STAT3 Inhibits CD103+ cDC1 Vaccine Efficacy in Murine Breast Cancer.

Conventional dendritic cells (cDCs) are a critical immune population, composed of multiple subsets, and responsible for controlling adaptive immunity and tolerance. Although migratory type 1 cDCs (CD103+ cDC1s in mice) are necessary to mount CD8+ T cell-mediated anti-tumor immunity, whether and how tumors modulate CD103+ cDC1 function remain understudied. Signal Transducer and Activator of Transcription 3 (STAT3) mediates the intracellular signaling of tumor-associated immunosuppressive cytokines, such as interleukin (IL)-10; thus, we hypothesized that STAT3 restrained anti-tumor immune responses elicited by CD103+ cDC1s. Herein, we show that in vitro-derived STAT3-deficient (Stat3∆/∆) CD103+ cDC1s are refractory to the inhibitory effects of IL-10 on Toll-like receptor 3 (TLR3) agonist-induced maturation responses. In a tumor vaccination approach, we found Stat3∆/∆ CD103+ cDC1s restrained mammary gland tumor growth and increased mouse survival more effectively than STAT3-sufficient CD103+ cDC1s. In addition, vaccination with Stat3∆/∆ CD103+ cDC1s elicited increased amounts of tumor antigen-specific CD8+ T cells and IFN-γ+ CD4+ T cells in tumors and tumor-draining lymph nodes versus phosphate-buffered saline (PBS)-treated animals. Furthermore, IL-10 receptor-deficient CD103+ cDC1s controlled tumor growth to a similar degree as Stat3∆/∆ CD103+ cDC1s. Taken together, our data reveal an inhibitory role for STAT3 in CD103+ cDC1 maturation and regulation of anti-tumor immunity. Our results also suggest IL-10 is a key factor eliciting immunosuppressive STAT3 signaling in CD103+ cDC1s in breast cancer. Thus, inhibition of STAT3 in cDC1s may provide an important strategy to improve their efficacy in tumor vaccination approaches and cDC1-mediated control of anti-tumor immunity.

Origin of Monocytes/Macrophages Contributing to Chronic Inflammation in Chagas Disease: SIRT1 Inhibition of FAK-NFκB-Dependent Proliferation and Proinflammatory Activation of Macrophages.

BACKGROUND: Trypanosoma cruzi (Tc) causes Chagas disease (CD) that is the most frequent cause of heart failure in Latin America. TNF-α+ monocytes/macrophages (Mo/Mφ) are associated with inflammatory pathology in chronic CD. In this study, we determined the progenitor lineage of Mo/Mφ contributing to inflammation and examined the regulatory role of SIRT1 in modulating the Mo/Mφ response in Chagas disease. METHODS AND RESULTS: C57BL/6 mice were infected with Tc, treated with SIRT1 agonist (SRT1720) after control of acute parasitemia, and monitored during chronic phase (150 days post-infection). Flow cytometry studies showed an increase in maturation of bone marrow hematopoietic stem cell (HSC)-derived Mo of proinflammatory and anti-inflammatory phenotype in acutely- and chronically-infected mice; however, these cells were not increased in splenic compartment of infected mice. Instead, yolk-sac-derived CD11b+ F4/80+ Mo/Mφ were increased in sinusoidal compartment of Chagas mice. The splenic CD11b+ F4/80+ Mo/Mφ of Chagas (vs. control) mice exhibited increased mRNA, protein, and surface expression of markers of proinflammatory phenotype (CD80+/CD64+ > CD200+/CD206+) associated with proinflammatory cytokines response (IL-6+TNF-α >> Arg-1+IL-10), and these were also detected in the myocardium of chronically infected mice. Infected mice treated with SRT1720 (vs. infected/untreated) exhibited decreased splenic expansion and myocardial infiltration of proinflammatory Mo/Mφ. SRT1720 did not alter the inherent capability of splenic Mo/Mφ of Chagas mice to respond to pathogen stimulus. Instead, SRT1720 dampened the Tc-induced increase in the expression and/or phosphorylation of focal adhesion kinase (FAK) and downstream transcription factors (Pu.1, c-Myb, and Runx1) involved in Mφ proliferation and migration and Notch1 involved in functional activation. Studies in cultured Mφ confirmed the agonistic effects of SIRT1 in controlling the Tc-induced, FAK-dependent increase in the expression of transcription factors and showed that SIRT1 agonist and FAK inhibitor abrogated the NF-κB transcriptional activity and inflammatory cytokine gene expression in Tc-infected Mφ. CONCLUSIONS: The proinflammatory Mo/Mφ of yolk sac origin drive the splenic and tissue inflammatory response in chronic CD. SRT1720 reprogrammed the Tc-induced FAK-dependent transcription factors involved in Mφ proliferation and proinflammatory activation in Chagas disease.

Pentose Phosphate Shunt Modulates Reactive Oxygen Species and Nitric Oxide Production Controlling Trypanosoma cruzi in Macrophages.

Metabolism provides substrates for reactive oxygen species (ROS) and nitric oxide (NO) generation, which are a part of the macrophage (Mφ) anti-microbial response. Mφs infected with Trypanosoma cruzi (Tc) produce insufficient levels of oxidative species and lower levels of glycolysis compared to classical Mφs. How Mφs fail to elicit a potent ROS/NO response during infection and its link to glycolysis is unknown. Herein, we evaluated for ROS, NO, and cytokine production in the presence of metabolic modulators of glycolysis and the Krebs cycle. Metabolic status was analyzed by Seahorse Flux Analyzer and mass spectrometry and validated by RNAi. Tc infection of RAW264.7 or bone marrow-derived Mφs elicited a substantial increase in peroxisome proliferator-activated receptor (PPAR)-α expression and pro-inflammatory cytokine release, and moderate levels of ROS/NO by 18 h. Interferon (IFN)-γ addition enhanced the Tc-induced ROS/NO release and shut down mitochondrial respiration to the levels noted in classical Mφs. Inhibition of PPAR-α attenuated the ROS/NO response and was insufficient for complete metabolic shift. Deprivation of glucose and inhibition of pyruvate transport showed that Krebs cycle and glycolysis support ROS/NO generation in Tc + IFN-γ stimulated Mφs. Metabolic profiling and RNAi studies showed that glycolysis-pentose phosphate pathway (PPP) at 6-phosphogluconate dehydrogenase was essential for ROS/NO response and control of parasite replication in Mφ. We conclude that IFN-γ, but not inhibition of PPAR-α, supports metabolic upregulation of glycolytic-PPP for eliciting potent ROS/NO response in Tc-infected Mφs. Chemical analogs enhancing the glucose-PPP will be beneficial in controlling Tc replication and dissemination by Mφs.

Macrophages Promote Oxidative Metabolism To Drive Nitric Oxide Generation in Response to Trypanosoma cruzi.

Trypanosoma cruzi is the causative agent of chronic chagasic cardiomyopathy. Why macrophages (mφs), the early responders to infection, fail to achieve parasite clearance is not known. Mouse (RAW 264.7) and human (THP-1 and primary) mφs were infected for 3 h and 18 h with T. cruzi TcI isolates, SylvioX10/4 (SYL, virulent) and TCC (nonpathogenic), which represent mφ stimulation and infection states, respectively. Mφs incubated with lipopolysaccharide and gamma interferon (LPS/IFN-γ) and with interleukin-4 (IL-4) were used as controls. We monitored the cytokine profile (using enzyme-linked immunosorbent assay [ELISA]), reactive oxygen species (ROS; fluorescent probes), nitric oxide (·NO; Griess assay), and metabolic state using a custom-designed mitoxosome array and Seahorse XF24 Analyzer. LPS/IFN-γ treatment of mφs elicited a potent increase in production of tumor necrosis alpha (TNF-α) at 3 h and of ROS and ·NO by 18 h. Upon SYL infection, murine mφs elicited an inflammatory cytokine profile (TNF-α ≫ TGF-ß + IL-10) and low levels of ·NO and ROS production. LPS/IFN-γ treatment resulted in the inhibition of oxidative metabolism at the gene expression and functional levels and a switch to the glycolytic pathway in mφs, while IL-4-treated mφs utilized oxidative metabolism to meet energy demands. SYL infection resulted in an intermediate functional metabolic state with increased mitoxosome gene expression and glycolysis, and IFN-γ addition shut down the oxidative metabolism in SYL-infected mφs. Further, TCC- and SYL-stimulated mφs exhibited similar levels of cell proliferation and production of TNF-α and ROS, while TCC-stimulated mφs exhibited up to 2-fold-higher levels of oxidative metabolism and ·NO production than SYL-infected mφs. Inhibiting ATP-coupled O2 consumption suppressed the ·NO generation in SYL-infected mφs. Mitochondrial oxygen consumption constitutes a mechanism for stimulating ·NO production in mφs during T. cruzi infection. Enhancing the oxidative metabolism provides an opportunity for increased ·NO production and pathogen clearance by mφs to limit disease progression.