Fecal Luminal Factors from Patients with Gastrointestinal Diseases Alter Gene Expression Profiles in Caco-2 Cells and Colonoids.

Previous in vitro studies have shown that the intestinal luminal content, including metabolites, possibly regulates epithelial layer responses to harmful stimuli and promotes disease. Therefore, we aimed to test the hypothesis that fecal supernatants from patients with colon cancer (CC), ulcerative colitis (UC) and irritable bowel syndrome (IBS) contain distinct metabolite profiles and establish their effects on Caco-2 cells and human-derived colon organoids (colonoids). The metabolite profiles of fecal supernatants were analyzed by liquid chromatography-mass spectrometry and distinguished patients with CC (n = 6), UC (n = 6), IBS (n = 6) and healthy subjects (n = 6). Caco-2 monolayers and human apical-out colonoids underwent stimulation with fecal supernatants from different patient groups and healthy subjects. Their addition did not impair monolayer integrity, as measured by transepithelial electrical resistance; however, fecal supernatants from different patient groups and healthy subjects altered the gene expression of Caco-2 monolayers, as well as colonoid cultures. In conclusion, the stimulation of Caco-2 cells and colonoids with fecal supernatants derived from CC, UC and IBS patients altered gene expression profiles, potentially reflecting the luminal microenvironment of the fecal sample donor. This experimental approach allows for investigating the crosstalk at the gut barrier and the effects of the gut microenvironment in the pathogenesis of intestinal diseases.

Differences in Metabolite Composition of Aloe barbadensis Mill. Extracts Lead to Differential Effects on Human Blood T Cell Activity In Vitro.

Aloe barbadensis Mill. (Aloe) is used for diverse therapeutic properties including immunomodulation. However, owing to the compositionally complex nature of Aloe, bioactive component(s) responsible for its beneficial properties, though thought to be attributed to polysaccharides (acemannan), remain unknown. We therefore aimed to determine the metabolite composition of various commercial Aloe extracts and assess their effects on human blood T cell activity in vitro. Peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated polyclonally in presence or absence of various Aloe extracts. T cell phenotype and proliferation were investigated by flow cytometry. Aloe extracts were analyzed using targeted 1H-NMR spectroscopy for standard phytochemical quality characterization and untargeted gas chromatography mass spectrometry (GC-MS) for metabolite profiling. Aloe extracts differing in their standard phytochemical composition had varying effects on T cell activation, proliferation, apoptosis, and cell-death in vitro, although this was not related to the acemannan content. Furthermore, each Aloe extract had its own distinct metabolite profile, where extracts rich in diverse sugar and sugar-derivatives were associated with reduced T cell activity. Our results demonstrate that all commercial Aloe extracts are unique with distinct metabolite profiles, which lead to differential effects on T cell activity in vitro, independent of the acemannan content.

Impaired Butyrate Induced Regulation of T Cell Surface Expression of CTLA-4 in Patients with Ulcerative Colitis.

Patients with ulcerative colitis (UC) have reduced intestinal levels of short-chain fatty acids (SCFAs), including butyrate, which are important regulators of host-microbiota crosstalk. The aim was therefore to determine effects of butyrate on blood and intestinal T cells from patients with active UC. T cells from UC patients and healthy subjects were polyclonally stimulated together with SCFAs and proliferation, activation, cytokine secretion, and surface expression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) were analyzed. Butyrate induced comparable, dose dependent inhibition of activation and proliferation in blood T cells and activation in intestinal T cells from UC patients and healthy subjects. However, surface expression of the inhibitory molecule CTLA-4 on stimulated blood and intestinal T cells was impaired in UC patients and was not restored following butyrate treatment. Furthermore, unlike intestinal T cells from healthy subjects, butyrate was unable to downregulate secretion of interferon gamma (IFNγ), interleukin (IL)-4, IL-17A, and IL-10 in UC patients. Although seemingly normal inhibitory effects on T cell activation and proliferation, butyrate has an impaired ability to reduce cytokine secretion and induce surface expression of CTLA-4 in T cells from UC patients with active disease. Overall, these observations indicate a dysfunction in butyrate induced immune regulation linked to CTLA-4 signaling in T cells from UC patients during a flare.

Fecal microbiota composition is linked to the postoperative disease course in patients with Crohn's disease.

BACKGROUND: The role of the fecal microbiota composition for the postoperative disease course of patients with Crohn's disease (CD) who have undergone ileocecal resection remains to be established. In this study, we investigated if the fecal microbiota composition, determined by a high throughput test quantifying a pre-selected set of bacteria, is associated with the postoperative disease course of CD patients. METHODS: Fecal samples were obtained from healthy subjects as well as from CD patients, 3-10 weeks and 1 year after ileocaecal resection. The fecal microbial composition was analyzed by Genetic Analysis GA-map Dysbiosis test, targeting ≥300 bacteria on different taxonomic levels. Postoperative disease status was assessed endoscopically according to Rutgeerts scoring system 1 year after surgery. Differences in fecal microbiota composition between groups were analyzed by multivariate factor analyses and cluster analysis. Microbial stability over time was determined using Bray-Curtis dissimilarity. RESULTS: One year after surgery, the fecal microbiota composition differed between CD patients (n = 21) and healthy subjects (n = 7). At this time point, the microbiota composition of CD patients was associated with disease course, clearly separating patients with disease relapse (n = 8) and patients in remission (n = 13). Further, the microbial within-patient stability was high during the first year after surgery, irrespective of disease course. CONCLUSION: The fecal microbiota composition of CD patients, analyzed by GA-map Dysbiosis test, is subject to little variation over time, and may potentially be used as a non-invasive diagnostic tool for the postoperative disease course.

The frequency of circulating integrin α4ß7+ cells correlates with protection against Helicobacter pylori infection in immunized mice.

BACKGROUND: Chronic Helicobacter pylori infection is the cause of peptic ulcers in a subpopulation of individuals and a risk factor for the development of gastric cancer. A vaccine against H pylori infection can prevent the acquisition of the infection and protect against reinfections. Clinical trials to date evaluating the efficacy of H pylori vaccines in human challenge models have shown moderate to poor protection with difficulties in predicting efficacy. Thus, while further studies are needed to design an effective vaccine, we also need to find relevant correlates for vaccine efficacy. OBJECTIVE: To find immune correlates to vaccine efficacy, the frequencies of neutrophils, eosinophils and inflammatory monocytes and CD4+ T-cell memory and mucosa homing integrin α4ß7+ cells were assessed by flow cytometry in the blood of mice after vaccination. MATERIALS AND METHODS: H pylori antigens and cholera toxin or the multiple mutant CT (mmCT) were administered via the sublingual (SL) and intragastric route (IG). The vaccinated mice were infected with H pylori strain SS1 bacteria, and colonization in the stomach and immune responses were evaluated. RESULTS: The H pylori vaccine was effective in reducing bacterial load in the stomach of mice and enhancing immune responses compared to unvaccinated infection controls. In the blood of mice after SL or IG route of vaccination, we observed changes in frequencies of innate and adaptive immune cell subsets compared to infection controls. Remarkably, the frequency of circulating mucosal homing α4ß7+ CD4+ T cells after vaccination correlated with low bacterial load in the stomach of individual mice irrespective of the immunization route. CONCLUSIONS: Our study shows that the innate and adaptive immune cell subsets can be measured in the blood after vaccination and that increased frequency of α4ß7+ CD4+ in the blood after immunization could be used as a predictive marker for the efficacy of vaccine against H pylori infection.

Immunopathogenesis of inflammatory bowel disease and mechanisms of biological therapies.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract with a multifactorial pathophysiology. Full comprehension of IBD pathology is still out of reach and, therefore, treatment is far from ideal. Nevertheless, components involved in IBD pathogenesis including environmental, genetic, microbial, and immunological factors are continuously being investigated and the improved knowledge contributes to the development of new therapies. In this article we review the aspects of the immunopathogenesis of IBD, with focus on mucosal immunity, and discuss mechanisms of action for current and emerging biological therapies.

Effects of Anti-TNFα Treatment on Mucosal Expression of IL-17A, IL-21, and IL-22 and Cytokine-Producing T Cell Subsets in Crohn's Disease.

T helper 17 (Th17) cells produce interleukin (IL) 17-A. In addition, Th17 cells produce IL-21 and IL-22. Th17 cells have a disease-promoting role in Crohn's disease (CD). We investigated the effects of anti-TNFα treatment on mucosal gene expression (qPCR) of IL-17A, IL-21, and IL-22 as well as on the frequency of lamina propria (LP) T cell subsets producing these cytokines (flow cytometry) in 12 active CD patients before and after 4 weeks of anti-TNFα treatment with adalimumab. At baseline, in inflamed mucosa we found increased gene expression of IL-17A and IL-22 but not IL-21 when compared to noninflamed mucosa. There were increased frequencies of IL-21-producing LP T cells but no differences in the frequencies of IL-17A- or IL-22-producing LP T cells when comparing inflamed versus noninflamed mucosa at baseline. There were no changes in the mucosal gene expression of IL-17A, IL-21, and IL-22 or the frequencies of IL-17A-, IL-21- and IL-22-producing LP T cell subsets between baseline and following 4 weeks of adalimumab initiation. Our results do not support the hypothesis that anti-TNFα treatment has an early effect on the mucosal levels of IL-17A, IL-21, and IL-22 or LP T cell production of these cytokines in CD.

Mucosal immune system of the gastrointestinal tract: maintaining balance between the good and the bad.

The gastrointestinal tract (GI tract) is a unique organ inhabited by a range of commensal microbes, while also being exposed to an overwhelming load of antigens in the form of dietary antigens on a daily basis. The GI tract has dual roles in the body, in that it performs digestion and uptake of nutrients while also carrying out the complex and important task of maintaining immune homeostasis, i.e., keeping the balance between the good and the bad. It is equally important that we protect ourselves from reacting against the good, meaning that we stay tolerant to harmless food, commensal bacteria and self-antigens, as well as react with force against the bad, meaning induction of immune responses against harmful microorganisms. This complex task is achieved through the presence of a highly efficient mucosal barrier and a specialized multifaceted immune system, made up of a large population of scattered immune cells and organized lymphoid tissues termed the gut-associated lymphoid tissue (GALT). This review provides an overview of the primary components of the human mucosal immune system and how the immune responses in the GI tract are coordinated and induced.

Reduced numbers of mucosal DR(int) macrophages and increased numbers of CD103(+) dendritic cells during anti-TNF-α treatment in patients with Crohn's disease.

OBJECTIVE: Anti-TNF-α treatment constitutes a mainstay in the treatment of Crohn's disease (CD), but its mechanisms of action are not fully understood. We aimed to investigate the effects of adalimumab, a human monoclonal TNF-α antibody, on macrophage (MQ) and dendritic cell (DC) subsets in mucosal biopsies and peripheral blood. MATERIAL AND METHODS: Intestinal biopsies and blood samples were obtained from 12 different CD patients both before and 4 weeks after the initiation of the induction of adalimumab treatment. Endoscopic disease activity was estimated by the Simple Endoscopic Score for Crohn's Disease. Biopsies were obtained from inflamed and non-inflamed areas. The numbers of lamina propria CD14 (+) DR(int) and CD14 (+) DR(hi) MQs, CD141(+), CD141(-) and CD103(+) DCs subsets, and circulating monocytes and DCs were analyzed using flow cytometry. RESULTS: At baseline, we observed higher numbers of DR(int) MQs and lower numbers of CD103(+) DCs in inflamed versus non-inflamed mucosa [843 vs. 391/10(5) lamina propria mononuclear cells (LPMCs) (p < 0.05) and 9 vs. 19 × 10(5) LPMCs (p = 0.01), respectively]. After four weeks of adalimumab treatment, the numbers of DR(int) MQs decreased [843 to 379/10(5) LPMCs (p = 0.03)], whereas the numbers of CD103(+) DCs increased [9-20 × 10(5) LPMCs (p = 0.003)] compared with baseline. In peripheral blood, no alterations were observed in monocyte or DC numbers between baseline and week 4. CONCLUSIONS: In CD, mucosal inflammation is associated with high numbers of DR(int) MQs and low numbers of CD103(+) DCs. This composition of intestinal myeloid subsets is reversed by anti-TNF-α treatment. These results suggest that DR(int) MQs play a pivotal role in CD inflammation.

Global mucosal and serum cytokine profile in patients with ulcerative colitis undergoing anti-TNF therapy.

BACKGROUND AND OBJECTIVE: The knowledge of the effects of anti-tumour necrosis factor (TNF) treatment on the global cytokine profile in patients with ulcerative colitis (UC) is limited. A better understanding of these mechanisms could improve the ability to select patients that should undergo the therapy. Therefore, the aim was to determine the global mucosal and serum cytokine profile before and during induction therapy with anti-TNF in UC patients. MATERIALS AND METHODS: In total, mucosal biopsies (n = 28) and serum samples (n = 42) were collected from UC patients (total n = 48) before anti-TNF therapy. At week 14 response to the therapy was evaluated and again mucosal biopsies (n = 14) and serum samples (n = 42) were collected. Quantitative real-time PCR was used to determine mucosal cytokine mRNA expression and the MSD MULTI-ARRAY assay system platform was used for analysis of cytokines in serum. The global cytokine profile was evaluated by multivariate factor analysis. RESULTS: At baseline, the global profile of mucosal cytokine mRNA expression and serum cytokines discriminated therapy responders from non-responders. Responders had lower mucosal mRNA expression of interleukin 1ß (IL-1ß), IL-17A, IL-6 and interferon γ (IFN-γ) than non-responders. Fourteen weeks after therapy start mucosal IL-1ß and IL-6 were down-regulated in therapy responders but not in non-responders. At week 14, serum levels of IL-6 were decreased in therapy responders whereas IFN-γ and IL-12p70 were increased in non-responders. CONCLUSIONS: Our data suggest that patients with a therapy failure have a more severe pro-inflammatory cytokine profile before start of anti-TNF treatment, which is less well suppressed by the treatment as compared to therapy responders.

Temporal stability of fecal metabolomic profiles in irritable bowel syndrome.

BACKGROUND: The potential of the fecal metabolome to serve as a biomarker for irritable bowel syndrome (IBS) depends on its stability over time. Therefore, this study aimed to determine the temporal dynamics of the fecal metabolome, and the potential relationship with stool consistency, in patients with IBS and healthy subjects. METHODS: Fecal samples were collected in two cohorts comprising patients with IBS and healthy subjects. For Cohort A, fecal samples collected during 5 consecutive days were analyzed by gas chromatography-tandem mass spectrometry (GC-MS/MS). For Cohort B, liquid chromatography-MS (LC-MS) was used to analyze fecal samples collected at week 0 (healthy and IBS) and at week 4 (patients only). Stool consistency was determined by the Bristol Stool Form scale. KEY RESULTS: Fecal samples were collected from Cohort A (seven healthy subjects and eight IBS patients), and Cohort B (seven healthy subjects and 11 IBS patients). The fecal metabolome of IBS patients was stable short-term (Cohort A, 5 days and within the same day) and long-term (Cohort B, 4 weeks). A similar trend was observed over 5 days in the healthy subjects of Cohort A. The metabolome dissimilarity was larger between than within participants over time in both healthy subjects and IBS patients. Further analyses showed that patients had greater range of stool forms (types) than healthy subjects, with no apparent influence on metabolomic dynamics. CONCLUSION & INFERENCES: The fecal metabolome is stable over time within IBS patients as well as healthy subjects. This supports the concept of a stable fecal metabolome in IBS despite fluctuations in stool consistency, and the use of single timepoint sampling to further explore how the fecal metabolome is related to IBS pathogenesis.

Faecal biomarkers for diagnosis and prediction of disease course in treatment-naïve patients with IBD.

BACKGROUND: Faecal biomarkers can be used to assess inflammatory bowel disease (IBD). AIM: To explore the performance of some promising biomarkers in diagnosing and predicting disease course in IBD. METHODS: We included 65 patients with treatment-naïve, new-onset Crohn's disease (CD), 90 with ulcerative colitis (UC), 67 symptomatic controls (SC) and 41 healthy controls (HC) in this prospective observational study. We analysed faecal samples for calprotectin (FC), myeloperoxidase (MPO), human neutrophil lipocalin (HNL), eosinophil cationic protein ECP and eosinophil-derived neurotoxin (EDN) and compared markers among groups. We assessed the diagnostic capability of biomarkers with receiver operating characteristic curves. Clinical disease course was determined for each patient with IBD and analysed the association with biomarkers by logistic regression. RESULTS: All markers were elevated at inclusion in patients with IBD compared with HC (p < 0.001) and SC (p < 0.001). FC (AUC 0.85, 95% CI: 0.79-0.89) and MPO (AUC 0.85, 95% CI: 0.80-0.89) showed the highest diagnostic accuracy in distinguishing IBD from SC. The diagnostic ability of biomarkers differed between IBD subtypes with the highest performance for FC and MPO in CD. The diagnostic accuracy was further improved by combining FC and MPO (p = 0.02). Levels of FC, MPO and HNL at inclusion were predictive of an aggressive disease course with MPO showing the strongest association (p = 0.006). CONCLUSIONS: This study provides new insight into the diagnostic and prognostic capability of neutrophil and eosinophil biomarkers in IBD and suggests that MPO, alone or in combination with FC, may add to the diagnostic power of faecal biomarkers.

Identification and validation of a blood- based diagnostic lipidomic signature of pediatric inflammatory bowel disease.

Improved biomarkers are needed for pediatric inflammatory bowel disease. Here we identify a diagnostic lipidomic signature for pediatric inflammatory bowel disease by analyzing blood samples from a discovery cohort of incident treatment-naïve pediatric patients and validating findings in an independent inception cohort. The lipidomic signature comprising of only lactosyl ceramide (d18:1/16:0) and phosphatidylcholine (18:0p/22:6) improves the diagnostic prediction compared with high-sensitivity C-reactive protein. Adding high-sensitivity C-reactive protein to the signature does not improve its performance. In patients providing a stool sample, the diagnostic performance of the lipidomic signature and fecal calprotectin, a marker of gastrointestinal inflammation, does not substantially differ. Upon investigation in a third pediatric cohort, the findings of increased lactosyl ceramide (d18:1/16:0) and decreased phosphatidylcholine (18:0p/22:6) absolute concentrations are confirmed. Translation of the lipidomic signature into a scalable diagnostic blood test for pediatric inflammatory bowel disease has the potential to support clinical decision making.

Fecal Supernatants from Patients with Crohn's Disease Induce Inflammatory Alterations in M2 Macrophages and Fibroblasts.

Intestinal macrophages and fibroblasts act as microenvironmental sentinels mediating inflammation and disease progression in Crohn's disease (CD). We aimed to establish the effects of fecal supernatants (FSs) from patients with CD on macrophage and fibroblast phenotype and function. FS were obtained by ultracentrifugation, and the metabolites were analyzed. Monocyte-derived M2 macrophages and fibroblasts were conditioned with FS, and secreted proteins, surface proteins and gene expression were analyzed. M2 macrophage efferocytosis was evaluated. Patients with CD (n = 15) had a skewed fecal metabolite profile compared to healthy subjects (HS, n = 10). FS from CD patients (CD-FS) induced an anti-inflammatory response in M2 macrophages with higher expression of IL-10, IL1RA and CD206 as compared to healthy FS (HS-FS) while the efferocytotic capacity was unaltered. CD-FS did not affect extracellular matrix production from fibroblasts, but increased expression of the pro-inflammatory proteins IL-6 and MCP-1. Conditioned media from M2 macrophages treated with CD-FS modulated gene expression in fibroblasts for TGFß superfamily members and reduced IL-4 expression compared to HS-FS. We show that M2 macrophages and fibroblasts react abnormally to the fecal microenvironment of CD patients, resulting in altered protein expression related to inflammation but not fibrosis. This implies that the gut microbiota and its metabolites have an important role in the generation and/or perpetuation of inflammation in CD.

Fecal Biomarkers of Neutrophil and Eosinophil Origin Reflect the Response to Biological Therapy and Corticosteroids in Patients With Inflammatory Bowel Disease.

INTRODUCTION: Fecal calprotectin (FC) is a noninvasive tool for examining response to biologics in inflammatory bowel disease (IBD), but its performance in relation to other novel fecal markers of various cellular origins is unknown. METHODS: We performed a prospective multicenter cohort study and included patients with active IBD who provided a fecal sample at initiation of biological therapy. Levels of FC, myeloperoxidase (MPO), human neutrophil lipocalin (HNL), and eosinophil-derived neurotoxin (EDN) were analyzed and related to clinical remission status at 3 months. Changes in levels of markers at 3 months were calculated, and the impact of concomitant use of corticosteroids at baseline was estimated. RESULTS: In patients achieving clinical remission (n = 27), a decrease in levels of FC ( P = 0.005), MPO ( P < 0.001), HNL ( P < 0.001), and EDN ( P < 0.001) was observed, whereas no significant decrease was seen in patients not achieving remission (n = 39). There was a significant difference in the change in the level of MPO ( P = 0.01) and HNL ( P = 0.02) between patients achieving clinical remission and those who did not, but changes in FC and EDN could not differentiate between these groups. Patients with concomitant systemic corticosteroids at inclusion had lower levels of HNL ( P = 0.01) and EDN ( P < 0.001) at baseline, compared with patients without corticosteroids. DISCUSSION: Fecal MPO, HNL, and EDN are all promising biomarkers for assessing the treatment outcome of biologics in patients with IBD. Fecal levels of EDN and HNL are significantly affected by corticosteroids indicating a greater sensitivity to the effects of corticosteroids compared with levels of FC and MPO.

Randomised controlled trial: effects of gluten-free diet on symptoms and the gut microenvironment in irritable bowel syndrome.

BACKGROUND: A gluten-free diet reduces symptoms in some patients with irritable bowel syndrome (IBS) through unclear mechanisms. AIMS: To assess the effects of gluten-free versus gluten-containing diet on symptoms and the gut microenvironment, and to identify predictors of response to the gluten-free diet in IBS METHODS: Twenty patients with IBS and 18 healthy controls (HC) followed a gluten-free diet during two 14-day intervention periods where they sprinkled either gluten (14 g/day) or rice flour powder over their meals. Primary outcomes included effects of the interventions on IBS symptoms (IBS-SSS) and bowel habits. Secondary outcomes included effects of gluten-free diet on faecal microbiota and metabolite profile. RESULTS: IBS symptoms improved during the gluten-free (p = 0.02), but not the gluten-containing period, with no difference between the interventions. IBS patients reported fewer loose stools during the gluten-free intervention (p = 0.01). Patients with IBS and HC presented distinct metabolite profiles based on the effects of the gluten-free diet (p < 0.001). True responders (reduced IBS-SSS by ≥50 solely after gluten-free period) and non-responders were discriminated based on the effects of the gluten-free diet on the microbiota (p < 0.01) and metabolite profiles (p < 0.001). The response to the gluten-free diet could be predicted by the metabolite profile before the intervention (p < 0.001). CONCLUSIONS: A gluten-free diet may influence symptoms in a subset of patients with IBS, with a particular effect on bowel habits. A gluten-free diet seems to impact the gut microenvironment. Responsiveness to the gluten-free diet may be predicted by the metabolite profile. CLINICALTRIALS: gov: NCT03869359.

Fecal luminal factors from patients with irritable bowel syndrome induce distinct gene expression of colonoids.

BACKGROUND: Alteration of the host-microbiota cross talk at the intestinal barrier may participate in the pathophysiology of irritable bowel syndrome (IBS). Therefore, we aimed to determine effects of fecal luminal factors from IBS patients on the colonic epithelium using colonoids. METHODS: Colon-derived organoid monolayers, colonoids, generated from a healthy subject, underwent stimulation with fecal supernatants from healthy subjects and IBS patients with predominant diarrhea, phosphate-buffered saline (PBS), or lipopolysaccharide (LPS). Cytokines in cell cultures and fecal LPS were measured by ELISA and mRNA gene expression of monolayers was analyzed using Qiagen RT2 Profiler PCR Arrays. The fecal microbiota profile was determined by the GA-map™ dysbiosis test and the fecal metabolite profile was analyzed by untargeted liquid chromatography/mass spectrometry. KEY RESULTS: Colonoid monolayers stimulated with fecal supernatants from healthy subjects (n = 7), PBS (n = 4) or LPS (n = 3) presented distinct gene expression profiles, with some overlap (R2 Y = 0.70, Q2  = 0.43). Addition of fecal supernatants from healthy subjects and IBS patients (n = 9) gave rise to different gene expression profiles of the colonoid monolayers (R2 Y = 0.79, Q2  = 0.64). Genes (n = 22) related to immune response (CD1D, TLR5) and barrier integrity (CLDN15, DSC2) contributed to the separation. Levels of proinflammatory cytokines in colonoid monolayer cultures were comparable when stimulated with fecal supernatants from either donor types. Fecal microbiota and metabolite profiles, but not LPS content, differed between the study groups. CONCLUSIONS: Fecal luminal factors from IBS patients induce a distinct colonic epithelial gene expression, potentially reflecting the disease pathophysiology. The culture of colonoids from healthy subjects with fecal supernatants from IBS patients may facilitate the exploration of IBS related intestinal micro-environmental and barrier interactions.

Combinatory analysis of immune cell subsets and tumor-specific genetic variants predict clinical response to PD-1 blockade in patients with non-small cell lung cancer.

Objectives: Immunotherapy by blocking programmed death protein-1 (PD-1) or programmed death protein-ligand1 (PD-L1) with antibodies (PD-1 blockade) has revolutionized treatment options for patients with non-small cell lung cancer (NSCLC). However, the benefit of immunotherapy is limited to a subset of patients. This study aimed to investigate the value of combining immune and genetic variables analyzed within 3-4 weeks after the start of PD-1 blockade therapy to predict long-term clinical response. Materials and methodology: Blood collected from patients with NSCLC were analyzed for changes in the frequency and concentration of immune cells using a clinical flow cytometry assay. Next-generation sequencing (NGS) was performed on DNA extracted from archival tumor biopsies of the same patients. Patients were categorized as clinical responders or non-responders based on the 9 months' assessment after the start of therapy. Results: We report a significant increase in the post-treatment frequency of activated effector memory CD4+ and CD8+ T-cells compared with pre-treatment levels in the blood. Baseline frequencies of B cells but not NK cells, T cells, or regulatory T cells were associated with the clinical response to PD-1 blockade. NGS of tumor tissues identified pathogenic or likely pathogenic mutations in tumor protein P53, Kirsten rat sarcoma virus, Kelch-like ECH-associated protein 1, neurogenic locus notch homolog protein 1, and serine/threonine kinase 11, primarily in the responder group. Finally, multivariate analysis of combined immune and genetic factors but neither alone, could discriminate between responders and non-responders. Conclusion: Combined analyses of select immune cell subsets and genetic mutations could predict early clinical responses to immunotherapy in patients with NSCLC and after validation, can guide clinical precision medicine efforts.

MEFV and NLRP3 Inflammasome Expression Is Attributed to Immature Macrophages and Correlates with Serum Inflammatory Proteins in Crohn´s Disease Patients.

Inflammasomes are intracellular protein complexes whose activation results in proinflammatory cytokines. Inflammasomes are implicated in Crohn´s disease (CD) pathogenesis, yet the contribution of inflammasomes in intestinal epithelial cells (IECs) versus lamina propria (LP) macrophages is poorly understood. Whether inflammasome expression in intestinal tissue reflects the serum inflammatory protein profile of patients is also not known. We aimed to determine the intestinal cell types where inflammasome expression is increased in CD and if they correlate with the serum protein profile. RT-PCR and NanoString nCounter technology were used to characterize inflammasome gene expression in CD patients and controls. The mucosa, LP and IEC cell fractions and FACS-sorted cells were analyzed. Proximity extension assay with a 92-protein panel was used to determine the serum inflammatory protein profile. Compositional analysis was used to correlate ileum inflammasome gene expression with intestinal mononuclear phagocyte populations. We show that NLRP3 and MEFV inflammasome sensors and downstream effector expression including IL-1ß are increased in inflamed mucosa of IBD patients and correlate with disease activity. Inflammasome gene expression increased with the abundance of immature intestinal macrophages, and increased IL-1ß released by CD LP cells correlated with immature macrophage frequency. Inflammasome gene expression was also increased in circulating monocytes, the precursors of immature intestinal macrophages. Finally, the serum inflammatory profile of CD patients correlates with ileal expression of genes related to NLRP3 and MEFV inflammasomes. Overall, we show that MEFV and NLRP3 inflammasome expression in CD intestine is attributed to the accumulation of immature macrophages and correlates with serum inflammatory proteins.

Downregulated Mucosal Autophagy, Alpha Kinase-1 and IL-17 Signaling Pathways in Active and Quiescent Ulcerative Colitis.

Background: Improved mucosal immune profiling in active and quiescent colonic inflammatory bowel disease (IBD) is needed to develop therapeutic options for treating and preventing flares. This study therefore aimed to provide a comprehensive mucosal characterization with emphasis on immunological host response of patients with active ulcerative colitis (UC active), UC during remission (UC remission) and active colonic Crohn's disease (CD active). Methods: Colonic biopsies from 47 study subjects were collected for gene expression and pathway analyses using the NanoString host-response panel, including 776 genes and 56 immune-related pathways. Results: The majority of mucosal gene expression and signaling pathway scores were increased in active IBD (n=27) compared to healthy subjects (n=10). However, both active IBD and UC remission (n=10) demonstrated decreased gene expression and signaling pathway scores related to autophagy, alpha kinase-1 and IL-17 signaling pathways compared to healthy subjects. Further, UC remission was characterized by decreased scores of several signaling pathways linked to homeostasis along with increased mononuclear cell migration pathway score as compared to healthy subjects. No major differences in the colonic mucosal gene expression between CD active (n=7) and UC (n=20) active were observed. Conclusion: This study indicates that autophagy, alpha kinase-1 and IL-17 signaling pathways are persistently downregulated in UC irrespective of disease activity. Further, UC patients in remission present a unique mucosal environment, potentially preventing patients from reaching and sustaining true homeostasis. These findings may enable better comprehension of the remitting and relapsing pattern of colonic IBD and guide future treatment and prevention of flares.