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Inhibition of CD40-induced N-Ras activation reduces leishmania major infection
Sushmita Chakraborty1, Ankita Srivastava1, Mukesh Kumar Jha1
1National Centre for Cell Science, Ganeshkhind, Pune 411007, India.
Abstract:
Leishmania major is a parasite that resides and replicates in macrophages. We previously showed that the parasite enhanced CD40-induced Raf-MEK-ERK signaling but inhibited PI3K-MKK-p38MAPK signaling to proleishmanial effects. As Raf and PI3K have a Ras-binding domain but exert opposite effects on Leishmania infection, we examined whether Ras isoforms had differential roles in Leishmania infection. We observed that L. major enhanced N-Ras and H-Ras expression but inhibited K-Ras expression in macrophages. L. major infection enhanced N-Ras activity but inhibited H-Ras and K-Ras activity. TLR2 short hairpin RNA or anti-TLR2 or anti-lipophosphoglycan Abs reversed the L. major-altered N-Ras and K-Ras expressions. Pam3CSK4, a TLR2 ligand, enhanced N-Ras expression but reduced K-Ras expression, indicating TLR2-regulated Ras expression in L. major infection. Whereas N-Ras silencing reduced L. major infection, K-Ras and H-Ras silencing enhanced the infection both in macrophages in vitro and in C57BL/6 mice. BALB/c-derived macrophages transduced with lentivirally expressed N-Ras short hairpin RNA and pulsed with L. major-expressed MAPK10 enhanced MAPK10-specific Th1-type response. CD40-deficient mice primed with these macrophages had reduced L. major infection, accompanied by higher IFN-γ but less IL-4 production. As N-Ras is activated by Sos, a guanine nucleotide exchange factor, we modeled the N-Ras-Sos interaction and designed two peptides from their interface. Both the cell-permeable peptides reduced L. major infection in BALB/c mice but not in CD40-deficient mice. These data reveal the L. major-enhanced CD40-induced N-Ras activation as a novel immune evasion strategy and the potential for Ras isoform-targeted antileishmanial immunotherapy and immunoprophylaxis.
Insights
Leishmania major infection manipulates Ras protein activity in macrophages, with N-Ras promoting infection and K-Ras/H-Ras inhibiting it. Targeting N-Ras offers a potential immunotherapy strategy against leishmaniasis.
Area of Science:
- Immunology
- Parasitology
- Cell Signaling
Background:
- Leishmania major parasites infect macrophages, modulating host cell signaling pathways.
- Previous studies indicated opposing roles for Raf-MEK-ERK and PI3K-MKK-p38MAPK pathways in Leishmania infection.
- Ras proteins, involved in cell signaling, possess Ras-binding domains and may play differential roles in infection.
Purpose of the Study:
- To investigate the differential roles of Ras isoforms (N-Ras, H-Ras, K-Ras) in Leishmania major infection within macrophages.
- To elucidate the involvement of Toll-like receptor 2 (TLR2) in regulating Ras isoform expression and activity during infection.
- To explore the therapeutic potential of targeting Ras isoforms for antileishmanial immunotherapy and immunoprophylaxis.
Main Methods:
- Analysis of Ras isoform expression and activity in macrophages infected with Leishmania major.
- Utilized TLR2 short hairpin RNA, anti-TLR2 antibodies, and anti-lipophosphoglycan antibodies to assess TLR2 involvement.
- Employed Ras isoform silencing (N-Ras, K-Ras, H-Ras) in vitro and in vivo (C57BL/6 and BALB/c mice).
- Generated N-Ras-specific immune responses using macrophages transduced with N-Ras short hairpin RNA and pulsed with MAPK10.
- Modeled N-Ras-Sos interaction and designed cell-permeable peptides targeting this interface.
Main Results:
- Leishmania major infection increased N-Ras and H-Ras expression and N-Ras activity, while decreasing K-Ras expression and activity.
- TLR2 signaling mediated the Leishmania-induced alterations in N-Ras and K-Ras expression.
- N-Ras silencing reduced infection, whereas K-Ras and H-Ras silencing enhanced infection in macrophages and mice.
- N-Ras targeted immunotherapy using peptides reduced Leishmania major infection in BALB/c mice, suggesting a novel immune evasion strategy.
Conclusions:
- Leishmania major hijacks CD40-induced signaling to activate N-Ras, representing a novel immune evasion mechanism.
- Differential regulation of Ras isoforms by Leishmania major influences parasite load.
- Targeting N-Ras offers a promising strategy for developing novel immunotherapies and immunoprophylactic interventions against leishmaniasis.
