Local tissue acidosis affects anti-tumor immunity. In contrast, data on tissue pH levels in infected tissues and their impact on antimicrobial activity is sparse. In this study, we assessed the pH levels in cutaneous Leishmania lesions. Leishmania major-infected skin tissue displayed pH levels of 6.7 indicating that lesional pH is acidic. Next, we tested the effect of low extracellular pH on the ability of macrophages to produce leishmanicidal NO and to fight the protozoan parasite Leishmania major. Extracellular acidification led to a marked decrease in both NO production and leishmanicidal activity of lipopolysaccharide (LPS) and interferon γ (IFN-γ)-coactivated macrophages. This was not directly caused by a disruption of NOS2 expression, a shortage of reducing equivalents (NAPDH) or substrate (L-arginine), but by a direct, pH-mediated inhibition of NOS2 enzyme activity. Normalization of intracellular pH significantly increased NO production and antiparasitic activity of macrophages even in an acidic microenvironment. Overall, these findings indicate that low local tissue pH can curtail NO production and leishmanicidal activity of macrophages.

IntroductionGlutamine deficiency is a well-known feature of the tumor environment. Here we analyzed the impact of glutamine deprivation on human myeloid cell survival and function.MethodsDifferent types of myeloid cells were cultured in the absence or presence of glutamine and/or with L-methionine-S-sulfoximine (MSO), an irreversible glutamine synthetase (GS) inhibitor. GS expression was analyzed on mRNA and protein level. GS activity and the conversion of glutamate to glutamine by myeloid cells was followed by 13C tracing analyses.ResultsThe absence of extracellular glutamine only slightly affected postmitotic human monocyte to dendritic cell (DC) differentiation, function and survival. Similar results were obtained for monocyte-derived macrophages. In contrast, proliferation of the monocytic leukemia cell line THP-1 was significantly suppressed. While macrophages exhibited high constitutive GS expression, glutamine deprivation induced GS in DC and THP-1. Accordingly, proliferation of THP-1 was rescued by addition of the GS substrate glutamate and 13C tracing analyses revealed conversion of glutamate to glutamine. Supplementation with the GS inhibitor MSO reduced the survival of DC and macrophages and counteracted the proliferation rescue of THP-1 by glutamate.DiscussionOur results show that GS supports myeloid cell survival in a glutamine poor environment. Notably, in addition to suppressing proliferation and survival of tumor cells, the blockade of GS also targets immune cells such as DCs and macrophages.