Fc gamma receptor 3A polymorphism and risk for HIV-associated cryptococcal disease

Abstract

Cryptococcus neoformans is one of the most common causes of fungal disease in HIV-infected persons, but not all of those who are infected develop cryptococcal disease (CD). Although CD4(+) T cell deficiency is a risk factor for HIV-associated CD, polymorphisms of phagocytic Fc gamma receptors (FCGRs) have been linked to CD risk in HIV-uninfected persons. To investigate associations between FCGR2A 131 H/R and FCGR3A 158 F/V polymorphisms and CD risk in HIV-infected persons, we performed PCR-based genotyping on banked samples from 164 men enrolled in the Multicenter AIDS Cohort Study (MACS): 55 who were HIV infected and developed CD and a matched control group of 54 who were HIV infected and 55 who were HIV uninfected. Using additive and allelic statistical models for analysis, the high-affinity FCGR3A 158V allele was significantly associated with CD status after adjusting for race/ethnicity (odds ratio [OR], 2.1; P = 0.005), as was the FCGR3A 158 VV homozygous genotype after adjusting for race/ethnicity, rate of CD4(+) T cell decline, and nadir CD4(+) T cell count (OR, 21; P = 0.005). No associations between CD and FCGR2A 131 H/R polymorphism were identified. In binding studies, human IgG (hIgG)-C. neoformans complexes exhibited more binding to CHO-K1 cells expressing FCGR3A 158V than to those expressing FCGR3A 158F, and in cytotoxicity assays, natural killer (NK) cells expressing FCGR3A 158V induced more C. neoformans-infected monocyte cytotoxicity than those expressing FCGR3A 158F. Together, these results show an association between the FCGR3A 158V allele and risk for HIV-associated CD and suggest that this polymorphism could promote C. neoformans pathogenesis via increased binding of C. neoformans immune complexes, resulting in increased phagocyte cargo and/or immune activation.

Importance: HIV-associated CD4(+) T cell deficiency is a sine qua non for HIV-associated cryptococcal disease (CD), but not all patients with CD4(+) T cell deficiency develop CD despite serological evidence of previous infection. At present, there are no biomarkers that predict HIV-associated CD risk. The goal of our study was to understand whether Fc gamma receptor (FCGR) polymorphisms that have been shown to portend CD risk in HIV-uninfected people are associated with CD risk in HIV-infected people. Such biomarkers could identify those who would benefit most from targeted prophylaxis and/or earlier treatment, particularly in sub-Saharan Africa, where there are nearly a million cases of HIV-associated CD annually. A biomarker of risk could also identify potential candidates for immunization, should there be a vaccine for Cryptococcus neoformans.

FIG 1

Total immunoglobulin levels in 164 human serum samples obtained from MACS. (A to C) Serum IgG1 (A), IgG2 (B), and IgG3 (C) levels were determined using radial immunodiffusion kits as described in the text. The y axis indicates concentrations in μg/ml for the groups depicted on the x axis. (D) Absorbance values shown for serum GXM-IgG levels. The white boxes represent the HIV⁺ CD⁺ group, the gray boxes represent the HIV⁺ CD⁻ group, and the black boxes represent the HIV^-CD⁻ group. Lines across each box show the median for each group, boundaries of the box represent the 25th and 75th percentiles, and lines extending from each box represent the 1st and 99th percentile. P values to compare medians between groups were calculated using the Mann-Whitney test. Asterisks (*), significant differences between the HIV-infected (HIV⁺ CD⁺ and HIV⁺ CD⁻) and HIV-uninfected (HIV⁻ CD⁻) groups; OD₄₀₅, optical density at 405 nm.

FIG 2

Binding of human Ig-C. neoformans immune complexes to CHO-K1 cell lines expressing FCGR3A 158F or 158V receptors. (A) Flow cytometric analysis of CHO cell lines expressing 158F (shaded plot) and 158V (dark line) receptors stained with CD16 MEM-154 antibody followed by FITC-conjugated goat anti-mouse IgG antibody. The histograms show the data for 50,000 events from each sample. (B) CHO cells were stained with APC-labeled antibodies recognizing fibronectin receptor and incubated with immune complexes comprising GFP-expressing C. neoformans and human immunoglobulins. The boxed insert (gate b) indicates a population that contains APC⁺ CHO cells. Free C. neoformans cells (gate c) and dead cells (gate a) were excluded. (C) APC⁺ CHO cells were further gated on the GFP channel to detect binding to precomplexed human Ig and GFP-expressing C. neoformans. The boxed insert indicates a population that contains CHO cells that bind immune complexes. Plots shown are representative of two independent experiments. (D) The proportions of CHO cells (expressing 158F or 158V receptors) binding to immune complexes comprising total normal human serum (0.1 µg/ml) and GFP-C. neoformans (10⁵) are shown as percentages. (E) CHO-K1 cells (expressing 158F or 158V) binding to immune complexes comprising human monomeric IgG subclasses (0.1 µg/ml) and GFP-C. neoformans (10⁵) are depicted as percentages for IgG1, IgG2, IgG3, and IgG4. An asterisk (*) indicates significant differences between groups (P < 0.05); octothorpes (#) indicate nearly significant differences between groups (P < 0.1) (Student’s t test).

FIG 3

Assessing anticryptococcal activity of NK cells. (A and B) NK cell lines expressing FCGR3A 158F (gray bars), FCGR3A 158V (black bars), or no FCGR3A (parental) (open bars) were incubated with C. neoformans strain 24067 for 24 h (A) and 48 h (B) at different effector/target (E/T) ratios as indicated on x axis. C. neoformans viability was analyzed by determining the number of CFU/ml as indicated on y axis. The numbers of C. neoformans cells at the start of the assay and at the indicated end times were included to account for the 10-to-500-fold growth of the organisms during the course of the assay. T = 0, zero time; Crypto alone, incubation in the absence of NK cells. Similar results were obtained using acapsular C. neoformans strain CAP67 (not shown). (C) Cytotoxicity assay performed by measuring LDH release. FCGR3A polymorphic NK cells (1 × 10⁵/well) were added to a 96-well round-bottom plate and coincubated with target cells (C. neoformans-infected CD14⁺ CD16⁺ monocytes) (1 × 10⁴/well) at 37°C and 5% CO₂ for 4 h in the presence of normal human serum (10 µg/ml) and pooled IgG (10 µg/ml) as indicated on the x axis. Percent cytotoxicity was calculated based on total content and spontaneous release of LDH. Values are shown as means ± standard errors of the means of the results from two different experiments. Asterisks (*), significant differences between groups (P < 0.05); n.s., not significant (Student’s t test).