Capsular specific IgM enhances complement-mediated phagocytosis and killing of Cryptococcus neoformans by methamphetamine-treated J774.16 macrophage-like cells

Abstract

Methamphetamine (METH) is a powerful and highly addictive stimulant that affects the central nervous system of users in the United States and worldwide, and its consumption is associated to the acquisition of HIV and AIDS-related infections. METH enhances cryptococcosis in mice, an opportunistic infection caused by the encapsulated fungus Cryptococcus neoformans. Due to its ability to survive within macrophages, C. neoformans is an ideal model to study pathogen-macrophage interactions. METH abrogates normal macrophage function, which might contribute to the higher rate and more rapid progression of infections in drug abusers. Hence, we investigated the role of complement and specific IgM to C. neoformans capsular polysaccharide on the function of J774.16 macrophage-like cells after exposure to METH. We found that complement and IgM significantly promotes complement-mediated phagocytosis of C. neoformans by J774.16 cells in comparison to co-incubation with complement alone. IgM enhances the expression of complement receptor 3 on the surface macrophages treated with the drug. Also, IgM-increased macrophage phagocytosis of C. neoformans may be associated with upregulation of GTPase-RhoA, a key regulator of the actin polymerization signaling cascade. Fungal cells incubated with complement and IgM in the presence of METH demonstrated higher number of cells per aggregate, a possible explanation for their enhanced ingestion by phagocytes. IgM increased killing of yeast cells by macrophages by inhibiting the alkalization of the phagosome and stimulating the intracellular production of nitric oxide. Together, our findings suggest that IgM stimulates the effector functions of macrophages against opportunistic pathogens in the setting of drug abuse.

Keywords: Complement; IgM; Infection; Methamphetamine; Nitric oxide; Phagocytosis.

Figure 1. Anti-polysaccharide capsule specific IgM and complement stimulates phagocytosis of C. neoformans by J774.16 macrophage-like cells after co-incubation with methamphetamine (METH). (A)

Light microscopy images of METH treated J774.16 cells incubated in presence of complement or complement + IgM (mAb 12A1, a capsular specific mAb) interacting with AIDS-associated yeast-like fungus C. neoformans. Images show activated macrophage-like cells with phagocytized yeast cells. Black arrows indicate C. neoformans cells outside of macrophages. White arrow heads denote yeast cells inside of macrophages. Scale bar: 10 μm. (B) The phagocytic indices (ratio of number of intracellular yeast cells to the number of macrophages counted) were determined after 2 h. Bars represent the means of four wells (100 cells per well) and error bars denote standard deviations (SDs). Symbols (*, #, ϕ, @) denote P-value significance (P<0.0001) calculated using analysis of variance analysis (ANOVA) and adjusted by use of the Bonferroni correction. *, #, ϕ, and @ indicate significantly higher fungal phagocytosis than in macrophages from untreated, 25 μM cytochalasin D (CytD, an inhibitor of actin polymerization and phagocytosis), 25 and 50 μM METH groups, respectively. Crosses (X) indicate P-value significance (P<0.0001) calculated using student’s t-test analysis. (C) Expression of CR3 (CD11b/CD18) and RhoA were determined by a western blot analysis. GAPDH was used as a control. (D) The levels of expression of CD11b, CD18, and RhoA were measured by determining the relative intensity ratios. Individual band intensities from the western blot in C were quantified using Image J software (US NIH). GAPDH was used to determine the relative intensity ratios shown in C. For panels B and D, symbols (*, #, ϕ) denote P-value significance (P<0.0001) calculated using analysis of ANOVA and adjusted by use of the Bonferroni correction. *, #, and ϕ indicate significantly higher fungal phagocytosis than in macrophages from untreated, 25 μM cytochalasin D (CytD, an inhibitor of actin polymerization and phagocytosis), and 25 μM METH groups. The experiments were performed twice with similar results obtained.

Figure 2. Specific IgM to C. neoformans polysaccharide capsule and complement promote fungal cell aggregation after incubation with METH. (A)

Immunofluorescent images of C. neoformans after co-incubation with METH and either PBS, complement, mAb 12A1 (IgM), or combination of complement and IgM, the cells were washed and incubated with mAb 18B7-FITC-conjugated goat anti-mouse IgG₁ stained to label the capsular polysaccharide. Scale bar, 10 μm. (B) Number of cell aggregates per field. Only clusters of ≥ 3 cryptococci were considered aggregates. (C) Cells per aggregate. Each aggregate was closely examined and the number of cells clustered was counted and recorded. For panels B and C, the number of cell aggregates per field and cells per aggregate was assessed after co-incubation of fungi with METH alone or METH with complement, IgM, or complement + IgM. Bars represent the means of multiple measurements and error bars denote SDs. Symbols (*, #, ϕ, @) denote P-value significance (P<0.0001) calculated using ANOVA and adjusted by use of the Bonferroni correction. *, #, ϕ, and @ indicate significantly higher than control, complement, IgM, and complement + IgM groups, respectively. The experiments were performed twice with similar results obtained.

Figure 3. Specific IgM to C. neoformans capsular polysaccharide and complement prevent METH-induced alkalization of phagosomes in J774.16 cells resulting in killing of the fungus

Macrophages were first allowed to phagocytize C. neoformans H99 cells for 2 h. Each well containing interacting cells was gently washed to get rid of fungal cells that were not phagocytize and incubated with growing medium supplemented with either PBS (untreated), Chlq (25 μM), or METH (25 or 50 μM) for 24 h. (A) For phagosomal pH determinations, the pH of phagosomes of J774.16 macrophage-like cells that contain C. neoformans yeast labeled with pH-sensitive and pH-insensitive probes was measured using a spectrofluorometer after treatment with PBS (untreated), Chlq (25 μM), or METH (25 or 50 μM). Bars represent the means of five measurements and error bars denote SDs. Symbols (*, %) denote P-value significance (P<0.0001) calculated using ANOVA and adjusted by use of the Bonferroni correction. * indicates significantly higher pH than in macrophages from untreated groups whereas % indicates significantly lower than in cells incubated with Chlq. Crosses (X) indicate P-value significance (P<0.01) calculated using student’s t-test analysis. The experiments were performed twice with similar results obtained. (B) For killing assay, phagocytic cells were lysed and fungal cells in the supernatant were plated and CFU were counted. Bars represent the means of four wells (three CFU counts per well) and error bars denote SDs. Bars represent the means of four wells (three CFU counts per well) and error bars denote standard deviations. Symbols (*, &, ≠) denote P-value significance (P<0.01) calculated using ANOVA and adjusted by use of the Bonferroni correction. * indicates significantly higher fungal killing than in macrophages from untreated groups. & and ≠ indicate significantly lower cryptococcal killing than in cells incubated with PBS and 25 μM METH, respectively. Crosses (X) indicate P-value significance (P<0.0001) calculated using student’s t-test analysis. For panels A and B, The experiments were performed twice with similar results obtained.

Figure 4. Anti-C. neoformans capsule specific IgM stimulates nitric oxide (NO) production by J774.16 cells after exposure to METH and interaction with C. neoformans

(A) NO production was quantified using the Griess method after J774.16 cells were treated with aminoguanidine (AG; iNOS inhibitor), PBS (untreated), Chlq (25 μM), or METH (25 or 50 μM). (B) Western blot analysis of inducible nitric oxide synthase (iNOS) of macrophages treated with AG, PBS, Chlq, or METH. (C) Quantitative measurements of individual band intensity in western blot gel in (B) for iNOS. GAPDH was used as a control to determine the relative intensity ratio. For panels A and C, bars represent the means of multiple measurements and error bars denote SDs. Symbols (&, %, ≠, @) denote P-value significance (P<0.0001) calculated using ANOVA and adjusted by use of the Bonferroni correction. &, %, ≠, and @ indicate significantly lower NO production and iNOS expression than in J774.16 cells incubated with PBS, Chlq, 25 and 50 μM METH groups, respectively. Crosses (X) indicate P-value significance (P<0.01) calculated using student’s t-test analysis. The experiments were performed twice with similar results obtained.