Effects of Cannabinoids on the Immune Response to Murine Systemic Candidiasis

描述（由申请人提供）：大麻的主要精神活性化合物（-9-四氢大麻酚（THC））已被证明可以抑制宿主对细菌、病毒和寄生虫感染的抵抗力，可能是通过中枢或外周大麻素受体CB1R或CB2R来实现的。CB1R在中枢神经系统中丰富，而CB2R主要在免疫细胞中表达。四氢大麻酚（THC）对嗜肺军团菌（l.p.）感染的影响已有深入研究。他们发现，THC抑制了辅助性T细胞因子1 (Th1)（即干扰素-3 (IFN- 3)），同时增加了Th2细胞因子（即白细胞介素-4 (IL-4)）。然而，大麻素对真菌感染的影响尚不清楚。本提案的目的是研究大麻素（THC和2-花生四烯醇甘油（2-AG））和CB2R对全身白色念珠菌（C.白色念珠菌）感染的影响。白色念珠菌是引起全身性念珠菌病的最常见原因，这种感染常见于免疫功能低下的个体（即艾滋病患者），他们可能会使用大麻来对抗呕吐和厌食症。全身性念珠菌病也可能发生在免疫能力强的个体（即留置导尿管的患者）。在本提案的第一个目的，我们将研究大麻素和CB2R在先天免疫应答念珠菌感染中的作用。在第二个目标中，我们将研究大麻素和CB2R对感染的适应性免疫反应的影响。为了实现第一个目标，我们将用载体或大麻素处理每只野生型（WT）或CB2R敲除（CB2R-/-）小鼠，并在18h后注射PBS或白色念珠菌（1x107酵母）。分别于感染后2h、8h和1 d采集血清、肾脏和脾脏标本。其他研究表明，急性期细胞因子（即白细胞介素-6 （IL-6）、肿瘤坏死- 1 （TNF- 1）和IFN- 3）在感染白色念珠菌的小鼠血清中升高。我们将分别使用酶联免疫吸附法和实时RT-PCR比较大麻素和载体处理小鼠的血清细胞因子和脾巨噬细胞细胞因子mRNA水平。为了实现本研究的第二个目的，WT或CB2R-/-小鼠将接受载体或大麻素，并在18h后注射PBS或白色念酵菌（0.75- 1x106酵母）。为了研究原代免疫应答，我们分别在1天、3天和7天后处死部分小鼠，测定血清中IFN-3和IL-4的水平。在第一次白色念珠菌剂量15天后，剩余的小鼠将接受更高剂量的白色念珠菌（1-2x107酵母）。其中一半的小鼠将被安置长达14天，以研究大麻素对记忆免疫反应的影响。其余小鼠在第二次酵母攻毒后2h、8h和1 d处死，分别测定血清和脾脏T细胞IFN-3和IL-4蛋白及mRNA水平。每天监测所有小鼠的体重和发病率。在不同时间点采集肾脏或脾脏，通过计数酵母菌菌落形成单位来评估感染水平。我们预计大麻素将改变先天和适应性免疫反应真菌感染。如果大麻素通过CB2R起作用，大麻素不会改变CB2R-/-小鼠的真菌感染。

DESCRIPTION (provided by applicant): The main psychoactive compound of marijuana, (-9-tetrahydrocannabinol (THC) has been shown to suppress host resistance to bacterial, viral and parasitic infections, presumably through the central or peripheral cannabinoid receptors, CB1R or CB2R, respectively. While CB1R is abundant in the central nervous system, CB2R is mainly expressed in immune cells. The effect of THC on Legionella pneumophila (L.p.) infection has been thoroughly studied by others. They found that THC suppressed T helper1 (Th1) cytokines (i.e interferon-3 (IFN- 3)) while increasing Th2 cytokines (i.e. interleukin-4 (IL-4)). However the effect of cannabinoids on fungal infections is unknown. The goal of the present proposal is to investigate the effects of cannabinoids (THC and 2-arachidonoylglycerol (2-AG)) and CB2R on a systemic Candida albicans (C. albicans) infection. C. albicans is the most common cause of systemic candidiasis, an infection that occurs frequently in immune compromised individuals (i.e., patients with AIDS), who may use marijuana to combat emesis and anorexia. Systemic candidiasis may also occur in immune competent individuals (i.e. patients with in-dwelling catheters). In the first aim of this proposal, we will investigate the role of cannabinoids and CB2R on the innate immune response to Candida infection. In the second aim, we will examine the effects of cannabinoids and CB2R on the adaptive immune response to the infection. To address the first aim, we will treat each wild type (WT) or CB2R knockout (CB2R-/-) mouse with vehicle or cannabinoids and 18h later will inject the mice with PBS or C. albicans (1x107 yeast). We will then collect serum, kidneys and spleens 2h, 8h and 1 day after the yeast infection. Others have shown that the acute phase cytokines (i.e. interleukin-6 (IL-6), tumor necrosis- 1 (TNF- 1) and IFN- 3) are elevated in the serum of C. albicans infected mice. We will compare serum cytokine and splenic macrophages cytokine mRNA levels from cannabinoid and vehicle treated mice using enzyme linked immunosorbent assay and real time RT-PCR, respectively. To address the second aim of this study, WT or CB2R-/- mice will receive vehicle or cannabinoids and 18h later will be injected with PBS or C. albicans (0.75- 1x106 yeast). To study the primary immune response, some of the mice will be sacrificed 1 day, 3 days and 7 days later to determine serum IFN-3 and IL-4 levels. Fifteen days after the 1st C. albicans dose, the remaining mice will receive a higher dose of C. albicans (1-2x107 yeast). Half of these mice will be housed for up to 14 days to investigate the effect of cannabinoids on the memory immune response. The remaining mice will be sacrificed 2h, 8h and 1 day after the 2nd yeast challenge to determine serum and splenic T cell IFN-3 and IL-4 protein and mRNA levels, respectively. Weight and morbidity will be monitored daily for all mice. Kidneys or spleens will be collected at the different time points to evaluate the level of infection by counting the yeast colony forming units. We expect that cannabinoids will alter the innate and adaptive immune responses to the fungal infection. If cannabinoids act via CB2R, cannabinoids will not alter the fungal infection in CB2R-/- mice. Public Health Relevance: Systemic Candida albicans yeast infections are common among AIDS, cancer and transplant patients, individuals who may use marijuana and related compounds, such as Marinol (delta-9-tetrahydrocannabinol (THC)), to combat emesis and anorexia. THC is known to suppress immunity to bacterial, viral and parasitic infections, but its effect on yeast infections is essentially unknown. The present proposal will investigate the effects of THC, its related compound 2-arachidonoylglycerol and the peripheral cannabinoid receptor on the immune responses to a systemic Candida albicans infection in mice.