Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNβ

In cutaneous leishmaniasis, Leishmania amazonensis activates macrophage double-stranded, RNA-activated protein kinase R (PKR) to promote parasite growth. In our study, Leishmania major grew normally in RAW cells, RAW-expressing dominant-negative PKR (PKR-DN) cells, and macrophages of PKR-knockout mice, revealing that PKR is dispensable for L. major growth in macrophages. PKR activation in infected macrophages with poly I: C resulted in parasite death. Fifty percent of L. major-knockout lines for the ecotin-like serine peptidase inhibitor (ISP2; Delta isp2/isp3), an inhibitor of neutrophil elastase (NE), died in RAW cells or macrophages from 129Sv mice, as a result of PKR activation. Inhibition of PKR or NE or neutralization of Toll-like receptor 4 or 2(TLR4 or TLR2) prevented the death of Delta isp2/isp3. Delta isp2/isp3 grew normally in RAW-PKR-DN cells or macrophages from 129Sv pkr(-/-), tlr2(-/-), trif(-/-), and myd88(-/-) mice, associating NE activity, PKR, and TLR responses with parasite death. Delta isp2/isp3 increased the expression of mRNA for TNF-alpha by 2-fold and of interferon beta (IFN beta) in a PKR-dependent manner. Antibodies to TNF-alpha reversed the 95% killing by Delta isp2/isp3, whereas they grew normally in macrophages from IFN receptor-knockout mice. We propose that ISP2 prevents the activation of PKR via an NE-TLR4-TLR2 axis to control innate responses that contribute to the killing of L. major.