Leishmania tropica infection induces immune responses through NOD2 pathway

INTRODUCTION: Leishmania tropica is the etiological agent of cutaneous leishmaniasis. Pattern recognition receptors such as toll-like receptors or nucleotide oligomerization domain-like receptors (NLR) have been associated with the disease. The role in innate immune responses against L. tropica of NOD2, a member of the NLR family, was investigated. MATERIALS AND METHODS: Interferon gamma (IFN-γ)-primed or unprimed immortalized mouse bone marrow macrophages (BMDM)-wild type (WT) or -NOD2 -/- were infected with L. tropica at different parasite/macrophage ratios (i.e. 2.5, 5, or 10:1), for 24h. Controls, such as medium alone, muramyldipeptide (MDP), or lipopolysaccharide (LPS), were included. Levels of cytokines or nitrite released into supernatants were measured through ELISA or Griess reagents. Levels of inducible nitric oxide synthase (iNOS) mRNA and protein were retrieved through Real-Time PCR and Western blot analyses, respectively. Also, unprimed BMDM- RIP2 -/- or -CARD9 -/- , two downstream components of NOD2 activation, were stimulated with the same ratios of L. tropica or controls, and the production of the pro-inflammatory cytokine TNF-a was compared with BMDM-WT. RESULTS AND CONCLUSIONS: Data showed that L. tropica did not induce the production of nitric oxide in unprimed BMDM-WT. L. tropica induced higher levels of nitric oxide in IFN-γ- primed BMDM-WT than in unstimulated cells. Stimulation due to L. tropica, or the control MDP (known to activate NOD2), was abrogated in the BMDM-NOD2 -/- , but not LPS, as so the expression levels of inducible nitric oxide synthase (iNOS) mRNA or protein. In addition, NOD2, RIP2 or CARD9 showed to be involved in the induction of TNF-a release from BMDM. These data suggest an involvement of NOD2 pathway in innate immune response to L. tropica infection.