Cynomolgus macaques naturally infected with Trypanosoma cruzi-I exhibit an overall mixed pro-inflammatory/modulated cytokine signature characteristic of human Chagas disease

Background: Non-human primates have been shown to be useful models for Chagas disease. We previously reported that natural T. cruzi infection of cynomolgus macaques triggers clinical features and immunophenotypic changes of peripheral blood leukocytes resembling those observed in human Chagas disease. In the present study, we further characterize the cytokine-mediated microenvironment to provide supportive evidence of the utility of cynomolgus macaques as a model for drug development for human Chagas disease. Methods and findings: In this cross-sectional study design, flow cytometry and systems biology approaches were used to characterize the ex vivo and in vitro T. cruzi-specific functional cytokine signature of circulating leukocytes from TcI-T. cruzi naturally infected cynomolgus macaques (CH). Results showed that CH presented an overall CD4+-derived IFN-γ pattern regulated by IL-10-derived from CD4+ T-cells and B-cells, contrasting with the baseline profile observed in non-infected hosts (NI). Homologous TcI-T. cruzi-antigen recall in vitro induced a broad pro-inflammatory cytokine response in CH, mediated by TNF from innate/adaptive cells, counterbalanced by monocyte/B-cell-derived IL-10. TcIV-antigen triggered a more selective cytokine signature mediated by NK and T-cell-derived IFN-γ with modest regulation by IL-10 from T-cells. While NI presented a cytokine network comprised of small number of neighborhood connections, CH displayed a complex cross-talk amongst network elements. Noteworthy, was the ability of TcI-antigen to drive a complex global pro-inflammatory network mediated by TNF and IFN-γ from NK-cells, CD4+ and CD8+ T-cells, regulated by IL-10+CD8+ T-cells, in contrast to the TcIV-antigens that trigger a modest network, with moderate connecting edges. Conclusions: Altogether, our findings demonstrated that CH present a pro-inflammatory/regulatory cytokine signature similar to that observed in human Chagas disease. These data bring additional insights that further validate these non-human primates as experimental models for Chagas disease

Cynomolgus macaques naturally infected with <i>Trypanosoma cruzi</i>-I exhibit an overall mixed pro-inflammatory/modulated cytokine signature characteristic of human Chagas disease

<div><p>Background</p><p>Non-human primates have been shown to be useful models for Chagas disease. We previously reported that natural <i>T</i>. <i>cruzi</i> infection of cynomolgus macaques triggers clinical features and immunophenotypic changes of peripheral blood leukocytes resembling those observed in human Chagas disease. In the present study, we further characterize the cytokine-mediated microenvironment to provide supportive evidence of the utility of cynomolgus macaques as a model for drug development for human Chagas disease.</p><p>Methods and findings</p><p>In this cross-sectional study design, flow cytometry and systems biology approaches were used to characterize the <i>ex vivo</i> and <i>in vitro T</i>. <i>cruzi</i>-specific functional cytokine signature of circulating leukocytes from TcI-<i>T</i>. <i>cruzi</i> naturally infected cynomolgus macaques (CH). Results showed that CH presented an overall CD4⁺-derived IFN-γ pattern regulated by IL-10-derived from CD4⁺ T-cells and B-cells, contrasting with the baseline profile observed in non-infected hosts (NI). Homologous TcI-<i>T</i>. <i>cruzi</i>-antigen recall <i>in vitro</i> induced a broad pro-inflammatory cytokine response in CH, mediated by TNF from innate/adaptive cells, counterbalanced by monocyte/B-cell-derived IL-10. TcIV-antigen triggered a more selective cytokine signature mediated by NK and T-cell-derived IFN-γ with modest regulation by IL-10 from T-cells. While NI presented a cytokine network comprised of small number of neighborhood connections, CH displayed a complex cross-talk amongst network elements. Noteworthy, was the ability of TcI-antigen to drive a complex global pro-inflammatory network mediated by TNF and IFN-γ from NK-cells, CD4⁺ and CD8⁺ T-cells, regulated by IL-10⁺CD8⁺ T-cells, in contrast to the TcIV-antigens that trigger a modest network, with moderate connecting edges.</p><p>Conclusions</p><p>Altogether, our findings demonstrated that CH present a pro-inflammatory/regulatory cytokine signature similar to that observed in human Chagas disease. These data bring additional insights that further validate these non-human primates as experimental models for Chagas disease.</p></div

<i>Ex vivo</i> frequency of high cytokine producers and overall signatures in macaques naturally infected with TcI <i>T</i>. <i>cruzi</i>.

<p><b>(</b>A) Gray-scale diagrams were used to compile the <i>ex vivo</i> frequency of high modulatory (gray square) and pro-inflammatory (black square) cytokine producers within <i>T</i>. <i>cruzi</i>-infected cynomolgus macaques (CH) and non-infected controls (NI). The global median values of cytokine⁺ cells were calculated, taken from the whole data universe (NI+CH, n = 26 non-human primates) and used as the cut-off mark to categorize each animal as “low” (white square) or “high” (gray square, black square) cytokine producers among NI and CH. Column statistics were run to quantify the frequency of high producers in each group. The biomarkers with frequencies above the 50^th percentile are highlighted by bold underline format. (B) Radar charts summarizing the modulatory (gray area) and pro-inflammatory (black area) cytokine signatures in a range of leukocyte subsets (monocytes, NK-cells, T-cells and B-cells) were plotted to evaluate the proportion of high producers within a given cell subpopulation. The frequencies of high producers confined outside the inner circle (50^th percentile) are underscored by bold underline format.</p

Ascendant cytokine signatures of <i>T</i>. <i>cruzi</i>-infected and non-infected macaques upon (TcI/TcIV)/<i>T</i>. <i>cruzi</i>-antigen recall <i>in vitro</i>.

<p>Bar charts and continuous lines were assembled together to characterize the differential impact of (A) TcI (NI = circle symbols; CH = Square symbols) and (B) TcIV (NI = triangle symbols; CH = inverted triangle symbols) <i>T</i>. <i>cruzi</i> antigen recall <i>in vitro</i> on the pro-inflammatory (black rectangle) and modulatory (gray rectangle) cytokine pattern of cynomolgus macaques naturally infected with <i>T</i>. <i>cruzi</i> (CH) and non-infected controls (NI). The ascendant constructions were used to identify the biomarkers with the most prominent contribution for high cytokine producers. The biomarkers with frequencies above the 50^th percentile line were tagged by rectangles to highlight the pro-inflammatory (black rectangle) and modulatory (gray rectangle) cytokine-producing leukocyte subset.</p

Overall cytokine signatures of <i>T</i>. <i>cruzi</i>-infected and non-infected macaques upon (TcI/TcIV)/<i>T</i>. <i>cruzi</i>-antigen recall <i>in vitro</i>.

<p>Radar charts were plotted to characterize the impact of (A) TcI and (B) TcIV <i>T</i>. <i>cruzi</i> antigen recall <i>in vitro</i> on the pro-inflammatory (black area) and modulatory (gray area) cytokine pattern of naturally infected cynomolgus macaques (CH) and non-infected controls (NI). The frequencies of high cytokine producers confined outside the inner circle (50^th percentile) are underscored by bold underline format.</p

Ascendant <i>ex-vivo</i> cytokine signatures and overlaid comparative analyses of macaques naturally infected with TcI <i>T</i>. <i>cruzi</i> and non-infected macaques.

<p>(A) Ascendant <i>ex vivo</i> cytokine signatures assembled to characterize the frequency of high producers within infected monkeys (CH) and non-infected controls (NI). Data are presented by bar charts and continuous lines (NI = circle symbols; CH = Square symbols). The biomarkers with frequencies above the 50^th percentile are highlighted by bold underline format and the ascendant construction was used to identify the biomarkers with the most prominent contribution for high cytokine producers. (B) Overlaid cytokine signature curves were plotted for comparative analyses of the overall <i>ex vivo</i> ascendant cytokine pattern of CH and NI. The frequencies of high producers confined above the 50^th percentile line were tagged by rectangles to highlight the pro-inflammatory (black rectangle) and modulatory (gray rectangle) cytokine-producing leukocyte subset.</p

Systems biology analysis of cytokine network upon (TcI/TcIV)/<i>T</i>. <i>cruzi</i> antigen recall <i>in vitro</i>.

<p>Correlation matrices for cytokine⁺ cells were built with significant indexes and circular layouts to characterize the differential impact of (A) TcI and (B) TcIV <i>T</i>. <i>cruzi</i> antigen recall <i>in vitro</i>. Biomarker networks for cynomolgus macaques naturally infected with <i>T</i>. <i>cruzi</i> (CH) and non-infected controls (NI) are displayed by clustered distribution of nodes for pro-inflammatory (black node) and modulatory (gray node) cytokine patterns. Biomarker networks are displayed by node sizes reflecting the number of neighborhood connections from 0 to 8, according to the scale provided in the figure. Significant correlations (p≤0.05) were represented by connecting edges to underscore strong positive (r > 0.68; thick black line), moderate positive (0.36 ≤ r ≤ 0.68; thin black line), strong negative (r < -0.68; thick gray dashed line), moderate negative (-0.68 ≤ r ≤-0.36; thin gray equal dashed line), as proposed by Taylor [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005233#pntd.0005233.ref023" target="_blank">23</a>].</p