Prophylactic interferon-γ and interleukin-17 facilitate parasite clearance in experimental visceral leishmaniasis.

BACKGROUND AND
OBJECTIVE: The synergy of interleukin (IL)-17 along with other pro-inflammatory cytokines is well known in various autoimmune and infectious diseases. A longitudinal study in the Sudanese population showed an association of IL-17 with the protection of kala-azar outbreak. The protective role of IL-17 is also known in terms of expansion of IL-17-producing cells in vaccine-induced immunity. However, the prophylactic role of IL-17 in visceral leishmaniasis has still not been validated. In the present study, we evaluated the prophylactic efficacy of IL-17A and interferon (IFN)-γ in Leishmania donovani-challenged Balb/c mice.
MATERIALS AND METHODS: Two doses of recombinant IL (rIL)-17A and/or IFN-γ were administered intraperitoneally after/at 1 week interval and then the mice were challenged with amastigote form of L. donovani. At 45 days of postchallenge, mice were sacrificed and evaluated for change in the body and organ weight, parasitic load in visceral organs, and fold change in gene expression of cytokines.
RESULTS: We observed that the prophylactic use of rIL-17A and IFN-γ alone or in combination significantly inhibited the parasitic load in visceral organs. Furthermore, pro-inflammatory cytokine gene expression increased up to 2-4-folds in mice treated with recombinant cytokines.
CONCLUSION: Our results suggest that prophylactic use of recombinant IFN-γ and IL-17A inhibits parasitic growth in visceral organs of L. donovani-challenged experimental mice model, especially through upregulation of pro-inflammatory cytokines' gene expression.

INTRODUCTION

Visceral leishmaniasis (VL) caused by protozoan parasite: Leishmania donovani is a disease of the reticuloendothelial system. If left untreated, in both humans and mice, the infection can spread from the skin to lymph nodes, liver, spleen, and bone marrow (BM), further leading to hepatomegaly, splenomegaly, and ultimately leads to death.[1] Effective immunity against parasite in VL is characterized by the emergence of a strong, parasite-specific Th1 response.[2] Interferon (IFN)-γ-inducible nitric oxide synthase (iNOS) and interleukin (IL)-12-STAT-4-mediated immunological pathways are important for effective parasite clearance on activation and induction of microbicidal functions of macrophage in VL.[3]

Recent studies have changed the Th1/Th2 cell dichotomy to Th1/Th2/Th3 after induction of Th17 (Th3) cell, a new T-cell subset in effector immunity.[4] The protective role of Th17 against extracellular bacterial and fungal infection is well established.[5] A recent study also demonstrated that Th17-IL23 pathway may play a major role in the induction of protective immunity against intracellular pathogens by regulating both innate and adaptive immune responses.[6] Both detrimental and protective role of IL-17 is known in different forms of clinical leishmaniasis. In cutaneous leishmaniasis, IL-17 is known for progression of disease in susceptible mice, wherein it induces neutrophil recruitment at disease site that persuades in disease progression. In VL also both protective and detrimental role of IL-17 is documented.[378] IL-17A (-/-) mice have been shown to have high resistance to VL infection.[3] Conversely, a human-based study in Sudanese population showed an increased level of IL-17 and IL-22 for protection against reexposure to VL.[9] Few recent studies have also reported the role of IL-17A as an important mediator of resistance against Leishmania infection, demonstrating that it acts synergistically with IFN-γ to promote parasite killing.[10] We have also observed a strong inverse correlation between the levels of IL-17A and IFN-γ with parasitic load at the local site (BM) in VL patients.[11] Hence, the overall findings pave the way to ponder the involvement of IL-17 in vaccine-induced protective immunity. Since there is no vaccine against any clinical forms of leishmaniasis, recombinant IL (rIL)-17 could be used along with vaccine candidate(s) to boost the protective immunity.[12] Moreover, a limited number of drugs used for the treatment of leishmaniasis are associated with high toxicity and unresponsiveness. Thus, to overcome these limitations and open a new avenue for protection and treatment from leishmaniasis, we have used recombinant IFN (rIFN)-γ and IL-17A cytokines for both prophylactic and adjunct therapy (along with suboptimal dose of amphotericin-B) in Leishmania mice model. Although the use of recombinant cytokine(s) as a prophylactic agent may not be a better alternative for protection from leishmaniasis, we performed in mice model to validate our earlier human-based findings. In the present study, we have used rIL-17A and IFN-γ alone or in combination as a prophylactic agent in a mice challenged model. The present study thus highlights the application of these recombinant cytokines as a prophylactic agent in Leishmania mice model. We show here that application of rIFN-γ and IL-17 resulted in parasitic clearance accompanied with increased Th1 types of cytokines gene expression, thus advocating that the use of recombinant cytokine(s) as a prophylactic agent may be beneficial for controlling of VL. Furthermore, this approach may also help in boosting the vaccine-induced immunity.

MATERIALS AND METHODS

Animal and parasites

Laboratory-bred Balb/c mice from the Animal House Facility of CDRI were used as the experimental host. The usage of mice was approved by the Institute's Animal Ethical Committee (protocol number IAEC/2016/T-7[12/16] dated November 04, 2016). The mice were used as per the National Institutes of Health guidelines. Clinical strains of L. donovani were obtained from the patient who was admitted at Balaji Utthan Sansthan, Patna, and cultured in our laboratory under the standard in vitro conditions as described elsewhere.[13] This strain was also established and maintained in mice model through the serial passage, that is, from amastigote to amastigote.

Prophylactic cytokine administration

Five groups containing six mice each were used for the study. These were treated prophylactically with recombinant cytokines (IL-17A and IFN-γ) alone or in combination [Table 1]. The mice in Group I (M1) were given phosphate-buffered saline (PBS) only (negative control) and the Group II (M2) mice were infected with amastigote parasite (positive control). Intraperitoneally, 10 μg/0.01 ml/animal of recombinant mouse cytokine(s) was administered in the study groups of mice: rIL-17A ([Group III; M3] [R & D System, Cat. No. 421-ML-025]), rIFN-γ ([Group IV; M4] [R & D System, Cat. No. 485-MI-100]), and both rIL-17A as well as rIFN-γ (Group V; M5). A booster dose of the same amount of recombinant cytokines was given intraperitoneally to all the mice of respective experimental groups (i.e., Groups M3–M5) at an interval of 7 days.

Table 1

Experimental conditions of mice

Group of mice	Conditions
M1 (negative control)	Without infection: Injected with only PBS
M2 (positive control)	Infected with amastigote parasite
M3	Injected with recombinant IL-17Day 0: 10 µg/0.01 mlDay 7: First booster dose (10 µg/0.01 ml)
M4	Injected with recombinant IFN-γDay 0: 10 µg/0.01 mlDay 7: First booster dose (10 µg/0.01 ml)
M5	Injected with recombinant IL-17 + IFN-γDay 0: 10 µg/0.01 mlDay 7: First booster doses (10 µg/0.01 ml)

IL: Interleukin, IFN: Interferon, PBS: Phosphate-buffered saline

Infection

Except naïve group (M1), all the groups of mice were challenged intravenously with 106 amastigotes of L. donovani after 7 days of the booster dose of prophylactic cytokine(s). Necropsies of all the mice were done at day 45 postchallenge (P.C.). Prophylactic efficacy was assessed in terms of parasitic load in the liver, spleen, and BM. Calculations 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:[14]

Primary culture of liver and splenic tissues

Liver and splenic extracts of different groups of mice were cultured in RPMI media to assess the parasitic load. Mice were dissected to isolate the liver and spleen. Isolated organs (liver/spleen) were washed 2–3 times with 1 × PBS-containing gentamycin antibiotic (2 mg/100 ml). Small pieces of tissue from the isolated organs were then transferred into flasks containing 10–15 ml of incomplete RPMI with gentamycin. Flasks were examined daily for up to 10–20 days for any moving promastigotes.

Immunological assay

Cytokine gene expression by real-time polymerase chain reaction

To quantitatively assess the mRNA expression of cytokines in experimental groups of mice, real-time polymerase chain reaction (PCR) was performed. Three individual mice were chosen randomly from each group and their spleen tissues were taken for RNA isolation. RNA isolation was carried out using Tri-reagent (Sigma-Aldrich: Cat No. 93289-100ML) at day 45 and quantified using GeneQuant (Bio-Rad). cDNA was synthesized from 1 μg of total isolated RNA using a first-strand cDNA synthesis kit (Fermentas: Cat No. K1622).

Real-time quantitative PCR (RT-PCR) was conducted as per the protocol described earlier.[7] Housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase, was used for normalization in all quantifications. To avoid contamination or nonspecific 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.[8] 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-ΔΔCT.[1516]

Statistical analysis

Statistical significance was determined using the unpaired Student's t-test using GraphPad Prism® for Windows, Version 7. Results were expressed as mean ± standard deviation (SD) and percentage inhibition (mean value of analyzing group/mean value of infected control group) ×100.

RESULTS

Change in body weight over time in mice treated with recombinant cytokine(s) was similar to that of control

The mice in study groups (M1 to M5) were used for evaluation of change in body weight and organ weight after prophylactic treatment with recombinant cytokine(s) on day 45 P.C. We observed a significant decrease in body weight of mice which were not treated with cytokine(s) before infection (M2; mean ± SD; 20 ± 1.93 g) as compared with uninfected control group of mice (M1; mean ± SD; 31.22 ± 3.73 g). Furthermore, we observed almost similar weight gain in mice, which was treated with cytokine(s) before infection (M3; mean ± SD; 27 ± 1.22 g), (M4; mean ± SD; 27 ± 2.26 g), and (M5; mean ± SD; 28 ± 2.18 g) as compared with uninfected control [Figure 1a]. In addition, on comparing the change in body weight, we observed significant gain in body weight in cytokine(s)-treated group of mice as compared to the untreated infected group of mice (M2) [Figure 1a]. Further, we also evaluated the groups of mice for change in weight of visceral organs (liver and spleen) but did not observe any significant change in weight of liver (approximately 1.2 ± 0.02 g; in all the study groups of mice) [Figure 1b]. However, on infection, a significant gain in spleen weight was observed in mice which were not treated with cytokine (M2; mean ± SD; 0.8 ± 0.14 g) [Figure 1c] compared with uninfected control (M1; mean ± SD; 0.24 ± 0.03 g). Similar spleen weight was also observed in cytokine-treated groups of mice as that of uninfected control.

Figure 1

In cytokines-treated group of mice change in body weight over time was similar to that of control: M1: The group of mice without any treatment uninfected. M2: Infected control group of mice. M3: Injected recombinant interleukin-17A. M4: Injected recombinant interferon-γ. M5: Both recombinant interleukin-17A and recombinant interferon-γ were injected. The recombinant cytokine(s) was given intraperitoneally. (a) Body weight in gram; (b) Liver weight in gram; and (c) weight of spleen in gram. Each bar represents the pooled data (mean ą standard deviation value) of five replicates

Significantly decreased parasitic load in visceral organs of mice treated with cytokine(s)

Parasitic load in visceral organs (liver, spleen, and BM) was evaluated to perceive the prophylactic, protective role of recombinant cytokine(s) in the study groups of mice. We observed significant percentage inhibition of parasitic load in visceral organs in recombinant cytokine-treated group of mice compared with infected control (M2) on day 45 P.C. in liver (M3 = 49.0%), (M4 = 56.6%), and (M5 = 55.6%) [Figure 2a]; spleen (M3 = 65.6%), (M4 = 69.7%), and (M5 = 71.2%) [Figure 2b]; and BM (M3 = 71.1%), (M4 = 73.7%), and (M5 = 73.3%) [Figure 2c]. Furthermore, we did not observe any parasitic growth in primary culture of liver and spleen tissues after 10 days of postculture, indicating that both the cytokines were sufficient to inhibit the parasitic growth in visceral organs when given alone or in combination.

Figure 2

Significantly decreased parasitic load in visceral organs of cytokine(s)-treated group of mice: Parasitic load in visceral organs: (a) Liver, (b) spleen, and (c) bone marrow; Parasite burden (number of amastigotes/1000 cell nuclei) in the groups of mice treated with recombinant cytokines (M3, M4, and M5) at day 45 postchallenge (M2: mean of infected was used to evaluate percentage inhibition of parasitic load in visceral organs, due to lack of parasitic infection in M1: negative controls group: infection was not observed). Each bar represents the pooled data (mean and standard deviation value) of five replicates

Th1 gene expression was upregulated in mice treated with recombinant cytokine(s)

The mRNA expression of Th1 (iNOS, tumor necrosis factor (TNF)-α, IL-2, and IL-12) cytokines was evaluated by real-time PCR on day 45 P.C. A maximum of 3–4-fold increase in expression level of iNOS gene was observed in the group of mice which were treated with a combination of recombinant cytokines (M5), whereas other recombinant cytokine-treated groups (M3 and M4) also showed marginal increase in iNOS expression (2–3 folds) when compared with infected controls [Figure 3a]. We observed a 4–5-fold increase in TNF-α level in combined recombinant cytokine-treated group (M5); however, in other groups of mice treated with either IFN-γ or IL-17 (M3 and M4, respectively), the expression level varied between 3 and 4 folds when compared with infected controls [Figure 3b]. A 2–3-fold increase in expression of early pro-inflammatory cytokines IL-2 and IL-12 was also observed when compared with an infected group on day 45 P.C. in all the treated groups (M3, M4, and M5) [Figure 3c and d].

Figure 3

Th1 gene expression was upregulated in mice treated with recombinant cytokine(s): The fold change in pro-inflammatory cytokine gene expression in treated groups (M3, M4, and M5) was compared with infected control group (M2). a) Inducible nitric oxide synthase (iNOS) gene expression was upregulated 3-4. b) Tumor necrosis factor-α (TNF-α) gene expression was upregulated 4-6 folds c) Interleukin-2 (IL-2) gene expression was upregulated 2-6 folds. d) Interleukin-12 (IL-12) gene expression was upregulated 2-3 folds in treated groups compared with untreated control

DISCUSSION

Leishmaniasis is known to cause an immunocompromised state of immunity with an imbalance of pro-inflammatory and anti-inflammatory cytokine milieu. Lack of effective CD4+/CD8+ T-cell responses underlies the disease progression that ultimately limits the therapeutic efficacy of antileishmanial drugs.[17] There is a persistent lack of vaccine against all clinical form of human leishmaniasis[18] has resulted in only chemotherapy-based treatment. Hence, there is an urgent need for prophylactic/or therapeutic vaccine alone or in combination with immune-chemotherapy agents.

Cytokines mediate a wide variety of biological activities, including inflammation induced by an immune response as well as tissue repair and remodeling of immune system. Excessive production of one arm of cytokine(s) (Th1/Th2) or the lack of another arm contributes to the immunopathogenesis. Restoring the optimal cytokine balance may have a therapeutic value which could be achieved either by blocking of excessive cytokine(s) or by induction of recombinant cytokine(s). Recombinant cytokines are being successfully used in different diseases such as IL-2 in cancer,[19] IFN-α in viral infection and cancer,[20] IFN-β in multiple sclerosis,[21] IFN-γ in cancer and osteoporosis,[22] and IL-11 in postchemotherapy-induced thrombocytopenia.[23] However, till date, there is no documented proof of the clinical use of rIL-17 in any disease. Thus, we wanted to explore the possibility of prophylactic protective role of rIL-17 in VL. In this study, we observed that the group of mice treated with rIL-17A showed an almost similar gain in body weight as that of IFN-γ-treated group. Moreover, the marginal gain in body weight was seen when we used both cytokines for treatment, which was comparable to normal control group of mice. The gain in body weight was most likely associated with prophylactic treatment of mice with recombinant cytokine(s). Furthermore, as with earlier findings, we also observed a significant decrease in body weight in infected groups of mice (M2; without treatment), indicating that active infection possibly perturbs the normal metabolic activity that restrains in normal weight gain over time.[24] Infection, however, did not influence any change in weight of liver [Figure 1b]. This is probably due to early resolution of disease in the liver associated with granuloma formation.[225] Furthermore, we observed that the group of mice treated with IL-17 alone showed decrease in parasitic burden by nearly 49%–71.1% (liver, spleen, and BM), which was marginally increased to 56.6%–73.7% when given along with IFN-γ; which further improved when we used both cytokines [Figure 2]. Moreover, we did not observe parasitic growth in primary culture of tissue taken from the spleen suggesting that the recombinant cytokine alone or in combination may be sufficient to completely control parasitic growth in host visceral organs. We hypothesize that prophylactic cytokine(s) may be able to boost the parasitic clearance. Furthermore, recombinant cytokines may also be used with immune modulator for effective control of parasite. In our other study of adjunct therapy, recombinant cytokine treatment showed more than 90% inhibition of parasitic growth in visceral organs; moreover, primary culture of tissues from visceral organs again did not show any parasitic growth. In addition, the dominance of Th1 cytokines gene expression was also observed (unpublished data).

IL-12 has been shown to induce IFN-γ production in IL-10-deficient mice and acts in synergism to have an additional paramount effect on parasitic clearance in tissue.[26] We observed that its level in treated groups of mice was raised; which is an indicative of skewing of Th2 cytokines toward Th1. The dominant effect of IFN-γ and TNF-α on macrophage parasitical response and other effector killing mechanisms is well documented.[2728] These cytokines were observed to be moderately expressed in infected mice and showed ≈ 2–3-fold increase in all the recombinant cytokine-treated groups of mice. Interestingly, we did not observe any significant increase of IL-4, IL-10, and transforming growth factor-β in prophylactic and in adjunct therapy group of mice (data not shown).

Based on the above findings, we propose that IFN-γ, as well as IL-17A, may contain the parasitic load in VL. This is possibly due to macrophage activation by IFN-γ which is further facilitated by IL-17A. Therefore, we suggest that prophylactic use of IFN-γ and IL-17A (alone or in combination) may synergize and can be used as prophylactic and adjunct therapy with low doses of conventional toxic drugs.

CONCLUSION

Prophylactic use of recombinant IFN-γ and IL-17A inhibits parasitic growth in visceral organs of L. donovani challenged experimental mice model, especially through the upregulation of pro-inflammatory cytokines' gene expression. Thus the application of these recombinant cytokine(s) as a prophylactic agent may help in controlling/protection from VL. Moreover, this approach may also help in boosting vaccine induced immunity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.