Characterization of the immune response in HIV-associated cryptococcal meningitis

Despite widespread roll out of anti-retroviral therapy (ART), cryptococcal meningitis (CM) remains a significant cause of morbidity and mortality among HIV-infected persons in sub-Saharan Africa. Host immune response is central to the pathogenesis, and not only influences susceptibility to infection, but clinical presentation and outcome. Animal studies suggest that macrophage activation is a crucial component of the host immune response but human studies are limited. This thesis aims to characterize the activation state of CSF macrophages and blood monocytes in persons with HIV-associated CM, assess the effect of ART on the immune response, and identify immune correlates of severe disease and poor outcome. A prospective cohort study was conducted in Cape Town, South Africa (Innate Immunity in Cryptococcal Disease (IICD)). Persons with HIV-associated CM were enrolled and the immune response in CSF and blood examined using flow cytometry and cytokine analysis. Soluble markers of macrophage activation (sCD14, sCD163) and the ex vivo responses of whole blood and monocytes to TLR ligands and cryptococcal preparations were also measured. Additional CSF and serum samples were obtained from the Cryptococcal Optimal ART timing (COAT) trial (South Africa and Uganda) to examine the immune consequences of earlier ART initiation in cryptococcal meningitis. In this trial, earlier ART was associated with increased mortality in CM compared with a deferred ART strategy (8 days vs. 5 weeks post CM diagnosis respectively). CSF flow cytometry showed that CD8 T cells were the most common cells in the CSF followed by neutrophils and CD4 T cells. Both CD14+ and CD14- CSF macrophages were identified and expressed a range of surface markers associated with different activation states. Cryptococci were identified using flow cytometry as CD45 negative cells; cryptococcal counts using flow cytometry showed a very strong correlation with quantitative culture. There was no suggestion that raised intracranial pressure occurred as a result of a pro-inflammatory response; instead, persons with high intracranial pressure had a greater CSF fungal burden, a significantly reduced CSF cellular infiltrate (particularly CD4 T cells, CD8 T cells and double negative T cells) and larger cryptococcal cells. Contrary to animal studies, there was no association between macrophage activation and fungal burden. ART appeared to have a more profound effect on the immune response at site of disease (CSF) compared to the blood. In the IICD study, ART was associated with an increased frequency of CD4 T cells and decreased frequency of CD8 T cells in the CSF, along with increased expression of CD206 and CD16 on the surface of CSF monocyte- macrophages, suggesting an alternatively activated (M2) phenotype. These changes were closely related to plasma HIV viral load, even in subjects not taking ART. In the COAT trial, participants who received earlier ART initiation were more likely to have a CSF cellular infiltrate by day 14 compared to those in the deferred ART arm (8 days vs. 5 weeks post CM diagnosis respectively). This primarily occurred among persons with an initial paucity of CSF cellular response, the same sub-group identified in the COAT trial to be at highest risk of mortality following early ART initiation, and the same group previous shown in cohort studies to be at increased risk of IRIS. Furthermore, participants in the Kampala earlier ART arm had increased sCD14 and sCD163 concentrations in their CSF at day 14 suggesting increased macrophage activation in the central nervous system (CNS). These findings are supportive of the hypothesis that the increased mortality associated with earlier ART initiation in the COAT trial was immunologically driven. Finally, immune correlates of fatal outcome were examined. Persons who died by week 12 in the IICD study were noted to have a lower proportion of double negative T cells and lower concentrations of IFN-γ in the CSF at baseline. Non-survivors also had evidence of increased immune activation in the blood, elevated IL-10, decreased monocyte HLA-DR expression, impaired monocyte TNF-α response to LPS, and decreased IFN-γ responses to LPS in whole blood. These features are consistent with those of monocyte anergy and the compensatory anti-inflammatory response syndrome. The cause for this immune signature among persons with HIV-associated cryptococcal meningitis was not clear and warrants further study; the downstream effects of HIV-1 induced immune activation or the immune modulatory effects of cryptococcal capsule are both possibilities