Authors

S Moulik¹; A Dighal¹; R Sengupta¹; D Mukhopadhyay¹; S Mukherjee¹; S J Choudhuri²; M Chatterjee¹;  ¹ Institute of PG Medical Education and Research, India;  ² Ranaghat SD Hospital, Kolkata, India

Discussion

Background: Indian Post kala-azar dermal leishmaniasis (PKDL) is the cutaneous aftermath of visceral leishmaniasis (VL) that manifests as macular or polymorphic PKDL in the ratio of 1:10. However, ensuing the ongoing active surveillance for PKDL as part of the Leishmaniasis elimination programme, a substantial increase in the proportion of macular PKDL has been established. The lesional distribution of polymorphic cases is predominantly in sun-exposed areas whereas it is disseminated in the macular variant, suggesting a differential role for Vitamin D. Accordingly, this study aimed to delineate the status of Vitamin D₃ expressing genes in peripheral blood and dermal lesions of patients with polymorphic vs. macular PKDL.

Methods: Patients clinically diagnosed with PKDL were recruited from active field surveys conducted in VL endemic districts of West Bengal. Blood and dermal biopsies were collected at disease presentation. Upon ITS-1 PCR positivity, parasite load was quantified, and mRNA expression of  Vitamin D receptor (VDR), 25-Hydroxyvitamin D₃ 1-alpha-hydroxylase (CYP27B1), LL-37 (cathelicidin) and β-actin was measured by qPCR in PBMC of PKDL cases (n≤20, macular: polymorphic, 1:1) and VL (n ≤ 6) along with reverse transcriptase-PCR from dermal biopsies (n≤10, macular: polymorphic 1:1). Plasma levels of 25(OH) Vitamin D₃ was measured by sandwich ELISA at disease presentation (n≤23, macular: polymorphic 11:12).

Results: As compared to healthy controls, plasma 1α,25-dihydroxyvitamin D3(1,25D₃) levels were significantly raised in the polymorphic variant and positively correlated with parasite load at disease presentation. In circulating monocytes, irrespective of the variant, the mRNA expression of VDR (responsible for nuclear signaling of 1,25D3), CYP27B1 (converts vitamin D to its active form, 1,25D3) and LL-37 was significantly increased, and they positively correlated with parasite load. Additionally, Plasma 1,25D₃ positively co related with mRNA expression of CYP27B1 and LL37. In dermal lesions, normal skin demonstrated expression of VDR but CYP27B1 and LL37 was not detectable. The mRNA expression of CYP27B1 was significantly raised in both variants, but an increase of LL37 was restricted to the polymorphic cases. The mRNA expression of Vitamin D related genes in circulating monocytes of VL patients was comparable with healthy individuals.

Conclusion: In PKDL, the enhanced polarization of monocytes-macrophages towards alternate activation accounted for the enhanced expression of Vitamin D related genes VDR, CYP27B1 and LL37 in both variants in their peripheral blood. Importantly, in dermal lesions which are the site of the disease, an increase in the mRNA expression of VDR