MECHANISM OF IL-12 INHIBITION BY CANDIDA ALBICANS

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DESCRIPTION (provided by applicant): Host response to Candida infection is a complex interplay between innate and adaptive immunity. The first line of defense against candidiasis is the innate immune response, which involves stimulation of proinflammatory cytokines like interleukin-12 (IL-12) and/or inhibition of anti-inflammatory cytokines (e.g., IL-10) by the host monocytes (MNs)/macrophages (MOs). Many pathogenic microbes overcome host immune response by suppressing IL-12 production. The overall hypothesis of the current proposal is that CA secretes a soluble glycoprotein, which inhibits production of IL-12 by host MNs/MOs, thereby helping the pathogen invade host tissues. In support of this hypothesis, we demonstrated that (i) CA cells inhibit IL-12 production by MNs (Publication 8,9, Appendix 2), (ii) inhibition of IL-12 production by CA culture supernatant is mediated by Secretory IL-12 Inhibitory Factor (CA-SIIF, Publication 10, Appendix 2), (iii) CA-SIIF is not the candidal phospholipase B (Plb1p) enzyme/protein, (iv) CA-SIIF is a heat-resistant, non-enzymatic glycoprotein of size >30 kDa, (v) CA-SIIF is Candida-specific, (vi) CA-SIIF inhibits IL-12 production by both murine and human MNs, (vii) intravenous (I.V.) injection of CA-SIIF in mice induces a reduction in the murine serum levels of IL-12, and (viii) the mechanism of CA-SIIF-mediated IL-12 inhibition involves the ERK MAPK signaling pathway (see Publication 10, Appendix 2). In the current proposal, we will purify and characterize CA-SIIF, and determine its mechanism/s of action under in vitro and in vivo conditions. Specific aims of the current proposal are: Aim I: (A) Purification and identification of CA-SIIF protein/s. (B) Characterization of the IL-12 inhibitory activity of purified CA-SIIF; Aim Il: (A) Construct a C. albicans delta casiif null mutant strain disrupted for CA-SIIF gene/s, and the corresponding revertant strain (SlIFr) with the CA-SIIF gene reintroduced. (B) Construct a C. albicans strain that produces recombinant CA-SIIF protein (FLAG-CASIIF) tagged to FLAG epitope; Aim IIl: Determine the mechanism by which CA-SIIF inhibits IL-12 production by MNs/MOs and dendritic cells; and Aim IV: (A) Determine whether a biologically relevant CA infection in vivo results in decreased IL-12 production. (B) Determine whether CA-SIIF inhibition of IL-12 is niche-specific. Data obtained from these studies will lead to a better understanding of the complex immune response to CA infection and may identify novel prevention/treatment strategies for candidiasis.