Induction of miR 21 impairs the anti-Leishmania response through inhibition of IL-12 in canine splenic leukocytes

Abstract

Visceral Leishmaniasis is a chronic zoonosis and, if left untreated, can be fatal. Infected dogs have decreased cellular immunity (Th1) and develop a potent humoral response (Th2), which is not effective for elimination of the protozoan. Immune response can be modulated by microRNAs (miRNAs), however, characterization of miRNAs and their possible regulatory role in the spleen of infected dogs have not been done. We evaluated miRNA expression in splenic leukocytes (SL) from dogs naturally infected with Leishmania infantum and developing leishmaniasis (CanL; n = 8) compared to healthy dogs (n = 4). Microarray analysis showed increased expression of miR 21, miR 148a, miR 7 and miR 615, and downregulation of miR 150, miR 125a and miR 125b. Real-time PCR validated the differential expression of miR 21, miR 148a and miR 615. Further, decrease of miR 21 in SL, by means of transfection with a miR 21 inhibitor, increased the IL-12 cytokine and the T-bet/GATA-3 ratio, and decreased parasite load on SL of dogs with CanL. Taken together, these findings suggest that L. infantum infection alters splenic expression of miRNAs and that miR 21 interferes in the cellular immune response of L. infantum-infected dogs, placing this miRNA as a possible therapeutic target in CanL.

miR-548d-3p Is Up-Regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth in Macrophages

“…These miRNAs presented below in sequence are related to activation or modulation of the immune system and may play a crucial role in Leishmania infection either in the control or progression of the infection. The following miRNAs may contribute to the progression of the infection: a) miR-21 seen up-regulated in dendritic cells, macrophages, inflammatory monocytes, polymorphonuclear neutrophils, and in the spleen and liver tissues after L. donovani infection and in splenic leukocytes from L. infantum -naturally infected dogs and related to the inhibition of IL-12 expression and impairment of the Th1 response implied in the control of the infection ( Melo et al., 2019 ; Varikuti et al., 2021 ); b) miR-302a-3p that regulates receptor activator of nuclear factor kappa-B ligand (RANKL) (a member of the tumor necrosis factor TNF superfamily) that regulates T-cell-dependent immune response and apoptosis ( Irwandi et al., 2018 ) and also up-regulated in in vitro L. braziliensis infection ( Souza et al., 2021 ); c) miR-372-3p that inhibits cell proliferation and apoptosis and seen up-regulated in L. braziliensis infection ( Chen et al., 2015 ; Souza et al., 2021 ). Otherwise, distinct miRNAs may act on the control of the infection: a) miR-340-5p that targets the IL-4, major Th2 cytokines secreted by CD4 + T cells, and this miRNA, different from observed with L. infantum , was down-regulated in in vitro L. donovani infection ( Kumar et al., 2020 ); b) miR-302b-3p that inhibits cell proliferation through the AKT pathway by targeting insulin-like growth factor 1 receptor (IGF-1R) ( Guo et al., 2017 ) and was also up-regulated in in vitro L. amazonensis and L. braziliensis infection ( Muxel et al., 2017 ; Souza et al., 2021 ); c) miR-373-3p that is implicated in the regulation of cell proliferation, apoptosis, senescence, migration, and cell invasion and was up-regulated in L. braziliensis infection ( Wei et al., 2015 ; Souza et al., 2021 ).…”

Highlighting the interplay of microRNAs from Leishmania parasites and infected-host cells

“…The transfection of infected-SL with a miR-21 inhibitor led to the increase of IL-12 cytokine and the T-box expressed in T cells (T-bet)/GATA-binding protein 3 (GATA-3) ratio (increasing Th1 profile population), and reduced Leishmania parasite load in infected-SL and revealed the interesting role of miR-21 in the inhibition of IL-12. These data highlighted that L. infantum infection changed the expression of miRNAs in L. infantum infected-PBMCs and -SL and that miRNAs including miR-21, miR-194, miR-371 and miR-150 interfered in the cellular immune response of L. infantum-infected dogs and also suggested such miRNAs as a plausible therapeutic target in CVL (Melo et al, 2019).…”

“…The differential expression pattern of miRNAs as well as their relationship with the immune response and parasite load have been recently investigated in PBMCs and splenic leucocytes (SL) of Canine VL (CVL)-infected dogs by L. infantum (Bragato et al, 2018a(Bragato et al, , 2018bMelo et al, 2019). In infected PBMCs, miR-21, miR-194, miR-424 and miR-451 showed a three-fold expression increase, miR-192, miR-371 and miR-503 denoted two-fold increase in their expression, whereas a two-fold decrease in miR expression level was detected for miR-150 and miR-574.…”

“…Similarly, microarray analyses indicated the enhanced expression level of miR-7, miR-21, miR-148a and miR-615, and the downregulation of miR-125a, miR-125b and miR-150 in infected-SL compared to control leucocytes. miR-148a targets genes involved in the regulation of apoptosis such as FAS and FAS ligand (FASLG) suggesting the role of this miRNA in the death of CD4 + and CD8 + cells in CVL-infected dogs (Melo et al, 2019). miR-615 targets ligand-dependant nuclear corepressor (LCoR), a derepressor of peroxisome proliferator-activated receptor gamma (PPARγ), which increases the phagocytic capacity of splenic macrophages (Jiang et al, 2011).…”

Evaluation of the cutaneous inflammatory cells in dogs with leishmaniosis and in dogs without the disease that were naturally infected by Leishmania infantum (syn. L. chagasi)

“…The transcription factor GATA3 is expressed by CD4+ Th2 cells and has been associated with type 2 immune responses, production of IL‐4, IL‐5 and IL‐13, downregulation of cell‐mediated and upregulation of humoral immunity 32,33,35,36,38,39 . However, GATA3 also is expressed by a low number of CD8+ cells, by double‐negative (CD4‐ and CD8‐) T cells and by some Treg cells 38 .…”