American tegumentary leishmaniasis (ATL) is an infectious parasitic disease caused by protozoa of the Leishmania genus. In the Americas, Brazil was the country with the highest number of reported cases (12,878) of ATL in 2022 (WHO, 2023). Patients with ATL can present a limited number of cutaneous lesions (one to 10), characterized into localized cutaneous leishmaniasis (LCL) or mucosal lesions, known as mucosal leishmaniasis (ML), in addition to other clinical forms that are less frequent (Goto and Lindoso, 2012). Host immune responses are decisive for the clinical outcome and can help in the successful treatment. These responses begin with the recognition of the protozoan by innate receptors that activate innate immune cells to produce cytokines, which modulate the acquired immune response that is responsible for the control of infection (Gollob et al., 2014). Among pro-inflammatory cytokines, tumor necrosis factor (TNF), interleukin (IL)-6, and IL-1β are produced early during infection. In addition, the pro-inflammatory cytokine IL-32 (Kim et al., 2005) is expressed in lesions of ATL patients (Galdino et al., 2014), and in mice infected with Leishmania (Viannia) braziliensis, the presence of IL32γ isoform contributed to the control of skin lesions (Gomes et al., 2017). Interferon-gamma (IFNγ), a crucial cytokine for macrophage activation, is produced by cells of innate and acquired immunity such as NK cells and type 1 helper T lymphocytes (Th1), respectively. This cytokine increases nitric oxide (NO) and reactive oxygen species (ROS) production to enable macrophages to kill the parasites (Liew et al., 1990; Novais et al., 2014; Scott and Novais, 2016; Toepp and Petersen, 2020). Moreover, IFNγ also induces IL-32 production by human macrophages (Montoya et al., 2014). Another pro-inflammatory cytokine IL-17 appears to play a role in the control (Novoa et al., 2011) as well as the immunopathogenesis of ATL or the progression of infection (Bacellar et al., 2009; Souza et al., 2013).

Vitamin D has been described as a regulator of immune responses during infectious diseases (Athanassiou et al., 2023). The vitamin D-dependent microbicidal activity of monocytes/macrophages has been described in Mycobacterium tuberculosis (Fabri et al., 2011; Montoya et al., 2014), Paracoccidioides brasiliensis (Guimarães de Matos et al., 2021), and Leishmania infection (Crauwels et al., 2019; Silva et al., 2020). In the vitamin D activation pathway, IFNγ induces STAT-1-dependent production of IL-15 in macrophages. This cytokine leads to upregulation of the CYP27B1 enzyme, which catalyzes the conversion of the inactive form of vitamin D [25(OH)D] into the active form [1,25(OH)₂D₃]. Additionally, IL-15 increases IL-32 production, which, in turn, induces CYP27B1 (Montoya et al., 2014). The active form of vitamin D activates the vitamin D receptor (VDR), which binds to VDR elements (VDREs) in the target genes, leading, e.g., to the production of antimicrobial peptides (AMPs). These AMPs, cathelicidin and β-defensin-2, act directly to control microorganisms. Crauwels et al. (2019) showed that vitamin D activates the transcription of the cathelicidin gene (CAMP) in vitro in human macrophages infected with Leishmania spp., contributing to infection control. Our group has demonstrated that IL-15 and IL-32 work together to increase the leishmanicidal activity of human macrophages through the production of ROS in a vitamin D-dependent manner (Silva et al., 2020). Furthermore, in ATL lesions of L. (V.) braziliensis-infected patients, transcriptional data demonstrated increase of cytokines and vitamin D pathway gene expression such as TNF (Christensen et al., 2016), IL1B (Amorim et al., 2019), IL32 (Galdino et al., 2014; Novais et al., 2015), IL15, IFNG, VDR, CYP27B1 (Novais et al., 2015), and IL17 (Katara et al., 2013).

The immune response can be affected by host genetic variability such as single-nucleotide polymorphisms (SNPs), which consist of exchanging one nucleotide for another, altering or not the amino acid sequence. Depending on the change in the immune genes, the expression and/or function of the protein can be affected, altering the immune responses and, consequently, the outcome of the diseases (Cabrera et al., 1995; Salhi et al., 2008). Concerning New World Leishmania studies, it has been demonstrated that SNPs in the CXCR1 and CXCR2 (Castellucci et al., 2010), FLI1 (Castellucci et al., 2011), TGFBR2, SMAD2, SMAD3, SMAD7 (Castellucci et al., 2012), COL1A1 (Almeida et al., 2015), IL1B (da Silva et al., 2019), and IFNG-AS1 (Castellucci et al., 2021) genes are associated with risk to ATL.

Shabandoust et al. (2020) evaluated polymorphisms (BsmI, TaqI, and FokI) in the VDR gene in patients with LCL infected with Leishmania tropica (Old World Leishmania), but no association with susceptibility or resistance to the disease was found. In our previous study, two SNPs in the IL32 gene were associated with protection or susceptibility to ATL (Dos Santos et al., 2020). Recently, Salem et al. (2023) identified, in Saudi patients, the association of polymorphisms of the VDR gene with parasite load and susceptibility to cutaneous leishmaniasis caused by Old World Leishmania.

This study first identified the expression of vitamin D pathway-associated genes as well as pro-inflammatory genes in patients with ATL. Further, as SNPs in genes of the components of the vitamin D pathway can influence the outcome of Leishmania infection, the present study aimed to evaluate some SNPs in genes of the vitamin D pathway (IL15, IL32, CYP27B1, and VDR), in association with clinical and treatment outcomes. The study can identify genetic markers of susceptibility or resistance to ATL as well as new candidate genes to be studied in the immune response against Leishmania.

To investigate a possible role of vitamin D in susceptibility or resistance to ATL, the present study searches for the expression of pro-inflammatory cytokines and components of the vitamin D pathway in lesions of patients with ATL in a less severe clinical form, LCL, and in the most severe form, ML. Further, SNPs in genes of two components of the vitamin D pathway (CYP27B1 and VDR) as well as in two cytokines related to this pathway (IL-15 and IL-32) were evaluated in cohorts of healthy individuals and ATL patients with LCL or ML. It was shown that in ML lesions, IL6, IFNG, and IL17 mRNA levels were higher than in LCL lesions, suggesting the involvement of these cytokines in the immunopathogenesis of ATL. The pro-inflammatory cytokines IL-6 and IL-17 have been found in ML lesions with necrotic areas infiltrated by neutrophils (Boaventura et al., 2010). The number of CD4⁺IL-17⁺ cells in ML lesions is higher than in CL (Bacellar et al., 2009). However, IL-17 also appears to participate in the pathogenesis of CL, contributing to the inflammatory infiltration at the site of infection (Bacellar et al., 2009). In tissue lesions of CL patients infected with L. tropica (Katara et al., 2013), high levels of Th17 lymphocyte markers were found. Together, data suggest that IL-6 and IL-17 can increase the inflammatory response contributing to tissue damage in ML and LCL patients, but with predominance in ML. These results are in agreement with our data showing a positive correlation between mRNA IL17 and IL6 levels in ML stronger than in LCL lesions.

The higher expression of IFNG in ML than in LCL lesions detected in the present study reflects the strong Th1 immune responses in ML caused by L. (V.) braziliensis (Bacellar et al., 2002; Silveira et al., 2009), and it is in accordance with the higher number of IFNγ-expressing cells detected in ML than in CL lesions (Faria et al., 2005). It is known that IFNγ and TNF-α, markers of Th1 response, are relevant cytokines to improve the macrophage leishmanicidal activity (Liew et al., 1990) but at the same time participate in tissue lesion (Bacellar et al., 2002). In our study, TNF mRNA was detected in low levels in both LCL and ML lesions, but it was positively correlated with IFNG, IL17, and IL6 more strongly in ML lesions than in LCL lesions. Altogether, the results strengthen the higher inflammatory profile detected in ML than in LCL.

The IL-32 is a pro-inflammatory cytokine known to induce CYP27B1 and VDR expressions, and this cytokine is part of a network with IFNγ and IL-15 to induce defense mechanisms against microorganisms (Montoya et al., 2014; Silva et al., 2020). In the current study, a stronger association between IFNG and IL15 mRNA levels was detected in LCL than in ML lesions. As these cytokines induce IL-32 production (Montoya et al., 2014), it was expected that IL32G mRNA levels were, in turn, associated with these cytokines. In fact, IL32G mRNA levels were weakly correlated with IFNG and IL15 mRNA levels in both types of lesions but were highly associated with IL15 expression, especially in ML than in LCL lesions. Both IL-15 and IFNγ are inducers of IL32γ in human macrophages, and they were strongly correlated in LCL as well as ML lesions. The axis IFNγ/IL-15/IL32γ is very important to the vitamin D-dependent microbicidal activity of human macrophages (Montoya et al., 2024; Silva et al., 2020). Concerning the mRNA levels of the components of the vitamin D pathway, VDR and CYP27B1 were associated with LCL lesions. We found a strong association between IL32G and VDR as well as IL17 and VDR expression levels in LCL and ML lesions, respectively. The data suggest that in mild leishmaniasis LCL, the lesions present a less inflammatory microenvironment with control of the parasites. However, patients with ML presented a strong positive correlation between IL32G and TNF, IL17, and IL6, thus indicating the participation of IL32γ in the inflammatory process. These results are in accordance with our previous results, where the association between TNF and IL32γ was shown only in patients with ML (Galdino et al., 2014). Together, these data suggest that IL-32/CYP27B1/VDR expression is related to microbicidal vitamin D-dependent mechanisms in LCL, contributing to protection against the parasites. Antimicrobial peptides such as cathelicidin and β-defensin-2, which are induced by active vitamin D in M1 macrophages, contribute to microorganism elimination (Montoya et al., 2014; Crauwels et al., 2019). However, in severe leishmaniasis, ML, IL32γ is highly associated with an inflammatory profile, which can be responsible for immunopathogenesis.

The link between vitamin D and IL32γ in ML is strengthened by the finding that the patients with sufficient serum levels of vitamin D expressed high levels of IL32G mRNA in the lesions, suggesting that vitamin D could increase IL32γ production. How vitamin D acts to control the expression of IL32 is not known and deserves further investigation. In the current study, IL32γ was associated with all pro-inflammatory cytokines evaluated in mucosal lesions, including IL-15. The cytokine IL-15, strongly associated with IL32γ in ML, is a pro-inflammatory cytokine that can control the growth and proliferation of T lymphocytes and NK cells as well as macrophage differentiation in M1/microbicidal profile, crucial for controlling intracellular microorganisms; however, it is also associated with the pathogenesis of chronic immune-mediated diseases (Waldmann, 2014; Yang et al., 2015). Few studies have evaluated IL-15 in leishmaniasis. In visceral leishmaniasis, it was shown that IL-15 is produced during the infection caused by Leishmania infantum (Milano et al., 2002) and increases the killing of this parasite (D’agostino et al., 2004; Oliveira et al., 2015). The cytokine IL-15 is also produced by monocytes cultured with New World Leishmania spp. antigens (Carrada et al., 2007; Lago et al., 2018), but its biological function was not evaluated in these studies. That IL-15 can be associated with parasite control is strengthened by the strong correlation between the IL15 and IFNG mRNA levels in LCL detected in our study. However, in addition to known cytokines involved in the immunopathogenesis of ATL, such as IFNγ, TNF, and IL32γ, our present data suggest that IL-15 should be especially considered in ML.

The expression of cytokines at the diagnosis time could be a marker of prognosis in ATL; however, despite high levels of IFNG, IL15, and IL32G mRNA in the early LCL lesions, these levels could not be associated with the therapeutic outcome in the present study. We detected 27.7% and 41.0% of therapeutic failure in LCL and ML patients, respectively, a lower rate than previously reported in Brazil for LCL (Carvalho et al., 2022), but in the same range for ML (30%–90% depending on the geographic area; Goto and Lindoso, 2012). Surprisingly, we found that the VDR mRNA levels in patients with LCL were higher in the failure outcome group. As the VDR mRNA levels are not only expressed in macrophages, where they are relevant for microbicidal activity, the increased levels of VDR in other cells present in the lesions can explain these results. It is known that vitamin D inhibits T helper lymphocyte cytokines, including Th1-derived cytokines required for control of Leishmania ssp. Even in macrophages, although vitamin D stimulates microbicidal activity, it inhibits pro-inflammatory cytokine production. In addition, vitamin D increases the production of anti-inflammatory/immunosuppressive cytokines, which were not evaluated in the present study (Ghaseminejad-Raeini et al., 2023). Thus, in the lesions, the immunomodulatory effects mediated by vitamin D–VDR on different immune cells can interfere with the outcome of the disease.

We also evaluated the SNPs in genes of the vitamin D pathway. No study has yet investigated SNPs in the IL15 gene in association with ATL outcomes. We found that allele A of IL15 rs10519613 was associated with high expression of CYP27B1. It was not associated with serum vitamin D levels nor with the therapeutic outcome of LCL. Although not in infectious diseases, there is a report of the association between the AA genotype of this SNP and hyperdiploidy, a clinical feature in acute lymphoblastic leukemia (ALL) (Rots et al., 2018), and allele A was associated with the risk of minimal residual disease–positivity (indicator for ALL) (Dawidowska et al., 2016). The allele A of this IL15 SNP was also correlated with the risk of type 1 diabetes (Zouidi et al., 2014). The IL15 rs10519613 is located at exon 6, 83 bp behind the stop codon, and the AA+AC genotypes and the allele A of this SNP were found as risk factors for coronary heart disease. Furthermore, the AC genotype and allele A were associated with high levels of IL-15 in acute form, and all subjects evaluated with the CC genotype presented lower levels of IL-15 (Gokkusu et al., 2010). Another genetic variation of the IL15 gene, the IL15 rs3775597, was selected in our evaluation due to the relevant frequency of the G variant allele in the American population, but there is yet no study that has investigated it in infectious or non-infectious disease outcomes. Only one report evaluated this SNP as a risk factor for hypertension but did not achieve conclusive results (Taylor et al., 2010). We demonstrated that in LCL, the GG IL15 rs3775597 genotype was associated with low VDR and CYP27B1 expressions. There was no association with vitamin D levels or with treatment outcomes in carriers of this SNP IL15. Despite this, we detected two variants of IL15 that were differentially associated with CYP27B1 expression. As IL-15 induces CYP27B1, in an IL-32-dependent manner (Montoya et al., 2014), these results suggest that the IL15 SNP rs10519613 allele A can be associated with a gain of function increasing CYP27B1 and favoring the control of the parasites in macrophages of LCL patients. However, IL15 SNP rs10519613 can interfere with the capacity of IL-15 to induce CYP27B1. In any situation, the data highlight a possible role of IL-15 in LCL. Concerning ML, the GG genotype of the IL15 rs3775597 variant was associated with low IL17 expression. This SNP is located in an intronic region, so the influence on cytokine production needs to be further investigated to better understand the mechanism by which this SNP can decrease IL17 expression, or whether this is an indirect effect, depending on other cytokines in ML lesions.

We had previously studied the IL32 rs1555001, but no significant association with ATL manifestations was found (Dos Santos et al., 2020) as well as with acute lung injury (Arcaroli et al., 2011) or with carotid artery calcified plaque in type 2 diabetes mellitus (Lehtinen et al., 2011). Now, with more patients in the cohort, we found that allele A was significantly associated with HC, which means that the risk of carriers of allele A not present in ML is significant. Thus, this can associate this SNP with mechanisms of resistance to develop ML. Previously, we showed that carriers of the AA genotype presented low production of IFNγ and IL-22 in cultures of peripheral blood mononuclear cells stimulated with L. braziliensis (Dos Santos et al., 2020). It is known that IL32γ and IFNγ play a role in infection control, but they exacerbate the responses linked to tissue damage present in ML (Ribeiro-De-Jesus et al., 1998; Oliveira et al., 2011; Galdino et al., 2014). As our previous data suggested, the genetic variant IL32 rs1555001 can reduce the production of IL32γ and IFNγ, and this can promote an environment that is less inflammatory and thus contribute to the resistance of ML development. However, IL-22 was found to be associated with control of inflammation and wound healing, but in a leishmaniasis model, this appears to be dependent on the level of tissue damage (Gimblet et al., 2015). The production of these immunomodulators, especially IL-22, in association with this IL32 genetic variation, needs to be further investigated.

Only two previous studies have investigated genetic variants related to vitamin D in association with leishmaniasis. One of them found no significant association between SNPs in the VDR gene with increased risk of L. tropica infection (Shabandoust et al., 2020), but another one demonstrated that the allele A of VDR rs7975232 (ApaI) was associated with susceptibility to CL in Saudi patients (Salem et al., 2023). We found that the presence of this allele was associated with low IL6 expression in the cutaneous lesions. Moreover, we reported in this study that the carriers of allele A with ML have more probability of therapeutic failure. The ApaI SNP, located in the intron 8 of the VDR, presents the C reference allele that changes to A and does not change the amino acid sequence of the VDR protein. However, the SNP could affect mRNA stability and VDR expression by LD (Mahto et al., 2018; Triantos et al., 2018). If TaqI (VDR rs731236) is in high LD with ApaI, its functional effect is the possible modification of one of the zinc fingers of the nuclear signaling heterodimer that binds to the VDREs located in the target genes (Mahto et al., 2018). We can speculate that possibly that in patients with ML carriers of the A variant allele, there is an LD between ApaI and another VDR SNP that compromises the binding of VDR to VDREs in DNA and, consequently, the production of antimicrobial peptides, favoring the parasite persistence and the therapeutic failure. In addition, as commented above, less stimulation of VDR can lead to high inflammation in lesions due to the missing control of pro-inflammatory cytokines by vitamin D in T lymphocytes (Ghaseminejad-Raeini et al., 2023). Previous data about this VDR SNP in association with treatment outcomes in other infectious diseases showed that the allele A of ApaI was considered the pretreatment genetic predictor of sustained hepatitis B surface antigen (HBsAg) and loss in hepatitis B early antigen (HBeAg) in patients with chronic hepatitis B with pegylated interferon (Peg-IFN) monotherapy (Shan et al., 2019). Furthermore, the VDR haplotype composed of the C allele of ApaI was a predictor of pegylated-interferon/ribavirin-based therapy failure in chronic hepatitis C (CHC) Caucasian patients (Baur et al., 2012; García-Martín et al., 2013), but not in CHC Asian patients (Hung et al., 2016). These data pointed out that, in contrast to the impact on treatment outcomes in hepatitis B and C, the altered allele A of ApaI could be a genetic marker of therapeutic failure in ML.

It is very difficult to reconcile all the results obtained in the current study, mainly those of mRNA expression versus SNPs and those with clinical/therapeutic outcomes. We confirmed that ML presents lesions with a stronger inflammatory profile than lesions of patients with LCL. In addition, we also confirmed our previous results about the strong connection between IL 32γ and TNF in ML and the role of vitamin D microbicidal pathway dependent on IL32γ and IL-15 in human macrophages. Here, the results suggest that for LCL, the axis IL32γ/IL-15/CYP27B1/VDR, although very important to control the parasites in human macrophages, can be affected by the effects of vitamin D on T lymphocyte cytokines in the lesions. This can be illustrated by two different IL15 SNPs that differentially affect the vitamin D pathway gene expression, CYP27B1 and VDR, whose expressions were associated with the high number of lesions and therapeutic failure, respectively. In addition, in ML, the axis IL32γ/TNF/IL-15/IFNγ/IL-6/IL-17 is highly inflammatory in the immunopathogenesis of the disease. In this pro-inflammatory environment, the VDR SNP was associated with therapeutic failure, which may be associated with the difficulty of vitamin D in controlling the inflammatory cytokines (Figure 6). This type of study is relevant to identify possible players and their connection to investigate their roles in ATL. A low number of studies on vitamin D, IL-32, and IL-15 have been published, and one of the difficulties is that the axis IL-15/IL32γ/vitamin D cannot be evaluated in a mouse model. Mouse does not present IL-32, and the β-defensin and cathelicidin genes are not controlled by vitamin D in these animals as in human beings. In addition, the IL-32 receptor was not yet identified. Together, these points can highlight the relevance of genetic studies on the cytokine/vitamin D pathway in ATL and other diseases.