MAPK Phosphotase 5 Deficiency Contributes to Protection against Blood-Stage yoelii 17XL Infection in Mice

This information is current as Qianqian Cheng, Qingfeng Zhang, Xindong Xu, Lan Yin, of September 28, 2021. Lin Sun, Xin Lin, Chen Dong and Weiqing Pan J Immunol 2014; 192:3686-3696; Prepublished online 14 March 2014; doi: 10.4049/jimmunol.1301863

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

MAPK Phosphotase 5 Deficiency Contributes to Protection against Blood-Stage Plasmodium yoelii 17XL Infection in Mice

Qianqian Cheng,* Qingfeng Zhang,* Xindong Xu,* Lan Yin,† Lin Sun,† Xin Lin,‡ Chen Dong,x and Weiqing Pan*,{

Cell-mediated immunity plays a crucial role in the development of host resistance to asexual blood-stage infection. However, little is known of the regulatory factors involved in this process. In this study, we investigated the impact of MAPK phosphotase 5 (MKP5) on protective immunity against a lethal Plasmodium yoelii 17XL blood-stage infection using MKP5 knockout C57BL/6 mice. Compared with wild-type control mice, MKP5 knockout mice developed significantly lower parasite burdens with prolonged

survival times. We found that this phenomenon correlated with a rapid and strong IFN-g–dependent cellular immune response Downloaded from during the acute phase of infection. Inactivation of IFN-g by the administration of a neutralizing Ab significantly reduced the protective effects in MKP5 knockout mice. By analyzing IFN-g production in innate and adaptive lymphocyte subsets, we observed that MKP5 deficiency specifically enhanced the IFN-g response mediated by CD4+ T cells, which was attributable to the increased stimulatory capacity of splenic CD11c+ dendritic cells. Furthermore, following vaccination with whole blood-stage soluble plasmodial Ag, MKP5 knockout mice acquired strongly enhanced Ag-specific immune responses and a higher level of protection against subsequent P. yoelii 17XL challenge. Finally, we found the enhanced response mediated by MKP5 deficiency http://www.jimmunol.org/ resulted in a lethal consequence in mice when infected with nonlethal P. yoelii 17XNL. Thus, our data indicate that MKP5 is a potential regulator of immune resistance against Plasmodium infection in mice, and that an understanding of the role of MKP5 in manipulating anti-malaria immunity may provide valuable information on the development of better control strategies for human malaria. The Journal of Immunology, 2014, 192: 3686–3696.

alaria, which is caused by intracellular protozoan parasites humoral responses (5, 6). However, accumulating evidence from field of the genus Plasmodium, remains a devastating public studies in humans strongly supports the view that protection in the M health problem resulting in high morbidity and mortality infected host will be enhanced by T cells specific for the malarial by guest on September 28, 2021 in tropical and subtropical regions (1–3). Of the two phases of the parasites (7–11). Furthermore, naive volunteers immunized with low malarial parasite’s life cycle in a human host, the asexual blood doses of blood-stage parasites showed proliferative T cell responses stage is pathogenic and responsible for the clinical symptoms and and increased resistance to subsequent challenge in the absence of syndromes. Moreover, the severity of the infection depends largely detectable Ab responses (12). These studies highlight the signifi- on the interplay between the parasite and the immunological cance of cell-mediated immunity to blood-stage malaria infections. responses of the host (4). Therefore, a better understanding of the Experimental rodent malaria models, which display many fea- mechanisms of the immune responses against the blood-stage tures in common with human malaria, are widely used to inves- parasites will greatly facilitate the development of effective tigate diverse aspects of the immune responses in vivo (13, 14). anti-malarial drugs and vaccines. Given the absence of MHC Moreover, gene knockout mouse models also represent a powerful molecules on the surface of erythrocytes, it has been accepted that tool to elucidate the direct role of a given molecule in resistance to protective immunity to blood-stage malaria depends mainly on malaria (15–17). Extensive studies on murine malaria models have established that CD4+ T cells are the major factor responsible for both the acquisition and maintenance of protective immunity *Institute of Infectious Disease and Vaccine Development, Tongji University School of Medicine, Shanghai 200092, China; †Department of Immunology, Tongji Univer- during blood-stage malaria (18–21). Additionally, the existence sity School of Medicine, Shanghai 200092, China; ‡Department of Molecular and of Ag-specific CD8+ T cells, the rapid activation of NK cells, Cellular Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030; xDepartment of Immunology, M.D. Anderson Cancer Center, Houston, TX NKT cells, macrophages, as well as the cross-talk between innate 77030; and {Department of Tropical Infectious Diseases, Second Military Medical and adaptive immune systems also appear to be involved in the University, Shanghai 200433, China protection against blood-stage malarial parasites (22–26). In par- Received for publication July 16, 2013. Accepted for publication February 11, 2014. ticular, the proinflammatory cytokines, such as IFN-g and TNF-a, This work was supported by National Natural Science Foundation of China Grant in these cellular responses are implicated as central mediators in 30430610 and by National Basic Research Program (973 Program) in China Grant limiting parasite replication and promoting parasite clearance 2007CB513100. during the infections (27–32), which also have to be tightly bal- Address correspondence and reprint requests to Prof. Weiqing Pan, Department of Tropical Infectious Diseases, Second Military Medical University, 800 Xiang Yin anced to avoid immune-mediated pathology (32–35). However, Road, Shanghai 200433, China. E-mail address: [email protected] the underlying mechanisms and associated regulators involved in Abbreviations used in this article: DC, dendritic cell; KO, knockout; MKP5, MAPK these processes, contributing to the differential resistance to ma- phosphotase 5; p.i., postinfection; pRBC, parasitized RBC; sAg, soluble Ag; uRBC, laria, remain to be fully elucidated. uninfected RBC; WT, wild-type. MAPK phosphotase 5 (MKP5), as an essential regulator of Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 MAPK signaling pathways, has been demonstrated to play im- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301863 The Journal of Immunology 3687 portant roles in the regulation of immune responses (36). Previous Sera collection and spleen cell culture in vitro studies have revealed that MKP5 negatively regulates At the indicated time points of infection, sera of KO and WT mice were the activities of the stress-activated MAPKs, JNK and p38 (37, isolated and stored at 220˚C until assayed for cytokine and Ab levels. For 38). Owing to the prominent roles of these MAPKs in immune in vitro cytokine production, pRBC lysate used as source of crude Ag was responses (39), the effects of MKP5 on the modulation of im- prepared by Percoll treatment, lysate from uninfected RBCs (uRBCs) used munity have been investigated in Mkp52/2 mice, which were as control was similarly prepared (40, 41). Single-cell suspensions of splenocytes were prepared as described previously (27). Briefly, the asep- generated through homologous recombination. In this animal tically removed spleens were pressed gently through a sterile fine-wire model, it has been shown that MKP5 is not essential for the de- mesh into RPMI 1640 supplemented with 10% heat-inactivated FBS (Life velopment of the immune system, but specifically dephosphor- Technologies, Carlsbad, CA) and 1% penicillin/streptomycin (Life Tech- ylates and inactivates the JNK signaling pathway in vivo (36). nologies). RBCs were lysed with NH4Cl lysis buffer (Beyotime Biotech- 2/2 nology, Jiangsu, China) and the viability of harvested cells, which was Compared with the wild-type (WT) controls, Mkp5 macro- consistently .90%, was determined by trypan blue exclusion. Aliquots phages could produce enhanced levels of proinflammatory cyto- (1 ml) of cell suspensions (5 3 106 live cells/ml) were plated in triplicate kines in response to LPS. In terms of effector T cell function, wells of 24-well flat-bottom tissue culture plates (Costar, Corning, NY) both Th1 and Th2 cells lacking MKP5 could produce signifi- and incubated in the presence of anti-CD3 (145-2C11; BioLegend, San cantly higher levels of cytokines after mitogen stimulation (36). Diego, CA) plus anti-CD28 (37.51; BioLegend) mAbs (positive control, both added at 1 mg/ml), pRBC lysate (1 3 106 pRBC equivalents/ml), Moreover, investigation of lymphocytic choriomeningitis virus uRBC lysate (1 3 106 RBCs/ml), or medium alone for 48 h at 37˚C with infection in MKP5-deficient mice showed that T cells produced 5% CO2. Supernatants were collected and stored at 280˚C until cytokine more effector cytokines in response to secondary infection, measurement was performed. which resulted in high rates of immune-mediated mortality, and Downloaded from Cytokine ELISAs and NO detection therefore implicated MKP5 as a crucial negative regulator of T cell–mediated immunity during viral infection (36). However, Cytokine levels in sera or cell culture supernatants were quantified by the importance of MKP5 in the development of host resistance to sandwich ELISA. For IFN-g, TNF-a, and IL-4, rat anti-mouse IFN-g (AN-18; BD Pharmingen, San Diego, CA), anti-mouse TNF-a (1F3F3D4; other pathogens has rarely been studied. eBioscience, San Diego, CA), and anti-mouse IL-4 (11B11; BD Phar- In the present study, the role of MKP5 on immune resistance to mingen) were used as capture mAbs. Biotinylated rat anti-mouse IFN-g blood-stage Plasmodium yoelii infection was evaluated in MKP5 (R4-6A2; BD Pharmingen), anti-mouse TNF-a (MP6-XT22 and MP6- http://www.jimmunol.org/ knockout (KO) and WT control C57BL/6 mice. We demonstrated XT3; eBioscience), and anti-mouse IL-4 (BVD6-24G2; BD Pharmingen) that MKP5 deficiency increased host resistance to the lethal were used as detection mAbs. Reactivity was visualized using 3,39,5,59- tetramethylbenzidine substrate, and OD values were read in a microplate P. yoelii 17XL infection with strongly reduced parasitemia and reader at 450 nm with a reference wavelength of 630 nm. The concen- prolonged survival, which was associated with the significantly trations of cytokines were calculated according to the standard curves enhanced protective IFN-g response during the acute phase of generated using murine recombinant cytokines (PeproTech, Rocky Hill, infection. We also showed an enhanced stimulatory capacity of NJ). Bioactive TGF-b and IL-10 levels were detected by using Platinum ELISA kits (eBioscience). Serum NO levels were measured with the splenic CD11c+ dendritic cells (DCs) to induce IFN-g production + Griess reaction kit according to the manufacturer’s instructions (Beyotime by responding CD4 T cells in the absence of MKP5. Further- Biotechnology). more, using a prime-boost immunization approach, we observed by guest on September 28, 2021 that MKP5 deficiency also had a profound effect on immunization- In vivo neutralization of IFN-g induced protective efficacy against P. yoelii 17XL challenge. Female KO mice received i.p. injections of 0.1 mg rat anti-mouse IFN-g However, the augmented proinflammatory response mediated by mAb (XMG1.2; BioLegend) 1 d prior to P. yoelii 17XL inoculation and MKP5 deficiency led to severe mortality in mice infected with the every other day thereafter. Treatments with equivalent volumes of normal rat IgG (Sigma-Aldrich, St. Louis, MO) and PBS were used as controls. nonlethal strain, P. yoelii 17XNL. Collectively, these data suggest that MKP5 has the potential to modulate host immunity against Intracellular cytokine staining for flow cytometry malaria infection, and the consequence depends largely on the Splenocytes were stimulated in vitro with 50 ng/ml PMA and 1 mg/ml parasite strains. ionomycin (Sigma-Aldrich) in the presence of 1 ml/ml brefeldin A (eBio- science) at 37˚C for 5 h, followed by staining with appropriate combina- tions of fluorochrome-conjugated Abs. All Abs were purchased from Materials and Methods BioLegend. Phenotypic characterization of cell populations was performed Mice by surface labeling with anti-mouse CD3 (17A2), anti-mouse CD4 (GK1.5), anti-mouse NK1.1 (PK136), anti-mouse TCRg/d (GL3), and Mkp5 + Breeding pairs of gene KO mice on the C57BL/6 background, anti-mouse CD8a (53-6.7). Cell populations were defined as CD4 T cells generated as previously described (36), were bred in the animal care (CD3+CD4+), CD8+ T cells (CD3+CD8+), NK cells (NK1.1+CD32), facility under specific pathogen-free conditions. WT control C57BL/ NKT cells (NK1.1+CD3+), and gd T cells (CD3+TCRg/d+). After fixa- 6 mice were purchased from Shanghai Laboratory Animal Center of tion with 1% paraformaldehyde, cells were permeabilized with 0.1% sa- the Chinese Academy of Sciences and maintained in the same fa- ponin (Sigma-Aldrich) and stained intracellularly with anti-mouse IFN-g cility. Female KO and WT mice (aged 6–8 wk) were used in all (XMG1.2) or isotype control Ab (RTK2071) before analyzing on a experiments. All procedures conducted on animals in this study were FACSCalibur (BD Biosciences, San Jose, CA). For detection of other approved by the Internal Review Board of Tongji University School cytokines produced by CD4+ T cells, anti-mouse IL-4 (11B11), anti-mouse of Medicine. IL-10 (JES5-16E3), and anti-mouse IL-17A (TC11-18H10.1) were used for intracellular staining. Data were analyzed using FlowJo software (Tree Parasite and experimental challenge Star, Ashland, OR). Blood-stage parasites of the highly virulent lethal strain P. yoelii 17XL Splenic DC/CD4+ T cell cocultures and the nonlethal variant P. yoelii 17XNL were originally obtained from the Department of Tropical Infectious Diseases of Second Mili- Splenic DCs from both KO and WT mice were separated 2 d after P. yoelii tary Medical University (Shanghai, China). Infections were initiated 17XL infection, according to the methods described previously (40, 42), by i.p. injection of 5 3 106 P. yoelii 17XL– or 1 3 105 P. yoelii and further purified by positive selection using biotinylated anti-CD11c 17XNL– parasitized RBCs (pRBCs) obtained from a homologous do- mAb (N418; BioLegend) in combination with BD IMag streptavidin nor, which had been previously infected with a frozen stock of para- particles (BD Biosciences), according to the manufacturers’ instructions. sites. Survival rates were monitored daily and parasitemia was assessed The purity was routinely $85% CD11c+ as confirmed by flow cytometry. by microscopic examination of Giemsa-stained thin smears of tail Splenic CD4+ T cells from uninfected KO and WT mice were enriched by blood. using biotinylated anti-CD4 mAb (RM4-5; BioLegend) in combination 3688 ROLE OF MKP5 IN MALARIA INFECTION with the streptavidin-labeled magnetic beads. The purity was routinely .95%. For coculture, 2 3105 purified DCs (designated as KO DCs and WT DCs) were incubated in triplicate with 4 3105 freshly purified CD4+ T cells (designated as KO CD4+ T and WT CD4+ T) in 96-well round- bottom plates (Nunc) at a final volume of 200 ml for 5 d at 37˚C. DCs incubated in medium alone were included as controls. The supernatants were collected and analyzed for IFN-g production by ELISA, as described above. P. yoelii 17XL soluble Ag preparation and immunization Whole blood-stage soluble plasmodial Ag (sAg) of P. yoelii 17XL was prepared according to previously described methods with minor mod- ifications (43–45). Briefly, blood (average 40–45% parasitemia) from infected C57BL/6 mice was harvested and lysed by treatment with 0.15% saponin for 10 min at 4˚C. Intact parasites were obtained by centrifugation at 10,000 3 g, washed extensively, and resuspended in PBS. After 3 cycles of rapid freezing and thawing, samples were briefly sonicated and the sAg fraction was recovered by centrifugation at 10,000 3 g for 15 min and frozen at 280˚C until used. For immunological and protection studies, groups of KO and WT mice were primed s.c. with 50 mg sAg (equivalent to 1 3 107 pRBCs) emulsified in CFA (Sigma-Aldrich), followed by two boost immunizations at 2-wk intervals, with the same doses of Ag in IFA. Control mice were immunized in a similar manner with adjuvant alone. Downloaded from Approximately 2 wk after the final immunization, mice were either chal- lenged with 5 3 106 P. yoelii 17XL pRBCs or sacrificed for analysis of cytokine and Ab responses.

Assessment of immune responses induced by immunization Cytokine production in vitro by spleen cells from P. yoelii 17XL sAg- http://www.jimmunol.org/ immunized and control mice was analyzed according to the described protocol. For evaluation of humoral responses, the levels of Ag-specific total IgG, as well as the major isotypes IgG1 and IgG2c, in prechallenge sera were determined by ELISA. The 96-well plates were coated with 100 ml sAg (20 mg/ml) overnight at 4˚C and subsequently blocked with 1% BSA in PBS for 1 h. Serum from each animal was assayed at a dilution of 1:100. Ag-specific Abs were detected using HRP-conjugated goat anti- mouse IgG (Promega, Madison, WI), anti-mouse IgG1 (SouthernBiotech, Birmingham, AL), and anti-mouse IgG2c (IgG2a b allotype) (Abcam, Cambridge, MA) with 3,39,5,59-tetramethylbenzidine as substrate. Serum samples were run in duplicate and absorbance was read at 450 nm with by guest on September 28, 2021 a reference wavelength of 630 nm. Ab levels in serum are expressed as FIGURE 1. Course of primary P. yoelii 17XL infection in MKP5 KO relative OD values. and WT C57BL/6 mice. Mice were infected i.p. with 5 3 106 pRBCs and A Statistical analysis the course of infection was followed by monitoring ( ) percentage survival and (B) parasitemia. Data shown are from one of three replicate experi- All analyses were performed using GraphPad Prism version 5.0 (GraphPad ments (n = 6 mice/group). Log-rank tests revealed a statistically significant 6 Software, La Jolla, CA). Data are presented as means SEM, unless difference in survival rates between KO and WT mice (**p , 0.01). otherwise stated. Statistical significance of differences between two groups Parasitemia values are presented as means 6 SEM (***p , 0.001). (KO versus WT) or two time points was assessed by unpaired Student t tests, except for survival. Kaplan–Meier curves of overall survival were compared by using the log-rank test. A p value ,0.05 was considered statistically significant. (Fig. 1B). Somewhat unexpectedly, all KO mice that survived acute infection displayed a recrudescent parasitemia in the chronic stage, followed by a decline in parasite burden, although the as- Results sociated and irreversible severe anemia led to the gradual death of Primary infection of MKP5 KO and WT C57BL/6 mice with mice. These results indicate that MKP5 deficiency contributes, at P. yoelii 17XL least in part, to protection against P. yoelii 17XL infection in Female KO mice and sex-matched WT littermates were infected C57BL/6 mice. i.p. with the lethal rodent malarial parasite P. yoelii 17XL as described in Materials and Methods. Survival rates and para- Enhanced IFN-g response in MKP5 KO mice during the early sitemia were monitored throughout the course of the infection. course of P. yoelii 17XL infection In both groups of mice, regardless of genetic background, par- The ability of KO mice to develop effective resistance to acute asites were detected in peripheral blood 2 d after inoculation. P. yoelii 17XL infection was consistent with the major biological However, WT controls exhibited a more severe course of in- function of MKP5 in regulating immune responses. To test the fection (Fig. 1). Whereas mice in the WT group suffered 100% hypothesis, a comparative analysis of the mediators that have been mortality within 1 wk postinfection (p.i.), none of the KO mice found to be essential for elimination of malarial parasites, as well died during this period and most (five of six) survived the in- as the major anti-inflammatory cytokines that are known to be im- fection for .20 d (p , 0.01) (Fig. 1A). Infection in WT mice portant components of the regulatory responses, was performed in resulted in rapidly ascending parasitemia, which peaked at 5 d KO and WT mice. p.i. with the levels in excess of 60%. Nevertheless, it was no- First, circulating levels of IFN-g, TNF-a, and NO, as well as table that KO mice developed significantly reduced parasite bur- TGF-b and IL-10, were quantified following the parasite inocu- dens (days 3–6 p.i.) than did their WT counterparts (p , 0.001), lation (Fig. 2). As shown in Fig. 2A, the most pronounced effect of with the peak parasitemia reaching only 40% at day 5 p.i. MKP5 deficiency observed was the large burst of serum IFN-g as The Journal of Immunology 3689 Downloaded from

FIGURE 2. Quantification of circulating cytokine and NO levels following primary P. yoelii 17XL infection in MKP5 KO and WT mice. Serum samples http://www.jimmunol.org/ were collected from KO and WT mice at days 0 (D0), 2 (D2), 3 (D3), and 5 (D5) of infection. Concentrations of (A) IFN-g and (B) TNF-a, as well as (D) TGF-b and (E) IL-10, were determined by ELISA. The levels of (C) NO were determined by the Griess reaction. Data shown are means 6 SEM (n = 3 or 4 mice/group) from one of two replicate experiments. #p , 0.05 for comparison between uninfected and infected mice; *p , 0.05, **p , 0.01 for com- parison between KO and WT mice. ND, not detectable.

early as day 3 p.i. (p , 0.05 versus day 0), reaching a maxi- 0.6904 versus 2.020 6 0.4707 ng/ml at day 5, p , 0.05) (Fig. 3A). mum at day 5 p.i., in parallel with the time of peak parasitemia. In contrast, no significant IL-4 secretion was detected in either KO In accordance with the previous observationoninfectionof or WT mice during the early phase (Fig. 3B). by guest on September 28, 2021 C57BL/6 mice with P. yoelii 17XL parasites (32), WT mice These results clearly suggest that, in the absence of MKP5, the also displayed gradually and moderately increased IFN-g pro- malaria-specific IFN-g response is significantly increased during duction in vivo. However, the levels were significantly lower theearlystageofP. yoelii 17XL infection, which might correlate than those detected in KO mice (0.587 6 0.099 versus 0.287 6 with the limitation of parasite replication in KO mice. 0.064 ng/ml at day 3, p , 0.05; 1.343 6 0.097 versus 0.721 6 0.097 ng/ml at day 5 p.i., p , 0.01). Because a previous report Protective role of IFN-g in the resistance of MKP5 KO mice to implied that macrophages were involved in the responses to primary P. yoelii 17XL infection primary malaria infections (41), and although our analysis also KO mice were injected either with normal rat IgG or with revealed slightly increased TNF-a, there were no apparent dif- XMG1.2, a rat anti-mouse IgG1 that has been found to block IFN-g ferences in TNF-a or NO levels between KO and WT mice up to activity in vivo (46, 47), prior to and during the early course day 5 p.i. (Fig. 2B, 2C). Owing to the 100% mortality of WT mice of infection with 5 3 106 pRBCs of P. yoelii 17XL. Negative within 1 wk of infection, we were unable to further compare this control animals were injected with PBS at the same time points. response, although the peak TNF-a and NO levels were noted at As expected, the course of infection in KO mice treated with day 10 p.i. in the KO group (data not shown). Additionally, con- rat IgG was similar to that resulting from PBS treatment (Fig. 4). sistent with the previous report (32), an extremely early TGF-b In contrast, treatment of KO mice with anti–IFN-g mAb greatly production within the first 2 d of infection was evident in both KO exacerbated the infection, with 75% mortality (three of four) as and WT mice (p , 0.05), and IL-10 levels were detectable at day early as that observed in WT mice treated with PBS. All mice 3 and increased at day 5 p.i. However, the amounts of the two succumbed to the infection by day 9 (Fig. 4A). Thus, neutrali- cytokines were comparable between the two groups of mice zation of IFN-g significantly increased the susceptibility of KO (Fig. 2D, 2E). mice to infection (p , 0.05 for anti–IFN-g treatment versus Next, we assessed IFN-g and IL-4 production by spleen cells rat IgG treatment in KO mice). Moreover, mice treated with following in vitro stimulation (Fig. 3). Although the responses of anti–IFN-g had significantly higher parasitemia at day 4 (p , naive cells were extremely low and similar to those of WT mice, 0.05) and peak parasitemia at day 5 p.i. (p , 0.01) compared cells harvested from infected KO mice were hyperresponsive in with rat IgG-treated mice, while the overall course of para- the presence of pRBC lysate, with remarkably elevated IFN-g sitemia was not significantly different from that observed in production at day 3 p.i. (p , 0.05) and peaked at day 5. Again, in WT mice administered with PBS (Fig. 4B). We obtained es- agreement with the serum cytokine profiles, spleen cells from KO sentially the same results in two successive experiments. These mice produced significantly greater amounts of IFN-g than did results clearly demonstrate the essential role of IFN-g in the de- those from their WT counterparts at the early time points (3.178 6 velopment of early defense against P. yoelii 17XL infection in 0.6895 versus 0.6163 6 0.2066 ng/ml at day 3, p , 0.05; 5.015 6 KO mice. 3690 ROLE OF MKP5 IN MALARIA INFECTION Downloaded from http://www.jimmunol.org/

FIGURE 3. IFN-g and IL-4 production in vitro by spleen cells from naive and P. yoelii 17XL–infected MKP5 KO and WT mice. Single-cell FIGURE 4. Protective role for IFN-g in the resistance of MKP5 KO suspensions of splenocytes were prepared at the indicated time points as mice against infection with P. yoelii 17XL. Groups of KO mice were by guest on September 28, 2021 described in Materials and Methods. Cells were cultured for 48 h in the injected i.p. with neutralizing anti–IFN-g or equivalent concentrations presence of anti-CD3 plus anti-CD28 (+), pRBC lysate (p), uRBC lysate ofnormalratIgGasacontrolAb1dbeforeinfectionandthenevery (u), or medium alone (m). The levels of (A) IFN-g and (B) IL-4 in culture other day to day 8. Animals treated with PBS were used as negative supernatants were analyzed by ELISA. Data are presented as means 6 controls. Mice were infected i.p. with 5 3 106 pRBCs and (A) survival SEM (n = 3 mice/group) from one of two replicate experiments. *p , 0.05 rates and (B) parasitemia were determined. Data are means 6 SEM (n = for comparison between KO and WT mice. 4–5 mice/group) from one of two successive experiments. *p , 0.05, ** p , 0.01 for comparison between anti–IFN-g and rat IgG treatments in KO mice. MKP5 deficiency enhances IFN-g response mediated by CD4+ T cells during acute P. yoelii 17XL infection showed that ab T cells (CD4+ TplusCD8+ T) were the dominating In previous studies it has been demonstrated that many lymphocyte producer of IFN-g during acute P. yoelii 17XL infection, account- subsets, including ab T, NK, NKT, and gd T cells contribute to the ing for nearly 81.13 and 70.81% of total IFN-g–secreting cells at IFN-g response in malaria (28, 29, 48–51). In this study, we used day 4 p.i., with .10 3 106 and 5.4 3 106 cells per spleen in KO FACS analysis to assess IFN-g production by these cell subsets and WT mice, respectively (Fig. 5C). Therefore, the comparative during the early acute stage of P. yoelii 17XL infection. To take analysis suggests that deficiency of MKP5 increases CD4+ T cell into account the increase in total spleen cells that occurs after IFN-g production during the early phase of infection, which may infection, we compared both the proportions and total numbers of account for the phenotypic differences between KO and WT mice. IFN-g+ cells in each of the gated subsets. As shown in Fig. 5, IFN-g Because the importance of immune responses generated by CD4+ production by the various cell subsets was simultaneously ele- T lymphocytes in protection against blood-stage malaria had been vated in both KO and WT mice at day 4 p.i., 1 d earlier than the demonstrated previously (20, 21), other cytokines produced by appearance of peak parasitemia. However, of all the lymphocyte CD4+ T cells were also analyzed. In contrast to the IFN-g re- subsets concerned, only splenic CD4+ T cells exhibited significant sponse, the proportions of IL-4–, IL-10–, and IL-17A–producing differences between the two groups, that is, 11.61- and 4.80-fold CD4+ T cells remained at basal or only slightly increased levels, higher levels of IFN-g than those of uninfected mice in the KO with no statistical differences detected between KO and WT and WT groups, respectively. Furthermore, both the frequency and groups (Fig. 5D), suggesting that strong Th2 and Th17 responses total number of IFN-g–producing cells were significantly higher were not induced during the early course of P. yoelii 17XL in- in KO mice than in WT mice (p , 0.01), whereas no significant fection. Taken together, these data reaffirm the pivotal role of differences were observed for the IFN-g+ CD8+ T, NK, NKT, and CD4+ T cell–mediated responses in immune defense against gd T cells between the two groups (Fig. 5A, 5B). Additionally, blood-stage malaria infection, and that the absence of MKP5 en- analysis of the relative contribution to the overall IFN-g response hanced Th1 response in mice during the early stages (36). The Journal of Immunology 3691 Downloaded from http://www.jimmunol.org/

FIGURE 5. Enhanced IFN-g response mediated by CD4+ T cells during the early phase of P. yoelii 17XL infection in MKP5 KO mice. (A)Spleen cells isolated from KO and WT mice at days 0 (D0) and 4 (D4) of infection were stained for surface markers and intracellular IFN-g following in vitro stimulation with PMA plus ionomycin and analyzed by FACS. Representative dot plots show the frequencies of IFN-g–producing cells contained in each gated subset. (B) Total numbers of splenic IFN-g+ CD4+ Tcells,IFN-g+ CD8+ Tcells,IFN-g+ NK cells, IFN-g+ NKT cells, and IFN-g+ gd T cells are compared between KO and WT mice. Data shown are means 6 SEM (n = 3 mice/group) from one of two replicate by guest on September 28, 2021 experiments. **p , 0.01. (C) Pie charts showing the relative contributions of ab T(CD4+ TplusCD8+ T cells), NK, NKT, gd T, and other subsets to the total number of IFN-g+ cells in KO and WT mice at day 4. (D) Representative dot plots showing the proportions of IL-4–, IL-10–, and IL-17A– producing cells in gated splenic CD4+ T cells.

Splenic DCs from KO mice support increased IFN-g Thus, these data support the view that the enhanced IFN-g– production by CD4+ T cells producing CD4+ T cell response in KO mice lies primarily on Given that APCs play a key role in shaping adaptive immune the increased ability of DCs to activate malaria-specific T cells responses, and several studies on mouse models of malaria have early after infection. provided evidence that splenic CD11c+ DCs activated in vitro or in vivo are potent stimulators of IFN-g production by naive CD4+ T cells (40, 52–55), we therefore used an in vitro priming approach to further investigate whether the phenotype observed in P. yoelii 17XL infection was linked to the impact of MKP5 deficiency on the stimulatory capacity of DCs, or a result of the direct regulation on T cell function. IFN-g levels in the single cultures of purified DCs from infected mice were nominal and comparable, excluding the possibility that the increased IFN-g was directly produced by KO DCs or the small population of contaminating cells copurified. In contrast, it was notable to observe that KO DCs cultured with either KO CD4+ T cells or WT CD4+ T cells produced significantly greater extent of IFN-g + (. 5-fold) than did the WT DCs with either KO CD4+ T cells FIGURE 6. IFN-g production in cocultures of splenic CD11c DCs and + or WT CD4+ T cells (KO DCs plus KO CD4+ T versus WT DCs CD4 T cells. Splenic DCs from day 2–infected KO and WT mice were + + purified by magnetic sorting and were cultured in medium alone or plus KO CD4 T cells, p , 0.05; KO DCs plus WT CD4 T cells + + , cocultured with naive CD4 T cells enriched from spleens of uninfected versus WT DCs plus WT CD4 T cells, p 0.01), whereas KO and WT mice, respectively. After 5 d of incubation, supernatants were + the levels were not significantly affected by CD4 T cells from assayed for IFN-g production by ELISA. Data are presented as means 6 either KO or WT mice (Fig. 6), indicating that DCs from KO mice SEM (n = 4–5 mice/group) from one of two replicate experiments. *p , are associated with the substantial increase in IFN-g production. 0.05, **p , 0.01. 3692 ROLE OF MKP5 IN MALARIA INFECTION

Immunization with malaria Ag enhances protection against bility that KO mice acquired more effective immune protection P. yoelii 17XL challenge in KO mice in response to immunization with malaria Ag than did the WT mice. In an attempt to investigate the modulatory effect of MKP5 on vaccination-induced protection, KO and WT mice were immu- Enhanced protective immune responses induced by nized with soluble P. yoelii 17XL Ag and subsequently chal- immunization in KO mice lenged with pRBCs obtained from a homologous donor. The Because a stronger IFN-g response is associated with increased re- courses of infection were monitoredfor5wkinAg-immunized sistance to acute blood-stage P. yoeli i 17XL during a primary in- and adjuvant control mice of both genotypes (Fig. 7A, 7B). fection, cellular immunity induced by anti-malaria vaccination was Similar to our previous results, both groups of control mice evaluated in the four groups of mice. Following immunization with suffered 100% mortality, although KO mice developed signif- P. yoeli i 17XL Ag, spleen cells from either KO or WT mice produced icantly lower levels of peak parasitemia than did their WT elevated levels of IFN-g in response to pRBC lysate compared with counterparts at day 5 p.i. (p , 0.01) and survived longer (p , the responses of the control groups. However, following immuniza- 0.05). In marked contrast, strong protection was conferred in tion with Ag, KO mice exhibited significantly increased (.7-fold) terms of highly reduced parasitemia and significantly increased parasite-specific IFN-g production compared with WT mice (36.10 6 survival in both KO and WT vaccinated mice (both p , 0.01 for 7.225 versus 4.915 6 1.172 ng/ml, p , 0.05) (Fig. 7C). Immuniza- comparison of survival rates). Interestingly, although the dif- tion with Ag also resulted in higher expression of IL-4 in the KO ference was not statistically significant, only 80% (four of five) group (0.8646 6 0.0546 versus 0.3688 6 0.0542 ng/ml, p , 0.01) of the immunized WT mice survived the challenge whereas (Fig. 7D). Furthermore, we observed that the total malaria-specific Ab 100% of KO mice recovered completely (Fig. 7A). More im- production, as well as that of IgG1 and IgG2c, was significantly in- Downloaded from portantly, it was noted that following immunization, the para- creased in immunized animals compared with the levels detected in sitemia in WT mice rose slowly to between 20 and 30%, peaked control mice prior to challenge. These data indicated the induction approximately at day 14, and subsided by day 32. In contrast, of humoral immune responses by immunization, and similar to the almost complete protection was achieved in the KO mice, all of cellular responses observed, the Ab levels were significantly higher in which developed extremely low-grade parasite burdens, with immunized KO mice than in their WT counterparts (p , 0.001 for the peak parasitemia reaching only 2.64% (2.420 6 1.288% total IgG, p , 0.001 for IgG1, and p , 0.01 for IgG2c) (Fig. 7E). http://www.jimmunol.org/ versus 26.47 6 2.806%, p , 0.001), and all animals in this Collectively, these findings indicate that in the absence of MKP5, group were found to be virtually free from parasites as early as immunization with the parasite Ag confers significantly enhanced day 18 after challenge (Fig. 7B). These results raise the possi- protective immunity against blood-stage P. yoelii 17XL challenge. by guest on September 28, 2021

FIGURE 7. Course of blood-stage P. yoelii 17XL challenge and protective responses elicited by immunization of MKP5 KO and WT mice. Groups of KO and WT mice were immunized and boosted twice with 50 mg soluble P. yoelii 17XL Ag emulsified in Freund’s adjuvant. Control mice were immunized with adjuvant alone. Two weeks after the last immunization, mice were either challenged i.p. with 5 3 106 pRBCs obtained from a homologous donor or sacrificed for detection of protective immunity induced by vaccination. (A) Survival rates and (B) parasitemia of Ag-immunized and adjuvant control mice of both genotypes were monitored after challenge. Spleen cells from groups of immunized and control mice were cultured in presence of anti-CD3 plus anti-CD28 (+), pRBC lysate (Ag), or medium alone (m). Levels of (C) IFN-g and (D) IL-4 in supernatants were quantified by ELISA. (E) Levels of total IgG, IgG1, and IgG2c specific for parasite Ag present in prechallenge sera of KO and WT mice were determined by ELISA. The levels of IgG isotypes were expressed as absorbance (OD450). All the results are expressed as means 6 SEM (n = 5 mice/group) from one of two replicate experiments. *p , 0.05, ** p , 0.01, *** p , 0.001 for comparison between Ag-immunized KO and WT mice. #p , 0.05, ##p , 0.01 for comparison between adjuvant control KO and WT mice. The Journal of Immunology 3693

Infection of MKP5 KO and WT mice with a nonlethal strain ences of any anti-inflammatory cytokines were observed between P. yoelii 17XNL infected KO and WT mice (Fig. 8E, 8F). These data suggest Previous study demonstrated that an early proinflammatory re- that, despite an accelerated clearance of parasites, the enhanced sponse is sufficient for the resolution of infection with nonlethal responses mediated by MKP5 deficiency might cause immuno- P. yoelii in C57BL/6 mice (32), which raised the hypothesis that pathology and consequently a lethal effect in mice when chal- MKP5 deficiency could induce exaggerated inflammation in this lenged with the nonlethal P. yoelii 17XNL. process. Therefore, we infected KO and WT mice with the non- lethal strain, P. yoelii 17XNL. As expected, most WT mice (75%) Discussion developed moderate parasitemia and recovered completely within MKP5 is recognized as an essential negative regulator of the JNK 4 wk. In contrast, although KO mice developed lower parasite signaling pathway that plays an important role in both innate and burdens over the entire course, all succumbed to the infection adaptive immune responses (36). Apart from the correlation with between days 15 and 21 p.i. (Fig. 8A, 8B). Then, we detected T cell–mediated immunity against secondary infection with lym- serum cytokine levels during the infection. Unlike the lethal phocytic choriomeningitis virus, little is known of the pathogenic P. yoelii 17XL challenge, both KO and WT mice showed a marked or protective roles of MKP5 in protozoan parasite infections. increase in IFN-g production as early as day 3 p.i. (p , 0.001), In the present study, we analyzed the influence of MKP5 on but the level dramatically increased at day 5 p.i. and peaked earlier the course of blood-stage P. yoelii 17XL (lethal) infection in an in KO mice, compared with WT controls (p , 0.05) (Fig. 8C). Mkp52/2 mouse model. As expected, our data provided in vivo Additionally, serum TNF-a levels in KO mice were greater than in evidence that MKP5 is involved in the regulation of host resis- WT mice at days 8 and 11 p.i. (p , 0.05) (Fig. 8D). No differ- tance to the parasite challenge. Both MKP5 KO and WT B6 mice Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. Course of infection and cytokine production in MKP5 KO and WT C57BL/6 mice infected with nonlethal P. yoelii 17XNL. Mice were infected i.p. with 1 3 105 pRBCs and the course of infection was followed by monitoring (A) percentage survival and (B) parasitemia. Serum levels of (C) IFN-g,(D) TNF-a,(E) TGF-b, and (F) IL-10 were determined by ELISA. Data are presented as means 6 SEM (n = 4 mice/group) from one of two replicate experiments. *p , 0.05, ***p , 0.001. 3694 ROLE OF MKP5 IN MALARIA INFECTION were found to be susceptible to primary infection. However, in more efficient control of parasitemia in MKP5-deficient mice, contrast to the WT controls, which succumbed rapidly to hyper- leading to the prolongation of survival during the early stage of parasitemia and acute anemia, KO mice displayed slowly increas- infection. On the basis of the stimulatory role of activated DCs in ing and significantly lower parasite burdens, with most exhibiting IFN-g production by naive CD4+ T cells in murine malaria models relatively prolonged survival after the initial acute phase. These (40, 53–55), as well as the demonstration that APCs from KO observations indicate that MKP5 deficiency has a significant effect mice are hyperresponsive to LPS and exhibit increased priming on the early control of P. yoelii 17XL infection. Additionally, capabilities (36), two possible mechanisms may account for the during the chronic stage, the parasite burdens in surviving KO differing IFN-g response derived from CD4+ T cells in the two mice fluctuated at relatively low levels (4.83–15.20%). However, groups of mice: one is the direct influence of MKP5 deficiency on the observation that these mice failed to recover from the infection T cell activity, the other is the indirect modulation on DC function. in the long term was somewhat unexpected, and the underlying By using an in vitro coculture approach to mimic the initial in- cause for the irreversible anemia in this model remains to be teraction between the parasite-activated DCs and naive CD4+ clarified. Given the potent immunoregulatory properties of MKP5, T cells, we found that splenic DCs from infected KO mice incu- it is reasonable to speculate that immunopathology may be linked bated with CD4+ T cells could produce higher levels of IFN-g, to the inability to resolve the chronic infection in KO mice. Taken whereas the different sources of CD4+ T cells did not affect the together, these results demonstrate that anti–P. yoelii 17XL im- response levels, suggesting that the ability of DCs to induce CD4+ munity is partially enhanced in the absence of MKP5, thus indi- T cell IFN-g production is significantly enhanced in the absence of cating that the MKP5-dependent pathway represents a potential MKP5 early after P. yoelii 17XL infection. Additionally, the ac- regulator of protection against malaria infection. tivation of parasite-specific CD8+ T cells observed in both KO and Downloaded from The ability to establish a precise balance between the proin- WT mice implied the existence of cross-presentation of malaria flammatory and regulatory immune responses is critical to guar- Ags by DCs (60, 61). antee the parasite clearance and successful resolution of malaria On the basis of the potential to induce a stronger protective (32, 33). Therefore, to elucidate the immunological mechanism un- response to primary blood-stage P. yoelii 17XL infection, we derlying the different resistance between KO and WT mice, we hypothesized that MKP5 deficiency may enhance immunization-

analyzed the effects of MKP5 deficiency on the key molecules induced protection against blood-stage malaria (62). To investi- http://www.jimmunol.org/ that might participate in the responses of the effective protection gate this possibility, we used soluble P. yoelii 17XL Ag emulsified against Plasmodium infection (20, 21, 32, 35, 56). Interestingly, in Freund’s adjuvant as an experimental malaria vaccine. Using we found that IFN-g production during the early stage of primary a prime-boost approach, we observed a significantly less severe P. yoelii 17XL infection was significantly elevated in KO mice course of Plasmodium challenge infection in KO mice, as dem- compared with their WT counterparts. Conclusive evidence for the onstrated by greatly reduced parasitemia and accelerated clear- pivotal role of IFN-g in controlling acute parasitemia and survival ance of parasites in comparison with WT immunized mice. As of mice is well established in Plasmodium chabaudi or Plasmo- previously reported, Mkp52/2 mice showed increased production dium berghei infections (27, 29, 57, 58), and studies in human of IFN-g and IL-4 in response to vaccination with keyhole limpet malaria also demonstrate that IFN-g is associated with protection hemocyanin in CFA (36). Similarly, we observed that spleen cells by guest on September 28, 2021 (49, 56, 59). However, the importance of this cytokine in P. yoelii from immunized KO mice produced significantly higher levels of infection remains a matter of controversy, which is likely to be IFN-g as well as Th2-polarized IL-4 in response to malaria Ag attributable to the differences in parasite virulence and technical than did their WT counterparts. Despite these differences, sig- approaches (30–32, 41). To address this controversy, we tested the nificantly higher levels of Ag-specific total IgG as well as Th1- definitive role of the increased IFN-g in protection by adminis- associated IgG2c and Th2-associated IgG1 responses were de- tering a neutralizing mAb to KO mice prior to and during the early tected in immunized KO mice than WT mice prior to infection. course of P. yoelii 17XL infection (22, 58). Our results clearly These observations indicate that, in the absence of MKP5, malaria- showed that anti–IFN-g treatment resulted in substantially higher specific cellular as well as humoral responses were significantly parasitemia in these mice and fatalities occurred much earlier than augmented by anti–P. yoelii 17XL immunization. As a consequence those treated with irrelevant Ab. Thus, our findings strongly of this, the protective efficacy against subsequent challenge was support the conclusion that optimal levels of proinflammatory enhanced in the murine host. Overall, these results have important cytokines are essential for the protective immunity against the implications not only for the regulation of MKP5 in T cell–medi- initial phase of primary P. yoelii 17XL challenge, and most im- ated immunity elicited by immunization, but also in extending its portantly, they point to an IFN-g–mediated regulatory role of immunoregulatory role to Ab-mediated immunity. Because selec- MKP5 in the immune resistance to this infection. tion of potent adjuvants capable of improving the immune efficacy Meanwhile, by phenotypic characterization of the cellular is an important aspect of malaria vaccine development, this study sources of IFN-g, we noted in both mouse strains that NK cells provides a piece of information that MKP5 may be a potential target contribute significantly to the earliest IFN-g response (within 2 d; in such application. However, there are many limitations of animal data not shown), whereas ab T lymphocytes represent the pre- models for testing malaria vaccines, and there is still much to be dominant contributors at later time points (e.g., day 4 p.i.). These learned about the biological effects of MKP5 signaling pathway in data are consistent with the previous observations on other humans, the safety issues must be carefully addressed. If possible, malaria models (28, 48) and suggest that there is little direct by reducing the likelihood of adverse effects, the transient down- effect of MKP5 on the kinetics of IFN-g production during the regulation approach performed by MKP5 antagonists at proper acute phase of infection. However, of all the lymphocyte subsets times might serve as part of the vaccination strategies to alleviate analyzed in this study, we found a specific and significant increase the burden of malaria. in the number of IFN-g–producing CD4+ T cells in infected KO MKP5 deficiency greatly enhances IFN-g-dependent early cell- mice compared with WT mice, whereas other cytokine responses in mediated protective response as well as vaccination-induced pro- these cells and IFN-g production by other cell types were minimally tection against the lethal Plasmodium yoelii 17XL infection. How- affected, which strongly suggest that the augmented IFN-g re- ever, considering the effect of the immunological balance on sponse mediated by CD4+ T cells may confer, at least partially, infection outcomes in hosts as demonstrated previously (32), we The Journal of Immunology 3695 further evaluated the influence of MKP5 deficiency on the non- receptor signaling in prevention of Th1-mediated immunopathology during malaria infection. J. Immunol. 185: 2482–2492. lethal strain P. yoelii 17XNL. Consistent with the finding that an 16. Ing, R., P. Gros, and M. M. Stevenson. 2005. Interleukin-15 enhances innate and early and strongly protective response is sufficient for controlling adaptive immune responses to blood-stage malaria infection in mice. Infect. infection in WT mice, we found the lack of MKP5 resulted in an Immun. 73: 3172–3177. 17. Li, C., I. Corraliza, and J. Langhorne. 1999. 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