Interleukin 23 receptor: Expression and regulation in immune cells

The importance of IL‐23 and its specific receptor, IL‐23R, in the pathogenesis of several chronic inflammatory diseases has been established, but the underlying pathological mechanisms are not fully understood. This review focuses on IL‐23R expression and regulation in immune cells.


Introduction
IL-23 was identified in 2000 based on the homology of its IL-23p19 subunit to IL-12p35 [1].Early data indicated that IL-23 enhanced IFN-γ production by memory CD4 + T cells; however, it was later shown that IL-23 also induced IL-17 production by these cells [2].The observation that IL-23p19 knock-out mice but not IL-12p40 knock-out mice were protected from brain autoimmune inflammation, in the EAE model [3], provided the first evidence that IL-23 was a key player in inflammatory diseases.Genomewide association studies subsequently demonstrated an important role of the IL-23 signaling pathway in the pathogenesis of several chronic inflammatory diseases, including Crohn's disease [4], axial spondyloarthritis [5], and psoriasis (Pso) [6].IL-23 therefore appeared to be an excellent pharmacological target for inflammatory diseases, a notion that has been validated in the clinics by the successful treatment of Pso, psoriatic arthritis, and Crohn's disease with IL-23 blockers [7,8].IL-23 signals via a heterodimeric receptor (in this review: IL-23R) composed of a specific subunit, IL23R, and the IL12Rβ1 subunit, which is shared with the IL-12 receptor [9].IL-23 signaling has been studied in particular in the context of Th17 cell differentiation and function, but more recent data have demonstrated high frequencies of IL-23R-positive "innatelike" T-cell populations, in particular, mucosal-associated invariant T cells and γδ T cells [10].Very recent data have demon-strated the important role of IL-23 signaling in infectious conditions.Patients with autosomal recessive IL-23R deficiency display Mendelian susceptibility to mycobacterial disease due to impaired IFN-γ production and, less frequently, to chronic mucocutaneous candidiasis due to impaired IL-17A/F production [11].Here, we focus on two questions: what are the immune cells that express the IL23R and how is IL23R expression acquired.
The IL-23R: Structure and signaling IL-23 is a heterodimeric cytokine formed by two disulfide-bound subunits: p19 (encoded by IL23A) and p40 (encoded by IL12B) [1].The latter subunit is shared with another inflammatory cytokine, IL-12 [12,13].IL-23 and IL-12 receptors also share a common chain: IL-12Rβ1 pairs with the IL23R chain to form the IL-23 receptor complex (IL-23R) that confers responsiveness to IL-23, or with IL-12Rβ2 to form the IL-12 receptor complex [14].Only human IL-23 exhibits affinity for the human IL-23R [9] and the interaction of both p40 with IL-12Rβ1 and p19 is required for competent signaling of IL-23R [15,16].Recently, the crystal structure of the human IL-23R has been solved.Bloch et al proposed a sequential assembly mechanism of the receptor complex in which the IL23R chain first binds to IL-23p19 subunit exclusively via its N-terminal domain.This induces a conformational change of the IL-23 heterodimer that enables the p40 subunit to bind to IL-12Rβ1 with high affinity and to recruit the complex [17].IL-12Rβ1 and IL23R lack intrinsic kinase activity and signal through the recruitment of JAK tyrosine kinases.IL-12Rβ1 recruits TYK2, whereas IL23R interacts with JAK2 and the latter association is indispensable for IL-23 signaling [18,19] (Fig. 1).The activation of Tyk2 and Jak2 by IL-23R promotes mainly STAT3, but also STAT1, STAT4 and STAT5 phosphorylation [9].In contrast, IL-12Rβ2-mediated signaling promotes predominantly STAT4 phosphorylation [20], but can also induce STAT1 and STAT5 phosphorylation [21].IL-23 receptor complex signaling has been extensively reviewed in [16].

The IL23R locus
In humans, the IL23R gene is located on chromosome 1 and is directly adjacent to the IL12RB2 gene.IL-12 stimulation was shown to induce IL23R expression [22], raising interesting questions about the coordinated versus exclusive regulation of these two genes in differentiating T-helper cells.Several IL23R isoforms may be produced by alternative splicing, resulting in premature termination and frameshifts that can generate receptors lacking the signaling components, or isolated ectodomains with the potential to act as decoy receptors [23][24][25].The functional significance of these isoforms is unclear, although several have been detected in human NK and T cells, and some are preferentially expressed in lung carcinoma cells [23].

Functional effect of coding genetic variants
A large number of genetic variants have been described at the IL23R locus (see [26] for an extensive review).The single nucleotide polymorphism rs11209026 is the best characterized functionally.This single nucleotide polymorphism has been associated with protection against the development of several CIDs, including CD, axSpA, Pso, and psoriatic arthritis [4,[27][28][29][30][31][32].The minor allele G to A variant introduces an amino acid change (Arg381Gln) in the intracellular domain of the IL-23R, in the proximity of the JAK2 kinase binding site.Human T cells carrying the minor allele show reduced IL-23-dependent STAT3 phosphorylation and IL-17 production, and subjects carrying this variant have decreased frequencies of circulating Th17 or IL-23R + T cells [33][34][35].Coffre et al. found that CD4 + T cells from SpA patients carrying the minor allele express lower levels of genes associated with both Th1 and Th17 differentiation and function, including IL17, RORC, IFNG, IL12RB2, and IL23R itself, supporting the protective role of this genetic variant [36].
Reduced STAT3 activation was also observed with two other protective coding variants (G149R, V362I) when expressed in Hela cells.In this case, reduced functionality of the variants could also be linked to reduced IL23R protein maturation and stability [37].

Immune cell subsets expressing the IL-23R
To fully understand the role of IL-23 in protective immunity and in disease, it is important to identify the cells that are responsive to this cytokine.Many of these studies have been performed in mouse models.IL-23R was identified in 2002 [9] and has been mostly studied in the context of Th17 cells [2].However, the important role of IL-23 in complex CID may suggest a broader role of this cytokine and more recently the attention has shifted to other immune cells, including innate-like T lymphocytes.

IL-23R in conventional T cells
Parham et al. [9] in 2002 described the presence of IL23R transcripts in T helper 1 cell clones.IL-23 became a focus of attention when it was shown to promote a distinct subset of murine T-helper cells producing IL-17 [2].IL-23 supports the expansion and survival of established murine Th17 cells [38] and Th17 clones express higher levels of IL-23R transcripts and IL-12Rβ1 than Th1 cells [39].IL-23 can upregulate its own receptor in TCRstimulated human CD4 + T cells and increase IL-22 secretion but is not required for IL-17A production from stimulated memory CD4 + T cells [40].
Although murine double-positive thymocytes express constitutively Il23r, conventional T cells in the periphery show IL-23R expression only upon activation [41,42].
In vitro, differentiation of naïve CD4 + T cells with IL-12 and IL-21 also induces high levels of IL-23R, which confers pathogenicity to Th1-like cells during intestinal inflammation [48].IL-23 also expanded IL-17-producing tissue-resident memory CD4 + T cells (TRM17) in the skin of mice infected with C. albicans.IL-23 blockade in mice and in psoriasis patients leads to depletion of skin TRM17 cells [49] T-follicular helper cells (Tfh) isolated from an EAE mouse model also expressed IL-23R and IL-17 after restimulation in vitro [38].IL-23 signaling induced a Tfh-IL-17 phenotype and promoted AID-mediated class-switch recombination [50].
A subset of regulatory T cells that expresses the transcription factor RORγt may also be responsive to IL-23 [51], although IL-23 signaling in this population may have different outcomes in different experimental settings, suggesting the need for further investigation.Murine intestinal Treg express higher levels of Il23r than Treg in other tissues [52], and expression of RORγt and IL-23R depends on the microbiota strain [53,54].In a colitis model, IL-23R signaling decreases Treg frequency in the gut and impairs their suppressive functions [52,55], possibly because of reduced expression of Foxp3 [56].However, Yang et al. [51] described the enhanced suppressive activity of the Foxp3 + RORγt + T-cell population in a colitis model, a difference that may depend on the different donor strains used for the transfer.IL-23 signaling also had a positive effect on Treg expansion in skin disease models, where IL-23 induced accumulation of Treg cells that express RORγt and IL-17, and treatment with anti-IL-23p19 reduced the number of skin Th17-like-Tregs [57].
CD4 + FOXP3 + T cells expressing IL23R along with RORC and IL17F were also detected in a Th17-like T-regulatory cell cluster in humans by single-cell RNA profiling [58].
IL-23 may also affect the function of type 1 regulatory T cells in inflammatory bowel disease patients, decreasing their IL-10 production [59].
Human memory CD161 + CD8 + T cells also respond to IL-23 [62].IL23R + IL-17A + CD8 + αβ T cells are enriched in synovial fluid from psoriatic arthritis patients, suggesting that these cells may support chronic inflammation [63].Similarly to what was observed for Th1 cells, enrichment of T-bet + IL-23R + CD8 + T cells was observed in the intestine and blood of Graft-versus-Host Disease patients [64].

IL-23R in innate-like T cells
Mucosal-associated invariant T cells (MAIT) constitutively express the IL-23R in both mice and humans, where it can be already detected in cord blood MAIT cells [65].Up to 85% of lung MAIT cells expressed IL-23R in naïve reporter mice [66].Il23r expression is further increased by TCR stimulation and addition of cytokines, such as IL-12, IL-18, and IL-15 [67].In mouse models, the expression of either T-bet or RORγt distinguishes MAIT1 from MAIT17 cells, the latter preferentially expressing Il23r [68].IL-23 signaling can also expand a MAIT-17/1 phenotype [66].These distinct subsets cannot be identified in humans, where IL23R constitutes a "core" gene for all MAIT cells [69][70][71].The expression of IL23R is higher in human tissue-resident (mucosal, lung, skin, liver and entheses) MAIT cells [10,66], [72][73][74].The enrichment of IL-23R expressing MAIT cells in synovial fluid from psoriatic arthritis and rheumatoid arthritis patients [75] and in peripheral blood of spondyloarthritis patients [10] suggests a role for IL-23R + MAIT cells in the pathogenesis of CID.
It has been shown that both murine and human γδ T cells constitutively express IL-23R [76,77].The expression of Il23r in murine γδ T cells has been extensively studied.γδ T cells constitute the major IL-23R-expressing subset in lymphoid organs of naïve mice [61,78] and produce IL-17 in response to IL-23 stimulation [76,79,80].Spleen γδ T cells from naive mice show low expression of Il23r mRNA and protein, but express IL-23R after antigen stimulation in vivo and have immunosuppressive activity on Th17 cells [81].Murine IL-23R + /IL-1R + γδ T cells were found to display a focused TCR repertoire, supporting a role for antigen recognition in the expansion of this subset [53].In the mouse, IL-23 responsive γδ T cells belong mainly to the Vγ4 + and Vγ6 + subsets [82] and may play a role in protection against infections [79,83].
In humans, IL-23R may be preferentially expressed on Vγ9Vδ2 cells that also express high levels of IFNγ and cytotoxic molecules [77].Human IL23R + γδ T cells may also play a role in inflammatory disease.Entheseal IL23R + γδ T cells express high levels of IL23R transcripts [84], and IL-23R + cells are enriched in the joints of spondylarthritis patients [85].IL-23R + γδ T cells were also increased in children with primary nephrotic syndrome [86].Like their murine counterpart, human γδ T cells were shown to produce IL-17 and IL-22 in response to IL-23 stimulation [85].
More recently, a T-cell population co-expressing γδ and αβ receptors were described in mice and humans.These cells expressed high levels of IL-23R and displayed a hyperinflammatory phenotype in response to IL-23 [87].
Murine and human NKT are other populations that express the Il-23R and can produce IL-17 (and IL-21) in response to IL-23 stimulation [62,85,88].In humans, a small fraction of NKT cells express IL-23R in peripheral blood.However, IL-23R + NKT cells are enriched in the synovial fluid of patients with spondylarthritis [85].IL-23 synergizes with IL-18 to promote IFNγ production in human NKT cells and to stimulate the proliferation of NKT and NK cells [89].

IL-23R in other innate lymphoid cells
Innate lymphoid cells (ILC) are mostly tissue-resident lymphoid cells that lack an antigen-specific receptor.ILCs were also found to express Rorc and Il23r and to be involved in the development of innate colitis [69,[90][91][92].ILCs can be assigned to different subsets (ILC1, ILC2, and ILC3) that mirror the Th1, Th2, and Th17 subsets of conventional T cells.The IL-23R is mainly expressed in the ILC3 subset, both in mice [69,70] and in humans [93,94], although IL-23 enhanced the differentiation and growth of both human ILC1 and ILC3 [95].
ILC3 cells in synovial tissue of spondylarthritis patients express RORC and IL23R and respond to IL-23 by expressing IL22 and CSF2, but not IL17A [93].

IL-23R in NK cells
IL23R transcripts were first detected in human NK clones and cell lines by Parham et al. [9].IL-23 treatment was shown to induce human NK activation and secretion of IFNγ [96].

IL-23R in B cells
B cells constitute about 10% of eGFP-IL-23R + cells in mouse spleen [61], but the role of IL-23 in this population has been poorly studied.
Primary B cells from healthy humans do not express IL-23R, which is upregulated in acute lymphoblastic leukemia B cells [97].IL-23 signaling in acute lymphoblastic leukemia B-cells induced apoptosis and inhibited proliferation, exerting antileukemic activity [97].Expression of the IL23R chain, in the absence of IL-12Rb1, was also reported in chronic lymphocytic leukemia B cells, and correlated with tumor progression [98].The role of IL-23 signaling in normal B-cell function is poorly investigated.Cell surface staining for IL-23R was also detected on plasma cells from healthy donors, which responded to IL-23 by increasing IgM secretion [99].

IL-23R in myeloid cells
Very little is known about IL-23R expression and function in myeloid cells.Parham et al. [9] found that human monocytes and dendritic cells (DC) express negligible levels of IL23R, however murine bone-marrow-derived (but not peritoneal) macrophages stimulated with LPS and IL-10 expressed Il23r in the absence of detectable Il12rb1, which could be induced by IFNγ stimulation.Inflammatory macrophages isolated from brains of mice with EAE, but not from healthy mice, expressed Il23r [3], and in a house dust mite model of airway inflammation, IL-23R positive cells in the lung were mainly macrophages and CD11 + dendritic cells [100].These data suggest that IL-23R in myeloid cells may be expressed only in particular conditions, consistent with the lack of expression by unstimulated human myeloid cells reported in databases [101,102].Surface expression of IL-23R was described for human monocyte-derived macrophages, which responded to IL-23 treatment with increased secretion of proinflammatory cytokines [103,104].Mycobacterial infection also increased the expression of IL-23R and IL-23 in human and murine macrophages [105].
Low levels of IL23R mRNA have been reported in human dendritic cells [9] and IL-23R levels were elevated in monocytederived DC from patients with HBV-related liver failure [106].IL-23 was described to have adjuvant effects on DC priming ability [107]; however, IL-23 signaling in DCs has not been further investigated.
Similarly, activated granulocytes have been reported to express IL-23R in inflammatory murine models of colitis or aspergillosis [108,109], but confirmation of the role of IL-23 in these cells is lacking.
A challenge for the characterization of IL-23R expression is the scarcity of reliable anti-IL23R antibodies and the fact that IL-23R is expressed only at low frequencies in most resting cells.

Transcriptional regulation of IL-23R
The transcriptional mechanisms regulating IL-23R expression have been mainly addressed in the context of murine Th17 cell differentiation (Table 1, Fig. 2).
Transcription factors of the AP1 family play an important role in Th17 determination and in Il23r transcription.In particular, JunB binds cooperatively with BATF and IRF4 to an AP1-IRF composite motif [110,111], which is also observed at the Il23r locus [112].BATF binding may be determinant to start IL23r expression, as it was shown to occur also at sites of closed chromatin, and was proposed to act as a pioneer factor in inducing chromatin remodeling by recruiting the boundary transcription factor CTCF [113].Further mechanistic studies will be required to precisely define the role of this pathway in inducing IL-23R expression.
Several of the Th17-inducing transcription factors may also affect IL23r transcription indirectly, for example by acting on the expression of Rorγt, which also binds to the Il23r locus and regulates its activation [114,115].Other factors, such as T-bet, ChIP-seq Knock-out EMSA [110] Irf4 Batf JunB Binding of Batf, Irf4, and Junb at Il23r promoter, intron 3, 6, and 7 was detected by ChIP seq in Th17 differentiating cells.
ChIP-seq [127] Rorγt Foxp3 Rorγt binds to the Il23r promoter and to introns 3 and 6 in Th17 cells.Foxp3 binding was observed in iTregs at intron 6 of Il23r.
ChIP-seq [115] p300 JunB Binding of JunB and p300 to the promoter and the introns 3, 6, and 7 of the Il23r locus.

Rbpj
Naive CD4 + T cells were sorted from CD4 Cre RBPJ flox/flox mice and differentiated under the Th17 polarizing condition.In Th17 cells, Rbpj binds the Il23r promoter, to a site (1100 bp upstream of the TSS) distinct from those bound by other factors indicated in this table.
ChIP-PCR [130] Batf Ctcf Ets1 Med12 Batf was shown to bind the Il23r locus (promoter and introns 2,3 and 6 in Th17 cells) and to pioneer the opening of the chromatin.Batf-induced binding of Ctcf, Ets1, and Med12 to the Il23r locus.
ChIP-seq Knock-out [114] Tbet Direct binding of T-bet to the Il23r promoter was shown in splenocytes from Va2.3/Vb8.2TCR-transgenic mice activated with myelin basic protein peptide.The binding induces decrease in Il23r expression.
ChIP-PCR Knock-down [116] Tbet T-bet binding was also observed at Il23r third intron in a transfer model of colitis and negatively regulates its expression. [117] Tbet T-bet binds to Il23r locus (introns 3 and 6) in murine Th1 cells.[118]
ChIP-seq [121] Clock Binding of Clock at the Il23r promoter in splenic γ/δ + T cells isolated from wild-type mice.
ChIP-seq [120] BRD4 BRD4 binds to the IL23R locus in CD4 + T cells activated through the TCR.A selective BET inhibitor reduced Il-23r expression in vitro and in a graft-versus-host disease model.

ChIP-seq
Inhibitor treatment [132] may display context-dependent effects on IL-23R expression: Tbet was shown to increase Il23r levels in an EAE model by direct binding to the Il23r promoter but to repress Il23r in Th17 cells in a colitis model [116,117].T-bet was reported to bind the Il23r gene also in Th1 cells, without affecting IL23R expression [118].FOSL proteins play a complex role in the regulation of Th17 differentiation and IL-23R expression.In the mouse, FOSL2 controlled the inflammatory response by reducing the expression of proinflammatory cytokines such as IL17A and IL17F; however, FOSL2 was also important for Th17 maintenance and survival since it increased Il23r, Il21, and Il12rb1 expression [112].Overexpression of FOSL1 and JUNB together promotes murine Th17 differentiation, increasing the expression of Il17, Rorc, and Il21.Consistently, inhibiting Fosl1 and Junb expression in vivo attenuated disease in an arthritis model [119].Overexpression of FOSL1 in CD4 + T cells from healthy donors led to an increase in IL-17-producing cells and IL-17A secretion [119].A recent study in humans showed that FOSL1, FOSL2, and BATF bind at the same site in the IL23R locus and at other Th17-associated genes.However, the study described a positive regulatory role of BATF but a negative role of FOSL proteins, since FOSL1 and/or FOSL2 overexpression decreased IL23R transcription.The authors suggested that BATF and FOSL proteins compete for binding to transcriptional partners and target genes [120].
Stat3 was also shown to directly regulate Il23r expression [45,121].Jain et al. [45] demonstrated that Il23r was downregulated in naive CD4 + T cells from STAT3 conditional KO mice, cultured under Th17 polarizing condition.Il23r expression was not affected in STAT1 KO mice.

Posttranscriptional regulation of IL23R expression
IL23R expression can be controlled posttranscriptionally by several miRs, adding another layer of complexity to IL-23R regulation.
Let-7f targets IL23R 3'UTR and its overexpression decreases IL23R levels in human CD4 + T and K562 cells.Let-7f levels can be regulated by estrogen signaling in humans and in mice.Th17 cells from female asthma patients had lower let-7f expression and higher IL-23R expression than Th17 from male patients, and let-7f inhibition further increased IL-23R levels.Administration of estrogen and progesterone to ovariectomized mice decreased let-7f levels and increased IL-23R and IL-17 expression [122].Consistently, Th17 cells from Esr −/− mice showed increased levels of let-7f and reduced IL-17 and IL-23R expression [123].Overexpression of let-7g in activated CD4 + T cells protected from severe disease in a mouse model of multiple sclerosis, reducing the secretion of proinflammatory cytokines and Il23r expression in Th17 cells [124].
The inhibitory effect of miR146b-5p on IL-23R cell surface expression in chronic lymphocytic leukemia cells is mediated by the downregulation of the IL-12Rb1 chain of the receptor [126].

Conclusion and perspectives
In the past decade, our understanding of the role of the IL-23/IL-23R pathway in health and disease has greatly advanced, resulting in the implementation of novel therapeutics of great efficacy for immune-mediated inflammatory diseases, such as psoriasis and psoriatic arthritis.However, much remains to be learned about IL-23 biological activity in health and disease.
The majority of the studies characterizing the IL-23/IL-23R pathway have focused on T lymphocytes, and primarily, on conventional CD4 + T cells.The biological functions of this pathway in other immune cells, such as innate lymphocytes, B cells, and myeloid subsets need to be further explored.The data available today indicate that most immune cells need to be activated in order to acquire responsiveness to IL-23.The stimuli and the transcriptional pathways that induce the IL-23R in the different cell types are variable, depend on the cell type and the environmental context, and are still poorly characterized.Additionally, future efforts should also focus on understanding the downstream effects of IL-23 signaling in the different cell types, and their role in disease settings.

Figure 1 .
Figure 1.IL-23R structure and expression on immune cell subsets.(a) Schematic representation of the IL-23 receptor complex and the IL-12 receptor complex.Abbreviations: Ig-like domain, immunoglobulin-like domain; CHR, cytokine-binding homology region; FnIII, Fibronectin III.(b) Expression of IL-23R in the main immune cell populations.Most studies have focused on T-cell populations.Created with BioRender.com

Table 1 .
Transcription factors that bind to the murine and human loci encoding IL-23R.seq analysis on murine naïve CD4 + cells differentiated under Th17 polarizing condition demonstrates binding of Irf4, Batf, and JunB to the AICEs motifs present in the promoter and intron 3 of the Il23r locus.

Figure 2 .
Figure 2. Transcription factors binding to the Il23r locus.(a) Alignment of the human and murine IL23R loci, obtained with the VISTA browser.Regions with >70% identity are marked in color: Conserved noncoding sequences (CNS) in salmon, 5 and 3 -UTR in light blue, and exons in purple.(b) Epigenetic marks tracks were obtained from the GEO database with the following GSM ID: ATAcseq, GSM4731624; H3K4me1, GSM4291301; H3K27ac, GSM4291309; and H3K4me3, GSM4291303.(c) Indication of transcription factors bound to the murine and to the human IL23R locus obtained from published ChIP data.