Deletion of the ST2 proximal promoter disrupts fibroblast-specific expression but does not reduce the amount of soluble ST2 in circulation.

IL-33 signals through ST2, which is expressed either as a full-length signaling receptor or a truncated soluble receptor that can suppress IL-33 activity. Previous data suggest that soluble ST2 mRNA in fibroblasts is coupled to a serum-inducible proximal promoter, while full-length ST2 expression in immune cells is directed from a distal promoter. In order to better understand the function of the alternative promoters and how they ultimately affect the regulation of IL-33, we generated a mouse in which the ST2 proximal promoter is deleted. Promoter deletion had no impact on ST2 expression in mast cells or their ability to respond to IL-33. In contrast, it resulted in a complete loss of both soluble and full-length ST2 mRNA in fibroblasts, which corresponded with both an inability to secrete soluble ST2 and a defect in IL-33 responsiveness. Importantly, in spite of the fibroblast defect, soluble ST2 concentrations were not reduced in the serum of naïve or allergen-exposed knockout mice. In summary, we found that ST2 promoter usage is largely cell-type dependent but does not dictate splicing. Moreover, the proximal promoter is not a major driver of circulating soluble ST2 under the conditions tested.

Identification of functional roles for both IL-17RB and IL-17RA in mediating IL-25-induced activities.

IL-25 (IL-17E) is a unique IL-17 family ligand that promotes Th2-skewed inflammatory responses. Intranasal administration of IL-25 into naive mice induces pulmonary inflammation similar to that seen in patients with allergic asthma, including increases in bronchoalveolar lavage fluid eosinophils, bronchoalveolar lavage fluid IL-5 and IL-13 concentrations, goblet cell hyperplasia, and increased airway hyperresponsiveness. IL-25 has been reported to bind and signal through IL-17RB (IL-17BR, IL-17Rh1). It has been demonstrated recently that IL-17A signals through a heteromeric receptor composed of IL-17RA and IL-17RC. We sought to determine whether other IL-17 family ligands also utilize heteromeric receptor complexes. The required receptor subunits for IL-25 biological activities were investigated in vitro and in vivo using a combination of knockout (KO) mice and antagonistic Abs. Unlike wild-type mice, cultured splenocytes from either IL-17RB KO or IL-17RA KO mice did not produce IL-5 or IL-13 in response to IL-25 stimulation, and both IL-17RB KO and IL-17RA KO mice did not respond to intranasal administration of IL-25. Furthermore, treatment with antagonistic mAbs to either IL-17RB or IL-17RA completely blocked IL-25-induced pulmonary inflammation and airway hyperresponsiveness in naive BALB/c mice, similar to the effects of an antagonistic Ab to IL-25. Finally, a blocking Ab to human IL-17RA prevented IL-25 activity in a primary human cell-based assay. These data demonstrate for the first time that IL-25-mediated activities require both IL-17RB and IL-17RA and provide another example of an IL-17 family ligand that utilizes a heteromeric receptor complex.

Distinct functional motifs within the IL-17 receptor regulate signal transduction and target gene expression.

IL-17 is the founding member of a novel family of proinflammatory cytokines that defines a new class of CD4+ effector T cells, termed "Th17." Mounting evidence suggests that IL-17 and Th17 cells cause pathology in autoimmunity, but little is known about mechanisms of IL-17RA signaling. IL-17 through its receptor (IL-17RA) activates genes typical of innate immune cytokines, such as TNFalpha and IL-1beta, despite minimal sequence similarity in their respective receptors. A previous bioinformatics study predicted a subdomain in IL-17-family receptors with homology to a Toll/IL-1R (TIR) domain, termed the "SEFIR domain." However, the SEFIR domain lacks motifs critical for bona fide TIR domains, and its functionality was never verified. Here, we used a reconstitution system in IL-17RA-null fibroblasts to map functional domains within IL-17RA. We demonstrate that the SEFIR domain mediates IL-17RA signaling independently of classic TIR adaptors, such as MyD88 and TRIF. Moreover, we identified a previously undescribed"TIR-like loop" (TILL) required for activation of NF-kappaB, MAPK, and up-regulation of C/EBPbeta and C/EBPdelta. Mutagenesis of the TILL domain revealed a site analogous to the LPS(d) mutation in TLR4, which renders mice insensitive to LPS. However, a putative salt bridge typically found in TIR domains appears to be dispensable. We further identified a C-terminal domain required for activation of C/EBPbeta and induction of a subset IL-17 target genes. This structure-function analysis of a IL-17 superfamily receptor reveals important differences in IL-17RA compared with IL-1/TLR receptors.

Interferon-gamma regulates susceptibility to collagen-induced arthritis through suppression of interleukin-17.

OBJECTIVE: The enhanced expression of experimental arthritis in the absence of interferon-gamma (IFNgamma) suggests that IFNgamma suppresses arthritis. Interleukin-17 (IL-17) is a pivotal T cell cytokine in arthritis, and in vitro studies have indicated that IFNgamma suppresses IL-17 production. We undertook this study to test the hypothesis that resistance to collagen-induced arthritis (CIA) in C57BL/6 (B6) mice is regulated by IFNgamma-mediated suppression of IL-17. METHODS: Wild-type (WT) B6 mice, IFNgamma-knockout (KO) B6 mice, and DBA/1 mice were immunized with type II collagen (CII) in Freund's complete adjuvant (CFA). Lymphocytes from immunized mice were analyzed for cytokine production ex vivo by intracellular staining or restimulation with CII and enzyme-linked immunosorbent assays. In vivo blockade of IL-17 was achieved with an anti-IL-17 monoclonal antibody (mAb). RESULTS: CII restimulation of T cells from CII/CFA-immunized mice resulted in an approximately 5-fold increase in IL-17 production in IFNgamma-KO B6 mice compared with WT B6 mice. Neutralization of IFNgamma increased IL-17 production in WT B6 mice, and neutralization of IL-4 had a synergistic effect. Interestingly, the prototypical CIA-susceptible strain DBA/1 also demonstrated a high IL-17 and a low IFNgamma cytokine profile compared with WT B6 mice. Administration of the anti-IL-17 mAb attenuated arthritis in DBA/1 mice and almost completely prevented expression of arthritis in IFNgamma-KO B6 mice. CONCLUSION: These results indicate that sensitivity of IFNgamma-deficient B6 mice to CIA is associated with high IL-17 production and that this cytokine is required for expression of arthritis in this strain.

Cutting edge: identification of a pre-ligand assembly domain (PLAD) and ligand binding site in the IL-17 receptor.

IL-17 is the hallmark cytokine of the newly described "Th17" lymphocyte population. The composition, subunit dynamics, and ligand contacts of the IL-17 receptor are poorly defined. We previously demonstrated that the IL-17RA subunit oligomerizes in the membrane without a ligand. In this study, computational modeling identified two fibronectin-III-like (FN) domains in IL-17RA connected by a nonstructured linker, which we predicted to mediate homotypic interactions. In yeast two-hybrid, the membrane-proximal FN domain (FN2), but not the membrane-distal domain (FN1), formed homomeric interactions. The ability of FN2 to drive ligand-independent multimerization was verified by coimmunoprecipitation and fluorescence resonance energy transfer microscopy. Thus, FN2 constitutes a "pre-ligand assembly domain" (PLAD). Further studies indicated that the FN2 linker domain contains the IL-17 binding site, which was never mapped. However, the FN1 domain is also required for high affinity interactions with IL-17. Therefore, although the PLAD is located entirely within FN2, effective ligand binding also involves contributions from the linker and FN1.

No defect in T-cell priming, secondary response, or tolerance induction in response to inhaled antigens in Fms-like tyrosine kinase 3 ligand-deficient mice.

BACKGROUND: Respiratory tract dendritic cells (DCs) are crucial for the regulation of immune responses to inhaled antigens. However, the precise function of the multiple DC subsets present in the lungs and the lung-draining lymph nodes is unknown. Fms-like tyrosine kinase 3 ligand (FLT3L) is a hematopoietic growth factor that drives the development of multiple subsets of DCs in the lymphoid organs. OBJECTIVE: We sought to study the contribution of DC subsets in the regulation of the balance between tolerance and immunity against respiratory antigens by using FLT3L knockout mice. METHODS: Phenotypic analysis of DC subsets in the airways and lungs of FLT3L knockout mice was performed. By using various experimental models, the role of FLT3L-dependent DCs in the priming of naive T cells, the presentation of inhaled antigen to previously primed T H 2 cells, and intranasal tolerance induction was addressed. RESULTS: FLT3L knockout mice display a 90% reduction in lung parenchyma DCs but a normal number of airway DCs and blood monocytes. FLT3L knockout mice had a normal induction of eosinophilic inflammation in response to intranasal administration of allergen. FLT3L-dependent DCs were not required for the presentation of inhaled antigen to previously primed T H 2 cells, and normal induction of T-cell tolerance in response to inhaled antigen was observed in FLT3L knockout mice. CONCLUSION: Airway DC development is independent of FLT3L. FLT3L-dependent DCs are not required for the development and maintenance of airway inflammation or for the induction of intranasal tolerance. Our results point to airway DCs as the major regulators of the balance between tolerance and immunity to inhaled antigens.

Paradoxical role of programmed death-1 ligand 2 in Th2 immune responses in vitro and in a mouse asthma model in vivo.

Programmed death-1 ligand 2 (PD-L2) is a ligand for programmed death-1 (PD-1), a receptor that plays an inhibitory role in T cell activation. Since previous studies have shown up-regulation of PD-L2 expression by Th2 cytokines, and asthma is driven by a Th2 response, we hypothesized that PD-L2 might be involved in regulation of the immune response in this disease. We have found that lungs from asthmatic mice had sustained up-regulation of PD-1 and PD-L2, with PD-L2 primarily on dendritic cells. Although addition of PD-L2-Fc in vitro led to decreased T cell proliferation and cytokine production, administration of PD-L2-Fc in vivo in a mouse asthma model resulted in elevated serum IgE levels, increased eosinophilic and lymphocytic infiltration into bronchoalveolar lavage fluid, higher number of cells in the draining lymph nodes, and production of IL-5 and IL-13 from these cells. Although PD-1 was expressed on regulatory T cells, PD-L2-Fc did not affect regulatory T cell activity in vitro. This study provides in vivo evidence of an exacerbated inflammatory response following PD-L2-Fc administration and indicates a potential role for this molecule in Th2-mediated diseases such as asthma.