Potential Utility of Protein Targets of Cysteine-S-Nitrosylation in Identifying Clinical Disease Status in Human Chagas Disease.

Trypanosoma cruzi (Tc) infection causes Chagas disease (ChD) presented by dilated cardiomyopathy and heart failure. During infection, oxidative and nitrosative stresses are elicited by the immune cells for control the pathogen; however, excess nitric oxide and superoxide production can result in cysteine S-nitrosylation (SNO) of host proteins that affects cellular homeostasis and may contribute to disease development. To identify the proteins with changes in SNO modification levels as a hallmark of ChD, we obtained peripheral blood mononuclear cells (PBMC) from seronegative, normal healthy (NH, n = 30) subjects, and from seropositive clinically asymptomatic (ChD CA, n = 25) or clinically symptomatic (ChD CS, n = 28) ChD patients. All samples were treated (Asc+) or not-treated (Asc-) with ascorbate (reduces nitrosylated thiols), labeled with the thiol-labeling BODIPY FL-maleimide dye, resolved by two-dimensional electrophoresis (total 166 gels), and the protein spots that yielded significant differences in abundance or SNO level at p-value of ≤ 0.05 t-test/Welch/BH were identified by MALDI-TOF/TOF MS or OrbiTrap LC-MS/MS. Targeted analysis of a new cohort of PBMC samples (n = 10-14/group) was conducted to verify the differential abundance/SNO levels of two of the proteins in ChD (vs. NH) subjects. The multivariate adaptive regression splines (MARS) modeling, comparing differences in relative SNO level (Asc-/Asc+ ratio) of the protein spots between any two groups yielded SNO biomarkers that exhibited ≥90% prediction success in classifying ChD CA (582-KRT1 and 884-TPM3) and ChD CS (426-PNP, 582-KRT1, 486-ALB, 662-ACTB) patients from NH controls. Ingenuity Pathway Analysis (IPA) of the SNO proteome dataset normalized to changes in protein abundance suggested the proteins belonging to the signaling networks of cell death and the recruitment and migration of immune cells were most affected in ChD CA and ChD CS (vs. NH) subjects. We propose that SNO modification of the select panel of proteins identified in this study have the potential to identify ChD severity in seropositive individuals exposed to Tc infection.

Gene Expression Profiling and Functional Characterization of Macrophages in Response to Circulatory Microparticles Produced during Trypanosoma cruzi Infection and Chagas Disease.

BACKGROUND: Chronic inflammation and oxidative stress are hallmarks of chagasic cardiomyopathy (CCM). In this study, we determined if microparticles (MPs) generated during Trypanosoma cruzi (Tc) infection carry the host's signature of the inflammatory/oxidative state and provide information regarding the progression of clinical disease. METHODS: MPs were harvested from supernatants of human peripheral blood mononuclear cells in vitro incubated with Tc (control: LPS treated), plasma of seropositive humans with a clinically asymptomatic (CA) or symptomatic (CS) disease state (vs. normal/healthy [NH] controls), and plasma of mice immunized with a protective vaccine before challenge infection (control: unvaccinated/infected). Macrophages (mφs) were incubated with MPs, and we probed the gene expression profile using the inflammatory signaling cascade and cytokine/chemokine arrays, phenotypic markers of mφ activation by flow cytometry, cytokine profile by means of an ELISA and Bioplex assay, and oxidative/nitrosative stress and mitotoxicity by means of colorimetric and fluorometric assays. RESULTS: Tc- and LPS-induced MPs stimulated proliferation, inflammatory gene expression profile, and nitric oxide (∙NO) release in human THP-1 mφs. LPS-MPs were more immunostimulatory than Tc-MPs. Endothelial cells, T lymphocytes, and mφs were the major source of MPs shed in the plasma of chagasic humans and experimentally infected mice. The CS and CA (vs. NH) MPs elicited >2-fold increase in NO and mitochondrial oxidative stress in THP-1 mφs; however, CS (vs. CA) MPs elicited a more pronounced and disease-state-specific inflammatory gene expression profile (IKBKB, NR3C1, and TIRAP vs. CCR4, EGR2, and CCL3), cytokine release (IL-2 + IFN-γ > GCSF), and surface markers of mφ activation (CD14 and CD16). The circulatory MPs of nonvaccinated/infected mice induced 7.5-fold and 40% increases in ∙NO and IFN-γ production, respectively, while these responses were abolished when RAW264.7 mφs were incubated with circulatory MPs of vaccinated/infected mice. CONCLUSION: Circulating MPs reflect in vivo levels of an oxidative, nitrosative, and inflammatory state, and have potential utility in evaluating disease severity and the efficacy of vaccines and drug therapies against CCM.