ST2 contributes to T-cell hyperactivation and fatal hemophagocytic lymphohistiocytosis in mice

Cytokine storm syndromes, such as familial hemophagocytic lymphohistiocytosis (FHL), are lethal disorders caused by uncontrolled, systemic immune activation. In the murine model of FHL, in which perforin-deficient (Prf1(-/-)) mice are infected with lymphocytic choriomeningitis virus (LCMV), disease is driven by overabundant interferon (IFN)γ-producing LCMV-specific CD8(+) T cells thought to arise from excessive antigen stimulation through the T-cell receptor. However, this paradigm is insufficient to explain several fundamental aspects of FHL, namely, the inability of many pathogenic antigens to induce hyperinflammation, and the previously identified role of MyD88 in the disease. We now show a novel role for the MyD88-dependent interleukin-33 (IL-33) receptor, ST2, in FHL. Expression of IL-33 and ST2 is upregulated in LCMV-infected Prf1(-/-) mice. Blockade of ST2 markedly improves survival of LCMV-infected Prf1(-/-) mice and reduces the severity of multiple disease parameters, including serum levels of IFNγ. This decrease in IFNγ corresponds to a reduction in both the frequency of IFNγ(+) LCMV-specific CD8(+) and CD4(+) T cells and the magnitude of IFNγ expression in these cells. These findings demonstrate that disruption of ST2 signaling in the murine model of FHL reduces T cell-mediated production of IFNγ and suggest a revised paradigm in which danger signals such as IL-33 are crucial amplifiers of immune dysregulation in FHL. Furthermore, this study provides evidence to support blockade of ST2 as a novel therapeutic strategy for FHL

G Protein bc-Subunit Signaling Mediates Airway Hyperresponsiveness and Inflammation in Allergic Asthma

Since the Gbc subunit of Gi protein has been importantly implicated in regulating immune and inflammatory responses, this study investigated the potential role and mechanism of action of Gbc signaling in regulating the induction of airway hyperresponsiveness (AHR) in a rabbit model of allergic asthma. Relative to non-sensitized animals, OVA-sensitized rabbits challenged with inhaled OVA exhibited AHR, lung inflammation, elevated BAL levels of IL-13, and increased airway phosphodiesterase-4 (PDE4) activity. These proasthmatic responses were suppressed by pretreatment with an inhaled membrane-permeable anti-Gbc blocking peptide, similar to the suppressive effect of glucocorticoid pretreatment. Extended mechanistic studies demonstrated that: 1) corresponding proasthmatic changes in contractility exhibited in isolated airway smooth muscle (ASM) sensitized with serum from OVA-sensitized+challenged rabbits or IL-13 were also Gbc-dependent and mediated by MAPK-upregulated PDE4 activity; and 2) the latter was attributed to Gbc-induced direct stimulation of the nonreceptor tyrosine kinase, c-Src, resulting in downstream activation of ERK1/2 and its consequent transcriptional upregulation of PDE4. Collectively, these data are the first to identify that a mechanism involving Gbc-induced direct activation of c-Src, leading to ERK1/2-mediated upregulation of PDE4 activity, plays a decisive role in regulating the induction of AHR an

IL-13-induced Gi-βγ signaling mediates ERK1/2-dependent upregulation of PDE4D mRNA transcripts in HASM cells.

<p>(<b><u>A</u></b>) IL-13 elicits temporal increases in PDE4D mRNA expression in HASM cells, with peak induction of transcripts detected at 12 hr. A representative experiment (<b><u>B</u></b>), and corresponding densitometric analysis of PDE4D mRNA expression expressed as a ratio of β-actin (<b><u>C</u></b>), demonstrate that IL-13 induced PDE4D transcripts at 12 hr is abrogated in HASM cells that are pretreated either with PTX, anti-Gβγ blocking peptide, or inhibitors of either MEK-ERK1/2 (U0126), c-Src tyrosine kinase (SU6656) or gallein, whereas pretreatment with inhibitors of p38 MAPK (SB202190) or JNK (SP600125) has no effect. Data are mean ± SE values from 4 experiments (**p<0.01).</p

Anti-Gβγ blocking peptide suppresses pulmonary inflammation in OVA-sensitized rabbits.

<p>Relative to controls (<b><u>A</u></b>), lungs isolated from antigen-challenged OVA-sensitized rabbits exhibit diffusely scattered patchy foci of inflammatory cell infiltration, including in peribronchial, perivascular, and parenchymal regions (<b><u>B</u></b>). Contrasting the lack of effect of pretreatment with MPS alone (<b><u>C</u></b>), inflammation is suppressed to a similar extent in antigen-challenged OVA-sensitized rabbits that were pretreated either with inhaled budesonide (<b><u>D</u></b>) or anti-Gβγ peptide (<b><u>E</u></b>), whereas pretreatment with gallein has relatively little anti-inflammatory effect (<b><u>F</u></b>). Representative photomicrographs (mag. ×100) are from 4 µM sections of H&E stained lung sections.</p

Anti-Gβγ blocking peptide prevents <i>in vivo</i> antigen-induced airway hyperresponsiveness in OVA-sensitized rabbits.

<p>Relative to OVA-challenged control (non-sensitized; n = 4) rabbits, Rrs responses to MCh are significantly increased at 24 hr following antigen challenge in OVA-sensitized rabbits (n = 4). This heightened bronchoconstrictor responsiveness to MCh is suppressed in OVA-sensitized rabbits that are treated either with inhaled anti-Gβγ peptide (1 mg/Kg; n- = 4) or budesonide (0.5 mg/Kg; n = 3) prior to antigen challenge. Data are mean ± SE values. ANOVA used for multiple comparisons of mean Rrs values. *p<0.05; **p<0.01. Note: a significant difference is also detected when using the nonparametric Kruskal-Wallis test with Dunn's post-test to compare the medians of the Rrs-max responses in the control vs. non-pretreated OVA sensitized rabbits (p<0.05), whereas no significant difference is detected between the control vs. OVA-sensitized animals that are pretreated with the anti-Gβγ blocking peptide.</p

Inhibition of ERK1/2, PDE4, or Gβγ signaling prevents induced changes in agonist responsiveness in OVA-serum-sensitized ASM tissues.

<p>Relative to vehicle- or control serum-exposed rabbit ASM tissues, tissues passively sensitized for ∼18 hr with OVA serum exhibit significantly increased contractility to ACh (<b><u>A</u></b>) and impaired relaxation to isoproterenol (<b><u>B</u></b>). Pre-treatment with either U0126, rolipram, or anti-Gβγ peptide prevents OVA serum-induced changes in ASM responsiveness. Data are mean ± SD values from 5–7 experiments. ANOVA used for multiple comparisons of mean Tmax values. *p<0.05; **p<0.01.</p

Inhibition Gβγ signaling prevents rolipram-sensitive changes in agonist responsiveness in IL-13-exposed rabbit ASM tissues.

<p>Relative to controls, ASM tissues sensitized with IL-13 (50 ng/ml×24 hr) exhibit significantly increased contractility to ACh (<b><u>A</u></b>) and impaired relaxation to isoproterenol (<b><u>B</u></b>). Inhibition of Gβγ signaling by pretreatment with anti-Gβγ blocking peptide or gallein, or inhibition of PDE4 activity with rolipram, prevents IL-13-induced changes in ASM responsiveness. Data are mean ± SD values from 5–6 experiments. ANOVA used for multiple comparisons of mean Tmax and Rmax values. *p<0.05; **p<0.01.</p