Infection with Francisella tularensis, the causative agent of the human disease tularemia, results in the overproduction of inflammatory cytokines, termed the cytokine storm. Excess metabolic byproducts of obesity accumulate in obese individuals and activate the same inflammatory signaling pathways as F. tularensis infection. In addition, elevated levels of leptin in obese individuals also increase inflammation. Since leptin is produced by adipocytes, we hypothesized that increased fat of obese females may make them more susceptible to F. tularensis infection compared with lean individuals. Lean and obese female mice were infected with F. tularensis and the immunopathology and susceptibility monitored. Plasma and tissue cytokines were analyzed by multiplex ELISA and real-time RT-PCR, respectively. Obese mice were more sensitive to infection, developing a more intense cytokine storm, which was associated with increased death of obese mice compared with lean mice. This enhanced inflammatory response correlated with in vitro bacteria-infected macrophage cultures where addition of leptin led to increased production of inflammatory cytokines. We conclude that increased basal leptin expression in obese individuals causes a persistent low-level inflammatory response making them more susceptible to F. tularensis infection and heightening the generation of the immunopathological cytokine storm.

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Preliminary data demonstrated that a greater percentage of female mice infected with F. tularensis succumbed to disease and developed symptoms faster than male mice. Increases in the serum inflammatory cytokine levels corresponded to this enhanced sensitivity in females consistent with a stronger inflammatory. Herein, we sought to understand the cellular basis for this sex difference in the inflammatory response between males and females. We used a combination of in vivo and ex vivo cellular analysis to determine differences in the cellular response to F. tularensis. Multiparametric cytokine analysis was used to quantify changes in serum and supernatant cytokines, real-time RT-PCR was used to measure cytokine mRNA expression in tissues, and multiparametric flow cytometry was used to discriminate cell populations responsive to infection. Despite a tendency for fewer T regulatory (Treg) cells, males had fewer inflammatory macrophage and an increase in regulatory macrophages. This corresponded with an overall decrease in the transcripts of IL-10, IL-5, and IL-4 in the tissues of mice succumbing to disease and an increase in IL-6 transcripts, a marker for the level of inflammation. The functional deficiency of regulatory macrophages and increase of M1 macrophages may enhance the inflammatory response to F. tularensis ultimately leading to decreased survival. Further investigations will focus on understanding the interactions between these myeloid populations with F. tularensis and other cells of the immune system. Development of therapeutics that activate or induce expansion of regulatory macrophages or that suppress the activity/activation of M1 macrophages may prevent death from this disease.

Francisella tularensis is intracellular bacteria which is the causative agent of the disease tularemia. Highly virulent in both humans and animals, it takes only as few as ten microorganisms to cause a lethal infection. The bacteria can enter via direct or indirect routes causing the activations of the host innate inflammatory response to ensue. The function of the bacteria once its entered the host cell is to be opsonized by phagocytic cell, survive and continue to proliferate in the host causing pathogenicity. The bacteria invade host dendritic cells, neutrophils, and predominately macrophages. This causes a mass inflammatory response resulting in the cytokine storm. Activation of natural killer T cells has been shown to suppress inflammation in in vitro functional assay studies. We know that select populations of NKT cells influence the cytokine storm at the onset of infection. Our hypothesis seeks to determine whether type one versus type two natural killer T cells are inhibiting inflammation. Based on our preliminary results we hypothesize that the type one NKT cells will show cell suppression.

Francisella tularensis is a pathogenic species and a causative agent of tularemia. This bacterium is incredibly infectious and often lethal in that F. tularensis causes the overproduction of pro-inflammatory cytokines. The objective of this study is to identify the innate immune receptors responsible for the production of inflammatory cytokines in response to F. tularensis infection. The study uses an immortalized C57BL/6 Bone Marrow Macrophage (BMMac) cell line as the control and knockout BMMac that are deficient in various innate immune receptors. The cells are passaged and seeded before infecting them with F. tularensis for 2 hours. The cells are treated with a high dose of gentamicin for 2 hours followed by a low dose of gentamicin for an incubation period of 24 hours. The supernatant from these cells are then collected and analysis processed by ELISA, an enzyme-linked immunosorbent assay, to measure production of inflammatory cytokines. These data were compared with the positive control of wild type BMMac and the negative control of uninfected BMMac cells. Analysis of these mutants reveal the involvement of innate immune receptors in triggering the pro-inflammatory cytokine storm in response to F. tularensis infection that leads to death of the host.