The molecular and histopathological investigations of TLR2 rs5743708 and TLR4 (rs4986790 and rs4986791) polymorphisms effects on cutaneous leishmaniasis lesions

Cutaneous leishmaniasis (CL), a zoonotic and neglected disease, is prevalent in numerous regions, particularly in tropical and sub-tropical countries. In Iran, endemic foci of leishmaniasis exist in specific regions, with zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania major being common in most rural areas. Toll-like receptors (TLRs) play a crucial role in both innate and adaptive immunities, and the investigation of TLR2 rs5743708 and TLR4 (rs4986790 and rs4986791) polymorphisms in parasitic diseases can have significant implications for patient treatment. In the present study, a total of 88 leishmaniasis patients using the patients' lesions from Khuzestan province health-treatment centers, Iran, including 50 cases (56.8%; Central region) and 38 cases (43.2%; Western region) underwent examination between the years 2022 and 2023. Two direct smears from the lesions of each patient were prepared and one of the smears was stained with Giemsa for parasitological examination. Among the 88 patients, the highest frequency was observed in the 21-30 years' age group (35.2%), while the lowest was in the 11-20 years' age group (10.2%). No statistically significant relationship was found between gender and age (P > 0.05). Following disease confirmation via microscopic examination, TLR2 rs5743708 and TLR4 (rs4986790 and rs4986791) polymorphisms in the patients were assessed using PCR-RFLP. Fragments of 264, 249, and 406 base pairs were successfully amplified, targeting the TLR2 and TLR4 genes, respectively. Out of the 88 leishmaniasis patients, 14 cases (15.9%) exhibited polymorphisms. Notably, all individuals in the polymorphism group carried both the TLR2 rs5743708 homozygous and the TLR4 rs4986791 heterozygous genotype combinations. There were no observations of TLR2 rs5743708 heterozygous, TLR4 rs4986790 heterozygous and homozygous and TLR4 rs4986791 homozygous genotypes within the polymorphism group. Biopsies from lesions for all contributors were prepared for histopathological examination. All patients with polymorphism showed larger lesions than patients without polymorphism (P < 0.05). Histophatological study showed abnormal cases in patients with polymorphism including mild hyperkeratosis, mild acanthosis, focal parakeratosis in the epithelium surface and mild hyperpigmentation of melanocytes in the basal layer. Furthermore, a strong infiltration of immune cells such as PMNs and a small number of lymphocytes was observed in the epidermal region of patients with polymorphisms. There was no statistically significant relationship between age and the quantity of lesions (P > 0.05). Additionally, some regions of the epidermal surface layer displayed pustule formation in patients with polymorphisms. No significant difference was discerned in the dermal layers of patients with polymorphisms compared to other patients. Considering that all patients with polymorphisms concurrently had TLR2 rs5743708 homozygous and TLR4 rs4986791 heterozygous genotype combinations, the anomalies observed in conjunction with histological changes in the skin lesions of patients with CL may plausibly be linked to the polymorphisms. Nonetheless, a more expansive dataset involving a larger population is imperative to comprehensively elucidate the pathogenesis of the Leishmania parasite and the potential impact of TLR2 rs5743708 and TLR4 (rs4986790 and rs4986791) gene polymorphisms in individuals afflicted with CL across diverse geographical locales.