Influence of Vector-Encoded Cytokines on Anti-Salmonella Immunity: Divergent Effects of Interleukin-2 and Tumor Necrosis Factor Alpha

Attenuated Salmonella strains are of interest as new vaccine candidates and as vectors of cloned genes of other organisms. Attenuated strains expressing specific cytokines were constructed as a means of manipulating the immune response in various disease settings. In the present study, interleukin-2 (IL-2)-expressing (GIDIL2) or tumor necrosis factor alpha (TNF-α)-expressing (GIDTNF) strains were compared with the parent strain (BRD509) for the effect of cytokines on anti-Salmonella immunity. Expression of IL-2 resulted in a rapid clearance of the organism soon after vaccination. The reduction in GIDIL2 CFU was 50- to 300-fold higher than that of BRD509 and correlated with a markedly decreased splenomegaly. Furthermore, no evidence for any significant activation, including upregulation of surface markers and production of nitric oxide (NO), was observed in spleens of GIDIL2-injected mice. In contrast, the host response to GIDTNF was marked by an early, strong, splenic cellular influx, but surprisingly, the degree of induced splenomegaly and NO secretion was only 50% of that observed in BRD509-treated mice. Despite this, bacterial colonization of the spleen in GIDTNF-immunized animals was either slightly decreased from or equivalent to that of the BRD509-treated group, suggesting the induction of additional antimicrobial mechanisms by TNF-α. In vivo protection studies demonstrated that, at limiting doses, GIDIL2 was inferior to GIDTNF and BRD509 in its capacity to protect against virulent challenge. At high doses, however, all three strains exhibited equal protective efficacy. These results demonstrate that the immune response against intracellular bacteria can be manipulated by pathogen-expressed cytokines and open the way for further fine tuning of immune responses not only to Salmonella strains themselves but also to the heterologous gene(s) carried by them.

减毒沙门氏菌菌株作为新型疫苗候选物及异源基因克隆表达载体，具有重要研究价值。研究人员已构建表达特定细胞因子的减毒菌株，用于在多种疾病情境下调控机体免疫应答。本研究中，我们将表达白细胞介素2（interleukin-2, IL-2）的GIDIL2菌株、表达肿瘤坏死因子α（tumor necrosis factor alpha, TNF-α）的GIDTNF菌株，与亲本菌株BRD509进行对比，以探究细胞因子对抗沙门氏菌免疫应答的调控作用。
表达IL-2的菌株在免疫接种后可快速清除体内的沙门氏菌：GIDIL2菌株的菌落形成单位（colony-forming unit, CFU）下降幅度较BRD509菌株高50~300倍，且与脾脏肿大程度显著减轻相关。此外，注射GIDIL2菌株的小鼠脾脏中未检测到显著的免疫细胞激活，包括表面标志物上调及一氧化氮（nitric oxide, NO）的生成。
与之相反，宿主对GIDTNF菌株的免疫应答以早期强烈的脾脏细胞浸润为特征，但令人意外的是，其诱导的脾脏肿大程度与NO分泌量仅为BRD509处理组小鼠的50%。尽管如此，经GIDTNF免疫的动物脾脏内的细菌定植量仅较BRD509处理组略有降低或基本持平，这表明TNF-α可诱导额外的抗菌免疫机制。
体内保护试验结果显示，在低免疫剂量下，GIDIL2对抗强毒沙门氏菌攻击的保护效果劣于GIDTNF与BRD509菌株；但在高剂量免疫时，三种菌株的保护效力并无显著差异。本研究结果证实，病原菌表达的细胞因子可调控机体针对胞内菌的免疫应答，这为进一步精准调控针对沙门氏菌菌株本身及其携带的异源基因的免疫应答提供了研究方向。