Uncoupling Protein 2 Negatively Regulates Mitochondrial Reactive Oxygen Species Generation and Induces Phosphatase-Mediated Anti-Inflammatory Response in Experimental Visceral Leishmaniasis

Abstract

To reside and multiply successfully within the host macrophages, Leishmania parasites impair the generation of reactive oxygen species (ROS), which are a major host defense mechanism against any invading pathogen. Mitochondrial uncoupling proteins are associated with mitochondrial ROS generation, which is the major contributor of total cellular ROS generation. In the present study we have demonstrated that Leishmania donovani infection is associated with strong upregulation of uncoupling protein 2 (UCP2), a negative regulator of mitochondrial ROS generation located at the inner membrane of mitochondria. Functional knockdown of macrophage UCP2 by small interfering RNA-mediated silencing was associated with increased mitochondrial ROS generation, lower parasite survival, and induction of marked proinflammatory cytokine response. Induction of proinflammatory cytokine response in UCP2 knocked-down cells was a direct consequence of p38 and ERK1/2 MAPK activation, which resulted from ROS-mediated inhibition of protein tyrosine phosphatases (PTPs). Administration of ROS quencher, N-acetyl-l-cysteine, abrogated PTP inhibition in UCP2 knocked-down infected cells, implying a role of ROS in inactivating PTP. Short hairpin RNA-mediated in vivo silencing of UCP2 resulted in decreased Src homology 2 domain-containing tyrosine phosphatase 1 and PTP-1B activity and host-protective proinflammatory cytokine response resulting in effective parasite clearance. To our knowledge, this study, for the first time, reveals the induction of host UCP2 expression during Leishmania infection to downregulate mitochondrial ROS generation, thereby possibly preventing ROS-mediated PTP inactivation to suppress macrophage defense mechanisms.

Antioxidant potential of CORM-A1 and resveratrol during TNF-α/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells

“…The fluorescence generated by DCF is proportional to the rate of carboxy-H 2 DCFDA oxidation, which is in turn indicative of the cellular oxidizing activity and intracellular ROS levels. DHR123 has been reported as a marker for mitochondrial ROS production (Jin et al, 2008;Basu Ball et al, 2011;Tiede et al, 2011). It is an uncharged and (dysfunctional) mitochondria (Zhou et al, 2011).…”

Leishmania donovani Exploits Host Deubiquitinating Enzyme A20, a Negative Regulator of TLR Signaling, To Subvert Host Immune Response

“…Virus titer was measured at 2 3 10 6 infectious units/ml. shRNA target sequence selection and nonrelevant control vector use was the same as reported elsewhere (29). For in vivo knockdown of A20, 50 ml of the 10003 vector concentrate of A20-specific shRNA construct was injected into spleen tissue in anesthetized BALB/c mice 3 d prior to infection.…”

Finding the Smoking Gun: Protein Tyrosine Phosphatases as Tools and Targets of Unicellular Microorganisms and Viruses

“…The observed increase is reduced by 62-70% in the mice with silenced UCP2. In the same animal model, the production of TNF-α is substantially increased, whereas the production of IL-10 is dramatically decreased when compared to the infected wild-type mice [71]. Macrophages derived from SHP-1 -/-mice display elevated iNOS induction, elevated NO generation, and more efficient killing of Leishmania [52].…”