Objectives: Cryptococcus neoformans is the most important fungal pathogen causing CNS-related mortality and morbidity world-wide. Treatment of C. neoformans CNS infections is very challenging. There is increasing evidence suggesting that paradoxical responses to therapy are mediated by patients’ own immune response to the fungus supported by beneficial effects of anti-inflammatory therapy. However, the use of broad anti- inflammatory (steroid) therapy is controversial because cryptococcosis is predominantly a disease of immunocompromised population. Using model of CNS- crytptococcosis in mice we will investigate CNS damage during cerebral cryptococcosis which we propose is a result of collaboration between myeloid cells with the CNS recruited T-cells. Our studies will define the mechanisms by which these cells are recruited to the CNS and cause detrimental CNS pathology. Hypothesis: CCR2-axis signaling recruits monocytes into the CNS, which further promotes T-cells recruitment and polarization facilitating fatal CNS damage. We further propose that Nitric oxide (NO) is central effector product of monocyte derived cells (MoC) activated by Th1 cells, which induces CNS pathology. To test distinct parts of this hypothesis in our mouse model we will: Aim 1. Determine whether CCR2-dependent MoC accumulation and activation drive CNS pathology. a) To establish the how CCR2-axis affects the kinetics of MoC accumulation and activation in the CNS during CME; b) To assess the contribution of the CCR2 axis/MoC to the development of CNS damage, neurological symptoms and mortality during CME; c)To provide additional mechanistic proof of concept that CCR2+ monocytes induce CNS injury in CME. Aim 2: Establish that CCR2+ MoC promote a Th1 response in the CNS during CME: a) To determine whether MoC produce CXCR3 ligands and to assess whether T-cell recruitment into the CNS is mediated via CXCR3-signaling; b) To determine whether CCR2+ MoC contribute to T-cell recruitment, activation, and polarization; c) To assess whether the adoptive transfer Th1 cells from the infected CCR2+/+ mice into CCR2-/- mice restores CME-associated damage. Aim 3. Determine whether iNOS/NO from CCR2+ MoC is required to mediate pathological CNS tissue damage observed during CME: a) To specifically test whether iNOS induction in CCR2+MoC mechanistically contribute to CNS pathology observed in CME; b) To determine whether IFNγ/IFNγR signaling in CCR2+ MoC is required to induce iNOS in these cells and the effect of IFNγ/IFNγR signaling on subsequent CNS damage. Research Plan and Methods: This proposal will utilize our novel mouse model of CM, which accurately recapitulates severe paradoxical immune responses experienced by patients with C. neoformans CNS infection. We have previously shown that ultra-polarized IFNγ-producing CD4+ T cells accumulate in the CNS and contribute to CNS pathology and mortality. In proposed work, we will use our in vivo model of C. neoformans CNS infection along with ...