MECHANISM OF IL-12 INHIBITION BY CANDIDA ALBICANS

描述（由申请人提供）：宿主对念珠菌感染的反应是先天免疫和适应性免疫之间复杂的相互作用。抗念珠菌病的第一道防线是先天免疫反应，包括宿主单核细胞(MNs)/巨噬细胞（MOs）刺激促炎细胞因子如白细胞介素-12 （IL-12）和/或抑制抗炎细胞因子（如IL-10）。许多病原微生物通过抑制IL-12的产生来克服宿主的免疫反应。目前建议的总体假设是CA分泌一种可溶性糖蛋白，抑制宿主MNs/MOs产生IL-12，从而帮助病原体侵入宿主组织。为了支持这一假设，我们证明了(i) CA细胞通过MNs抑制IL-12的产生（出版物8,9，附录2），（ii） CA培养上清抑制IL-12的产生是由分泌IL-12抑制因子（CA- siif，出版物10，附录2）介导的，（iii） CA- siif不是念珠菌磷脂酶B （Plb1p）酶/蛋白，（iv） CA- siif是一种耐热的，大小为bbb30 kDa的非酶糖蛋白，(v) CA- siif是念珠菌特异性的，（vi） CA-SIIF通过小鼠和人的MNs抑制IL-12的产生，（vii）小鼠静脉注射CA-SIIF诱导小鼠血清IL-12水平的降低，（viii） CA-SIIF介导的IL-12抑制机制涉及ERK MAPK信号通路（见出版物10，附录2）。在目前的提案中，我们将纯化和表征CA-SIIF，并确定其在体外和体内条件下的作用机制。当前提案的具体目标是：目标一：(A) CA-SIIF蛋白/s的纯化和鉴定。(B)纯化的CA-SIIF抑制IL-12活性的表征；目的1:(A)构建CA-SIIF基因/s中断的白色念珠菌δ casiif零突变株，并重新引入CA-SIIF基因的相应的反向菌株（SlIFr）。(B)构建一株白色念珠菌，产生重组CA-SIIF蛋白（FLAG- casiif），标记FLAG表位；目的ii：确定CA-SIIF抑制MNs/MOs和树突状细胞产生IL-12的机制；目的IV:(A)确定体内生物相关的CA感染是否会导致IL-12产生降低。(B)确定CA-SIIF对IL-12的抑制是否具有利基特异性。从这些研究中获得的数据将有助于更好地理解CA感染的复杂免疫反应，并可能确定新的假丝酵母菌病预防/治疗策略。

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.