MECHANISM OF IL-12 INHIBITION BY CANDIDA ALBICANS

描述（由申请方提供）：宿主对念珠菌感染的反应是先天免疫和适应性免疫之间的复杂相互作用。对抗念珠菌病的第一道防线是先天免疫应答，其涉及刺激促炎细胞因子如白介素-12（IL-12）和/或抑制抗炎细胞因子（例如，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应答。目前建议的总体假设是CA分泌可溶性糖蛋白，其抑制宿主MN/MO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细胞抑制MN产生IL-12，（出版物8，9，附录2），（ii）CA培养物上清液对IL-12产生的抑制是由分泌型IL-12抑制因子介导的（CA-SIIF，出版物10，附录2），（iii）CA-SIIF不是念珠菌磷脂酶B（Plb 1 p）酶/蛋白，（iv）CA-SIIF是耐热的，大小>30 kDa的非酶促糖蛋白，（v）CA-SIIF是胞浆特异性的，（vi）CA-SIIF抑制鼠和人MN的IL-12产生，（vii）静脉内（I. V.）在小鼠中注射CA-SIIF诱导鼠血清IL-12水平的降低，和（viii）CA-SIIF介导的IL-12抑制的机制涉及ERK MAPK信号传导途径（参见出版物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。目的I：（A）CA-SIIF蛋白的纯化和鉴定。(B)纯化的CA-SIIF的IL-12抑制活性的表征;目的II：（A）构建C.白色念珠菌delta casif无效突变株的CA-SIIF基因被破坏，以及相应的回复突变株（SlIFr），其中CA-SIIF基因被重新引入。(B)构造一个C.目的III：确定CA-SIIF抑制MN/MO和树突细胞产生IL-12的机制;和目的IV：（A）确定体内生物学相关的CA感染是否导致IL-12产生减少。(B)确定IL-12的CA-SIIF抑制是否是小生境特异性的。 从这些研究中获得的数据将导致更好地了解CA感染的复杂免疫反应，并可能确定新的预防/治疗念珠菌病的策略。

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.