(C) Mean fluorescence intensity (MFI) of CD69 expression on NK cells

(C) Mean fluorescence intensity (MFI) of CD69 expression on NK cells. animal model have demonstrated that CD4+T cells, especially Th1 type of response are the crucial component in mediating protective immunity in both primary and secondary infections with (Su and Caldwell, 1995; Williams et al., 1997; Morrison and Caldwell, 2002; Gondek et al., 2009). SB269652 In particular, memory CD4+T cells can persist for a long time, proliferate rapidly and secrete cytokines such as IFN- during secondary chlamydial infection (Igietseme et al., 1993; Morrison and Morrison, 2000; Stary et al., 2015). Other immune components including antibodies and CD8+T cells also involved in partial protection of the host against chlamydial reinfection (Starnbach et al., 2003; Morrison and Morrison, 2005; Li and McSorley, 2015). NK cells are a predominant component of innate immune system, and also play an important role in host to combat against chlamydial infections. As a frontline responder, NK cells can contribute to host defense by cytotoxicity and cytokine-mediated effector functions without prior sensitization (Vivier et al., 2008). Besides acting as important innate effector, NK cells can regulate adaptive immune responses during primary bacterial and viral infection settings (Lodoen and Lanier, 2006; Cook et al., 2014; Crouse et al., 2015). In primary chlamydial infection, NK cells have been demonstrated to exert immunoregulatory function in adaptive immunity. In particular, NK cells promote Th1 responses by modulating dendritic (DC) function (Jiao et al., 2011; Shekhar et al., 2015). Furthermore, we have recently reported that the protective effect of NK cells is closely related to its ability to maintain a Th1/Treg and Th17/Treg balance (Li et al., 2016). However, the role of these cells in the memory associated immunity to secondary chlamydial infection is poorly understood. Recently, several reports highlighted that NK cells contribute to the SB269652 protective memory responses upon secondary infection (Alexandre et al., 2016; Habib et al., 2016; Zheng et al., 2016). For example, NK cell-depleted mice showed less resistant to rechallenge along with impairment of protective recall responses (Habib et SB269652 al., 2016). Moreover, during re-infection, activated NK cells were the major producers of early IFN- and promoted protective memory CD8+T cell response (Alexandre et al., 2016). Furthermore, NK cellCderived IFN- played a necessary role in the proliferation and activation of CD8+T cells, especially in inducing secondary CD8+T cell responses against HBV (Zheng et al., 2016). Here, we have addressed the effect of NK cells on modulating memory T cells response to respiratory infection with and during secondary infection. Enhanced Tregs and Th2 response with decreased levels of Nigg strain was used for this study. The culture of the organism was performed as described previously (Wang et al., 2012). The purified elementary bodies (EBs) were prepared by density gradient centrifugation and then stored at ?80C for future use. The same stock was used for all of the experiments. Mice Six to eight-week-old male C57BL/6 mice were purchased from Vital River Laboratory (Beijing, China). Animal experimental studies were conducted in accordance with a protocol approved by the Animal Care and Use Committee of Shandong University. NK Cell Depletion NK cells were depleted by intravenous injection with anti-asialo GM1 (Wako Chemicals, Richmond, VA). At 1 day before and 1 day after secondary infection, 20 l anti-asialo GM1 or normal rabbit IgG (isotype control) antibodies were used and followed by 10 l of a dose of every 3 days. The depletion efficiency of NK cells was confirmed by flow cytometric assay. Mice Infection Protocol and Quantification Mice were inoculated intranasally with (1 103 inclusion-forming units, IFUs) in 40 l PBS, and then the secondary infection was performed with the same dose of the organism after 8 weeks of primary infection. For the determination of growth loads by infection on Hep-2 cells. Cell Isolation and Cytokine Detection Mononuclear cells of spleen and lung tissues were separated as described previously (Peng et al., 2014). Briefly, spleens were aseptically removed, homogenized and treated with RBC lysis buffer (ebioscience). For the preparation of lung mononuclear cells, lung tissues were teased apart and digested with collagenase XI (1 mg/ml). After digestion, cells were resuspended in 35% Percoll solution (Pharmacia) and followed by erythrocytes CD221 lysis. All the cells were suspended with complete RPMI medium containing 10%.

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