| The intracellular parasite Neospora caninum has been known as a major cause of reproductive failure in cattle. It is indispensable that the host system is biased in favor of a type 1 immune response prior to initiation and generation of an effective acquired immunity. It has been documented that N. caninum (Nc) elicits a strong type 1 immune response, however, the mechanisms by which this parasite induces type 1 immune responses and associated immunopathology remain to be elucidated. The studies presented in this dissertation characterized the underlying mechanisms fostering a Th1 immune response by a group of Nc-derived "alarmins", the high-mobility group box-1 (HMGB1) proteins.Upregulation of type 1 immune responses and activation of TLR9 by N. caninum in the presence of CpG: Our studies showed that the Nc tachyzoites (NcTz) induced production of nitric oxide (NO) by macrophages and there was no synergistic effect between the parasite and bacterial lipopolysaccharide (LPS). Interestingly, live or killed NcTz or Nc lysate drastically enhanced the ability of CpG to activate the Toll-like receptor 9 (TLR9), although by themselves had little effect. This suggest that there is some compentent in NcTz to activation of TLR9 and induce type 1 immune responses.Identification, cloning, expression, purification and localization of N. caninum high mobility group box 1 proteins : An Nc genome-wide search revealed 7 genes encoding proteins containing the HMGB motif, 3 of which, designated NcHMGB1a, NcHMGB1b and NcHMGB1c, code for the HMGB1 box B with moderate sequence homology among themselves. Cloning, expression, immunoblotting, and immunohistochemistry confirmed the presence of these proteins in the parasite with an apparent molecular mass of 5 to 18KDa. Only NcHMGB1a appeared to be secreted. So NcHMGB1 can present in intracellular and extraceller.Upregulation of inflammatory cytokines by recombinant N. caninum HMGB1: Recombinant NcHMGB1 (rNcHMGB1) induced production of tumor-necrosis factor alpha (TNF-alpha) in a dose-dependent manner and all 3 isoforms stimulated interleukine-12 (IL-12) by dendritic cells. This suggest NcHMGB1 can induce type 1 immune responses.Characterization of N.caninum HMGB1s as parasitic alarmins responsible for CpG- associated activation of TLR9 and regulation of inflammatory cytokine production: Due to their high content in lysine, NcHMGB1 bound to DNA efficiently at 1.7±0.2μg/μg recombinant protein, which was shown by the Hoechst DNA assay and gel shift assay. Next generation DNA sequencing of the bacterial DNA fragment isolated from rNcHMGB1 showed that NcHMGB1 bound to the entire E. coli genome. rNcHMGB1 alone or rNcHMGB1 plus CpG did not activate TLR9. However, DNase treatment to remove bacterial DNA fragment from rNcHMGB1 prior to addition of nuclease resistant CpG significantly augmented activation of TLR9. All 3 isofoms of NcHMGB1 were similarly potent in enhancing CpG activity with an optimal dose ratio of 1μg NcHMGB1 to 0.5μg CpG. Anti-rNcHMGB1 sera partially blocked the ability of NcHMGB1 to assist CpG in activating TLR9. rNcHMGB1+CpG also stimulated high level production of IL-6 by dendritic cells in a dose-dependent fashion. In vivo, rNcHMGB1a plus CpG stimulated high levels of IL-6 production 2h post injection and CCL2 6h post injection. The result showed that NcHMGB1 is the important compentent in NcTz to activation of TLR9 and induce type 1 immune responses.These studies demonstrated that NcHMGB1 in part mediates activation of host innate immune responses by N. caninum through activation of TLR9. The results suggest that the unbound ("empty") NcHMGB1 may act as an¨alarmin〃during infection by itself or by non-specifically binding to immunogenic DNA such as CpG, activating host TLR9 and promoting an overall type 1 immunity. Further studies of this class of alarmins may facilitate in-depth understanding of the host-parasite interaction and aid in development of counter-measures for the control of infection-associated immunopathology.
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