IL1RAP Blockade With a Monoclonal Antibody Reduces Cardiac Inflammation and Preserves Heart Function in Viral and Autoimmune Myocarditis

Abstract

Background: Currently, there are no therapies targeting specific pathogenic pathways in myocarditis. IL (interleukin)-1 blockade has shown promise in preclinical studies and case reports. We hypothesized that blockade of IL1RAP (IL-1 receptor accessory protein), a shared subunit of the IL-1, IL-33, and IL-36 receptors, could be more efficient than IL-1 blockade alone.

Methods: We induced coxsackievirus B3 (CVB3)-mediated or experimental autoimmune myocarditis (EAM) in BALB/c mice, followed by treatment with an Fc (fragment crystallizable)-modified mIgG2a mouse anti-mouse IL1RAP monoclonal antibody (mCAN10). Myocarditis severity and immune infiltration were assessed by histology and flow cytometry. Cardiac function was measured by echocardiography. We used spatial transcriptomics (Visium 10× Genomics) to compare the gene expression landscape in the hearts of mCAN10-treated versus control mice.

Results: IL1RAP blockade reduced CVB3 and EAM severity. In EAM, the treatment prevented deterioration of cardiac function, measured on day 42 post-disease induction (left ventricular ejection fraction: 56.5% versus 51.0% in isotype controls [P=0.002] and versus 51.4% in mice treated with anti-IL-1β antibodies alone [P=0.003]; n=10-11 mice per group). In the CVB3 model, mCAN10 did not impede viral clearance from the heart and significantly lowered the numbers of CD4⁺ (cluster of differentiation 4) T cells (P=0.025), inflammatory Ly6C⁺CCR2⁺ (lymphocyte antigen 6 complex, locus C/C-C motif chemokine receptor 2) monocytes (P=0.038), neutrophils (P=0.001) and eosinophils (P<0.001) infiltrating the myocardium. The spatial transcriptomic analysis revealed reduced canonical IL-1 signaling and chemokine expression in cardiac immune foci in CVB3-infected mice treated with IL1RAP blockade.

Conclusions: Blocking IL1RAP reduces acute CVB3 myocarditis and EAM severity and preserves cardiac function in EAM. We conclude that IL1RAP blockade is a potential therapeutic strategy in viral and autoimmune myocarditis.

Keywords: eosinophils; flow cytometry; monocytes; myocarditis; therapeutics.

Figure 1.

IL1RAP (interleukin-1 receptor accessory protein) expression in the heart and bone marrow during acute viral myocarditis. A, Histograms of IL1RAP expression levels on immune cell subsets in naive or coxsackievirus B3 (CVB3)–infected (day 10) mouse hearts as assessed by flow cytometry. Cells were stained with anti-IL1RAP-Alexa Fluor 647 or isotype control to establish fluorescence baseline. The x axis is the fluorescence intensity of the anti-IL1RAP Alexa Fluor 647 signal. The y axis is the cell counts normalized to mode. B, Flow cytometry histograms indicating the expression of IL1RAP in bone marrow populations during naive state and CVB3 myocarditis (day 10). C, IL1RAP MFI (baseline isotype fluorescence subtracted) for each of the indicated heart and bone marrow populations during acute CVB3 myocarditis (day 10). Frequency of IL1RAP⁺ cells within each population is indicated by the color of the points. D, Dot plots showing the naive and CVB3 myocarditis expression of Il1r1 (encoding IL1R1) and Il1rap (encoding IL1RAP) in various cardiac cell populations utilizing a publicly available single-cell RNA sequencing data set (GSE174458). E and F, UMAP of publicly available single-cell RNA sequencing data sets showing the expression of IL1RAP in the healthy human heart (E; ERP123138) and bone marrow (F; GSE75478) populations. CLP indicates common lymphoid progenitor; CM, cardiomyocyte; CMP, common myeloid progenitor; Eos, eosinophil; Fibro, fibroblast; GMP, granulocyte-monocyte precursor; HSC, hematopoietic stem cell; Lin, lineage; LK, Lineage^negSca-1⁻c-Kit⁺ precursor; LSK, Lineage^negSca-1⁺c-Kit⁺ precursor; mDC, monocyte-derived dendritic cell; MFI, mean fluorescence intensity; Mo/Mac, monocyte/macrophage; MPP, multipotent progenitor; Neutro, neutrophil; SMC, smooth muscle cell; Tcm, T central memory cell; Teff, T effector cell; Tn, T naive cell; and UMAP, uniform manifold analysis and projection.

Figure 2.

IL1RAP (interleukin-1 receptor accessory protein) blockade by mCAN10 reduces disease severity in acute CVB3 myocarditis. A, Acute CVB3-myocarditis experimental timeline. All mice were infected with CVB3 virus on day 0 and treated with mCAN10 (n=10), isotype control (n=10), IL1Ra (interleukin-1 receptor antagonist; n=10), or PBS (n=10). Mice were given a loading dose of 20 mg/kg mCAN10 or isotype intraperitoneally on day 0 and were subsequently treated with either of the 2 antibodies at a 10-mg/kg dose biweekly. IL1Ra was administered subcutaneously at 25 mg/kg daily. An equal volume (100 μL) of PBS was administered subcutaneously daily as control. Mice were euthanized on day 10 post-infection. B, Myocarditis was scored on H&E–stained sections as described in Methods. Comparison of myocarditis severity score among treatment groups (n=10 mice per group, 3 sections per mouse; Kruskal-Wallis test). Data are presented as mean±SD. C, Representative H&E-stained heart sections of all treatment groups. Top, 1×, scale bar=200 µm; bottom: 20×, scale bar=50 µm. D, Representative M-mode echocardiography strips. x axis: large ticks=0.1 s. y axis: ticks=1 mm. E, Left ventricular ejection fraction for each treatment group. Data are presented as mean±SD. F, Left ventricular end-systolic volume. Data are presented as mean±SD. G, Percent body weight change, intergroup differences analyzed by 2-way ANOVA, the P value for interaction between treatment and time is shown on graph. H and I, Heart weight/body weight and spleen weight/body weight ratios, respectively. B, E, F, H, and I, Kruskal-Wallis test with Dunn multiple pairwise comparisons. P values are presented only where the difference between the groups was significant or where there was a trend. All other intergroup comparisons were nonsignificant. CVB3 indicates coxsackievirus B3; H&E, hematoxylin and eosin; IVS, interventricular septum; LV, left ventricle; LVIDd, left ventricular internal diameter, diastole; LVIDs, left ventricular internal diameter, systole; and LVPW, left ventricular posterior wall.

Figure 3.

mCAN10 reduces cardiac immune populations during acute viral myocarditis. A through H, Mice were infected with CVB3 and treated with mCAN10, isotype control, IL1Ra (interleukin-1 receptor antagonist), or PBS for 10 days. Flow cytometry was used to quantify immune populations in the heart, expressed as cell numbers per milligram of cardiac tissue (mCAN10, IL1Ra, and PBS, n=10; isotype, n=8). F, Average composition of immune cell populations for each treatment group. Bar size is proportional to cell number per milligram of the heart tissue. 1 cm²=40 cells/mg. A through H, Data are representative of 2 independent experiments, only 1 of which is shown. A through E, G, and H, Kruskal-Wallis test with Dunn multiple pairwise comparisons. P values are presented only where the difference between the groups was significant. All other intergroup comparisons were nonsignificant. CVB3 indicates coxsackievirus B3; ILC2, innate lymphoid cells type 2; and NK, natural killer.

Figure 4.

mCAN10 decreases inflammatory leukocyte precursors in the bone marrow during acute viral myocarditis. Mice were infected with CVB3 and treated with mCAN10, isotype control, IL1Ra (interleukin-1 receptor antagonist), or PBS for 10 days (n=10 per group). A, Diagram of bone marrow progenitor population differentiation. B through I, Flow cytometry was used to quantify bone marrow precursor populations, expressed as cell numbers per femur, at day 10 post-infection. J, Average composition of bone marrow progenitor cell populations for each treatment group. Size is proportional to cell number per femur, 1 cm²=6.01×10⁴ cells/mg. Kruskal-Wallis test with Dunn multiple pairwise comparisons. P values are presented only where the difference between the groups was significant. All other intergroup comparisons were nonsignificant. CLP indicates common lymphoid progenitor; CMP, common myeloid progenitor; CVP3, coxsackievirus B3; GMP, granulocyte-monocyte precursor; HSC, hematopoietic stem cell; Lin, lineage; LK, Lineage^negSca-1⁻c-Kit⁺ precursor; LSK, Lineage^negSca-1⁺c-Kit⁺ precursor; MEP, megakaryocyte-erythrocyte precursor; and MPP, multipotent progenitor.

Figure 5.

mCAN10 reduces inflammatory gene signaling in cardiac immune foci. A, UMAP clustering of spatial transcriptomics gene sequencing spots of acute viral myocarditis heart slides from each treatment group (each group, n=1 slide). The dashed line indicates the cluster corresponding to manually annotated inflammatory foci spots. B, Visualization of expression of selected genes and pathways in cardiac tissue during acute viral myocarditis. Immune foci are recognized in H&E stains and circled in red dashed lines, which were overlaid on the spatial gene expression images. Scale bar=1 mm. C through E, Quantification of spatial gene expression data. C, Percentage of Ccr2-expressing pixels in the delineated immune foci. D and E, Quantification of gene or gene pathway expression levels. Data are represented as mean±SD. F, SPADE analysis was utilized to compare 10× Genomics Visium spatial transcriptomic data sets between H&E-stained slides from mCAN10-treated and isotype-treated mice on day 10 post-infection. The SPADE analysis focused on 4 molecular pathways: IL (interleukin)-1 downstream signaling, type 1 IFN (interferon) downstream signaling, proinflammatory response, and MHC-II antigen presentation. The plots show genes differentially downregulated in mCAN10-treated immune foci-specific spots or fibroblast-specific spots relative to their isotype-treated counterparts, stratified by the molecular pathway. Genes differentially expressed in immune foci-specific spots are shown in red, and genes differentially expressed in fibroblast-specific spots are shown in green. P values are presented only where the difference between the groups was significant. All other intergroup comparisons were nonsignificant. H&E indicates hematoxylin and eosin; MCH-II, major histocompatibility complex class II; SPADE, Spatial Deconvolution for Domain Specific Cell-Type Estimation; and UMAP, uniform manifold approximation and projection.

Figure 6.

IL1RAP (interleukin-1 receptor accessory protein) blockade reduces the severity of experimental autoimmune myocarditis and preserves heart function. A, EAM was induced by immunization with αMyHC (α-myosin heavy-chain peptide) in CFA on days 0 and 7. The mCAN10 and anti-IL (interleukin)-1β antibodies, their respective control isotypes, or PBS were administered intraperitoneally twice a week starting on day 7, at the time of the booster immunization (n=10 per group). B, Change in left ventricular EF from baseline to days 28 and 42 post-induction, 2-way repeated measures ANOVA (P<0.001). The P values above the data points refer to comparisons between the mCAN10 and anti-IL-1β treatment groups and their respective isotype controls by Fisher LSD post hoc test. Data are presented as mean±SD. C and D, Comparison of EF ratio between groups at days 28 and 42 post-EAM induction, expressed as the proportion of baseline EF. E, Myocardial infiltration of CD68⁺ (cluster of differentiation 68) macrophages on day 21. F, Myocardial infiltration of Ly6G⁺ (lymphocyte antigen 6 complex, locus G) neutrophils on day 21. G, For the delayed treatment experiment, treatment was started on day 14 post-induction using the same treatment regimen as in A (mCAN10, n=11; isotype and PBS groups, n=10). H, Comparison of myocarditis severity score between treatment groups on day 42. I, Comparison of myocardial fibrosis among treatment groups on day 42, expressed as the percentage of left ventricular area. J, Comparison of EF ratio between groups, expressed as the proportion of baseline EF, on days 28 and 42 post-EAM induction. Data are presented as individual data points and median value for each group, except for B. Between-group comparisons were performed by the Kruskal-Wallis test with Dunn multiple comparisons post hoc test. P values are presented only where the difference between the groups was significant or where there was a clear trend. All other intergroup comparisons were nonsignificant. CD68 indicates cluster of differentiation 86; CFA, complete Freund adjuvant; EAM, experimental autoimmune myocarditis; EF, ejection fraction; LSD, least significant difference; and Ly6G, lymphocyte antigen 6 complex locus G6D.