PMC

ST2 blockade suppresses the immune response rather than LCMV infection. WT and Prf1^−/− mice were infected with LCMV, treated with either α-ST2 or control antibodies, and analyzed on day 8 p.i.; n = 4 mice per group. Analyzed by 2-way ANOVA; genotype (WT vs Prf1^−/−), ^###P < .001; treatment (control vs α-ST2), ^§§§P < .001; interaction between genotype and treatment, ***P < .001. (A) Splenic LCMV titer. Dotted line indicates limit of detection of plaque assay. Representative of 2 independent experiments. pfu, plaque-forming units. (B) Serum IFNγ level. Representative of 3 independent experiments.

Proposed model of FHL pathophysiology. In the traditional paradigm (left circle, connected by black arrows), antigen-presenting cells (APCs) and LCMV-infected cells present viral antigens to LCMV-specific T cells, activating them to secrete IFNγ. IFNγ acts back on APCs to enhance their ability to present antigen, setting up a positive feedback loop. In Prf1^−/− mice, T cells are unable to eliminate APCs via perforin, resulting in a loss of negative regulation that enables feed-forward amplification of inflammation. In our revised model, IL-33 signaling (right circle, connected by white arrows) further amplifies this vicious cycle. Tissue damage and cell death leads to the release of IL-33, which acts either directly or indirectly on LCMV-specific T cells to further promote their production of IFNγ. Elevated IFNγ exacerbates immunopathology, leading to further release of IL-33 from dying cells.

MyD88 is required for the development of FHL2 in mice. Prf1^−/− mice (n = 3) and Prf1^−/−Myd88^−/− mice (n = 3) were infected with LCMV and analyzed 10 days postinfection for signs of FHL2. Representative of 3 independent experiments. Analyzed by Student 2-tailed t test. (A) Spleen weight expressed as a ratio of total body weight. (B) Platelet counts and hemoglobin levels from peripheral blood. (C) Representative hematoxylin and eosin–stained liver sections, original magnification ×100. (D) Serum IFNγ levels. (E) Frequencies of splenic LCMV-specific T cells stained with gp33 major histocompatibility complex class I tetramer (tetr). *P < .05; ***P < .001.

The frequency and IFNγ production capacity of IFNγ⁺ LCMV-specific T cells are reduced in FHL2 mice receiving ST2 blockade. WT and Prf1^−/− mice were infected with LCMV, treated with either α-ST2 antibodies (open circles) or control antibodies (closed circles), and assessed on day 8 p.i. (n = 12-13 mice per group, data pooled from 3 independent experiments). Analyzed by linear mixed-effects model to account for baseline variability between experimental replicates: treatment and genotype were modeled as fixed effects, and experiment was treated as a random effect. The number symbol indicates significance of genotype (WT vs Prf1^−/−); the paragraph symbol indicates treatment (control vs α-ST2); and the asterisk indicates interaction between genotype and treatment. Representative flow plots gated on live CD90.2⁺CD8⁺ T cells (A) or CD90.2⁺CD4⁺ T cells (C), showing intracellular IFNγ expression in response to in vitro gp33 (A) or gp61 (C) peptide stimulation. Summary data showing frequencies of IFNγ⁺ LCMV-specific CD8⁺ T cells (B) and CD4⁺ T cells (D). Summary data showing median IFNγ fluorescence intensity (MFI) of CD8⁺ T cells (E) and CD4⁺ T cells (F) producing IFNγ in response to restimulation with LCMV peptides. MFI is normalized to WT control mean for each experiment. ^§§§P < .001; ^###P < .001; ^##P < .01; ^#P < .05; *P < .05.

ST2 blockade decreases numbers of LCMV-specific effector CD8⁺ and CD4⁺ T cells in FHL2 mice. WT and Prf1^−/− mice were infected with LCMV, treated with either α-ST2 antibodies (open circles) or control antibodies (closed circles), and assessed on day 8 p.i. Analyzed by linear mixed-effects model to account for baseline variability between experimental replicates: treatment and genotype were modeled as fixed effects, and experiment was treated as a random effect (eg, intercept only). The number symbol indicates significance of genotype (WT vs Prf1^−/−); the paragraph symbol indicates treatment (control vs α-ST2); and the asterisk indicates interaction between genotype and treatment. Representative flow plots gated on live CD90.2⁺CD8⁺ T cells (A) or CD90.2⁺CD4⁺ T cells (C), showing effector/memory phenotyping by CD44 and CD62L expression. Numbers of splenic CD44^hiCD62L^loCD127^lo effector CD8⁺ T cells (B) or CD4⁺ T cells (D). Data pooled from 4 independent experiments, n = 17 to 18 mice per group. (E) Representative flow plots gated on live CD90.2⁺CD8⁺ T cells, showing gp33 major histocompatibility complex (MHC) class I tetramer (tetr) staining. (F) Numbers of splenic gp33-specific CD8⁺ T cells (n = 13-14 mice per group, data pooled from 3 independent experiments). (G) Representative flow plots gated on live CD90.2⁺CD4⁺ T cells, showing gp66 MHC class II tetramer staining. (H) Numbers of splenic gp66-specific CD4⁺ T cells (n = 9-10 mice per group, data pooled from 2 independent experiments). *P < .05; ^§§§P < .001; ^##P < .01; **P < .01; ^§§P < .01; ^###P < .001.

Splenic expression of IL-33 is enhanced in FHL2 mice, and expression of ST2 is increased in mice and in patients with FHL2. Spleens and livers from Prf1^−/− and WT mice were analyzed before and after LCMV infection. (A) Expression of Il33 at day 0 (Uninf) and at 7 days postinfection (Inf) (n = 4 mice per group). Analyzed by 2-way ANOVA. The number symbol indicates significance of genotype (WT vs Prf1^−/−); the double dagger symbol indicates LCMV (Uninf vs Inf); and the asterisk indicates interaction between genotype and LCMV. ^##P < .01; ^‡‡‡P < .001; *P < .05. (B) Immunohistochemical staining of IL-33 in Prf1^−/− mice. Original magnification ×200. Representative of 4 mice per group. (C) IL-33 immunohistochemistry in livers of LCMV-infected Prf1^−/− mice 8 days p.i., with or without in vivo administration of ST2-blocking antibody. Representative of 4 mice per group. Original magnification as indicated. (D) Expression of Il1rl1 (ST2 gene), analyzed as in panel A; *P < .05; ^‡‡‡P < .001; ^#P < .05; **P < .01; ^##P < .01. (E) Expression of IL1RL1 in peripheral blood mononuclear cells from pediatric patients with FHL2 (n = 3), patients with systemic juvenile idiopathic arthritis (sJIA, n = 18), and healthy control subjects (control 1, n = 33; control 2, n = 29). Data are combined from 2 published data sets (GSE26050 [triangles] and GSE21521 [diamonds]).^, Analyzed by 1-way ANOVA; ***P < .001 by Dunnett multiple comparison posttest comparing FHL2 patients to all other groups. ANOVA, analysis of variance; AU, arbitrary units; mRNA, messenger RNA; p.i., postinfection.

ST2 blockade reduces morbidity and mortality of FHL2 mice. Prf1^−/− mice were infected with LCMV to induce FHL2 and treated with either α-ST2 or control antibodies. (A) Survival of α-ST2-treated mice (n = 9) and control mice (n = 10). Representative of 2 independent experiments. Analyzed by log-rank (Mantel-Cox) test. LCMV-infected WT mice (n = 4) are included for visual comparison. (B) Body weight of α-ST2-treated mice and control mice. Symbols represent mean ± standard error of the mean of 9 to 10 mice. The dagger symbol indicates time points at which control mice died and were excluded from subsequent weight analysis. Representative of 2 independent experiments. Analyzed by linear mixed-effects model to allow for missing data due to mouse mortality: treatment and body weight were modeled as fixed effects, and individual mice were treated as a random effect to account for baseline variability between animals (eg, intercept only). Significance of interaction term (α-ST2 vs control over time) is indicated. LCMV-infected WT mice (n = 4) are included for visual comparison. (C) Body weight of Prf1^−/− mice withdrawn from α-ST2 treatment and switched to control antibody at day 18 p.i. or receiving continued α-ST2 treatment. Symbols represent mean ± standard error of the mean of 4 to 5 mice. The † symbol indicates the time point at which 1 withdrawal (w/d) mouse died and was excluded from subsequent weight analysis. Analyzed by linear mixed-effects model as in panel B; significance of interaction term (α-ST2 vs w/d over time) is indicated. Platelet counts (D) and hemoglobin levels (E) in peripheral blood of α-ST2-treated Prf1^−/− mice (n = 9) and control Prf1^−/− mice (n = 10) 2 days prior to infection and 8 days p.i. Representative of 2 independent experiments. Analyzed by repeated-measures 2-way ANOVA; significance of interaction term (α-ST2 vs control over time) is indicated. Ferritin (F) and soluble CD25 (sCD25) levels (G) in serum 8 days p.i. Data pooled from 2 independent experiments, n = 7 to 8 mice per group. (H-M) Liver pathology in α-ST2-treated Prf1^−/− mice (n = 4) and control Prf1^−/− mice (n = 4) 8 days p.i. Representative of 2 independent experiments. Analyzed by Student 2-tailed t test. (H) Liver weight, expressed as a ratio of total body weight. (I) Total numbers of intrahepatic leukocytes. (J) Numbers of lobular inflammatory foci per ×20 objective high-power field (hpf), enumerated from hematoxylin and eosin (H&E)-stained tissue sections. Total numbers of intrahepatic CD8⁺ T cells (K) and CD4⁺ T cells (L), as measured by flow cytometry. (M) Representative H&E-stained liver sections, original magnification ×200. Severity of microvesicular steatosis was assessed on day 8 p.i. using a standardized scoring system as follows: 0, absent; 1, 1% to 20% of area per ×20 objective hpf; 2, 21% to 40%; 3, 41% to 60%; 4, 61% to 80%; 5, 81% to 100%. *P < .05; **P < .01; ***P < .001.