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We previously demonstrated that an attenuated strain of Salmonella enterica serovar Typhimurium, engineered to express IL-2 (strain GIDIL2), is cleared more rapidly than its parental, non-cytokine-expressing, strain (designated BRD509) from the reticuloendothelial system of susceptible BALB/c mice. This early clearance correlated with the induction of a strong innate immune response within a few hours of administration of GIDIL2 organisms. In the present study, we wished to assess the contribution of LPS recognition to GIDIL2-induced immune responses using Toll-like receptor 4 (TLR4) mutant C3H/HeJ mice. In contrast to LPS responder mice, both BRD509 and GIDIL2 strains persisted at higher levels in LPS non-responder animals. However, the GIDIL2 bacterial loads in the peritoneal cavity and spleen, recovered over a period of 21 days post infection, were consistently lower than the corresponding CFUs of the BRD509 strain. Direct evidence for the induction of innate immunity was shown by demonstrating increased NK cell cytotoxicity, NOS2 gene expression, and nitric oxide synthesis by peritoneal cells obtained as early as 2h after infection with GIDIL2, but not BRD509, organisms. Unlike BALB/c mice, however, these responses failed to afford any protection against virulent challenge in C3H/HeJ mice. Taken together, our data demonstrate that despite the induction of innate immune responses by IL2-expressing organisms, this was not sufficient to induce protection in TLR4 mutant mice.

Previously, we demonstrated that an attenuated Salmonella strain expressing IL-2 (designated GIDIL2) is cleared from the circulation at a much faster rate than the non-cytokine-expressing parental strain (designated BRD509). These findings suggested that IL-2 expression led to the rapid induction of innate immune responses that, in turn, accounted for the accelerated rate of bacterial clearance. In the present study, the mechanism by which this early antibacterial response is mediated was investigated. We demonstrate that as early as 2 h after infection with GIDIL2, but not BRD509, peritoneal excudate cells exhibited enhanced NK cytotoxic activity and upregulated NOS2 mRNA and NO production. This early response coincided with an enhancement of GIDIL2 clearance from the peritoneal cavity, first evidenced 22 h post-infection. Moreover, it conferred a high level of protection against virulent Salmonella challenge given within 16-20 h of GIDIL2 administration. These findings highlight the importance of innate immunity in the control of early bacterial proliferation and demonstrate the rapidity by which these responses are induced following bacterial entry., (Copyright 2003 Elsevier Science Ltd.)

Attenuated Salmonella strains have shown excellent efficacy as mucosal vaccine delivery systems. In the present report, several recombinant strains of Salmonella enterica serovar Typhimurium, engineered to express defined murine cytokines, were used to study their potential immunoregulatory capacity in the mouse model of typhoid fever. Specifically, recombinant strains expressing IL-2 (known as GIDIL2) or TNF-alpha (GIDTNF) were compared with the parental, non-cytokine-secreting, strain (BRD509) for their ability to induce a variety of immune responses in susceptible BALB/c mice. Our findings indicate that bacterially-expressed cytokines are functional in vivo and do induce a unique pattern of responses, quite distinct from that induced by BRD509 organisms. Both the type and magnitude of specific immune parameters were affected. These included the capacity to induce an inflammatory response resulting in a state of profound splenomegaly and hepatomegaly, activation of individual immune cells (particularly macrophages and other myeloid lineage cells), and the induction of nitric oxide (NO) secretion. Furthermore, a structural analysis using light as well as electron microscopy was undertaken to examine the host cellular response to infection with the different bacterial strains. The results indicate that cytokine expression by the invading pathogen can dramatically influence host immunity from a very early stage following infection. These findings may well have important consequences for the potential utilization of bacterial vector-encoded cytokines in immunoregulation in different disease settings.