Mitochondrial function and gastrointestinal diseases.

Mitochondria are dynamic organelles that function in cellular energy metabolism, intracellular and extracellular signalling, cellular fate and stress responses. Mitochondria of the intestinal epithelium, the cellular interface between self and enteric microbiota, have emerged as crucial in intestinal health. Mitochondrial dysfunction occurs in gastrointestinal diseases, including inflammatory bowel diseases and colorectal cancer. In this Review, we provide an overview of the current understanding of intestinal epithelial cell mitochondrial metabolism, function and signalling to affect tissue homeostasis, including gut microbiota composition. We also discuss mitochondrial-targeted therapeutics for inflammatory bowel diseases and colorectal cancer and the evolving concept of mitochondrial impairment as a consequence versus initiator of the disease.

Acute gastrointestinal permeability after traumatic brain injury in mice precedes a bloom in Akkermansia muciniphila supported by intestinal hypoxia.

Traumatic brain injury (TBI) increases gastrointestinal morbidity and associated mortality. Clinical and preclinical studies implicate gut dysbiosis as a consequence of TBI and an amplifier of brain damage. However, little is known about the association of gut dysbiosis with structural and functional changes of the gastrointestinal tract after an isolated TBI. To assess gastrointestinal dysfunction, mice received a controlled cortical impact or sham brain injury and intestinal permeability was assessed at 4 h, 8 h, 1 d, and 3 d after injury by oral administration of 4 kDa FITC Dextran prior to euthanasia. Quantification of serum fluorescence revealed an acute, short-lived increase in permeability 4 h after TBI. Despite transient intestinal dysfunction, no overt morphological changes were evident in the ileum or colon across timepoints from 4 h to 4 wks post-injury. To elucidate the timeline of microbiome changes after TBI, 16 s gene sequencing was performed on DNA extracted from fecal samples collected prior to and over the first month after TBI. Differential abundance analysis revealed that the phylum Verrucomicrobiota was increased at 1, 2, and 3 d after TBI. The Verrucomicrobiota species was identified by qPCR as Akkermansia muciniphila, an obligate anaerobe that resides in the intestinal mucus bilayer and produces short chain fatty acids (e.g. butyrate) utilized by intestinal epithelial cells. We postulated that TBI promotes intestinal changes favorable for the bloom of A. muciniphila. Consistent with this premise, the relative area of mucus-producing goblet cells in the medial colon was significantly increased at 1 d after injury, while colon hypoxia was significantly increased at 3 d. Our findings reveal acute gastrointestinal functional changes coupled with an increase of beneficial bacteria suggesting a potential compensatory response to systemic stress after TBI.

Effectiveness of Upadacitinib for Patients With Acute Severe Ulcerative Colitis: A Multicenter Experience.

INTRODUCTION: A significant proportion of patients with acute severe ulcerative colitis (ASUC) require colectomy. METHODS: Patients with ASUC treated with upadacitinib and intravenous corticosteroids at 5 hospitals are presented. The primary outcome was 90-day colectomy rate. Secondary outcomes included frequency of steroid-free clinical remission, adverse events, and all-cause readmissions. RESULTS: Of the 25 patients with ASUC treated with upadacitinib, 6 (24%) patients underwent colectomy, 15 (83%) of the 18 patients with available data and who did not undergo colectomy experienced steroid-free clinical remission (1 patient did not have complete data), 1 (4%) patient experienced a venous thromboembolic event, while 5 (20%) patients were readmitted. DISCUSSION: Upadacitinib along with intravenous corticosteroids may be an effective treatment for ASUC.

Inflammatory Bowel Disease in Appalachian Kentucky: An Investigation of Outcomes and Health Care Utilization.

BACKGROUND: Rural residence has been associated with a lower incidence of inflammatory bowel disease (IBD) but higher health care utilization and worse outcomes. Socioeconomic status is intrinsically tied to both IBD incidence and outcomes. Inflammatory bowel disease outcomes have not been investigated in Appalachia: a rural, economically distressed region rife with risk factors for both increased incidence and unfavorable outcomes. METHODS: Hospital inpatient discharge and outpatient services databases were utilized to assess outcomes in patients diagnosed with either Crohn's disease (CD) or ulcerative colitis (UC) in Kentucky. Encounters were classified by patient residence in Appalachian or non-Appalachian counties. Data were reported as crude and age-adjusted rates of visits per 100,000 population per year collected in 2016 to 2019. National inpatient discharge data from 2019, stratified by rural and urban classification codes, were utilized to compare Kentucky to national trends. RESULTS: Crude and age-adjusted rates of inpatient, emergency department and outpatient encounters were higher in the Appalachian cohort for all 4 years observed. Appalachian inpatient encounters are more frequently associated with a surgical procedure (Appalachian, 676, 24.7% vs non-Appalachian, 1408, 22.2%; P = .0091). In 2019, the Kentucky Appalachian cohort had significantly higher crude and age-adjusted rates of inpatient discharges for all IBD diagnoses compared with national rural and nonrural populations (crude 55.2; 95% CI, 50.9-59.5; age-adjusted 56.7; 95% CI, 52.1-61.3). CONCLUSIONS: There is disproportionately higher IBD health care utilization in Appalachian Kentucky compared with all cohorts, including the national rural population. There is a need for aggressive investigation into root causes of these disparate outcomes and identification of barriers to appropriate IBD care.

The Kentucky Appalachian IBD population experiences increased health care utilization, with increased rates of inpatient admissions, emergency department, and outpatient visits compared with non-Appalachian Kentuckians. Kentucky Appalachian rates of inpatient admissions are higher compared with national rates, controlling for rural residence.

Study Transportation of Drugs within Newly Established Murine Colon Organoid Systems.

The development of 3D organoids of the small intestine is a tremendous breakthrough in drug development and biological research. However, the development of colonic organoids (i.e., colonoids) is particularly challenging due to a lack of simple, cost-effective protocols for colonoid cultivation. Here, intestinal homogenates are described as a supplement to the culture medium for maintaining and replicating colonic stem cells. Colonoids generated by this cultivation protocol demonstrate substantial proliferation and differentiation (3 months). There is a similarity between cultured colonoids and primary colon tissue regarding structure and functionality. To evaluate the functionality of colonoids, permeability testing is performed with suspensions of 4 and 40 kDa fluorescein isothiocyanate-dextran (FITC-DEX). It is observed that neither can permeate the healthy epithelial barrier. The P-glycoprotein receptor, a vital drug efflux pump mitigating potential drug toxicity, is functionally manipulated, as evidenced by its inhibition function by verapamil and monitoring uptake of Rhodamin 123. In addition, Forskolin treatment which affects chloride transport results in organoid swelling; this confirms the functional expression of the CFTR transporter in the colonoids. This protocol to generate colonoids is promising for high-throughput drug screening, toxicity testing, and oral drug development.

Chronic opioid use is associated with early biologic discontinuation in inflammatory bowel disease.

BACKGROUND: Chronic opioid use is associated with poorer clinical outcomes in inflammatory bowel disease. AIMS: To investigate an association between chronic opioid use and persistence with biologic agents in management of inflammatory bowel disease. METHODS: A total of 16 624 patients diagnosed with inflammatory bowel disease and receiving a first-time biologic prescription from 2011 to 2016 were identified retrospectively from the Truven MarketScan Database. A cohort of 1768 patients were identified as chronic opioid users utilising outpatient prescription claims. Utilisation patterns of biologic therapies were assessed from inpatient administration and outpatient claims data, including persistence calculations. Information on healthcare utilisation and common comorbidities was also collected. A Cox regression model was constructed to assess the hazard of chronic opioid use on early discontinuation of biologic therapy controlling for disease severity. RESULTS: A mean 1.5 different biologic agents were utilised by inflammatory bowel disease patients with chronic opioid use (vs 1.37 in the comparator group; P < 0.0001). A lower proportion of the chronic opioid use cohort persisted on biologic therapies to the end of the study period (16.2% vs 33.5% P < 0.0001). Inflammatory bowel disease patients with chronic opioid use utilised more healthcare resources and had a higher rate of comorbidities than the reference cohort. Patients with chronic opioid use were 23% more likely (hazard ratio 1.23; 95% CI [1.16-1.31]) to be non-persistent with biologic therapy while accounting for relevant markers of disease acuity. CONCLUSIONS: Chronic opioid use is associated with increased hazard of biologic discontinuation in inflammatory bowel disease. Symptoms of opioid withdrawal may mimic IBD flares thereby leading providers to inappropriately switch biologic therapies and compromise disease control.

Vedolizumab Dose Escalation Improves Therapeutic Response in a Subset of Patients with Ulcerative Colitis.

BACKGROUND: The Gemini trial failed to detect a significant difference in response rate for patients with ulcerative colitis (UC) randomized to standard (every 8 week) vedolizumab dosing vs escalated (every 4 week) dosing. Subsequent real-world data imply the Gemini trial design may have obscured a benefit of escalated dosing. AIMS: We investigated outcomes after vedolizumab dose escalation for patients with UC. We also explored potential clinical predictors of dose escalation requirement. METHODS: In this retrospective study, we included patients with UC who received vedolizumab between 1/2017-1/2019. We compared rates of clinical response (decrease in partial Mayo score by ≥ 2) and remission (partial Mayo < 2) for standard vs escalated dosing. RESULTS: Among the 90 patients reviewed, 52 achieved and maintained remission on standard dosing. The average time to remission with standard dosing was 33.3 ± 6.6 weeks. After an average of 56.3 ± 7.4 weeks standard dosing, 24 patients (22 "partial responders" and 2 "non-responders") were dose-escalated. Of the 22 "partial responders" dose-escalated, 10 (45%) achieved remission, 10 (45%) achieved further improvement. Neither "non-responder" demonstrated further clinical benefit. Prior anti-tumor necrosis factor (anti-TNF) biologic exposure predicted dose escalation requirement (p = 0.008). Patients requiring dose escalation had more severe disease at baseline as measured by both full Mayo (p = 0.009) and partial Mayo scores (p = 0.01). CONCLUSIONS: We show dose escalation benefited patients with UC who exhibit a "partial response" to standard dosing. Early vedolizumab dose escalation should be considered in both patients with severe disease and those with prior anti-TNF experience.

Ustekinumab dose escalation improves clinical responses in refractory Crohn's disease.

BACKGROUND: Clinicians often utilize off-label dose escalation of ustekinumab (UST) in Crohn's disease (CD) patients with disease refractory to standard dosing. Previous studies report mixed results with dose escalation of UST. METHODS: A retrospective observational study of 143 adult patients with CD receiving UST over a 33-month time period was conducted. Patients receiving UST at standard dosage for a minimum of 16 weeks were included in the analysis. Primary outcomes collected were clinical response [Physician Global Assessment Score (PGA) by >1] and remission (PGA = 0). Changes in clinical parameters were calculated for dose-escalated patients beginning with the time of dose switch (~42 weeks) and compared with a group of patients who were classified as "failing" standard dosing at 42 weeks who were not dose escalated. RESULTS: Dose escalation improved PGA by 0.47 ± 0.19 compared with patients remaining on every 8 weeks dosing (Q8 week), who worsened by 0.23 ± 0.23 (p < 0.05). Dose escalation decreased CRP 0.33 ± 0.19 mg/L and increased serum albumin 0.23 ± 0.06 g/dL (p < 0.05). Surprisingly, disease duration and prior CD surgeries inversely correlated with the need for dose escalation. CONCLUSION: Our results support UST Q4 week dose escalation for selected CD patients who fail to achieve remission on standard Q8 week dosing. Dose escalation improves clinical outcomes, prevents worsening disease severity, and positively impacts CRP and albumin levels. Together these data indicate that clinicians should attempt Q4 week UST dosing in refractory CD patients before switching to an alternative class of biologic therapy.

Doublecortin-like kinase 1 promotes hepatocyte clonogenicity and oncogenic programming via non-canonical ß-catenin-dependent mechanism.

Chronic liver injury is a risk factor for cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms that regulate the decision between normal injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a tumor stem cell marker, is induced during cirrhosis and HCC. Here, we demonstrate that DCLK1-overexpressing primary human hepatocytes formed spheroids in suspension cultures. Spheroids derived from DCLK1-overexpressing hepatoma cells showed high level expression of active ß-catenin, α-fetoprotein, and SOX9, suggesting that DCLK1 overexpression induces clonogenicity and dedifferentiated phenotypes in hepatoma cells. DCLK1 overexpression in hepatoma cells also increased phosphorylation of GSK-3ß at Ser9. This was associated with an induction of a 48-kDa active ß-catenin with a preserved hypophosphorylated N-terminus that interacted with nuclear TCF-4 resulting in luciferase reporter activity and cyclin D1 expression. DCLK1 downregulation inhibited 48-kDa ß-catenin expression. The proteasome inhibitor bortezomib did not block the 48-kDa ß-catenin, instead, caused a threefold accumulation, suggesting a proteasome-independent mechanism. Liver tissues from patients with cirrhosis and HCC revealed epithelial co-staining of DCLK1 and active ß-catenin, and cleaved E-cadherin. Repopulated DCLK1-overexpressing primary human hepatocytes in humanized FRG mouse livers demonstrated active ß-catenin. In conclusion, DCLK1 regulates oncogenic signaling and clonogenicity of hepatocytes by a novel non-canonical/atypical ß-catenin-dependent mechanism.

Double Biologic Therapy for Refractory Stricturing Crohn's Disease: A Successful Case of Deep Remission with Ustekinumab and Vedolizumab.

Stricturing Crohn's disease (CD) is a severe phenotype that presents unique challenges to therapeutic management. Emerging literature suggests that anti-TNF monoclonal antibody (mAb) therapies are inadequate for preventing progression to stricture. We hereby present a case of a patient with refractory CD who required multiple surgical resections despite several anti-TNF treatment regimens. Subsequent surgical complications were avoided after changing to combination vedolizumab and ustekinumab therapies every 4 weeks. This case argues for a tailored approach to CD therapy based on disease phenotype and demonstrates that combination therapy with ustekinumab and vedolizumab is a viable option for patients with stricturing disease.

SIRT2 Contributes to the Regulation of Intestinal Cell Proliferation and Differentiation.

BACKGROUND AND AIMS: Intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. Disruption of this homeostasis is associated with disorders such as inflammatory bowel disease (IBD). We investigated the role of Sirtuin 2 (SIRT2), a NAD-dependent protein deacetylase, in intestinal epithelial cell (IEC) proliferation and differentiation and the mechanism by which SIRT2 contributes to maintenance of intestinal cell homeostasis. METHODS: IECs were collected from SIRT2-deficient mice and patients with IBD. Expression of SIRT2, differentiation markers (mucin2, intestinal alkaline phosphatase, villin, Na,K-ATPase, and lysozyme) and Wnt target genes (EPHB2, AXIN2, and cyclin D1) was determined by western blot, real-time RT-PCR, or immunohistochemical (IHC) staining. IECs were treated with TNF or transfected with siRNA targeting SIRT2. Proliferation was determined by villus height and crypt depth, and Ki67 and cyclin D1 IHC staining. For studies using organoids, intestinal crypts were isolated. RESULTS: Increased SIRT2 expression was localized to the more differentiated region of the intestine. In contrast, SIRT2 deficiency impaired proliferation and differentiation and altered stemness in the small intestinal epithelium ex vivo and in vivo. SIRT2-deficient mice showed decreased intestinal enterocyte and goblet cell differentiation but increased the Paneth cell lineage and increased proliferation of IECs. Moreover, we found that SIRT2 inhibits Wnt/ß-catenin signaling, which critically regulates IEC proliferation and differentiation. Consistent with a distinct role for SIRT2 in maintenance of gut homeostasis, intestinal mucosa from IBD patients exhibited decreased SIRT2 expression. CONCLUSION: We demonstrate that SIRT2, which is decreased in intestinal tissues from IBD patients, regulates Wnt-ß-catenin signaling and is important for maintenance of IEC proliferation and differentiation.

Vitamin C and B3 as new biomaterials to alter intestinal stem cells.

Vitamin C (ascorbic acid) and vitamin B3 (niacin) have been extensively studied since the 20th century. In the area of stem cell biology, vitamin C has shown its direct impact toward homeostasis and epigenetic changes (D'Aniello et al., Stem Cells International, 2017, 1-16). Vitamin B3 aids in maintaining healthy intestinal homeostasis and reducing gut inflammation by participating in the rapamycin signaling pathway (Kumar et al., The American Journal of Physiology-Gastrointestinal and Liver Physiology, 2013). In this study, vitamin C and vitamin B3 (600 and 1,200 µg/mL) have been explored as potential new biomaterials to study their effects on four types of intestinal stem cells which are isolated from mice bearing different microbiota. We observed that C3H ASF and 129 ASF IL-10 are more sensitive towardB7 600 µg/mL vitamin B3 and 1,200 µg/mL vitamin C. The lowest growth rate and viability for all types of organoids was with 1,200 µg/mL vitamin C. From quantitative polymerase chain reaction analysis (qPCR analysis), MUC2 was upregulated for 129 ASF and C3H Conv when exposed to 600 µg/mL and 1,200 µg/mL vitamin C. It suggests that large amounts of glycoprotein may be produced after adding high concentrations of vitamin C. Since inflammatory bowel disease has low level of MUC2, this finding may be helpful in restoring mucosal health by upregulating the MUC2 gene while altering patient's microbiota (Sibila et al., Annals of the American Thoracic Society, 2016). These results are expected to have a positive translational impact because this bottom-up strategy would be instrumental in developing Vitamin C and B3 based orally available therapeutic strategies and formula for advancing the fields of gastrointestinal regenerative medicine.

Correction: Validation of near infrared fluorescence (NIRF) probes in vivo with dual laser NIRF endoscope.

[This corrects the article DOI: 10.1371/journal.pone.0206568.].

The mitochondrial retrograde signaling regulates Wnt signaling to promote tumorigenesis in colon cancer.

Cancer cells are known to upregulate aerobic glycolysis to promote growth, proliferation, and survival. However, the role of mitochondrial respiration in tumorigenesis remains elusive. Here we report that inhibition of mitochondrial function by silencing TFAM, a key transcription factor essential for mitochondrial DNA (mtDNA) replication and the transcription of mtDNA-encoded genes, markedly reduced tumor-initiating potential of colon cancer cells. Knockdown of TFAM significantly decreased mitochondrial respiration in colon cancer cells; however, the cellular levels of ATP remained largely unchanged as a result of increased glycolysis. This metabolic alteration rendered cancer cells highly susceptible to glucose deprivation. Interestingly, upregulation of glycolysis was independent of hypoxia-inducible factor-1 (HIF1) as TFAM knockdown cells fail to stabilize HIF1α under hypoxic conditions. Moreover, knockdown of TFAM results in decreased expression of genes-associated cancer stem cells downstream of Wnt/ß-catenin signaling. Metabolic analysis reveals that the level of α-ketoglutarate (α-KG) was significantly upregulated in TFAM knockout cells. Silencing of prolyl hydroxylase domain-containing protein 2 (PHD2), a α-KG-dependent dioxyenase, rescued the expression of target genes of both HIF1α and Wnt/ß-catenin. Furthermore, intestinal-specific knockout of TFAM prevents tumor formation in Apc-mutant mouse models of colon cancer. Taken together, our findings identify a novel role of mitochondria-mediated retrograde signaling in regulating Wnt signaling and tumor initiation in colon cancer.

Blocking NF-κB Activation in Ly6c+ Monocytes Attenuates Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease affecting premature infants with intestinal inflammation and necrosis. The neonatal intestinal inflammatory response is rich in macrophages, and blood monocyte count is low in human NEC. We previously found that NF-κB mediates the intestinal injury in experimental NEC. However, the role of NF-κB in myeloid cells during NEC remains unclear. Herein, inhibitor of kappaB kinase ß (IKKß), a critical kinase mediating NF-κB activation, was deleted in lysozyme M (Lysm)-expressing cells, which were found to be Cd11b+Ly6c+ monocytes but not Cd11b+Ly6c- macrophages in the dam-fed neonatal mouse intestine. NEC induced differentiation of monocytes into intestinal macrophages and up-regulation of monocyte recruitment genes (eg, L-selectin) in the macrophage compartment in wild-type mice, but not in pups with IKKß deletion in Lysm+ cells. Thus, NF-κB is required for NEC-induced monocyte activation, recruitment, and differentiation in neonatal intestines. Furthermore, pups with Lysm-IKKß deletion had improved survival and decreased incidence of severe NEC compared with littermate controls. Decreased NEC severity was not associated with an improved intestinal barrier. In contrast, NEC was unabated in mice with IKKß deletion in intestinal epithelial cells. Together, these data suggest that recruitment of Ly6c+ monocytes into the intestine, NF-κB activation in these cells, and differentiation of Ly6c+ monocytes into macrophages are critical cellular and molecular events in NEC development to promote disease.

Effects of six common dietary nutrients on murine intestinal organoid growth.

The intestinal epithelium of the gastrointestinal (GI) tract constantly renews itself to absorb nutrients and provide protection for the body from the outside world. Since the intestinal epithelium is constantly exposed to various chemicals and dietary components, it is critical to determine which constituents promote or inhibit intestinal epithelium health and growth rate. Intestinal organoids, three-dimensional miniature models of the intestines, represent an ex vivo tool to investigate intestinal physiology and growth patterns. In this study, we measured the growth rates of murine intestinal organoids exposed to various concentrations of different dietary constituents. Results indicate that caffeic acid inhibited organoid growth in a concentration-dependent manner, curcumin exhibited variable effectiveness, and vitamin C had no effect on organoid growth.

Beta-catenin cleavage enhances transcriptional activation.

Nuclear activation of Wnt/ß-catenin signaling is required for cell proliferation in inflammation and cancer. Studies from our group indicate that ß-catenin activation in colitis and colorectal cancer (CRC) correlates with increased nuclear levels of ß-catenin phosphorylated at serine 552 (pß-Cat552). Biochemical analysis of nuclear extracts from cancer biopsies revealed the existence of low molecular weight (LMW) pß-Cat552, increased to the exclusion of full size (FS) forms of ß-catenin. LMW ß-catenin lacks both termini, leaving residues in the armadillo repeat intact. Further experiments showed that TCF4 predominantly binds LMW pß-Cat552 in the nucleus of inflamed and cancerous cells. Nuclear chromatin bound localization of LMW pß-Cat552 was blocked in cells by inhibition of proteasomal chymotrypsin-like activity but not by other protease inhibitors. K48 polyubiquitinated FS and LMW ß-catenin were increased by treatment with bortezomib. Overexpressed in vitro double truncated ß-catenin increased transcriptional activity, cell proliferation and growth of tumor xenografts compared to FS ß-catenin. Serine 552-> alanin substitution abrogated K48 polyubiquitination,  ß-catenin nuclear translocation and tumor xenograft growth. These data suggest that a novel proteasome-dependent posttranslational modification of ß-catenin enhances transcriptional activation. Discovery of this pathway may be helpful in the development of diagnostic and therapeutic tools in colitis and cancer.

Intestinal organoids containing poly(lactic-co-glycolic acid) nanoparticles for the treatment of inflammatory bowel diseases.

Inflammatory bowel disease (IBD) causes inflammation to the gastrointestinal tract. Local administration of anti-inflammatory drugs such as 5-aminosalicylic acid (5-ASA) can alleviate the symptoms of IBD. The application of nanoparticles for IBD treatment in direct rectal administration showed high drug availability and treatment efficacy. However, relying on size-dependent adsorption of smaller particles is not sufficient for making the formulation capable of targeting. Intestinal organoids can improve the functionality of the nanoparticles due to their ability to adsorb small nanoparticle inside the lumen and attach to the damaged area. In this study, intestinal organoids were used as carriers of 5-ASA-loaded poly(lactic-co-glycolic acid) nanoparticles. The nanoparticle sizes, confirmed by scanning electron microscopy, were 200-300 nm and the zeta potential were negative. The nanoparticles did not have any noticeable pernicious effect on organoid growth and viability. After mixing the nanoparticles with Matrigel and organoids, Rhodamine B loaded inside the nanoparticles was highly detected inside the organoid's lumen after 3 days by confocal fluorescent microscopy and no longer detected in the lumen after day 4. It may be attributed to the ability of the lumen to digest particles. Thus, the organoid Trojan horse system is a possible approach for delivering drugs to inflamed areas. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 876-886, 2018.