Paradoxical Immune Responses in Non-HIV Cryptococcal Meningitis

<div><p>The fungus <i>Cryptococcus</i> is a major cause of meningoencephalitis in HIV-infected as well as HIV-uninfected individuals with mortalities in developed countries of 20% and 30%, respectively. In HIV-related disease, defects in T-cell immunity are paramount, whereas there is little understanding of mechanisms of susceptibility in non-HIV related disease, especially that occurring in previously healthy adults. The present description is the first detailed immunological study of non-HIV-infected patients including those with severe central nervous system (s-CNS) disease to 1) identify mechanisms of susceptibility as well as 2) understand mechanisms underlying severe disease. Despite the expectation that, as in HIV, T-cell immunity would be deficient in such patients, cerebrospinal fluid (CSF) immunophenotyping, T-cell activation studies, soluble cytokine mapping and tissue cellular phenotyping demonstrated that patients with s-CNS disease had effective microbiological control, but displayed strong intrathecal expansion and activation of cells of both the innate and adaptive immunity including HLA-DR+ CD4+ and CD8+ cells and NK cells. These expanded CSF T cells were enriched for cryptococcal-antigen specific CD4+ cells and expressed high levels of IFN-γ as well as a lack of elevated CSF levels of typical T-cell specific Th2 cytokines -- IL-4 and IL-13. This inflammatory response was accompanied by elevated levels of CSF NFL, a marker of axonal damage, consistent with ongoing neurological damage. However, while tissue macrophage recruitment to the site of infection was intact, polarization studies of brain biopsy and autopsy specimens demonstrated an M2 macrophage polarization and poor phagocytosis of fungal cells. These studies thus expand the paradigm for cryptococcal disease susceptibility to include a prominent role for macrophage activation defects and suggest a spectrum of disease whereby severe neurological disease is characterized by immune-mediated host cell damage.</p></div

Brain immunofluorescence of autopsy specimens from patients who died of s-CNS cryptococcosis.

<p>Fluorescent macrophage marker (CD68) and fungal marker calcofluor white (CFW) was used to stain cells within the peri-vascular brain parenchyma. Magnification at 10x with inset and scale bar set at 100 μm for 10x images.</p

<i>Ex vivo</i> cryptococcal antigenic stimulation by pulsed autologous mature dendritic cells (mDCs) co-cultured with T lymphocytes from CSF or blood demonstrates compartmentalization of immune responses in a subgroup of 8 s-CNS and 5 non-CNS patients.

<p>(A) Sum of cytokine-producing IFN-γ+, TNF-α+, and IFN-γ+/TNF-α+ CD4+ and CD8+ T lymphocytes (after mDC presentation of MP and Crypto). Events are normalized to fluorescent beads. B) IFN-γ production of activated CD4+ and CD8+ T lymphocytes after Crypto presentation by mDCs. Tc = T lymphocyte; Crypto = glass bead-fractured, heat-killed <i>C</i>. <i>neoformans</i> strain H99; MP = <i>C</i>. <i>neoformans</i> mannoprotein; No Ag = no antigen (un-loaded mDCs). Open circles and bars are representative of non-CNS (Pulmonary) cases, filled circles and bars of s-CNS (CNS) cases. Error bars specify minimum to maximum values. *0.01≤p<0.05; **0.001≤p<0.01; ***0.0001≤p<0.001.</p

Immunophenotyping demonstrates increased absolute counts of CSF circulating activated HLA-DR+ CD4+ and CD8+ as well as—B-cells in s-CNS patients.

<p>Absolute numbers and proportions of activated CD4+ and CD8+ T lymphocytes (HLA-DR+),—and B lymphocytes were assessed in fresh CSF or blood by flow cytometry as described in Materials and Methods in patients with s-CNS disease (N = 17), non-CNS disease (N = 6), and healthy donors (HD; N = 11). Proportions are calculated from the total CD45+ cells. *0.01≤p<0.05; **0.001≤p<0.01; ***0.0001≤p<0.001.</p

Intrathecal cytokine and chemokine profiles reveal elevated IFN-γ and related inflammatory responses in cryptococcosis patients with s-CNS disease compared with non-CNS disease and healthy donors (HD).

<p>CSF was analyzed using Luminex assays according to the Materials and Methods section. (a) IFN-γ, (b) CXCL10 (IP-10), (c) IL-18, (d) TNF-α, (e) IL-10, (f) IL-4, (g) IL-6, (h) IL-13, (i) MCP-1/CCL2, (j) MIP-1α/CCL3, (k) MCP-3/CCL7, and (l) MIP-3β/CCL19. Cytokine and chemokine concentrations were expressed as log₁₀ picogram per milliliter (pg/ml) in s-CNS and non-CNS patients and HD. IL-8, GM-CSF, M-CSF, IL-12p40, IL-17, and IFN-α2 are not shown (without statistically significant differences among case groups). Error bars represent minimum to maximum values. *0.01≤p<0.05; **0.001≤p<0.01; ***0.0001≤p<0.001; ****p<0.0001.</p

Brain immunofluorescence of autopsy specimens from patients who died of s-CNS cryptococcosis demonstrates macrophage and T-cell tissue infiltration.

<p>Fluorescent macrophage markers (CD68) and M1 marker (iNOS) and M2 marker (CD200R1) were used to stain cells within the peri-vascular brain parenchyma. DAPI was used for nuclear localization. Magnification at 10x with inset and scale bar set at 100 μm for 10x images.</p

Immunophenotyping demonstrates increased absolute CSF cytotoxic (CD56dim) and immunoregulatory (CD56bright) NK cell populations, monocytes (Mono), and both myeloid (MyDC) and plasmacytoid (PlDC) dendritic cells in CSF of s-CNS patients.

<p>Absolute numbers and proportions of cell types were quantified by flow cytometry in CSF and blood as described in the Materials and Methods section in patients with s-CNS disease (N = 17), non-CNS disease (n = 6), and healthy donors (HD; N = 11). Proportions are calculated from the total CD45+ cells. *0.01≤p<0.05; **0.001≤p<0.01; ***0.0001≤p<0.001.</p

Brain biopsy specimens from patients with s-CNS cryptococcosis demonstrate macrophage and T-cell tissue infiltration.

<p>A) Diagnostic specimens obtained from two s-CNS patients were stained with macrophage markers CD 68 and CD163 and T-cell marker CD3. B) Immunofluorescence of brain biopsy of one patient after staining with macrophage marker CD68 and M1 marker (iNOS) and M2 marker (CD200R1), fungal stain, calcofluor white (CFW) and nuclear stain, 4',6-diamidino-2-phenylindole (DAPI). Magnification at 10 x with inset at 20x and scale bar set at 100 μm for 10x images.</p

Brain immunohistochemistry staining of brain autopsy specimens from patients who died of s-CNS cryptococcosis demonstrate macrophage and T-cell tissue infiltration.

<p>Autopsy specimens from meninges (#1, #2), Virchow-Robin space (#3, #4) or within brain parenchyma (#2, #5) were stained with macrophage markers CD 68 and CD163 and T-cell markers CD3, CD4 and CD8. Magnification at 10x with inset at 20x and scale bar set at 100 μm for 10x images.</p

Steroid-responsiveness of s-CNS cryptococcosis in previously healthy patients.

<p>A) Patient 1: Left panel, Enhanced T1 (upper row) and FLAIR-weighted MRI images (lower row) pre-steroid (pre) and 22 days post (22 d post) steroid treatment. White arrowheads point to increased (bright) signal within the sulci on enhanced FLAIR imaging. Right panel, Ten day average Glasgow coma score (GCS) (•) +/- SEM (N > 20 for each value) and CSF cryptococcal latex antigen (*). Red arrow indicates time of initiation of treatment with IFN-γand black arrow indicates initiation of steroid therapy. B) Patient 2: Enhanced T1 (upper row) and FLAIR (lower row) MRI images are displayed, corresponding to the indicated day of admission (d). White arrows point to areas of edema surrounding the basal ganglia lesions. Days 1 and 34 were prior to steroid course #1 and #2, respectively. Day 24 and 66 were post steroid course #1 and #2, respectively.</p