Innate Determinants of Microbial Immunity

National Institute Of Allergy And Infectious Diseases

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Abstract

We have been studying innate recognition pathways involved in the response to mycobacteria. This topic is of importance not only for the identifying host resistance mechanisms to pathogenic species such as Mycobacterium tuberculosis (MTb)but also for understanding the potent adjuvant effects of attenuated and dead mycobacteria in promoting immune responses to proteins, unrelated pathogens and tumors. In work published this year (see Scientific Advance), we demonstrated that the dependence of host resistance to Mtb on the TLR/IL-1R adaptor MyD88 is largely due to a requirement for IL-1 rather than TLR signaling and identified a major role for IL-1beta in this response. In the same study we established that host resistance does not involve caspase-1 or ASC, two important components of the inflammasome, a protein complex usually required for processing of pro-IL-1beta into the bioactive cytokine. In more recent studies we have extended these findings to demonstrate an additional major role for IL-1alpha in host resistance to Mtb. In addition we have developed intracellular cytokine staining techniques for identifying IL-1alpha and beta producing cells and begun to characterize a major population of IL-1 secreting myeloid cells in the lungs of infected mice. Although mycobacteria and their products have long been known to display potent adjuvant activity, the innate recognition pathways through which they act are still poorly understood. We have been studying the role of TLR/IL-1R signaling in the T cell immunostimulatory activity of the mycobacteria in Complete Freunds Adjuvant (CFA). Mice immunized with OVA in CFA develop strong OVA-specific Th1 and Th17 responses and this cytokine polarization (as opposed to T cell priming) is totally MyD88-dependent. Surprisingly, IL-1beta/IL-1R rather than TLR (or IL-18R) signaling appears to account for this MyD88 requirement. Pro-IL-1beta mRNA was detected in the skin of both WT and MyD88 KO mice at the CFA injection site although its induction was delayed in the latter animals. This argued that TLR signaling is also not essential as the signal one for IL-1beta induction. To investigate IL-1beta processing we immunized mice deficient in ASC and caspase-1 and observed defective Th1/Th17 responses thereby implicating a major role for the inflammasome in CFA-induced immune polarization. Biochemical fractionation of mycobacteria indicated that peptidoglycan is the primary stimulus for inflammasome activation and suggested that some of the constituent muramyl peptides involved may operate through a Nod1/Nod2 independent pathway. Together these experiments demonstrate a major role for IL-1beta and the inflammasome in the adjuvant activity of mycobacteria and reveal the involvement of TLR independent pathways in both IL-1beta induction and processing. Dendritic cells (DCs) are thought to play a major role in initiation of immune responses to intracellular pathogens in part through their production of the important pro-inflammatory cytokine IL-12. In the case of infection with the intracellular protozoan Toxoplasma gondii the CD8alpha+ subset was initially proposed by us as the major source of this cytokine, and interestingly this response occurred in the absence of priming signals mediated by IFN-gamma that had been previously described as required for DC IL-12 responses. Since these early experiments involved injection of parasite extract (STAg) rather than live infection, it was possible that different populations of DCs requiring IFN-gamma priming were important in the response to the intact parasite. As introduced in last years report, to address this question we infected IL-12/23p40 reporter mice with cysts of an avirulent T. gondii strain and analyzed the IL-12 producing cells at the peritoneal infection site. Unexpectedly, we found that the major IL-12p40 producing cells were CD11b+CD8- DCs and that their response was dependent on the presence of both NK cells and IFN-gamma. By means of cell transfer experiments we determined that these DC derive from monocyte precursors and that both IFN-gamma and MyD88 control their functional maturation. Together these observations establish CD11b+CD8- monocyte-derived DCs as the major IL-12 producing cells in response to T. gondii and reveal a dichotomy in the requirement for IFN-gamma in the priming of CD8alpha+ vs CD8alpha- DCs