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获得性免疫之间的复杂相互作用。对抗念珠菌病的第一道防线是先天免疫反应，它涉及宿主单核细胞(MN)/巨噬细胞(MO)刺激促炎细胞因子如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。目前提出的总体假设是CA分泌一种可溶性糖蛋白，抑制宿主MNS/MOS产生IL-12，从而帮助病原体入侵宿主组织。(2)CA培养上清液抑制IL-12的产生是由分泌性IL-12抑制因子(CA-SIIF，第10号出版物，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是一种耐热的非酶糖蛋白，大小为GT；30 kDa，(V)CA-SIIF是念珠菌特异性的，(Vi)CA-SIIF抑制小鼠和人MNS产生IL-12，(Vii)静脉注射(Vii)注射CA-SIIF可降低小鼠血清IL-12水平，(Viii)CA-SIIF抑制IL-12的机制涉及ERK MAPK信号通路(见第10号出版物，附录2)。在目前的方案中，我们将纯化和鉴定CA-SIIF，并在体外和体内条件下确定其作用机制/S。本研究的具体目标是：(A)CA-SIIF蛋白的纯化和鉴定/S。(B)纯化的CA-SIIF抑制IL-12活性的鉴定；目的：(A)构建CA-SIIF基因缺失突变株/S，以及重新导入CA-SIIF基因的相应突变株(SLIFR)。(B)构建能产生标记FLAG表位的重组CA-SIIF蛋白(FLAG-CASIIF)的白色念珠菌菌株；目的III：确定CA-SIIF抑制MNS/MOS和树突状细胞产生IL-12的机制；以及目的IV：(A)确定体内生物相关的CA感染是否导致IL-12产生减少。(B)确定CA-SIIF对IL-12的抑制是否具有特定的生态位。从这些研究中获得的数据将有助于更好地了解CA感染的复杂免疫反应，并可能确定新的预防/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.