Islamuddin Mohammad, Chouhan Garima, Farooque Abdullah, Dwarakanath Bilikere S, Sahal Dinkar, Afrin Farhat
Parasite Immunology Laboratory, Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi, India.
Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Timarpur, Delhi, India.
PLoS Negl Trop Dis. 2015 Jan 8;9(1):e3321. doi: 10.1371/journal.pntd.0003321. eCollection 2015 Jan.
In the absence of vaccines and limitations of currently available chemotherapy, development of safe and efficacious drugs is urgently needed for visceral leishmaniasis (VL) that is fatal, if left untreated. Earlier we reported in vitro apoptotic antileishmanial activity of n-hexane fractions of Artemisia annua leaves (AAL) and seeds (AAS) against Leishmania donovani. In the present study, we investigated the immunostimulatory and therapeutic efficacy of AAL and AAS.
METHODOLOGY/PRINCIPAL FINDINGS: Ten-weeks post infection, BALB/c mice were orally administered AAL and AAS for ten consecutive days. Significant reduction in hepatic (86.67% and 89.12%) and splenic (95.45% and 95.84%) parasite burden with decrease in spleen weight was observed. AAL and AAS treated mice induced the strongest DTH response, as well as three-fold decrease in IgG1 and two-fold increase in IgG2a levels, as compared to infected controls. Cytometric bead array further affirmed the elicitation of Th1 immune response as indicated by increased levels of IFN-γ, and low levels of Th2 cytokines (IL-4 and IL-10) in serum as well as in culture supernatant of lymphocytes from treated mice. Lymphoproliferative response, IFN-γ producing CD4+ and CD8+ T lymphocytes and nitrite levels were significantly enhanced upon antigen recall in vitro. The co-expression of CD80 and CD86 on macrophages was significantly augmented. CD8+ T cells exhibited CD62Llow and CD44hi phenotype, signifying induction of immunological memory in AAL and AAS treated groups. Serum enzyme markers were in the normal range indicating inertness against nephro- and hepato-toxicity.
CONCLUSIONS/SIGNIFICANCE: Our results establish the two-prong antileishmanial efficacy of AAL and AAS for cure against L. donovani that is dependent on both the direct leishmanicidal action as well as switching-on of Th1-biased protective cell-mediated immunity with generation of memory. AAL and AAS could represent adjunct therapies for the treatment of leishmaniasis, either alone or in combination with other antileishmanial agents.
由于缺乏疫苗以及现有化疗方法存在局限性，对于未经治疗会致命的内脏利什曼病（VL）而言，迫切需要研发安全有效的药物。此前我们报道了黄花蒿叶（AAL）和种子（AAS）的正己烷馏分对杜氏利什曼原虫具有体外凋亡抗利什曼活性。在本研究中，我们调查了AAL和AAS的免疫刺激作用及治疗效果。
方法/主要发现：感染后10周，对BALB/c小鼠连续10天口服给予AAL和AAS。观察到肝脏（86.67%和89.12%）和脾脏（95.45%和95.84%）中的寄生虫负荷显著降低，脾脏重量减轻。与感染对照组相比，经AAL和AAS处理的小鼠诱导出最强的迟发型超敏反应（DTH），IgG1水平降低了三倍，IgG2a水平升高了两倍。细胞因子珠阵列进一步证实了Th1免疫反应的激发，表现为经处理小鼠血清以及淋巴细胞培养上清液中IFN-γ水平升高，Th2细胞因子（IL-4和IL-10）水平降低。体外抗原再次刺激后，淋巴细胞增殖反应、产生IFN-γ的CD4+和CD8+ T淋巴细胞以及亚硝酸盐水平显著增强。巨噬细胞上CD80和CD86的共表达显著增加。CD8+ T细胞表现出CD62Llow和CD44hi表型，表明在AAL和AAS处理组中诱导了免疫记忆。血清酶标志物在正常范围内，表明对肾毒性和肝毒性无作用。
结论/意义：我们的结果证实了AAL和AAS对杜氏利什曼原虫具有双重抗利什曼作用，这既依赖于直接的杀利什曼原虫作用，也依赖于开启以Th1为主的保护性细胞介导免疫并产生记忆。AAL和AAS可单独或与其他抗利什曼药物联合作为治疗利什曼病的辅助疗法。
