Evasion of Innate Immune Responses: Evidence for Mannose Binding Lectin Inhibition of Tumor Necrosis Factor Alpha Production by Macrophages in Response toBlastomyces dermatitidis

Serum factors, including mannose binding lectins (MBL), influence innate responses to microbes. Little is known about the effects of serum factors or MBL on the interaction ofBlastomyces dermatitidis , a pulmonary fungal pathogen, with macrophages or on tumor necrosis factor alpha (TNF-α) production. Since macrophage production of TNF-α is an important innate immune response, we examined a mouse peritoneal macrophage (PM) cell line (RAW) and resident PM from CD-1 mice to study TNF-α production by PM stimulated with heat-killed (HK) or liveB. dermatitidis yeast cells. Mouse serum and heat-inactivated mouse serum inhibited TNF-α production 94% when macrophages were stimulated byB. dermatitidis , whereas mouse immunoglobulin G (IgG) did not have this effect. HKB. dermatitidis incubated with serum and then washed also failed to stimulate significant TNF-α production by PM. By the sandwich immunofluorescent antibody (IFA) method with anti-mouse MBL (MBL-A or -C), we showed that serum MBL bound toB. dermatitidis . When serum was absorbed with HKB. dermatitidis or liveB. dermatitidis , absorbed serum failed to significantly inhibit TNF-α production by RAW cells plusB. dermatitidis , and immunoblotting showed that absorbed serum was depleted of MBL-C. If serum was absorbed with liveB. dermatitidis , unbound serum was eluted, and bound serum factor(s) (BS) was released with guanidine buffer, BS inhibited TNF-α production by PM plusB. dermatitidis in a concentration-dependent manner. BS contained MBL-C, which boundB. dermatitidis , as shown by IFA assay. 1,3-β-Glucan stimulated TNF-α production by PM, and this was inhibited by mouse serum. Treatment ofB. dermatitidis with anti-1,3-β-glucan antibody inhibited TNF-α production by PM. With anti-1,3-β-glucan antibody, we showed by IFA assay thatB. dermatitidis contained 1,3-β-glucan. In an IFA study withB. dermatitidis , serum with an anti-mouse IgG conjugate did not result in fluorescence, yet serum blocked IFA staining ofB. dermatitidis by anti-1,3-β-glucan IgG antibody. This indicated that non-IgG serum factors binding toB. dermatitidis prevented access to 1,3-β-glucan by anti-1,3-β-glucan antibody. These results suggest that the mechanism of inhibition of the innate proinflammatory immune response of PM toB. dermatitidis is mediated by serum MBL binding toB. dermatitidis at 1,3-β-glucan sites or sterically masking 1,3-β-glucan sites, thus preventing 1,3-β-glucan stimulation of PM for TNF-α production.