Anti-granulocyte-macrophage colony-stimulating factor autoantibodies are a risk factor for central nervous system infection by Cryptococcus gattii in otherwise immunocompetent patients

Abstract

Cryptococcosis is caused by either Cryptococcus neoformans or C. gattii. While cryptococcal meningoencephalitis is caused mostly by C. neoformans in immunocompromised patients, the risk factors remain unclear for patients with no known immune defect. Recently, anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies were detected in the plasma of seven "immunocompetent" cryptococcosis patients, and the cryptococcal strains from these patients were reported as C. neoformans (three strains), C. gattii (one strain), and Cryptococcus (three strains not identified to the species level). We identified all three strains that had not been identified to the species level as C. gattii. Notably, the three strains that were reported as C. neoformans but were unavailable for species confirmation originated from Sothern California and Thailand where C. gattii is endemic. Most clinical laboratories designate C. neoformans without distinguishing between the two species; hence, these three strains could have been C. gattii. Since C. gattii infects more immunocompetent patients than C. neoformans, we pursued the possibility that this antibody may be more prevalent in patients infected with C. gattii than in those infected with C. neoformans. We screened the plasma of 20 healthy controls and 30 "immunocompetent" patients with cryptococcal meningoencephalitis from China and Australia (multiple ethnicities). Anti-GM-CSF autoantibodies were detected only in the plasma of seven patients infected by C. gattii and one healthy volunteer and in none infected by C. neoformans. While plasma from these C. gattii patients completely prevented GM-CSF-induced p-STAT5 in normal human peripheral blood mononuclear cells (PBMCs), plasma from one healthy volunteer positive for anti-GM-CSF autoantibodies caused only partial blockage. Our results suggest that anti-GM-CSF autoantibodies may predispose otherwise immunocompetent individuals to meningoencephalitis caused by C. gattii but not necessarily to that caused by C. neoformans. IMPORTANCE Cryptococcal meningoencephalitis is the most serious central nervous system (CNS) infection caused by Cryptococcus neoformans or C. gattii. Cryptococcus primarily infects immunocopromised patients but is also sporadically encountered in otherwise "immunocompetent" patients with no known risk. In a recent study, anti-GM-CSF autoantibodies were detected in the plasma of seven otherwise immunocompetent patients with cryptococcal meningoencephalitis. Four of seven (57%) cryptococcal isolates from these patients were identified as C. gattii, while three strains were unavailable for species confirmation. We collected plasma from 30 otherwise healthy patients with CNS cryptococcosis in China and Australia (multiethnic) and analyzed the samples for the presence of anti-GM-CSF autoantibodies. The results suggest that anti-GM-CSF autoantibodies are a risk factor for CNS infection by C. gattii but not C. neoformans. GM-CSF may have a specific role in host defense against C. gattii, thereby elevating the importance of determining the level of anti-GM-CSF autoantibodies which can impact clinical management.

FIG 1

Anti-GM-CSF autoantibodies in plasma (1:100 dilution). The fluorescence intensity of the anti-GM-CSF autoantibodies in each healthy individual (n = 20), each CNS cryptococcosis (CNSC) patient (n = 21) in China, and each CNS cryptococcosis patient (n = 9) in Australia was plotted as a function of antibody concentration.

FIG 2

Inhibitory function of anti-GM-CSF autoantibodies. (A) Normal peripheral blood mononuclear cells (PBMCs) were incubated with 10% plasma of each anti-GM-CSF autoantibody-positive CNS cryptococcosis (CNSC) patient (n = 7) or of each anti-GM-CSF autoantibody-negative healthy volunteer (n = 5) in the presence or absence of GM-CSF (10 ng/ml) for 30 min and production of p-STAT5 was evaluated by flow cytometry. The stimulation index (ratio of stimulated/unstimulated geometric mean channels) was calculated. aab, autoantibodies. (B) Evaluation of the inhibition of p-STAT5 production by the plasma from one healthy control in China who was positive for anti-GM-CSF autoantibodies. Normal PBMCs were left unstimulated or were stimulated by 10 ng/ml·GM-CSF (cerebrospinal fluid) in the presence of one representative healthy plasma sample negative for anti-GM-CSF autoantibodies (Healthy/Aab−), of one healthy plasma sample positive for anti-GM-CSF autoantibodies (Healthy/Aab+), or of one representative CNS cryptococcosis patient’s plasma sample positive for anti-GM-CSF autoantibodies (CNSC/Aab+), and p-STAT5 production was measured by flow cytometry by intracellular staining for p-STAT5.

FIG 3

Anti-GM-CSF autoantibodies containing plasma blunts the response of PBMCs to GM-CSF stimulation. (A) A representative dose-response curve was depicted by measuring p-STAT5 production in normal PBMCs with 10% plasma from a patient or from a healthy individual under conditions of stimulation at increasing concentrations (between 0.001 ng/ml and 10 µg/ml) of GM-CSF. The concentration of GM-CSF required to phosphorylate 50% of STAT5 (EC₅₀) was 2,298 ng/ml (R² = 0.9998) and 0.6609 ng/ml (R² = 0.9990) for the patient’s plasma and for the normal plasma, respectively. (B) The concentration of GM-CSF required to phosphorylate 50% of STAT5 (EC₅₀) was determined from the dose-response curves generated for each of the anti-GM-CSF autoantibody-positive CNS cryptococcosis (CNSC) patients’ plasma samples (n = 7), plasma from anti-GM-CSF autoantibody-negative healthy volunteers (n = 5), and plasma from an anti-GM-CSF autoantibody-positive healthy volunteer (n = 1). Aab, anti-GM-CSF autoantibodies.