Host-parasite Responses Outcome Regulate the Expression of Antimicrobial Peptide Genes in the Skin of BALB/c and C57BL/6 Murine Strains Following Leishmania major MRHO/IR/75/ER Infection

Abstract

Background: Different background of immunity responses determine resistance or susceptibility of certain mouse strains to Leishmania major infection. Some have been well known previously. Antimicrobial peptides (AMPs) such as cathelicidins and defensins are unique fragments of innate immunity system with well-known effects against the invasion pathogens. Despite their outstanding roles and being of extensive cases of cutaneous leishmaniasis (CL) caused by L. major, they have been less studied in Leishmania fields. The aim of present study was to determine whether these components play a role in the protection of skin against Leishmania infections.

Methods: The animal model of Leishmania infection was established by the subcutaneous inoculation of 5×10⁶(parasites/ml) from the stationary phase of L. major promastigotes to BALB/c and C57BL/6 mice from January 2016 to August 2016 in Kerman Province, southeast of Iran. After 1, 3 and 7 d of post-infection (PI), the samples needed for detecting of the mRNA levels of mouse beta-defensin (mBD)-1, mBD2, mBD3, mBD4, mBD6, cathlin-related antimicrobial peptide (CRAMP), interleukin (IL)-10, IL-12 and parasite load were taken under standard methods.

Results: The findings related to cytokines profiles in BALB/c (↑IL-10, ↓IL-12) and C57BL/6 mouse strains (↓IL-10, ↑IL-12) demonstrated that immunity system has been accurately activated during CL caused by L. Major parasites. We also observed a significant up-regulation of all aforementioned AMPs genes in BALB/c mice at selected times compared to another strain.

Conclusion: CL occurred in BALB/c mice in spite of the fact that the expression of AMPs was higher than the other strain. AMPs genes are well expressed to provide defense against the parasites that have increased and escaped from immunity system but cannot create an absolute protection.

Keywords: Antimicrobial peptides; Cathelicidin; Cathelin-related antimicrobial peptide; Cytokine; Defensin; Leishmania major; Mouse β-defensin.

Fig. 1:

Melt curve from qPCR of mBD1gene of one reaction

Fig. 2:

Standard curve. Amplification of kDNA from L. major with serial dilutions ranging from 10⁶ to 10¹ parasites per reaction

Fig. 3:

Different AMPs gene expression in mouse strains after infection with L. major parasites. BALB/c and C57BL/6 mice were infected by the stationary phase of L. major promastigotes at the base of their tails. Skin samples were taken from both strains. The expression levels of AMPs were separately detected against their controls for both strains. The obtained results in the BALB/c mice (white bar) compared to the C57BL/6 mice (black bar) at selected times. (A) CRAMP expression. (B) The expression levels of mBD1. (C) The mBD2 gene expression. (D) The mBD3 expression. (E) The mRNA levels of mBD4. (F) The mBD6 gene expression. Data were shown as mean ± SEM and P<0.05 considered as significance level

Fig. 4:

Detection of cytokine expression. BALB/c and C57BL/6 mice were infected by the stationary phase of L. major promastigotes at the base of their tails. Macrophages were isolated from both strains at selected times of PI. The expression levels of cytokines were separately detected against their controls for both strains. The expression levels of IL-10 and IL-12 in BALB/c mice (white bar) were compared to C57BL/6 mice (black bar). (A) IL-10 expression. (B) IL-12 expression. Data were expressed as mean ± SEM and P<0.05 considered as significant level