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

Abstract

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.

Antigen-Based Nano-Immunotherapy Controls Parasite Persistence, Inflammatory and Oxidative Stress, and Cardiac Fibrosis, the Hallmarks of Chronic Chagas Cardiomyopathy, in A Mouse Model of Trypanosoma cruzi Infection

“…Splenocytes from mice were suspended in color-free RPMI-1640 medium (5 × 10 5 cells/100 µL), and incubated with or without TcL (25 µg/mL) at 37 • C, 5% CO 2 for 48 h. For the measurement of intracellular markers, brefeldin A (10 µg/mL, BD Biosciences) was added to splenocyte cultures for the final 4 h of incubation to block protein secretion. Cells were then labeled with antibodies for surface markers, fixed and permeabilized (as above), and utilized for intracellular staining of cytokines or specific markers [21]. Fluorescence-conjugated antibodies were purchased from BD Biosciences (APC hamster anti-mouse CD3ε clone 145-2C11, BV510 rat anti-mouse CD4 clone RM4-5, BUV395 rat anti-mouse CD8 clone 53-6.7, BV711 rat anti-mouse interferon γ (IFN-γ) clone XMG1.2, BV650 rat anti-mouse CD62L clone MEL-14, BV786 rat anti-mouse CD44 clone IM7, and BV421 rat anti-mouse CD25 clone 7D4), eBiosciences (PE rat anti-mouse granzyme clone NGZB), and Thermo Fisher Scientific (Waltham, MA; FITC rat anti-mouse perforin clone eBioMAK-D).…”

“…Tissue sections from heart and skeletal muscle (10 mg each, n = 5 mice per group) were homogenized and total DNA was purified by using a DNeasy Blood and Tissue kit (69504, Qiagen, Germantown, MD). A real-time qPCR was performed on an iCycler thermal cycler with SYBR Green super-mix (Bio-Rad), 50 ng of total DNA, and oligonucleotides specific for Tc18SrDNA sequence and murine Gapdh sequence [21]. The threshold cycle (C t ) values for Tc18SrDNA were normalized to Gapdh reference sequence.…”

PARP1-cGAS-NF-κB pathway of proinflammatory macrophage activation by extracellular vesicles released during Trypanosoma cruzi infection and Chagas disease

“…PARP1-cGAS-NF-κB pathway in macrophages color). Finally, slides were rinsed in 1X PBS buffer and mounted in VectaMount AQ Aqueous Mounting Medium (H-5501, Vector Laboratories) [45].…”

The Versatility of Sirtuin-1 in Endocrinology and Immunology

“…Tc infection can lead to enlargement of the liver, spleen and lymph node and in the case of severe disease can lead to inflammation of the brain and heart and fatal accumulation of fluid around the heart. In vivo models of CD show that administration of a SIRT1 activator (SRT1720) suppresses oxidative stress and inflammation associated with chronic Tc infection (Wan et al, 2016(Wan et al, , 2019. Wan et al (2019) demonstrated that SRT1720 had no significant effect on the total population of splenic cells but caused a significant decrease in subpopulations of monocytes and Macs in the spleen of chronically infected mice and a decrease in the CD80 + /CD64 + M1 phenotype Chagas mice.…”

“…In vivo models of CD show that administration of a SIRT1 activator (SRT1720) suppresses oxidative stress and inflammation associated with chronic Tc infection (Wan et al, 2016(Wan et al, , 2019. Wan et al (2019) demonstrated that SRT1720 had no significant effect on the total population of splenic cells but caused a significant decrease in subpopulations of monocytes and Macs in the spleen of chronically infected mice and a decrease in the CD80 + /CD64 + M1 phenotype Chagas mice. Interestingly, investigators demonstrated that SRT1720 administration had no effect on parasite survival and persistence as measured by parasite DNA in splenic monocytes/macrophages, while there was a significant reduction in the mono/mac ratio in the spleen and heart and an improvement of left ventricular function in Chagas mice.…”