonatal mice. Finally, primed neonatal and adult mice were protected from colonization of the Peyer's patches, weight loss and mortality after a lethal infection in adulthood, demonstrating the development of long-lived protective memory responses at the intestinal interface. Together, these results indicate that both B and T cell responses, in particular Th1 and Th17 associated immunity, are important for the development of long lasting immunity to this pathogen in early life. Third, infection of neonatal mice with a Y. enterocolitica strain deleted of the anti-inflammatory protein YopP led to massive infiltration and/or accumulation of innate phagocytes in the intestine and MLN. This effect was not detectable in infected adult mice. Thus, we have identified a novel negative regulator of intestinal inflammation which might be valuable in preventing or ameliorating inflammatory conditions. This model system has revealed the unprecedented potential of neonatal mice to develop protective inflammatory innate and adaptive immunity at mucosal surfaces. The combined results presented here demonstrate that neonatal mice may be well equipped to mount robust innate and adaptive intestinal inflammatory responses that are highly protective toward Y. enterocolitica. These findings have implications for understanding how pediatric intestinal adaptive immune responses develop in response to naturally occurring gastroenteric pathogens and offer a new biological platform for development of vaccines aimed at improving mucosal and systemic immunity in early life.

Keywords

Enteropathogenic Bacteria; Granulocytes; Development Of Gut Immunity; Intestinal Immunity; Myeloperoxidase; Yersinia Outer Proteins

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