In a different study, FopA, another outer membrane protein, was shown to protect mice against FT LVS infection, and antibodies played an important role. Moreover, it is Tulathromycin B possible that Tul4 may play a role in the adherence of FT to lung cells, and therefore, mucosal IgA anti-Tul4 antibodies may limit bacterial colonization and promote clearance. In this regard, IgA antibodies to FT have been shown to be important for protection against respiratory tularemia. Tul4 is a TLR2 agonist. Therefore, in addition to the adjuvant effects of GPI, it is possible that Tul4 might also act in potentiating innate and/or adaptive immune responses. In this regard, it has been shown that innate immune responses induced via several TLRs can protect against a lethal FT challenge : however, for optimal protection, TLR agonists need to be administered 1�C3 days before infection. Furthermore, since TLRs influence adaptive immune responses; it is possible that Tul4mediated TLR signaling might provide additional priming for the development of an antigen-specific response. This possibility gains support from the evidence that Tul4 induces a robust antibody response even in the absence of an adjuvant, and from previous studies demonstrating the importance of TLR2 in host responses to F. tularensis. Cytokines are important players of immune responses, and several cytokines, including TNF-a, IFN-c, IL-6, IL-12, IL-17A and IL-10, have been shown to be relevant in the host defense against FT infection. However, our studies did not reveal a specific cytokine pattern associated with protection seen in DnaK+Tul4+GPI immunized mice. In this regard, no difference was observed in the level or pattern of the IFN-c response among all experimental groups of mice, even though IFN-c has been shown to be a critical cytokine in host responses against FT infection. Moreover, between 3�C5 days after infection, the levels of IL-12p70, TNF-a and IL-6 were higher in mice immunized with one of the antigens than in mice immunized with both DnaK and Tul4, a pattern similar to that seen with the other cytokines assessed. These findings suggest that in vivo, protection against FT infection likely occurs due to the presence and interactions of numerous inflammatory mediators. Molecules like IP-10, CXCL1, RANTES and G-CSF that were evaluated in the present study are also critical participants in immune responses. For instance, RANTES, IP-10 and CXCL1, are chemotactic cytokines that belong to the chemokine family and play active roles in recruiting leukocytes to inflammatory sites. Furthermore, G-CSF, besides being a growth factor, is a cytokine that stimulates the survival, proliferation, differentiation and function of neutrophils, in addition to its involvement in the regulation of the PI3K/Akt signaling pathway. Finally, the observed changes in cytokine levels between 3 to 5 days after infection are perhaps important for the observed protection, since a differential modulation of the cytokines during this period was observed between infected mice immunized with each antigen and mice immunized with the LOUREIRIN-B vaccine preparation containing both antigens. Five days post i.n. infection of mice with FT LVS, the relative bacterial burden in the livers and spleens was roughly similar to that observed in the lungs of non-immunized mice. This finding indicates that there was a notable systemic dissemination of the bacteria, as has been reported earlier. However, there was a significant decrease in the relative bacterial burden in the lungs, livers and spleens of immunized compared to non-immunized mice.