Leishmaniasis is an ongoing global public health problem despite efforts towards its control and elimination over the last decade. It is a disease complex with clinical manifestations ranging from systemic visceral leishmaniasis (VL) caused by Leishmania donovani complex, cutaneous leishmaniasis (CL) with skin restricted lesions caused by Leishmania tropica complex, and mucocutaneous leishmaniasis (MCL) involving mucocutaneous membrane caused by Leishmania braziliensis complex (1). The varied disease outcomes are determined by the infecting parasite species/subspecies and the host immune response. In the Indian subcontinent, classical systemic infection caused by anthroponotic L. donovani is the prominent disease form accounting for more than half the world’s disease burden in known and newer endemic regions in India, Nepal, Bangladesh, Bhutan, and Sri Lanka (2–5). CL cases are lesser in number with L. tropica/L. major as the causative agent in India, Nepal, and Bangladesh (6–8). More recently, the disease is emerging as an unusual cutaneous manifestation caused by L. donovani in the newer endemic sites in Sri Lanka, Nepal, and India (9–11). The emergence of such atypical CL poses a newer challenge to the ongoing disease-control program and thus needs to be studied to understand the atypical disease etiology. So far, there are few reports on genetic analysis of parasite isolates from atypical CL cases endemic in Sri Lanka, Nepal, and the states of Kerala and Himachal Pradesh (HP) in India, identified as L. donovani genetic variants distinct from the classical VL L. donovani isolates (9–12).

In regions of the emergence of dermotropic L. donovani, it is imperative to understand the immune response elicited by the parasite variants to further understand the unusual disease outcome. Immunology of VL and CL diseases caused by visceralizing versus dermotropic parasite species is well studied in a classical setting. Leishmania species-specific antigens prime and shape up the T-cell response, crucial in determining the course of the disease progression. In experimental models, a polarized Th1/Th2 helper T-cell response is seen with a Th1 skewed protective response in C57BL/6 resistant mice and a Th2-dominating disease progressive response in the susceptible BALB/c mice, while in humans, a mixed Th1/Th2 response is demonstrated (13, 14). Interestingly, the disease outcome is presently understood as a more complex phenomenon such that discrete parasite-specific antigens induce differential T-cell subset-specific inflammatory, anti-inflammatory, and/or regulatory cytokines that together fine-tune the disease outcome in terms of differential parasite load, dissemination, and persistence leading to a gradation of typical VL and CL manifestations (14–22).

Immune response in classical human VL cases is marked by elevated levels of multiple pro-inflammatory cytokines IFN-γ, TNF-α, IL-12, and IL-17 along with high expression of immune-regulatory cytokines IL-10 and TGF-β that counteract the macrophage activation with enhanced parasite survival and disease progression (23–25). Importantly, a range of cutaneous manifestations are seen in humans with differences in the number and nature of lesions, parasite load and persistence, and localized versus diffuse lesions with varying timelines of healing. The heterogeneity in CL disease phenotypes is associated with differences in lesional and systemic host immune environments. The gradation of T-cell immune response in a non-healing to healing lesional phenotype varies from a mixed Th1/Th2 to a Th1-dominant response with the key modulation exerted by the regulatory cytokines IL-10 and TGF-β (19). The regulatory cytokine axis controls the host antiparasitic response by differential modulation of pro-inflammatory and anti-inflammatory cytokine levels that may exhibit as subclinical infection to mild-to-moderate disease to extreme CL phenotypes (19, 26). In classical CL cases, susceptibility is represented by non-healing lesions with low IFN-γ and high IL-4 as well as IL-10 production and vice versa in cases with healing lesions, highlighting the significance of IFN-γ/IL-10 ratio in driving disease phenotype and severity (17, 27–29).

With this background, the immune status of patients designated as “Atypical CL cases” caused by L. donovani is not yet explored substantially with only a few preliminary reports on lesional cytokine patterns in atypical CL patients from Sri Lanka (30–32). These reports imply a Th1-biased lesional cytokine environment with co-expression of Th2 cytokines such that the levels of the IFN-γ correlate with the healing versus non-healing lesion duration. In this regard, systemic immune correlates of the atypical CL patients are not yet understood. Thus, the manifestation of L. donovani-mediated atypical CL (LdCL) needs to be urgently explored from an immunological perspective with the potential to explain the unusual disease phenotype. In the present work, we investigated systemic immune-cytokine expression profile and parasite-specific immunoglobulin G (IgG) and IgG subclasses expression patterns in LdCL patients from the endemic state of HP (11). The study provides new knowledge on host immune variables as determinants of restricting a classically visceralizing L. donovani to localized cutaneous lesions unlike the systemic infection in northeast India. In turn, the study brings out the significance of the fine-tuning of the host immune response modulated by the L. donovani variants circulating in the study region in driving the cutaneous outcome.

Leishmaniasis initiates as a skin infection and progresses as varied disease outcomes, driven by the infecting Leishmania species/strains and heterogeneous host immune modality. The initial host immune response elicited by distinct parasite determinants set the stage for differential parasite persistence, dissemination, and immunopathogenesis that together determine the gradation of visceral and cutaneous manifestations. Further, with Leishmania being an intracellular pathogen, the total of T-cell immunity comprising heterogeneous T-cell subsets with discrete effector cytokines direct the course of the disease ranging from non-symptomatic to mild-to-severe VL and CL phenotypes (14, 16, 17, 21, 22). With substantial insight into the host immune correlates that underline the classical visceral and cutaneous manifestation, scarce reports are available on host immune correlates in L. donovani atypical CL disease emerging in known and newer endemic sites in the Indian subcontinent (10, 30–32, 35, 36). Knowledge on comparative host immune response in LdCL versus typical VL is important to define the key immune checkpoints that can modulate the disease towards atypical phenotype (9–12, 37). In this regard, no data are available on the systemic cytokine correlates in atypical CL cases with few reports on RT PCR-based lesional cytokine environment in Sri Lankan atypical CL cases. Our finding on systemic cytokine expression in atypical HPCL patients is in line with a Th1-dominating lesional cytokine response reported earlier in Sri Lankan CL patients with elevated levels of plasma IFN-γ, TNF-α, and IL-12 along with a low-to-undetectable expression of IL-4 (30–32). With the knowledge that IFN-γ is the key Th1 cytokine critical in the defense against the intracellular parasite, a robust Th1 response in HPCL patients suggests an enhanced parasite killing activity that could result in parasite restriction to localized skin lesions (17, 21). In contrast, the Th2-specific IL-4 is a disease-promoting cytokine and counteracts protective Th1 response, which further supports dominance of Th1 parasite killing systemic response in HPCL patients exhibiting a marginal increase in IL-4 (17, 21). Importantly, with the Th1 protective response similar to the typical VL cases albeit with cytokine-specific quantitative differences, the expression pattern of additional cytokines could possibly explain the dermotropic manifestation in HPCL cases.

In this regard, Th17/Th22-specific pro-inflammatory cytokines are important regulators of parasite multiplication and ensuing tissue inflammation that determine heterogeneous outcomes in VL and CL (15, 19, 38–40). The Th17–Th22 antiparasitic cytokine axis works in a cross-regulatory loop such that Th17 effector cytokines lead to tissue inflammation if over-produced, and Th22 effector response is understood more as tissue-protective (41–43). Th17 effector function itself exhibits heterogeneity with an inflammatory Th17 pathogenic response that synergizes with parasite-killing Th1 activity or a regulatory-type Th17 response that works to maintain immune homeostasis (41, 44). Additionally, Th22 effector cytokines crosstalk with the Th17 cells via the common pool of IL-22 effector cytokine ( Figure 5 ). IL-22 is antiparasitic with a predominant role in tissue repair along with the potential to mediate the inflammatory response in conjunction with IL-17 (17, 42, 43). Thus, differential expression of Th17 and Th22 cytokines drives the degree of microbicidal activity, host tissue inflammation, and repair in varied CL and VL manifestations. A basal Th17 and an enhanced Th22 response in atypical CL patients could thus manifest antiparasitic and tissue-protective responses albeit with limited inflammation in the presence of marginal IL-17 level. Additionally, an enhanced IL-1β itself can induce parasite-killing NO production similar to TNF-α and can suppress IL-4 activity, while IL-6 can skew the IL-22-mediated antiparasitic and tissue repair response. The said cytokine pattern can possibly explain the development of the localized cutaneous lesions associated with low parasite load and restricted tissue damage (41, 42). In CL, the Th17/IL-17 and Th22/IL-22 axes are reported to be parasite species-dependent such that varying spectra of lesional and systemic Th17/Th22 cytokine expression pattern correlate with heterogeneous phenotypes of healing to non-healing cutaneous lesions (19). This can possibly implicate the threshold of host Th17/Th22 cytokine pattern induced by an antigenic pool of L. donovani variant in the study region to be responsible for the atypical localized cutaneous manifestation.

HPCL patients exhibited a near-around cytokine pattern in comparison with the VL cases with respect to Th1/Th17/Th22 cytokines, although with dramatically different disease outcomes. The phenomenon can be explained on the basis of the difference in the expression levels of the regulatory cytokine IL-10. IL-10 is demonstrated to be the key immunoregulatory cytokine that pins down the protective function and regulates inflammation mediated by the Th1/Th17/Th22-specific cytokines, thereby modulating the parasite load and host immuno-pathological consequences as shown in Figure 5 . The regulatory role of IL-10 in varied spectra of visceral and cutaneous disease manifestations is mediated by its differential induction by the infecting parasite species/strains and the host genetics (17–19, 24, 45, 46).

Classical visceral disease is associated with elevated levels of IL-10 that mediate systemic parasite dissemination with varying degrees of tissue damage correlating with disease severity (23, 47–52). The higher IL-10 expression in the BVL patient group, in line with reports on classical VL, could render the Th1/Th17/Th22 cytokine axis ineffective in parasite killing and could lead to visceral dissemination and aggravated host tissue damage (24, 25, 41, 48, 53, 54). In the cutaneous disease, a varied expression of IL-10 lesser than the levels in typical VL disease is reported, with a role in limiting parasite load as well as persistence along with the extent of tissue damage in varying gradations of lesion types and severity (17, 19, 26, 46, 55, 56). With this understanding, a significant downregulation of IL-10 in HPCL patients compared with the VL group suggests an effective parasite-killing Th1 axis that could limit the systemic parasite circulation supported by the detection of faint L. donovani-specific amplicons with lack of any VL like diagnosis (11). The central role of IL-10 proposed in modulating VL to CL outcome in the HPCL patients is strengthened by the reports on typical VL and CL diseases, wherein the phenotypic heterogeneity from systemic to cutaneous disease with healing to non-healing lesions correlates with the ratio of protective IFN-γ to regulatory IL-10 levels (19, 24) ( Figure 5 ). Also, suppressed TGF-β expression in the atypical CL cohort compared with the typical VL patients signifies an enhanced parasite killing activity of host cells.

IL-27 regulates IFN-γ-mediated Th1 protection, enhances IL-10-mediated regulatory response, and also functions as a negative regulator of Th17 inflammatory response (54, 57–64). A trend towards an increased IL-27 expression in HPCL could imply its role in an altered Th1–Treg–Th17 axis such that localized cutaneous lesions with limited cutaneous pathology and parasite load are manifested in corroboration with the few CL-specific reports available (60, 65). IL-27 expression in the BVL patients was comparable with that in the control group in contrast to the existing reports for elevated IL-27 in VL patients, possibly due to the small sample size with variable reads (54, 66).

Assessment of parasite-specific humoral response in terms of the elevated IgG and differential titers of Leishmania-specific IgG subclasses is another important immune correlate of the disease phenotype. With a lack of understanding of the protective role of antibodies, augmented IgG antibody response against a parasite is reported for both VL and CL with variation in IgG subclasses driven by cytokine-mediated isotype class/subclass switching (67–70). In typical VL and CL cases, SLA-specific IgG along with IgG1 and IgG3 have been shown to be enhanced with variable reports on IgG2 and IgG4 expression patterns (34, 71–74). With the lack of such study for the atypical CL cases, we elucidated a robust parasite-specific IgG response in CL patients from HP with values higher than the typical VL patients. Interestingly, the relative increase in total IgG levels in HPCL versus BVL cases majorly represented enhanced IgG2 and IgG3 levels with decreased IgG1 levels. The pattern of the differential IgG1, IgG2, and IgG3 levels in HPCL versus BVL patients possibly represents differences in the complement of B cell-specific immunodominant antigens in the CL- and VL-causing L. donovani variants (68, 70). This is reflected in the poor rK39 seroreactivity of atypical CL patients in the study area along with similar findings with Sri Lankan CL cases (11, 33). It is important to mention that IgG/IgG subclass evaluation in the present study was done using SLA prepared using a standard VL-derived L. donovani culture. An enhanced antibody titer suggests the presence of a large number of common immuno-dominant antigens in the VL-causing L. donovani variant and CL-causing L. donovani variant along with antigenic variations showing up in terms of differences in levels of IgG subclasses and rK39 reactivity (11). With IL-10 determining the IgG1 and IgG3 isotype switching, variation in levels of IL-10 in HPCL and BVL patients could potentially result in differences in the levels of IgG subclasses with a role in downstream effector functions potentially associated with different disease outcomes.

In summary, an enhanced IFN-γ/IL-10 ratio in atypical CL cases could potentially explain the localized cutaneous phenotype over the visceral disease. IL-27 could potentially cross-regulate the Th1/Th17/Th22-mediated spectrum of immune effector function associated with heterogeneous disease outcomes as depicted in Figure 5 . Importantly, we can conclude that IL-10 is the key immune checkpoint that potentially modulates the disease phenotype towards a cutaneous manifestation. With a decrease in IL-10, an effective Th1/Th17/Th22 response would ensue that could restrict the L. donovani variant to the skin and modulate the disease progression towards a cutaneous phenotype in the HPCL cases over a visceral phenotype that is classically associated with high circulatory IL-10 levels. An altered pool of immunogens, evident from the multiple genetic changes within the coding genomic sequences in the atypical L. donovani isolates in comparison with the classical wild-type L. donovani, could possibly fine-tune the systematic immune response towards a cutaneous phenotype. Importantly, for an understanding of the mechanism of IL-10-mediated atypical cutaneous phenotype, the cause–effect axis needs to be explored further. In-depth analysis of the immune cells with discrete cytokine expression profiles, possibly mediating the observed cytokine changes, needs to be studied by deep flow cytometry profiling. Advances in the understanding of T-cell immunity in terms of the T-cell subsets that produce specific cytokines along with the target cells with cognate cytokine receptors can give major leads towards elucidating atypical cases from an immunological perspective. Such findings will be relevant with the preposition for rationalized immune therapeutics and vaccine development against the atypical disease per se along with the visceral and cutaneous diseases. Thus, more studies for unraveling the mechanism of the key cytokine players involved in atypical CL manifestation are required. An animal model mimicking atypical CL can provide a workable model to further dissect out the immune checkpoints that drive the unusual disease phenotype along with a better definition of the complex immune environment that drives VL and CL.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Indira Gandhi Medical College, Approval no. HFW (MS) G-5 (Ethics)/2014-10886; Central University of Punjab, Approval no. CUPB/IEC/2016/034; and Medical Research Centre, Muzaffarpur, Bihar, India Approval no. Dean/2015-16/EC/364. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

LT collected the samples, performed the experiments, and analyzed the data. PM helped with the figures. VS and AN provided the CL samples. AJ provided infrastructural support to execute the experiments. SBC, OP, and SS provided the VL samples. OP and AJ critically revised the manuscript. MJ contributed to the concept and design of the study, data analysis, drafting of the article, and final approval of the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by Collaborative Research Project Grant from ICGEB, Trieste (Project CRP/IND19-01, GP#163, Central University of Punjab) to MJ. LT was funded with a doctoral fellowship, Central University of Punjab, Bathinda. PM is supported by DBT Senior Research Fellowship.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.