Systemic cytokines, chemokines and growth factors reveal specific and shared immunological characteristics in infectious cardiomyopathies

Abstract

Heart disease is a major cause of death worldwide. Chronic Chagas cardiomyopathy (CCC) caused by infection with Trypanosoma cruzi leading to high mortality in adults, and rheumatic heart disease (RHD), resulting from infection by Streptococcus pyogenes affecting mainly children and young adults, are amongst the deadliest heart diseases in low-middle income countries. Despite distinct etiology, the pathology associated with both diseases is a consequence of inflammation. Here we compare systemic immune profile in patients with these cardiopathies, to identify particular and common characteristics in these infectious heart diseases. We evaluated the expression of 27 soluble factors, employing single and multivariate analysis combined with machine-learning approaches. We observed that, while RHD and CCC display higher levels of circulating mediators than healthy individuals, CCC is associated with stronger immune activation as compared to RHD. Despite distinct etiologies, univariate analysis showed that expression of TNF, IL-17, IFN-gamma, IL-4, CCL4, CCL3, CXCL8, CCL11, CCL2, PDGF-BB were similar between CCC and RHD, consistent with their inflammatory nature. Network analysis revealed common inflammatory pathways between CCC and RHD, while highlighting the broader reach of the inflammatory response in CCC. The final multivariate model showed a 100% discrimination power for the combination of the cytokines IL-12p70, IL-1Ra, IL-4, and IL-7 between CCC and RHD groups. Thus, while clear immunological distinctions were identified between CCC and RHD, similarities indicate shared inflammatory pathways in these infectious heart diseases. These results contribute to understanding the pathogenesis of CCC and RHD and may impact the design of immune-based therapies for these and other inflammatory cardiopathies that may also share immunological characteristics.

Keywords: Chagas disease; Inflammation; Pathology; Rheumatic heart disease; Systemic immune profile.

Fig. 1.. Comparative analysis of plasma cytokines levels between the study groups.

The groups evaluated were CONTROL (n = 12), CCC (Chagas cardiomyopathy, n = 38) and RHD (rheumatic heart disease, n = 17). p values <0.05 were considered statically significant. * p <0.05, ** p <0.005 *** and **** p <0.0001. Plasma soluble cytokine levels were measured using the Bio-Plex ProTM Human Cytokine Standard 27-plex Kit and results are expressed in MFI, as described in Materials and Methods. IL: Interleukin. TNF: Tumor necrosis factor. IFN: Interferon Gamma. IL-1Ra: Interleukin-1 receptor antagonist.

Fig. 2.. Comparative analysis of levels of plasma chemokines (A) and growth factors (B) between the study groups.

The groups evaluated were CONTROL (n = 12), CCC (Chagas cardiomyopathy, n = 38) and RHD (rheumatic heart disease, n = 17). p values <0.05 were considered statically significant. * p <0.05, ** p <0.005 *** and **** p <0.0001. Plasma soluble cytokine levels were measured using the Bio-Plex ProTM Human Cytokine Standard 27-plex Kit and results are expressed in MFI, as described in Materials and Methods. CCL: chemokine (C-C motif) ligand. CXCL: chemokine (C-X-C motif) ligand. G-CSF: Granulocyte colony-stimulation factor. GM-CSF: Granulocyte macrophage colony-stimulating factor. PDGF: Platelet- derived growth factor. VEGF: vascular endothelial growth factor. FGF basic: Fibroblast growth factor.

Fig. 3.. General profile of the evaluated soluble factors represented by radar graphs and tSNE analysis.

Representative analysis by radar chart with the median plasma levels of (A) cytokines, (B) chemokines and (C) growth factors in samples from CONTROL group (orange line), patients with CCC (green line) or RHD (purple line). (D) Generated tSNE map, showing the stratification between the different groups evaluated after overlapping on the islands formed by the algoritm. (E) Clusters detected by the FlowSOM analysis and projected on the tSNE map to identify the distribution within the groups evaluated. (F) Heat map representation of the relative intensity expression of the molecules performed by the cluster explorer algorithm.

Fig. 4.. Network and pathway interactions in Chagas and rheumatic herat diseases.

Protein-protein network interactions were performed considering the molecules altered in CCC and RHD as compared to CONTROL group. Top panels of (A) and (B) show the networks resulting of the analysis for CCC and RHD, respectively. Dark and light green represent rub nodes. Different colors distinguish nodes with different numbers of interactions. Bottom tables show the pathways found using the generated by the Kyoto Encyclopedia of Genes and Genomes (KEGG) algorithm, showing general common pathways between CCC and RHD (grey rows), the correspondence between the top ten hits for CCC in the top 20 for RHD (yellow rows) and the pathways exclusive for CCC (red rows).

Fig. 5.. Cluster analysis of the soluble immune mediators.

(A) Heatmap representation of the plasma levels of all soluble mediators evaluated; and individual analysis by group of molecules: (B) cytokines, (C) chemokines, and (D) growth factors in samples from CONTROL group (n = 12), patients with CCC (n = 38) or RHD (n = 17). Both rows and columns are clustered using correlation distance and average linkage. Light gray indicates higher plasma levels, while dark grey indicates lower plasma level.

Fig. 6.. Identification of predictors of immunological segregation between heart diseases.

(A) Violin Plot depict the relationship of each molecule in the study groups (Chagas cardiac and RHD); p-values are shown in bottom graph (B) marginal distribution of the selected covariates and their correlations. In the main diagonal of the figure, graphs of the marginal distribution (smoothed) of each covariate are presented, while below the diagonal, scatter plots are represented for each pair. Above the diagonal, the estimated correlations are shown. Coefficient values S0 are shown on the side. Graph on the right-hand side shows the discrimination between CCC (diamonds) and RHD (triangles) patients, based on the expression of IL-12p70, IL-1Ra, IL-4 and IL-7. Y-axis shows the predicted probability of RHD and X-axis displays the individual patient ID.