J Arthropod-Borne Dis, March 2021, 15(1): 133–142 A Miramin-Mohammadi et al.: Immune Responses in … 133 http://jad.tums.ac.ir Published Online: March 31, 2021 Original Article Immune Responses in Cutaneous Leishmaniasis: in vitro Thelper1/Thelper2 Cy- tokine Profiles Using Live Versus Killed Leishmania major Akram Miramin-Mohammadi 1 ; Amir Javadi 2 ; Seyyed Ebrahim Eskandari 1 ; Mahmood Nateghi-Rostami 3 ; *Ali Khamesipour 1 1Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran 2Department of Social Medicines, Qazvin University of Medical Sciences, Qazvin, Iran 3Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran *Corresponding author: Dr Ali Khamesipour, E-mail: Ali.khamesipour@gmail.com (Received 29 May 2019; accepted 14 Mar 2021) Abstract Background: Recovery from cutaneous leishmaniasis (CL) leads to protection against further lesion development. In contrast, vaccination using killed parasites does not induce enough protection; the reason(s) is not currently known but might be related to different immune response induced against live versus killed parasites. In this study, Th1/Th2 cyto- kine profiles of CL patients were evaluated against live versus killed Leishmania major. Methods: In this study peripheral blood mononuclear cells (PBMC) of the volunteers with active CL lesion (CL), histo- ry of CL (HCL) and healthy volunteers were cultured and stimulated with live or killed Leishmania major, the superna- tants were collected and levels of IFN-γ, IL-5 and IL-10 were titrated using ELISA method. Results: The results showed that IFN-γ levels in CL patients (p< 0.001) and HCL volunteers (p< 0.005) are signifi- cantly higher when stimulated with live than stimulated with killed L. major. IFN-γ production in PBMC volunteers with CL and HCL stimulated with live or heat-killed L. major was significantly (p< 0.001) higher than in unstimulated ones. The level of IL-5 in CL patients (p< 0.005) and HCL volunteers (p< 0.001) are significantly lower when stimulat- ed with live than killed L. major. There was no significant difference between the levels of IL-10 in PBMC stimulated with either live or killed L. major. Conclusion: It is concluded that using live Leishmania induces a stronger Th1 type of immune response which justify using leishmanization as a control measure against CL. Keywords: Cutaneous leishmaniasis; Live Leishmania; Heat-killed Leishmania; In vitro Immune response Introduction Leishmaniasis is a neglected tropical disease (1) caused by different Leishmania species and transmitted by the bite of various Phlebotomus species. The disease is endemic in 102 coun- tries, it is estimated that 10th of the world pop- ulation is at risk to contract one form of the disease, worldwide at least 12 million are af- fected and 1.5–2 million is the annual incidence. Over 90% of VL occurs in six countries of Bangladesh, Ethiopia, Brazil, India, Sudan, and South Sudan and 70–75% of CL cases occur in 10 countries including Afghanistan, Alge- ria, Brazil, Iran, Peru, Ethiopia, North Sudan, Costa Rica, Colombia and Syria. The burden of the disease (DAILYs) is reported to be 3.3 million (2, 3). Leishmaniasis is a major public health problem in some endemic foci, infec- tion with Leishmania parasites depends upon the Leishmania species causes the disease and the host genetic background which governs the immune response. The outcome of exposure to Leishmania might be asymptomatic or might induce a range of clinical manifestations from a simple self-healing skin lesion to non-heal- ing form of CL, diffuse CL, mucocutaneous leishmaniasis, a fatal systemic disease and post Copyright © 2020 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2021, 15(1): 133–142 A Miramin-Mohammadi et al.: Immune Responses in … 134 http://jad.tums.ac.ir Published Online: March 31, 2021 kala-azar dermal leishmaniasis (PKDL). Cur- rent available measures are not practically ef- fective to control leishmaniasis; there is am- ple evidence to prove that in principle devel- opment of a vaccine against leishmaniasis is possible. Numerous Leishmania antigens have been introduced as potential vaccine candidate and a few of first-generation vaccines have been tested in human clinical trials, but so far no vaccine is available against any form of hu- man leishmaniasis. Control measures are not effective and chemotherapy accompanies draw- backs and is not always effective. In experi- mental murine models of leishmaniasis, con- trol of infection and induction of protection require generation of Th1 type of immune re- sponse represented by production of a high lev- el of IFN-γ in the absence of Th2 response, in most of the mouse strains like C57BL/6 mice, infected with Leishmania major induce a self- healing lesion and upon healing the animals are protected against further lesion develop- ment, so protection accompanies development of Th1 response, whereas in susceptible BALB/c mice infected with L. major induces progres- sive lesions which eventually generalized and kills all the mice, the progressive disease oc- curs with development of a Th2 type of im- mune response with high levels of IL-4, IL-5 and IL-10 and no IFN-γ production (4, 5). In human leishmaniasis recovery from CL induc- es by natural infection or Leishmanization (LZ) usually accompanies with protection against subsequent lesion development. Leishmaniza- tion is an inoculation of live virulence Leish- mania intradermally into susceptible individu- als to induce artificial CL lesion, LZ showed to be an effective control measure against CL. The surrogate marker(s) of cure and protection in human leishmaniasis is not well known, but in some studies, a correlation between the gen- eration of Th1 type of immune response and protection is shown (6-13). In this study, vol- unteers with active of CL lesion, volunteers with a history of CL and healthy volunteers were recruited. Blood samples were collected and stimulated with either live or killed L. ma- jor and Th1/Th2 cytokine profile was checked. Materials and Methods Ethical considerations and Study population The study was carried out in leishmaniasis clinic of Center for Research and Training in Skin Diseases and Leprosy, Tehran Univer- sity of Medical Sciences (CRTSDL, TUMS) during March 2017 to March 2019. The study proposal was approved by the Ethical Com- mittee of Tehran University of Medical Sci- ences. The study groups are as follow: (i) 16 parasitologically proven patients with active CL lesion(s), (ii) 25 volunteers with history of CL, (iii) 15 healthy volunteers from non-en- demic areas with no history of leishmaniasis. The lesion of every CL patient and volunteers with history of CL were parasitologically con- firmed by direct smear, and/or culture, PCR was used to identify the Leishmania species. Isolation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMC) were isolated from a heparinized blood sample which was collected from each volunteer by using Ficoll- Hypaque density centrifugation. Parasites and Antigens Leishmania major (MRHO/IR/75/ER) was isolated from the spleen of an infected BALB/c mouse, the parasites were cultured in NNN me- dia and sub-cultured in complete RPMI media supplemented with 10% FCS and penicillin/ streptavidin. The promastigotes were harvest- ed at stationary phase, washed 3 times, and re- suspended in PBS and then divided into two parts; one part was used as it was to stimulate PBMCs (5×105 live promastigotes/well), and the second part was killed by boiling for 5 minutes and checked microscopically to as- sure that the parasites are killed, the killed Leish- mania was used to stimulate PBMCs (the same number as live promastigote, 5×105/well). J Arthropod-Borne Dis, March 2021, 15(1): 133–142 A Miramin-Mohammadi et al.: Immune Responses in … 135 http://jad.tums.ac.ir Published Online: March 31, 2021 Blood sampling collection Heparinized blood samples (10mL) were col- lected from every volunteer, blood samples were diluted 1:1 using RPMI. The diluted blood samples were overlaid gradually with Ficoll- Hypaque (30–40% of blood volume) in 50ml disposable centrifuge tubes. Isolated PBMC was washed 3 times using PBS and resuspended in RPMI containing 10% heat- inactivated fetal calf serum, 100U/ml penicillin and 100μ/ml/ ml streptomycin. Peripheral blood mononuclear cell culture The cell number was adjusted to a final con- centration of 1×106PBMC/ml, the cells were cultured in triplicates in U-bottom 96 well cul- ture plates (2×105 cells in 200μl volume/well) for 72h at 37 °C in 5% CO2, the PBMCs were stimulated with either PHA(5μg/ml), live par- asites (5×105/well) or heat-killed L. major (5× 105/well), or culture media alone with no stim- ulation as a negative control. After 72 hours of incubation, 150μl of the supernatants were carefully collected from each well and the trip- licates were pooled and kept at -80 °C until used. Cytokine assay The level of IL-5, IL-10, and IFN-γ were titrated in culture supernatants using ELISA method (Biosciences, USA) according to the manufacture’s recommendation. Briefly, the plates were coated with anti-IFN-γ/IL-5/IL-10 mAb in PBS, pH 7.4, and incubated at 4 °C overnight. After blocking the wells using buff- er containing PBS plus 0.05% (v/v) Tween 20, supernatants were added to each well. Biotin- labeled mAb in incubation buffer was added to each well and as enzyme streptavidin-HRP was used. The reaction was developed using 3, 3′, 5, 5′-tetramethylbenzidine (TMB) sub- strate and stopped with 0.5 M H2SO4 solu- tion. The plates were washed after each step of incubation using PBS +0.05% (v/v) Tween20. The plates were read at 450nm using a reader (BioTek, USA). The mean optical densities (ODs) of triplicate cultures were compared with the standard curves prepared using recombi- nant IL-5, IL-10, and IFN-γ. The cytokine lev- els represent the differences between the ODs of test and background wells. The results are expressed in pg/ml (mean +SD) of triplicates experiments (11, 12). Statistical analysis Shapiro-Wilk test was used to evaluate the normality of numerical variables. The Quan- titative variables were expressed as mean ±SD (standard deviation), and qualitative variables as a percentage. Chi-square test and Fisher’s exact test was used to examine the relation be- tween qualitative variables. Pair T-test /Wil- coxon signed-rank test was used to the inter- group comparison. One-way ANOVA with a followed post-hoc test (Bonferroni) was used to multiple comparisons between the groups. P-value less than 0.05 was considered statisti- cally significant. The SPSS Version 16 (SPSS Inc., Chicago, IL, USA) software was used for all statistical analyses. Results The background information of the volun- teers including gender, age and, cutaneous leishmaniasis status are presented in Table 1. In healthy volunteers, the mean +SD of IFN-γ production in PBMC stimulated with live or heat-killed parasites versus unstimulated were (408.5+198.2), (369.5+199.07) and (179.7+ 88.1), respectively. There was a significant dif- ference in IFN-γ production in PBMC of healthy volunteers stimulated with live L. major (p< 0.001), heat-killed L. major (p< 0.005) vs. un- stimulated, there was no significant difference in IFN-γ production in PBMC of healthy vol- unteers stimulated with heat-killed L. major or live L. major (Fig. 1A), In active lesion of CL volunteers the mean +SD of IFN-γ pro- duction in stimulated with live or heat-killed L. major vs. unstimulated were (1,953.2+ 1,032.8), (1,157.0+678.3) and (220+182), re- spectively. IFN-γ production in PBMC volun- teers with active lesion stimulated with live or J Arthropod-Borne Dis, March 2021, 15(1): 133–142 A Miramin-Mohammadi et al.: Immune Responses in … 136 http://jad.tums.ac.ir Published Online: March 31, 2021 heat-killed L. major was significantly (p< 0.001) higher than with non-stimulated (Fig. 1a), IFN-γ production in PBMC of volunteers with ac- tive lesion stimulated with live L. major was significantly (p< 0.001) higher than stimulat- ed with heat-killed L. major (Fig. 1a). In vol- unteers with history of CL, the mean +SD of IFN-γ production in PBMC stimulated with live L. major, heat-killed L. major and unstim- ulated were (2337.4+879), (1729.4+597.1), and (209+107), respectively. IFN-γ production in PBMC stimulated with live or heat-killed L. major was significantly (p< 0.001) higher than unstimulated PBMC, there was a significantly higher IFN-γ (p< 0.005) production in PBMC of history of CL volunteers stimulated with live L. major in comparison with PBMC stim- ulated with heat-killed L. major (Fig. 1a). In healthy volunteers, the mean +SD of IL-5 pro- duction in stimulated with live or killed L. ma- jor and unstimulated were (157.3+53.9), (191.2+ 86.6) and (160.3+36.8), respectively. There was no significant difference in IL-5 production in PBMC of healthy volunteers stimulated with live or killed L. major and unstimulated PBMC, There was no significant difference in IL-5 pro- duction in PBMC of healthy volunteers stim- ulated with live L. major or heat-killed L. ma- jor (Fig. 1b). In PBMC collected from volun- teers with active CL lesion, the mean +SD of IL-5 production in PBMC stimulated with live, heat-killed L. major or unstimulated were (187.8+69.1), (272.6+113.5) and (198.7+97.5), respectively. There was no significant differ- ence in IL-5 production in PBMC of active lesion CL volunteers stimulated with live L. ma- jor compared with unstimulated PBMC, but there was a significant difference (p< 0.05) in IL-5 production in PBMC of volunteers with active CL lesion stimulated with killed L. ma- jor compared with unstimulated PBMC. IL-5 production in PBMC of volunteers with active lesion stimulated with live L. major was sig- nificantly (p< 0.005) lower than PBMC stim- ulated with heat-killed L. major (Fig. 1b). In volunteers with a history of CL, the mean +SD of IL-5 production in PBMC stimulated with live, heat-killed L. major and unstimulated were (133.4+50.4), (196.7+68.8) and (148.6+ 675), respectively. There was no significant differ- ence in IL-5 production in PBMC of volunteers with a history of CL stimulated with live L. ma- jor in comparison with unstimulated but there was a significant difference (p= 0.005) in IL- 5 production in PBMC of volunteers with his- tory of CL stimulated with heat-killed L. ma- jor in comparison with unstimulated (Fig. 1b). IL-5 production in PBMC of volunteers with history of CL stimulated with live L. major was significantly (p< 0.001) lower than that of PBMC stimulated with heat-killed L. major (Fig. 1B). In healthy volunteers, the mean+SD of IL-10 production in PBMC of volunteers stim- ulated with live L. major, heat-killed L. major and unstimulated were (272.2+180.2), (263.4+ 236.4) and (212.3+160.2), respectively. There was no significant difference in IL-10 produc- tion in PBMC of healthy volunteers stimulat- ed with live L. major, heat-killed L. major and unstimulated PBMC, there was no significant difference in IL-10 production of PBMC of healthy volunteers stimulated by heat-killed L. major and PBMC stimulated with live L. ma- jor (Fig. 1c). In volunteers with active CL le- sion, the mean +SD of IL-10 production in PBMC stimulated with live L. major, heat- killed L. major and unstimulated were (451.1+ 291.1), (464.4+264.6) and (286.6+28.1), re- spectively. There was no significant differ- ence in IL-10 production in PBMC of volun- teers with active CL lesion stimulated with live L. major, and unstimulated but there was a sig- nificant difference (p< 0.05) in IL-10 produc- tion in PBMC of volunteers with active CL lesion stimulated with heat-killed L. major or unstimulated PBMC, there was no significant difference in IL-10 production in PBMC of vol- unteers collected from active CL lesion stimu- lated with live L. major or killed L. major (Fig. 1c). In volunteers with history of CL, the (mean +SD) of IL-10 production in PBMC stimulated with live L. major, heat-killed L. major and un- J Arthropod-Borne Dis, March 2021, 15(1): 133–142 A Miramin-Mohammadi et al.: Immune Responses in … 137 http://jad.tums.ac.ir Published Online: March 31, 2021 stimulated PBMC were (359.2+120.4), (365.3+ 198.6) and (241+98), respectively. There was no significant difference in IL-10 production in PBMC of volunteers history of CL stimulated with heat-killed L. major or live L. major with unstimulated, there was no significant differ- ence in IL-10 production in PBMC of volun- teers with a history of CL stimulated with live L. major or killed L. major (Fig. 1c). There was a significant difference (p< 0.001) between the IFN-γ levels in supernatants of PBMC col- lected from volunteers with active CL lesion and PBMC of healthy volunteers stimulated with heat-killed or live L. major (Fig. 1a). There was a significant (p< 0.001) difference between the IFN-γ levels in supernatants of PBMC of vol- unteers with history of CL and PBMC of healthy volunteers stimulated with heat-killed or live L. major (Fig. 1a). There was a significant dif- ference between the IFN-γ levels in superna- tant of PBMC of volunteers with history of CL and volunteers with active CL lesion stimu- lated with heat-killed L. major (p< 0.005) and but there was no significant difference between the IFN-γ levels in supernatant of PBMC of volunteers with history of CL and volunteers with active CL lesion stimulated with live L. major (Fig. 1a). There was a significant dif- ference between the IL-5 levels in supernatants of PBMC of volunteers with active lesion and PBMC of healthy volunteers stimulated with heat-killed L. major (p< 0.005) but there was no significant difference between the IL-5 levels in supernatants of PBMC of volunteers with active lesion and that of healthy volunteers stim- ulated with live L. major (Fig. 1b). There was a significant (P< 0.005) difference between the IL-10 levels in supernatants of PBMC of vol- unteers with active lesion and that of healthy volunteers stimulated with live or heat-killed L. major. But there was no significant differ- ence between the IL-10 levels in supernatants of PBMC of volunteers with a history of CL lesion and that of volunteers with active le- sion stimulated with live or heat-killed L. ma- jor. There was no significant difference between the IL-10 levels in supernatants of PBMC of volunteers with active lesion and that of vol- unteers with history of CL stimulated with live L. major or heat-killed L. major (Fig. 1c). Table 1. Characteristic of the volunteers HCL Cases Active lesion Healthy Number of volunteers 25 16 15 Mean of Age (Year) 32 33.6 45 Gender (M/F) 16.9 13.3 13.2 Number of lesion 30 51 0 Position of the lesion Upper limb 11 12 - Lower limb 2 2 - Trunk 1 1 - Face 4 2 - Mean duration of the lesion (months) 5.4 2 - J Arthropod-Borne Dis, March 2021, 15(1): 133–142 A Miramin-Mohammadi et al.: Immune Responses in … 138 http://jad.tums.ac.ir Published Online: March 31, 2021 Fig. 1. Cytokine levels (pg/ml) in culture supernatants of peripheral blood mononuclear cell (PBMC) of different vol- unteers (healthy, active cutaneous leishmaniasis( CL) lesion, History of CL) stimulated with Live L. major or Killed L. major, (a) IFN-γ level in culture, (b) IL-5 level in culture, (c) IL-10 level in culture a c b J Arthropod-Borne Dis, March 2021, 15(1): 133–142 A Miramin-Mohammadi et al.: Immune Responses in … 139 http://jad.tums.ac.ir Published Online: March 31, 2021 Discussion Recovery from leishmaniasis induced by natural infection or leishmanization leads to the development of lifelong protection against further disease development. Leishmanization is the most effective control measure against CL (14). Experiences with vaccination using killed Leishmania vaccines showed limited protection. It seems that the quantity/quality of immune re- sponse which generated in vaccines receiving killed Leishmania vaccine apparently is not sim- ilar to the immune response generated against live Leishmania. Although the reason of the dif- ferent immune response against live vs. killed Leishmania is not well known but one reason might be that likely live Leishmania is present- ed by both endogenous and exogenous antigen presentation pathways but killed Leishmania is only presented by the exogenous pathway. Studies on murine model of leishmaniasis in- dicated that parasite persistence plays a crucial role in the induction of a strong and long-term immunity, another factor such as differences in the nature of the early inflammatory and/or immune responses to live vs. killed parasites might be important, animals inoculated with live Leishmania induced more IFN-γ and less IL-4 than those which were injected with killed Leish- mania (it was shown that infection with live parasites, (virulent or avirulent) leads to exten- sive activation and IFN-γ production by both CD4+ and CD8+ T cells while injection with killed parasites only lead to activation of CD4+ T cells. This activation of CD8+ T cells may play a role in the regulation of the early immune response to live vs. killed parasites. Live Leish- mania promastigotes showed to directly acti- vate primary NK cells to produce IFN-γ in the absence of IL-12 and professional antigen pre- senting cells while Killed parasites unable to do so, this means that live Leishmania activates NK cells by direct mechanism but killed Leish- mania do not activate NK cells. In another study, C57BL/6 mice which were clinically cured from L. major lesion, skin-resident CD4+ T cells were found in the skin far from the site of the pri- mary infection and were able to enhance pro- tection against a later challenge by producing IFN-γ and recruiting circulating T cells to the skin. It was shown that live and dead parasites can differ in their ability to induce cellular re- sponses in healthy donors as defined by IFN-γ production and cell proliferation (15-25). His- torically, leishmanization showed to be highly efficacious control measure against CL, in con- trary, the results of several clinical trials using different Leishmania species killed by various ways showed that killed Leishmania does not induce immune response strong enough to pro- tect against natural infection. In this study, the Th1/Th2 profile of volunteers with a history of CL and volunteers with active cutaneous leish- maniasis lesions were evaluated against live L. major in comparison with killed L. major. IFN- γ production in PBMC of volunteers with ac- tive lesion (p< 0.001) and a history of CL (p< 0.005) stimulated with live L. major was sig- nificantly higher than stimulated with heat-killed L. major. IL-5 production in PBMC of volun- teers with active lesion (p< 0.005) and volun- teers with history of CL (p< 0.001) stimulated with live L. major was significantly lower than PBMC stimulated with heat-killed L. major. There was no significant difference in IL-10 production in PBMC of volunteers with active CL lesion stimulated with live L. major and killed L. major. IL-5 and IL-10 in the superna- tant of PBMC collected from volunteers with active lesion were significantly (p< 0.005) high- er than healthy volunteers stimulated by live or killed L. major. The level of IFN-γ in vol- unteers with a history of CL was higher than active lesion but not significantly, and the lev- el of IFN-γ in volunteers with active lesions was significantly higher (p< 0.001) than healthy vol- unteers, this means that Th1 response which is necessary for protection against leishmaniasis is induced upon recovery. IL-5 level in volun- teers with active lesion(s) was more than vol- J Arthropod-Borne Dis, March 2021, 15(1): 133–142 A Miramin-Mohammadi et al.: Immune Responses in … 140 http://jad.tums.ac.ir Published Online: March 31, 2021 unteers with a history of CL. The results of this study showed that the intensity of Th1 type of immune response is significantly higher in PBMC stimulated with live Leishmania than stimulated with killed Leishmania, the current results showed why natural infection/l eish- manization induces protection but vaccination with killed Leishmania does not induce enough protection. This study is in agreement with pre- vious studies carried out in which it was shown that responses to live and dead promastigotes are different in the induction of cytokines and response to live in which response to live par- asites was parasites produced significantly more IFN-γ than the group of animals injected with killed stronger than dead ones (20). In another study, it was shown that the group of mice in- oculated with live Leishmania parasites pro- duced significantly more IFN-γ than the group of animals injected with killed parasites (25). 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