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. 2016 Jun 16;1(9):e85851.
doi: 10.1172/jci.insight.85851.

Cardiac myosin-Th17 responses promote heart failure in human myocarditis

Jennifer M Myers  1 , Leslie T Cooper  2 , David C Kem  3 , Stavros Stavrakis  4 , Stanley D Kosanke  5 , Ethan M Shevach  6 , DeLisa Fairweather  2   7 , Julie A Stoner  8 , Carol J Cox  1 , Madeleine W Cunningham  1
Affiliations

Cardiac myosin-Th17 responses promote heart failure in human myocarditis

Jennifer M Myers et al. JCI Insight. .

Abstract

In human myocarditis and its sequela dilated cardiomyopathy (DCM), the mechanisms and immune phenotype governing disease and subsequent heart failure are not known. Here, we identified a Th17 cell immunophenotype of human myocarditis/DCM with elevated CD4+IL17+ T cells and Th17-promoting cytokines IL-6, TGF-β, and IL-23 as well as GM-CSF-secreting CD4+ T cells. The Th17 phenotype was linked with the effects of cardiac myosin on CD14+ monocytes, TLR2, and heart failure. Persistent heart failure was associated with high percentages of IL-17-producing T cells and IL-17-promoting cytokines, and the myocarditis/DCM phenotype included significantly low percentages of FOXP3+ Tregs, which may contribute to disease severity. We demonstrate a potentially novel mechanism in human myocarditis/DCM in which TLR2 peptide ligands from human cardiac myosin stimulated exaggerated Th17-related cytokines including TGF-β, IL-6, and IL-23 from myocarditic CD14+ monocytes in vitro, and an anti-TLR2 antibody abrogated the cytokine response. Our translational study explains how an immune phenotype may be initiated by cardiac myosin TLR ligand stimulation of monocytes to generate Th17-promoting cytokines and development of pathogenic Th17 cells in human myocarditis and heart failure, and provides a rationale for targeting IL-17A as a therapeutic option.

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Figures

Figure 1
Figure 1. Th17 immunophenotype contributes to myocarditis and dilated cardiomyopathy (DCM).
(A) Peripheral blood mononuclear cells (PBMCs) from myocarditis/DCM subjects (n = 28) (baseline blood sample) or healthy controls (n = 26) were stained for CD4, CD3, and IL-17 and analyzed using a flow cytometer. Mann-Whitney, P = 0.0008. (B) Representative CD3+CD4+IL-17+ Th17 FACS diagrams are shown from myocarditis/DCM (n = 2) and a healthy control (n = 1). (C) Th1 (IFN-γ+) cells were not significantly high in myocarditis/DCM as a group in baseline blood samples. PBMCs from myocarditis/DCM subjects (n = 15) and healthy controls (n = 10) were stained for CD4, CD3, and IFN-γ and analyzed using FACS. Mann-Whitney, P = 0.72. (D) IL-17A in myocarditis/DCM males (n = 27) was significantly elevated at baseline compared to myocarditis/DCM females (n = 14) and trended toward a significant interaction by case/control status. Healthy controls: males (n = 16), females (n = 8). Three male and 4 female myocarditis/DCM samples had undetectable IL-17A and were placed on the graph halfway between zero and the lowest detectable value of 1.5 pg/ml. Two-way ANOVA, P = 0.029 (gender by group interaction, P = 0.15). FACS analysis was performed on fresh PBMCs, which were analyzed immediately upon receiving the blood sample. Only 1 sample per time point was analyzed by FACS and compared to isotype controls. Cytokine analysis was performed in triplicate to determine the serum cytokine concentration.
Figure 2
Figure 2. Th17 immunophenotype in human myocarditis and dilated cardiomyopathy (DCM) contributes to heart failure.
Th17 percentages in peripheral blood mononuclear cells (PBMCs) were compared among myocarditis/DCM subjects with left heart failure (LHF) (n = 4), right heart failure (RHF) (n = 5), or no heart failure (No HF) (n = 7), as determined by clinical observations and compared to heart failure determined by New York Heart Association (NYHA) class. (A) Elevated Th17 cells were associated with heart failure. Mann-Whitney, P = 0.0061 (adjusted alpha level: 0.0167). (B) Th17 cells were significantly elevated in peripheral blood of NYHA class III and IV (n = 3) heart failure compared to class I and II (n = 10) heart failure in myocarditis/DCM. Mann-Whitney, P = 0.0223. (C) IL-17A was significantly elevated at 6 months in NYHA class III and IV (n = 9) heart failure compared to NYHA class I and II (n = 18) heart failure in myocarditis/DCM. Mann-Whitney, P = 0.0236 (adjusted alpha level: 0.0167). Healthy controls, n = 27. (D) IL-17A was significantly elevated at 12 months in NYHA class III and IV (n = 7) compared to NYHA class I and II (n = 16) heart failure in myocarditis/DCM. Mann-Whitney, P = 0.044 (adjusted alpha level: 0.0167). Healthy controls, n = 27. (E) IL17A+ myocarditis heart biopsies (n = 11) with representative IL-17A+ cells indicated with arrows. Of 11 different biopsies, 45% contained IL17A+ cells. As controls, alkaline phosphatase–conjugated secondary antibody and PBS-treated biopsies were negative. The total original magnification for magnified panels was ×200 (20× objective × 10× eyepiece). FACS analysis was performed on fresh PBMCs, which were analyzed immediately upon receiving the blood sample. Cytokine analysis was performed in triplicate to determine the serum cytokine concentration. Single-biopsy specimens were stained once with anti–IL-17A antibody, but compared to biopsy sections cut serially from the same piece of tissue. Controls for the biopsy tissues were taken from the serial sections of the same piece of tissue.
Figure 3
Figure 3. Th17 cytokines associate with myocarditis/dilated cardiomyopathy (DCM).
(A–C) Cytokine concentrations in myocarditis/DCM subjects in baseline blood sample (n = 41) and healthy controls (n = 32) were assessed using human ELISA kits and reported as pg/ml (adjusted alpha level: 0.0167). (A) IL-6 was significantly elevated in myocarditis/DCM. Mann-Whitney, P < 0.0001. (B) TGF-β1 was significantly elevated in myocarditis/DCM. Mann-Whitney, P = 0.0001. (C) IL-23 was significantly elevated in myocarditis/DCM. Mann-Whitney, P = 0.0001. (D) Significantly elevated GM-CSF was found in 6-month myocarditis/DCM blood samples (n = 27) versus healthy controls (n = 25). GM-CSF (pg/ml) was assessed using human ELISA kits. Mann-Whitney, P = 0.0336. (E) Myocarditis/DCM patients (n = 11) demonstrated significantly elevated intracellular GM-CSF+CD4+CD3+ cell percentages. Peripheral blood mononuclear cells (PBMCs) from myocarditis/DCM (n = 11) subjects and healthy controls (n = 12) were stained for CD4, CD3, and GM-CSF and analyzed using a flow cytometer. Mann-Whitney, P = 0.0006. FACS analysis was performed on fresh PBMCs, which were analyzed immediately upon receiving the blood sample. Only 1 sample per time point was analyzed by FACS and compared to isotype controls. Cytokine analysis was performed in triplicate to determine the serum cytokine concentration.
Figure 4
Figure 4. Th17 cytokines associate with myocarditis/dilated cardiomyopathy (DCM) and may prevent recovery.
(A) Significantly elevated IL-6 in nonrecovered myocarditis/DCM 12-month blood samples (n = 5) versus healthy controls (n = 27). IL-6 (pg/ml) was assessed using human ELISA kits (Mabtech). Mann-Whitney, P < 0.0001 (baseline Mann-Whitney, P = 0.031). (B) Significantly elevated IL-17A in nonrecovered 6-month myocarditis/DCM blood samples (n = 5) versus healthy controls (n = 27). Three myocarditis/DCM samples demonstrated undetectable IL-17A and were placed on the graph halfway between zero and the lowest detectable value of 1.5 pg/ml. Mann-Whitney, P = 0.019. (C) Significantly elevated TGF-β1 in nonrecovered 12-month myocarditis/DCM blood samples (n = 5) versus healthy controls (n = 27). n = 6 at baseline, but one patient did not survive and n = 5 for 6-month and 12-month blood samples. Mann-Whitney, P = 0.0076 (baseline Mann-Whitney, P = 0.024). Cytokine analysis was performed in triplicate to determine the serum cytokine concentration.
Figure 5
Figure 5. Significantly elevated IL-6 levels were progressive and associated with heart failure in men.
(A) IL-6 levels in myocarditis/dilated cardiomyopathy (DCM) males (n = 27) tended to be elevated compared to female myocarditis/DCM (n = 14) in the baseline blood sample. (B) IL-6 levels in myocarditis/DCM males (n = 18) tended to be elevated compared to female myocarditis/DCM (n = 10) in 6-month blood samples. (C) IL-6 levels in myocarditis/DCM males (n = 15) were significantly elevated compared to female myocarditis/DCM (n = 8) in 12-month blood samples. ANOVA, P = 0.0077. (D) IL-6 levels were elevated and highly significant in male NYHA class I–II heart failure (n = 10) compared to female NYHA class I–II heart failure (n = 6) in myocarditis/DCM in 12-month blood samples. ANOVA, P < 0.0001. Healthy controls: males (n = 19), females (n = 13). Cytokine analysis was performed in triplicate to determine the serum cytokine concentration.
Figure 6
Figure 6. Human cardiac myosin (HCM) is a major immune stimulator of myocarditis/dilated cardiomyopathy (DCM) CD14+ monocytes.
(A–D) Peripheral blood mononuclear cells (PBMCs) isolated from myocarditis/DCM subjects at baseline blood sample or from normal healthy individuals were cultured with HCM, HCM peptide S2-16, HCM peptide S2-28, or HCM peptides S2-16/S2-28 for 24 hours. After 24 hours, supernatants were collected and assayed by ELISA for TGF-β1, IL-6, IL-23, or IL-1β. P value indicates comparisons among myocarditis/DCM subjects and controls for 5 treatment conditions (media, +HCM, +S2-16, +S2-28, +S2-16/S2-28). (A) TGF-β1 production from CD14+ monocytes was significantly increased in myocarditis/DCM, as the TGF-β1 response to treatment with HCM TLR ligands differed between cases (n = 40) and controls (n = 16). Treatment by group interaction, repeated-measures ANOVA, P < 0.0001. (B) IL-6 production from CD14+ monocytes was significantly increased in myocarditis/DCM, as the IL-6 response to treatment differed between the cases (n = 33) and controls (n = 16). Treatment by group interaction, repeated-measures ANOVA, P = 0.0075. (C) IL-23 production from CD14+ monocytes was significantly increased in myocarditis/DCM, as the IL-23 response to treatment differed between cases (n = 11) and controls (n = 6). Treatment by group interaction, repeated-measures ANOVA, P < 0.0001. (D) IL-1β production from CD14+ monocytes was significantly increased in myocarditis/DCM, as the IL-1β response to treatment differed between cases (n = 34) and controls (n = 16). Treatment by group interaction, repeated-measures ANOVA, P = 0.0003. (E) Representative FACS analysis of PBMCs from myocarditis/DCM patients demonstrated that nearly 90% of cells producing TGF-β and IL-6 were CD14+ monocytes. FACS analysis was performed on fresh PBMCs, which were analyzed immediately upon receiving the blood sample. Only 1 sample per time point was analyzed by FACS and compared to isotype controls. Cytokine analysis was performed in triplicate to determine the cytokine concentration.
Figure 7
Figure 7. Anti-TLR2 antibody blocks production of Th17-promoting cytokines from myocarditis/dilated cardiomyopathy (DCM) monocytes.
(A–C) Peripheral blood mononuclear cells (PBMCs)/CD14+ monocytes were preincubated with antibodies shown in the graph prior to addition of human cardiac myosin (HCM) S2-16 and S2-28 peptide TLR ligands. Cytokines were assessed after 24 hours using a human ELISA kit. P values indicate comparisons between all treatment conditions. (A and B) Anti-TLR2 antibody significantly blocked IL-6 production in myocarditis/DCM subjects (n = 2). (C) Anti-TLR2 significantly blocked TGF-β1 production in a myocarditis/DCM subject (n = 1). Mean IL-6 and TGF-β measures significantly differed from controls and were significantly reduced among anti-TLR2 treatment groups. A mixed-effects ANOVA model was fit to account for the correlation among the repeated measures made on each specimen (n = 3). Pairwise testing between pairs of treatments was performed using Tukey’s method to adjust for multiple comparisons. ANOVA overall test and Tukey’s comparison, P < 0.0001.
Figure 8
Figure 8. Decreased Tregs are an important immunological feature of myocarditis/dilated cardiomyopathy (DCM).
(A) CD4+FOXP3+CD25+ Treg percentages were significantly decreased in peripheral blood of myocarditis/DCM subjects at baseline blood sample. Peripheral blood mononuclear cells (PBMCs) from myocarditis/DCM subjects (n = 28) and healthy individuals (n = 19) were stained for CD4, FOXP3, and CD25 and analyzed using a flow cytometer. Mann-Whitney, P = 0.0006. (B) CD4+FOXP3+ Treg percentages were significantly decreased in myocarditis/DCM peripheral blood. Mann-Whitney, P = 0.0027. (C) Representative CD4+FOXP3+CD25+ Treg FACS diagram from a single individual in each group is shown and was found to be similar to other individuals (not shown).
Figure 9
Figure 9. In myocarditis/dilated cardiomyopathy (DCM), low Tregs correlated with high TGF-β1 and IL-6 levels produced from peripheral blood mononuclear cells (PBMCs)/CD14+ monocytes stimulated with human cardiac myosin (HCM) TLR ligands.
When Tregs were low in peripheral blood, PBMCs/CD14+ monocytes responded to cardiac myosin TLR ligands with significantly elevated cytokines IL-6 and TGF-β1 proportionately to the lowered Treg percentages, as shown by the inverse correlation (n = 28). (A) TGF-β1 production from PBMCs/CD14+ monocytes after a 24-hour exposure to TLR ligand S2-16 versus CD4+FOXP3+ Treg percentage. P = 0.0065. (B) TGF-β1 production from PBMCs/CD14+ monocytes after a 24-hour exposure to TLR ligand S2-28 versus CD4+FOXP3+ Treg percentage. P = 0.0208. (C) IL-6 production from PBMCs/CD14+ monocytes after a 24-hour exposure to TLR ligand S2-16 versus CD4+FOXP3+ Treg percentage. P = 0.0255. (D) IL-6 production from PBMCs after a 24-hour exposure to TLR ligand S2-28 versus CD4+FOXP3+ Treg percentage. P = 0.0203.
Figure 10
Figure 10. Diagram of pathogenic mechanisms in the Th17 immunophenotype in human myocarditis.
As cardiac myosin is released from damaged heart, cardiac myosin functions as a danger signal and acts as a TLR2 ligand in human myocarditis, leading to exaggerated responses from CD14+ monocytes promoting a Th17 immunophenotype. Anti-TLR2 blockade prevents Th17 responses from CD14+ myocarditic monocytes. Reduced Tregs identify with a Th17 immunophenotype in human myocarditis as well as the cytokines TGF-β, IL-6, IL-1β, GM-CSF, and IL-23. The IL-23 receptor (IL-23R) on Th17 cells and the production of GM-CSF from Th17 cells all were part of the phenotype as shown in our study. Th17/CD14+ monocyte responses are linked to heart failure and disease progression through mechanisms of fibrosis related to TGF-β production.
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