Mice with genetic deletion of group VIA phospholipase A2β exhibit impaired macrophage function and increased parasite load in Trypanosoma cruzi-induced myocarditis

Trypanosoma cruzi infection, which is the etiological agent of Chagas disease, is associated with intense inflammation during the acute and chronic phases. The pathological progression of Chagas disease is influenced by the infiltration and transmigration of inflammatory cells across the endothelium to infected tissues, which are carefully regulated processes involving several molecular mediators, including adhesion molecules and platelet-activating factor (PAF). We have shown that PAF production is dependent upon calcium-independent group VIA phospholipase A(2)β (iPLA(2)β) following infection of human coronary artery endothelial cells (HCAECs) with T. cruzi, suggesting that the absence of iPLA(2)β may decrease the recruitment of inflammatory cells to the heart to manage parasite accumulation. Cardiac endothelial cells isolated from iPLA(2)β-knockout (iPLA(2)β-KO) mice infected with T. cruzi demonstrated decreased PAF production compared to that by cells isolated from wild-type (WT) mice but demonstrated increases in adhesion molecule expression similar to those seen in WT mice. Myocardial inflammation in iPLA(2)β-KO mice infected with T. cruzi was similar in severity to that in WT mice, but the iPLA(2)β-KO mouse myocardium contained more parasite pseudocysts. Upon activation, macrophages from iPLA(2)β-KO mice produced significantly less nitric oxide (NO) and caused less T. cruzi inhibition than macrophages from wild-type mice. Thus, the absence of iPLA(2)β activity does not influence myocardial inflammation, but iPLA(2)β is essential for T. cruzi clearance

Charting the future of cancer health disparities research: A position statement from the American Association for Cancer Research, the American Cancer Society, the American Society of Clinical Oncology, and the National Cancer Institute

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138314/1/caac21404_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138314/2/caac21404.pd

Th17 cells are more protective than Th1 cells against the intracellular parasite Trypanosoma cruzi

Th17 cells are a subset of CD4+ T cells known to play a central role in the pathogenesis of many autoimmune diseases, as well as in the defense against some extracellular bacteria and fungi. However, Th17 cells are not believed to have a significant function against intracellular infections. In contrast to this paradigm, we have discovered that Th17 cells provide robust protection against Trypanosoma cruzi, the intracellular protozoan parasite that causes Chagas disease. Th17 cells confer significantly stronger protection against T. cruzi-related mortality than even Th1 cells, traditionally thought to be the CD4+ T cell subset most important for immunity to T. cruzi and other intracellular microorganisms. Mechanistically, Th17 cells can directly protect infected cells through the IL-17A-dependent induction of NADPH oxidase, involved in the phagocyte respiratory burst response, and provide indirect help through IL-21-dependent activation of CD8+ T cells. The discovery of these novel Th17 cell-mediated direct protective and indirect helper effects important for intracellular immunity highlights the diversity of Th17 cell roles, and increases understanding of protective T. cruzi immunity, aiding the development of therapeutics and vaccines for Chagas disease

IL-17A alone is not responsible for the Th17-mediated enhanced protective effects.

<p>(A-B) <i>T</i>. <i>cruzi</i>-specific Th17 cells were co-transferred with CD8⁺ T cells into RAG KO mice (5/group) prior to <i>T</i>. <i>cruzi</i> challenge. Neutralizing anti-IL-17A or control IgG1 antibodies were injected intraperitoneally every 48 hours. IL-17A neutralization did not reduce protection as measured by both parasitemia (A) and survival (B). *p<0.001 by two-tailed Student t test, **p<0.01 by log-rank test compared with negative controls. (C-D) Polyclonal CD8⁺ T cells were transferred intravenously (i.v.) into RAG KO mice prior to <i>T</i>. <i>cruzi</i> infection. Either control AdV or IL-17 AdV was injected 1 day prior to infection and 7 days post-infection at 5x10⁹ PFU i.v. Protection was measured by parasitemia 18 days post-infection (C) and survival (D).</p

Enhanced protection by Th17 cells involves more potent qualitative and quantitative helper effects for the development of <i>T</i>. <i>cruzi</i>-specific CD8⁺ T cells.

<p>RAG KO mice were adoptively transferred with polyclonal CD8⁺ T cells and Th1 or Th17 cells and then infected systemically with <i>T</i>. <i>cruzi</i>. (A) Greater absolute numbers of CD8⁺ T cells were recovered from mice co-adoptively transferred with Th17 cells at 7 days post-infection. (B) The mean fluorescence intensity (MFI) of T-bet expression in these same CD8⁺ T cells was increased over mice receiving CD8⁺ T cells alone or CD8⁺ T cells with Th1 cells, as measured by ICS. (C) Co-culture of sub-optimally stimulated CD8⁺ T cells with activated Th17 cell SN <i>in vitro</i> also induced higher T-bet expression than co-culture with Th1 cell SN. (D-E) Antigen-specific total and CD8⁺ T cell responses were studied 7 days post-challenge by IFN-γ ELISPOT (D) and intracellular cytokine staining (E), respectively. Similar results were detected in multiple experiments.</p

<i>T</i>. <i>cruzi</i>-specific Th17 cells provide T helper effects and directly activate CD8⁺ T cells through a soluble mediator.

<p>(A) Th17 cells required co-adoptive transfer with polyclonal CD8⁺ T cells to confer optimal immunity as measured by parasite burden. **p<0.01, ***p<0.001 by two-tailed Student t test. (B) Th17 cells transferred alone were not able to protect mice from <i>T</i>. <i>cruzi</i>-related death, indicating that Th17 cells protect through helper effects on CD8⁺ T cells. **p<0.01 by Mantel-Cox log-rank test. (C) CFSE-labeled, polyclonal CD8⁺ T cells were sub-optimally activated with plate-bound α-CD3 (1 μg/ml) and increasing numbers of dendritic cells. CD8⁺ T cells were cultured with or without Th17 cells for 5 days. Proliferation was measured by CFSE dilution. (D) CD8⁺ T cells were sub-optimally activated with α-CD3 as in C (without added dendritic cells), in the presence of Th17 cells, Th17 SNs or Th17 cells plus IL-17A neutralizing antibody. CD8⁺ T cell proliferation, CD44 expression and MIP-1α/IFN-γ production were measured 5 days later by Flow Cytometry and ICS and shown as fold increases compared with α-CD3 activation alone. (E) Purified Th17 cytokines were individually added to sub-optimally α-CD3 activated CD8⁺ T cells and markers of activation were analyzed 5 days later. (F) High levels of IL-21 could be detected by ELISA in Th17 cell SNs, but not in Th1 cell SNs. Representative results from multiple experiments are shown.</p

Th17 cells can inhibit <i>T</i>. <i>cruzi</i> intracellular growth in macrophages <i>in vitro</i> by inducing NADPH oxidase via IL-17A.

<p>PEMs were infected with trypomastigotes for 3 hours (MOI = 5). <i>T</i>. <i>cruzi</i>-specific TCR Tg Th1 or Th17 cells were then added to macrophages 1:25. Two days later, slides were Giemsa stained and intracellular parasites counted microscopically. Th1 and Th17 cells (A), as well as IFN-γ or IL-17A alone (B) were able to decrease the number of infected macrophages. *p<0.001 compared to medium alone by two-tailed Student t test. (C) Neutralizing antibodies directed against IFN- γ or IL-17A were able to abolish the direct protection provided by Th1 and Th17 cells, respectively. (D-E) The effects of the iNOS inhibitor L-NIL on Th1- and Th17-induced NO production and <i>T</i>. <i>cruzi</i> protection are shown. **p<0.001 by two-tailed Student t test. (F+G) BMDMs from wild type (WT) and gp91^phox KO mice were infected with <i>T</i>. <i>cruzi</i> for 3 hours (MOI = 10), then cytokines IFN-γ and IL-17A were added. Two days later, NO was measured (F) and intracellular parasites were enumerated (G). **p<0.001 by two-tailed Student t test. These results were reproduced in multiple experiments.</p

Th17 cell-derived IL-21 can help expand and activate protective CD8⁺ T cells.

<p>(A) WT or IL-21R KO CD8⁺ T cells were sub-optimally stimulated with plate-bound α-CD3 as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005902#ppat.1005902.g004" target="_blank">Fig 4C and 4E</a>. ICS assays indicated the addition of Th17 cell SNs could help activate WT CD8⁺ T cells but not IL-21R KO CD8⁺ T cells. Shown are fold increases compared to WT or IL-21R KO CD8⁺ T cells cultured with sub-optimal α-CD3 alone, without Th17 cell SNs. (B) Total spleen cells (TSCs) recovered from infected RAG KO mice adoptively transferred with Th17 cells and IL-21R KO CD8⁺ T cells had diminished responses to TS stimulation compared to mice receiving Th17 cells and WT CD8⁺ T cells prior to challenge, as measured by IFN-γ ELISPOT. (C) Mice transferred with Th17 cells (n = 4) and IL-21R KO CD8⁺ T cells (n = 8) were unable to control infection, as indicated by increasing parasitemia over time. **p<0.01 by two-tailed Student t test. (D) Transfer of IL-21R KO CD8⁺ T cells failed to protect mice from <i>T</i>. <i>cruzi</i>-mortality. *** p<0.001 by Mantel-Cox log-rank test.</p

Social Isolation and Mortality in US Black and White Men and Women

Social isolation is associated with higher mortality in studies comprising mostly white adults, yet associations among black adults are unclear. In this prospective cohort study, we evaluated whether associations of social isolation with all-cause, cardiovascular disease, and cancer mortality differed by race and sex. Adults enrolled in Cancer Prevention Study II in 1982/1983 were followed for mortality through 2012 (n = 580,182). Sex- and race-specific multivariable-adjusted hazard ratios and 95% confidence intervals were estimated for associations of a 5-point social isolation score with risk of death. Social isolation was associated with all-cause mortality in all subgroups (P for trend ≤ 0.005); for the most isolated versus the least isolated, the hazard ratios were 2.34 (95% confidence interval (CI): 1.58, 3.46) and 1.60 (95% CI: 1.41, 1.82) among black men and white men, respectively (P for interaction = 0.40) and 2.13 (95% CI: 1.44, 3.15) and 1.84 (95% CI: 1.68, 2.01) among black women and white women, respectively (P for interaction = 0.89). The association did not differ between black men and black women (P for interaction = 0.33) but was slightly stronger in white women than in white men (P for interaction = 0.01). Social isolation was associated with cardiovascular disease mortality in each subgroup (P for trend < 0.03) but with cancer mortality only among whites (P for trend < 0.0001). Subgroup differences in the influence of specific social isolation components were identified. Identifying and intervening with socially isolated adults could improve health outcomes

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

<div><p><i>Trypanosoma cruzi</i> infection is controlled but not eliminated by host immunity. The <i>T</i>. <i>cruzi</i> trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4⁺ and CD8⁺ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against <i>T</i>. <i>cruzi</i> challenge. We generated a new TS vaccine in which the immunodominant TS CD8⁺ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8⁺ T cell epitope enhance subdominant pathogen-specific CD8⁺ T cell responses. More specifically, CD8⁺ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8⁺ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for <i>T</i>. <i>cruzi</i> vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.</p></div