Adjunct Therapy of rIFN-γ and rIL-17A along with Sub- optimal Dose of Amphotericin-B effectively Control the Leishmania donovani Parasitic Growth in Infected Mice

Containment/dissemination of Leishmaniasis depends on the dominance of Th1/ Th2 immunity. IFN-γ and IL-17A are well known for protection from leishmaniasis. Synergistic effects of these two cytokines are also known in various autoimmune diseases. However, the therapeutic, as well as adjunct therapeutic use of rIL-17A and rIFN-γ in combination with sub-optimal dose of amphotericin-B (AmpB) is still not validated in visceral leishmaniasis. In the present study, we have evaluated the adjunct therapy in a mouse model of VL. After twenty-one days of post infection, in the therapeutic group, mice were intra-peritoneally injected with two doses of recombinant cytokines at one week interval. In adjunct therapeutic groups of mice, immune components were primed for three days with recombinant cytokine(s) followed by injection of sub-optimal dose of AmpB. Body weight, parasitic load in visceral organs and fold change in cytokines’ gene expression was evaluated. We observed significant gain in body weight, inhibition of parasitic load in visceral organs {(liver; 71.7% 95%), (spleen; 70%-88.7%) (Bone marrow; 46.6 -87.1%)}; significant up regulation in fold change of proinflammatory cytokine(s) gene expression (TNF-γ,iNOS, IL-2 and IL-12) as well as marginal increase of anti-inflammatory cytokine(s) gene expression (IL-4, IL-10&TGF-γ} in adjunct therapeutic groups of mice. Our results suggest that though the therapeutic use of recombinant cytokine(s) is not the best option; however, use of recombinant cytokine(s) along with suboptimal dose of amphoterocin-B to reduce drug toxicity could have a way for better treatment options.


INTRODUCTION
Visceral leishmaniasis (VL), also known as kala-azar (black fever) is a disease of reticuloendothelial system caused by Leishmaniadonovani and Leishmania infantum 1,2 . The clinical manifestations caused by these organisms are usually not distinguishable. However, the decisions for treatment usually do not require species identification. The infection spreads from skin (site of infected insect bite) to visceral organs, especially spleen, liver and bone marrow which additionally progress in spleenomegaly and hepatomegaly that ultimately leads to death, if not treated 3 . In experimental Leishmania donovani infection mice model susceptibility (BALB/c) and resistant (C57BL/6J) in mice acquire via a macrophageactivating, Th1 cell-dependent mechanism governed by different interdigitating cytokines 4 . Use of recombinant cytokine-based therapies, over the past few decades, boosts the clinical relevance, especially in the anticancer immune response [5][6][7] . Recombinant cytokines milieu virtually regulates the innate and adaptive immunity, including the imitation, execution and extinction of pathogen through the induced immune response.
Cytokines are known for their pleiotropic functions so that they function locally or at a distance to suppress or enhance the immunity 8,9 . Cytokine/cytokine-combinations that selectively induce the dominance of Th1 response might be useful for VL treatment option. Strong effective immunity against VL parasite is characterised by the emergence of strong parasite-specific Th1 response. Interferon (IFN)-γ and its major inducer IL-12 dictate the blood circulating T cells and monocytes to assemble at the site of infection and engulf the infected macrophages 10,11 . Typically Th1 activating cytokines accompanied with tumor necrosis factor induces profound inflammation and activate inducible nitric oxide synthase (iNOS) that leads to parasite killing in infected macrophages 12,13 . However, the dominance of IL-4 and IL-10 alone or in additive are responsible for dissemination of parasite into visceral organs 14,15 . Murray et.al., first of all, paved the pathway for the use of cytokines as therapy in visceral leishmaniasis. They reported the killing of parasite in the liver of infected mice with the use of anti-IL-10 receptor antibody (anti-IL-10R mAb) through an inducible nitric oxide synthase-dependent mechanism 16 .
The available options for the treatment of visceral leishmaniasis are associated with severe problems like adverse effect, low efficacy, long treatment period and the development of resistance 17,18 . Hence there is an urgent need for an alternative treatment option for visceral leishmaniasis. In this context, we designed and executed this study in a mice model; in which we have studied three different aspects of the use of recombinant cytokine(s) for an alternative treatment options. i) Prophylactic use of recombinant cytokine(s) for protection from leishmaniasis ii) Therapeutic use of recombinant cytokine(s) once the disease establishes and iii) use of sub-optimal dose of amphotericin-Balong with recombinant cytokine(s) to reduce the drug induced toxicity.
We did not observe better response of recombinant cytokine(s) as a prophylactic or as a therapeutic agent. However, in adjunct therapy, we observed significant results (in terms of parasite clearance in visceral organs, increased proinflammatory cytokines response and decreased/ marginal increase of anti-inflammatory cytokine response). These findings may help in redefining the treatment module in the treatment of visceral leishmaniasis and decreasing the drug induced toxicity (especially amphotericin-Binduced toxicity). Furthermore, this study can also be done in the drug unresponsive cases.

MATERIALS AND METHODS Animal and parasites
Mice from Animal House Facility of CDRI were used as experimental hosts. This study was compliance with Ethical standard. Clinical strains of Leishmania donovani parasite were taken from patients those who were admitted for diagnosis and treatment of kala-azar in Balaji Utthan Sansthan (BUS), Patna. The clinical isolate of parasite were further cultured aseptically in our laboratory under the standard in vitro conditions as described elsewhere 19 . Further, grown parasite was injected into mice to establish and maintained in mice model with specific infectivity. Serial passages were done to maintain the amastigote to amastigote.

Mice and Infection
A total of 60 mice were utilized in this study. A 0.1ml of inoculum containing 10 6 amastigotes/ml was injected intravenously through tail vein to 52 naive Balb/c mice (25-30 g in body weight) {instead of 45 infected mice required for this study} with a sterile 26 gauge needle while rest of the 8 animals were kept as an uninfected control {instead of 5 uninfected mice required for this study} (Table 1 & 2). The magnitude of infection in infected animals was assessed 21 days later by necropsy of two mice from infected group. Treatment Ten groups containing five mice each were used for the study. These were treated with different recombinant cytokines alone and in various combinations with suboptimal dose of AmpB. The mice of uninfected controls (M1) were given PBS only (negative Control) and the rest groups of mice were infected with 1x10 6 amastigote parasites through the tail vein. No. 485-MI-100 and Group M8= rIL-17A+ rIFN-γ) was administered via intra peritoneal in respective group of mice. After seven days of the first dose, the second dose of the same amount of recombinant cytokines was again injected. After 10 days of second dose (i.e. day 45) mice were sacrificed and evaluated for study parameters (Table 1).

Adjunct therapeutic groups
From day 21 of post challenge, adjunct therapy was initiated in respective groups. In this, groups of mice where the only conventional drug, AmpB was used for therapy: two doses of optimal (5µg/mice) and sub-optimal (1µg/mice) AmpB (on day 21 and 28 post challenge respectively) was administered in M10 and M11 group of mice respectively. In groups of mice where sub-optimal dose of AmpB along with recombinant cytokine(s) used for therapy: Three days prior to sub-optimal dose of AmpB administration, mice were sensitized with recombinant cytokine(s) (GroupM12= rIL-17A; Group M13=rIFN-γ and Group M14= rIL-17A+ rIFN-γ). After 15 days of the second dose of drug (i.e. day 45) mice were sacrificed and evaluated for study parameters (Table 2).

Parasitic Burden Post Treatment
Necropsy of all the mice was done after 10 days post-treatment to assess the parasitic burden in spleen, liver and bone marrow. Therapeutic efficacy was assessed in terms of the parasitic load in each organ. Calculations for parasitic density were done as the number of amastigotes/1000 cell nuclei in each organ as compared to untreated controls (M2). Percentage inhibition was assessed in comparison to the infected control by following formula 20

Immunological assay Cytokine gene expression by Real time-PCR
To assess the quantitative analysis of mRNA expression of various cytokines and inducible NO synthase (iNOS) in spleen cells of various experimental groups of mice, Real-time PCR was performed. Spleen tissues were taken for RNA isolation. RNA isolation was carried out using Tri-reagent (Sigma-Aldrich: Cat No. 93289-100ML) after day 10 post treatment and quantified by using Gene-quant (Bio-Rad). cDNA was synthesized from 1µg of total isolated RNA using a first-strand cDNA synthesis kit (Fermentas: Cat No. K1622). Real-time quantitative polymerase chain reaction (RT-PCR) was conducted as per the protocol described earlier 20 . Housekeeping gene GAPDH was used for normalization in all quantifications. To avoid contamination or non-specific reactions, a no-template cDNA was included. Cycle threshold (CT) values obtained indicated the number of PCR cycles which are required for the fluorescence signal to exceed the detection threshold value (background noise). Comparative CT method was used to calculate the differences in gene expression 21 . The fold change in the expression of genes in recombinant cytokine treated groups as compared to that of the infected group was determined by the formula fold change in expression 2-∆∆CT 21 .

Therapeutic use of recombinant cytokines (IL-17A and IFN-γ) after infection has no effect on change in body and organ weight of mice
Analysis for change/gain in body and organ weight during the course of treatment is an important indicator for evaluation of efficacy of any intervention 22

Therapeutic use of recombinant cytokine(s) inhibit parasitic growth in visceral organs of Leishmania donovani infected mice
After infection, leishmania parasites from the local site (tissue) migrate through the lymph nodes to the internal organs (especially, liver, spleen and bone marrow). Hence, we compared the parasitic load in visceral organs in treated group of mice (M6, M7 and M8) and compared with non-treated group of mice (M2). In liver: significant decrease (49% to 56.6%) in parasitic load was observed in cytokine(s) treated groups;   2.6 ± 0.35)} compared with infected non-treated control group (Group M2) (Figure 2A III). Gene expression of IL-12: In treated groups of mice relative fold expression of IL-12 was up-regulated (1.3 to 2.6 times); {(Group M6; mean ± SD: 1.3 ± 1.0), (Group M7; mean ± SD: 1.8 ± 0.6) and (Group M8; mean ± SD: 1.6 ± 1.1)} compared with infected non-treated control group (GroupM2) (Figure 2A  IV). The up-regulation of relative gene expression was not significant probably due to small sample size.

Change in anti-inflammatory gene expression in infected mice upon therapeutic use of recombinant cytokine(s)
Similar to pro-inflammatory cytokine(s) gene expression, relative fold expression of antiinflammatory cytokines gene expression was also evaluated in infected after cytokine treated groups. Gene expression of IL-4: In treated groups of mice relative fold expression of IL-4 was almost

Adjunct therapy of recombinant cytokines (IL-17A and IFN-γ) along with sub-optimal dose of AmpB, influence significant improvement in body weight but not change in organ weight
Change/gain in body and organ weight was evaluated in groups of mice used for conventional drug therapy after infection: optimal (Group M10:AmpB; 5µg/mice) and suboptimal (Group M11: 1µg/mice) dose of drug, alone, and sub-optimal dose of drug along with recombinant cytokines (rIL-17A and rIFN-γ) alone or in combination (Adjunct therapy; M12, M13 & M14). After twenty-one (21) days of infection, therapy was started. Two doses of drugs and recombinant cytokines were given after a oneweek interval. In order to prime the immune components to the fullest function of drug, recombinant cytokine(s) was given three (3) days prior to drug infusion in adjunct therapy groups of mice. After forty-five (45) days of infection body weight of mice was taken before sacrifice and organ weight was taken after sacrifice. Significant change in body weight was observed in all the group of mice (M10 to M14) when compared with body weight at zero-day ( Figure 3A I). When  Further, we evaluated the parasitic load in visceral organs (especially, liver, spleen and bone marrow) of the study groups of mice (M1, M2, and M10 to M14) to see the effectiveness of drugs (optimal and suboptimal) alone or in combination with recombinant cytokine (suboptimal dose of drug). The parasitic load was compared with infected non-treated control group of mice (M2). In liver: significant decrease in parasitic load in optimal drug treated group (Group M10; mean± SD; 569 ±328.3; 90.53%) andin sub-optimal drug treated group (Group M11; mean± SD; 2675 ±124.3; 55.4%). Moreover, in sub-optimal drug along with recombinant cytokine(s) treated groups showed significant reduction in parasitic load:

DISCUSSION
The anti-leishmania drugs are the only treatment option for visceral Leishmaniasis till date 24,25 . This treatment option is always associated with adverse effect on the host like cost, toxicity, longer treatment duration, drug resistance etc 24,25 . Even though several research advances has been made to overcome the associated problems; drug resistance among VL patients is emerging as a major challenge. Researchers are continuously searching for alternative therapy and developing short duration combination therapy along with reducing drug dose in order to overcome the drug resistance 24 .

Journal of Pure and Applied Microbiology
In the present study, author(s) interested in evaluating a new prophylactic and therapeutic strategy with recombinant cytokine(s) for better treatment of visceral leishmaniasis. Change in body weight during the course of treatment has been recognised as a relevant determinant for clinical outcome of the infection with leishmaniasis 22,26 . Galindo et al. in 2013, showed the gain in body weight of hamsters with progression of disease 26 . However, we observed no significant change in body weight in both prophylactic and therapeutic groups of mice when compared with control group with the course of treatment. This shows that the recombinant cytokine(s) have some role in amelioration of disease. In our findings, we did not observe any change in the weight of visceral organs among the study groups compared with controls. Additionally, significant gain in spleen weight was observed in untreated group of mice. This is an indicative of profound parasitemia in leishmaniasis in untreated group of mice. This further substantiates our finding of the role of recombinant cytokine(s) in disease management. Sudharshan et al. in 2011 27 has shown that a significant correlation between parasitic load and clinical outcome in patients undergone treatment with L-AmpB. Mary et al. in 2006 proposed the persistence of more than one parasite/ml of blood after treatment is associated with high risk of relapse 16 . Few more studies were also shown an increase of more than 10 parasite/ml of blood after treatment is a strong indicative of relapse 16,27,28 . In our study, we observed significant inhibition of parasitic load in visceral organs in both therapeutic and adjunct therapeutic group of mice with the use of recombinant cytokine(s). When we evaluated the sub-optimal dose of AmpB with cytokine(s) primed group of mice, we observed more than 95% of parasitic clearance.
Furthermore, balance and dynamic change of pro-inflammatory and anti-inflammatory cytokines may help in predicting the clinical outcome of disease 14,29 . On one hand increased proinflammatory response amplifies the inflammatory reactions that trigger the immune response to overcome the disease; on the other hand this heightened pro-inflammatory response may cause collateral tissue damage. Simultaneously the antiinflammatory cytokines limit the pro-inflammatory cytokine response to prevent the collateral damage of tissue 30 . However, an excessive down regulation of pro-inflammatory cytokines may favour disease progression. Several reports show that the Th1 type of pro-inflammatory cytokine response (especially interferon-γ, tumor necrosis factor-±, interleukin-2, interleukin-12 and iNOS) are the crucial factors in the initiation of protective immunity against visceral leishmaniasis. In our study, we did not find any clear cut cytokine dominance in therapeutic group of mice. Moreover, in adjunct therapeutic group of mice, we observed a clear shift toward Th1 type of cytokine. Furthermore, we also observed increased expression of anti-inflammatory cytokine genes (especially IL-4, IL-10 and TGF-β). Probably this over expression of anti-inflammatory cytokine(s) was the activation of compensatory mechanism to limit the over expressed pro-inflammatory response. This was further confirmed with significant parasitic inhibition in visceral organs of this group.
Based on the above findings we propose that use of recombinant IFN-γ as well as IL-17A may curtain the parasitic load in visceral leishmaniasis. This is possibly due to Th1 induced macrophage activation of IFN-γ/IL-12 axis, which is further facilitated by IL-17A. Therefore we suggest that both therapeutic, as well as adjunct therapeutic use of recombinant cytokine(s) is/are beneficial in redefining the disease treatment. However, the adjunct therapeutic use of recombinant cytokine(s) shows much better outcome as it reduces the parasitic load in visceral organs and skews the cytokine milieu dominantly towards Th1.