Deciphering site 3 interactions of interleukin 12 and interleukin 23 with their cognate murine and human receptors.

Interleukin (IL)-12 and IL-23 belong to the IL-12 type family and are composite cytokines, consisting of the common ß subunit p40 and the specific cytokine α subunit p35 and p19, respectively. IL-12 signals via the IL-12Rß1·IL-12Rß2 receptor complex, and IL-23 uses also IL-12Rß1 but engages IL-23R as second receptor. Importantly, binding of IL-12 and IL-23 to IL-12Rß1 is mediated by p40, and binding to IL-12Rß2 and IL-23R is mediated by p35 and p19, respectively. Previously, we have identified a W157A substitution at site 3 of murine IL-23p19 that abrogates binding to murine IL-23R. Here, we demonstrate that the analogous Y185R site 3 substitution in murine and Y189R site 3 substitution in human IL-12p35 abolishes binding to IL-12Rß2 in a cross-species manner. Although Trp157 is conserved between murine and human IL-23p19 (Trp156 in the human ortholog), the site 3 W156A substitution in hIL-23p19 did not affect signaling of cells expressing human IL-12Rß1 and IL-23R, suggesting that the interface of murine IL-23p19 required for binding to IL-23R is different from that in the human ortholog. Hence, we introduced additional hIL-23p19 substitutions within its binding interface to hIL-23R and found that the combined site 3 substitutions of W156A and L160E, which become buried at the complex interface, disrupt binding of hIL-23p19 to hIL-23R. In summary, we have identified substitutions in IL-12p35 and IL-23p19 that disrupt binding to their cognate receptors IL-12Rß2 and IL-23R in a murine/human cross-species manner.

Functional analyses of the interaction of chicken interleukin 23 subunit p19 with IL-12 subunit p40 to form the IL-23 complex.

This study represents the first description of the cloning of chicken IL-23p19 (ChIL-23α) and the function of the IL-23 complex in birds. Multiple alignment of ChIL-23α with other known IL-23α amino acid sequences revealed regions of amino acid conservation. The homologies of ChIL-23α, IL-12p35, and similar mammalian subunits ranged between 26% and 42%. ChIL-23α consisted of four exons and three introns; similar to those in humans and mice, and limited conservation of synteny between the human and chicken genomes was observed. Using bioinformatics tools, we identified the NF-κB, C/EBPα-ß, c-Jun, c-Rel, AP-1, GATA-1, and ER promoter sites in ChIL-23α. Moreover, IL-23α mRNA was more highly expressed than IL-12p40 and IL-12p35 mRNA in several organs of chickens infected with Salmonella. In addition, ChIL-23 complex are associated with IL-23R, IL-12Rß1 receptors; activate the JAK2/TYK2, STAT1/3, SOCS1 genes, and induced proinflammatory cytokines in immune cells. Collectively, these results indicate that ChIL-23 is a member of the IL-12 family, has proinflammatory properties related to IL-23R and IL-12Rß1 receptor expression, and activates the JAK/STAT signaling pathway that results in the interaction of ChIL-23α with ChIL-12p40 to form the novel ChIL-23 complex. Our results provide novel insights into the regulation of immunity, inflammation, and immunopathology.

p19-targeted ABD-derived protein variants inhibit IL-23 binding and exert suppressive control over IL-23-stimulated expansion of primary human IL-17+ T-cells.

Interleukin-23 (IL-23), a heterodimeric cytokine of covalently bound p19 and p40 proteins, has recently been closely associated with development of several chronic autoimmune diseases such as psoriasis, psoriatic arthritis or inflammatory bowel disease. Released by activated dendritic cells, IL-23 interacts with IL-23 receptor (IL-23R) on Th17 cells, thus promoting intracellular signaling, a pivotal step in Th17-driven pro-inflammatory axis. Here, we aimed to block the binding of IL-23 cytokine to its cell-surface receptor by novel inhibitory protein binders targeted to the p19 subunit of human IL-23. To this goal, we used a combinatorial library derived from a scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived p19-targeted variants, called ILP binders. From 214 clones analyzed by ELISA, Western blot and DNA sequencing, 53 provided 35 different sequence variants that were further characterized. Using in silico docking in combination with cell-surface competition binding assay, we identified a group of inhibitory candidates that substantially diminished binding of recombinant p19 to the IL-23R on human monocytic THP-1 cells. Of these best p19-blockers, ILP030, ILP317 and ILP323 inhibited IL-23-driven expansion of IL-17-producing primary human CD4+ T-cells. Thus, these novel binders represent unique IL-23-targeted probes useful for IL-23/IL-23R epitope mapping studies and could be used for designing novel p19/IL-23-targeted anti-inflammatory biologics.

A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice.

Interleukin-12 family cytokines have emerged as critical regulators of immunity with some members (IL-12, IL-23) associated with disease pathogenesis while others (IL-27, IL-35) mitigate autoimmune diseases. Each IL-12 family member is comprised of an α and a ß chain, and chain-sharing is a key feature. Although four bona fide members have thus far been described, promiscuous chain-pairing between alpha (IL-23p19, IL-27p28, IL-12/IL-35p35) and beta (IL-12/IL-23p40, IL-27/IL-35Ebi3) subunits, predicts six possible heterodimeric IL-12 family cytokines. Here, we describe a new IL-12 member composed of IL-23p19 and Ebi3 heterodimer (IL-39) that is secreted by LPS-stimulated B cells and GL7(+) activated B cells of lupus-like mice. We further show that IL-39 mediates inflammatory responses through activation of STAT1/STAT3 in lupus-like mice. Taken together, our results show that IL-39 might contribute to immunopathogenic mechanisms of systemic lupus erythematosus, and could be used as a possible target for its treatment.

TLR3 drives IRF6-dependent IL-23p19 expression and p19/EBI3 heterodimer formation in keratinocytes.

Interferon regulatory factor (IRF) family members impart cell-type specificity to toll-like receptor (TLR) signalling, and we recently identified a role for IRF6 in TLR2 signalling in epithelial cells. TLR3 has a well-characterized role in wound healing in the skin, and here, we examined TLR3-dependent IRF6 functions in human keratinocytes. Primary keratinocytes responded robustly to the TLR3 agonist poly(IC) with upregulation of mRNAs for interferon-ß (IFN-ß), the interleukin-12 (IL-12) family member IL-23p19 and the chemokines IL-8 and chemokine (C-C motif) ligand 5 (CCL5). Silencing of IRF6 expression enhanced poly(IC)-inducible IFN-ß mRNA levels and inhibited poly(IC)-inducible IL-23p19 mRNA expression in primary keratinocytes. Consistent with these data, co-transfection of IRF6 increased poly(IC)-inducible IL-23p19 promoter activity, but inhibited poly(IC)-inducible IFN-ß promoter activity in reporter assays. Surprisingly, poly(IC) did not regulate IL-12p40 expression in keratinocytes, suggesting that TLR3-inducible IL-23p19 may have an IL-23-independent function in these cells. The only other IL-12 family member that was strongly poly(IC) inducible was EBI3, which has not been shown to heterodimerize with IL-23p19. Both co-immunoprecipitation and proximity ligation assays revealed that IL-23p19 and EBI3 interact in cells. Co-expression of IL-23p19 and EBI3, as compared with IL-23p19 alone, resulted in increased levels of secreted IL-23p19, implying a functional role for this heterodimer. In summary, we report that IRF6 regulates a subset of TLR3 responses in human keratinocytes, including the production of a novel IL-12 family heterodimer (p19/EBI3). We propose that the TLR3-IRF6-p19/EBI3 axis may regulate keratinocyte and/or immune cell functions in the context of cell damage and wound healing in the skin.

Downregulation of microRNA-107 in intestinal CD11c(+) myeloid cells in response to microbiota and proinflammatory cytokines increases IL-23p19 expression.

Commensal flora plays an important role in the development of the mucosal immune system and in maintaining intestinal homeostasis. However, the mechanisms involved in regulation of host-microbiota interaction are still not completely understood. In this study, we examined how microbiota and intestinal inflammatory conditions regulate host microRNA expression and observed lower microRNA-107 (miR-107) expression in the inflamed intestines of colitic mice, compared with that in normal control mice. miR-107 was predominantly reduced in epithelial cells and CD11c(+) myeloid cells including dendritic cells and macrophages in the inflamed intestines. We demonstrate that IL-6, IFN-γ, and TNF-α downregulated, whereas TGF-ß promoted, miR-107 expression. In addition, miR-107 expression was higher in the intestines of germ-free mice than in mice housed under specific pathogen-free conditions, and the presence of microbiota downregulated miR-107 expression in DCs and macrophages in a MyD88- and NF-κB-dependent manner. We determined that the ectopic expression of miR-107 specifically repressed the expression of IL-23p19, a key molecule in innate immune responses to commensal bacteria. We concluded that regulation of miR-107 by intestinal microbiota and proinflammatory cytokine serve as an important pathway for maintaining intestinal homeostasis.

CD40 ligand-triggered human dendritic cells mount interleukin-23 responses that are further enhanced by danger signals.

Interleukin (IL)-23 is a heterodimeric cytokine composed of the IL-23-specific subunit p19 and the p40 subunit which also constitutes part of IL-12. IL-23 propagates development of Th17 cells, a novel T cell subset which produces IL-17 but no interferon-gamma or IL-4. For both, IL-23 and IL-23-driven IL-17, a crucial role in autoimmune diseases such as experimental autoimmune encephalomyelitis, collagen-induced arthritis, and colitis is well accepted. Recent studies indicate that there is also a role for IL-23 and IL-17 in tumorigenesis, promoting tumor growth and vascularization, and affecting tumor incidence. We show that human CD14(+) peripheral blood monocyte-derived dendritic cells (DC), as used for clinical applications in anti-tumor immunization strategies, produce high amounts of IL-23. CD40-triggering of immature and mature DC but not of primary monocytes induced a rapid expression of high levels of IL-23, free p40, and minor levels of IL-12. Upon stimulation of DC subsets with a variety of different danger signals such as single stranded and double stranded RNA, bacterial components or viral infections, IL-23 expression pattern was analyzed. Interestingly, co-stimulation with CD40L enabled IL-23 expression by DC subsets towards danger signals to which they have been unresponsive upon single stimulation. Furthermore, we detected two novel splice variants of the IL-23-specific subunit p19 that could be associated with the regulation of IL-23 expression. Data presented here might have an impact on DC-based cancer vaccination strategies and contribute to a better understanding of the complex regulation of the heterodimeric cytokine IL-23.

Crystal structures of the pro-inflammatory cytokine interleukin-23 and its complex with a high-affinity neutralizing antibody.

Interleukin (IL)-23 is a pro-inflammatory cytokine playing a key role in the pathogenesis of several autoimmune and inflammatory diseases. We have determined the crystal structures of the heterodimeric p19-p40 IL-23 and its complex with the Fab (antigen-binding fragment) of a neutralizing antibody at 2.9 and 1.9 A, respectively. The IL-23 structure closely resembles that of IL-12. They share the common p40 subunit, and IL-23 p19 overlaps well with IL-12 p35. Along the hydrophilic heterodimeric interface, fewer charged residues are involved for IL-23 compared with IL-12. The binding site of the Fab is located exclusively on the p19 subunit, and comparison with published cytokine-receptor structures suggests that it overlaps with the IL-23 receptor binding site.

The structure of interleukin-23 reveals the molecular basis of p40 subunit sharing with interleukin-12.

Interleukin (IL)-23 is a recently identified member of the IL-12 family of heterodimeric cytokines that modulate subpopulations of T helper cells, and both IL-12 and IL-23 are attractive targets for therapy of autoimmune diseases. IL-23 is a binary complex of a four-helix bundle cytokine (p19) and a soluble class I cytokine receptor p40. IL-12 and IL-23 share p40 as an alpha-receptor subunit, yet show only 15% sequence homology between their four-helix cytokines p19 and p35, respectively, and signal through different combinations of shared receptors. In order to elucidate the structural basis of p40 sharing, we have determined a 2.3-A crystal structure of IL-23 for comparison to the previously determined structure of IL-12. The docking mode of p19 to p40 is altered compared to p35, deviating by a 'tilt' and 'roll' that results in an altered footprint of p40 on the A and D helices of the respective cytokines. Binding of p19 to p40 is mediated primarily by an arginine residue on helix D of p19 that forms an extensive charge and hydrogen-bonding network with residues at the base of a pocket on p40. This 'arginine pocket' is lined with an inner shell of hydrophobic interactions that are ringed by an outer shell of polar interactions. Comparative analysis indicates that the IL-23 and IL-12 complexes 'mimic' the network of interactions constituting the central arginine pocket despite p19 and p35 having limited sequence homology. The majority of the structural epitopes in the two complexes are composed of unique p19 and p35 pairwise contacts with common residues on p40. Thus, while the critical hotspot is maintained in the two complexes, the majority of the interfaces are structurally distinct and, therefore, provide a basis for the therapeutic targeting of IL-12 versus IL-23 heterodimer formation despite their use of a common receptor subunit.