P121 Effect of corticosteroids on the host innate immune response and in vitro growth characters during dermatophyte infection.
Authors: Sharma Arunima , Singh Shreya , Dogra Sunil , Narang Tarun , Saikia Biman , Saikia Uma Nahar , Chakrabarti Arunaloke , Kaur Harsimran , Ghosh Anup , Rudramurthy Shivaprakash M. 
Published: September 2022

Abstract

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM

Objective
During the current epidemic of dermatophytosis, dermatologists in India are noticing atypical clinical presentations of dermatophytosis lesions. Though fixed drug combinations of topical application containing corticosteroid-antifungal-antibacterial drugs are attributed to this phenomenon, it is still not clear about its exact role. Corticosteroids alleviate itching but do not clear off dermatophyte infection from the skin surfaces, which may lead to a relapse of dermatophytosis. Therefore, we analyzed the effect of corticosteroids on host immune response and pathogen in vitro during dermatophyte infection.

Methodology
Patients (n = 15) were recruited in three groups; proven cases of dermatophytosis with a history of corticosteroid usage for &gt;30 days (Group A), dermatophytosis with no history of corticosteroid usage for &gt;30 days (Group B), and patients without dermatophytosis and expected to have normal skin (Group C). Skin biopsies were collected and subjected to scanning electron microscopy (SEM) and cytokine expression study. All in vitro experiments were performed with HaCaT Keratinocytes cell line co-cultured with Trichophyton mentagrophytes complex conidia isolated from dermatophytosis (n = 4) and standard strain (n = 1, ATCC 18748). Biopsies were fixed in 2.5% glutaraldehyde and dehydrated through (50%-100%) ethanol gradient. RT-PCR expression of pro-inflammatory cytokines (IL-6, TNF-a, IL-1β, IL-1α, IFN-ϒ, TLR-2, and TLR-4) from skin biopsies and HaCaT cells were conducted using beta-actin as reference gene. The viability and Cell-cycle analysis of HaCaT cells in the presence and absence of clobetasol propionate (0.05% w/w) was performed by MTT assay and Propidium Iodide (PI) staining via flow cytometry, respectively. Growth kinetics of dermatophytes was performed for 96 h in presence and absence of corticosteroid. Expression of sulfite efflux pump gene (ssu1) and pH response gene (pacC), involved in virulence of Trichophyton mentagrophytes complex clinical isolates from classical and atypical lesions (n = 3 each) as well as standard ATCC 18748 was studied by RT-PCR. All results were statistically analyzed using GraphPad Prism 6 software.

Results
SEM results showed skin atrophy in skin biopsies from patients with steroid usage. Relative gene expression (2-Δct) of pro-inflammatory cytokines from skin biopsies was significantly reduced in IL-6, IL-1β, IFN-ϒ, TLR-2 (P-values = .001; .005; .004; .001) in steroid-modified tinea group. Similarly, a difference was observed in keratinocytes in vitro. According to in vitro analysis clobetasol propionate treatment significantly arrests HaCaT cells in the S/G2M phase (P-value = .04). Corticosteroid slows down the growth of dermatophytes in the presence of corticosteroid. A significant upregulation was observed in ssu1 during co-culture of dermatophytes with HaCaT cells as well when co-culture was treated with corticosteroid as compared to the culture alone whereas significant changes were not observed with pacC in similar conditions.

Conclusion
Increased atrophy caused by corticosteroids allows dermatophytes to thrive on the intact keratin when steroid pressure is removed. Reduced cytokine response, viability, and S-phase arrest in host correlate with delayed clearance of dermatophyte infection from skin. Delayed growth of dermatophytes in the presence of corticosteroids and upregulation of ssu1 in dermatophytes when co-cultured with keratinocytes and corticosteroid correlates with recurrent infection. In addition, increased production of sulfite ions that degrade keratin may lead to the formation of widespread lesions.