Mutation in IL36RN impairs the processing and regulatory function of the interleukin-36-receptor antagonist and is associated with DITRA syndrome.

The identification of loss-of-function mutations of the IL36RN gene encoding the interleukin-36 receptor antagonist (IL-36Ra) in generalized pustular psoriasis (GPP) emphasized the key role of this pathway in skin innate immunity and systemic inflammation. It has been previously shown in vitro that removal of the N-terminal amino acid IL36Ra (M1) is critical to its biological activity, but the in vivo contribution of this processing remains unknown. We report herein a new homozygous (c4G>T, pV2F) missense IL36RN mutation segregating in a family with three GPP-affected patients. The V2F mutation does not alter IL-36Ra protein expression but was devoid of any antagonist activity. Mass spectrometry showed that the V2F IL-36Ra mutant retains its first N-terminal methionine. These results provide the first in vivo demonstration that removal of N-terminal methionine of native IL-36Ra is a mandatory step to reach optimal antagonist activity and to prevent sustained skin and systemic inflammation in humans.

Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis.

BACKGROUND: Generalized pustular psoriasis is a life-threatening disease of unknown cause. It is characterized by sudden, repeated episodes of high-grade fever, generalized rash, and disseminated pustules, with hyperleukocytosis and elevated serum levels of C-reactive protein, which may be associated with plaque-type psoriasis. METHODS: We performed homozygosity mapping and direct sequencing in nine Tunisian multiplex families with autosomal recessive generalized pustular psoriasis. We assessed the effect of mutations on protein expression and conformation, stability, and function. RESULTS: We identified significant linkage to an interval of 1.2 megabases on chromosome 2q13-q14.1 and a homozygous missense mutation in IL36RN, encoding an interleukin-36-receptor antagonist (interleukin-36Ra), an antiinflammatory cytokine. This mutation predicts the substitution of a proline residue for leucine at amino acid position 27 (L27P). Homology-based structural modeling of human interleukin-36Ra suggests that the proline at position 27 affects both the stability of interleukin-36Ra and its interaction with its receptor, interleukin-1 receptor-like 2 (interleukin-1 receptor-related protein 2). Biochemical analyses showed that the L27P variant was poorly expressed and less potent than the nonvariant interleukin-36Ra in inhibiting a cytokine-induced response in an interleukin-8 reporter assay, leading to enhanced production of inflammatory cytokines (interleukin-8 in particular) by keratinocytes from the patients. CONCLUSIONS: Aberrant interleukin-36Ra structure and function lead to unregulated secretion of inflammatory cytokines and generalized pustular psoriasis. (Funded by Agence Nationale de la Recherche and Société Française de Dermatologie.).

Interleukin-36 (IL-36) ligands require processing for full agonist (IL-36α, IL-36ß, and IL-36γ) or antagonist (IL-36Ra) activity.

IL-36α, IL-36ß, and IL-36γ (formerly IL-1F6, IL-1F8, and IL-1F9) are IL-1 family members that signal through the IL-1 receptor family members IL-1Rrp2 (IL-1RL2) and IL-1RAcP. IL-36Ra (formerly IL-1F5) has been reported to antagonize IL-36γ. However, our previous attempts to demonstrate IL-36Ra antagonism were unsuccessful. Here, we demonstrate that IL-36Ra antagonist activity is dependent upon removal of its N-terminal methionine. IL-36Ra starting at Val-2 is fully active and capable of inhibiting not only IL-36γ but also IL-36α and IL-36ß. Val-2 of IL-36Ra lies 9 amino acids N-terminal to an A-X-Asp motif conserved in all IL-1 family members. In further experiments, we show that truncation of IL-36α, IL-36ß, and IL-36γ to this same point increased their specific activity by ∼10(3)-10(4)-fold (from EC(50) 1 µg/ml to EC(50) 1 ng/ml). Inhibition of truncated IL-36ß activity required ∼10(2)-10(3)-fold excess IL-36Ra, similar to the ratio required for IL-1Ra to inhibit IL-1ß. Chimeric receptor experiments demonstrated that the extracellular (but not cytoplasmic) domain of IL-1Rrp2 or IL-1R1 is required for inhibition by their respective natural antagonists. IL-36Ra bound to IL-1Rrp2, and pretreatment of IL-1Rrp2-expressing cells with IL-36Ra prevented IL-36ß-mediated co-immunoprecipitation of IL-1Rrp2 with IL-1RAcP. Taken together, these results suggest that the mechanism of IL-36Ra antagonism is analogous to that of IL-1Ra, such that IL-36Ra binds to IL-1Rrp2 and prevents IL-1RAcP recruitment and the formation of a functional signaling complex. In addition, truncation of IL-36α, IL-36ß, and IL-36γ dramatically enhances their activity, suggesting that post-translational processing is required for full activity.

Differing roles for short chain fatty acids and GPR43 agonism in the regulation of intestinal barrier function and immune responses.

Inflammatory bowel disease (IBD) is associated with a loss of intestinal barrier function and dysregulated immune responses. It has been shown that short chain fatty acids (SCFAs) are protective in IBD and that GPR43 mediates the protective effects of SCFAs. In this study, we investigated the effects of SCFAs in comparison to highly specific GPR43 agonists on human intestinal epithelial and immune cells. Our results confirm that SCFAs are enhancers of barrier function in intestinal epithelial cells. Additionally, SCFAs also displayed potent immunoregulatory properties based upon the ability to inhibit LPS-induced cytokine production in PBMC, and human T cell proliferation and cytokine production. Unexpectedly, and in contrast to the current belief, specific GPR43 agonists failed to exhibit similar barrier enhancing and anti-inflammatory properties. These findings demonstrate that SCFA possess broad protective functions in IBD and agonizing GPR43 alone is unlikely to be beneficial in patients.

Essential role of PI3Kdelta and PI3Kgamma in thymocyte survival.

Class 1 phosphoinositide 3-kinases (PI3Ks), consisting of PI3Kalpha, beta, gamma, and delta, are a family of intracellular signaling molecules that play important roles in cell-mediated immune responses. In thymocytes, however, their role is less clear, although PI3Kgamma is postulated to partially contribute to pre-TCR-dependent differentiation. We now report that PI3Kdelta, in conjunction with PI3Kgamma, is required for thymocyte survival and ultimately for T-cell production. Surprisingly, genetic deletion of the p110delta and p110gamma catalytic subunits resulted in a dramatic reduction in thymus size, cellularity, and lack of corticomedullary differentiation. Total thymocyte counts in these animals were 27-fold lower than in wild-type (WT) controls because of a diminished number of CD4+ CD8+ double-positive (DP) cells and were associated with T-cell depletion in blood and in secondary lymphoid organs. Moreover, this alteration in the DP population was intrinsic to thymocytes, because the reconstitution of p110gammadelta-/- animals with WT fetal liver cells restored the proportions of all thymocyte populations to those in WT controls. The observed defects were related to massive apoptosis in the DP population; TCRB expression, pre-TCR selection, and generation of DP cells appeared relatively unperturbed. Thus, class 1 PI3Ks work in concert to protect developing thymocytes from apoptosis.

The role of endothelial PI3Kgamma activity in neutrophil trafficking.

Phosphoinositide 3-kinase gamma (PI3Kgamma) in neutrophils plays a critical role in the directed migration of these cells into inflamed tissues. In this study, we demonstrate the importance of the endothelial component of PI3Kgamma activity relative to its leukocyte counterpart in supporting neutrophil interactions with the inflamed vessel wall. Despite the reconstitution of class-Ib PI3K function in neutrophils of p110gamma-/- mice, we observed a 45% reduction in accumulation of these cells in an acute lung injury model. Mechanistically, this appears to result from a perturbation in selectin-mediated adhesion as manifested by a 70% reduction in wild-type (WT) neutrophil attachment to and 17-fold increase in rolling velocities on p110gamma-/- microvessels in vivo in response to tumor necrosis factor alpha (TNFalpha). This alteration in adhesion was further augmented by a deficiency in p110delta, suggesting that the activity of both catalytic subunits is required for efficient capture of neutrophils by cytokine-stimulated endothelium. Interestingly, E-selectin-mediated adhesion in p110gamma-/-) mice was impaired by more than 95%, but no defect in nuclear factor kappa B (NF-kappaB)-induced gene expression was observed. These findings suggest a previously unrecognized partnership between class-I PI3Ks expressed in leukocytes and endothelium, the combination of which is required for the efficient trafficking of immunocompetent cells to sites of inflammation.

Mechanisms and implications of phosphoinositide 3-kinase delta in promoting neutrophil trafficking into inflamed tissue.

The phosphoinositide 3-kinase (PI3K) catalytic subunit p110 delta is expressed in neutrophils and is thought to play a role in their accumulation at sites of inflammation by contributing to chemoattractant-directed migration. We report here that p110 delta is present in endothelial cells and participates in neutrophil trafficking by modulating the proadhesive state of these cells in response to tumor necrosis factor alpha (TNF alpha). Specifically, administration of the selective inhibitor of PI3K delta, IC87114, to animals reduced neutrophil tethering to and increased rolling velocities on cytokine-activated microvessels in a manner similar to that observed in mice deficient in p110 delta. These results were confirmed in vitro as inhibition of this isoform in endothelium, but not neutrophils, diminished cell attachment in flow. A role for PI3K delta in TNF alpha-induced signaling is demonstrated by a reduction in Akt-phosphorylation and phosphatidylinositol-dependent kinase 1 (PDK1) enzyme activity upon treatment of this cell type with IC87114. p110 delta expressed in neutrophils also contributes to trafficking as demonstrated by the impaired movement of these cells across inflamed venules in animals in which this catalytic subunit was blocked or genetically deleted, results corroborated in transwell migration assays. Thus, PI3K delta may be a reasonable therapeutic target in specific inflammatory conditions as blockade of its activity reduces neutrophil influx into tissues by diminishing their attachment to and migration across vascular endothelium.