c mice

Experimental Parasitology 134 (2013) 341–348

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Effect of IL-22 on DNA vaccine encoding LACK gene of Leishmania major in BALB/c mice Hajar Ziaee Hezarjaribi a, Fatemeh Ghaffarifar a,⇑, Abdolhosein Dalimi a, Zohreh Sharifi b, Ogholniaz Jorjani c a b c

Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran Laboratory Science Research Center, Golestan University of Medical Sciences, Gorgan, Iran

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 It was shown that IL-22 brought

Survival rate of immunized and control groups of mice during 30 weeks after the challenge with 2  106 promastigotes of Leishmania major.

about Th1 cytokine responses.  IFN-c level in the LACK + IL-22 group was significantly higher than LACK group.  IL-4 level in the LACK + IL-22 group was significantly lower than LACK group.  Results show effectiveness of IL-22 in survival rate.

a r t i c l e

i n f o

Article history: Received 29 June 2012 Received in revised form 15 March 2013 Accepted 17 March 2013 Available online 27 March 2013 Keywords: Leishmania major DNA vaccine pcLACK IL-22

a b s t r a c t In the present study, the effect of IL-22 together with the plasmid encoding LACK (Leishmania homolog of receptors for activated C-kinase) gene of Leishmania major on the trend of leishmaniasis in BALB/c mice was evaluated. Evaluation of the cellular and humoral immunity was performed by measurement of IL-4 and IFN-c, culture of splenocytes and MTT assay, and measurement of total IgG, IgG1, and IgG2a in the control and immunized groups. Clinical evaluations were also carried out by measurement of the lesion size, survival rate, and body weight of mice. Comparison of the mean size of lesions in the LACK and LACK + IL-22 groups demonstrated that the mean size of lesions of the two groups was significantly different from week four (p < 0.05). The survival rate at day 170 after challenge for the PBS, pcDNA3 (empty plasmid), pcLACK (pcDNA3 containing LACK gene), and pcLACK + IL-22 groups were 20%, 40%, 60%, and 80%, respectively. According to the results of IFN-c, IL-4, total IgG, IgG1, and IgG2a measurement and the MTT assay, IL-22 obviously caused an increase in IFN-c production and a decrease in IL-4 production before and after the challenge (p < 0.05). The results showed the effectiveness of IL-22 in DNA vaccine. It showed that IL-22 brought about Th1 cytokine responses and high survival rate of mice. Ó 2013 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Address: Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-331, Tehran, Iran. Fax: +98 (21) 82884555. E-mail address: [email protected] (F. Ghaffarifar). 0014-4894/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2013.03.012

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1. Introduction Leishmaniasis is a widespread disease, reported from almost all countries of the world. So far, no effective vaccine or drug for control of the disease, and appropriate chemical method for definite fight against the vector is available (Reed, 2001; Duthie et al., 2012; Wolff et al., 1990). In past decades, the results of numerous studies about the appropriate vaccine have been publicly released. DNA vaccines are powerful methods for induction of the specific cellular and humoral immune response. Sometimes, these vaccines are used in combination with plasmids encoding exogenous cytokines (such as IL-6, IL-7, or IL-12) or chemokines as the regulators of the immune response, the immune stimulator molecules against the antigen encoded by the DNA (Bhopale, 2003; Kofta and Wedrychowicz, 2001; Hoseinian Khosroshahi et al., 2012). The LACK (Leishmania homolog of receptors for activated C-kinase) antigen of Leishmania major (L. major) is a 36-KDa protein, consisting of 313 amino acids, and is present in the promastigote and amastigote of different strains of Leishmania (Kemp et al., 1994; Antonio et al., 2006; Perez-Jimenez et al., 2006; Maillard and Launois, 2001). The immunogenicity of DNA of LACK/p36 against leishmaniasis was confirmed (Lopez-Fuertes et al., 2002; Ahmed et al., 2004; Tapia and Perez-Jimenez, 2003; Gurunathan and Sacks, 1997). The LACK antigen, that is highly conserved among related Leishmania species, is expressed in both promastigote and amastigote forms of the parasite (Mugneau et al., 1995). LACK is a target for the early anti-parasite immune response (Julia and Glaichenhaus, 1999). In a study comparing different DNA vaccine candidates, it was demonstrated that the most promising gene is LACK (Ahmed et al., 2004). LACK vaccination trials using protein or DNA vectors show protection against cutaneous L. major infections by a protective Th1 response (Mugneau et al., 1995; Afonso et al., 1994). Cocktail DNA vaccine containing LACK gene increased the cellular response and survival rate and induced protection against infection with L. major in the mice (Ghaffarifar et al., 2012). IL-22 is a cytokine belonging to the IL-10 family, is an effective inhibitor of cellular immunity and a potent enhancer of humoral immunity (Dumoutier et al., 2001). IL-22 is secreted by Th-17, particularly natural killer (NK) cells, Th22 (CCR10+), and Th1 cells

immediately after activation of IL-4. IL-22 activates JAK1, TYK2, STAT1, STAT3, STAT5, and the MAP kinase pathway (Conti et al., 2003; Igwa et al., 2006; Dumoutier et al., 2001; Nagem et al., 2002). It has been shown that cutaneous keratinocytes have receptors for this cytokine. In the skin, IL-22 induces antimicrobial peptides, enhances the proliferation and inhibits the differentiation of keratinocytes, and plays a role in wound healing and innate immunity mechanism. The role of IL-22 in skin disorders such as atopic eczema and allergic contact dermatitis is unknown (Boniface et al., 2005). The IL-22 produced by NK cells inhibits the growth of Mycobacterium tuberculosis via activation of macrophages and increases the phagolysosome activity (Dhiman et al., 2009). In a study, it was demonstrated that the people with higher levels of IL-22 have a higher level of resistance to human Kala-azar. It was reported that Leishmania donovani stimulates the differentiation of Th17 cells for production of IL-22 and IFN-c, and IL-22 has a protective effect against human Kala-azar (Pitta et al., 2009). The factors related to definite treatment and eradication of L. major and also control of its arthropod vector are not still determined. Furthermore, the role of IL-22 in development of protection and innate immunity mechanisms particularly for intracellular bodies has been recently confirmed. Therefore, the current study was performed to know whether IL-22 influences the DNA vaccine effect or not. 2. Materials and methods 2.1. Plasmid construction We used a recombinant pcDNA3 expression plasmid that contained LACK gene (939 bp) and encodes a 36-kDa antigen. The plasmid was constructed in the Department of Parasitology, Tarbiat Modares University, Tehran, and its expression was previously evaluated by Jorjani et al. (2012). Primary and mass culture of the plasmid containing LACK gene and pcDNA3 was carried out in TOP10 bacterium on the LB Broth culture medium (LB Broth, Miller, Luria–Bertani, Germany). The plasmid was extracted by the endo-free plasmid purification Mega Kit (QIAGEN, Germany). 2.2. Preparation and injection of IL-22 Murine recombinant IL-22 protein (Cat. Number 582-ML; R&D) was prepared in sterile PBS containing 1% BSA according to the manufacturer’s protocol. IL-22 (5 ng per g body weight of mice in 100 ll) was injected in the quadriceps muscle using 30-gauge insulin syringes (Ziaee Hezarjarib et al., 2012). Since BLAB/c mice are susceptible to promastigotes of L. major, we bought 60 female inbred BALB/c mice with the age of eight weeks from the Razi Vaccine and Serum Research Institute. The mice were divided into four groups, two control and two experimental groups (15 mice in each group). Each group was divided to three subgroups, five mice in each subgroup: one subgroup was vaccinated without being infected by the parasite (20 mice) and the second and third subgroups received vaccination and then were challenged by the parasite (40 mice). 2.3. Vaccination

Fig. 1. Mean values of body weight (g) in vaccinated groups in three doses with three-week intervals with pcLACK (50 lg in 100 ll), pcLACK (50 lg in 100 ll) plus one dose of IL-22 (5 ng per g body weight of mice), and control groups, PBS (100 ll) and pcDNA3 (50 lg in 100 ll), as second control group, 7 and 30 weeks after challenge in comparison with uninfected control mice.

Sixty female BALB/c mice of eight-week age were divided into four groups. Group 1 received PBS (100 ll) in three doses with three-week intervals, as first control group; group 2 received pcDNA3 (50 lg in 100 ll) in three doses with three-week intervals, as second control group; group 3 received pcLACK (50 lg in 100 ll,

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Fig. 2. Mean and SD of lesion size (mm) in vaccinated groups in three doses with three-week intervals with pcLACK (50 lg in 100 ll), pcLACK (50 lg in 100 ll) plus one dose of IL-22 (5 ng per g body weight of mice), and control groups, PBS (100 ll) and pcDNA3 (50 lg in 100 ll), as second control group, seven weeks after the challenge with 2  106 L. major promastigotes. pcDNA3 = Empty plasmid. LACK = pcDNA3 plasmid containing LACK gene. LACK22 = pcDNA3 plasmid containing LACK gene + IL22.

Fig. 3. Survival rate of vaccinated groups in three doses with three-week intervals with pcLACK (50 lg in 100 ll), pcLACK (50 lg in 100 ll) plus one dose of IL-22 (5 ng per g body weight of mice), and control groups, PBS (100 ll) and pcDNA3 (50 lg in 100 ll), as second control group, during 30 weeks after the challenge with 2  106 promastigotes of Leishmania major. pcDNA3 = Empty plasmid. LACK = pcDNA3 plasmid containing LACK gene. LACK-22 = pcDNA3 plasmid containing LACK gene + IL22.

eluted in PBS) in three doses with three-week intervals and group 4 received pcLACK (50 lg in 100 ll, eluted in PBS) in three doses with three-week intervals, plus one dose of IL-22 (5 ng per g body weight of mice) prior to the challenge. The injection was done in the quadriceps muscle (I.M.) using 30-gauge insulin syringes (Kellum, 1986). 2.4. Challenge with promastigotes of L. major Seven weeks after the last immunization, second and third subgroups were infected experimentally with L. major. The mice were challenged with 2  106 of stationary phase of L. major promastigotes (standard strain MRHO/IR/75/ER). The promastigotes were inoculated subcutaneously in the tail base of the mice. 2.5. Evaluation of humoral immunity Blood sampling was performed four times; time zero (before vaccination), three weeks after the second immunization, seven

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weeks after the third vaccination, and seven weeks after the challenge. The sampling was performed by non-heparinized capillary tubes from the retro-orbital plexus of mice. The blood sera were collected for evaluation of the humoral immunity, and kept at 20 °C. Evaluation of humoral immunity was performed on the sera obtained from immunized and control mice by measurement of total IgG using ELISA test. Moreover, the antibody subtypes (IgG1 and IgG2a) were measured by Mouse Monoclonal Antibody Isotyping Reagent Kit (SigmaÒ, USA). For ELISA test, at first we prepared SLA (Soluble L. major Antigen). The SLA was prepared from L. major according to the method of Scott (Scott et al., 1987). Briefly, promastigotes of L. major were washed three times in cold PBS (pH 7.5). Then, 1 mM phenyl methyl sulfonyl fluoride (PMSF) was added to the promastigotes pellet, and the suspensions were frozen and thawed six times in PBS, and then sonicated at 4 °C with 20 cycles at 2-s blasts, followed by centrifugation at 100,000g for 2 h at 4 °C. Contained SLA was passed through a 0.22-lm filter for sterilization and stored at 70 °C until being used (Scott et al., 1987). The protein concentrations were measured by the Bradford method (Bradford, 1976). In ELISA test, 20 lg/ml of SLA Ag and 1/10 dilution of vaccinated mice sera were used. 2.6. Extraction of lymphocytes from mouse spleen To evaluate IL-4 and IFN-c levels, the cell supernatant of spleen lymphocytes obtained from immunized and control (challenged and unchallenged) mice was used. Splenocytes were cultured according to the following method. In different groups, the five control mice and immunized mice were killed before the challenge and seven weeks after the challenge. Lymphocytes were extracted from the spleen under sterile condition and cultured in RPMI 1640 (Gibco, BRL, Maryland, USA) and 20% FCS (Gibco, BRL, Maryland, USA) plated in duplicate in 24-well plates (Nalge-Nunc International, Rochester, NY, USA) at 2  106 cells/ml, then allowed to multiply for 72 h in the medium alone (control group), in the presence of SLA with concentration of 40 lg/ml, or 10 lg/ml of PHA (Sigma–Aldrich, Deisenhofen, Germany) as mitogen and incubated at 37 °C and 5% CO2. After that, the cell supernatant was slowly collected at hour 72. Finally, the supernatant was aliquoted in 300-ll volumes in vials and was kept at 70 °C until cytokine assay (Farnandez-Botran and Vetyickaz, 2001). 2.7. Cytokine assay In vaccinated subgroup without challenge (seven weeks after the third vaccination) and in vaccinated subgroup with challenge (seven weeks after the challenge), the cytokine assay was performed. IFN-c and IL-4 levels of the cellular supernatant prepared from the spleen lymphocytes of immunized and control mice were measured by the ELISA method, using U-CyTech biosciences (Netherlands). According to the standard measures included in the kit, the standard diagrams were drawn and the samples were evaluated. 2.8. MTT assay for evaluation of lymphocyte proliferation MTT assay was used for evaluation of lymphocyte proliferation as follows. First, 100 ll of the splenocyte suspension from each mouse was added to each well of a 96-well plate (i.e., 3  105 cells per well) and cultured in three wells. Then, 40 lg/ml of the SLA was added to each well. The final volume of each well reached 200 ll by adding RPMI + FBS 10% The cultured plates were kept in a 5% CO2 incubator at 37 °C for 72 h. After the incubation period, 200 ll lymphocyte and 20 ll of the MTT solution (5 mg/ml) were added to each well. The plate

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A

Indicates statistically significant differences between the marked group with PBS and pcDNA3 control groups according to the Mann-Whitney test results (P<0.05). Indicates statistically significant difference between the marked group with the other groups according to the Mann-Whitney test results (P<0.05).

B

Indicates statistically significant difference between the marked group with the other groups according to the Mann-Whitney test results (P<0.05). Fig. 4. Mean and SD values of INF-c (A) and IL-4 (B) (pg/ml) produced by splenocyte culture treated with 40 lg/ml of the SLA for 72 h from control and immunized mice in both; vaccinated subgroup without challenge (seven weeks after the third vaccination) and in vaccinated subgroup seven weeks after the challenge, by ELISA kit.

was again kept in the 5% CO2 incubator at 37 °C for 4 h (until the Formosan crystals appear). Without stirring the content, the cell suspension was slowly centrifuged and the supernatant was completely removed. Then, 100 ll of DMSO was added to each well. The absorbance values of the samples were read at 540 nm by ELISA reader (Verma et al., 2010).

using SPSS software. The mean and standard deviation values were calculated to describe the data and to calculate the central indices and distribution of the data. The non-parametric independent groups were compared using Mann–Whitney test.

3. Results 2.9. Measurement of lesion size and weight of mice

3.1. Measurement of weight in immunized animals

After the challenge and emergence of the lesion at the tail base of mice, the size of lesions was weekly measured with a digital caliper, such that the sum of length and width of the lesion was divided by two and considered as the size of the lesion. During the experiment, the body weight of mice was weekly measured with a digital balance. The survival rates of the vaccinated and control mice were weekly measured after the parasite challenge.

The mean weight of mice in the pcLACK, and pcLACK + IL-22 groups within 7–30 weeks after the challenge showed a regular increase in comparison with the control groups (Fig. 1). The mean weight in the pcLACK and pcLACK + IL-22 groups indicated that the mean increase in the pcLACK + IL-22 group was higher than that in the pcLACK group.

2.10. Statistical analysis

3.2. Lesion status

The results of measurement of IFN-c, IL-4, total IgG, MTT, IgG1, and IgG2a, and also lesion size and survival of mice were analyzed

Lesions with the mean size of 0.2 mm occurred 44 days after the challenge in the pcLACK + IL-22 groups, while in the pcDNA3, PBS,

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A

Indicates statistically significant differences between the marked group with PBS and pcDNA3 control groups according to the Mann-Whitney test results (P<0.05). Indicates statistically significant difference between the marked group with the other groups according to the Mann-Whitney test results (P<0.05).

B

According to the Mann-Whitney test results, there isn’t any statistically significant difference between all groups (P>0.05). Fig. 5. Mean and SD of OD values of total IgG (A), IgG1 (B), and IgG2a (C) in sera obtained from mice in three steps (after second vaccination, after third vaccination, and after challenge with L. major) in different groups. (A) Total IgG detected by ELISA in sera of immunized mice in three steps (after second vaccination, after third vaccination, and after challenge) (values are expressed as mean ± SD). (B) IgG1 detected by ELISA in sera of immunized mice in three steps (after second vaccination, after third vaccination, and after challenge) (values are expressed as mean ± SD). (C) IgG2a detected by ELISA in sera of vaccinated mice in three steps (after second vaccination, after third vaccination, and after challenge) (values are expressed as mean ± SD).

and pcLACK groups, lesions with the mean size of 2.55, 2.82 and 1.08 mm were observed 30 days after the challenge, respectively. Comparison of the mean size of lesions in the LACK and LACK + IL-22 groups demonstrated that the mean size of lesions of the two groups was significantly different 4 weeks after the challenge (p < 0.05). The mean and SD of lesion size in vaccinated and control groups 7 weeks after the challenge is shown in Fig. 2.

3.3. Evaluation of survival rate Before day 51, survival rate was 100% in all the groups. The survival rate was 80% in day 51 of the PBS and pcDNA3 groups, day 72 for the LACK group, and day 163 for the LACK + IL-22 group. On day 170, the survival rates were 20% and 40% for the PBS and pcDNA3 groups, respectively. On the same day, the survival rates were 60% and 80% for the LACK and LACK + IL-22 groups, respectively. The

mortality rates were 100% for the PBS and pcDNA3 groups in days 184 and 191 after the challenge, respectively (Fig. 3). 3.4. Measurement of cytokine assay According to the results obtained from the Mann–Whitney test, in different groups, the level of IFN-c changed significantly during the experiment (p < 0.05) (Fig. 4A). Furthermore, the LACK + IL-22 group was compared with the LACK group in the mean levels of IFN-c in both subgroups without and with challenge. The results indicated that although the mean level of IFN-c in the LACK + IL-22 group increased after the challenge, the two groups were significantly different in this respect (p < 0.05) (Fig. 4A). According to the results obtained from the Mann–Whitney test and comparison of the mean levels of IL-4 in both subgroups without and with challenge, the mean IL-4 levels in the LACK + IL-22

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C

Indicates statistically significant differences between the marked group with PBS and pcDNA3 control groups according to the Mann-Whitney test results (P<0.05). Indicates statistically significant difference between the marked group with the other groups according to the Mann-Whitney test results (P<0.05). Indicates statistically significant difference between the marked group with PBS control group according to the Mann-Whitney test results (P<0.05). Fig. 5. (continued)

Fig. 6. Mean and SD of MTT colorimetric assay (OD) for spleenocyte culture treated with 40 lg/ml of the SLA for 72 h in vaccinated groups in three doses with three-week intervals with pcLACK (50 lg in 100 ll), pcLACK (50 lg in 100 ll) plus one dose of IL-22 (5 ng per g body weight of mice), and control groups, PBS (100 ll) and pcDNA3 (50 lg in 100 ll), as second control group, without and after the challenge. indicates statistically significant difference between the marked group with PBS control group according to the Mann–Whitney test results (p < 0.05). indicates statistically significant differences between the marked group with PBS and pcDNA3 control groups according to the Mann–Whitney test results (p < 0.05). indicates statistically significant difference between the marked group with pcLACK group according to the Mann– Whitney test results (p < 0.05).

group was significantly lower than those in the LACK group (p < 0.05) (Fig. 4B).

3.5. Evaluation of the level of the anti-Leishmania major total IgG, IgG1, and IgG2a Mean and standard deviation of OD values of total IgG, IgG1, and IgG2a in time zero before vaccination were 0.098 ± 0.04, 0.14 ± 0.03, and 0.15 ± 0.04, respectively. Mean and standard deviation of OD values of total IgG, IgG1, and IgG2a in different immunized and control groups after the second and third vaccination and after the challenge are provided in (Fig. 5A–C).

As shown by the Mann–Whitney test, the LACK and LACK + IL22 groups were significantly different in the mean level of IgG2a in step 2 (p < 0.05). However, the two groups were not significantly different in IgG1 level in any step. The difference between the two groups was statistically significant in total IgG only in step 3 (p < 0.05) (Fig. 5A–C).

3.6. Evaluation of MTT assay In all the groups, mean levels of MTT after the challenge were higher than the corresponding values before the challenge (Fig. 6). The results of the Mann–Whitney test and comparison of means indicated that the mean level of MTT in the LACK + IL-22 and LACK

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groups were significantly different after the challenge (p < 0.05) (Fig. 6).

4. Discussion Considering the results of IFN-g, IL-4, total IgG, IgG1, and IgG2a, it was observed that IL-22 obviously leads to increased IFN-c production and decreased IL-4 production. It was shown that IL-22 brought about Th1 cytokine responses. Furthermore, high survival rate, decreased mortality, significant weight gain, and reduced lesion size in mice that received IL-22 indicate the role of this cytokine in prevention of cutaneous leishmaniasis. Therefore, it is of great importance to choose an appropriate strategy for vaccination, which enhances the immunogenicity of T cells and produces protective cellular immunity response against the intracellular pathogens. Moreover, in design of new vaccines, we considered production of vaccines that stimulate a specific type of T-helper response (either Th1 or Th2) (Ivory and Chadee, 2004; Liu, 2003; Boniface et al., 2005). In the current study, IL-22 was used for the first time at the dose of 5 ng per g body weight of mice; since in evaluation of IL-22 at doses of 5 and 10 ng per g body weight of mice, in previous study we concluded that the 5-ng dose has a better effectiveness (Ziaee Hezarjarib et al., 2012). Also, in some studies, side effects have been reported for some doses of IL-22 (Boniface et al., 2005). It can be stated that the local effects of IL-22 in the skin in cutaneous leishmaniasis showed good results and healing of the lesion. Today, it has been confirmed that skin keratinocytes have receptors for IL-22. Furthermore, in a study, it was demonstrated that IL-22 induces antimicrobial peptides in the skin, and also enhances the proliferation of keratinocytes and inhibits their differentiation, and plays a role in the innate immunity mechanism and wound healing (Boniface et al., 2005). The mean weight of mice in the LACK and LACK + IL-22 groups had a regular increase in comparison with the control groups of PBS and pcDNA3, and the difference was statistically significant (p < 0.05). In other words, using LACK + IL-22 was more effective than LACK in improvement of the weight of mice. Therefore, it can be mentioned that IL-22 performed better in improvement of weight of mice. Since determination of Th1 and Th2 pattern is a main in vivo evaluation (Bretscher et al., 1997; Mauel, 2002), we measured the IL-4 and IFN-c levels. If the Th2 response is established, IL-4, IL-5, and IL-10 would be secreted. When IL-10 is produced, it has an inhibitory effect on the macrophages and leads to exacerbation of the infection. By producing cytokines such as IFN-c, IL-22, and TNF-b2, the Th1 cells are mediators of healing. The IFN-c produced by these cells is the most powerful cytokine for activation of macrophages, which in turn leads to production of nitric oxide (NO) and killing of the parasite (Munder et al., 1998; Rodriguez and Whitton, 2000; Webb et al., 1998). The IL-4 level in the LACK + IL-22 group was significantly lower than that in the LACK group (p < 0.05). Furthermore, the study conducted by Gurunathan et al. confirmed that when the immune response moves toward the Th2 response and IL-4, the disease is aggravated (Gurunathan and Sacks, 1997). It should be considered that IL-4 has an inhibitory effect on the function of macrophages (Gurunathan and Sacks, 1997; Sasaki et al., 2003; Rodriguez and Whitton, 2000; Mcsorley et al., 1996; Scott, 1989). With regard to the humoral immunity response, the mean levels of IgG1 in LACK + IL-22 and LACK groups were similar to those in the control groups, and in any of the steps, the groups were not significantly different in this respect. With regard to the IgG2a level, LACK + IL-22 is higher than LACK (p < 0.05). This is while

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mouse serum collection for the first and second steps was performed before administration of IL-22. This shows the effectiveness and role of IL-22 in stimulation of the humoral immune system in groups with the LACK gene. Moreover, the difference between the pcLACK and pcLACK + IL22 groups in the MTT level was statistically significant only after the challenge (p < 0.05). This indicates the role of IL-22 in stimulation of the immune system against the parasite. 5. Conclusions In this study, the results showed that in the LACK + IL-22 brought about Th1 cytokine responses. IFN-c level in the LACK + IL-22 group was significantly higher than that in the LACK group. IL-4 level in the LACK + IL-22 group was significantly lower than that in the LACK group. The results showed effectiveness of IL-22 DNA vaccine containing pcLACK in increasing the survival rate. Therefore, we suggest further research on the effect of IL-22 on other types of DNA vaccine to understand more about the effectiveness of this cytokine. Conflict of interest The authors declare no conflict of interest. Acknowledgments This study was performed as a PhD thesis and supported by the Tarbiat Modares University. The project was authorized by the Ethic Committee of Tarbiat Modares University. References Afonso, L.C., Scharton, T.M., Vieira, L.Q., Wysocka, M., Trinchieri, G., Scott, P., 1994. The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science 263, 235–237. Ahmed, S.B., Bahloul, C., Robbana, C., Askri, S., Dellagi, K., 2004. A comparative evaluation of different DNA vaccine candidates against experimental murine Leishmaniasis due to L. major. Vaccine 22, 1631–1639. Antonio, E., Coelho, F., Tavares, C., Fernandes, A., Lima, K., Silva, C., Rodriguesjr, J., 2006. Mycobacterium hsp65 DNA entrapped into TDM-loaded PLGA microspheres induces protection in mice against Leishmania (L) major infection. Parasitol. Res. 98, 568–575. Bhopale, G.M., 2003. Development of a vaccine for Toxoplasmosis: current status. Microb. Infect. 5, 457–462. Boniface, K., Bernard, F.X., Garcia, M., Gurney, A.L., Lecron, J.C., Morel, F., 2005. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes. J. Immunol. 174, 3695–3702. Bradford, M.M., 1976. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Bretscher, P.A., Ogunremi, O., Menon, J., 1997. Distinct immunological states in murine cutaneous leishmaniasis by immunizing with different amounts of antigen: the generation of beneficial, potentially harmful, harmful and potentially extremely harmful states. Behring. Inst. Mitteilungen. 98, 1–11. Conti, P., Kempuraj, D., Frydas, S., Kandere, K., Boucher, W., Letourneau, R., Madhappan, B., Sagimoto, K., Christodoulou, S., Theoharides, T.C., 2003. IL-10 subfamily members: IL-19, IL-20, IL-22, IL-24 and IL-26. Immunol. Lett. 88, 171–174. Dhiman, R., Indramohan, M., Barnes, P.F., Nayak, R.C., Paidipally, P., Rao, L.V., Vankayalapati, R., 2009. IL22 produced by human NK cells inhibits growth of Mycobacterium tuberculosis by enhancing phagolysosomal fusion. J. Immunol. 183, 6639–6645. Dumoutier, L., Leemans, C., Lejeune, D., Kotenko, S.V., Renauld, J.C., 2001. Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. J. Immunol. 167, 3545–3549. Duthie, M.S., Raman, V.S., Piazza, F.M., Reed, S.G., 2012. The development and clinical evaluation of second-generation leishmaniasis vaccines. Vaccine 30, 134–141. Farnandez-Botran, R., Vetyickaz, V., 2001. Methods in Cellular Immunology, 2nd ed. CRF Press LNC. Ghaffarifar, F., Jorjani, O., Sharifi, Z., Dalimi, A., Hassan, Z., Tabatabaie, F., Khoshzaban, F., Ziaei Hezarjaribi, H., 2012. Enhancement of immune response induced by DNA vaccine cocktail expressing complete LACK and TSA genes against Leishmania major. APMIS 121, 290–298.

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