Humans are triploblastic and thus, develop a tri-laminar embryo upon gastrulation, consisting of ectoderm, endoderm, and mesoderm. Several groups have cultured PSCs in a feeder free matrix-coated surface and promoted differentiation by adding growth factors and inhibitors to this 2D system, while other groups applied inductive media to a 3D system, after the generation of a sphere of PSC cells known as an embryoid body (EB). Protocols applying defined time periods and concentrations of exposure to inductive agents have allowed for the differentiation of endo-, ecto-, or mesodermal lineages and specific cell types. For example, activin signaling leads to 80% efficient induction of SOX17+/GSC+/FOXA2+/MIXL1+ definitive endoderm in hESC cultures after 5 days of differentiation in 100 ng/ml activin A (D'Amour et al., 2005). In another seminal study, sequential treatment of high-density undifferentiated monolayer hESC cultures with activin A for 24 h and bone morphogenetic protein 4 (BMP4) for 4 days consistently yielded >30% cardiomyocytes (Laflamme et al., 2007). For neural induction of hESCs, the growth factors insulin, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were added in a chemically defined medium giving rise to up to 90% PAX6+ neural progenitors (Joannides et al., 2007). These studies have proved that while a spontaneously differentiating mass of PSC can generate varying proportions of endo- and ecto- and mesoderm, targeting signaling pathways prominent in the development of an organ of interest in a temporal fashion can promote that particular lineage. Over the years, researchers have tested various combinations of cell signaling modifiers to achieve enrichment of tissue-specific cell types in a heterogenous population of differentiated PSC progeny (reviewed by Murry and Keller, 2008). Moreover, the level of enrichment varies with the iPSC or ESC used. iPSC that are reprogrammed from a somatic cell type originating from an organ and are differentiated to a cell type of the same organ, show higher efficiencies owing to epigenetic memory of the source (Hiler et al., 2015). This highlights the need for standardization of protocols with certain PSC lines for certain lineages.

Unlike other organs, few protocols have been established for the kidney. The kidney exhibits a remarkable architectural complexity coupled with the presence of at least 26 different specialized cells (Al-Awqati and Oliver, 2002). The kidney is mesodermal in origin, arising from the intermediate mesoderm (IM). Arising from IM, the ureteric bud (UB) interacts with an adjacent cell population, the metanephric mesenchyme (MM). Through reciprocal interactions, the ureteric bud will generate the ureteric tree giving rise to the ureter, renal pelvis and collecting ducts, while progenitors within the metanephric mesenchyme will generate nephrons (Grobstein, 1953; Saxén and Sariola, 1987). Based on earlier studies that highlighted important molecules and pathways that drive mesodermal and nephron differentiation in model organisms, initial studies of differentiation toward the renal lineage were performed on mouse ESC EBs treated with media containing serum together with multiple combinations of factors including activin A, BMP4, BMP7, retinoic acid (RA), leukemia inhibiting factor (LIF), and glial-derived neurotrophic factor (GDNF) or UB-derived conditioned media. These protocols led to the generation of cells expressing markers of differentiation, e.g., Pax2 (kidney tubules), Aquaporin-2 (collecting duct principal cells), Wt1 (metanephric mesenchyme and podocytes), or Ksp-Cadherin (distal nephron tubules), within EBs, which provided evidence of successful renal lineage induction (Kobayashi et al., 2005; Bruce et al., 2007; Vigneau et al., 2007; Morizane et al., 2009; Ren et al., 2010; Nishikawa et al., 2012). Although a renal identity was achieved in the examples mentioned before, the desired cell types could not be isolated owing to low and varying frequencies of occurrence and their functionality was not demonstrated.

Meanwhile, genetic lineage tracing demonstrated that the induced Six2-expressing cap mesenchyme represents a nephron progenitor population that gives rise to all cell types of the nephron (Kobayashi et al., 2008). Also, the intermediate mesodermal origin of kidney cells was confirmed when Mugford et al., used molecular fate mapping to demonstrate that the majority of cell types within the metanephric kidney arise from an Osr1-expressing population within the intermediate mesoderm (Mugford et al., 2008). These new findings were considered in differentiation protocols to distinguish the exact mesodermal cell sub-type required to enrich cultures of PSC-derived renal progeny. For instance, Mae et al developed a robust protocol using activin A and Wnt-agonist CHIR99021 for 2 days and sequential treatment with BMP7 and CHIR99021 for 8 days to obtain 90% OSR1+ cells (Mae et al., 2013). Despite such an efficient protocol, the dependence on OSR1 as a population identifier created ambiguity, since even though OSR1 is expressed in the intermediate mesoderm, it is also expressed in the earlier mesoderm prior its subdivision into paraxial and intermediate domains (Guillaume et al., 2009). Moreover, OSR1 is expressed in both the intermediate mesoderm and lateral plate (James and Schultheiss, 2003; Wilm et al., 2004).

Additional protocols were developed to induce different cell types within the nephron. Human PSC derived podocytes expressing podocin, nephrin, and synaptopodin were generated from EBs using treatment with Activin A, RA, and BMP7 and plating on gelatin (Song et al., 2012). In another study, ∼30% AQP1+ proximal tubule cells were obtained by treating a monolayer PSC culture with media containing RA, BMP2, and BMP7 for 20 days (Narayanan et al., 2013). Recent reports demonstrated stepwise induction of UB and/or MM through systematic induction of primitive streak alone, followed by intermediate mesodermal specification (Xia et al., 2013, 2014; Lam et al., 2014; Taguchi et al., 2014; Takasato et al., 2014). These studies performed thorough characterization of cell types obtained at every stage, focusing on obtaining PAX2+ GATA3+ LHX1+ UB cells (Xia et al., 2013) and SIX2+ PAX2+ OSR1+−GDNF+ HOX11+ WT1+ MM cells (Taguchi et al., 2014). Takasato et al. were able to generate UB and MM between 14 and 18 days, whereas Lam et al generated SIX2+ SALL1+ WT1+ cap mesenchyme by 8 days and showed the potency of PAX2+ LHX1+ imtermediate mesodermal cells to generate tubule structures that express Lotus lectin and Ksp-Cadherin after 9 days of differentiation (Lam et al., 2014; Takasato et al., 2014).

Together, these important studies provided proof of principle that most if not all components of the kidney can be induced from PCS. The PSC-derived UB cells can be utilized to generate a ureteric tree or PSC-derived MM cells can be coaxed to produce S-shaped bodies that undergo proximal distal patterning initiating tubulogenesis, giving rise to fetal nephrons in culture. It would also be interesting to use cells derived from the Xia protocol to generate UB and Taguchi protocol to obtain MM and investigate if they interact in a similar manner as these cell types do in vivo. Since most kidney diseases involve the damage and loss of podocytes or hypertrophy of tubular epithelial cells, these cell types have a high priority of being derived. On the other hand, the big picture of nephron reconstruction requires other specialized cells including mesangial cells, glomerular endothelial cells, epithelial cells of the loop of Henle, principal cells and intercalated cells that have not yet been procured from PSCs. Induction of terminal differentiation, recapitulation of the architectural context and building of functional nephrons of the kidney are key challenges to be mastered.

## Morphogenesis and Patterning

The development of an organism involves not only differentiation of cells, but also their morphogenesis and appropriate patterning to form the architectural context of tissues and organs (Gilbert, 2000). Essentially, cells need to communicate with each other and their microenvironment, by means of growth factors, morphogens, cell adhesion molecules, and mechanoreceptors. The elegant experiments of Holtfreter and Townes studying Xenopus embryo re-aggregation patterns and organization indicated that a whole system of attraction and repulsion phenomena operates between various cell types during development and that information on this system will yield valuable information concerning the segregation of tissues during organogenesis. Holtfreter used the term "tissue affinity" (Gewebeaffinität) to describe this force (Holtfreter, 1939, 1944; Townes and Holtfreter, 1955). Steinberg's differential adhesion hypothesis supported the ideas of Holtfreter with thermodynamic principles (Steinberg, 1970) providing a partial explanation of the self-organizing properties of tissues. Embryonic tissue re-aggregation and adult organ-derived cells have demonstrated this property, e.g., in liver, lung, eye, brain, and thyroid gland (Weiss and Taylor, 1960; Grover, 1961a,b; Ishii, 1966; Hilfer et al., 1968; Stefanelli et al., 1977). The observation of such tissue affinity and self-assembly among PSC-derived progeny flags the success of recapitulating natural embryological behavior in vitro.

Toward this extent, one of the first reports of such a phenomenon in mouse PSC-differentiated cells came from the Sasai lab. The appearance of multi-layered cortical neuroepithelium in serum-free embryoid body-like aggregates in suspension culture, showed a similar pattern as observed during development. This remarkable finding could also be reproduced in hESCs (Eiraku et al., 2008). Further examples of selforganizing tissue morphogenesis were the generation of optic cups from mouse ESCs (Eiraku et al., 2011) and of neural

retina from human ESCs (Nakano et al., 2012). Lancaster et al., developed 3-dimensional self-organizing cerebral organoids from human ESCs, which develop distinct interconnected brain regions (Lancaster et al., 2013). Spence et al. developed self-organizing intestinal organoids from human ESCs, which recapitulated cell types and the stem cell compartment of the intestine (Spence et al., 2011).

Partial self-organization of mouse embryonic kidney cells upon their re-aggregation after dissociation was first achieved by Unbekandt and Davies (2010). The novelty of this system was the introduction of a ROCK inhibitor, which prevented the dissociation-induced apoptosis within single cell suspensions and facilitated a significant recovery of re-aggregated tissues. The Unbekant re-aggregation method has since then been used as a test system to check the capacity of cells (e.g., PSC-derived cells) to integrate into forming tubules or glomeruli of the mouse nephron. While this method proves the property of test cells to contribute to kidney formation, it cannot provide proof of self-organization of PSC-derived renal progenitors. To this end, cases of kidney organogenesis from PSC have been reported by Takasato et al., where 18 days of differentiation of PSC seeded initially on matrigel, develop an ECAD+ ureteric epithelium surrounded by clumps of SIX2+ WT1+ PAX2+ MM cells or JAG1+ CDH6+ renal vesicles (Takasato et al., 2014). Lam et al. also observed appearance of tubulelike structures positive for Lotus lectin (a proximal tubule marker) from SIX2+ cap mesenchyme cell cultures, obtained on day 7 of their differentiation procedure, upon treatment with CHIR99021 (Lam et al., 2014). This was reminiscent of induced metanephric mesenchyme that responds to Wnt signals to undergo mesenchymal-epithelial transition and form renal vesicles in vivo (Park et al., 2007; Schmidt-Ott et al., 2007). Meanwhile, the group of Nishinakamura had also obtained evidence of a slightly different nature. They used PSC in the form of EBs for a differentiation protocol that took 8.5 days in mouse ESCs and 14 days in human iPSCs, resulting in SIX2+ WT1+ SALL1+ PAX2+ MM cells that could give rise to tubules and podocytes when induced by mouse embryonic spinal cord (Taguchi et al., 2014). These studies are evidence that a systematic mirroring of embryonic kidney development in PSC derivatives can lead to the formation of organo-typical structures, as summarized in **Table 1**. This brings us a step closer to develop nephrons in vitro, as these structures can be coaxed under concentration gradients of growth factors to boost tubulogeneis. Yet, there is clearly a need to enhance protocols to achieve full maturation of nephrons that have on one hand a filtering unit and on the other hand an optimal spatial orientation of tubules and trigger their functionality in terms of electrolyte transport.

A niche for organogenesis was created in post-blastocyst mutant mouse embryos that are genetically precluded from developing a particular organ by Kobayashi et al. The group generated a chimeric animal where injected PSC-derived cells colonized this developmental niche and compensated for the developmental defect to form a donor-induced organ in vivo. They injected rat wild-type PSCs into Pdx1−/− mouse blastocysts, generating normally functioning rat pancreas in Pdx1−/− mice. These data constituted proof of principle for interspecific blastocyst complementation and for generation of organs derived from donor PSCs using a xenogenic environment in vivo (Kobayashi et al., 2010). On similar lines, the same group injected mouse PSCs in Sall1-deficient mice, since Sall1 is expressed in the metanephric mesenchyme–derived structures in the developing kidney. The kidneys of Sall1−/− chimeric mice were almost entirely composed of EGFP-marked mouse iPSCderived cells. These iPSC-derived kidneys of Sall1−/− chimeric mice were grossly normal in shape and size. Bladders of these mice were filled with urine, indicating that the iPS-derived kidneys were functional and appropriately connected to the lower urinary tract (Usui et al., 2012). It can be anticipated that human organs can similarly be developed in animal hosts.

## REVISITING RENAL DEVELOPMENT—MATURATION OF NEPHRONS

Kidney organoids obtained in vitro are comparable to E12.5– E13.5 metanephric mouse kidneys, where in, the ureteric tree

has branched and the cap mesenchyme undergoes mesenchymalto-epithelial transition into renal vesicles, which elongate and undergo patterning to form comma-shaped and S-shaped bodies that are scattered in the cortex. In parallel, the renal stroma, derived from Foxd1+ cells and Flk+ cell derived vasculature, are also contributing to development of kidney architecture (Hatini et al., 1996; Robert et al., 1996; Abrahamson et al., 1998). Formation of renal vasculature is a combination of angiogenesis and vasculogenesis. The lateral branch of the aorta invades the kidney at E12.5 and becomes the renal artery that has 3–4 branches by E13.5. Around E17.5, the arterial tree extends until the cortex due to strong VEGFA signals from developing podocytes in the glomerular zone, leading to the formation of afferent arterioles. Although it has been observed that at around E13-14, endothelial cells migrate into the cleft of glomeruli to form a capillary network, the source of these cells remains elusive (Herzlinger and Hurtado, 2014). Lineage tracing of Tie1/LacZ E11 metanephroi transplanted into a nephrogenic cortex has shown that endothelial precursors exist before the onset of nephrogenesis since the donor tissue showed transgene-expression in glomerular capillary loops (Loughna et al., 1997). A cKIT+ cell population originating from the aortagonad-mesonephros hemangioblasts has also been observed during E10.5–E11.5 that are distinct from Foxd1+ stromal cells (Schmidt-Ott et al., 2006), the fate of these cells has not been examined. Foxd1+ stromal derivatives differentiate into peritubular capillary endothelial cells that can be observed around E18.5 (Sims-Lucas et al., 2013) and also secrete factors required for normal nephron and vascular differentiation (Das et al., 2013; Hum et al., 2014).

Rymer et al. performed in utero embryonic intra-cardiac injection of tomato lectin (TL) to label perfused blood vessels at different stages of development (E11.5–E17). They found that perfused blood vessels were closely linked to mature nephron structures, while the nephrogenic zone did not show evidence of blood flow. This suggests that terminal differentiation is linked to an oxygen-rich environment, while multipotent nephron progenitors exist under low oxygen conditions (Rymer et al., 2014). This finding is potentially important with respect to tissue engineering, since strategies to establish blood flow and oxygenation within organoids may provide a critical step toward generating mature renal tissues.

## BRINGING BLOOD INTO AN ORGANOID—VASCULARIZATION STRATEGIES

PSC-derived tissue specific cell types irrespective of being generated in a 2- or 3-dimensional culture are functionally immature despite having morphological properties of the desired cell type. Essential functions of the kidney, including glomerular filtration, tubular reabsorption, or hormone secretion, essentially dependent on the presence of a vasculature. In addition, the diffusion limit of oxygen in organ cultures is around 150µm, while the organoids that are currently being developed range from 1 to 2 mm, raising an issue of oxygen deprivation in the core regions gradually leading to tissue decay.

Learning from embryonic vascular development, each organ has progenitors distributed in the primordia that develop into the vasculature of the respective organ. Growth factors acting on these progenitors, including VEGF (Tufro et al., 1999), stem cell factor (SCF) (Schmidt-Ott et al., 2006), and Ang1 (Loughna et al., 1997; Woolf et al., 2009) can be supplemented into culture media to enrich the differentiated progeny. Xinaris et al. used 5-day-organoids derived from E11.5 mouse kidney, dissociated into single cells, and used a protocol involving VEGF treatment and transplantation of these cells under the kidney capsule of athymic rats. This facilitated vascularization with blood vessels from donor (mouse) origin, in vivo viability for at least 3 weeks, and glomerular maturation characterized by formation of capillary walls with fenestrated endothelium, podocytes with foot processes and slit diaphragms. Moreover, FITC-conjugated albumin injections were recovered in proximal tubules suggesting successful glomerular filtration and tubular reabsorption in these allografts (Xinaris et al., 2012).

Another approach that could enhance vascularization of organoids that are considerably thick is in vitro addition of vascular and perivascular cells. Formation of micro-capillary like structures by endothelial cells prior to transplantation accelerates anastomosis with host vasculature. The first report of in vitro prevascularized tissues was the generation of a skin equivalent by culturing dermal fibroblasts, keratinocytes and human umblical veil endothelial cells (HUVECs) (Black et al., 1998; Tonello et al., 2003). Stevens et al constructed "tri-cell" cardiac patches containing hESC-derived cardiomyocytes, HUVECs, and mouse embryonic fibroblasts (MEFs) in 1:1:0.5 ratios, respectively (cardio-HUVEC-MEF patches). In sharp contrast to cardio-HUVEC patches, addition of MEFs to human cardiomyocytes and HUVECs resulted in the formation of human CD31-positive endothelial cell networks that morphologically resembled a vascular plexus (Stevens et al., 2009). Similar success has been reported in generating a human liver bud where PSC-derived hepatic progenitors, human mesenchymal stromal cells(hMSC) and HUVECs were co-cultured in vitro prior to cranial transplantation in immune-deficient mice (Takebe et al., 2013). As a development to the previous study, Takebe et al. also developed kidney buds by substituting PSC-derived hepatic progenitors with E13.5 kidney derived dissociated cells that could form glomerular like micro-tissues after 8 days of transplantation followed by quick vascularization (Takebe et al., 2015).

A step closer toward a vascularized organoid is the use of scaffolds that are pre-vascularized with HUVECs with or without perivascular cells, followed by seeding of dissociated cells of the organoid or just PSC-derived sorted progenitors. Acellular porcine or rodent kidney matrices have been repopulated with HUVECs lining vessel walls and fetal kidney cells in tubular and glomerular compartments; orthotopic transplantation of the reseeded kidney in single nephrectomized rats showing blood flow through the vasculature and urine production (Song et al., 2013). Miller et al. printed rigid 3D filament networks of carbohydrate glass in the engineered tissue to generate cylindrical networks that can be lined by endothelial cells and perfused, offers an independent extravascular tissue. Initially, a mixture of glucose, sucrose, and dextran was developed such that it formed a glass when cooled and optically transparent such that it could be used in conjunction with photo-cross-linkable materials. The carbohydrate glass was then extruded through a heated syringe into an interconnected microfluidic vascular network allowed to cool, and a scaffold was cast around it in a mixture of mouse fibroblast cells and photo-cross-linkable ECM pre-polymer. When immersed in water, the carbohydrate quickly dissolved, leaving a hollow network in its place (Miller et al., 2012). Bio-printing and micro-fabrication of cells embedded in hydrogel in a pattern generated from knowledge of renal architecture is an emerging technology that is still in its infancy. Currently, acellular adult kidneys and bio-printing are methods that may provide architectural features similar to that of an adult organ, and hold a promising future for creating vascularized transplantable organs in vitro. Current limitations of bioprinting are dimensional, with minimal sizes approaching 30–50µm, and the availability of adequate materials. Extracellular matrix printing seems to provide some benefit over other polymers in terms of cell survival and differentiation. Its combination with shape controllable polymers, for example to print preformed scaffolds that are then filled with matrix containing cells or cell clusters will perhaps lead to readily generated 3-dimensional and properly tubulized tissues. What remains to be figured out is which cell type can be chosen to repopulate such a prevascularized-scaffold. Among various other candidates, would several PSC-derived organoids occupy the matrix and develop a nephron-vascular system or is it sufficient to use a cocktail of PSC-derived terminal cell types and allow them to anchor to the scaffold and attain function?

The simplest method to vascularize the kidney organoid would involve connection to an extrinsic vascular supply. This was first demonstrated by Sariola et al., when they grafted mouse embryonic kidney explants on to chorio-allantoic-membrane (CAM) of a quail and maintained them for 5–10 days. The grafted kidney gave rise to well- developed convoluted secretory tubules and highly branched collecting ducts. Definitive glomeruli were also identified in these grafts by the presence of efferent tubules, visceral and parietal epithelium, capillary tuft, and Bowman's space. These CAM grafts appeared histologically comparable to the 14- 15 -day in vivo embryonic metanephros (Preminger et al., 1980). Following this example, Taguchi et al placed mouse-PSCderived MM cell mass induced by mouse spinal cord beneath the kidney capsule of immunodeficient mice. When harvested after a week, tubulogenesis and glomerular formation was seen as expected and around 84.6% of the glomeruli showed integration of vessels from the host. This demonstrates maturation of the induced EB in presence of a vascular supply (Taguchi et al., 2014).

Ectopic organogenesis has been extended to several sites in vivo, since the kidney capsule restricts space availability for the maturating renal primordia. Transplantation of E15 rat metanephroi into omentum of non-immunosuppressed adult rats resulted in a normal kidney structure and function following uretero-uterostomy, surviving as long as 32 weeks (Hammerman, 2002). A recent study by the Lagasse group has reported the successful organogenesis of the kidney using the mouse lymph node as a developmental niche. Some of their findings include the survival of neo-kidneys until the 12th week of transplantation, engraftment and maturation of metanephroi although fluid waste accumulation resulted in graft degeneration. Moreover, nephrectomy of the mice resulted in accelerated organogenesis suggesting a compensatory response (Francipane and Lagasse, 2015). To determine whether the fetal kidney could provide a suitable niche for MSCs to generate renal tissue, Yokote and Yokoo (2013) injected GDNF-expressing human MSCs into rodent whole-embryo cultures at the point of budding. MSC were shown to integrate into the developing fetal kidney forming a chimeric organ leading to a possibility that depletion of animal cells in this chimeric kidney after transplantation into the omentum of human patients would allow further development of the kidney only from human cells (Yokoo and Kawamura, 2009; Yokote and Yokoo, 2013). The result that we aim for in any case, is a kidney that when perfused with blood/ equivalent of body fluids can filter larger proteins and reabsorb electrolytes to secrete a urine concentrate into a functional ureter. Further molecular and cellular investigations of the afore-mentioned experiments of in vivo vascularization of kidney organoids (listed in **Table 2**)

can provide valuable lessons on how the vascular networks are established and can be transferred to in vitro experiments conducted on scaffolds, increasing our chances to develop a nephron-vascular unit.

#### OUTLOOK

The options for generating vascularized tissues suitable for in vitro assays or for regenerative medicine have increased greatly in the last decade. Vascularization is achievable in vitro by means of preformed natural or artificial printed or casted scaffolds or pre-established endothelialized networks, which can then be populated with tissue cells, depending on the application. The populating cells may lead to further remodeling and capillary formation, which could be directed using switchable expression of angiogenic factors. Alternatively, supplying tissues, cells or organoids undergoing morphogenesis with vascular or lymphatic precursor cells may be useful to enable the tissue to autonomously generate vascularity or to allow faster vascularization post transplantation. The perfusion of the vascular system in vitro will become feasible with advanced microfluidics devices and bioreactors for larger organs.

In addition, vascularization of preformed avascular organoids is achievable in vivo, by transplantation either ectopically or at the organ specific target site. The aggregation of pre-formed smaller functional tissue units or organoids may be a technique to build larger organs. These smaller units could be either avascular or preconditioned with endothelial cells or suitable scaffolds to quickly vascularize the larger aggregates. The provision of larger vessel adapters to allow direct connectivity to the host's vascular net will provide blood supply early after transplantation. Finally, the generation of embryonic or adult human organs in vivo would allow complete vascularization after transplantation to environmental niches that permit continuing organogenesis. These niches could be provided in vitro and in vivo. Based on this approach, growth of pre-vascular fetal human kidneys in transgenic kidney deficient mice may be used to generate kidney primordia for transplantation and vascularization in adult

#### REFERENCES

human hosts. The establishment of a fully humanized kidney in vivo, including human derived vasculature and innervation may also be achieved using hMSC in an animal model as demonstrated by Yokote et al. (2015). Similarly, adult kidneys could be generated in donor animals that promote development of fully humanized organs and transplanted or used in perfusion systems.

Striking advances have also been achieved regarding the toolbox for generating human cells that are useful to generate tissue specific organoids. This includes the establishment of well characterized adult and fetal tissue derived primary differentiated and precursor cells, as well as of PSC-derived endothelial, lymphangiogenic, and organ specific cells. Endothelial cells and vasculatures have tissue-specific characteristics, for example in the kidney glomerulus they are highly fenestrated and differentiate in close association with podocytes. Whether iPSCderived endothelial cells are able to acquire the tissue specific phenotype needs to be investigated. The design of cells with tailored function is another option where the basic technologies are already known. It can be expected that design of cells will advance to the tissue and organoid level including cells with vascularizing functions.

Applications of organoids, which model function and structure of the kidney, are not restricted to understanding human kidney development. They are applicable to study the pathogenesis of genetic diseases as patient –derived PSC can be used for organoid generation. These are equally applicable for drug testing as PSC can be stratified according to patient cohorts and organoids functionally characterized in vitro as well as in vivo post grafting. Finally, organoids may be applicable in regenerative medicine to replace or repair functional tissue units.

#### ACKNOWLEDGMENTS

This work was funded by the German Research Foundation [Emmy-Noether grant (to KS), Research Unit 667 grant (to KS), and Research Unit 1368 grant (to KS)] and the Urological Research Foundation.

Ishii, K. (1966).  Cytologia 31, 89–98. doi: 10.1508/cytologia.31.89

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Hariharan, Kurtz and Schmidt-Ott. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Exosomes as renal inductive signals in health and disease, and their application as diagnostic markers and therapeutic agents

#### Mirja Krause, Anatoliy Samoylenko and Seppo J. Vainio\*

Biocenter Oulu, Infotech Oulu, Developmental Biology Lab, Faculty of Biochemistry and Molecular Medicine, Center for Cell Matrix Research, University of Oulu, Oulu, Finland

Cells secrete around 30–1000 nm membrane-enclosed vesicles, of which members of the subgroup between 30 and 100 nm are termed exosomes (EXs). EXs are released into the extracellular space and are widely present in body fluids and incorporated mRNA, miRNA, proteins, and signaling molecules. Increasing amounts of evidence suggest that EXs play an important role not only in cell-to-cell communication but also in various physiological and disease processes. EXs secreted by kidney cells control nephron function and are involved in kidney diseases and cancers. This makes them potential targets for diagnostic and therapeutic applications such as non-invasive biomarkers and cell-free vaccines and for use as drug delivery vehicles. This review provides an overview on the known roles of EXs in kidney development and diseases, including renal cancer. Additionally, it covers recent findings on their significance as diagnostic markers and on therapeutic applications to renal diseases and cancers. The intention is to promote an awareness of how many questions still remain open but are certainly worth investigating.

#### Edited by:

Tomer Avidor-Reiss, University of Toledo, USA

#### Reviewed by:

Bas W. M. Van Balkom, University Medical Center Utrecht, Netherlands Gloria Alvarez-Llamas, Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, Spain

#### \*Correspondence:

Seppo J. Vainio<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cell Growth and Division, a section of the journal Frontiers in Cell and Developmental Biology

> Received: 01 July 2015 Accepted: 05 October 2015 Published: 20 October 2015

#### Citation:

Krause M, Samoylenko A and Vainio SJ (2015) Exosomes as renal inductive signals in health and disease, and their application as diagnostic markers and therapeutic agents. Front. Cell Dev. Biol. 3:65. doi: 10.3389/fcell.2015.00065 Keywords: extracellular vesicles, exosomes, kidney development, diagnostic markers, therapeutics, renal disease, renal cancer

## INTRODUCTION

Cells from various organisms, including all the eukaryotes and many prokaryotes, release extracellular vesicles of different types into their environment. The term exosomes (EXs) describes those vesicles which are of endosomal origin. They are small (30–100 nm) membrane-enclosed vesicles which are secreted by various cell types, and can be found in biological fluids such as blood, semen, saliva, and urine (reviewed by Raposo and Stoorvogel, 2013; Yanez-Mo et al., 2015). Initially these were considered to take part in the cell's waste management but evidence has accumulated that they are instrumental in intercellular and system (humoral)-level communication in organisms. The striking property of EXs is that they can transfer important compounds such as membrane and cytosolic proteins, lipids, mRNA, miRNA (Valadi et al., 2007), and even DNA between cells. Thus, they provide a novel but apparently evolutionary ancient platform for cell-to-cell and tissue interactions (Valadi et al., 2007).

Even though the putative developmental role of EXs remains in most respects unclear, it is emerging that at least many of the key developmental signaling pathways are coordinated by EXs (Raposo and Stoorvogel, 2013; Urbanelli et al., 2013). Kidney cells, for instance, appear to release vesicles presumably in a developmentally regulated manner, and the currently available data suggest that EXs may initiate and regulate organogenesis. In addition, they may also take part in processes such as regeneration and the development of diseases such as cancer. Interestingly, kidneyderived vesicles may also have an impact on the cells of certain other organs (Grange et al., 2011) and even the immune system. The aim of this review is to summarize the roles that have been demonstrated for EXs in kidney development and disease, especially in kidney tumorigenesis.

## EXOSOMES AS RENAL INDUCTIVE SIGNALS, DIAGNOSTIC MARKERS, DISEASE, AND THERAPEUTIC AGENTS

#### Biogenesis and Signal Transduction

The biogenesis of EXs is a complex intracellular process that forms part of the endosomal cell sorting machinery, in which an array of regulatory factors has been identified (Stoorvogel et al., 2002; Théry et al., 2002; Kowal et al., 2014). Upon the initiation of EX assembly an early endosome is formed by an inward budding of the cellular membranes via endocytosis. In association with this budding the endosomal membrane undergoes a second inward folding to generate further smaller vesicles inside the endosomal lumen. The process is collectively called the formation of multivesicular bodies (MVB; **Figure 1**).

The machinery known as the Endosomal Sorting Complex Required for Transport (ESCRT) is involved in the formation of MVBs, although it also entails an ESCRT-independent mechanism involving proteins such as tetraspanins and lipids (for details, see recent reviews by Hanson and Cashikar, 2012; Henne et al., 2013; Colombo et al., 2014; Kowal et al., 2014). The assembled MVBs can then fuse either with lysosomes, leading to their degradation, or alternatively with the cellular plasma membrane (PM), which releases their vesicles, called from here on exosomes (EXs), into the extracellular milieu of the cell. The fusion of MVBs with the PM was first described in the hematopoietic system, namely in differentiating red blood cells (Pan and Johnstone, 1983), and thereafter in several other cell types such as B-cells, T-cells, dendritic cells (Escola et al., 1998; Zitvogel et al., 1998; Blanchard et al., 2002), mast cells (Raposo et al., 1997), and yeast cells (Henne et al., 2013). The biogenesis of EXs has also been described in epithelial cells, including those of the kidney (Knepper and Pisitkun, 2007), being the cells that line the renal tubule lumen (Pisitkun et al., 2004).

On their release EXs interact with a recipient cell in several ways. A schematic overview of the biogenesis of EXs and how they enter target cells and interact with them is depicted in **Figure 1**. As one mode of interaction, EXs bind to the target cell via membrane receptors such as the Major histocompatibility complex (MHC) that mediates binding to T-cells (Denzer et al.,

FIGURE 1 | Biogenesis and uptake of exosomes. Inward budding of the membrane of an endosomal vesicle in the cytosol of the donor cell leads to the formation of multivesicular bodies (MVBs). These can either turn into lysosomes, whereupon their content will be degraded, or fuse with the plasma membrane, releasing their content into the extracellular environment. These vesicles are called exosomes. By contrast, larger microvesicles are formed when the cell membrane directly buds outwards, giving rise to micelles. Exosomes can interact with recipient cells in various ways. Signaling can be initiated by an antigen-antibody interaction in the recipient cell or by the activation of a receptor on the cell membrane of the target cell. Exosomes can also be taken up by endocytosis or fuse directly with the plasma membrane. Upon fusion their cargo is released into the cytosol of the target cell.

2000; Nolte-'t Hoen et al., 2009). Another way in which EXs bind is via recognition of the ligands on their lipid bilayer by means of specific cell surface receptors. Indeed, many common ligand/receptor pairs such as recognition by integrins or tetraspanins have been identified. The capacity of an EX to bind to a given cell depends to a great deal, however, on the cellular content, and also the characteristics of the donor cell from which the EXs originated. The most prominent binding mechanisms have been reviewed recently by Colombo et al. (2014) and are therefore, not reviewed here. Besides receptormediated binding, the entry of EXs into a target cell may involve endocytosis (Morelli et al., 2004), phagocytosis (Feng et al., 2010; Christianson et al., 2013; Yanez-Mo et al., 2015), or pinocytosis (Parolini et al., 2009; Théry et al., 2009; Yanez-Mo et al., 2015). These processes can also be receptor-mediated, aided by several other proteins such as dynamin (Fitzner et al., 2011) and clathrin (Mulcahy et al., 2014; Tian et al., 2014), which regulate entry. In the case of kidney-derived cells such as COS-7, the cells seem to internalize EX through lipid raft-mediated endocytosis which is negatively regulated by caveolin-1 (Svensson et al., 2013). It is currently poorly understood whether EX uptake mechanisms are common or specific to each cell type (Gildea et al., 2014). Furthermore, there is also some debate as to how widely certain EXs can influence cell behavior (White et al., 2006; Svensson et al., 2013).

## Exosomes in Kidney Physiology

The currently available data suggest that the extracellular vesicles are coupled to normal development and various diseases. Given that EXs contain key regulatory signals such as mRNA and miRNA and can transfer them from one cell type to another, they may have a profound influence on target cell homeostasis (Montecalvo et al., 2012; Stoorvogel, 2012; Tomasoni et al., 2012; Zhang et al., 2015b). The capacity of EXs to transport miRNA is of particular importance when considering participation in developmental control in general (Tang et al., 2007). Even though it still remains unknown whether EXs indeed play a crucial role in morphogenesis, the current results suggest that at least urinary EXs, besides being involved in the secretion of senescent proteins as exocytic waste (Pisitkun et al., 2004; Knepper and Pisitkun, 2007) may also have other roles (van Balkom et al., 2011; Fang et al., 2013). In the light of current data, the systemic serum containing EXs cannot cross the kidney filter within the glomerulus under normal conditions (Pisitkun et al., 2004; Gildea et al., 2014), but whether this changes in the presence of kidney anomalies remains open.

Proteomic studies of urinary EXs have identified proteins that characterize certain nephron segments such as the glomerulus and Henle's loop. A summary is given in **Table 1** (Pisitkun et al., 2004; Miranda et al., 2010; Dear et al., 2013). The available data suggest that most, if not all, of the nephron cell types have the capacity to secrete vesicles. EXs may play an important role in mediating cell-to-cell communication along the nephron with potential significance for kidney performance, and it has been shown that the function of the EXs within the nephron may be the adaptation of nephron function to changes in physiology, i.e., in homeostasis. This possibility is based on the observation that different segments of the nephron secrete and take up EXs differentially (Dimov et al., 2009). Renal EXs can also transfer functional molecules such as aquaporin-2 between cells (Street et al., 2011), although their uptake capacity becomes restricted in the fully matured adult kidney. This is probably caused by accumulation of the tubular Tamm-Horsfall protein, which prevents contacts between EXs and the tubular luminal cells unless this protein is degraded locally (van Balkom et al., 2011). Hence, the luminal epithelial cells of the nephron apparently secrete the EXs found in urine. Furthermore, proteins which are specifically associated with the urinary bladder and prostate gland have also been found in urine (Musante et al., 2014), although further studies are needed to provide conclusive evidence of this.

When considering the embryonic kidney and the potential of EXs for taking part in its developmental programming, many of the proteins known to control organogenesis, including growth factors (see the recent review by Krause et al., 2015) are in fact found in the EXs that have been characterized from a variety of cell lines (**Table 2**). The Wnt-family members and their signal transduction pathway are critical for kidney development, and interestingly, several Wnt proteins and their downstream factors such as β-catenin are associated with EXs and can also mediate activation of the pathway (**Table 2**; Zhang et al., 2015a). It can be speculated that a panel of key developmental signals may also be associated with and transported to the target cells via EXs during kidney development.

If this is the case, then EXs constitute a new critical mechanism in the control of kidney development by transferring and integrating key inductive signals. Thus, their presence during kidney development should be explored further: Which cells secrete EXs under which environmental conditions, and do these EXs populations differ one from another? Further analysis should then reveal details of their content, transport mechanisms and physiological roles during kidney development.

The putative role of EXs has recently been explored in Madin-Darby canine kidney cells (MDCK; Kwon et al., 2014). When these cells are subjected to the hepatocyte growth factor (HGF) their proliferation is stimulated and eventually tubular cysts form. Changes in the expression of a specific G protein-coupled receptor, GPRC5B, are associated with EXs being secreted by these cells. This protein is also up-regulated in tubulogenesis, while the exosomal delivery of GPRC5B induces extracellular signal-regulated kinase 1/2 (Erk1/2). As the GPRC5B is expressed in the ureteric bud of the embryonic kidney, this may suggest that it also plays a role in organogenesis.

## The Role of Exosomes in Kidney Regeneration and Diseases

While the role of kidney-derived EXs in physiological processes remains poorly investigated, EXs have been found to exercise beneficial or adverse functions in the development of several kidney diseases (Borges et al., 2013a; Fang et al., 2013). For example, vesicles derived from mesenchyme stem cells (MSCs) or endothelial stem cells can promote kidney regeneration (Borges et al., 2013a). The positive impact of MSCs on both acute and chronic kidney injury (AKI and CKI) was first attributed to

Data compiled from Pisitkun et al. (2004), Gonzales et al. (2009), Miranda et al. (2010), Dear et al. (2013), and Musante et al. (2014) and the Online Mendelian. Inheritance in Man OMIM® website (http://www.omim.org/, see MIM numbers for reference).

TABLE 2 | Genes of importance during kidney development found in exosomes of various origins.

their role in directly replacing renal tubular cells, but later it became clear that these cells rather provide paracrine support for endogenous regeneration (Biancone et al., 2012). At present the role of MSCs has been assigned in part to the secretion of EXs (Camussi et al., 2010; Biancone et al., 2012; Borges et al., 2013a), e.g., in that MSC-derived EXs can enhance regeneration of the rat kidney epithelium when injured by ischemia-reperfusion (Gatti et al., 2011). This EX-mediated recovery involves cell-to-cell transfer of mRNAs and/or microRNAs and may be connected with the inhibition of renal cell apoptosis and the stimulation of tubular epithelial cell proliferation. Moreover, by reducing the acute injury, the EXs also protected the rats from later CKI development 6 months after the operation (Gatti et al., 2011).

Other studies have reported a regenerative potential in microvesicles (MVs) produced by bone marrow MSCs in glycerol-induced (Bruno et al., 2009), cisplatin-induced (Bruno et al., 2012), and gentamicin-induced (Reis et al., 2012) AKI models via a mechanism dependent on RNA delivery. Here the protective effect of EXs was mainly ascribed to an increase in surviving tubular cell proliferation (Bruno et al., 2009; Reis et al., 2012) and a decrease in tubular epithelial cell apoptosis (Bruno et al., 2009, 2012; Reis et al., 2012). These effects are thought to occur via the up-regulation of anti-apoptotic genes and down-regulation of genes involved in the execution phase of cell apoptosis (Bruno et al., 2012).

It has also been found that the development of chronic tubular injury is inhibited by multiple injections of MVs, while the effect of a single injection was not sufficient to prevent CKI (Bruno et al., 2012). Like bone marrow MSC-derived EXs, human umbilical cord MSC-derived EXs also demonstrated a protective effect on cisplatin-induced nephrotoxicity in vivo and in vitro, whereas human lung fibroblast-derived EXs did not (Dorronsoro and Robbins, 2013; Zhou et al., 2013a). Zhou et al. (2013a) demonstrated that these EXs can reduce cisplatin-mediated renal oxidative stress and apoptosis in rats in vivo and increase the proliferation of renal tubular epithelial NRK-52E cells in culture. They also showed that human umbilical cord MSC-derived EXs can reduce Bax (bcl-2-like protein 4) level and increase Bcl-2 (B-cell lymphoma 2) in order to inhibit apoptosis and stimulate Erk1/2, thereby increasing proliferation after cisplatin-induced injury in the kidney. Another group has shown that EXs isolated from peripheral blood-derived endothelial progenitor cells can prevent AKI in an ischemia-reperfusion rat model (Cantaluppi et al., 2012). In this case the miRNAs that modulate proliferation, angiogenesis and apoptosis were found to be responsible for the protective effects of EXs.

Kidney epithelial cells are another source of the EXs involved in kidney regeneration. It was found that administration of the epithelium-derived exosomal ATF3 (activating transcription factor 3) mRNA attenuates ischemia/reperfusion-induced kidney injury by inhibiting monocyte chemotactic protein-1 (MCP-1) induced macrophage infiltration (Chen et al., 2014b). While all the above-mentioned studies demonstrate beneficial effects of EXs on kidney regeneration, it was found on the other hand that EXs produced by injured proximal tubular epithelial cells in a murine model of hypoxic kidney fibrosis after unilateral ureteral obstruction can initiate tissue repair/regenerative responses and activate fibroblasts, leading to fibrosis (Borges et al., 2013b). Fibroblast proliferation and the production of matrix proteins were particularly dependent on EXs delivering TGF-β1 (transforming growth factor β1) mRNA (Borges et al., 2013b).

Interestingly, certain in vitro studies published more than 20 years ago demonstrated that renal brush border-derived MVs about 100 nm in diameter can induce and promote calcium oxalate crystallization (Nagasawa et al., 1992; Anderson et al., 2010), which is one of the features of nephrolithiasis (kidney stone formation), a pathological kidney condition leading to fibrosis and chronic renal failure (Anderson et al., 2010).

Since, the endothelial cells are connected to the control of blood flow, pressure and clotting, they are prime targets when considering the development of EX-based therapies, and also the treating of renal diseases (van Balkom et al., 2011). It has been shown that circulating levels of endothelial-derived MVs are significantly higher in chronic renal failure patients than in healthy subjects (Faure et al., 2006). Moreover, blood levels of these MVs correlate with endothelial dysfunction and arterial stiffness in end-stage renal failure hemodialysis patients (Amabile et al., 2005). In contrast to endothelium-derived MVs, those produced by platelets or erythrocytes do not seem to be connected with endothelial dysfunction (Amabile et al., 2005). In another study it was found that the number of endothelialderived microparticles was inversely correlated with brachial artery and aortic laminar shear stress values in end-stage renal disease patients with a high cardiovascular risk (Boulanger et al., 2007).

It may be assumed that the action of cell-damaging agents such as low shear stress and increased arterial stiffness contributes to endothelial apoptosis through a substantial release of endothelial MVs (Amabile et al., 2005; Boulanger et al., 2007). The pathogenetic effects of EXs and other circulating microparticles in promoting vascular calcification and sclerosis in chronic kidney disease (CKD) are nevertheless not yet clearly defined (Anderson et al., 2010; Fang et al., 2013). Rather surprisingly, Neal et al. (2011) found that the levels of circulating miRNAs in patients with different stages of chronic kidney failure, including those receiving hemodialysis treatment, were reduced by comparison with patients having mild renal impairment or normal renal function. This observation might be explained by the fact that many circulating miRNAs are bound to Argonaute 2-containing protein complexes (Arroyo et al., 2011) or to high-density lipoproteins (Vickers et al., 2011) rather than within EXs. It has been demonstrated in a rat model of CKD induced by 5/6 nephrectomy that the administration of conditioned medium from embryonic MSC has a therapeutic effect, whereas MSC-derived EXs tested in the same experimental setting showed no protective effect on the kidney (van Koppen et al., 2012).

## Exosomes in Kidney Cancer

Although tumor cells secret large amounts of various MVs that enter the blood and other body fluids (Lee et al., 2011; Azmi et al., 2013), the isolation of cancer EXs from patients remains a serious problem due to the lack of specific markers that can distinguish cancer-derived from non-cancer-derived EXs. One such marker that has been identified recently is glypican-1, detected on EXs derived from the serum of patients with pancreatic cancer but not on EXs from healthy subjects or subjects with chronic pancreatitis (Melo et al., 2015). Various biological roles have been proposed for EXs in cancer, such as the expulsion of key proteins and miRNA from cells, the removal of anti-cancer drugs and the release of signaling and regulatory molecules (Lee et al., 2011; Azmi et al., 2013). The properties of EXs may also enable them to take part in the control of cell proliferation, cell survival, angiogenesis, metastasis and immune response (Azmi et al., 2013; Fang et al., 2013). EX-released factors promote stromal remodeling and hypoxia-mediated epithelial mesenchyme transformation, which is critical for the evolution of cancer (Nieto, 2011). EXs can also stimulate the proliferation of fibroblasts by causing a desmoplastic reaction, and they can induce immune escape mechanisms by suppressing antigen-specific immune responses and by up-regulating immunosuppressive cell differentiation and the functioning of these cells (Azmi et al., 2013; Minciacchi et al., 2015). For these reasons EXs form a critical aspect of tumorigenesis.

Much less is known at present about the roles of EXs in kidney tumorigenesis, however. The relevant findings are that the vesicles released by renal carcinoma stem cells (rCSCs) derived from a tumor-bearing patient can trigger angiogenesis and promote lung metastasis when studied with the SCID (immunocompromised severe combined immunodeficient) mouse model (Grange et al., 2011). Grange et al. (2011) showed that rCSCs are secreted by a subset of tumor-initiating cells characteristically expressing the mesenchymal stem cell marker CD105, and that EXs derived from these rCSCs were able to stimulate the growth and invasiveness of normal HUVEC (human umbilical vein endothelial cells). In addition, these EXs increased the formation of capillary-like structures in culture and in induced vessel formation when cells treated with them were grafted into SCID mice.

More detailed molecular characterization of the CD105 positive EXs pointed to significant differences in mRNA and miRNA content as compared with EXs that were negative for CD105. Consistent with their properties, the rCSC-derived EXs have mRNAs that encode for proangiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), angiopoietin-1, ephrin A3, and matrix metalloproteinases (MMP)-2 and -9. Similarly, these were reported to be absent from the CD105-negative tumor cellderived EXs. In addition to these findings, miRNAs that have been reported to be involved in biological processes such as the control of transcription, cell adhesion and cell proliferation were enriched in CD105-positive EXs (Grange et al., 2011, 2014).

Another indication that EXs derived from kidney cancer cells may be involved in the cancerous state and its severity, including the formation of metastases, came from the work of Tauro et al. (2013), who performed a proteomic analysis by comparing EXs released by normal and oncogenic H-Ras (21D1) transfected MDCK cells. While the control and 21D1-MDCK cell-derived EXs were similar in their morphology, the 21D1 derived EXs had high levels of proteases, annexins, integrins, and other secreted proteins which are typically associated with the formation of premetastatic niches. The ability of primary cancer to make changes in normal tissue located in a pre-metastatic niche prior to the arrival of metastasizing cells is an important feature that facilitates sustained cancer growth (Wels et al., 2008).

The role of EXs in the "education" of normal cells toward a pro-metastatic phenotype has been demonstrated not only for renal cell carcinoma but also for other cancers, including melanomas (Peinado et al., 2012) and breast cancer (Fong et al., 2015). Elsewhere, the transfection of MDCK cells with oncogenic H-Ras has been found to induce the release of EXs that contain factors known to control nuclear assembly, transcription, splicing, and translation. The most abundant protein in the 21D1-derived EXs was the Y-box-binding protein (YBX1), which is a DNA and RNA- binding transcription factor involved in DNA replication, DNA repair, transcription, pre-mRNA splicing, and mRNA translation (Eliseeva et al., 2011).

 Like the rCSC-derived EXs (Grange et al., 2011) the 786-0-derived ones promote tubulogenesis in HUVECs (Human umbilical vein endothelial cells; Zhang et al., 2013), and it has been speculated that the pro-angiogenic effect of these renal EXs may be mediated via down-regulation of hepaCAM (hepatic and glial cell adhesion molecule), a hepatocyte cell adhesion molecule, and up-regulation of VEGF. In turn, it has been shown that the EXmediated down-regulation of hepaCAM expression is in effect mediated by Akt phosphorylation connected with the enhanced renal carcinoma cell proliferation (Jiang et al., 2014). Collectively, the 786-0-derived EXs enhance cell migration, invasion and chemokine receptor type 4 (CXCR4) and MMP-9 expression and concurrently reduce the adhesion of 786-0 cells (Chen et al., 2014a).

A comprehensive report on the influence of EXs on renal carcinoma 786-0 cells has been published recently by Du et al. (2014), who investigated the putative effects of EXs released by human Wharton's jelly mesenchyme stem cells (hWJ-MSCs). These EXs promoted cell proliferation, cell migration, and progression of the cell cycle from G0/G1 to the S phase and the HGF/c-Met, Akt, and Erk1/2 pathways in these cells. Moreover, the hWJ-MSCs-derived EXs stimulated tumorigenesis in the 786- 0-cells and also enhanced tumor size. At the molecular level, the EXs induced cyclin D1, MMP-2, and MMP-9 expression in a BALB/c nude mouse model (Du et al., 2014). Meanwhile, RNase pre-treatment abrogated these exosomal effects, indicating that the RNA delivered via EXs serves as a crucial mediator (Du et al., 2014).

Little is known as to whether the cells in various organs take up the cancer-derived EXs selectively. Rana et al. (2012) found that small differences in the EX-tetraspanin complexes that originated from different rat tumor cell lines greatly influenced the cell type to which the EXs were targeted in vitro or in vivo. When tetraspanin-8+ EXs were monitored after 24 h of injection they had been taken up by the pancreas and lung cells, whereas certain large vessels and the kidneys showed a lower abundance of such EXs. Moreover, the liver and gut cells were for the most part negative. These data thus suggest some target cell selectivity among EXs that are secreted in vivo.

Interestingly, EXs engineered to express a chimeric tetraspanin-8 in which the N-terminal region was swapped for a domain from the CD9 protein were readily taken-up by kidney cells but not by other organs, whereas EXs engineered to contain tetraspanin-8 that was fused to an integrin β4 was targeted preferentially to lung, kidney and gut cells. It is significant that also within an organ, EXs seem to be taken up preferentially by specific cell types depending on the exosomal protein content. One indication of this is the fact that tetraspanin-8; integrin β+ fusion EX products could be identified in only the kidney glomeruli (Rana et al., 2012).

There are several reports stating that EXs produced by kidney tumor cells are coupled to cancer-associated immune suppression (Yang et al., 2013; Diao et al., 2015; Gu et al., 2015). As an example of this, EXs purified from human kidney adenocarcinoma ACHN cells inhibited proliferation and induced apoptosis of Jurkat-immortalized T-cells while reducing in vitro interleukin-2 (IL-2), IL-6, IL-10 and interferonγ production (Yang et al., 2013). In the light of these findings, the authors proposed that the Fas ligand within the tumorderived EXs must be responsible for exosomally induced T-cell death.

There is considerable evidence that EXs play a role in renal tumor progression in vivo. The survival of mice inoculated with renal adenocarcinoma Renca cells, which go on to generate tumors, was reduced to some degree in the presence of Rencaderived EXs in the assay (Gu et al., 2015), and similar data have been reported by Diao et al. (2015), leading them to propose that the heat shock protein HSP70 present in EXs derived from cancer cells promotes the immunosuppressive activity of myeloid-derived suppressor cells (MDSCs), possibly via an increase in Stat3, a signal transducer and activator of transcription 3-phosphorylation.

Besides suppressing the immune system, EXs can also do exactly the opposite. Certain tumor-derived EXs induce an immune response. This suggests that EXs may even offer opportunities for developing individualized tumor immunotherapies (Greening et al., 2015). Given the fact that tumor-derived EXs also contain immunosuppressive molecules that reduce their immunogenicity, it is important to learn in detail how these properties are regulated, opening new avenues for valuable therapeutics. Along these lines, EXs that are secreted by IL-12-expressing RC-2 human renal cancer cells exhibited greater anti-tumor effects than EXs derived from "wild type" renal cells or cytocine-supplemented IL-12 alone (Zhang et al., 2010). Such IL-12-containing EXs efficiently induced cell proliferation, the release of interferon-gamma and the specific cytotoxic effects of T-cells derived from cultured human peripheral blood cells.

It is also of interest that the mice that had tumors derived either from the mouse myeloid leukemia line WEHI3B or renal carcinoma Renca cells survived longer if they had been vaccinated beforehand with the EX-loaded dendritic cells from the tumor (Gu et al., 2015). In other words, the tumor cell-derived EXs seemed to be superior to the tumor lysates as a source of antigen. Interestingly, the immunosuppressive features of EXs do not detract from their capacity to serve as an antigen source for the dendritic cells (Gu et al., 2015). These properties make the EXs very promising components for the development of novel cancer therapies.

## Kidney/Urinary Exosomes as Diagnostic Biomarkers and Therapeutic Agents

EXs have a great potential for use as valuable diagnostic biomarkers, especially in the case of monitoring kidney malfunction. Since, many reviews have been published on the analysis of urinary EXs and their potential as diagnostic markers for kidney disease, injury, and transplant rejection, this topic will be covered in the present review only briefly (Knepper and Pisitkun, 2007; Dimov et al., 2009; Dear et al., 2013; Properzi et al., 2013; Musante et al., 2014; Ranghino et al., 2014; Salih et al., 2014).

As discussed, the urinary EX components can be assigned to specific nephron segments, the glomerulus, the proximal/distal tubule, Henle's loop and the collecting duct (**Table 1**; Pisitkun et al., 2004; Miranda et al., 2010; Dear et al., 2013; Musante et al., 2014). Many of these proteins can be associated with certain diseases, but they are not all necessarily linked directly to the kidney. The level of GPRC5B in the urinary EXs correlates with AKI, so that its values are elevated in cases of AKI by comparison with normal healthy subjects (Kwon et al., 2014), making GPRC5B a candidate diagnostic marker for AKI. Furthermore, levels of Fetuin-A were also found to be elevated in patients suffering from AKI (Zhou et al., 2006).

The review by Ranghino et al. (2014) summarizes several suitable urinary exosomal biomarkers for glomerular and tubular damage, including Wilms Tumor 1 Homolog (WT1), ATF3, and Neutrophil Gelatinase-Associated Lipocalin (NGAL). WT1 was found in urinary exosomes of patients who suffer from focal segmental glomerulosclerosis (FSGS; Zhou et al., 2008, 2013b) and in most diabetic patients (Kalani et al., 2013). ATF3 is another marker for AKI alongside Fetuin A and GPRC5B (Zhou et al., 2008). NGAL levels were elevated in patients with delayed graft function after kidney transplantation (Alvarez et al., 2013), and several protein markers have also been found in cases of diabetic nephropathy (Zubiri et al., 2014) and prostate (Lu et al., 2009; Mitchell et al., 2009) and urine bladder cancer (Smalley et al., 2008; Blackwell et al., 2014).

Some markers that classify a person as healthy have also been identified, and these may be of diagnostic value in cases of kidney regeneration. Promonin-1 (CD133) serves as such a marker, for example, as it is lost in the urine at the end stage of renal disease (Dimuccio et al., 2014), and additional markers have been defined for a panel of kidney diseases that include diabetic nephropathy (Musante et al., 2014), cardio-renal syndrome (Gonzalez-Calero et al., 2014), autosomal-dominant polycystic kidney disease (ADPKD; Fang et al., 2013; Ben-Dov et al., 2014) and Gitelman's and Bartter syndromes (Corbetta et al., 2015), as well as for following the organ-acceptance after a kidney transplantation (Alvarez et al., 2013).

In 2007 the group led by Jan Löttvall found that EXs carry different functional RNA species (Valadi et al., 2007). Renal mRNA levels have been used in the past as prognostic markers for kidney diseases (Eikmans et al., 2003), but this required an invasive kidney biopsy. Analysis of the protein and RNA content of urinary EXs provides a non-invasive alternative for evaluating changes in renal gene and protein expression, and it has been found that several exosomal microRNAs isolated from urine are suitable markers for certain kidney diseases. One study showed that exosomal miR-145 and miR-130a levels were elevated in patients with diabetic nephropathy, while levels of miR-155 and miR-424 were down-regulated (Barutta et al., 2013). Further studies with a larger group of patients would be necessary to confirm these results.

Several miRNA markers of CKD/renal fibrosis have been identified. Levels of exosomal miR-29a, miR-29c, miR-200b, and miR-200c were down-regulated in patients with moderate-tosevere fibrosis (Lv et al., 2013), but not in cases with mild fibrosis. The same paper also demonstratesthat miR-29c provides indicators of renal function and the histological degree of fibrosis, making it the most prominent candidate for a biomarker of CKD, while (Lv et al., 2014) showed that the exosomal mRNA level of CD2-associated protein (CD2AP) was down-regulated in CKD patients, and even more so in patients with a more severe disease. Both of these reports not only identified disease markers, but also demonstrated that the level of miRNA/mRNA present in urinary EXs provides an opportunity to define the progression of the disease.

Protocols for analyzing exosomes and isolating RNA from them still have to be optimized further in order to yield unbiased, reliable results. Furthermore, it still needs to be ascertained whether levels of exosomal mRNA can also reflect levels of the proteins used as biomarkers for kidney diseases. The same RNA and protein markers as were used in the past when a kidney biopsy was performed might not apply to exosomal RNA and protein levels, as is supported by the fact that little equivalence was found between the exosomal and cellular RNAs of exosome-producing cells (Skog et al., 2008; Mittelbrunn et al., 2011; Koppers-Lalic et al., 2014). This implies an underlying mechanism for the targeted loading of certain RNA species into EXs, in contrast to the loading of the most abundant cellular RNA into EXs in order to discard it. Future studies will have to elucidate the sorting mechanisms responsible for this process. Nonetheless, it is this fact that makes EXs especially interesting, as mRNAs and miRNAs can influence gene expression in the recipient cell and providing the EXs with their therapeutic potential.

One therapeutic strategy would be that RNA-bearing EXs would deliver their cargo to specific malfunctioning target cells and could restore damaged or deregulated protein production. Several studies have shown that exosomes shuttle functional miRNA and influence the gene expression levels of target cells (Pegtel et al., 2010; Montecalvo et al., 2012; Chen et al., 2014c).

Another promising feature of EXs is to serve as a noncytotoxic drug delivery system. The challenge still lies in loading the drugs onto the EXs without imperiling their biological properties (Suntres et al., 2013). Different methods for loading a defined cargo onto EXs have been established, as recently reviewed (Johnsen et al., 2014). This is a possible way of developing novel therapeutics to treat various diseases. Some examples exist of studies which are currently in clinical trial phase I.

One such investigation concerns the potential application of EXs to deliver curcumin (which has proved to have biological activity) to colon cancer tissue (clinical trial no. NCT01294072), while another uses EXs as a vaccine, administering dendritic cellderived EXs (CSET 1437) loaded with antigen to lung cancer patients in order to activate their innate and adaptive immunity during therapy (clinical trial no. NCT01159288). Furthermore, the potential application of EXs to the treatment of kidney diseases is being explored. As described in Section The Role of Exosomes in Kidney Regeneration and Diseases above, various groups have shown that MSCs have a paracrine effect on acute and chronic kidney diseases (Gatti et al., 2011; van Balkom et al., 2011; Fang et al., 2013) in which soluble factors such as those associated with the secreted vesicles have a positive influence on cell behavior and promote the initiation of recovery. The potential of EXs for use in cell-free therapy applications has been summarized recently (Vishnubhatla et al., 2014) and will not be discussed here in detail. It is clear, however, that EXs from various cells such as MSCs and cancer cells have a great potential as novel therapeutic tools, and they can be expected to have a significant impact on the development of diagnostics and new treatments, for kidney diseases among others.

## CONCLUSIONS

It has become generally accepted for the moment that EXs are present in a wealth of body fluids and are not only a cellular waste system as was thought earlier. Collectively, they may represent a newly identified but apparently ancient humoral system controlling homeostasis and disease, and they also provide a useful bank of biomarkers for a variety of diseases and may raise the value of urine as a non-invasive diagnostic component in medical practice. The analysis of urinary EXs not only provides us with prognostic disease markers, but might also make it possible to differentiate between diseases which display similar symptoms. Furthermore, certain data have shown that it is possible to estimate the severity of a disease, and hence its progression. This could enable non-invasive monitoring of responses to treatment and also make it possible to look for complications after kidney transplantations. The use of EXs as therapeutics and vaccines seems to be safe and effective due to their target specificity and their lack of cytotoxicity. This and the fact that a specific cargo can be loaded into them also make them very promising candidates as novel drug delivery systems. This is still something of a challenge, however, and better protocols need to be developed. More research will be necessary to ensure the safety of the resulting medical applications.

A schematic overview of the signaling role of EXs in relation to the kidney is presented in **Figure 2**. It is known that EXs play a role in kidney diseases and renal cancer, but many of the signaling molecules found during nephrogenesis occur in association with EXs, leading us to conclude that they not only participate in disease processes but also play a putative role in developmental processes. Which machinery regulates these processes during embryonic kidney development still remains largely an open

abnormal kidney conditions appear, exosomes can be released from the abnormal cells to be subsequently taken up by healthy cells, which will then be transformed and might even become abnormal as well. It has been reported that exosomes released from transformed kidney cells can initiate cancer in lung cells (Grange et al., 2011; Rana et al., 2012) and lead to tumor-promoted immune suppression in certain immune cells (Yang et al., 2013; Diao et al., 2015; Gu et al., 2015). Kidney cells and transformed kidney cells also take up the released vesicles again (dotted lined arrows). Most of the means of signaling depicted here are still not very well-characterized or understood.

question, but the accumulating evidence is starting to point toward a critical role for EXs in inductive signaling in the kidney. Future work should be targeted toward elucidating the normal physiological roles of EXs in vivo in defined disease models and advancing our knowledge of the precise mechanisms by which they promote renal diseases and cancer.

#### AUTHOR CONTRIBUTIONS

MK drafted the manuscript and prepared the figures and tables. AS helped with writing the manuscript and designing the figures. AS and SV critically reviewed and revised the manuscript.

#### REFERENCES

All three authors read and approved final version of the manuscript.

## FUNDING

This work was supported by the Academy of Finland (206038, 121647, 250900, and 260056), Centre of Excellence grant 2012–2017 of the Academy of Finland (251314), the Sigrid Jusélius Foundation and the European Community's Seventh Framework Program, FP7/2009, under grant agreement 305608 (EURenOmics: European Consortium for High-Throughput Research in Rare Kidney Diseases).

survival in a lethal model of acute kidney injury. PLoS ONE 7:e33115. doi: 10.1371/journal.pone.0033115

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Krause, Samoylenko and Vainio. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Developing a functional urinary bladder: a neuronal context

#### Janet R. Keast\*, Casey J. A. Smith-Anttila and Peregrine B. Osborne

*Department of Anatomy and Neuroscience, University of Melbourne, Melbourne, VIC, Australia*

The development of organs occurs in parallel with the formation of their nerve supply. The innervation of pelvic organs (lower urinary tract, hindgut, and sexual organs) is complex and we know remarkably little about the mechanisms that form these neural pathways. The goal of this short review is to use the urinary bladder as an example to stimulate interest in this question. The bladder requires a healthy mature nervous system to store urine and release it at behaviorally appropriate times. Understanding the mechanisms underlying the construction of these neural circuits is not only relevant to defining the basis of developmental problems but may also suggest strategies to restore connectivity and function following injury or disease in adults. The bladder nerve supply comprises multiple classes of sensory, and parasympathetic or sympathetic autonomic effector (motor) neurons. First, we define the developmental endpoint by describing this circuitry in adult rodents. Next we discuss the innervation of the developing bladder, identifying challenges posed by this area of research. Last we provide examples of genetically modified mice with bladder dysfunction and suggest potential neural contributors to this state.

Keywords: micturition, continence, pelvic ganglion, dorsal root ganglion, inferior hypogastric plexus, visceral afferent, neural development, organogenesis

## Understanding the Endpoint: Innervation of the Adult Urinary Bladder

#### Overview of Bladder Innervation

The urinary bladder functions to collect and store urine so that it can be released at behaviorally appropriate times. These alternating phases of continence and micturition are precisely controlled by a hierarchical system of neural controls in the spinal cord and brain (Andersson and Wein, 2004; Beckel and Holstege, 2011; de Groat and Yoshimura, 2015). This circuitry connects with the bladder via a system of direct and indirect peripheral neural pathways summarized in **Figure 1**. In each component of this circuit, nerves involved in bladder control are intermingled with many other nerve types, as discussed in detail below.

This review will provide an overview of the features that govern the organization and function of bladder innervation in laboratory animals and humans. In general, the neural connectivity of the nerve supply to the bladder tissues is similar across species. However, it should be noted that while most contemporary studies use rodents, it was previously more common to use cats, which have larger nerves and are easier to study with neurophysiological techniques. Nevertheless, it is

#### Edited by:

*Sunder Sims-Lucas, University of Pittsburgh, USA*

#### Reviewed by:

*Martin C. Michel, Boehringer Ingelheim Pharma GmbH & Co. KG, Germany Kenneth A. Walker, University of Pittsburgh, USA*

#### \*Correspondence:

*Janet R. Keast, Department of Anatomy and Neuroscience, University of Melbourne, Parkville, Melbourne, VIC 3010, Australia<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cell Growth and Division, a section of the journal Frontiers in Cell and Developmental Biology*

Received: *30 June 2015* Accepted: *14 August 2015* Published: *01 September 2015*

#### Citation:

*Keast JR, Smith-Anttila CJA and Osborne PB (2015) Developing a functional urinary bladder: a neuronal context. Front. Cell Dev. Biol. 3:53. doi: 10.3389/fcell.2015.00053*

**Abbreviations:** Ach, acetylcholine; DRG, dorsal root ganglion; NAd, noradrenaline (norepinephrine); NF200, neurofilament protein, 200 kd; TRPV1, transient receptor potential vanilloid receptor 1.

the spinal cord and make connections in the spinal cord and with the urinary bladder tissues. In rodents these DRG neurons are concentrated in spinal levels L1–L2 ("lumbar") and L6–S1 ("sacral"). They are activated by various physiological and pathophysiological changes within the bladder wall and signal to neurons in the spinal cord and, in turn, the brain. Integrative centers in the brainstem determine whether sympathetic (red) or parasympathetic (blue) pathways are activated to promote, respectively, continence or voiding. These pathways comprise two types of neurons, the first of which are preganglionic neurons in the intermediolateral nucleus (IML) of the spinal cord and the second are postganglionic neurons with cell bodies in the mixed pelvic ganglia. The preganglionic neurons release acetylcholine (ACh) to excite postganglionic neurons in pelvic ganglia that then release different transmitters to tissues in the bladder wall (ACh, NAd [noradrenaline] and ATP). Sympathetic preganglionic neurons also traverse the inferior mesenteric ganglion (not shown) before they reach the pelvic ganglion. Vascular innervation is not shown but is likely to arise from sympathetic chain rather than pelvic ganglion neurons. These autonomic motor pathways are coordinated with sphincter activity, mediated by somatic neurons in the spinal cord (not shown) that directly innervate the striated muscle of the external urethral sphincter. The axons from these neurons are not shown as they do not traverse the pelvic ganglion but project in the pudendal nerve.

clear that rodents have limitations for studying some specific questions relevant to human clinical urology, such as urethral sphincter control. As species differences cannot be discussed in detail here, the reader is referred to recent reviews that discuss this and other species differences in detail (Andersson and Wein, 2004; Thor and de Groat, 2010; Andersson et al., 2011).

Despite the paucity of data on the development of urogenital innervation and mechanisms of its remodeling in adulthood, the importance of this area of the nervous system is illustrated by the many clinical problems that arise when the nervous system is perturbed. For example, various types of pelvic surgery performed for tumor removal unavoidably damage nearby nerves, resulting in incontinence and sexual dysfunction (Mauroy et al., 2003; Cornu et al., 2014; Wit and Horenblas, 2014; Laterza et al., 2015). Understanding factors that drive growth of these connections during development could identify strategies for their repair in adults. Conversely, chronic cystitis is accompanied by bladder hyperinnervation (Dickson et al., 2006; Boudes et al., 2013) that may contribute to hyperactivity and pain. In this case, identifying growth inhibitory or chemorepulsive factors involved in bladder nerve development may provide targets for reducing aberrant growth and dysfunction in adults.

The nerves of the bladder can be divided into two classes sensory and autonomic—that differ in their anatomy, chemistry, and function (Jänig, 2008). There are multiple subtypes of sensory and autonomic nerves, as discussed below. As the bladder fills, the bladder wall relaxes to accommodate increasing volume and sympathetic nerves constrict the bladder base and urethra to promote continence. In cats, sympathetic innervation of the detrusor may contribute to continence (Vaughan and Satchell, 1992; Khadra et al., 1995) including by modulating transmission in parasympathetic ganglion neurons (de Groat and Saum, 1972), but this remains a debatable area in rodent research. Continence continues until a threshold tension of the bladder wall is detected by low-threshold mechanosensors (sensory neurons). This can trigger voiding but in healthy adults is dependent on the overriding neural controls from the brain. When suitable to initiate voiding, bladder contraction is activated in coordination with relaxation of the bladder base and urethra, and opening of the external urinary sphincter (striated muscle). This "voluntary" sphincter component of bladder control sets it apart from most other areas of autonomic function (e.g., cardiovascular regulation) that are activated in direct response to changing environmental and bodily needs but are "involuntary" (Jänig, 2008). The innervation of the external sphincter (urethral rhabdosphincter) originates from somatic motor neurons in the spinal cord and will not be discussed further here.

#### Sensory Innervation of the Bladder

Sensory axons form a rich network in most bladder tissues (Gabella, 1995, 1999; Gabella and Davis, 1998; Forrest et al., 2014). They distribute uniformly in the smooth muscle (detrusor) of the bladder wall, but near the urothelium become heavily concentrated at the base and neck—where some axons also penetrate the deeper layers of urothelial cells (Gabella and Davis, 1998; Gillespie et al., 2006; Forrest et al., 2014). This suggests signaling from the urothelium to sensory nerves, which is important in both health and disease (Kanai, 2011; Birder and Andersson, 2013), is spatially restricted. Sensory axons are also found within many blood vessels in the bladder wall (Gabella and Davis, 1998; Forrest et al., 2014) where their role is unknown.

As in other internal organs, sensory axons in the bladder terminate in simple, free endings (Gabella, 1995, 1999; Gabella and Davis, 1998; Forrest et al., 2014). Although often regarded as structurally unspecialized, different branching patterns and terminal structures occur but these variations are relatively subtle in comparison with the large, complex structures at the terminals of specialized sensory nerves in skin and skeletal muscle (e.g., Pacinian and Ruffini corpuscles). These small structural variations have not yet been correlated with particular sensory modalities in the bladder.

Sensory axons in the bladder originate from distant neuronal cell bodies in the lumbosacral dorsal root ganglia (DRG) that lie close to each side of the spinal cord (**Figure 1**). After leaving the DRG, their axons pass through the pelvic autonomic ganglia en route to the bladder, then intermingle with the bladderprojecting motor axons until they reach the bladder wall. Sensory and motor axons cannot be distinguished at this point unless immunolabeled for specific neural markers. As most bladder sensory axons originate from the sacral DRG these neurons are better characterized and more commonly experimentally manipulated than those originating from upper lumbar DRG (Kanai, 2011; Gonzalez et al., 2014; de Groat and Yoshimura, 2015). Given only a minority of neurons in DRG of both spinal levels project to the bladder (Robinson and Gebhart, 2008; de Groat and Yoshimura, 2009), assessing their molecular changes during disease or injury states raises a technical challenge. Anatomical tracing strategies [e.g., retrograde tracer micro-injection into the bladder wall Yoshimura et al., 2003; Forrest et al., 2013; Gonzalez et al., 2014] have been critical for visually separating bladder sensory neurons to define their properties.

Physiological studies recording electrical activity within single axons entering the bladder wall or at their terminals during different types of bladder manipulation (e.g., stretch) have subclassified them by transduction properties (Zagorodnyuk et al., 2007, 2010; Xu and Gebhart, 2008). The predominant class acts as low-threshold mechanosensors that respond to tension and contraction. However, other classes of low-threshold and high-threshold mechano- and chemo-sensitive nerves can be recruited, especially in response to physical or chemical signals of impending bladder damage (Kanai, 2011; Skryma et al., 2011). These latter signals may be interpreted as sensations of discomfort or pain, but normally the organism does not perceive activity in these neural pathways.

Bladder sensory nerves are either lightly myelinated (Adelta) fibers or unmyelinated (C-fibers) (Kanai, 2011; de Groat and Yoshimura, 2015). Most of the A-delta class comprises low-threshold mechanosensors whereas most C-type fibers are higher threshold sensors that respond to diverse types of stimuli (polymodal), including damage. Measurements of action potential conduction velocity distinguish these two classes, but molecular properties are also useful indicators. For example, the majority of C-fiber bladder sensory neurons express the nociceptive transducer ion channel, TRPV1 (transient receptor potential vanilloid receptor 1), as well as the neuropeptides, calcitonin gene-related peptide and substance P (Arms and Vizzard, 2011; Skryma et al., 2011; Forrest et al., 2013). Functional studies have also used capsaicin-sensitivity to identify activity attributed to bladder sensory neurons that express TRPV1 (de Groat and Yoshimura, 2009; Michel and Igawa, 2015). Conversely, the 200 kD neurofilament protein (NF200) distinguishes the cell bodies of sensory neurons with myelinated axons (Lawson et al., 1993), including the Adelta bladder afferents (Forrest et al., 2013). However, the latter has limited use in the identifying their terminals where neurofilament levels are low (Forrest et al., 2014). Also, NF200 is not diagnostic of sensory axons in the bladder where many autonomic axons express this protein (Forrest et al., 2014).

#### Motor Innervation of the Bladder

The bladder can show myogenic activity that is independently driven by detrusor smooth muscle, but the effectors for the coordinated activity required by continence and micturition are parasympathetic and sympathetic autonomic neurons (Beckel and Holstege, 2011; de Groat and Yoshimura, 2015). Autonomic neurons also control vascular tone in the bladder (Michel and Igawa, 2015).

In the bladder, autonomic axons innervate the same tissues as the sensory axons, albeit in different density—in the detrusor and vasculature, autonomic axons are more prevalent than sensory axons, whereas the converse is true for the urothelium (Gabella and Davis, 1998; Gabella, 1999; Gillespie et al., 2006; Forrest et al., 2014). Although less prevalent than sensory axons, autonomic axons show a similar gradient of urothelial innervation toward the bladder base and neck (Dickson et al., 2006).

Parasympathetic axons release acetylcholine as a transmitter and in some tissues and species release a co-transmitter, ATP (Jänig, 2008). This is the dominant axon type in the detrusor, where they cause muscle contraction during micturition (de Groat and Yoshimura, 2015). Sympathetic axons release noradrenaline (norepinephrine) and potentially also ATP but are scarce in the detrusor, only being found in small numbers in the base and neck of the bladder where they have a role in continence (Kihara and de Groat, 1997; Gosling et al., 1999; Forrest et al., 2014; de Groat and Yoshimura, 2015). A small population of parasympathetic axons lies close to the urothelium, where they are closely associated with sensory terminals (Dickson et al., 2006; Gillespie et al., 2006). The dominant population of sympathetic axons supplies the vasculature (Gabella, 1999; Forrest et al., 2014), where they likely cause constriction.

Most of the autonomic axons innervating the bladder originate from neuron cell bodies in the nearby pelvic ganglia (**Figure 1**) (Keast and de Groat, 1989). Sympathetic axons innervating the bladder vasculature most likely originate from neurons in the sympathetic chain (paravertebral) ganglia, as is the case for sympathetic vasoconstrictor axons in other organs (Jänig, 2008). The pelvic ganglia of rodents are functionally equivalent to the much more dispersed inferior hypogastric plexus in humans (Keast, 1999; Shoja et al., 2013). Rodent pelvic ganglia comprise paired clusters of thousands of neurons lying close to the uterine cervix and lateral lobes of the prostate, and contain many cell types. First, they contain neurons that each innervate a distinct region of the urogenital tract and lower bowel (Keast and de Groat, 1989; Kepper and Keast, 1995). Therefore, as discussed for identifying bladder sensory neurons in DRG, strategies such as retrograde tracing are valuable. Second, these target-defined neurons comprise both sympathetic and parasympathetic classes (Keast, 1995, 1999; Kanjhan et al., 2003; Jobling and Lim, 2008). Therefore, during development axons from two different regions of the spinal cord converge on the ganglia to synapse with the appropriate type of ganglion neuron. This mixture of sympathetic and parasympathetic neurons in one ganglion occurs nowhere else in the body. In addition to the pelvic ganglia, in rodents a few autonomic ganglion neurons are embedded in the bladder wall (Gillespie et al., 2006; Forrest et al., 2014), albeit are less common than intramural neurons in human bladder (Gilpin et al., 1983).

### Development of Bladder Innervation

Sensory and autonomic axons innervating the bladder mostly originate from neuronal cell bodies that are some distance from the bladder tissues although some neuronal cell bodies appear transiently in the bladder wall during development and early postnatal life, with very few remaining by adulthood (Zvarova and Vizzard, 2005). Their properties more closely resemble autonomic than sensory neurons (Zvarova and Vizzard, 2005; Forrest et al., 2014) but their function is unknown. Therefore, to understand developmental mechanisms, it is necessary to identifying the relevant neuronal populations within DRG and pelvic ganglia. This includes defining the cues that promote growth and survival, determine guidance and appropriate synaptic targeting, and specify appropriate signaling properties (e.g., ion channel expression, transmitter synthesis). Progress in most of these areas remains limited. Bladder tissues express a typical array of neurotrophic factors (Vizzard, 2000; Vizzard et al., 2000; Gonzalez et al., 2014), and many neurotrophic factor receptors have been identified in neurons within the pelvic ganglia and bladder sensory neurons of the lumbosacral DRG (Murray et al., 2004; Hiltunen et al., 2005; Palma and Keast, 2006; Vizzard, 2006; Forrest et al., 2013; Gonzalez et al., 2014), but their roles in development of bladder innervation have not been defined.

Nerves grow into the bladder tissues well before birth. Axons enter the human fetal urinary bladder by 13 weeks postconception and their density across each tissue type increases from that time (Kimmel and Mc, 1958; Klück, 1980; Gilpin et al., 1983; Dixon and Jen, 1995; Jen et al., 1995; Dixon et al., 1997, 1998, 1999). In these studies, immunohistochemistry for neuronal markers such as neuropeptides confirms that each of the major chemical classes of axons is likely to be present prior to birth. Moreover, neuronal cell bodies with transmitter chemistry similar to rodent pelvic ganglion neurons are embedded within the bladder wall and on its surface (Gilpin et al., 1983; Dixon et al., 1997); it is likely they are extensions of the inferior hypogastric plexus.

Our understanding of the timing and mechanisms of bladder innervation during embryonic development remains limited. In rodents, neural activation of the detrusor is evident just a few days after birth, but the properties of these contractions (including transmitter and receptor type) continue to change over the first couple of weeks (Levin et al., 1981; Maggi et al., 1984; Kruse and De Groat, 1990; Iuchi et al., 1994; Sann et al., 1997). Numerous images on the GUDMAP database (www.gudmap.org) (Harding et al., 2011) show that in embryonic mice, axons first reach the outer region of the detrusor by embryonic day (E) 14–15, and the urothelium by E18. At this time, axons can be distinguished by immunohistochemical markers that in adults would indicate parasympathetic, sympathetic and sensory axons. However, these may not be reliable markers in developing systems, where gene expression patterns may be quite dynamic. For example, tyrosine hydroxylase identifies adult noradrenergic sympathetic axons, but in development this enzyme is transiently expressed by a much broader neuron population (Rohrer, 2011). There is some early transient expression of cholinergic markers (Rohrer, 2011), and neuropeptide expression may be dynamic. Moreover, if expression of a particular neural marker is absent, it is tempting (but possibly inaccurate) to infer that the axon itself is not yet present.

Bladder-projecting sensory and autonomic neurons comprise only a minority of the neurons in pelvic ganglia and DRG, therefore studying their developmental mechanisms requires their identification (or separation) from surrounding neurons. Whereas, retrograde labeling can be conducted as simple recovery surgery in adult animals, comparable studies cannot be performed in the living embryo. Tracing nerve tracts in vitro by applying lipophilic dyes to fixed tissues (Ratcliffe et al., 2006) would be a valuable approach. This may also enable topographic maps to be constructed for developing bladderprojecting neurons, as performed previously for different types of neurons in pelvic ganglia of adult rats (Keast, 1999). In addition to correlating with expression patterns of transmitters, intriguingly expression patterns of transcription factors have been reported in developing mouse pelvic ganglia (Wiese et al., 2012), some of which may correlate with bladder-specific pathways. In parallel with the order of organ maturation, is also possible that bladder-projecting pelvic ganglion neurons differentiate and mature much earlier than those innervating reproductive organs. Therefore, to determine the mechanism by which the bladder becomes innervated, each target tissue within the bladder must be examined, a range of ganglion systems investigated and the neurons within those ganglia that project to bladder identified. Immunohistochemical tools enable distinction of some elements within this system, but without a specific molecular phenotype yet identified for bladderprojecting neurons, progress in defining the route and source of connections will be constrained. Understanding development of mechanisms by which nerves communicate with non-neuronal cells within the bladder, such as glial cells and interstitial cells (Gabella, 1995, 1999; McCloskey, 2011) is also important.

#### Genetic Models of Bladder Dysfunction

Animal models provide valuable insights into the factors contributing to particular bladder phenotypes and the mechanisms driving development of normal bladder innervation. Here we highlight some examples of mouse models with dysfunction due to enlarged bladders. We will discuss these in the context of possible neuronal impairment derived from loss of sensory transduction (to detect distension) or motor control (to enable contraction and emptying).

Deletion of the nicotinic acetylcholine receptor Chrna3, or the combined deletion of Chrnb2 and Chrna4, result in a bladder phenotype known as megacystis (Xu et al., 1999a,b). This is characterized by extreme bladder enlargement, overflow incontinence and bladder infection with urinary stones. These features are likely driven by neuronal dysfunction, as nicotinic receptors are expressed by autonomic ganglion neurons and required for transmitting the excitatory message from spinal neurons to ganglion cells, and hence to the organs. Specifically, Chrna3 and Chrnb4 are highly expressed in pelvic ganglia (Park et al., 2006; Girard et al., 2013) and are upregulated in parasympathetic pelvic ganglion neurons in a surgical obstruction model of the urethra (Chung et al., 2015). While not excluding other mechanisms, these reports are consistent with the bladder dysfunction being driven by an inability to void, resulting in distension and hypertrophy of the bladder.

#### References

Andersson, K. E., Soler, R., and Füllhase, C. (2011). Rodent models for urodynamic investigation. Neurourol. Urodyn. 30, 636–646. doi: 10.1002/nau. 21108

This model could provide additional insights into the impact of autonomic inactivity (rather than neuronal loss) on upstream components of the reflex circuitry. The effects of prolonged distension on sensory neurons could reveal mechanisms relevant to a number of clinical obstructive conditions.

A more bladder-specific disruption has been seen with "megabladder" mice (Singh et al., 2007; McHugh, 2014), a phenotype caused by randomly inserting a transgene into chromosome 16 that subsequently, along with a portion of chromosome 16, translocated to chromosome 11 downstream of myocardin. Profound changes, including severe distension and thinning of bladder wall were reported in utero, with the bladder almost completely lacking detrusor muscle; less dramatic changes occur in lamina propria and urothelium. This severe problem was restricted to lower urinary tract; muscle development was normal in the gastrointestinal and respiratory tracts, and vascular system. The innervation of these bladder tissues has not yet been examined but could reveal if and how a normal detrusor determines normal bladder innervation patterns. Furthermore, the phenotype was more severe in male, providing a platform to study sex differences in neuronal development.

A different outcome occurred after deleting FGFR2 (fibroblast growth factor receptor 2) from the bladder mesenchyme, leading to thinner detrusor muscle but in this case a thickened, collagen-enriched lamina propria (Walker et al., 2015). This impacted on muscle contractility in response to cholinoceptor and purinoceptor agonists and compliance of the bladder wall. Innervation density was not examined, but their decreased intervoiding interval in cystometry studies provides further encouragement to examine sensory nerve density and threshold for activation in these animals.

#### Conclusions

This review highlights the primary features of bladder innervation in the adult in order to demonstrate the gaps to be filled in understanding the development of this anatomically and functionally complex nerve supply. Defining the developmental mechanisms in this system may also reveal strategies to drive regrowth, targeting and functional recovery of bladder nerves in the adult.

#### Acknowledgments

This work was supported by the National Institutes of Health (5U01 DK094479 to JK) and the National Health and Medical Research Council of Australia (1003512 to PO and JK, 1022941 to JK).

Andersson, K. E., and Wein, A. J. (2004). Pharmacology of the lower urinary tract: basis for current and future treatments of urinary incontinence. Pharmacol. Rev. 56, 581–631. doi: 10.1124/pr.56.4.4

Arms, L., and Vizzard, M. A. (2011). Neuropeptides in lower urinary tract function. Handb. Exp. Pharmacol. 202, 395–423. doi: 10.1007/978-3-642-16499-6\_19

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Keast, Smith-Anttila and Osborne. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Interleukin-22 Signaling in the Regulation of Intestinal Health and Disease

Olivia B. Parks 1 , Derek A. Pociask 1, 2, Zerina Hodzic1 , Jay K. Kolls 1, 2 and Misty Good1, 3 \*

1 Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, 2 Department of Pediatrics, Richard King Mellon Foundation Institute for Pediatric Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, 3 Division of Newborn Medicine, Department of Pediatrics, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

#### Edited by:

Eiman Aleem, Phoenix Childrens Hospital and University of Arizona College of Medicine-Phoenix, USA

#### Reviewed by:

Lauren A. Zenewicz, The University of Oklahoma Health Sciences Center, USA Régis Josien, Nantes University Medical School, France

> \*Correspondence: Misty Good<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cell Growth and Division, a section of the journal Frontiers in Cell and Developmental Biology

Received: 14 August 2015 Accepted: 14 December 2015 Published: 13 January 2016

#### Citation:

Parks OB, Pociask DA, Hodzic Z, Kolls JK and Good M (2016) Interleukin-22 Signaling in the Regulation of Intestinal Health and Disease. Front. Cell Dev. Biol. 3:85. doi: 10.3389/fcell.2015.00085 Interleukin (IL)-22 is a member of the IL-10 family of cytokines that has been extensively studied since its discovery in 2000. This review article aims to describe the cellular sources and signaling pathways of this cytokine as well as the functions of IL-22 in the intestine. In addition, this article describes the roles of IL-22 in the pathogenesis of several gastrointestinal diseases, including inhibition of inflammation and barrier defense against pathogens within the intestine. Since many of the functions of IL-22 in the intestine are incompletely understood, this review is meant to assess our current understanding of the roles of IL-22 and provide new opportunities for inquiry to improve human intestinal health and disease.

Keywords: interleukin-22, gastrointestinal tract, epithelial cells, barrier defense

### INTRODUCTION

Since its discovery in 2000 (Dumoutier et al., 2000a), interleukin-22 (IL-22) has been widely studied for its diverse roles in cell proliferation, tissue regeneration, cellular defense, and inflammation. IL-22 is expressed by inflammatory cells in a number of tissues in the body including the lungs, liver, kidneys, thymus, pancreas, breast, gut, skin, and synovium, reviewed in Dudakov et al. (2015). The focus of this review article is on the distinct roles of IL-22 within the intestine. One of the main functions of IL-22 is to support and maintain the gastrointestinal (GI) epithelial barrier as well as to facilitate barrier defense mechanisms against bacterial pathogens such as Clostridium difficile (Hasegawa et al., 2014), Citrobacter rodentium (Zheng et al., 2008; Muñoz et al., 2015), and Toxoplasma gondii (Muñoz et al., 2015). Studies have shown that regulation of IL-22 is an important component of many diseases including graft-versus-host disease (GVHD) (Munneke et al., 2014), inflammatory bowel disease (IBD), specifically Crohn's disease (Wolk et al., 2007; Schmechel et al., 2008; Souza et al., 2013), ulcerative colitis and the experimental model of ulcerative colitis dextran sodium sulfate (DSS)-induced colitis (Sugimoto et al., 2008) as well as acute polymicrobial sepsis (Weber et al., 2007). The diverse effects of IL-22 against several inflammatory conditions implicate IL-22 as a promising therapeutic target for these types of GI-related illnesses. The purpose of this review article is to describe the distinct roles of IL-22 in the regulation of health and disease in the intestine.

## CELLULAR SOURCES OF IL-22

IL-22 is a member of the IL-10 family and has recently been grouped into a smaller subset of cytokines called the IL-20 subfamily, which is comprised of IL-19, IL-20, IL-22, IL-24, and IL-26 (Ouyang et al., 2011; Rutz et al., 2014). This family was identified based on their cellular targets and ability to recognize similar receptor subunits.

Many different types of immune cells produce IL-22. In lymphoid tissues, αβ and γδ T cells, innate lymphoid cells (ILCs; Cella et al., 2009; Colonna, 2009; Cella et al., 2010; Sonnenberg et al., 2011a; Hanash et al., 2012; Lee et al., 2012; Spits et al., 2013; Korn et al., 2014) and NK T cells (NKTs) all produce IL-22. In addition to the IL-22 produced in lymphoid tissue, macrophages, neutrophils (Zindl et al., 2013; Lee et al., 2015), dendritic cells (Mann et al., 2014), and fibroblasts (Ikeuchi et al., 2005) have also been identified as sources for IL-22; however, these nonlymphoid sources produce less IL-22 relative to the lymphoid sources. Depending on the tissue type, other non-traditional cell types can produce IL-22. For example, in the gastrointestinal tract of mice with experimental colitis, neutrophils become activated and are subsequently capable of expressing IL-22 (Zindl et al., 2013; Lee et al., 2015). In a mouse model of DSS-induced colitis, Zindl et al. demonstrated that depletion of Ly6G/C+ neutrophils/dendritic cells using RB6-8C antibody resulted in reduced colonic levels of IL-22, and additionally, these isolated colonic neutrophils were capable of producing IL-22 after IL-23 stimulation (Zindl et al., 2013). In a mouse model of infectious colitis, Lee and colleagues found the CD11b+ Ly6C+ Ly6G+ subset of neutrophils were the main source of IL-22 secretion (Lee et al., 2015). This type of non-traditional expression of IL-22 can be found in patients with diseases outside the GI tract such as rheumatoid arthritis (RA), where fibroblasts have been found to contribute to the production of IL-22 (Ikeuchi et al., 2005). Accordingly, cellular sources of IL-22 are numerous, and the main production sites of IL-22 in lymphoid-derived cells are described below:

## αβ T Cells

IL-22 production has been demonstrated by three subtypes of T helper (Th) cells: Th1, Th17, and Th22. The prototypical IFNγ producing Th1 cells can produce IL-22 without additional growth factors during effector function. However, naive T cells require the presence of transforming growth factor-β (TGF-β) to drive either the formation of Th17 cells, which can then produce IL-22, and/or T regulatory cells (Tregs; Littman and Rudensky, 2010). The cytokine IL-6 drives the expression of retinoic acid-related orphan receptor-γt (RORγt) and IL-23, both of which are critical for Th17 cells to produce IL-22 (Zhou et al., 2007). The regulation of Th17 cells is further controlled by changes in the concentration of TGF-β (Zhou et al., 2008). When the concentration of TGF-β is low, Th17 cells are induced via IL-23-stabilization of RORγt (Zhou et al., 2008). However, when TGF-β is present in high concentrations, expression of the IL-23 receptor is inhibited, reducing the expression of both RORγt and IL-22, while also activating the transcription factor forkhead box P3 (FoxP3; Zhou et al., 2008). This leads to the expansion of Tregs. Thus, high concentration of TGF-β promotes

Th22 cells also produce IL-22 and were first identified in the skin as a helper T cell population expressing CCR6, CCR4, and CCR10 (Duhen et al., 2009; Trifari et al., 2009). Th22 cells are classified as a subset of CD4+ helper T cells characterized for their production of IL-22, IL-13, and tumor necrosis factor (TNF)-α, but not IL-17, IFN-γ, or IL-4 (Duhen et al., 2009). Th22 cells are further described by their inability to produce T-bet (a transcription factor known to control IFN-γ) and negligible expression of RORγt (a Th17 and IL-22 transcription factor; Duhen et al., 2009). However, the development of CD4+ T cells capable of producing IL-22 is dependent on the aryl hydrocarbon receptor (AhR) and T-bet (Basu et al., 2012). Moreover, a recent study has demonstrated that activation of signal transducer and activator of transcription factor 3 (STAT3) was required for IL-22 production by Th22 cells and was responsible for effective host clearance of infectious colitis (Backert et al., 2014).

## γδ T Cells

γδ T cells are found in the intraepithelial lymphocyte (IEL) compartment of the intestine and secrete a variety of cytokines, including a significant amount of IL-22 (Sutton et al., 2009). γδ and αβ T cells share two features in common: (1) expression of RORγt and (2) expression of IL-22 in the presence of IL-23 (Martin et al., 2009; Sutton et al., 2009; Mabuchi et al., 2011; Mielke et al., 2013). Certain types of γδ T cells express Toll-like receptors (TLRs) and can directly interact with specific pathogen products (Martin et al., 2009; Crellin et al., 2010).

γδ T cells have roles in limiting the translocation of pathogens such as Salmonella typhimurium and T. gondii in the intestinal epithelial tissue (Edelblum et al., 2015). The γδ IELs contribute to the regulation and maintenance of gut homeostasis (Fuell et al., 2015). Studies have shown that mice deficient in γδ IELs (T Cell Receptor δ −/− mice) have structural differences within their intestine compared to wild-type littermates, including altered intestinal glycosylation and glycan antennae (Fuell et al., 2015). These structural differences can contribute to a lack of mucosal protection in the gut of mice deficient in γδ IELs, indicating γδ IELs and γδ T cells play a role in intestinal host defense (Edelblum et al., 2015; Fuell et al., 2015).

## Innate Lymphoid Cells (ILCs)

ILCs contribute to the innate and adaptive immunity of the intestine and can be found at mucosal surfaces or in cryptopatches, which are found in the lamina propria of the small intestine beneath the intestinal crypts (Diefenbach, 2012). ILCs are characterized by their lymphoid morphology, absence of cytotoxic capacity, and lack of B or T cell receptors as reviewed in Spits and Cupedo (2012) and Spits et al. (2013). ILCs are a relatively recent addition to the immune cell family and are grouped based on their functional characteristics and expression of cytokines and transcription factors (Spits et al., 2013). Group 1 ILC (ILC1s), defined by their expression of the transcription factor T-bet, produce the Th1 cytokine IFN-γ and are critical to host response of intracellular infections (Klose et al., 2014). Group 2 ILCs (ILC2s) are dependent on the transcription factor GATA3 to produce the Th2 cytokines IL-5 and IL-13 (Hoyler et al., 2012) and are important in helminth infection (Fallon et al., 2006). Group 3 ILC (ILC3s) are defined by their expression of RORγt, production of the Th17 cytokines IL-17 and IL-22, and involvement in defense against extracellular bacterial or fungal infections (Sonnenberg et al., 2011b; Gladiator et al., 2013; Edelblum et al., 2015) as described in **Table 1** (Eberl et al., 2015).

Moreover, CD4+ T cells can regulate ILC production of IL-22 as well as downstream production of antimicrobial peptides (AMPs) in an IFN-γ dependent manner (Korn et al., 2014). The AMPs evaluated in this study were regenerating islet derived 3 (Reg3) γ and β, members of the C-type lectin family (Gallo and Hooper, 2012), which are induced by IL-22 (Kolls et al., 2008; Zheng et al., 2008). Reg3β has been reported to kill Escherichia coli and may be involved in creating a niche of invading pathogens, specifically S. typhimurium (Stelter et al., 2011). In contrast, the related protein Reg3γ, which binds to peptidoglycan, has bactericidal activity against Gram-positive bacteria (Cash et al., 2006) and maintains a physical space between luminal bacteria and the epithelium (Vaishnava et al., 2011).

ILC3s can be found in the small and large intestinal mucosa, Peyer's patches and gut-associated lymphoid tissue (GALT; Cella et al., 2014). ILC3s include lymphoid tissue-inducer (LTi) cells and ILC3s that can be activated to express IL-22 by IL-23 through natural cytotoxicity receptor (NCR)+ and NCR− ILC3s (Spits and Cupedo, 2012; Spits et al., 2013). In both humans and mice, NCR+ ILC3s do not produce IFN-γ, which promote NK cell function, differing these cells from typical NK cells (Colonna, 2009; Cella et al., 2010; Spits et al., 2013). In addition, IL-15 and RORγt are essential for the development and maturation of NCR+ ILC3s (Satoh-Takayama et al., 2008; Cella et al., 2009; Luci et al., 2009; Sanos et al., 2009).

#### Natural Killer T Cells

All Natural Killer T (NKT) cells develop and mature in the thymus where they diverge from other cell types at the CD4+CD8+ double positive thymocyte stage (Mebius et al., 2001; Bendelac et al., 2007; Possot et al., 2011). Before this thymocyte stage, the αβ T cell receptor (TCR) of undifferentiated cells must recombine to bind to a CD1d molecule to progress developmentally (Bendelac et al., 2007). The NKT cell developmental cycle has unique stages of development (Bendelac et al., 2007; Godfrey et al., 2010) based on the NKT cells expression of CD24, CD44, or NK1.1 as well as the transcription factors PLZF, c-Myc, Egr2, RelA, or T-bet (Bendelac et al., 2007; Godfrey et al., 2010). NKT cells that produce IL-22 are also known to express CCR6, IL-23R, RORγt, paralleling γδ T cells, Th17 cells, and ILC3s (Bendelac et al., 2007; Godfrey et al., 2010). In addition, NKT cells produce IL-22 in the presence of IL-23 (Rachitskaya et al., 2008; Doisne et al., 2011; Moreira-Teixeira et al., 2011; Paget et al., 2012). Accordingly, NKT cells are similar to ILCs and γδ T cells in that these cell types can produce IL-22 without transcriptional activator interferon regulatory factor 4 (IRF4) signaling, whereas αβ T cells require IRF4 signaling to initiate IL-22 expression (Raifer et al., 2012). However, IL-22-producing NKT cells have been found to require interaction from TCR-CD1d to induce production of IL-22 (Doisne et al., 2011). Many details surrounding NKT cells that produce IL-22 are not well understood. A study has suggested that a stage during NKT cell development called "stage 0" (CD24+CD44loNK1.1−) is crucial to the maturation of these cells and is hypothesized to be controlled by RORγt (Benlagha et al., 2005); however, further studies are needed to completely understand the role of NKT cells in IL-22 production.

#### POSITIVE AND NEGATIVE REGULATORS OF IL-22

IL-22 is mainly produced by Th17 cells, ILCs, γδ T cells, and NKT cells (Ouyang et al., 2011). Th17 cells produce IL-22 in response to IL-6 and TNF-α in the setting of inflammation and trauma (Liang et al., 2006; Zhang et al., 2011). However, IL-6 can independently initiate the expression of IL-22 (Liang et al., 2006; Zheng et al., 2007). Il-22 gene expression, initially found only in the thymus and brain (Dumoutier et al., 2000a,b; Sabat et al., 2014), has been discovered in the gastrointestinal tract, liver, lung, skin, pancreas, and spleen (Sabat et al., 2014). IL-22 expression and production is positively and negatively regulated by several molecules including IL-23, IL-7, the Notch signaling pathway, the AhR, IL-22 binding protein (IL-22BP), IL-25, and IL-1β. Our continued study of these molecules can aid in the understanding IL-22, particularly in the setting of gastrointestinal disease.

#### Interleukin-23

IL-23 is one of the main inducers of the expression and production of IL-22 (Kastelein et al., 2007). During differentiation of Th17 cells, IL-23 enhances IL-22 expression, which leads to increased expression of the IL-23 Receptor (IL-23R). This results in enhanced interaction between IL-23 and its receptor, and consequently, increased IL-22 production. Activated dendritic cells (DCs) and macrophages, in response to microbial stimulation, are important sources of IL-23 (Langrish et al., 2004). IL-23 production by DCs is regulated by the lymphotoxin-β receptor (LTβR; Tumanov et al., 2011). IL-23 production by activated DCs via LTBR indirectly results in increased IL-22 by RORyt+ ILCs (Tumanov et al., 2011).

However, there are other cellular sources of IL-23. DSSinduced epithelial injury results in activation of LTβR signaling, which promotes intestinal mucosal healing by triggering production of IL-23 from intestinal epithelial cells (Macho-Fernandez et al., 2015). This ultimately results in increased IL-22 production by RORyt+ ILC3s, particularly CCR6+Tbet- CD4 and CD4+ LTi cells (Macho-Fernandez et al., 2015).

#### Interleukin-7

IL-7, a critical cytokine for the maintenance, development, and proliferation of αβ and γδ T cells, also acts to positively regulate the production of IL-22 (Cella et al., 2010). It is unlikely that the cytokine IL-7 directly regulates the expression of IL-22; however, IL-7 is necessary for the stable expression of RORγt, a transcription factor important in IL-22 expression (Vonarbourg et al., 2010). RORγt expression regulates the differentiation of cells that produce IL-22 to achieve the optimal conditions for the production of IL-22 (Nurieva et al., 2007; Qiu et al., 2012). These observations would suggest that IL-7 acts in an expansion role to promote the expression of IL-22 by many types of cells. However, IL-7 is not directly required for functional IL-22 cytokine to be produced in tissues (Peschon et al., 1994; von Freeden-Jeffry et al., 1995; Nurieva et al., 2007; Vonarbourg et al., 2010; Qiu et al., 2012).

## Notch

Notch-induced stimulation of CD4+ T cells increases the production of IL-22 within the intestine, which is important for epithelial cell proliferation and differentiation (Murano et al., 2014). Overexpression of Hes1, a Notch target gene, enhanced IL-22-induced STAT3 expression in a human intestinal epithelial cell line (Murano et al., 2014). However, in Notch-deficient mice, IL-22 signaling and production was eliminated (Alam et al., 2010; Murano et al., 2014). Lee et al. demonstrated that the Notch receptor was induced by the AhR (Lee et al., 2012), which promotes the development of ILCs and Th17 cells, ultimately leading to increased IL-22 production.

## Aryl Hydrocarbon Receptor (AhR)

The AhR induces IL-22 in one of two ways. AhR can either (1) directly regulate IL-22 gene expression and cytokine production or (2) regulate the production and development of ILC3 and Th17 cells (Veldhoen et al., 2008; Lee et al., 2012; Qiu et al., 2012). AhR is found in the cytoplasm complexed with heat shock protein 90 (Hsp90; Esser et al., 2009), and once activated, the AhR complex can translocate to the nucleus where AhR can act as a transcription factor (Tsuji et al., 2014). Various ligands, physical stress, cyclic AMP, and calcium (Ca2+) can all activate AhR. It is thought that AhR ligands derived from gut microbiota are not required for the development of ILCs (Veldhoen et al., 2008; Esser et al., 2009; Qiu et al., 2012). However, these ligands are thought to be an essential component to initiate the transcription of IL-22 (Denison and Nagy, 2003; Oesch-Bartlomowicz et al., 2005; McMillan and Bradfield, 2007; Nguyen and Bradfield, 2008; Puga et al., 2009; Lee et al., 2012; Zelante et al., 2013; Lowe et al., 2014).

## Interleukin-1β

IL-1β is an influential cytokine, capable of activating NKT cells, ILC3s, and Th17 cells to produce IL-22 (Sutton et al., 2009; Doisne et al., 2011; Paget et al., 2012; Chen et al., 2013; Lee et al., 2013; Monteiro et al., 2013). Macrophages, DCs, neutrophils, B and T cells, endothelial cells, and epithelial cells all produce IL-1β, making this ligand a diverse molecule in cellular pathways (Sims and Smith, 2010). IL-1β production sustains expression of IL-22 in vitro, promotes the expansion of NK cells, specifically, human stage three immature NK cells, in conjunction with IL-15 from secondary lymphoid tissue DCs, and inhibits these NK cells from differentiating into IFN-γ-producing cells (Sutton et al., 2009). Of note, in IL-1 receptorhi immature NK cells, IL-1β must be constantly present in order for continual expression of IL-22. This is in direct contrast to IL-23, which only needs to be present to initiate the IL-22 signaling pathway so that IL-22 production continues in the absence of IL-23 (Hughes et al., 2010).

## Negative Regulation of IL-22

There are several molecules that negatively regulate IL-22 expression. IL-22 Binding Protein (IL-22BP, also known as IL-22RA2) is a soluble receptor that is able to regulate IL-22 bioactivity, which has a 1000 times higher binding affinity for IL-22 compared to the IL-22 receptor (IL-22RA1) complex (Weiss et al., 2004; Wolk et al., 2007). Epithelial expression of IL-25 is able to repress IL-22 production by RORyt+ ILCs (Weiss et al., 2004; Wolk et al., 2007). Transforming growth factor-β (TGF-β) has several functions, specifically with respect to Th17 cells, RORγt, IL-23R, and IL-22 (Mangan et al., 2006; Zhou et al., 2007; Morishima et al., 2009). TGF-β is essential for the differentiation of Th17 cells and influences RORγt and IL-23R expression in various tissues (Morishima et al., 2009). In IL-22 signaling, TGF-β acts in a dose-dependent fashion to regulate the expression of IL-22 (Zheng et al., 2007; Volpe et al., 2009; Rutz et al., 2011; Penel-Sotirakis et al., 2012). However, the cytokine IL-23 is able to overcome the effects of TGF-β on IL-22, thereby increasing production of IL-22 (Volpe et al., 2009; Rutz et al., 2011). Furthermore, both the transcription factor c-Maf (an inhibitor of TGF-β, IL-22, IL-27) and the inducible costimulator (ICOS) pathways can influence IL-22 production (Bauquet et al., 2009; Paulos et al., 2010; Rutz et al., 2011). A recently described and poorly understood cytokine member of the IL-1 family, IL-38, can also regulate IL-22 production. At low concentrations, IL-38 can prevent the production of IL-22, while at high concentrations, IL-38 promotes the production of IL-22 (Tortola et al., 2012; van de Veerdonk et al., 2012). However, further studies are required to determine the signaling pathways involved with IL-38 and their biologic effects.

### IL-22 Binding Protein

IL-22RA2, alternatively called IL-22BP, has been found throughout the body. It is a soluble secreted receptor with a binding structure similar to the membrane bound IL-22RA1 (Dumoutier et al., 2001; Wu et al., 2008). This structural homology allows IL-22BP to bind to IL-22, thereby inhibiting the binding of IL-22 to its receptor complex and consequently preventing IL-22 signaling (Wu et al., 2008). Interestingly, it has been observed that IL-22BP levels decrease with significant increases in IL-22 (Weiss et al., 2004; Wolk et al., 2007). IL-22BP levels increase only after persistently high levels of IL-22, indicating that IL-22BP has a regulatory role after the initial effects of the elevated IL-22 levels have been established (Wolk et al., 2007; Sugimoto et al., 2008; Huber et al., 2012). In the intestine, IL-22BP is highly expressed in colonic dendritic cells (Huber et al., 2012). When tissue damage in the intestine is detected, the NLRP3 or NLRP6 inflammasomes down-regulate IL-22BP via activation of IL-18 (Huber et al., 2012). Additionally, Martin et al. discovered a subset of conventional dendritic cells, lamina propria CD103+CD11b+ DCs, in both lymphoid and non-lymphoid tissues as a significant source of IL-22BP (Martin et al., 2014). Additionally, eosinophils within the human intestine were also identified as an important source of IL-22BP (Martin et al., 2015).

## IL-22 RECEPTOR AND SIGNALING

The IL-22 receptor is a type 2 cytokine receptor comprised of the heterodimeric complex with IL-22RA1 and IL-10R2 (IL-10Rβ; Kotenko et al., 1997, 2001; Xie et al., 2000; Dumoutier et al., 2000c; Li et al., 2004). While IL-10R2 is constitutively expressed in cells throughout the body, IL-22RA1 is expressed almost exclusively in epithelial tissues (Wolk et al., 2004). Due to this specificity of IL-22RA1, it is hypothesized that this receptor has a defining role in facilitating the innate immunity of epithelial cells (Zheng et al., 2007, 2008). Interestingly, IL-22 has been shown to have no affinity for IL-10R2 and rather a very high affinity to bind to IL-22RA1 (Logsdon et al., 2002, 2004; Li et al., 2004; Wolk et al., 2004; Jones et al., 2008; Yoon et al., 2010). Binding of IL-22 to IL-22RA1 increases its affinity for IL-10R2 (Logsdon et al., 2002; Li et al., 2004; Logsdon et al., 2004; Wolk et al., 2004; Jones et al., 2008; Yoon et al., 2010), suggesting a stepwise process in the binding (Bleicher et al., 2008).

IL-22 signals through the IL-22 receptor complex, leading to activation of Janus kinase 1 (Jak1) and non-receptor protein tyrosine kinase 2 (Tyk2; Lejeune et al., 2002). This primarily leads to tyrosine residue phosphorylation of STAT3; however, STAT1 and STAT5 have also been shown to be activated by IL-22 (Lejeune et al., 2002). STAT3 activation in the intestinal epithelium is responsible for immune homeostasis as well as wound healing in an IL-22 dependent manner (Pickert et al., 2009). Several additional pathways are involved in IL-22 signaling, including Mitogen Activation Protein Kinase (MAPK), Akt (Sekikawa et al., 2010), and p38 pathways (Andoh et al., 2005).

## IL-22 REGULATION OF HEALTH AND DISEASE: GASTROINTESTINAL TRACT