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Opioids are the most prescribed analgesics for pain in Inflammatory Bowel Diseases (IBD), however the consequences of opioid use on IBD severity is not well defined. We evaluated the histopathology of gut biopsy from opioid using and non-opioid using IBD patients. Significantly higher histological score with severe pathological damage including disturbed architecture of the villi and crypts, edema, and increased inflammatory cell infiltrates were observed in opioid using when compared to non-opioid using IBD patients. To determine the detrimental effect of opioids on IBD severity we investigated the consequence of hydromorphone, the most prescribed opioid for pain management in IBD, in a murine model of dextran-sodium-sulfate (DSS)-induced colitis. We show that hydromorphone and DSS independently induced epithelial barrier dysfunction, bacterial translocation, disruption of tight junction proteins organization and increased intestinal and systemic inflammation, which were exacerbated in mice receiving hydromorphone in combination with DSS. Hydromorphone-treated DSS mice exhibited significant microbial dysbiosis with an expansion of Proteobacteria and Verrucomicrobia and reduced proportion of Firmicutes. Predictive metagenomic analysis of the gut microbiota revealed high abundance in the bacterial communities associated with virulence, antibiotic resistance, toxin production and inflammatory properties. We further observed that hydromorphone modulates tight junction protein organization in a myosin light chain kinase (MLCK)-dependent manner. Treatment with ML-7, an selective MLCK inhibitor, ameliorates the detrimental effects of hydromorphone on DSS induced colitis and thus decrease severity of IBD. These findings warrant a careful evaluation of the potential detrimental effects of prescription opioids on IBD severity and should be prescribed cautiously.
The United States is estimated to consume 80% of the global opioid supply, in spite of it constituting approximately 5% of the world’s population (1). Opioids are commonly used in the treatment of pain and associated symptoms of many diseases including abdominal pain secondary to IBD. The uninterrupted use of opioids has been linked with adverse outcomes, including death. Attaining the required degree of analgesia often leads to tolerance due to dose escalation. A large body of studies has shown that morphine treatment induces gut barrier disruption and dysfunction of the immune system, which results in bacterial infection and ultimately sepsis (2, 3). Lately, prescription of hydromorphone for IBD pain control and associated symptoms including diarrhea has increased as it has been reported to be stronger with fewer side effects. However, the impact of hydromorphone has not been properly evaluated on alteration in gut microbiota, inflammation and severity of disease in IBD.
IBD including ulcerative colitis (UC) and Crohn’s disease (CD) affects 1.6 million people with an annual incidence of 70,000 new cases in the United States alone (4). Although the pathogenesis of IBD remains complex, immune dysfunction, host genotype, environmental exposure, lifestyle factors and more recently compositional changes in enteric microbiome are increasingly implicated in disease pathophysiology (5, 6). The interactions between intestinal epithelial damage and microbial dysbiosis are proving to be most potent host factors in the pathogenesis of disease (7, 8). IBD is associated with severe morbidity and impaired quality of life owing to its chronic nature and high recurrence, and represents a substantial socioeconomic burden in the United States costing approximately over two billion dollars annually (4, 9, 10).
IBD pathogenesis involves damage to gut epithelial layer and dysregulation of immune response to intestinal microbes (5). Among various animal models, DSS-induced colitis is most extensively utilized and best-described murine model of IBD (11–13). In DSS model, mice are treated with 3-5% DSS in their drinking water for 5-9 days to induce acute colitis (14, 15), which provides a model of acute intestinal injuries that permits clinical monitoring of colitis (16). The macroscopic signs as well as microscopic observations in a murine model, recapitulate many aspects of IBD in humans (11).
The intestinal microbiota is associated with fundamental physiological processes (17). The gut microbiota mostly consists of Gram-positive Firmicutes and Gram-negative Bacteroidetes, which are conserved in humans and mice (18). Gut microbial dysbiosis is commonly associated with IBD. There is growing evidence that dysregulated immune response against commensal bacteria lead to IBD and the enormous variety of gut flora contributes to the heterogeneity of the disease. IBD patients show higher numbers of Proteobacteria and lower numbers of Firmicutes with reduced functional diversity compared to healthy individuals (19).
IBD is an independent risk factor in heavy opioid users. Patients with IBD have abdominal pain that is not infrequently medicated with opioids especially in the hospital setting. Approximately 15% of IBD patients are on chronic opioids and an estimated 5% of individuals become dependent, which is associated with a significant risk of mortality (20). Previous studies in our laboratory show that morphine causes disruption of intestinal tight junction proteins organization, gut barrier dysfunction, induces bacterial translocation and modulates immune responses (3). Opioid use has been linked with increased complications, increased hospitalizations, and decreased quality of life in IBD patients (21) but in clinical care it is difficult to assess whether opioid use is associated with more inflamed patients or opioids worsen intestinal inflammation.
Based on our previous studies, we hypothesized that prescription opioids will worsen intestinal inflammation and thus severity of IBD. We will investigate the integrity of intestinal tight junction proteins organization, gut barrier dysfunction and microbial dysbiosis as potential underlying mechanisms. To test our hypothesis, we quantified the degree of inflammation in patients with IBD on regular doses of opioids versus patients that are not on opioids. To further extrapolate the observed clinical data and to determine if opioids can independently impact the severity of disease, we investigated the consequence of the prescription opioid, hydromorphone, on gut barrier dysfunction, disruption of tight junction proteins organization on intestinal epithelial cells, microbial dysbiosis, and intestinal as well as systemic inflammation in a mouse model of IBD. Tight junction proteins maintain intestinal barrier integrity. The tight junction include paracellular proteins such as Zona occludens-1 (ZO-1) and transmembrane protein such as claudin-1 which assist to seal the paracellular pathway between adjacent intestinal epithelial cells. Previous studies have shown that tight junction disruption is initiated by the phosphorylation of myosin light chain (MLC), which mainly depends on activation of MLCK. It has been shown that there is strong association between gut barrier dysfunction and TLR-activation (3). However, the mechanisms underlying disruption of tight junction integrity after hydromorphone treatment is not well defined. Disruption of intestinal tight junction barrier function has severe consequences including bacterial translocation from the gut leading to immune activation and inflammation. Therefore, we for the first time elucidated the mechanism by which hydromorphone alter intestinal tight junctions organization in a murine model of IBD. Our results indicate that hydromorphone induced MLCK dependent disruption of intestinal tight junction proteins mediates epithelial barrier dysfunction, which in turn increase severity of IBD. Selective inhibition of MLCK activation attenuates opioid induced exacerbation of IBD. The results of this study provide a new perspective on the effect of opioids on IBD.
IBD patients on opioids demonstrate increased intestinal inflammation in spite of medical therapy
To determine the consequences of opioid use in IBD, we investigated the clinical symptoms of IBD in patients that regularly use opioids and non-opioid using patients. Our results demonstrate increased levels of inflammation in opioid using IBD patients compared with patients that are not on opioids but on similar medical therapy. We evaluated hematoxylin and eosin (H&E) stained sections of the terminal ileum biopsies from opioid using and non-opioid using CD patients. A total of 5 CD patients using chronic opioids for analgesia were identified and 5 CD patients not using opioids served as the control group. Histopathological analysis showed Irregularity, blunting or broadening of the villi and crypts in the control group, which were further progressed up to villous atrophy, complete loss of villi, crypt distortion, shortening of glands and granular hyperplasia in opioid using CD patients (Figure 1). There was severe epithelial erosion, mucosal ulceration, edema, tissue destruction and increased inflammatory cell infiltrates in opioid using CD patients compared to control group (Figure 1). The histologic score in opioids using CD patients was significantly higher compared to patients that were not on opioids (Figure 1).
Hydromorphone induces weight loss, intestinal permeability and bacterial translocation in a dose dependent manner
We next asked whether hydromorphone treatment perturb fundamental aspects of IBD pathogenesis in mice. The animals were treated with clinically relevant doses of hydromorphone to determine the most effective treatment dose of hydromorphone for subsequent experiments in the DSS-induced colitis mouse model.
To determine the effect of hydromorphone on change in body weight, the initial and final body weight was measured for all mice in five experimental groups. No mortality was observed in either of the groups for the duration of the study. Hydromorphone treatment resulted in significant loss in body weight in a dose dependent manner. All mice in the treatment groups showed significant weight loss compared to control group and the highest weight loss was recorded with the highest dose of hydromorphone (Figure 2A).
To investigate the consequence of hydromorphone on intestinal permeability, we assessed gut leakiness by measuring the leakage of orally administered Rhodamine B using in-vivo imaging system (IVIS). The lower doses of hydromorphone showed significant fluorescence level compared to control, and the highest fluorescence was recorded at highest dose of hydromorphone compared all other groups. The representative images of Rhodamine B fluorescence in In-vivo from each group and quantitative measurement of fluorescence intensity are shown in Figure 2B. The fluorescence level in blood plasma samples are shown in Figure 2C. The results show significant increase in gut permeability following hydromorphone treatment which was dose dependent. Highest permeability was seen at highest dose of hydromorphone compared to control mice.
To determine the consequence of gut permeability as bacterial translocation from gut to systemic compartment of the body, liver was isolated, homogenized and cultured on blood agar plate overnight in aerobic conditions. The bacterial colonies were counted as colony forming units (CFU) and normalized per mg of total protein of liver tissue homogenate. The hydromorphone treated mice showed significant bacterial translocation compared to control mice (Figure 2D). The highest number of colonies was observed in mice treated with the highest dose of hydromorphone.
Hydromorphone worsens clinical symptoms of colitis
We next asked whether hydromorphone worsens clinical symptoms of colitis and increase severity of the disease in a mouse model of IBD. To evaluate the effect of hydromorphone on severity of colitis, the most effective dose of hydromorphone (15mg/kg/day) was used for further study in a DSS mouse model. All groups with DSS treatment exhibited an increase in diarrhea and rectal bleeding from day 3 post-DSS until completion of experiment. No mortality was observed in either of the groups for the duration of the study. All animals in the treatment groups showed significant loss in body weight compared to control, and the weight loss was significantly higher in hydromorphone plus DSS treated group compared to all other treatment groups (Figure 3A). No significant difference in DSS consumption was found between the groups.
On day 7, colonoscopy was performed to evaluate colon characteristics. The representative images of colonoscopy examination from each group are shown in Figure 3B. The colonoscopy examination shows that hydromorphone plus DSS treated mice display greater loss in translucency, fibrin, granularity, and mucosal vascular pattern (MVP) with increased bleeding and diarrhea compared to hydromorphone and DSS alone. Colonoscopy score was evaluated based on different colon characteristics, and hydromorphone plus DSS treated mice showed significantly higher score compared to control and all other treatment groups (Figure 3B). Colon length measurement is a useful assessment of colitis and considered a marker of inflammation. The representative images of colon length shortening, an indicator of the severity of colitis, following five days after DSS administration are shown in Figure 3C. The colon length of all mice in each group was measured in centimeter (cm) is shown in Figure 3C. A significant decrease in colon length was observed in hydromorphone plus DSS treated animals compared to control and individually treated groups (Figure 3C).
Hydromorphone enhances gut permeability and bacterial translocation in DSS treated mice
To determine the effect of hydromorphone and DSS on intestinal barrier function, we assessed gut permeability by oral administration of 70-kDa Rhodamine B and measuring the leakage of Rhodamine B after 4 h by IVIS. The representative in-vivo images and quantitative measurement of Rhodamine B fluorescence intensity show that gut permeability was increased in treated mice compared to control. Significantly higher levels of Rhodamine B fluorescence were observed in all treated groups and it was highest in hydromorphone plus DSS treated mice (Figure 4A).
We hypothesized that compromised gut permeability in treated mice may play a role in intestinal bacterial translocation systemically. Increase in the translocation of bacteria was observed in the liver of animals that were treated with either hydromorphone or DSS alone but significantly high level of CFU was observed in animals that were treated with both hydromorphone and DSS when compared to individually treated groups (Figure 4B).
Hydromorphone exacerbates DSS induced intestinal inflammation and immune cell infiltration
We next evaluated histologically the H&E stained sections of the distal colon in untreated and treated animals. No histopathological changes were found in the control group whereas hydromorphone and DSS treated animals showed extensive mucosal damage with large number of inflammatory cell infiltrates, mucus discharge and architectural abnormalities in crypts. In the hydromorphone plus DSS treated animals, inflammatory immune cells infiltration was widespread, and epithelial damage was evident with complete crypt disappearance, and mucosal erosion in some areas (Figure 4C), resulting in the highest score in histological scoring by microscopic analysis. The occurrence of IBD was corroborated based on histological damage and inflammatory infiltrate into the colon. In the hydromorphone and DSS group, preparations showed grade 0, 1, and 2 lesions, which was graded based on colon characteristics and presented as histological score. The histologic score in hydromorphone plus DSS treated animals was significantly higher compared to control and individually treated group (Figure 4C). We next determined the cell type of inflammatory infiltrate into the colon by immunofluorescence staining of colon sections. The representative images of our Immunofluorescence staining analysis show intestinal inflammation and infiltration of immune cells into the colon. Notably, 5 days after the DSS challenge, macrophage mobilization and accumulation in the colon was higher in hydromorphone plus DSS treated mice compared to and individually treated animals (Figure 4D).
Hydromorphone increases systemic pro-inflammatory cytokine levels and worsens inflammation in DSS-induced colitis
We next asked whether hydromorphone induces cytokines production systemically and further increase level of cytokines in DSS treated mice. The levels of cytokines were measured in the supernatant of liver homogenate using ELISA. Hydromorphone and DSS treated mice displayed significantly elevated levels of IL-6 (Figure 4E) and IL-17A (Figure 4F) and the levels were highest in hydromorphone plus DSS treated animals.
Hydromorphone induces microbial dysbiosis and decreases bacterial diversity in DSS treated mice
The results of our initial study support the hypothesis that hydromorphone perturbs gut homeostasis. Therefore, further we investigate whether these perturbations increase the severity of DSS-induced colitis. The intestinal content was collected for 16S rDNA sequencing to study gut microbiota. We determined gut microbial changes in each group of mice to establish a link between microbial dysbiosis, hydromorphone, and DSS- induced colitis in mice. The principal component analysis was used to evaluate composition of the intestinal microbiota. Correlations between the resulting changes in microbial composition and susceptibility to DSS-induced colitis identified signature bacterial families regulating the severity of colitis. Alpha-diversity analysis, which describes observed number of bacterial species richness within community, was done by Chao 1 using QIIME workflow. The result shows significant reduction in alpha diversity and species richness in the gut bacterial community in the hydromorphone plus DSS treated group compared to control and individually treated mice (Figure 5A). Beta diversity analysis, which describes observed number of species richness between community, was done by the principal component analysis (PCoA) on resulting distance matrices to generate three- dimensional plots using QIIME. The result shows distinct clustering of the microbial community based on the treatment groups, suggesting that all four-treatment group contain distinct microbial communities (Figure 5B). The unweighted UniFrac distance was calculated using standardized galaxy and QIIME (22) at the Minnesota Supercomputing Institute (University of Minnesota, Minneapolis, MN, USA). The result shows significant unifrac distance between DSS alone and hydromorphone plus DSS treated group (Figure 5C), suggesting that treatment with hydromorphone mediating the reduction in bacterial species richness and contributing to distinct bacterial communities.
Hydromorphone alters microbial composition at the phyla and lower taxonomic level
Microbial composition analysis was performed on phylum to genus level to identify the bacterial taxa, which are altered and may enhance severity of colitis in hydromorphone treated DSS mice. Global analysis of the microbiota at phyla level demonstrated a total of 10 bacterial phyla, of which 4 were more abundant including Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia and 6 were less abundant including Actinobacteria, Deferribacteres, Tenericutes, cyanobacteria and TM7. Hydromorphone treatment significantly altered the composition of gut microflora in DSS treated mice. The bacterial phylum Firmicutes was underrepresented and Proteobacteria and Verrucomicrobia were overrepresented in hydromorphone plus DSS treated animals compared to control (Figure 5D). On the other hand, the OTU abundance analysis shows significant decrease in Firmicutes and significant increase in Proteobacteria, and Verrucomicrobia phyla in hydromorphone plus DSS treated mice compared to control and individually treated mice (Figure 5D). The progression of colitis did not affect Bacteroidetes significantly.
Classification of the OTUs at the lower taxonomical levels resulted in identification of many taxa. At the family level, taxonomic analyses show increase abundance of Bacteroidaceae, Porphyromonadaceae, Enterococcaceae, Enterobacteriaceae, Verrucomicrobiaceae, and Peptostreptococcaceae whereas reduced abundance of Odoribacteraceae, Rikenellaceae, S24-7, Lactobacillaceae, Lachnospiraceae and Ruminococcaceae in hydromorphone treated DSS mice compare to control mice (Figure 5E). Bacteroidaceae expanded to represent the major bacterial family. The abundance of Enterobacteriaceae correlated with the magnitude of colitis. Taxonomic analysis at the genus level shows expansion of Bacteroides, Parabacteroides, Enterococcus, Turicibacter, Ruminococcus, Sutterella, Bilophila, and Akkermansia whereas depletion of Adlercreutzia, Odoribacter, AF12, Lactobacillus, and Anaerostipes in hydromorphone plus DSS treated mice compare to control mice (Figure 5F). The analysis indicated that Shutterella accounted for the observed increase of Enterobacteriaceae, Bacteroides for Bacteroidaceae and Akkermansia for Verrucomicrobiaceae. The species level analyses show elevation of Bacteroides acidifaciens, Ruminococcus gnavus, Akkermansia muciniphila whereas contraction of Mucispirillum schaedleri, Lactobacillus reuteri in hydromorphone treated DSS mice compare to control mice.
Hydromorphone induces functional pathogenicity and anti-microbial resistance
Phylogenetic marker (16S rRNA) gene profiling is a crucial technique to study microbial communities but does not provide indication about functional consequences of the community. To further measure the functional composition or phenotypes following intestinal microbial dysbiosis, predicted metagenomic functional analysis was performed using a BugBase software package. BugBase analyses is a computational approach that relies on 16S rRNA marker gene sequencing data and a database of reference genomes. The analysis show that hydromorphone induces functional phenotypic alterations in gut microbiota in DSS treated mice, which results in loss of protective and rise in harmful bacterial species. There was decrease in anaerobic bacterial population (Figure 6A) and significant decrease in Gram positive bacterial population (Figure 6B) was observed in hydromorphone plus DSS treated mice compared to control mice. Furthermore, there was a significant increase in facultative anaerobic (Figure 6C), Gram negative (Figure 6D), pathogenic (Figure 6E), mobile genetic elements (MGEs) containing (Figure 6F), biofilm forming (Figure 6G) and stress tolerant bacteria (Figure 6H) in hydromorphone plus DSS treated group compared to control and other treatment groups (Figure 6). These results implicate that reduced abundance of anaerobic and an expansion of facultative anaerobic bacteria is associated with intestinal inflammation (23). Expansion in Gram-negative bacteria and their products induce strong inflammatory response in the gut. Increase in pathogenic bacteria promote infection in the host and deplete commensal microbiota by exploiting nutrients and signals derived from the microbiota (24, 25). The MGEs containing bacteria are responsible for the development of bacterial virulence, antibiotic resistance and production of toxins (26). Hydromorphone plus DSS treatment trended towards bacterial communities that are higher in biofilms forming characteristics and associated with enhanced resistance to phagocytosis and antimicrobial agents (27, 28). Thus, these pathogenic or functionally altered commensal bacteria collectively result in enhanced inflammation and increase level of inflammatory cytokines such as IL-6 (Figure 4E) and IL-17A (Figure 4F).
Hydromorphone in combination with DSS up-regulates Mu Opioid Receptor (MOR) and Toll Like Receptor (TLR) expression
Recent studies show increased expression of MOR and its implication in DSS induced colitis (29) and IBD (30). Hydromorphone is a derivative of morphine, which mediate its effects via MOR. Therefore, we determined whether hydromorphone and DSS treatment increase expression of MOR in colonic tissues of mice. Total RNA was isolated from these cells and processed for qPCR. Our results showed that hydromorphone increased DSS induced mRNA levels of MOR (Figure 7A) compared to control and individually treated groups. Previous studies from our laboratory have shown that morphine induced gut microbial dysbiosis and bacterial translocation are mediated by TLR signaling. To determine whether TLR expression on colonic cells is a potential mechanism by which hydromorphone and DSS modulates barrier function, we measured TLR2 and TLR4 expression on colonic cells by qPCR. Results show that hydromorphone plus DSS treatment significantly up-regulated mRNA levels of TLR2 (Figure 7B) and TLR4 (Figure 7C) compared to control and hydromorphone alone treated groups, which was consistent with observed human studies (31, 32).
Hydromorphone modulates intestinal tight junction proteins organization in a MLCK-dependent manner
Previous studies have demonstrated an association between TLR activation and disruption of tight-junction protein organization in the intestine, which results in gut barrier dysfunction, bacterial translocation and inflammation (3). Similarly, our findings showing significantly increased expression of TLRs in the intestine of hydromorphone plus DSS treated mice prompted us to study the role of tight-junction proteins disruption as a potential mechanism. Our results show that in control group, the paracellular tight junction protein ZO-1 localized on the apical side of the membrane with a continuous and intact organization (Figure 8E). In contrast, H and DSS treated either alone or in combination showed disrupted and disorganized localization of ZO-1 with loss of bright green spots. The smooth arc-like ZO-1 transformed into a complex series of irregular undulations with thinner and more serpentine morphology suggesting impaired recruitment of the protein to the membrane (Figure 8E). The trans-membrane protein claudin-1 also localized on the apical side of the epithelium in control mice however, its organization was seen to be disrupted in H and DSS treated either alone or in combination (Figure 8F). Our results indicate that hydromorphone treatment impacts the distribution and organization of tight junction proteins, resulting in increased intestinal permeability. Quantification of the tight junction protein intensity also showed significant reduction in the intensity of the tight junction proteins in either hydromorphone and DSS group treated alone or in combination.
Next we determined the mechanism of how modulation of TLR expression by hydromorphone disrupts tight junction protein organization. Recent studies have shown that activation of MLCK induces phosphorylation of MLC, resulting in the internalization of associated tight junction proteins such as ZO-1 and claudin-1 (Ref). To validate the role of MLCK in tight junction modulation, animals were injected with MLCK inhibitor ML-7 (2mg/kg; BID) prior to hydromorphone and DSS treatment. Disruption of ZO-1 and claudin-1 were significantly decreased in the ML-7 pre-treated hydromorphone and DSS groups and organization of tight junction proteins were similar to that in the control group (Figure 8E, F).
MLCK inhibition with ML-7 attenuates hydromorphone induced severity of colitis in DSS treated mice
We next asked whether inhibition of MLCK activity by ML-7 attenuates the detrimental effects of hydromorphone on clinical symptoms of colitis and severity of the disease in a mouse model of IBD. The effect of ML-7 on symptoms of colitis was evaluated in an independent experiment in which the effect of hydromorphone on DSS induced colitis was similar as described previously (Figure 3 & 4). We demonstrate that inhibition of MLCK activity by ML-7 attenuated severity of colitis in H+DSS treated group (Figure 8).
We also measured the body weight loss and found that hydromorphone and DSS group showed significant weight loss (Figure 8A), which was similar to what was described previously (Figure 3A). The H+DSS group exhibited significant weight loss compared to hydromorphone and DSS alone. The weight loss was significantly reversed by ML-7 treatment in the H+DSS group (Figure 8A).
The colon length of all mice in each group was measured in cm and the representative images of colon length are shown (Figure 8B). All DSS treated animals showed significant colon length shortening, which was similar to what was shown previously (Figure 3C). A significant decrease in colon length was observed in hydromorphone plus DSS treated group compared to control and individual treatment groups, which was significantly reversed by ML-7 treatment (Figure 8B).
Next, we evaluated bacterial translocation in the liver. All animals in hydromorphone and DSS group show bacterial translocation, which was reduced partially by ML-7 treatment. The number of CFU was markedly higher in H+DSS combined group than that in the control and separately treated group, which was significantly reduced by ML-7 treatment (Figure 8C). The result indicated that ML-7 protects the intestinal mucosal barrier in murine IBD model particularly in the context of opioid treatment..
In histological evaluation, histological score was significantly higher in H+DSS group compared to control and separately treated groups mainly in the severity of mucosal, epithelial cells, crypts damage and inflammatory cells infiltration. Moreover, the protective effects of MLCK inhibitor ML-7 were significantly marked in H+DSS+ML-7 treated group, which showed normal appearance with little damage or inflammation compared to control group (Figure 8D).
We next asked whether ML-7 treatment attenuate hydromorphone induced cytokines production in DSS treated mice. Hydromorphone and DSS treated mice displayed significantly elevated levels of IL-6 and IL-17A and the levels were highest in the hydromorphone plus DSS treated animals as shown previously (Figure 4E & F). The level of cytokines were significantly lower in ML-7 treated groups (Figure 8G & H).
The aim of this study was to evaluate the consequences of opioid exposure on severity of IBD. Several clinical reports implicate increased complications, increased hospitalizations, and decreased quality of life in IBD patients that are on opioids however it is not clear if these patients require opioids because of greater underlying inflammation or opioid use drives inflammation leading to greater complications. Our findings show more histological damage such as villi and crypt distortion, epithelial erosion, infiltration and/or recruitment of immune cells into the intestine, which results in an increased level of inflammation in IBD patients that are on opioid compared to non-opioid using patients (Figure 1). The histologic score in opioids using IBD patients was significantly higher compared to patients that are not on opioids but on similar medical therapy. Our findings are consistent with previous study showing association of opioids with decreased quality of life in IBD patients (21).
To determine if opioids use is driving the increased severity of IBD, we next investigate effect of hydromorphone on severity of the disease in a murine model of IBD by evaluating various clinical symptoms of DSS-induced colitis. Our results show that use of hydromorphone exacerbated DSS induced colitis in mice. Indeed, hydromorphone alone was responsible for inducing intestinal permeability, bacterial translocation (Figure 2) and alteration in gut microbiota (Figure 5). Treatment with hydromorphone increased severity of colitis in DSS treated mice, which was demonstrated by significantly decreased body weight, Increased colonoscopy score and shortening of colon length (Figure 3). It is well established that DSS induces inflammation by damaging intestinal epithelial monolayer and allowing the dissemination of bacteria and their products into the peripheral tissues. Hydromorphone treatment further Increases DSS induced gut permeability, bacterial translocation and pro-inflammatory cytokines level (Figure 4). The clinical symptoms were associated with the presence of significant increase of histological damage in hydromorphone plus DSS treated mice, which was similar to histological findings that were seen in opioids using IBD patients (Figure 1). Our observed clinical symptoms are consistent with previous studies showing shortening of the colon, characteristic intestinal histology and increase in inflammatory infiltrate of immune cells leading to pro-inflammatory cytokine production (12, 33).
Usually acute colitis is induced by using 3-5% DSS for 5-9 days treatment (14, 15, 34). In our model, we show that using lower concentration of DSS (3%) for a short duration (5 days) induces milder acute colitis, which is a more relevant model for studying the impact of opioid on severity of DSS-induced colitis. Here, we show that hydromorphone and DSS independently altered gut microbiota but in combination a significantly greater microbial dysbiosis was observed with exacerbation in the severity of colitis. Hydromorphone treatment resulted in reduced gut bacterial diversity and induced dysbiosis by contraction in Firmicutes phyla and expansion in Proteobacteria and Verrucomicrobia phyla in DSS treated mice (Figure 5). Microbial dysbiosis resulted in dysfunction of commensal microbiota and increase in pathogenic, mobile genetic elements containing and biofilm forming bacteria (Figure 6).
We and others have shown that DSS-induced colitis in mice resembles IBD in humans because of several similar features such as clinical symptoms, altered gut microbiota, inflammatory markers and histopathological changes (11). Therefore, based on our results, we conclude that hydromorphone use for pain management increases the severity of IBD. We also found an association between opioid use and intestinal inflammation in IBD patients. Our findings of opioid use an indicator of more severe disease are in agreement with previous reports showing narcotic use is associated with worse disease activity and diminished quality of life in IBD patients (21).
The healthy gut microbiota is known to be stable over time. However, diseases associated with immune responses drive the microbial community to an imbalanced unstable state (35). Alpha diversity analysis of gut microbiota in the hydromorphone treated DSS mice showed reduced bacterial species richness. The PCoA showed that all four groups were composed of distinct microbial communities, which clustered separately according to the treatment group. Our findings of alpha and beta diversity are in agreement with previous report showing decreased fecal species richness and distinct microbial communities after induction of colitis (36, 37). Normally in a healthy gut, the most abundant phyla Firmicutes and Bacteroidetes dominate the microbial community, while Proteobacteria, Verrucomicrobia, Actinobacteria, and TM7 are less abundant. Our taxonomic analysis show that the dominant bacterial species were drastically altered, which was characterized by a significant decrease in Firmicutes and an increase in the relative abundance of Proteobacteria, and Verrucomicrobia in hydromorphone plus DSS treated animals, which is consistent with other reports in IBD patients (37, 38). We observed significant differences between hydromorphone and non-hydromorphone groups at the taxonomic level after induction of colitis and found that specific taxa were associated with these groups. Of interest, Bacteroides, Enterococcus, Sutterella, and Akkermansia were enriched whereas Lactobacillus and Lachnospiraceae were decreased in the hydromorphone group before induction of colitis indicating their strong association with hydromorphone exposure. The changes in abundance of these bacteria at phylum and genus levels were markedly increased in hydromorphone plus DSS treatment group. Therefore, we can speculate that hydromorphone induced changes in these taxa may have specific roles in the susceptibility and severity of DSS-induced colitis. The expansion of Sutterella is associated with degradation of secretory IgA and mucosal damage (37). The enriched level of Akkermansia Muciniphila, a putative host-derived mucin degrader, has been linked with inflammation and contribute to exacerbation of colitis (39). The expansion of Proteobacteria is associated with intestinal inflammation.
We further determined the functional consequences following alteration of gut microbiota. Our predicted metagenomic functional analysis showed significant increase in pathogenic, MGEs containing, biofilm forming, facultative anaerobic and Gram negative bacteria whereas decrease in anaerobic and Gram positive bacteria in hydromorphone plus DSS treated mice compared to all other groups. Studies have shown that pathogenic bacteria promote their development and virulence by using nutrients and regulatory signals produced by commensal microbiota thereby promoting infection (40). Biofilms forming bacteria adhere to the surface and increases their metabolic efficiency. Increased Biofilms forming bacteria offer horizontal gene transfer, produce toxins, show enhanced resistance to phagocytosis and antimicrobial agents (27, 28). MGEs containing bacteria transfer DNA fragments, which play important roles in the development of bacterial virulence, antibiotic resistance and production of toxins (26). Proteobacteria especially Enterobacteriaceae can cause metabolic dysfunctions of the microbiota and affect the production of bacterial byproducts such as short-chain fatty acids (SCFAs), thus impacting mucosal immune response (35). The mucosal inflammatory environment favors the growth of Gram negative facultative anaerobes Proteobacteria at the cost of obligate anaerobes Firmicutes. Enterobacteriaceae can utilize oxygen or nitrate for their aerobic and anaerobic respiration respectively. Thus, expansion of these communities outcompete commensal bacteria by reducing the availability of oxygen (41) and deplete Firmicutes that depend on fermentation for their development (35). Enterobacteriaceae are also known to possess MGEs, which are responsible for genetic variability and gene transfer in bacterial strains and might contribute to their strength to outcompete other commensal gut microbiota (35). Furthermore, there is reduced abundance of anaerobic and Gram positive bacteria (Firmicutes) indicating perturbation to gut homeostasis. The above findings implicate that hydromorphone treated commensal gut microbiota might be less likely to outcompete pathogenic bacteria and prevent their colonization in the gut of DSS treated mice. Based on above findings, we propose that an increased prevalence of Proteobacteria and Verrucomicrobia may be a useful diagnostic signature marker of dysbiosis and severity of IBD.
It is still unclear whether alterations in the gut microbial composition represent cause or consequence of host inflammation and state of the disease. Hydromorphone induced changes in gut microbiota are crucial determinants in the susceptibility to experimental colitis in a murine model of IBD. The mechanism by which hydromorphone induces microbial dysbiosis and exacerbate severity of colitis is not fully clear. Hydromorphone is a derivative of morphine, which mediate its effects via MOR. Hydromorphone and DSS separately increases MOR expression and it was higher in hydromorphone plus DSS treated mice (Figure 7A). Here we show that microbial dysbiosis is also associated with mobilization of inflammatory immune cells into the colon (Figure 4D), which play an important role in production of cytokines and thus in inflammation. Recent studies have also shown the association of increased MOR expression with IBD through increased migration of circulatory immune cells to the site of inflammation (2, 30), consistent with our findings. Based on these results we speculate that hydromorphone plus DSS treatment may exert its immune-modulatory effects via MOR signaling. The role of TLRs is well established in mucosal pathogenic complications. Previously our lab has shown that morphine induced microbial dysbiosis and gut bacterial translocation are mediated by TLR signaling (3, 22). Others have shown that DSS can stimulate TLRs and modulates tight junction proteins leading to disrupted epithelial barrier, which results in gut permeability and subsequent inflammation (42). To determine, if hydromorphone play a role in the severity of colitis via modulation of TLRs, we determined TLR2 and TLR4 mRNA expression in colon tissues. We found significantly higher level of TLR2 (Figure 7B) and TLR 4 (Figure 7C) expression in hydromorphone plus DSS treated mice compared to control and hydromorphone alone. Our findings are corresponding with the observations of previous studies showing high level of TLR expression in colon biopsy of IBD patients (31, 32) and in DSS treated mice (43, 44) compared to controls. In our study, TLR2 expression level was higher compared to TLR4 expression. The possible reason could be TLR2 interacts with peptidoglycan, a component of all bacterial cell walls, as well as additional constituents of Gram-positive bacteria and fungi. A recent study also demonstrated that gram-positive bacteria trigger colitis by inducing monocyte/macrophage recruitment into the colon (18), which is similar to our findings. Our results implicate that hydromorphone, a MOR selective ligand, play a role in the progression of DSS induced colitis through activation of MOR and TLRs signaling pathways. We can hypothesize that both hydromorphone and DSS, alter gut microbial composition, which initiate recruitment and local proliferation of immune cells into the colon to create inflammatory environment. Modulation of TLRs expression by hydromorphone plus DSS play a role in gut permeability and bacterial translocation.
We show strong relationship between increase in TLR expression and disruption of tight-junction protein organization in the intestine, which is consistent with previous observations (3). Tight junction proteins have been shown to seal the gap between gut epithelial cells and play an important role in preventing potential pathogen invasion. Distribution of TJ proteins is involved in modulating intestinal permeability. Therefore, we determined intestinal epithelial TJ protein expression in this study. We show that hydromorphone treatment significantly disrupted tight junction protein organization in gut epithelial cells, which results in compromised intestinal barrier function, bacterial translocation and inflammation. We speculate that increased intestinal inflammation and proinflammatory cytokines play a feedback role on the intestinal epithelium activating the MLCK signaling pathway to cause further barrier dysfunction and thus increase severity of colitis. Some of the reports have shown that an increased intestinal permeability was partly regulated by MLCK upregulation and increasing phosphorylation of MLC. Therefore, we evaluated the effect of MLCK inhibitor ML-7 on the detrimental effect of hydromorphone on severity of DSS induced colitis. The administration of ML-7 prevented the intestinal barrier dysfunction by inhibiting MLCK protein expression and increasing intestinal epithelial TJ expression. We also show that ML-7 treatment ameliorated the clinical symptoms of colitis including weight loss, colon length shortening, histological intestinal damage and inflammation in H+DSS+ML7 group. These results indicate that the beneficial effects of ML-7 on the intestinal mucosal barrier may suppress the intestinal inflammatory pathology and subsequently attenuate colonic inflammation.
The impaired intestinal barrier function and inflow of commensal bacteria are prerequisites for chronic inflammation, which results in increased severity of DSS induced colitis. We show that the severity of colitis was significantly attenuated by selective inhibition of MLCK activation using ML-7 (Figure 9). The results of this study suggest that use of opioids for pain management is a risk factor in the co-morbidities associated with IBD by potentially altering the gut microbiome. The restoration of gut homeostasis by modulating the gut microbiota could be a relevant therapeutic strategy.
We retrospectively identified IBD patients followed at the Crohn’s and Colitis Center of the University of Miami from 2013 to 2017. Information on disease type (CD or UC), opioid use, prescription medication, demographic and clinical history was extracted from the medical record of patients. The intake visits of 10 CD patients were evaluated based on collected database. A total of 5 CD patients using chronic prescription opioids for analgesia were identified and included in the study. A total of 5 CD patients that were not using opioids but on similar medical therapy served as the control group.
We utilized pathogen-free wild-type C57BL/6 male mice (Charles Rivers Laboratories International, Inc., USA), ages 8-10 weeks old. Mice were kept under standardized conditions at 22±2℃ and 50% humidity, under a 12-hour light/dark cycle and were maintained with ad lib access to standard chow and tap water. Acclimatization to the laboratory conditions occurred for at least 7 days before experimental inclusion (18, 39).
The animals were treated with clinically relevant doses of hydromorphone 0.53mg-1.2mg/kg/day which was calculated to be equivalent to 2.5-15mg/kg/day in mice (45) by intraperitoneal injection (IP). After 48 hours of treatment, the mice were euthanized. In this manner, the treatment dose of hydromorphone for subsequent experiments in the DSS colitis model was determined. Further mice were treated with saline (Control), hydromorphone (H), DSS (DSS), and hydromorphone plus DSS (H+DSS). While hydromorphone was continued, at day 3, acute colitis was induced by oral administration of 3% DSS (molecular weight; MW 40 kDa: MP Biomedicals, Soho, OH, USA) dissolved in drinking water for 5 days. The milder DSS for short duration treatment model is a powerful means of evaluating the effect of hydromorphone on the severity of colitis in a murine model of IBD. To evaluate the effect of MLCK inhibitor ML-7 on intestinal tight junction organization and severity of colitis in hydromorphone and DSS treated mice, the most widely using dose of ML-7 (2 mg/kg; BID) was used. Animals were injected with ML-7 overnight before hydromorphone and/or DSS treatment (Ref). The untreated mice served as control and received normal drinking water (18, 46).
Measurement of body weight and colon length
The mice body weight was measured on day 0 (initial weight) and day 7 (final weight) and the weight loss was determined. After seven days, mice were euthanized and the entire colon length was removed and measured in cm to determine the colon length of control and treated mice (11, 46).
Endoscopic examination of colon
On day 7, the endoscopy was performed to evaluate DSS-induced colonic destruction using Coloview system (Karl Storz Veterinary Endoscopy, Tuttlingen, Germany). The endoscopic procedure was viewed and digitally recorded. Colonoscopic score was given by assessing different colonic features including translucency, presence of fibrin, granularity, mucosal vascular pattern (MVP) loss, stool characteristic and presence of blood. For each category, a score of 0 indicated normal, 1 indicated mild change, 2 indicated moderate change and 3 indicated severe change. The combined colonoscopic score ranged from 0 (normal appearance) to 18 (severe damage and inflammation) was determined in similar fashion as mentioned (11).
Gut permeability assay
To evaluate the gut leakiness, mice were gavaged with Rhodamine B isothiocyanate-dextran (MW 70kDa, Sigma-Aldrich) at the dose 600mg/kg body weight of mouse.After 3-4 h, fluorescence intensity was measured using Xenogen live IVIS. The fluorescence images were digitally recorded. The quantification of the fluorescence intensity was done using Living Image software. Fluorescence intensity in plasma of the mice was measured using a Fluorometer (Thermo Scientific™) with an excitation/emission wavelength of 570/590 nm.
To examine the impact of opioids on severity of IBD in humans, we included 10 patients with confirmed cases of CD and complete assessment of medical history. Based on medical record, a total of 5 patients using opioids and 5 patients not using opioids were identified. To examine intestinal inflammation and pathological damage, histological analysis was performed using terminal ileum biopsy sample of CD patients. Biopsy specimens were fixed with 10% formaldehyde overnight, embedded in paraffin, sectioned at 5 μm, mounted on clean glass slides, and stained with H&E for histological evaluation. The H&E stained samples were randomized and coded to perform unbiased histological evaluation. The slides were analyzed in a blinded fashion by a pathologist using light microscopy. Each sample was given a histological score based on cumulative scores specified to different histological features including degree of epithelial damage or tissue destruction, loss of crypt and villi architecture, inflammatory cell infiltration, and ulceration or edema. For each category, a score of 0 indicated normal appearance, a score of 1 indicated mild change, and a score of 2 indicated severe change, encompassing greater than 50% of the high-power field. The combined histological score ranged from 0 (normal appearance) to 8 (extensive tissue damage and inflammatory cell infiltration) was determined in similar fashion as mentioned in previous studies (18, 46).
To further extrapolate our observation, we investigated the consequence of hydromorphone on mouse model of IBD. Mouse tissue harvested from the distal colon (1-cm from the rectum) was cut into segments, and stained with H&E for histological evaluation and each sample was assigned a histological score as described above for histological evaluation.
After necropsy, liver tissue was harvested and homogenized in sterile PBS using 100μm cell strainers (BD Biosciences) following aseptic techniques. Homogenized tissue was then plated on blood agar plates and incubated at 37°C overnight in aerobic conditions. Bacterial colonies were counted as CFU and normalized for varying protein concentrations in the tissue homogenate.
Cytokine level measurement
Liver tissue was homogenized and suspended in PBS containing protease inhibitors (sigma). Supernatants were collected after centrifugation (10,000 rpm for 5 min) and the levels of cytokines such as IL-6 and IL-17A were determined using ELISA (ebiosciences) (18).
The fixed colon sections were deparafﬁnized with xylene and rehydrated through a series of graded alcohols, and then processed for antigen retrieval. These processed sections of colonic tissue were used for macrophage and tight junction staining. For macrophage staining, tissues were incubated with FITC-conjugated anti-mouse F4/80 antibody (BD Biosciences, United States) at a 1:100 dilution in PBS for overnight at 4℃, followed by counterstained with DAPI. The intestinal sections were stained for ZO-1 and claudin-1, two proteins integral to the formation of epithelial tight-junction. For tight junction staining, tissue sections were incubated with polyclonal rabbit antibody against ZO-1 or claudin-1 (Invitrogen) in PBS with 1% bovine serum albumin (BSA) for overnight at 4℃. After washing, sections were incubated with rhodamine phalloidin (Invitrogen) and secondary Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Invitrogen) for 1 hour at room temperature. After washing thrice in PBS, sections were mounted under coverslips using ProLong Gold antifade reagent with DAPI (Invitrogen). Sections were imaged and photographed using a laser confocal microscope (Leica Microsystems, Germany). Image J RG2B software was used to quantify the intensity of green fluorescence and normalized to blue fluorescence (DAPI).
Microbiota analysis using bacterial 16s rDNAamplification andmiseq250sequencing
Enteric content (fecal material) was collected from distal colon region and frozen on dry ice. The DNA was isolated using Power-soil/fecal DNA isolation kit (Mo Bio Laboratories, Inc.) as per manufacturer’s specifications. All DNA samples were quantified using Qubit® Quant-iT dsDNA Broad- Range Kit (Invitrogen, Life Technologies, Grand Island, NY) and submitted to the University of Minnesota Genomic Center for sequencing of bacterial 16S V5-V6 rDNA region (47). Resulting sequences were then searched against the Greengenes reference database of 16S sequences, clustered at 97% by UCLUST (closed-reference OTU picking). The microbiome data was analysed using QIIME at the phyla and lower taxonomic level (22).
Microbiome phenotype analysis
Predicted metagenomic functional analysis based on microbial communities using 16s rDNA marker gene sequences was performed (48) by using a BugBase software package, which is based on the softwares like PICRUSt, QIIME (49) and KEGG metabolic OTUs from the GreenGenes reference database (50).
Quantitative real-time polymerase chain reaction (qPCR)
Total RNA from colon tissue was isolated using TRIzol (Invitrogen) method. The cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The changes in host gene expression were measured using gene specific primers for MOR, TLR2, TLR4, and GAPDH from Invitrogen. qPCR was performed using LightCycler® 96 Real-Time PCR System (Roche) and mRNAs were quantified using SYBR green Master Mix as directed by the manufacturer’s protocol. All samples were run in triplicate, and relative mRNA expression levels were determined after normalizing all values to the unaffected housekeeping gene GAPDH as the reference.
All results are presented as mean ± SEM. Data was analyzed using GraphPad Prism 6 statistical software and P<0.05 was considered as statistically significant. Statistical comparison between two groups was analyzed using Mann–Whitney U test. Statistical comparison among more than two groups was analyzed using one-way ANOVA when effect of one factor being compared and two-way ANOVA was performed when two factors were analyzed followed by Tukey’s multiple comparisons test.
All human studies were conducted with the approval of the Institutional Review Boards at the University of Miami and Jackson Memorial Hospital (Miami, FL, USA). The written informed consent was received from participants prior to inclusion in the study. Participants are identified by provided number and not by their name. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC), University of Minnesota and University of Miami. The experiments were performed in compliance with the institutional laws and guidelines.
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