Interleukin‐22 in urinary tract disease – new experimental directions

Interleukin (IL)‐22 is expressed by immune cells in the urinary tract and IL‐22 receptor is expressed in urothelium and renal tubule cells. IL‐22 can be specifically targeted in the urinary tract or conditionally depleted in mice and targeted therapeutically in humans.

Interleukin (IL)-22 is a cytokine produced by immune cells with bimodal functionality depending on the tissue it is expressed in and the context of its expression. IL-22 has antimicrobial properties and can limit bacterial infections but also drive pro-inflammatory responses that may have deleterious consequences for disease progression. Conversely, IL-22 can also promote epithelial cell repair, regeneration and restoration of mucosal barrier function. IL-22 may also alter the microbiota, which has broad implications for the maintenance of homeostasis at mucosal surfaces colonised by commensal microorganisms, such as the gastrointestinal or the urinary tract. A critical limitation in the exploration of IL-22 function, at sites such as in the urinary tract, is a lack of appropriate experimental tools. Studies using newly available, genetically modified reporter mice have led to the identification of the cellular source and location of IL-22, while Cre-lox expressing transgenic mice permit specific targeting of IL-22 in the urinary tract. With a greater understanding of the role of IL-22 in the urinary tract, repurposing of emerging novel human immunotherapies that either inhibit or activate IL-22 may create new opportunities for the treatment of urinary tract diseases.

IL-22 AT MUCOSAL SURFACES
IL-22 is an IL-10 family cytokine produced mainly by immune cells of the lymphoid lineage including group 3 innate lymphoid cells (ILC3), mucosal-associated invariant T cells (MAIT), natural killer T cells (NKT), T helper 22 cells (Th22) and cdT cells 1 (Figure 1a). However, macrophages or neutrophils can also produce IL-22 under certain circumstances, such as in a model of dextran sodium sulphate-induced colitis, in which IL-22-expressing neutrophils contribute to epithelial repair in the colon. 2 IL-22 signals through the IL-22 receptor (IL-22R), which is composed of two heterodimeric subunits, IL-22R1 (encoded by Il22ra1 in mice) and IL-10R2. 1 IL-22 has a high affinity for IL-22R1, and while it has no affinity for IL-10R2, the IL-22-IL-22R1 complex has a strong binding affinity for the IL-10R2 subunit (Figure 1a). IL-22R1 expression is restricted to renal tubular epithelial cells in mice. 3 Its expression in the bladder epithelium is unreported, but it is typically expressed in epithelial surfaces.
Indeed, IL-22 primarily targets nonhaematopoietic epithelial cells at mucosal barrier sites (e.g. lung, skin, gastrointestinal tract), where it promotes proliferation of epithelial cells and tissue regeneration after injury. 2 IL-22 regulates host microbial defences through induction of antimicrobial peptides, such as the Reg family of proteins during Citrobacter rodentium infection. 4 Studies in the gastrointestinal tract demonstrate that IL-22, expressed by ILCs, helps to maintain containment of commensal bacterial strains, and in the absence of these cells, specific microbes can breach the gastrointestinal barrier. 5 Given that IL-22 is produced at sites of inflammation, it may either mediate a physiologic response to repair local tissue damage or it may contribute to inflammatory lesions. 1 Therefore, caution is needed when interpreting the impact of IL-22 in disease models and there is a clear need for organ-and tissue-specific tools to accurately assess its function.

IL-22 IN THE KIDNEY
The role of IL-22 in the kidney is reasonably well studied (reviewed in Weidenbusch et al. 6 ). Infiltrating immune cells secrete IL-22, inducing progressive kidney remodelling following unilateral ureteral obstruction, which augments renal tubular epithelial integrity and epithelial barrier function. 7 IL-22 accelerates kidney regeneration and ameliorates renal ischaemiareperfusion injury, 3,8 suggesting, overall, that IL-22 function in the kidney is most likely tissue-protective and that restoring optimal IL-22 function may be beneficial in certain kidney diseases. Interestingly, however, endogenous IL-22 does not play a role in glomerulonephritis, 9 highlighting that IL-22 function in the kidney is disease-dependent.  reliance upon antibody specificity for the intracellular cytokine, although nonspecific fluorescent leakage of reporter mice is still a concern. In addition to identifying the cells producing IL-22, localisation of cytokineproducing cells is possible using 3-dimensional imaging of optically transparent IL-22 reporter mouse tissue in high resolution with 2-photon fluorescent microscopy. 11 Finally, reporter mice can be used for real-time in vivo imaging and tracking of IL-22 + cell migration into organs of the urinary tract.

NEW EXPERIMENTAL DIRECTIONS FOR
IL-22-deficient and IL-22 receptor-deficient mice are key to providing information on the impact of a lack of IL-22 signalling systemically. However, because of the complex multifaceted and contextspecific nature of IL-22 signalling, systemic deletion will likely provide confounding results. IL-22 is also critical for the maintenance and regulation of the gastrointestinal microbiota, 4,5 which is known to impact multiple diseases of the urinary tract both clinically and experimentally. To improve future experimental design and limit unintentional off-target effects of IL-22 systemically and on the microbiota, mice that specifically lack the IL-22 receptor in bladder or kidney epithelial cells should be created. This can be achieved by crossing IL-22 receptor-floxed ( fl/fl ) mice (Il22ra1 fl/fl , The Jackson Laboratory, USA) with the mouse uroplakin 2 (Upk2) promoter driving Cre recombinase ( Cre ) expression (Upk2 Cre , The Jackson Laboratory) in the bladder urothelium or by crossing the Il22ra1 fl/fl mouse with Cre recombinase under the control of the mouse cadherin 16 (Cdh16) promoter (Cdh16 Cre , The Jackson Laboratory) resulting in loss of IL-22 receptor expression in the renal tubules of adult mice (Figure 1b). Il22ra1 fl/fl mice could also be crossed with tamoxifen-induced (ERT2) Cre mice to control when IL-22 is disrupted in a given urinary tract disease model, with the caveat that tamoxifen-mediated deletion is not restricted solely to the urinary tract.

EMERGING IL-22 IMMUNOTHERAPIES -POTENTIAL FOR THE TREATMENT OF URINARY TRACT DISEASES
There are novel human immunotherapies in the clinical trial that either inhibit or enhance human IL-22 activity (Figure 1c). Clinical safety and pharmacokinetic studies are completed for both of these therapies, and both are in phase IIa trials for human diseases. 12,13 Fezakinumab (ILV-094, Pfizer) is a human anti-IL-22 monoclonal antibody that blocks IL-22 and was developed for the treatment of moderate-to-severe atopic dermatitis. 12 IL-22 IgG2-Fc (F-652, Generon BioMed) is a human recombinant IL-22 developed to promote IL-22-mediated tissue repair and regeneration in the context of graft-vs-host disease and alcoholic hepatitis. 13 To facilitate the translation of emerging immunotherapies targeting IL-22 in human urinary tract diseases, the critical first step will be to provide proof-ofprincipal studies in experimental mouse models of urinary tract disease, using tools that restrict the manipulation of IL-22 specifically to the urinary tract. In this way, it will be apparent whether the addition or inhibition of IL-22 is beneficial to the host response to urinary tract diseases.

CONCLUSION
Without a clearly defined role for IL-22 in the urinary tract using experimental approaches, it will be difficult to justify whether inhibition or enhancement of IL-22 function in humans with urinary tract disease will be of therapeutic benefit. Strategies to specifically target these emerging immunotherapies to the urinary tract will be required as targeting IL-22 systemically may have unwanted off-target effects on other systems; however, the bladder, for example, is uniquely suited for local delivery via catheterisation. Careful consideration of the impact of IL-22 therapies on the microbiota and its subsequent impact on urinary tract disease will also need to be carefully considered in future clinical trial design.