G protein-coupled receptor kinase-2-deficient mice are protected from dextran sodium sulfate-induced acute colitis

Abstract

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase and plays a key role in different disease processes. Previously, we showed that GRK2 knockdown enhances wound healing in colonic epithelial cells. Therefore, we hypothesized that ablation of GRK2 would protect mice from dextran sodium sulfate (DSS)-induced acute colitis. To test this, we administered DSS to wild-type (GRK2^+/+) and GRK2 heterozygous (GRK^+/−) mice in their drinking water for 7 days. As predicted, GRK2^+/− mice were protected from colitis as demonstrated by decreased weight loss (20% loss in GRK2^+/+ vs. 11% loss in GRK2^+/−). lower disease activity index (GRK2^+/+ 9.1 vs GRK2^+/− 4.1), and increased colon lengths (GRK2^+/+ 4.7 cm vs GRK2^+/− 5.3 cm). To examine the mechanisms by which GRK2^+/− mice are protected from colitis, we investigated expression of inflammatory genes in the colon as well as immune cell profiles in colonic lamina propria, mesenteric lymph node, and in bone marrow. Our results did not reveal differences in immune cell profiles between the two genotypes. However, expression of inflammatory genes was significantly decreased in DSS-treated GRK2^+/− mice compared with GRK2^+/+. To understand the mechanisms, we generated myeloid-specific GRK2 knockout mice and subjected them to DSS-induced colitis. Similar to whole body GRK2 heterozygous knockout mice, myeloid-specific knockout of GRK2 was sufficient for the protection from DSS-induced colitis. Together our results indicate that deficiency of GRK2 protects mice from DSS-induced colitis and further suggests that the mechanism of this effect is likely via GRK2 regulation of inflammatory genes in the myeloid cells.

INTRODUCTION

The intestinal tract, one of the largest mucosal-lined surfaces, is essential in creating and maintaining the barrier between the luminal environment and the host. Damage to the integrity of this barrier can lead to various inflammatory processes and pathologies, one of which is inflammatory bowel disease (IBD) (7, 11, 30, 42). IBD is a multifactorial disease that results from dysregulated immune response and loss of integrity of the epithelial cells lining the intestinal barrier. Response of the immune system to foreign antigens especially from the intestine must be regulated to maintain gut homeostasis. Modern therapies for the treatment of IBD include both anti-inflammatory as well as immunosuppressive therapies. Given that IBD has diverse etiology, these therapies are not effective for a significant proportion of patients and/or treatment responsiveness can decrease over time (3). Therefore, further understanding of IBD pathogenesis is critical for the identification of novel therapeutic targets and treatment regimes.

G protein-coupled receptor kinases (GRKs) are serine/threonine kinases originally identified for their key role in G protein-coupled receptor (GPCR) life-cycle by their ability to phosphorylate G protein-coupled receptors. There are seven GRKs grouped into three subfamilies based on their sequence similarities: GRK1, composed of GRK1 (rhodopsin kinase) and GRK7 (cone opsin kinase); GRK2, including GRK2 and GRK3; and GRK4, made up of GRK4, GRK5, and GRK6 [reviewed in (36)]. Although all GRKs were thought to phosphorylate GPCRs equally, recent studies demonstrate that the GRK2/3 and the GRK5/6 subfamilies differ in their ability to phosphorylate inactive GPCRs with GRK5/6 being able to phosphorylate GPCRs in both the active and inactive conformations (17). Although expression of specific GRKs can be tissue-restricted (e.g., GRK1 in eye), GRK2 is ubiquitously expressed and is capable of interacting with a variety of GPCRs. Its critical role in regulating GPCRs in the context of cardiac function is well established and GRK2 is a major therapeutic target for cardiac disease (9). Although its role in GPCR phosphorylation is a critical physiological mechanism in receptor regulation, a number of nonreceptor substrates have also been identified in different cell types. This diverse set of substrates and binding partners allows for GRK2 to regulate many cellular processes including inflammatory gene expression and cellular migration (5).

The ability of GRK2 to regulate inflammation is related to its role in modulating inflammatory signaling pathways in immune and nonimmune cells. GRK2 has been shown to interact with NF-κB and MAPK pathways [reviewed in (36)] as well as localize to the mitochondria to regulate reactive oxygen species (34). In addition, GRK2 levels have been shown to be modulated during inflammatory disease processes prompting several laboratories including ours to examine the role of GRK2 in inflammatory diseases with animal models. This has been primarily achieved by using heterozygous GRK2 knockout mice (homozygous are lethal) as well as cell type-targeted GRK2 transgenic and knockout mice (25, 28, 41). Using these models, in previous studies we demonstrated that myeloid-specific GRK2 is an important regulator of inflammation in sepsis pathogenesis (26, 27). Similar critical roles of GRK2 in atherosclerosis, arthritis, and autoimmune encephalomyelitis have been demonstrated by other groups (23, 37, 39). Even though the role of GRK2 in inflammatory bowel disease is not known, in recent studies, our laboratory demonstrated a key role for GRK2 in intestinal epithelial wound healing (35). Because wound healing is an important aspect in the pathogenesis of IBD, in this study we examined the role of GRK2 in acute colitis, using a dextran sodium sulfate (DSS)-induced colitis model in mice. Our studies highlight a critical role for GRK2 in mediating acute colitis and suggests GRK2 as a potential drug target for IBD.

MATERIALS AND METHODS

Mice.

GRK2 heterozygous mice (C57BL6 background) were obtained from Jackson Laboratories (kindly donated by Dr. Robert Lefkowitz, Duke University) and were bred at Michigan State University by breeding GRK2 heterozygous mice with wild-type (WT) C57BL6 mice. Note that homozygous knockout of GRK2 mice is embryonically lethal (10). GRK2 floxed mice were kindly provided by Dr. Gerald Dorn II (Washington University School of Medicine, St. Louis, mo (19). These mice were backcrossed to C57BL6 background for more than 12 generations and bred to LysMCre mice (C57BL6 background from Jackson Laboratories) to generate myeloid-cell specific GRK2 knockout (GRK2^{fl/fl+LysMcre} mice). We have previously demonstrated that in these mice, GRK2 is knocked out specifically in myeloid cells but not in other tissues (27). All mice were bred and housed in a specific-pathogen-free facility and were maintained at 22–24°C with 50% humidity a 12 h light dark cycle. Mouse chow and water were provided ad libitum. All experiments (with exception of the initial experiments in females) were performed with age-matched male mice between 8 and 12 wk of age. All animal procedures were approved by the Michigan State University Institutional Animal Care and Use Committee and conformed to National Institutes of Health guidelines.

DSS-induced colitis model.

Colitis was induced in mice with DSS as previously described (15, 16, 32). In brief, mice were administered DSS (3.5–4.25% wt/vol) in their drinking water for the indicated times. During DSS treatment, mice were examined daily and scored for disease activity index (DAI) under the following criteria: stool consistency (1, loose), blood present in stool (1, mild; 2, gross), ruffled hair coat (0 or 1), crusty eyes (0 or 1), hunched posture (0 or 1), and weight loss (1, 0–5%; 2, 5–10%; 3, 10–15%). At the time of harvest splenic weight, colon length, and colon weight were additionally measured.

Sample preparation.

Following DSS treatment, at the appropriate time, mice were euthanized by CO₂ asphyxiation. Spleen, mesenteric lymph nodes (MLN), and bone marrow were collected and processed as previously described (15, 16, 32). In short, spleen and MLN were homogenized, treated with red blood cell lysis buffer, filtered through 40 μm nylon mesh, and counted for stimulation or flow cytometry analysis. Bone marrow cells were analyzed as previously described (15, 16, 32). At harvest, after removal of fecal content, colon lengths and weights were noted, and 5 mm segments of colon were flash-frozen for mRNA and protein isolation or prepared for histology. The remaining colon was processed as previously described for leukocyte isolation (15, 16, 32). In short, the colon was cut into 5 mm segments and incubated in epithelial dissociation buffer at 25°C with gentle agitation for 30 min. These segments were further cut into 1 mm segments and incubated for 1 h in 0.5 mg/ml collagenase-D. Finally, the pieces were strained through a 100 μm filter and loaded onto 80:40 Percoll gradients. Cells were ultimately collected from the interface and used as leukocyte fraction after a wash in phosphate-buffered saline.

Flow cytometry.

Tissue/cell processing and flow cytometry analysis were done as described before (15, 16, 32). Briefly, processed cells were incubated with fcγR blocking antibody to block nonspecific binding and were then surface-stained with antibody cocktail and washed with staining buffer (phosphate-buffered saline with sodium azide and bovine calf serum). When required, intracellular staining was performed by fixing the cells with fixation buffer (eBioscience) and permeabilized and washed with perm buffer (phosphate-buffered saline with sodium azide and saponin, eBioscience). The antibodies used against cell surface markers were CD11b, F4/80, Ly6G, CD3, CD4, CD8; intracellular cytokines IL-10 and TNF-α were assessed with respective antibodies. All antibodies were obtained from eBiosciences and used as per the manufacturer’s instructions. All cells were run on LSR II, and the data were analyzed with FlowJo software.

Cytokine/chemokine measurements.

Cytokines were measured from plasma and ex vivo culture supernatants with enzyme-linked immunosorbent assay kits from eBiosciences as described (15, 16, 32, 33) and as per manufacturer’s protocol.

RT-PCR.

RNA extraction and quantitative RT-PCR analysis was done as described before (35). For determination of relative levels of RNA transcripts, RNA was isolated from snap-frozen tissue with the Qiagen RNeasy mini kit following manufacturer’s protocol. Reverse transcription was performed on 1 μg of RNA for single-stranded cDNA synthesis. Quantitative real-time PCR was performed for various genes of interest and the cDNA was amplified with SYBR Green Pro Master Mix with hypoxanthine-guanine phosphoribosyltransferase as the normalization control as described before (18).

Histopathology.

Colon tissue was harvested, Swiss-rolled, and fixed overnight in 10% formalin followed by 70% ethanol. The tissue was then embedded in paraffin, sectioned, and stained with hematoxylin and eosin and was analyzed in a blinded fashion by a board-certified pathologist (P. C. Lucas). Histologic sections were scored on a well-established composite scoring system (15, 22). Histologic activity scores were assigned as follows: scores were the sum of the following features: 1) degree of inflammation in lamina propria (score 0–3); 2) goblet cell loss (score 0–2); 3) abnormal crypts (score 0–3); 4) presence of crypt abscesses (score 0–1); 5) mucosal erosion and ulceration (score 0–1); 6) submucosal spread or transmural involvement (score 0–3); 7) number of neutrophils counted at 40 magnification (score 0–4). When values from the seven parameters are totaled, the overall histologic activity score (colitis score) can range from 0 to 17.

Statistical analysis.

All values are represented as means ± SE. All data were tested for statistical significance by unpaired Student’s t-test (two sample comparisons) and analysis of variance with Tukey post hoc test (>2 sample comparisons). All analysis was done with Prism GraphPad software. A P value of < 0.05 was considered significant.

RESULTS

GRK2 heterozygous mice are protected from DSS-induced colitis.

This study was designed to investigate the role of GRK2 in the onset and progression of colitis. Based on recent work from our laboratory (35) we hypothesized that GRK2 would play an important regulatory role in the onset and progression of colitis. To test this, we treated male GRK2^+/− and WT (GRK2^+/+) mice with 4.25% DSS in their drinking water and assessed various clinical signs of colitis until euthanasia at day 8. Following DSS treatment, GRK2^+/+ mice lost significant body weight beginning at day 4 and displayed severe signs of disease as measured by Disease Activity Score (indicated by loose and bloody stool, hunched posture, crusty eyes, ruffled hair, etc., see materials and methods for complete description) (Fig. 1, A and B). Compared with the GRK2^+/+ mice, GRK2^+/− mice were significantly protected from clinical signs of colitis including weight loss and DAI.

Fig. 1.

G protein-coupled receptor kinase (GRK)2^+/− mice are protected from acute dextran sodium sulfate (DSS)-induced colitis in male and female mice. Wild-type (GRK2^+/+)and GRK2 heterozygous mice (GRK2^+/−) were administered either water or 4.25% DSS in their drinking water ad libitum for 6 days to induce colitis, switched to water on day 7 and euthanized on day 8. A: body weight in male mice over the course of the experiment expressed as % starting weight. B: disease activity index in male mice observed over the course of the experiment. C: body weight in female mice. D: disease activity index measured in female mice over the course of the experiment. n = 9–11 for males; n = 6–7 for females, ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data pooled from 3 independent experiments for males.

To determine if there are any sex differences in the role of GRK2 in colitis pathogenesis, we administered DSS to female GRK2^+/+ and GRK2^+/− mice. Interestingly, and as noted in previous studies (2), female mice showed delayed onset of colitis in WT mice. Similar to males, female GRK2^+/− mice were protected from the clinical signs of colitis (Fig. 1, C and D). These results suggest that while there may be differences in the kinetics of colitis pathogenesis in males and females, GRK2 knockdown is protective in colitis. Because the phenotypic differences were more striking in the males, we focused on males in our subsequent studies.

Consistent with the clinical signs, DSS treatment caused significant colon shortening in GRK2^+/+ mice, as compared with water treatment (Fig. 2, A and B). This was significantly attenuated in GRK2^+/− mice (longer colons compared with WT). Interestingly, the GRK2^+/− mice had enlarged spleens at the time of harvest despite their overall protection (Fig. 2C). Finally, to assess potential differences in histological damage, we examined the colon sections for pathological damage induced during colitis development. Unlike gross morphology and clinical signs, the two genotypes had comparable levels of histological damage (Fig. 2D). Although the microscopic damage to the colon was similar at this time of harvest, together these results support a negative role for GRK2 in the clinical onset and progression of DSS-induced colitis in mice.

Fig. 2.

Gross and histological assessments of colitis in wild-type and G protein-coupled receptor kinase (GRK)2 heterozygous mice subjected to dextran sodium sulfate (DSS) treatment: GRK2^+/+ and GRK2^+/− mice were subjected to DSS treatment as described in Fig. 1. A: quantification of colon lengths at time of harvest. B: representative images of colons at time of harvest. C: spleen weight taken at time of harvest. D: histological images of proximal colon; all images were taken at the same magnification, and scale bars represent 200 μm each. n = 6–7, ± SE. *P < 0.05, **P < 0.01. Data pooled from 2 independent experiments.

GRK2 regulates inflammatory genes in the colon but not cellular infiltration.

To further test if the phenotype of the GRK2^+/− mice is reflected at the level of gene expression for inflammatory cytokines, we examined expression of cytokines and chemokines in the colon from GRK2^+/+ and GRK2^+/− mice subjected to DSS-induced colitis. Despite similar histological damage, the expression of inflammatory mediators, including components of innate cytokines, chemokines, and T cell effectors, was significantly inhibited in the GRK2^+/− compared with the GRK2^+/+ mice (Fig. 3). In particular, we observed that levels of IL-12p40 and MIP2, as well as the IL-6-to-IL-10 ratio, were significantly lower in GRK2^+/− mice as compared with GRK2^+/+ mice. Although GM-CSF was also lower in the GRK2^+/− mice compared with GRK2^+/+, it did not reach statistical significance. Interestingly, GRK2^+/− mice also had lower expression of T cell cytokines, including IL-17A as well as transcription factor GATA3, indicating that there may be a decrease in the Th17 and Th2 responses in these mice. This is consistent with decreased clinical disease manifestation in the GRK2^+/− mice. These results suggest that indeed GRK2 functionally regulates inflammatory gene expression in the colon following DSS-induced colitis.

Fig. 3.

Assessment of inflammatory genes in the colon following dextran sodium sulfate (DSS)-induced colitis. RNA was extracted from proximal colon segments of G protein-coupled receptor kinase (GRK)2^+/+ and GRK2^+/− mice after DSS treatment as described in Fig. 1, subjected to complementary DNA synthesis and quantitative RT-PCR to quantify the expression of indicated genes. n = 6–7 for DSS-treated animals. *P < 0.05; **P < 0.01.

Because GRK2 is a critical regulator of chemokine receptors (GPCRs), it is also possible that deficiency of GRK2 may lead to altered immune cell chemotaxis, as shown in GRK6 knockout mice (4), and that this could account for phenotype differences in colitis. To test this, we examined the levels of different immune cell types within the colonic lamina propria and MLNs as well as the populations of innate and adaptive leukocytes within the bone marrow. DSS treatment of WT mice led to a significant increase in granulocytes and CD4⁺ and CD8⁺ T-lymphocytes in the lamina propria compared with the water-treated WT mice. Although we did not observe any major changes in the MLN cellular population, bone marrow exhibited an increase in granulocytes but a decrease in CD4⁺ and CD8⁺ T-lymphocytes in the DSS-treated WT mice. Interestingly, when compared between DSS-treated GRK2^+/+ and GRK2^+/− mice, we observed no significant differences in the numbers of macrophages, dendritic cells, and granulocytes as well as CD3+, CD4+, and CD8+ T cells in the colonic lamina propria (Fig. 4A) and MLNs (Fig. 4B). Similarly, there were no differences seen in the lymphocyte, macrophage, or dendritic cell populations in the bone marrow of the two genotypes treated with DSS. There was, however, a significant difference in the number of granulocytes detected in the WT mice treated with DSS when compared with the GRK2^+/− mice (Fig. 4C).

Fig. 4.

G protein-coupled receptor kinase (GRK)2 does not influence immune population in the colonic lamina propria, mesenteric lymph node, and bone marrow following dextran sodium sulfate (DSS) treatment. GRK2^+/+ and GRK2^+/− mice were subjected to DSS treatment as described in Fig. 1 and the respective tissues processed, cell collected and the immune cells quantified using flow cytometry as described in the methods. Cellular populations in the colonic lamina propria (A), mesenteric lymph node (B), and bone marrow (C) of GRK2^+/+ and GRK2^+/− mice expressed as fold WT H20. n = 6–7 per genotype. Data pooled from 2 independent experiments. *P < 0.05; **P < 0.01.

Reviewing the onset of colitis in these animals, we observed differences between the two groups as early as day 4 of DSS treatment and therefore questioned if there would be differences in immune infiltration between the two genotypes at this early time point after DSS treatment. To that end, we examined the composition of immune cells in the colonic lamina propria, MLN, and bone marrow at 4 days after DSS treatment. Similar to the 8-day time point, there were no detectable differences in the number of innate or adaptive leukocytes in the colonic lamina propria, MLN, and the bone marrow (data not shown). Together these data suggest that the protection from DSS induced colitis in GRK2^+/− mice is likely independent of immune infiltration into the colonic lamina propria and MLNs and also potentially independent of changes in bone marrow immune populations.

GRK2 knockout in myeloid cells is sufficient to protect against DSS-induced colitis.

To begin to dissect the cell-specific mechanisms by which GRK2 regulates DSS-induced colitis, we focused on myeloid-specific GRK2 knockout mice. We chose to knock out GRK2 in this cell type/lineage because of the significant changes in cytokine expression that we observed in the GRK2^+/− DSS mice. Previously, our laboratory demonstrated that GRK2 levels in these animals are significantly reduced specifically in the macrophages and neutrophils (27). WT and myeloid-specific GRK2 knockout (GRK2^MyeKO) mice were treated with DSS and various clinical, gross, and histological parameters of colitis assessed. Much like in the GRK2^+/− mice, we observed that the GRK2^MyeKO animals were protected from colitis-induced weight loss (Fig. 5A) and DAI score (Fig. 5B) when compared with WT animals. This protection from DSS-induced colitis was further substantiated by significantly shorter colons in the WT mice compared with the GRK2^MyeKO animals (Fig. 5C). Similar to previous experiments, spleens from GRK2^MyeKO were larger than their WT counterparts (Fig. 5C). Colon histopathology of the GRK2^MyeKO animals showed a significantly improved colitis score and protection from damage, compared with the WT animals (Fig. 5D).

Fig. 5.

Myeloid-specific G protein-coupled receptor kinase (GRK)2 knockout mice are protected from acute dextran sodium sulfate (DSS)-induced colitis. WT and myeloid-specific GRK2 knockout (GRK2^LysM) mice were administered water or 3.25% DSS in their drinking water ad libitum and euthanized at the indicated time point. A: percentage of body weight in mice over the course of the experiment expressed as % starting weight. B: disease activity index in mice observed over the course of the experiment. C: quantification of the colon lengths and spleen weight taken at time of harvest. D: representative images of colon histology taken at time of harvest and pathology score for WT and GRK2^LysM mice. Mice given water had scores of 0 and are not included in the graph. All images were taken at the same magnification and scale bars represent 200 μm each. Means ± SE n = 7–12. Data pooled from 2 independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001.

Together these data suggest that GRK2 critically regulates the pathogenesis of colitis and that depletion of myeloid-specific GRK2 is sufficient for the observed phenotype. Importantly, the effect of GRK2 in colitis progression is likely independent of immune cell infiltration and dependent on the regulation of inflammatory pathways in myeloid-derived cells.

DISCUSSION

In this study, we demonstrate that mice lacking one allele of GRK2 exhibit decreased gross and systemic manifestations of colitis following DSS treatment, despite having similar histologic features of colitis and similar patterns of colonic immune cell infiltration. These results suggest that the likely effect of GRK2 in colitis is independent of immune infiltration per se and dependent on functional aspects of inflammatory gene expression. However, it is possible that the time points we examined are insufficient to make this conclusion. It is also possible that complete deletion of GRK2 is necessary for observing any differences in immune infiltration. Despite this lack of difference in immune infiltration, GRK2^+/− mice were still protected from DSS-induced colitis, suggesting that deletion of one allele GRK2 gene is sufficient for the overall phenotype. Based on inflammatory gene expression data in the colon from GRK2^+/− mice, we also examined if GRK2 expression specifically in the myeloid compartment is important in colitis pathogenesis. Our results indeed reveal an important role for myeloid-specific GRK2 in DSS-induced colitis. Together, these data clearly demonstrate that GRK2 is detrimental to the onset and progression of colitis and its role in the myeloid population is a key component in the colitogenic potential of this kinase.

Immune infiltration is one of the hallmarks of colitis, and the hyperactivation and prolonged duration of immune activation are a primary cause of damage to the intestinal lining (12, 31). Given the influence of GRKs in regulating GPCRs such as chemokine receptors, we posited that the role of GRK2 in colitis pathogenesis is based, at least in part, on regulation of immune cell infiltration into the colon. Indeed, other GRKs and especially GRK6 are implicated in influencing colitis development through regulation of chemotaxis. For example, the presence of GRK6 expression is protective against DSS-induced colitis through regulation of CD4+ T cell and granulocyte infiltration (4). However, as the role for these proteins continues to expand and the number of nonreceptor substrates increase, it is becoming clear that these proteins can influence the disease pathogenesis independently of their effect on immune infiltration. Recently, we showed that GRK2 regulates TNF-α signaling in intestinal epithelial cells and that this effect of GRK2 may influence its role in a wound healing model in vitro and in vivo (35). In earlier studies, we also demonstrated that GRK2 regulates TLR signaling in macrophages, particularly production of inflammatory cytokines (27). In other studies, we have shown that GRK5 regulates inflammation and pathogenesis of polymicrobial sepsis without affecting cellular infiltration in mice (24). Thus, it is clear that GRKs have functions that are independent of GPCRs and can influence biological functions, especially in the immune system, that are independent of chemotaxis (9, 36).

Although we did not observe alterations in immune infiltration, we did find significant fluctuations in key pro- vs. anti-inflammatory cytokine ratios, especially IL6:IL10. In addition, other cytokines/chemokines, including IL12p40 and MIP2, known to be associated with the onset and severity of colitis, were significantly altered in the GRK2 heterozygous mice (1, 13, 29, 43). These changes in key cytokines are generally consistent with the overall phenotype observed in the GRK2^MyeKO mice, suggesting that GRK2 regulation of myeloid cells likely is important for the observed phenotype. Interestingly, several genes differentially regulated in the GRK2^+/− mice are also associated with T cell differentiation. One of the genes that was significantly increased in the WT mice (compared with GRK2^+/− mice) was GATA3, a Th2 lineage commitment transcription factor (44). Recent work has shown that increased levels of GATA3 can accelerate acute DSS-induced colitis (21). Another T cell cytokine significantly increased in the WT animals (compared with GRK2^+/−) was IL-17A. Produced by a unique subset of memory T cells, IL-17A has broad impact on the immune system and has been shown to stimulate fibroblasts, endothelial cells, macrophages, and epithelial cells to secrete proinflammatory mediators such as IL-1, IL-6, TNF-α, MMPs, and many others (6). Recent work on IL-17 has shown that this cytokine can be both pathogenic or protective depending on the source of the cytokine (14). The source of IL-17 and whether it is contributing to increased inflammation and damage or trying to restore the epithelial barrier in our mice remains unknown at this time. While our study investigated the infiltration of CD3+, CD4+, and CD8+ T lymphocytes, the specific lineages and differentiated cell types of T cells were not investigated. Future studies will focus on this as well as on generating T cell-specific GRK2 knockout to study the role of GRK2 in intrinsic lymphocyte signaling in the context of colitis development.

We observed that knockout of GRK2 in the myeloid population was sufficient to confer the protection from acute DSS-induced colitis. Although GRK2 has been implicated in other inflammatory systems it has never been investigated in the context of acute colitis. Work done in models such as hyperalgesia demonstrates that the effect of GRK2 is through alterations in myeloid IL-10 production (40). Other models including sepsis (26) and endotoxemia (27) have shown that knocking down GRK2 in the myeloid cells generally exacerbates inflammation, potentially via negative regulation of NFκB1p105 leading to increases in IL6:IL10 ratios during the onset of inflammation. This is in contrast to the gene expression seen in the current colitis model where the IL6:IL10 ratio was significantly decreased in the knockout mice compared with the WT mice. These results highlight a diverse role of GRK2 in different models of inflammation and demonstrate that the response seen in these mice may be differentially regulated depending on the disease context and tissue microenvironment.

IBD has conventionally been treated with nonspecific immune suppression and more recently targeted therapies against specific inflammatory cytokines or pathways such as TNF-α. However, because of the diversity of this disease a large number of patients often do not respond to certain types of therapies or they eventually develop tolerance to treatments due to the chronic administration of these therapies (20). Therefore, identifying new therapies is important for overcoming the limitations of currently approved treatments. GRK2 offers a new targetable option for the management of IBD. Development of inhibitors of this kinase is currently being pursued in part for the role of GRK2 in heart failure. One promising inhibitor is the SSRI paroxetine (Paxil), which is approved by the Food and Drug Administration and demonstrated to potently inhibit GRK2 both in vitro and in vivo (38). In addition to paroxetine, there are a considerable number of additional GRK2 inhibitors currently in development (8). Therefore, it is a realistic possibility that therapeutics could target pathways regulated by GRK2 and potentially treat IBD in the future.

In summary, this study demonstrates an important role for GRK2 in regulating mucosal inflammation under colitic conditions. This is likely not due to dysregulation in immune infiltration but rather due to alterations in inflammatory gene expression, possibly in the myeloid population. Further work will determine the mechanism by which GRK2 influences these responses and provide insight into the specific cells involved in the onset and pathogenesis of DSS-induced colitis.

GRANTS

We gratefully acknowledge the support from NIH (grants HL-095637, AI-099404, and AR-056680 to N. Parameswaran).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.D.S., L.R.M., and N.P. conceived and designed research; M.D.S., H.J.K., and T.L. performed experiments; M.D.S., H.J.K., P.C.L., L.R.M., and N.P. analyzed data; M.D.S., P.C.L., L.R.M., and N.P. interpreted results of experiments; M.D.S., P.C.L., and N.P. prepared figures; M.D.S. and N.P. drafted manuscript; M.D.S., H.J.K., T.L., P.C.L., L.R.M., and N.P. edited and revised manuscript; M.D.S., H.J.K., T.L., P.C.L., L.R.M., and N.P. approved final version of manuscript.