Combination of Mycobacterium indicus pranii and Heat-Induced Promastigotes Cures Drug-Resistant Leishmania Infection: Critical Role of Interleukin-6-Producing Classical Dendritic Cells

Abstract

The major issues in available therapeutic modalities against leishmaniasis are cost, toxicity, and the emergence of drug resistance. The aim of this work was to develop a successful therapeutic adjuvant against drug-resistant Leishmania donovani infection by means of combining Mycobacterium indicus pranii with heat-induced promastigotes (HIP). One-month postinfected BALB/c mice were administered subcutaneously with M. indicus pranii (108 cells) and HIP (100 μg) for 5 days. Spleens were harvested for flow cytometric and reverse transcriptase PCR analysis. The antileishmanial effect of the combination strategy was associated with induction of a disease-resolving Th1 and Th17 response with simultaneous downregulation of CD4+ CD25+ Foxp3+ (nTreg) cells and CD4+ CD25− Foxp3− (Tr1) cells in the spleen. The significant expansion of CD4+ TCM (CD4+ CD44hi CD11ahi CD62Lhi) cells was a further interesting outcome of this therapeutic strategy in the context of long-term protection of hosts against secondary infection. Toll-like receptor 2 (TLR2) was also found instrumental in this antiparasitic therapy. Induced interleukin-6 (IL-6) production from expanded CD11c+ CD8α+ (cDC1) and CD11c+ CD11b+ (cDC2) dendritic cells (DCs) but not from the CD11b+ Ly6c+ inflammatory monocytes (iMOs), was found critical in the protective expansion of Th17 as evidenced by an in vivo IL-6 neutralization assay. It also promoted the hematopoietic conversion toward DC progenitors (pre-DCs) from common dendritic cell progenitors (CDPs), the immediate precursors, in bone marrow. This novel combinational strategy demonstrated that expansion of Th17 by IL-6 released from CD11c+ classical DCs is crucial, together with the conventional Th1 response, to control drug-resistant infection.

Deletion of Glutamine Synthetase Gene Disrupts the Survivability and Infectivity of Leishmania donovani

“…Ld GS (++/++) were found efficient over both Ld GS (−/−) and Ld GS (+/−) as evidenced in comparison to the wild type parasite infection at one-month post infected animals [inhibition only by 23.5 ± 15.05% and 23.97 ± 16.21% in the spleen and liver, respectively] as -represented in ( Figure 3D I and II). The presence of Leishmania -kDNA in respect to Mϕ-GAPDH determined the degree of infection, in vivo ( Dey et al., 2020 ). Expression of kDNA further confirmed the result that Ld GS (−/−) parasites had low infection rate, in vivo , as evidenced from the significantly reduced expression of Leishmania -kDNA in Ld GS (−/−) infected animals in comparison to Ld GS (+/+) at both 15 days and 30 days post infection against normalized equal expression of Mϕ-GAPDH ( Figure 3E ).…”

“…The experimental groups (5 animals in each group; 4-6 weeks age; weighing approximately 20g) were infected, i.v., with 2 × 10 7 promastigotes of Ld GS (+/+) , Ld GS (+/−) , Ld GS (+/−/+) , Ld GS (−/−) , Ld GS (−/−/+) , Ld GS (++/++) , and Ld : psp (Vc). Four-week post-infected animals were sacrificed, amastigotes were calculated from the spleen and liver Giemsa-micrographs under phase contrast microscope (Zeiss Axioscope), and parasite burden was extrapolated using Stauber’s formula as described earlier ( Dey et al., 2020 ). The survival percentage of promastigotes maintained under specific antibiotic surveillance was monitored in the flow cytometer (BD FACS Verse) using 7-Aminoactinomycin D (7-AAD) before in vivo infection ( Sultana et al., 2018 ).…”

“…The mRNA was extracted from the spleens of the infected animals and converted to cDNA. The expression of kinetoplast DNA (kDNA) was evaluated by semi-quantitative PCR ( L. donovani kDNA; Forward: 5′-AAATCGGCTCCGAGGCGGGAAAC-3′ and Reverse: 5′- GGTACACTCTATCAGTAGCAC-3′), normalized against equal expression of house-keeping gene GAPDH (murine GAPDH; Forward: 5´-GAGCCAAACGGGTCATCATC-3´ and Reverse: 5´-CCTGCTTCACCACCTTCT TG-3´) ( Dey et al., 2020 ). The densitometric quantification was analyzed by Image Lab software and represented as mean ± SE from 3 animals per group.…”

Short-Course Treatment With Imipramine Entrapped in Squalene Liposomes Results in Sterile Cure of Experimental Visceral Leishmaniasis Induced by Antimony Resistant Leishmania donovani With Increased Efficacy

“…Interestingly Liposomal-imipramine treatment was associated with decrease in IL-10 production irrespective of the nature of the input infection ( Figure 3A , III). It has been recently reported that IL-6 plays an important role in rendering protective immune response against Sb R LD mediated infection by lowering IL-10 level ( Dey et al., 2020 ). In the present work, we have also observed that there is a strong suppression of IL-10 level, which might result from increased IL-6 level, as oral imipramine treatment increased IL-6 level in Sb R LD mediated infection, and this in all probability will be further enhanced with liposomal imipramine treatment as observed in the increased suppression level of IL-10 in presence of liposomal-imipramine ( Figure 3A , iv)…”

A mushroom derived ‘carbohydrate‐fraction’ reinstates host‐immunity and protects from Leishmania donovani infection

“…For nitric oxide (NO) estimation, uninfected and infected peritoneal macrophages (10 5 cells) were treated with increasing concentrations of Ah f-Car and kept for 60 hours. 17 Supernatants from uninfected (UIM) and infected controls (IM) along with treated cells (50 µg/ mL, 75 µg/mL, and 100 µg/mL) were collected and absorbance was measured at 540 nm in a microplate reader (iMARK, Bio-Rad) after incubation with Greiss reagent (Sigma).…”