Understanding cytokine immunobiology is central to the development of rational therapies for destructive inflammatory diseases such as rheumatoid arthritis (RA) and periodontitis. The classical interleukin-1 (<em>IL</em>-1) family cytokines, <em>IL</em>-1alpha and <em>IL</em>-1beta, as well as <em>IL</em>-18, play key roles in inflammation. Recently, other members of the <em>IL</em>-1 family have been identified. These include six cytokines whose genes are located downstream of the genes for <em>IL</em>-1alpha and <em>IL</em>-1beta on chromosome 2 (<em>IL</em>-1F5-10) and also <em>IL</em>-33, which is the ligand for ST2, a member of the <em>IL</em>-1R/Toll-like receptor (TLR) receptor superfamily. <em>IL</em>-1F6, <em>IL</em>-1F8 and <em>Il</em>-<em>1F9</em> are agonists and, along with their receptor <em>IL</em>-1Rrp2, are highly expressed in epithelial cells suggesting a role in immune defence in the skin and the gastrointestinal (GI) tract including the mouth. Synovial fibroblasts and articular chondrocytes also express <em>IL</em>-1Rrp2 and respond to <em>IL</em>-1F8, indicating a possible role in RA. <em>IL</em>-33 is associated with endothelial cells in the inflamed tissues of patients with RA and Crohn's disease, where it is a nuclear factor which regulates transcription. <em>IL</em>-33 is also an extracellular cytokine: it induces the expression of T helper 2 (Th2) cytokines in vitro and in vivo as well as histopathological changes in the lungs and GI tract of mice. Therapeutic agents which modify <em>IL</em>-1 cytokines (e.g. recombinant <em>IL</em>-1Ra) have been used clinically and others are at various stages of development (e.g. anti-<em>IL</em>-18 antibodies). This review highlights the emerging data on these novel <em>IL</em>-1 cytokines and assesses their possible role in the pathogenesis and therapy of destructive inflammatory disorders such as RA and periodontitis.

Interleukin 1 (<em>IL</em>-1) plays a prominent role in immune and inflammatory reactions. Our understanding of the <em>IL</em>-1 family has recently expanded to include six novel members named <em>IL</em>-1F5 to <em>IL</em>-1F10. Recently, it was reported that <em>IL</em>-<em>1F9</em> activated NF-kappaB through the orphan receptor <em>IL</em>-1 receptor (<em>IL</em>-1R)-related protein 2 (<em>IL</em>-1Rrp2) in Jurkat cells (Debets, R., Timans, J. C., Homey, B., Zurawski, S., Sana, T. R., Lo, S., Wagner, J., Edwards, G., Clifford, T., Menon, S., Bazan, J. F., and Kastelein, R. A. (2001) J. Immunol. 167, 1440-1446). In this study, we demonstrate that <em>IL</em>-1F6 and <em>IL</em>-1F8, in addition to <em>IL</em>-<em>1F9</em>, activate the pathway leading to NF-kappaB in an <em>IL</em>-1Rrp2-dependent manner in Jurkat cells as well as in multiple other human and mouse cell lines. Activation of the pathway leading to NF-kappaB by <em>IL</em>-1F6 and <em>IL</em>-1F8 follows a similar time course to activation by <em>IL</em>-1beta, suggesting that signaling by the novel family members occurs through a direct mechanism. In a mammary epithelial cell line, NCI/ADR-RES, which naturally expresses <em>IL</em>-1Rrp2, all three cytokines signal without further receptor transfection. <em>IL</em>-1Rrp2 antibodies block activation of the pathway leading to NF-kappaB by <em>IL</em>-1F6, <em>IL</em>-1F8, and <em>IL</em>-<em>1F9</em> in both Jurkat and NCI/ADR-RES cells. In NCI/ADR-RES cells, the three <em>IL</em>-1 homologs activated the MAPKs, JNK and ERK1/2, and activated downstream targets as well, including an <em>IL</em>-8 promoter reporter and the secretion of <em>IL</em>-6. We also provide evidence that <em>IL</em>-1RAcP, in addition to <em>IL</em>-1Rrp2, is required for signaling by all three cytokines. Antibodies directed against <em>IL</em>-1RAcP and transfection of cytoplasmically deleted <em>IL</em>-1RAcP both blocked activation of the pathway leading to NF-kappaB by the three cytokines. We conclude that <em>IL</em>-1F6, <em>IL</em>-1F8, and <em>IL</em>-<em>1F9</em> signal through <em>IL</em>-1Rrp2 and <em>IL</em>-1RAcP.

<em>IL</em>-36α (<em>IL</em>-1F6), <em>IL</em>-36β (<em>IL</em>-1F8), and <em>IL</em>-36γ (<em>IL</em>-<em>1F9</em>) are members of the <em>IL</em>-1 family of cytokines. These cytokines bind to <em>IL</em>-36R (<em>IL</em>-1Rrp2) and <em>IL</em>-1RAcP, activating similar intracellular signals as <em>IL</em>-1, whereas <em>IL</em>-36Ra (<em>IL</em>-1F5) acts as an <em>IL</em>-36R antagonist (<em>IL</em>-36Ra). In this study, we show that both murine bone marrow-derived dendritic cells (BMDCs) and CD4(+) T lymphocytes constitutively express <em>IL</em>-36R and respond to <em>IL</em>-36α, <em>IL</em>-36β, and <em>IL</em>-36γ. <em>IL</em>-36 induced the production of proinflammatory cytokines, including <em>IL</em>-12, <em>IL</em>-1β, <em>IL</em>-6, TNF-α, and <em>IL</em>-23 by BMDCs with a more potent stimulatory effect than that of other <em>IL</em>-1 cytokines. In addition, <em>IL</em>-36β enhanced the expression of CD80, CD86, and MHC class II by BMDCs. <em>IL</em>-36 also induced the production of IFN-γ, <em>IL</em>-4, and <em>IL</em>-17 by CD4(+) T cells and cultured splenocytes. These stimulatory effects were antagonized by <em>IL</em>-36Ra when used in 100- to 1000-fold molar excess. The immunization of mice with <em>IL</em>-36β significantly and specifically promoted Th1 responses. Our data thus indicate a critical role of <em>IL</em>-36R ligands in the interface between innate and adaptive immunity, leading to the stimulation of T helper responses.

<em>IL</em>-1F6, <em>IL</em>-1F8, and <em>IL</em>-<em>1F9</em> and the <em>IL</em>-1R6(RP2) receptor antagonist <em>IL</em>-1F5 constitute a novel <em>IL</em>-1 signaling system that is poorly characterized in skin. To further characterize these cytokines in healthy and inflamed skin, we studied their expression in healthy control, uninvolved psoriasis, and psoriasis plaque skin using quantitative RT-PCR and immunohistochemistry. Expression of <em>IL</em>-1F5, -1F6, -1F8, and -<em>1F9</em> were increased 2 to 3 orders of magnitude in psoriasis plaque versus uninvolved psoriasis skin, which was supported immunohistologically. Moreover, treatment of psoriasis with etanercept led to significantly decreased <em>IL</em>-1F5, -1F6, -1F8, and -<em>1F9</em> mRNAs, concomitant with clinical improvement. Similarly increased expression of <em>IL</em>-1F5, -1F6, -1F8, and -<em>1F9</em> was seen in the involved skin of two mouse models of psoriasis. Suggestive of their importance in inflamed epithelia, <em>IL</em>-1α and TNF-α induced <em>IL</em>-1F5, -1F6, -1F8, and -<em>1F9</em> transcript expression by normal human keratinocytes. Microarray analysis revealed that these cytokines induce the expression of antimicrobial peptides and matrix metalloproteinases by reconstituted human epidermis. In particular, <em>IL</em>-1F8 increased mRNA expression of human β-defensin (HBD)-2, HBD-3, and CAMP and protein secretion of HBD-2 and HBD-3. Collectively, our data suggest important roles for these novel cytokines in inflammatory skin diseases and identify these peptides as potential targets for antipsoriatic therapies.

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

<em>IL</em>-17C is a functionally distinct member of the <em>IL</em>-17 family that binds <em>IL</em>-17 receptor E/A to promote innate defense in epithelial cells and regulate Th17 cell differentiation. We demonstrate that <em>IL</em>-17C (not <em>IL</em>-17A) is the most abundant <em>IL</em>-17 isoform in lesional psoriasis skin (1058 versus 8 pg/ml; p < 0.006) and localizes to keratinocytes (KCs), endothelial cells (ECs), and leukocytes. ECs stimulated with <em>IL</em>-17C produce increased TNF-α and KCs stimulated with <em>IL</em>-17C/TNF-α produce similar inflammatory gene response patterns as those elicited by <em>IL</em>-17A/TNF-α, including increases in <em>IL</em>-17C, TNF-α, <em>IL</em>-8, <em>IL</em>-1α/β, <em>IL</em>-1F5, <em>IL</em>-<em>1F9</em>, <em>IL</em>-6, <em>IL</em>-19, CCL20, S100A7/A8/A9, DEFB4, lipocalin 2, and peptidase inhibitor 3 (p < 0.05), indicating a positive proinflammatory feedback loop between the epidermis and ECs. Psoriasis patients treated with etanercept rapidly decrease cutaneous <em>IL</em>-17C levels, suggesting <em>IL</em>-17C/TNF-α-mediated inflammatory signaling is critical for psoriasis pathogenesis. Mice genetically engineered to overexpress <em>IL</em>-17C in KCs develop well-demarcated areas of erythematous, flakey involved skin adjacent to areas of normal-appearing uninvolved skin despite increased <em>IL</em>-17C expression in both areas (p < 0.05). Uninvolved skin displays increased angiogenesis and elevated S100A8/A9 expression (p < 0.05) but no epidermal hyperplasia, whereas involved skin exhibits robust epidermal hyperplasia, increased angiogenesis and leukocyte infiltration, and upregulated TNF-α, <em>IL</em>-1α/β, <em>IL</em>-17A/F, <em>IL</em>-23p19, vascular endothelial growth factor, <em>IL</em>-6, and CCL20 (p < 0.05), suggesting that <em>IL</em>-17C, when coupled with other proinflammatory signals, initiates the development of psoriasiform dermatitis. This skin phenotype was significantly improved following 8 wk of TNF-α inhibition. These findings identify a role for <em>IL</em>-17C in skin inflammation and suggest a pathogenic function for the elevated <em>IL</em>-17C observed in lesional psoriasis skin.

The <em>IL</em>-1 family members <em>IL</em>-36α (<em>IL</em>-1F6), <em>IL</em>-36β (<em>IL</em>-1F8), and <em>IL</em>-36γ (<em>IL</em>-<em>1F9</em>) and the receptor antagonist <em>IL</em>-36Ra (<em>IL</em>-1F5) constitute a novel signaling system that is poorly understood. We now show that these cytokines have profound effects on the skin immune system. Treatment of human keratinocytes with <em>IL</em>-36 cytokines significantly increased the expression of CXCL1, CXCL8, CCL3, CCL5, and CCL20, potent chemotactic agents for activated leukocytes, and <em>IL</em>-36α injected intradermally resulted in chemokine expression, leukocyte infiltration, and acanthosis of mouse skin. Blood monocytes, myeloid dendritic cells (mDC), and monocyte-derived DC (MO-DC) expressed <em>IL</em>-36R and responded to <em>IL</em>-36. In contrast, no direct effects of <em>IL</em>-36 on resting or activated human CD4(+) or CD8(+) T cells, or blood neutrophils, could be demonstrated. Monocytes expressed <em>IL</em>-1A, <em>IL</em>-1B, and <em>IL</em>-6 mRNA and <em>IL</em>-1β and <em>IL</em>-6 protein, and mDC upregulated surface expression of CD83, CD86, and HLA-DR and secretion of <em>IL</em>-1β and <em>IL</em>-6 after treatment with <em>IL</em>-36. Furthermore, <em>IL</em>-36α-treated MO-DC enhanced allogeneic CD4(+) T cell proliferation, demonstrating that <em>IL</em>-36 can stimulate the maturation and function of DC and drive T cell proliferation. These data indicate that <em>IL</em>-36 cytokines actively propagate skin inflammation via the activation of keratinocytes, APC, and, indirectly, T cells.

The <em>IL</em>-1 family of cytokines, which now includes 11 members, is well known to participate in inflammation. Although the most recently recognized <em>IL</em>-1 family cytokines (<em>IL</em>-1F5-11) have been shown to be expressed in airway epithelial cells, the regulation of their expression and function in the epithelium has not been extensively studied. We investigated the regulation of <em>IL</em>-1F5-11 in primary normal human bronchial epithelial cells. Messenger (m)RNAs for <em>IL</em>-1F6 and <em>IL</em>-<em>1F9</em>, but not <em>IL</em>-1F5, <em>IL</em>-1F8 or <em>IL</em>-1F10, were significantly up-regulated by TNF, <em>IL</em>-1β, <em>IL</em>-17 and the Toll-like receptor (TLR)3 ligand double-stranded (ds)RNA. mRNAs for <em>IL</em>-1F7 and <em>IL</em>-1F11 (<em>IL</em>-33) were weakly up-regulated by some of the cytokines tested. Notably, mRNAs for <em>IL</em>-1F6 and <em>IL</em>-<em>1F9</em> were synergistically enhanced by the combination of TNF/<em>IL</em>-17 or dsRNA/<em>IL</em>-17. <em>IL</em>-<em>1F9</em> protein was detected in the supernatant following stimulation with dsRNA or a combination of dsRNA and <em>IL</em>-17. <em>IL</em>-1F6 protein was detected in the cell lysate but was not detected in the supernatant. We screened for the receptor for <em>IL</em>-<em>1F9</em> and found that lung fibroblasts expressed this receptor. We found that <em>IL</em>-<em>1F9</em> activated mitogen-activated protein kinases and the transcription factor NF-κB in primary normal human lung fibroblasts. <em>IL</em>-<em>1F9</em> also stimulated the expression of the neutrophil chemokines <em>IL</em>-8 and CXCL3 and the Th17 chemokine CCL20 in lung fibroblasts. These results suggest that epithelial activation by TLR3 (e.g., by respiratory viral infection) and exposure to cytokines from Th17 cells (<em>IL</em>-17) and inflammatory cells (TNF) may amplify neutrophilic inflammation in the airway via induction of <em>IL</em>-<em>1F9</em> and activation of fibroblasts.

In recent years, different genes and proteins have been highlighted as potential biomarkers for psoriasis, one of the most common inflammatory skin diseases worldwide. However, most of these markers are not only psoriasis-specific but also found in other inflammatory disorders. We performed an unsupervised cluster analysis of gene expression profiles in 150 psoriasis patients and other inflammatory skin diseases (atopic dermatitis, lichen planus, contact eczema, and healthy controls). We identified a cluster of IL-17/tumor necrosis factor-α (TNFα)-associated genes specifically expressed in psoriasis, among which IL-36γ was the most outstanding marker. In subsequent immunohistological analyses, IL-36γ was confirmed to be expressed in psoriasis lesions only. IL-36γ peripheral blood serum levels were found to be closely associated with disease activity, and they decreased after anti-TNFα-treatment. Furthermore, IL-36γ immunohistochemistry was found to be a helpful marker in the histological differential diagnosis between psoriasis and eczema in diagnostically challenging cases. These features highlight IL-36γ as a valuable biomarker in psoriasis patients, both for diagnostic purposes and measurement of disease activity during the clinical course. Furthermore, IL-36γ might also provide a future drug target, because of its potential amplifier role in TNFα- and IL-17 pathways in psoriatic skin inflammation.

The airway epithelium responds to microbial exposure by altering expression of a variety of genes to increase innate host defense. We aimed to delineate the early transcriptional response in human primary bronchial epithelial cells exposed for 6 h to a mixture of <em>IL</em>-1beta and TNF-alpha or heat-inactivated Pseudomonas aeruginosa. Because molecular mechanisms of epithelial innate host defense are not fully understood, the open-ended expression-profiling technique SAGE was applied to construct gene expression profiles covering 30,000 genes: 292 genes were found to be differentially expressed. Expression of seven genes was confirmed by real-time qPCR. Among differentially expressed genes, four classes or families were identified: keratins, proteinase inhibitors, S100 calcium-binding proteins, and <em>IL</em>-1 family members. Marked transcriptional changes were observed for keratins that form a key component of the cytoskeleton in epithelial cells. Expression of antimicrobial proteinase inhibitors SLPI and elafin was elevated after microbial or cytokine exposure. Interestingly, expression of numerous S100 family members was observed, and eight members, including S100A8 and S100A9, were among the most differentially expressed genes. Differential expression was also observed for the <em>IL</em>-1 family members <em>IL</em>-1beta, <em>IL</em>-1 receptor antagonist, and <em>IL</em>-<em>1F9</em>, a newly discovered <em>IL</em>-1 family member. Clustering of differentially expressed genes into biological processes revealed that the early inflammatory response in airway epithelial cells to <em>IL</em>-1beta-TNF-alpha and P. aeruginosa is characterized by expression of genes involved in epithelial barrier formation and host defense.

Interleukin-1 (<em>IL</em>-1) is a proinflammatory cytokine that signals through the Type I <em>IL</em>-1 receptor (<em>IL</em>-1RI). Novel <em>IL</em>-1-like cytokines were recently identified. Their functions in lung disease remain unclear. Interleukin-1 family member-9 (<em>IL</em>-<em>1F9</em>) is one such <em>IL</em>-1-like cytokine, expressed in the lungs of humans and mice. <em>IL</em>-<em>1F9</em> signals through <em>IL</em>-1 receptor-related protein 2 (<em>IL</em>-1Rrp2/<em>IL</em>-1RL2), which is distinct from <em>IL</em>-1RI. We sought to determine if <em>IL</em>-<em>1F9</em> acts as a proinflammatory cytokine in lung disease. <em>IL</em>-<em>1F9</em> protein was increased in lung homogenates of house dust mite-challenged A/J mice compared with controls, and expression was seen in airway epithelial cells. The intratracheal administration of recombinant mouse <em>IL</em>-<em>1F9</em> increased airway hyperresponsiveness and induced neutrophil influx and mucus production, but not eosinophilic infiltration in the lungs of mice. In addition, <em>IL</em>-1α protein levels in bronchoalveolar lavage fluid, chemokines, and chemokine-receptor mRNA expression in the lungs were increased after the instillation of intratracheal <em>IL</em>-<em>1F9</em>. Consistent with these changes, NF-κB transcription factor activity was increased in the lungs of mice challenged with <em>IL</em>-<em>1F9</em> and in a macrophage cell line treated with <em>IL</em>-<em>1F9</em>. These data suggest that <em>IL</em>-<em>1F9</em> is upregulated during inflammation, and acts as a proinflammatory cytokine in the lungs.

<em>IL</em>-36 is the common name for the three <em>IL</em>-1 family members <em>IL</em>-36α, <em>IL</em>-36β, and <em>IL</em>-36γ, formerly known as <em>IL</em>-1F6, <em>IL</em>-1F8, and <em>IL</em>-<em>1F9</em>, respectively. <em>IL</em>-36 appears to have pro-inflammatory activities; however, the physiological function of these cytokines remains unknown. Expression of <em>IL</em>-36 by keratinocytes implies its possible involvement in innate immune responses in the skin. We observed that, of the three <em>IL</em>-36 isoforms, human keratinocytes express high levels of <em>IL</em>-36γ. <em>IL</em>-36γ mRNA expression was dramatically induced by the Toll-like receptor ligands polyinosinic-polycytidylic acid (poly(I:C)) and flagellin. Surprisingly, the <em>IL</em>-36γ protein was released by cells treated with poly(I:C), but remained intracellular in cells treated with flagellin only. poly(I:C), but not flagellin, induced cell death and caspase-3/7 activation. Inhibition of caspase-3/7 and caspase-1 blocked extracellular release of <em>IL</em>-36γ from poly(I:C)-treated cells. Furthermore, caspase-1 inhibition prevented poly(I:C)-induced caspase-3/7 activation. Interestingly, transcription of the gene <em>IL</em>36G was dependent on caspase-1, but not caspase-3/7, activation. This demonstrates that the pathways leading to <em>IL</em>36G transcription and caspase-3/7 activation branch after caspase-1. This divergence of the pathways allows the cells to enter a state of de novo protein synthesis before committing to pyroptosis. Overall, our observations suggest that <em>IL</em>-36γ may be an alarmin that signals the cause, e.g., viral infection, of cell death.

We report for the first time that expression of the novel <em>IL</em>-1 cytokine receptor <em>IL</em>-1Rrp2 (<em>IL</em>-1R6) is unique to DCs within the human myelomonocytic lineage. <em>IL</em>-1Rrp2 was expressed by monocyte-derived dendritic cells (MDDCs) which was dose-dependently increased by <em>IL</em>-4 and correlated with increased numbers of differentiated MDDCs. Human plasmacytoid DCs also express <em>IL</em>-1Rrp2 but the receptor is not expressed by either myeloid DC type 1 (mDC1) or mDC2 cells. We also show that <em>IL</em>-1F8 or <em>IL</em>-<em>1F9</em> cytokines, which signal through <em>IL</em>-1Rrp2, induce maturation of MDDCs, as measured by increased expression of HLA-DR and CD83 and decreased expression of CD1a. Furthermore, <em>IL</em>-1F8 stimulated increased CD40 and CD80 expression and <em>IL</em>-18 and <em>IL</em>-12 p70 production by MDDCs, which induced proliferation of IFN-γ-producing CD3(+) lymphocytes (indicative of inflammatory Th1 subsets). <em>IL</em>-1F8 and <em>IL</em>-1F2 were equipotent in their ability to stimulate <em>IL</em>-18 secretion from MDDCs but <em>IL</em>-1F8 was not as potent as <em>IL</em>-1F2 in stimulating secretion of <em>IL</em>-12p70 from MDDCs or inducing lymphocyte proliferation Therefore, <em>IL</em>-1Rrp2 expression by some DC subsets may have an important function in the human immune response in vivo via its role in differentiation of inflammatory Th1 lymphocytes.

By concerted action in dendritic (DC) and T cells, T-box expressed in T cells (T-bet, Tbx21) is pivotal for initiation and perpetuation of Th1 immunity. Identification of novel T-bet-regulated genes is crucial for further understanding the biology of this transcription factor. By combining siRNA technology with genome-wide mRNA expression analysis, we sought to identify new T-bet-regulated genes in predendritic KG1 cells activated by IL-18. One gene robustly dependent on T-bet was IL-36γ, a recently described novel IL-1 family member. Promoter analysis revealed a T-bet binding site that, along with a κB site, enables efficient IL-36γ induction. Using knock-out animals, IL-36γ reliance on T-bet was extended to murine DC. IL-36γ expression by human myeloid cells was confirmed using monocyte-derived DC and M1 macrophages. The latter model was employed to substantiate dependence of IL-36γ on endogenous T-bet in human primary cells. Ectopic expression of T-bet likewise mediated IL-36γ production in HaCaT keratinocytes that otherwise lack this transcription factor. Additional experiments furthermore revealed that mature IL-36γ has the capability to establish an inflammatory gene expression profile in human primary keratinocytes that displays enhanced mRNA levels for TNFα, CCL20, S100A7, inducible NOS, and IL-36γ itself. Data presented herein shed further light on involvement of T-bet in innate immunity and suggest that IL-36γ, besides IFNγ, may contribute to functions of this transcription factor in immunopathology.

Similarity in structure and sequence homology has led to the identification of new members of the interleukin-1 (<em>IL</em>-1) ligand and receptor superfamilies. <em>IL</em>-1F6, <em>IL</em>-1F8 and <em>IL</em>-<em>1F9</em> have been shown to signal through <em>IL</em>-1R-related protein 2 and <em>IL</em>-1 receptor accessory protein leading to activation of NFkappaB, while <em>IL</em>-1F7 and <em>IL</em>-1F10 interact with the <em>IL</em>-18 receptor and the soluble <em>IL</em>-1 receptor type I respectively. In contrast, identification of a biological role for <em>IL</em>-1F5 has remained elusive, with conflicting data relating to its possible ability to antagonize <em>IL</em>-<em>1F9</em>-stimulated activation of NFkappaB in Jurkat cells transfected with <em>IL</em>-1R-related protein 2. In this study, we set out to investigate a possible role for <em>IL</em>-1F5 in the brain and report that it antagonizes the inflammatory effects of <em>IL</em>-1beta and lipopolysaccharide (LPS) in vivo and in vitro including the inhibitory effect on long-term potentiation (LTP) in rat hippocampus. We demonstrate that <em>IL</em>-1F5 induces <em>IL</em>-4 mRNA and protein expression in glia in vitro and enhances hippocampal expression of <em>IL</em>-4 following intracerebroventricular (i.c.v.) injection. The inhibitory effect of <em>IL</em>-1F5 on LPS-induced <em>IL</em>-1beta is attenuated in cells from <em>IL</em>-4-defective (<em>IL</em>-4-/- mice). Our findings suggest that <em>IL</em>-1F5 mediates anti-inflammatory effects through its ability to induce <em>IL</em>-4 production and that this is a consequence of its interaction with the orphan receptor, single Ig <em>IL</em>-1R-related molecule (SIGIRR)/TIR8, as the effects were not observed in SIGIRR-/- mice. In contrast to its effects in brain tissue, <em>IL</em>-1F5 did not attenuate LPS-induced changes, or up-regulated <em>IL</em>-4 in macrophages or dendritic cells, suggesting that the effect is confined to the brain.

BACKGROUND

A number of studies have challenged the T-cell-centred pathogenetic view of psoriasis by the finding that epithelium-expressed genes are intimately involved in the inflammatory process. Interleukin (IL)-17 is an important inflammatory mediator in skin psoriasis.

OBJECTIVE

IL-17 is known to act on keratinocytes and we were interested in its impact on the expression of pro- and anti-inflammatory IL-1 family members.

METHODS

We compared human keratinocytes derived from epidermal stem cells of hair follicles plucked from patients with psoriasis and healthy individuals using quantitative reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays.

RESULTS

In the presence of <em>IL</em>-17, psoriasis-derived keratinocytes showed a significantly higher induction of the proinflammatory <em>IL</em>-1 family members <em>IL</em>-1F6 and <em>IL</em>-<em>1F9</em>, but not of anti-inflammatory members <em>IL</em>-1F5, <em>IL</em>-1F7 or <em>IL</em>-1F3 compared with keratinocytes from healthy individuals. Both basal and <em>IL</em>-17-induced levels of <em>IL</em>-1F2 and <em>IL</em>-1F1 were found to be significantly lower in psoriasis keratinocytes.

CONCLUSIONS

As keratinocytes were derived from epidermal stem cells of the hair follicles and were obtained from nonlesional sites, differences found are likely to present an intrinsic feature of psoriasis epithelium. Our data support the significance of IL-1 family members as therapeutic targets in psoriasis conditions.

We have previously reported that direct injection of dendritic cells (DC) engineered to express the Type-1 transactivator Tbet (i.e., DC.Tbet) into murine tumors results in antitumor efficacy in association with the development of structures resembling tertiary lymphoid organs (TLO) in the tumor microenvironment (TME). These TLO contained robust infiltrates of B cells, DC, NK cells, and T cells in proximity to PNAd+ blood vessels; however, they were considered incomplete, since the recruited B cells failed to organize into classic germinal center-like structures. We now report that antitumor efficacy and TLO-inducing capacity of DC.Tbet-based i.t. therapy is operational in peripheral lymph node-deficient LTA-/- mice, and that it is highly dependent upon a direct Tbet target gene product, <em>IL</em>-36γ/<em>IL</em>-<em>1F9</em>. Intratumoral DC.Tbet fails to provide protection to tumor-bearing <em>IL</em>-36R-/- hosts, or to tumor-bearing wild-type recipient mice co-administered rm<em>IL</em>-1F5/<em>IL</em>-36RN, a natural <em>IL</em>-36R antagonist. Remarkably, the injection of tumors with DC engineered to secrete a bioactive form of m<em>IL</em>-36γ (DC.<em>IL</em>36γ) also initiated therapeutic TLO and slowed tumor progression in vivo. Furthermore, DC.<em>IL</em>36γ cells strongly upregulated their expression of Tbet, suggesting that Tbet and <em>IL</em>-36γ cooperate to reinforce each other's expression in DC, rendering them competent to promote TLO formation in an "immunologically normalized," therapeutic TME.

The recently discovered <em>IL</em>-<em>1F9</em> (<em>IL</em>-1H1) is a putative member of the interleukin (<em>IL</em>)-1 family of cytokines that has been shown to activate nuclear factor-kappa B (NFkappaB) in Jurkat cells transfected with the orphan receptor <em>IL</em>-1 receptor-related protein (<em>IL</em>-1Rrp)2. The aim of the present study was therefore to investigate expression of <em>IL</em>-1Rrp2 and to determine if <em>IL</em>-<em>1F9</em> induces known <em>IL</em>-1 signaling pathways in the different cell types of the mouse brain in culture. Messenger RNA for <em>IL</em>-1Rrp2 was not detected in primary neurones by RT-PCR, but significant constitutive expression was found in mixed glial cells, particularly in astrocytes and microglia, which was strongly decreased by exposure to bacterial lipopolysaccharide (LPS). LPS induced the release of <em>IL</em>-6, and activated NFkappaB and the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated protein kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) in microglial cultures. <em>IL</em>-1beta induced release of <em>IL</em>-6 and activated NFkappaB, p38, JNK and ERK1/2 in mixed glial cultures, which was completely abolished in the presence of <em>IL</em>-1 receptor antagonist (<em>IL</em>-1ra). When injected intracerebroventrically in the rat, <em>IL</em>-1beta increased core body temperature, and reduced body weight and food intake. In contrast, <em>IL</em>-<em>1F9</em> failed to induce any of these responses either in vivo or in vitro. These results demonstrate that glial cells may be a target for the new ligand <em>IL</em>-<em>1F9</em>, since high expression of <em>IL</em>-1Rrp2 mRNA was detected in these cells. However, <em>IL</em>-<em>1F9</em> failed to induce any of the classical <em>IL</em>-1beta responses, suggesting that it may trigger alternative pathway(s).

Viral respiratory infections activate the innate immune response in the airway epithelium through Toll-like receptors (TLRs) and induce airway inflammation, which causes acute exacerbation of asthma. Although increases in <em>IL</em>-17A expression were observed in the airway of severe asthma patients, the interaction between <em>IL</em>-17A and TLR activation in airway epithelium remains poorly understood. In this study, we demonstrated that <em>IL</em>-17A and polyI:C, the ligand of TLR3, synergistically induced the expression of proinflammatory cytokines and chemokines (G-CSF, <em>IL</em>-8, CXCL1, CXCL5, <em>IL</em>-<em>1F9</em>), but not type I interferon (IFN-α1, -β) in primary culture of normal human bronchial epithelial cells. Synergistic induction after co-stimulation with <em>IL</em>-17A and polyI:C was observed from 2 to 24 hours after stimulation. Treatment with cycloheximide or actinomycin D had no effect, suggesting that the synergistic induction occurred without de novo protein synthesis or mRNA stabilization. Inhibition of the TLR3, TLR/TIR-domain-containing adaptor-inducing interferon β (TRIF), NF-κB, and IRF3 pathways decreased the polyI:C- and <em>IL</em>-17A/polyI:C-induced G-CSF and <em>IL</em>-8 mRNA expression. Comparing the levels of mRNA induction between co-treatment with <em>IL</em>-17A/polyI:C and treatment with polyI:C alone, blocking the of NF-κB pathway significantly attenuated the observed synergism. In western blotting analysis, activation of both NF-κB and IRF3 was observed in treatment with polyI:C and co-treatment with <em>IL</em>-17A/polyI:C; moreover, co-treatment with <em>IL</em>-17A/polyI:C augmented IκB-α phosphorylation as compared to polyI:C treatment alone. Collectively, these findings indicate that <em>IL</em>-17A and TLR3 activation cooperate to induce proinflammatory responses in the airway epithelium via TLR3/TRIF-mediated NF-κB/IRF3 activation, and that enhanced activation of the NF-κB pathway plays an essential role in synergistic induction after co-treatment with <em>IL</em>-17A and polyI:C in vitro.

Our previous studies revealed that REIC/Dkk-3 was expressed various tissues, including skin keratinocytes. The aim of the present study was to identify the factors that regulate the expression of the dickkopf Wnt signaling pathway inhibitor 3 (REIC/Dkk‑3) tumor suppressor gene in normal human skin keratinocytes (NHKs). Several growth factors and cytokines that have previously been reported to be involved in the growth and differentiation of keratinocytes were screened as potential regulators. Western blot analysis was performed using protein from NHKs cultured with/without various factors including the epidermal growth factor, tumor necrosis factor‑α, transforming growth factor‑β, interleukin (<em>IL</em>)‑<em>1F9</em>, <em>IL</em>‑6, <em>IL</em>‑8 and Ca2+. The results indicated that only TNF‑α downregulated REIC/Dkk‑3 expression in NHKs. Subsequently, TNF‑α was confirmed to reduce the expression levels of REIC/Dkk‑3 in mouse skin tissue and hair culture models. TNF‑α‑mediated downregulation of REIC/Dkk‑3 expression in NHKs was abrogated by the addition of a TNF‑α‑specific antibody. In conclusion, the results indicate that TNF‑α downregulates REIC/Dkk‑3 expression in normal skin keratinocytes.