Ex Vivo Cytokine Profiling of Cryptococcus neoformans Strains Suggests Strain-Specific Immune Modulation: A Cross-Sectional Study

Abstract

Cryptococcal meningitis (CM) remains a major cause of mortality among people living with human immunodeficiency virus (HIV), especially in sub-Saharan Africa. The interplay between fungal genotype and host immune response is critical in determining disease outcome. We conducted ex vivo cytokine profiling using peripheral blood from HIV-positive and HIV-negative adults stimulated with heat-inactivated whole-cell antigens from two Cryptococcus neoformans strains: the reference strain H99 and the genetically distinct UgCl377 clinical strain. These strains differ at multiple loci, including the CNAG_04922 gene. Luminex-based quantification revealed that H99 induced significantly higher levels of CD40-ligand, IL-10, IL-12p70, IL-13, IL-15, and IL-33. These cytokines reflect pro-inflammatory, Th2, and regulatory responses, suggesting robust immune activation. In contrast, the UgCl377 strain elicited a dampened cytokine profile. While this study does not isolate the effect of CNAG_04922 alone, it demonstrates that whole-cell antigens from genetically distinct strains of C. neoformans elicit differential cytokine responses. These findings provide a foundation for future mechanistic studies using purified proteins or isogenic strains.

Keywords: cnag_04922; cryptococcal meningitis; cryptococcosis; cryptococcus neoformans; cytokine response; gene alleles; host-pathogen interaction; pro-inflammatory cytokines; single nucleotide polymorphism; tnf-alpha.

Figure 1. Study Flow Diagram

This flow diagram outlines the experimental flow: From blood collection from HIV-positive and HIV-negative participants → preparation of heat-inactivated Cryptococcus neoformans strains (H99 and UgCl377) → whole-blood stimulation → Luminex-based cytokine quantification.

Figure 2. Comparative Analysis of Cytokine Expression Levels Between H99 and UgCl377 Antigens

Median cytokine levels (pg/mL) are shown with interquartile ranges (IQR) for each group. H99 generally induced higher cytokine responses compared to UgCL377 across all groups. Statistically significant differences between groups are indicated by asterisks (*), based on Mann-Whitney U tests (p < 0.05). Significant upregulation in response to H99 was observed for the following cytokines: CD40-ligand (p = 0.047) and IL-10 (p = 0.028). These results highlight a stronger pro-inflammatory and immunomodulatory cytokine response to H99 compared to UgCl377, suggesting distinct immune activation pathways induced by the two antigens.

Figure 3. Cytokine Levels by HIV Status

This figure presents box plots of six cytokines (IL-10, IL-12p70, IL-13, IL-15, IL-33, CD40L) measured in response to stimulation with two different Cryptococcus neoformans strains, H99 and UgCl377, stratified by HIV status (HIV+ vs. HIV-).

Figure 4. Fold Change in Cytokine Responses: H99 Versus UgCl377

All cytokines were significantly elevated in response to H99 stimulation compared to CNAG_04922_377. IL-10, IL-13, and IL-33 showed the largest fold increases (>2.5-fold). P-values indicate statistically significant differences for all six cytokines (p < 0.05).

Figure 5. Radar Plot: Cytokine Responses to H99 Versus UgCl377

The radar plot visualizes the relative expression levels of six cytokines - CD40L, IL-10, IL-12p70, IL-13, IL-15, and IL-33 - following stimulation with two Cryptococcus neoformans strains: H99 (blue) and UgCl377 (red). The plot shows consistently higher responses to H99: Across all six cytokines, H99 elicited stronger cytokine responses compared to UgCl377, as reflected by the consistently larger area covered by the blue plot. Marked differences were noted in IL-12p70, IL-13, and IL-15. The widest separation between H99 and UgCl377 was seen in IL-12p70, IL-13, and IL-15, indicating a pronounced fold increase in response to H99. HIV+ participants: H99 (blue) consistently induced higher cytokine levels than UgCl377 (red). IL-12p70 and IL-13 showed the greatest differences, suggesting stronger Th1/Th2 responses from H99. HIV- participants: A similar trend was observed: H99 > UgCl377 across all cytokines. Here, cytokine levels were generally lower than in HIV+ participants, particularly for IL-15 and IL-33, possibly indicating an attenuated immune profile.