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The cytokine IL-22 is primarily expressed by Th17 CD4 T cells and is highly upregulated during chronic inflammatory diseases. IL-22 receptor expression is absent on immune cells, but is instead restricted to the tissues, providing signaling directionality from the immune system to the tissues. However, the role of IL-22 in inflammatory responses has been confounded by data suggesting both pro- and ant-inflammatory functions. Herein, we provide evidence that during inflammation IL-22 plays a protective role in preventing tissue injury. Hepatocytes from mice deficient in IL-22 are highly sensitive to the detrimental immune response associated with hepatitis. Additionally, IL-22 expressing Th17 cells can provide protection during hepatitis in IL-22 deficient mice. On the other hand, IL-17, which is co-expressed with IL-22 and can induce similar cellular responses, has no observable role in liver inflammation. Our data suggest that IL-22 serves as a protective molecule to counteract the destructive nature of the immune response to limit tissue damage.
Chronic inflammation of the liver inflicted by long-term infections such as hepatitis B or C viruses or alcohol abuse often leads to liver disease and cancer later in life. Such inflammation is associated with T cell activation and the secretion of numerous pro-inflammatory cytokines, such as IFNγ, that damage hepatocytes. In addition to the pro-inflammatory factors secreted by T cells, protective cytokines, such as IL-10, allow for more precise regulation of highly destructive T cell responses by down-regulating the activities of a variety of cells of the immune system. On the other hand, regulatory mechanisms are needed to regulate tissue responses during inflammation. T cells secrete IL-22, a cytokine that specifically signals to the tissues and is a good candidate to mediate protective functions.
IL-22 was discovered as a gene upregulated by CD4 T cells upon activation which shares 22% amino acid sequence identity with IL-10; it was thus originally named IL-10-related T cell-derived inducible factor (IL-TIF) (Dumoutier et al., 2000a; Dumoutier et al., 2000b). However, unlike IL-10, IL-22 expression appears to be limited to T cells, NK cells and NK T cells since IL-22 mRNA cannot be detected in resting or activated monocyte-derived cells or B cells (Wolk et al., 2004). In vivo, within 2 hours of LPS injection of mice, IL-22 mRNA is upregulated in many organs, including the spleen, liver, kidneys and lung (Dumoutier et al., 2000b). IL-22 has been found in diseased tissues from patients with different chronic inflammatory diseases that involve infiltrating activated T cells, such as rheumatoid arthritis, psoriasis and inflammatory bowel disease (Andoh et al., 2005; Ikeuchi et al., 2005; Wolk et al., 2004). However, this correlation between IL-22 expression and inflammation has been further confounded by contrary data indicating both pro- and anti-inflammatory roles for the cytokine. Most recently Zheng et al. showed that IL-22 is important for mediating IL-23 induced dermal inflammation in a mouse model of psoriasis indicating a pro-inflammatory role (Zheng et al., 2007).
IL-22 signals through a heterodimer of the receptor chains IL-22R and IL-10Rβ(Kotenko et al., 2001). Since IL-10Rβ is broadly expressed by many different cell types, IL-22R expression is the limiting component which determines IL-22 responsiveness of cells. IL-22R is expressed strongly in the liver, as well as the skin, lungs, pancreas, and other peripheral tissues (Aggarwal et al., 2001; Wolk et al., 2004). Resting and activated T cells do not express IL-22R and IL-22 stimulation has no observable effects on T cells (Wolk et al., 2004). Extensive screening of different cell lines has revealed that only cells which express IL-22R respond to IL-22 suggesting that there is no alternate receptor that can mediate IL-22 signaling (Wolk et al., 2005). Thus, it appears that the effects of IL-22 are limited to peripheral tissues and not on cells of the immune system.
IL-22 stimulation of IL-22 receptor expressing cells, such as hepatocytes, results in activation of Stat3 signaling pathways (Boniface et al., 2005; Lejeune et al., 2002; Nagalakshmi et al., 2004; Radaeva et al., 2004; Wolk et al., 2004; Xie et al., 2000). Stat3 signaling leads to the induction of genes involved in many diverse processes, including apoptosis, cell cycle progression, and angiogenesis that contribute to both wound healing and cancer (Dauer et al., 2005). Stat3 is critical to proper mouse development and Stat3 deficient mice die early in embryogenesis (Takeda et al., 1997). Although liver-specific Stat3 deficient mice have no abnormalities in liver development, they do have a reduced ability to recover from liver damage (Li et al., 2002). IL-22 activation of Stat3 in hepatocytes results in upregulation of expression of acute phase reactants, such as serum amyloid A (SAA), α1-antichymotrypsin, and haptoglobin and in vitro IL-22 protects hepatocytes from serum starvation-induced apoptosis (Dumoutier et al., 2000b; Radaeva et al., 2004). Thus, IL-22-mediated activation of Stat3 may play an important role in hepatocyte survival in vivo. A previous study suggested that IL-22 may play a protective role during acute liver inflammation (Radaeva et al., 2004). Injection of recombinant IL-22 prior to treatment with several different inflammatory molecules reduced the amount of hepatocyte damage in the liver. In addition, injection of IL-22 neutralizing antibodies immediately after the inflammatory molecules, exacerbated hepatic injury.
The same subset of CD4 T cells that express IL-22, also express IL-17 (Liang et al., 2006). IL-17 is highly expressed by a newly defined subset of helper T cells, Th17 cells (Weaver et al., 2006). Distinct from Th1 or Th2 cells, differentiation of these cells is dependent on the presence of both TGF-β and pro-inflammatory cytokines such as IL-6 and TNFα (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006). Both IL-17 and IL-22 are upregulated in the lesions of patients with various chronic inflammatory diseases, such as psoriasis, inflammatory bowel disease and rheumatoid arthritis (Andoh et al., 2005; Chabaud et al., 1998; Fujino et al., 2003; Liang et al., 2006; Teunissen et al., 1998; Wolk et al., 2004). Moreover, these cytokines have been shown to have additive in vitro effects on the induction of antimicrobial peptides in keratinocytes or cytokines by colon myofibroblasts (Andoh et al., 2005; Liang et al., 2006). These data suggest that IL-22 and IL-17 may serve similar roles in vivo during inflammation.
In this study, we show that IL-22 is important in vivo for limiting hepatocyte damage during acute liver inflammation. Under specific-pathogen free conditions IL-22 deficient mice have normal immune systems and liver pathology. However, these mice are extremely susceptible to liver damage during conA-mediated hepatitis and thus, IL-22 protects hepatocytes from the destructive effects of activated T cells. We show that IL-22 expressing Th17 cells, but not IL-22 deficient Th17 cells, can provide protection to hepatocytes during hepatitis. However, although IL-17, like IL-22, is induced during conA-mediated hepatitis, our data suggest that IL-17 does not play an observable role in disease pathogenesis in this model. Therefore, although IL-22 and IL-17 are co-expressed by inflammatory T cells, these cytokines can have distinct functions in the host inflammatory response.
To investigate the in vivo role of IL-22 during liver inflammation, we generated IL-22 deficient mice (Supplemental Figure 1). IL-22 deficient mice were viable, fertile, and pups were born at the expected Mendelian ratio. Upon histological examination of different organs, including the liver, spleen, kidney, skin, kidney, pancreas, and thymus, we observed no abnormalities at 8 weeks of age (Supplemental Figure 2). We also compared the total numbers and activation status of different immune cell subsets; CD4 T cells, CD8 T cells, NK T cells, and NK cells, in the spleens and livers of IL-22 deficient and wild-type littermates and found no significant differences (data not shown). After TCR stimulation, CD4 T cells from wild-type but not IL-22 deficient mice, had detectable levels of IL-22 mRNA or secreted protein (Supplemental Figure 3).
Since IL-22 is highly expressed by CD4 T cells, as well as NK cells and NK T cells, we wanted to determine whether IL-22 deficiency resulted in any in vivo defects in innate or adaptive immunity. To analyze this we used the infection model of Listeria monocytogenes in which both innate and adaptive immune responses have been well characterized. This facultative intracellular Gram-positive bacterium induces both NK cell and CD4 T cell responses. NK cells play a role in the clearance of bacteria by secreting IFNγ that activates macrophages. L. monocytogenes infection also generates strong L. monocytogenes-specific CD4 T cell responses. To examine innate immunity, we therefore infected IL-22 deficient and wild-type littermate control mice with 5×104 CFU of wild-type L. monocytogenes. Three days post-infection, the peak of the innate immune response, we observed no difference in bacterial loads in the spleens and livers between IL-22 deficient and wild-type mice (Supplemental Figure 4). To examine the antigen-specific T cell response, we infected wild-type and IL-22 deficient mice with 1×104 CFU of recombinant strain of L. monocytogenes that expresses ovalbumin (rLM-ova). At seven days post-infection both IL-22 deficient and wild-type mice had an increased percentage of activated cells (CD44high, CD62Llow) in their spleens compared to uninfected mice (Figure 1C). To quantitate the antigen-specific response, lymphocytes from the spleens and livers of infected mice were stimulated in vitro with the MHC class II-restricted L. monocytogenes-derived epitope, LLO190–201, or the MHC class I-restricted epitope, OVA257-261. Both IL-22 wild-type and deficient mice generated similar percentages of LLO190–201 specific cells in their spleens and livers (Figure 1D), as well as OVA257–261 specific cells (Supplemetnal Figure 5). Thus, IL-22 deficient mice appear to have normal innate and adaptive immune responses against the pathogen L. monocytogenes.
IL-22 is expressed by effector CD4 T cells, NK cells, and NK T cells, the populations of which are all enriched in the mouse liver. However, these cells do not express IL-22R. As reported in the literature, IL-22R mRNA expression was not detectable in immune cell subsets, including CD4 T cells, macrophages, and B cells, but these cells did express IL-10Rα and IL-10Rβ mRNA (Figure 2A). On the other hand, as previously shown by other studies, hepatocytes expressed high levels of IL-22R and IL-10Rβ mRNA, whereas IL-10Rα mRNA expression was undetectable (Figure 2A). Stimulation of the murine hepatocyte cell line FL38B with recombinant murine IL-22, but not IL-10, resulted in activation of Stat3 and Akt as observed by increased phosphorylation of these two signaling molecules (Figure 2B and D). As expected, recombinant murine IL-10, but not IL-22, induced Stat3 activation in bone-marrow derived macrophages (Figure 2C). These data indicate that murine hepatocytes are a target of IL-22, but not IL-10.
Hepatitis is a complex of diseases of the liver in which activated T cells are frequently responsible for mediating hepatocyte damage. An experimental model of this disease is conA-mediated hepatitis, in which the lectin concanavalin A (conA) is injected intravenously into mice and is selectively retained by the liver. This stimulates the CD4 and NK T cells in a polyclonal manner, inducing a rapid T cell-mediated liver disease due to upregulation of both IFNγ and FasL (Takeda et al., 2000; Tiegs et al., 1992; Toyabe et al., 1997). Injection of mice with conA also leads to rapid induction of IL-22 mRNA in liver lymphocytes (Figure 3). To examine the role of IL-22 in acute liver inflammation, we injected IL-22 deficient, IL-22 heterozygous and IL-22 wild-type control mice with conA. After injection with 10 μg/g mouse conA, some IL-22 deficient mice (32%) and IL-22 heterozygous mice (25%) succumbed to mortality before 18 hrs post-injection, however no wild-type mice ever exhibited any outward signs of morbidity. At greater doses of conA, 20 μg/g mouse, 100% of IL-22 deficient and heterozygous mice died by 18 hrs post-injection, whereas IL-22 wild-type mice survived.
To examine if this mortality was associated with increased levels of liver damage, we examined serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as liver histology. After conA treatment, compared to wild-type or heterozygous mice, IL-22 deficient mice had significantly higher levels of ALT and AST in their sera, indicative of liver damage (Figure 4C and Supplemental Figure 6). No differences were observed in the ALT levels of untreated mice indicating that the IL-22 deficient mice do not have liver disease under non-experimental conditions (Figure 4B). Upon examination of liver pathology, IL-22 deficient mice injected with conA had more extensive lesions, as well as a greater number of lesions in their livers (Figure 4A). Lesions were most commonly found on the perimeter of the lobes, especially the left and median lobes. Mice heterozygous for the IL-22 gene had an intermediate phenotype between IL-22 deficient and IL-22 wild-type mice and thus were excluded from the remainder of our studies.
IL-22 deficient mice have more extensive liver damage in response to conA-mediated hepatitis than wild-type mice. Since IL-22 is expressed by T cells, along with the potent cytokines that mediate disease such as IFNγ, we examined if IL-22 deficient and wild-type control mice have equal immune responses to conA-mediated hepatitis. IL-22 deficient and wild-type control mice had equal induction in the liver of IFNγ, TNFα, IL-4, IL-15, IL-6 and IL-10 mRNA six hours after conA administration, as well as IFNγ and TNFα levels in their sera (Figure 5A and 5B). In addition, CD4 T cells from the spleens and livers of wild-type and IL-22 deficient mice both rapidly upregulated surface expression of the early activation marker CD69 (Figure 5C). Thus, the increased level of liver damage observed in IL-22 deficient mice after conA administration is not due to an increased immune response in these mice, but instead suggested that it may be due to increased susceptibility of hepatocytes to the destructive immune response.
An alternate hypothesis to explain the increased hepatic lesions in IL-22 deficient mice during inflammation is that these hepatocytes have a greater intrinsic susceptibility to apoptosis. To examine if hepatocytes from IL-22 deficient mice were more susceptible to apoptosis independent of inflammation, and hence in the absence of IL-22, we compared hepatocyte responses to an activating anti-Fas Ab (clone Jo2). Injection of mice with this Ab leads to hepatocyte apoptosis since these cells are particularly sensitive to Fas-FasL mediated killing (Ogasawara et al., 1993). Injection of mice with anti-Fas Ab did not induce IFNγ mRNA in the liver, unlike conA injection (Figure 6C). However, Ab injection did induce equal levels of TNFα in the livers of IL-22 wild-type and deficient mice (Figure 6D). As expected, IL-22 mRNA was not detected in the spleen or liver after anti-Fas Ab treatment (data not shown). IL-22 deficient and wild-type control mice both had comparable sera ALT levels 6 hrs post-injection (Figure 6B). In addition, we observed no difference in hepatocyte injury between IL-22 deficient and control mice (Figure 6A). Thus, in the absence of IFNγ-induced inflammation, wild-type and IL-22 deficient mice had similar levels of liver damage, indicating that hepatocytes of IL-22 deficient mice are not more intrinsically susceptible to apoptosis.
The susceptibility of IL-22 deficient mice to conA-mediated hepatitis is reminiscent of the phenotype observed in IL-6 deficient mice. IL-6 deficient mice also have greater hepatocyte damage after conA-mediated hepatitis than wild-type mice, which correlates with reduced levels of Stat3 activation in hepatocytes of IL-6 deficient mice (Hong et al., 2002; Tagawa et al., 2000). Furthermore, IL-6 is an important cytokine in the in vitro differentiation of IL-22-expressing Th17 cells (Liang et al., 2006; Zheng et al., 2007). In the absence of TGF-β or when there are only very low amounts of TGF-β, IL-6 preferentially leads to differentiation of Th17 cells expressing substantially greater levels of IL-22 than IL-17, whereas IL-17 expression is greater than that of IL-22 in the presence of higher levels of TGF-β (Zheng et al., 2007). Therefore to investigate if IL-6 is required in vivo for the induction of IL-22 during conA-mediated hepatitis, we compared IL-22 expression between wild-type and IL-6 deficient mice. By two hours post-conA injection, IL-6 mRNA was induced in the livers of wild-type mice and decreased by 4 hours (Figure 7). As expected, no IL-6 mRNA was detected in IL-6 deficient mice. At both 2 and 4 hrs post-conA injection, there was no significant difference in IL-22 mRNA induction between IL-6 deficient and wild-type control mice. Thus, IL-6 is dispensable in vivo for induction of IL-22 in the liver during hepatitis.
Both IL-22 and IL-17 are expressed by inflammatory T cells and both cytokines are upregulated in the lesions of chronic inflammatory diseases such as psoriasis, inflammatory bowel disease and rheumatoid arthritis (Andoh et al., 2005; Chabaud et al., 1998; Fujino et al., 2003; Liang et al., 2006; Teunissen et al., 1998; Wolk et al., 2004). Recent studies have observed that the effects of IL-22 on cells are similar to the effects of IL-17, and showed that stimulation of cells with both cytokines has an additive effect. Both cytokines induce expression of antimicrobial peptides in keratinocytes, as well as cytokine secretion by colon myofibroblasts (Andoh et al., 2005; Liang et al., 2006). As for IL-22, stimulation of some cell types with IL-17 activates Akt and Stat3 (Hwang et al., 2004; Subramaniam et al., 1999). As we had observed for IL-22, IL-17 mRNA was induced in the spleen and liver upon conA injection (Figure 8A) and IL-17A protein was detected in the sera (Figure 8B). Thus, we hypothesized that IL-17 may also play a role during acute liver inflammation.
IL-17 deficient mice, however, exhibited no difference in liver injury compared to wild-type mice 18 hrs post-conA injection. Both groups had similarly elevated ALT levels, as well as limited necrotic lesions in their livers (Figure 8C and D). To determine if IL-17 could play a role in hepatitis in the absence of IL-22, we generated IL-22/IL-17 double deficient mice. ConA injection of these mice led to extensive hepatocyte injury (Figure 8C and D). However, this damage was not significantly different from that observed in IL-22 deficient mice. These data suggest that IL-17 does not protect from liver damage during hepatitis, nor does it appear to increase the damage, and thus, IL-17 appears to play no role in hepatocyte damage during conA-mediated hepatitis.
Since we did not observe differences in liver damage between wild-type and IL-17 deficient mice, or between IL-22 deficient mice and IL-22 and IL-17 double deficient mice, we investigated the in vitro hepatocyte response to IL-17. To determine if hepatocytes respond to IL-17, we stimulated FL38B cells with IL-22 or IL-17 and examined if there was subsequent Stat3 and/or Akt activation. As previously observed, IL-22 induced both Stat3 and Akt phosphorylation (Figure 8E). However, hepatocytes did not respond in this way to IL-17 stimulation. Therefore, unlike the protective role of IL-22 on hepatocytes during liver inflammation, IL-17 expressed in the liver during hepatitis does not appear to play a role in hepatocyte responses to inflammation. Although these cytokines are co-expressed by activated T cells, and have similar effects on keratinocytes and colon myofibroblasts, IL-22 and IL-17 have distinct roles during acute liver inflammation.
To determine if IL-22-expressing CD4 T cells are able to provide protection to hepatocytes during hepatitis, we employed an adoptive transfer model. Naïve IL-22 wild-type or deficient CD4 T cells were differentiated into effector cells under Th17-polarizing conditions for 5 days. Compared to restimulated cells cultured under neutral (Th0) conditions, restimulation of these cells resulted in 1000-fold induction of IL-22 and over 10,000-fold induction of IL-17, as well as low levels of IFNγ (Figure 9A). IL-22 wild-type and deficient cells expressed equal levels of IL-17 and IFNγ. 5×106 non-restimulated Th17 cells, either IL-22 wild-type or deficient, were transferred into IL-22 deficient host mice and then mice were injected with conA to induce hepatitis. IL-22 deficient mice that received wild-type cells had significantly reduced serum levels of ALT and AST at 6 and 18 hrs post-conA (Figure 9B and C). These data indicate that IL-22 expressing Th17 cells are able to provide protection to hepatocytes during conA-mediated hepatitis.
IL-22 has been most commonly described as a pro-inflammatory cytokine due to its expression in lesions of patients with chronic inflammatory diseases and its induction of pro-inflammatory cytokines such as IL-6, IL-8 and TNFα (Andoh et al., 2005; Brand et al., 2006). IL-22 has recently been shown to be an important mediator in vivo for dermal inflammation (Zheng et al., 2007). However, other data suggest that IL-22 has a less direct inflammatory role and instead induces expression of genes associated with antimicrobial defense and cellular differentiation (Boniface et al., 2005; Wolk et al., 2004; Wolk et al., 2006). Our data indicate that in vivo IL-22 also functions to protect hepatocytes from the destructive effects of immune cells in the inflamed liver. In an acute hepatitis model, mice deficient in IL-22 exhibited extensive liver damage compared to wild-type mice.
IL-22 stimulation of hepatocytes leads to activation of both Stat3 and Akt signaling pathways. Triggering both of these well-described pathways leads to activation of cell survival genes that are involved in promoting cell-cycle progression, cellular transformation, and in preventing apoptosis, as well as specifically involved in liver regeneration (Haga et al., 2005; Li et al., 2002). IL-22 protects hepatocytes from in vitro serum starvation in a Stat3-dependent manner, potentially through induction of anti-apoptotic proteins such as Bcl-2 and Bcl-xL (Radaeva et al., 2004). IL-22 also has mitogenic activities and induces expression of such proteins as c-myc and cyclin D1 (Radaeva et al., 2004), however this activity probably plays little role in our rapid phenotype during conA-mediated hepatitis. The role of Akt in hepatocytes is less well elucidated, but has been shown to be compensatory for Stat3 in its absence (Haga et al., 2005). We speculate that these signaling pathways may synergize to induce expression of anti-apoptotic factors. During conA-mediated hepatitis, we found that mRNA of the anti-apoptotic Bcl-2 was induced approximately 1000-fold in the liver (data not shown). However in IL-22 deficient mice, this induction was attenuated and only about 200-fold. Thus, IL-22 itself may not induce pro-inflammatory responses, but instead is an important component of the inflammatory response to limit tissue damage.
IL-22 has homology to IL-10 and its heterodimeric receptor shares use of the IL-10Rβ chain. Due to the mutual exclusive expression of IL-10Rα and IL-22R, no known cell type is responsive to both cytokines. IL-10 is a master regulator of the immune response which mediates down-regulation of pro-inflammatory cytokine expression in macrophages, T cells and other cells of the immune system (Moore et al., 2001). IL-10 is responsible for dampening the immune response which indirectly protects the host from potentially detrimental immunopathologic effects. On the other hand, IL-22 has no observable effects on cells of the immune response but instead acts primarily on the tissues (Wolk et al., 2004). In a sense, during inflammation IL-22 directly protects the host’s tissues from the destructive immune response. Thus, both IL-10 and IL-22 may have evolved to safeguard the host from a potentially overwhelming immune response by acting on immune and tissue cells, respectively.
IL-22 may be structurally homologous to IL-10, but IL-22 has recently been shown to be co-expressed with a structurally unrelated cytokine, IL-17. These two cytokines are expressed by the newly identified Th17 CD4 T cell subset (Chung et al., 2006; Liang et al., 2006). IL-6 acts as a sufficient cytokine for the in vitro differentiation of IL-22 expressing Th17 cells contrary to the requirement of additional TGF-β for the optimal expression of IL-17 (Zheng et al., 2007). However, our data provide evidence that IL-6 is not required for the in vivo generation of IL-22 expressing cells since IL-6 deficient mice express IL-22 during conA-mediated hepatitis. We have shown herein that IL-22 expression by these cells can mediate hepatocyte protection during hepatitis.
As for IL-22, IL-17 has been described to be a pro-inflammatory cytokine, however, like IL-22, much remains to be elucidated about the in vivo role of this cytokine in inflammation. IL-17 induces expression of IL-6 and IL-8, is important for neutrophil infiltration and is critical for full disease pathogenesis of the highly inflammatory experimental autoimmune encephalitis model (Fossiez et al., 1996; Hofstetter et al., 2005; Komiyama et al., 2006; Stark et al., 2005; Yao et al., 1995). Although IL-22 and IL-17 have been shown to have similar effects on keratinocytes and colon myofibroblasts, we have shown that they do not have parallel effects in the liver. Unlike IL-22 deficient mice, IL-17 deficient mice have no difference in their response to conA-mediated hepatitis although conA does induce IL-17 expression. This may be due to redundancy in the effects of IL-17 with IL-17F, which is also induced in the liver during conA-mediated hepatitis (data not shown). However, hepatocytes do not appear to respond to IL-17, although IL-17RA and IL-17RC mRNA can be detected in the liver and hepatocyte cell lines (data not shown). IL-22R and IL-17RA expression are not overlapping. IL-22R is expressed in the tissues and absent from cells of hemapoetic origin. On the other hand, IL-17RA is expressed in both the tissues, and by cells of the immune system, such as T cells and macrophages (Yao et al., 1995). Therefore, Th17 cells secreting both IL-22 and IL-17 may target different cell subsets during an immune response. In this way, IL-22 may act on the tissues and upregulate expression of pro-survival genes, whereas IL-17 may serve a role in directing the immune response.
IL-22 and IFNγ appear to have little direct effects on each other. The genes encoding these cytokines are situated in tandem on mouse chromosome 10 (Dumoutier et al., 2000a); however it does not appear that the two cytokines are co-expressed (Liang et al., 2006). Moreover, under several different stimulatory conditions our experiments show that IL-22 deficient mice express as much IFNγ as wild-type mice and IFNγ deficient CD4 T cells express equivalent amounts of IL-22 mRNA as wild-type cells (data not shown). Hepatocytes express both IL-22R and IFNγR, however they potentially respond differently to these cytokines since IFNγ preferentially activates Stat1, whereas IL-22 mainly activates Stat3. In keratinocytes, IFNγ or IL-22 stimulation results in very different expression profiles (Wolk et al., 2006). IFNγ stimulation results in upregulation of MHC related molecules, cytokines, chemokines and their receptors, whereas IL-22 stimulation leads to upregulation of expression of genes encoding antimicrobial defensins and proteins that increase cellular motility. Our data suggest that during conA-mediated hepatitis when both cytokines are induced, IL-22 protects hepatocytes from the destructive effects of IFNγ.
IL-22 may have promise as a potential therapeutic for chronic inflammatory diseases. Treatment with recombinant cytokine or gene therapy delivery of IL-22 may alleviate tissue destruction during inflammatory responses. Experimental delivery of IL-22 has been efficacious in treating autoimmune disorders such as experimental autoimmune myocarditis in rats (Chang et al., 2006; Pan et al., 2004). Suppressing the immune system via anti-inflammatory treatments such as TNFα inhibitors can lead to unwanted dampening of the immune response, weakening its ability to respond to infection. On the other hand, IL-22 is an ideal therapeutic candidate since it will specifically affect tissue responses and not have direct effects on the immune response.
Chronic inflammation is also linked to the development of cancer (Karin et al., 2006). Sustained tissue damage and oxidative DNA damage from toxic immune infiltrates and damage-induced proliferation can act as tumor promoters. Epidemiological studies show that patients with many different chronic inflammatory diseases are at increased risk for developing cancer in those inflamed organs (Lu et al., 2006). Chronic HBV or HCV infection, as well as long-term alcohol abuse, lead to hepatocyte damage that ultimately results in liver cirrhosis and most hepatocellular carcinomas (HCCs) form in cirrhotic livers. In addition, HBV or HCV chronic carriers have a 50 to 60% lifetime risk of developing HCCs (Blum, 2005). HCC is one of the most common cancers world-wide and is also responsible for a high number of cancer-related deaths. Since most HCCs occur in liver disease patients, it will be very beneficial to develop preventative therapies aimed at interfering with HCC development in these predisposed patients. During acute liver inflammation, IL-22 protects hepatocytes from injury possibly through Stat3-mediated upregulation of pro-survival and proliferative responses. IL-22 may also contribute to limiting damage during chronic inflammation and recombinant IL-22 therapy may prevent development of HCCs. However, IL-22 may have opposing short-term and long-term effects in the liver. Expression of IL-22 during chronic inflammation may allow for survival of damaged hepatocytes that are precursors for HCCs and therefore may promote cancer. Future studies will examine the role of IL-22 in chronic inflammation and the development and treatment of liver cancer. IL-22 may prove to be an important target for developing new drugs and treatments to combat liver diseases.
IL-22−/− mice were generated by the VelociGene approach as described (Valenzuela et al., 2003). Briefly, embryonic stem cells were targeted with the construct indicated in Supplementary Figure 1. A pZEN6 cassette was constructed, in which a reporter lacZ gene was placed in tandem with a neomycin resistance gene flanked by loxP sites and driven by a promoter that allowed for positive selection in both bacterial and mammalian cells. The pZEN6 cassette was ligated to double-stranded oligonucleotides and used for the generation of a bacterial artificial chromosome-based targeting vector. To generate chimeric mice deficient in IL-22, correctly targeted embryonic stem cells derived from the 129/Sv×C57BL/6 F1 background carrying the IL-22 construct were injected into BALB/c blastocysts, which were then implanted in CD1 pseudopregnant foster mothers. Male chimeras were bred with C57BL/6 to screen for germ-line-transmitted offspring. Mice bearing the targeted IL-22 allele were screened by PCR. The derived heterozygous mice were backcrossed onto the C57BL/6 genetic background for 10 generations. IL-22+/− mice were intercrossed to generate IL-22 deficient and wild-type control mice. IL-17A deficient mice on a C57BL/6 stain (N9) were kindly provided by Dr. Yoichiro Iwakura of the University of Tokyo, Japan (Nakae et al., 2002). IL-22 and IL-17 double deficient mice were generated by intercrossing the progeny of an IL-22 deficient to IL-17 deficient cross. IL-6 deficient mice were obtained from The Jackson Laboratories (Bar Harbor, ME) (Kopf et al., 1994). C57BL/6 mice from the National Cancer Institute (Frederick, MD) were used for cytokine induction experiments. Mice within experiments were age- and sex-matched. Mice were cared for in accordance with institutional animal care and use committee-approved protocols at the Yale University School of Medicine animal facility.
To examine innate immunity, mice were intravenously injected via the lateral tail vein with 5×104 CFU wild-type L. monocytogenes (strain 10403S). To quantitate bacterial burden, at day 3 post-infection spleens and livers were homogenized in RPMI 1640, then lysed with 0.5% Triton-X100, serially diluted in PBS, then plated in triplicate onto brain heart infusion agar plates. To examine the adaptive response, mice were intravenously injected with 1×104 CFU recombinant L. monocytogenes expressing ovalbumin (rLM-ova) as described previously (Foulds et al., 2002). To assess T cell responses, L. monocytogenes-infected mice were euthanized at day 7 post-infection, as well as uninfected control mice. Lymphocyte activation was measured by direct ex vivo surface stain with fluorescently-conjugated anti-CD4 (clone RM4-5), anti-CD44 (clone IM7) and anti-CD62L (clone MEL-14) Abs (all from BD Pharmingen; San Jose, CA), followed by FACS analysis. To quantitate antigen-specific CD4 T cell responses, lymphocytes from the spleens and livers were incubated with GolgiStop (BD Pharmingen) for 5 hrs in the presence or absence of the peptide LLO190–201. Cells were then surface stained for CD4, and intracellularly stained for IFNγ (clone XMG1.2) using the Cytofix/Cytoperm kit (BD Pharmingen) according to the manufacturer’s protocol.
Mice were injected intravenously via the lateral tail vein with 10 μg/g of conA (Sigma; St. Louis, MO) in PBS. Mice were euthanized at the indicated time post-injection.
Mice received 1 μg/g mouse of anti-Fas Ab (clone Jo2) (BD Pharmingen) without preservatives by intraperitoneal injection (Ogasawara et al., 1993).
Spleens and livers were aseptically removed from euthanized mice, placed into cold Bruff’s media, and passed through a wire-mesh screen. To prepare the spleen lymphocytes, RBCs were lysed with ACK buffer. Splenocytes were resuspended in complete Bruff’s medium containing 5% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Aliquots were diluted in 0.1% trypan blue/PBS to calculate the number of viable cells per spleen. Lymphocytes were isolated from the liver as previously described (Kuniyasu et al., 2005). Briefly, liver homogenate was incubated with 100 U/ml collagenase (Sigma; St. Louis, MO) and 20 μg/ml DNase I (Sigma) for 40 min at 37 °C. To remove hepatocytes, homogenates were centrifuged at 300 rpm for 3 min, and then supernatants were centrifuged at 1500 rpm for 10 min. The cells were resuspended in 1 ml complete media and 4 ml of 30% OptiPrep (Axis-Shield; Oslo, Norway) in a sodium phosphate buffer and 1 ml of media was carefully layered on top. Cells were centrifuged at 2700 rpm for 20 min. The top layer and interface were harvested as the liver lymphocyte population.
Sera were harvested from mice at the indicated times post-injection. TNFα (eBioscience), IFNγ (BD Pharmingen), and IL-17A (Southern Biotech; Birmingham, AL) ELISAs were performed according to the manufacturers’ protocols.
Cells were stained with fluorescently-conjugated Abs in 1% BSA/PBS and subsequently fixed in 2% paraformaldehyde. The following Abs were used: CD44 (clone IM7), CD62L (clone MEL-14), CD69 (clone H1.2F3) and CD4 (clone RM4-5) (all Ab from BD Pharmingen). Cells were analyzed using a FACSCalibur (BD Biosciences; San Jose, CA) and data were analyzed by FlowJo v. 6.1 (TreeStar, Inc., Ashland, OR).
Organs were removed and fixed in 4% paraformaldehyde overnight at 4 °C, then embedded in paraffin, sectioned, and stained with H&E. Slides were prepared at the Yale University Program for Critical Technologies in Molecular Medicine, Department of Pathology.
Serum ALT and AST levels were quantitated by colorimetric ALT and AST enzyme assays (Biotron Diagnostics, Hemet, CA) using a modified manufacturer’s protocol scaled to a 96 well plate format.
RNA from cells or organs was isolated using Trizol reagent (Invitrogen; Carlsbad, CA) according to the manufacturer’s protocol. RNA was subjected to reverse transcriptase using Superscript II (Invitrogen) with oligo dT primer according to the manufacturer’s protocol. cDNA was semi-quantitated using commercially available primer/probe sets (Applied Biosystems; Foster City, CA) and the Δ ΔCT method. HPRT was included as an internal control for all samples.
FL38B cells (ATCC CRL2390) were grown in F12K media supplemented with 10% FBS, 2 mM glutamine, 1.5 g/L sodium bicarbonate, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were stimulated for 20 min with the indicated concentration of recombinant murine IL-22 (R&D Systems; Minneapolis, MN), IL-10 (BD Pharmingen) or IL-17 (eBioscience; San Diego, CA). Cell lysates were separated under reducing conditions on 4% to 12% gradient gel using the NuPAGE electrophoresis system (Invitrogen). Gels were transferred to Immobolin P membrane (Millipore; Billerica, MA), blocked with 5% dry milk in PBS with 0.01% Tween. Blots were then incubated overnight at 4 °C with one of the following primary Ab: anti-phospho-Stat3 Tyr705 (polyclonal), anti-Stat3 Ab (polyclonal), and anti-phospho-Akt Ser473 (clone 587F11) (all from Cell Signaling Technology; Danvers, MA) or anti-β-actin (Santa Cruz Biotechnology; Santa Cruz, CA). Blots were washed, incubated with appropriate secondary antibodies conjugated to horse radish peroxidase, and then developed using chemiluminescent substrate (Pierce, Rockford, IL) and film.
Naïve sorted CD4 T cells (CD4+ NK1.1− CD25− CD44low CD62Lhigh) were cultured in complete Bruff’s media on plates coated with 10 μg/ml α-CD3 Ab and 1 μg/ml α-CD28 Ab. The media was supplemented with the following Abs and cytokines for the following differentiation conditions: Th0, 10 μg/ml α-IFNγ Ab (clone XMG) and 10 μg/ml α-IL-4 Ab (clone 11B11); Th17, 1 ng/ml TGF-β (R&D Systems), 20 ng/ml IL-6 (BD Pharmingen), 10 ng/ml IL-1β (BD Phamringen), 10 ng/ml TNFα (BD Pharmingen), 10 μg/ml α-IL-4 Ab and α-IFNγ Ab. On day 5, cells were extensively washed and then restimulated for 5 hrs with 80 μM PMA and 1μM ionomycin. RNA was harvested and RT-PCR was performed as described above.
IL-22 +/+ or −/− cells were cultured under Th17 conditions as described above. On day 5 cells were extensively washed with PBS, and then 5×106 cells were intraperitoneally injected into IL-22 deficient mice. 6 to 12 hours later, mice were injected intravenously with 10 μg/g conA.
LAZ is supported by NRSA Immunology Training Grant 2-T32-AI07019-29. RAF is an Investigator of the Howard Hughes Medical Institute. The authors would like to thank Drs. Yisong Y. Wan and Sean Stevens for critical reading of the manuscript, Dr. Elizabeth A. Jones for help with back-crossing mice, and Ms. Linda Evangelisti and Cindy Hughes for culturing ES cells and generating chimeric mice, respectively.
Conflict of Interest
LAZ and RAF declare no conflict of interest. GDY, DMV, AJM and MK were employees of Regeneron Pharmaceuticals at the time this work was performed.
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