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The gut wall’s potential as a partner for precision oncology in immune checkpoint treatment

Open AccessPublished:May 07, 2022DOI:https://doi.org/10.1016/j.ctrv.2022.102406

      Highlights

      • Gut microbiota impacts tumor response to immune checkpoint inhibitors (ICI).
      • The potential role of the gut wall regarding the tumor response to ICI has received little attention.
      • Selective markers can define gut wall integrity and the intestinal immune system.
      • Clinical interventions are available that modify key components of the gut wall.
      • The gut wall’s potential to improve ICI efficacy should be further explored.

      Abstract

      The gut wall is the largest immune organ and forms a barrier through which gut microbiota interact with the immune system in the rest of the body. Gut microbiota composition plays a role in the strength and timing of the anticancer immune response on immune checkpoint inhibitors (ICI). Surprisingly, the effects of gut wall characteristics, such as physical barrier integrity, permeability, and activity and composition of the intestinal immune system, on response to ICI has received little attention. Here, we provide an overview of markers to characterize the gut wall and interventions that can modulate these gut wall characteristics. Finally, we present a future perspective on how these gut wall markers and interventions might be utilized and studied to improve ICI treatment strategies.

      Graphical abstract

      Keywords

      Introduction

      Tumor cells escape destruction by the immune system by exploiting mechanisms that suppress an anticancer immune response. [
      • Hanahan D.
      Hallmarks of cancer: new dimensions.
      ] An important mechanism is the activation of immune checkpoints, which are the ‘brakes’ in the immune system that prevent inappropriate cytotoxic T-cell activation. Immune checkpoint inhibitors (ICI) can release these immune brakes and thus trigger a durable anticancer immune response. These medicines have dramatically improved patient outcomes across numerous tumor types. [
      • Chen D.S.
      • Mellman I.
      Elements of cancer immunity and the cancer–immune set point.
      ] Unfortunately, most patients with cancer still do not benefit from ICI as they fail to obtain a durable response. [
      • Haslam A.
      • Prasad V.
      Estimation of the percentage of US patients with cancer who are eligible for and respond to checkpoint inhibitor immunotherapy drugs.
      ]
      A complex set of tumor and patient characteristics is emerging that govern the anticancer immune response's strength and timing. [
      • Chen D.S.
      • Mellman I.
      Elements of cancer immunity and the cancer–immune set point.
      ,
      • Blank C.U.
      • Haanen J.B.
      • Ribas A.
      • Schumacher T.N.
      Cancer immunology. The “cancer immunogram”.
      ] Together, these characteristics determine the 'cancer-immune set point', the threshold that must be surpassed in a patient to trigger an anticancer immune response to ICI. [
      • Chen D.S.
      • Mellman I.
      Elements of cancer immunity and the cancer–immune set point.
      ] The gut microbiota and microbial metabolites can influence the cancer-immune set point. [
      • Frankel A.E.
      • Coughlin L.A.
      • Kim J.
      • Froehlich T.W.
      • Xie Y.
      • Frenkel E.P.
      • et al.
      Metagenomic shotgun sequencing and unbiased metabolomic profiling identify specific human gut microbiota and metabolites associated with immune checkpoint therapy efficacy in melanoma patients.
      ,
      • Mager L.F.
      • Burkhard R.
      • Pett N.
      • Cooke N.C.A.
      • Brown K.
      • Ramay H.
      • et al.
      Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy.
      ,
      • Smith M.
      • Dai A.
      • Ghilardi G.
      • Amelsberg K.V.
      • Devlin S.M.
      • Pajarillo R.
      • et al.
      Gut microbiome correlates of response and toxicity following anti-CD19 CAR T cell therapy.
      ] Pilot studies showed that transplantation of fecal microbial samples obtained from patients responding to ICI into patients not responding to ICI can lead to tumor response in initially non-responding patients after reintroduction of ICI. [
      • Baruch E.N.
      • Youngster I.
      • Ben-Betzalel G.
      • Ortenberg R.
      • Lahat A.
      • Katz L.
      • et al.
      Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients.
      ] This has sparked a series of clinical trials currently investigating whether modulation of gut microbiota through fecal microbial transplantation (FMT), probiotics, and specific diets can enhance tumor response to ICI. [
      • Baruch E.N.
      • Youngster I.
      • Ben-Betzalel G.
      • Ortenberg R.
      • Lahat A.
      • Katz L.
      • et al.
      Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients.
      ,
      • McQuade J.L.
      • Daniel C.R.
      • Helmink B.A.
      • Wargo J.A.
      Modulating the microbiome to improve therapeutic response in cancer.
      ]
      The gut microbiota interacts with the immune system in the rest of the body through the gut wall, which encompasses the intestinal mucosa and submucosa, in which immune cells are located which reflect the physical gut barrier. The gut is the primary site of interaction between the host and the outside world and harbors over 70% of the body's immune cells. [
      • Vighi G.
      • Marcucci F.
      • Sensi L.
      • Di Cara G.
      • Frati F.
      Allergy and the gastrointestinal system.
      ] Surprisingly, the relationship between ICI response, integrity, and permeability of the physical gut barrier and the activity of the intestinal immune system has been largely overlooked. So far, we know that the intestinal immune system becomes activated during ICI treatment, and adequate baseline intestinal epithelial cell function, reflected by high plasma citrulline levels (≥20 μM), is associated with a favorable anticancer immune response to ICI. [
      • Berman D.
      • Parker S.M.
      • Siegel J.
      • et al.
      Blockade of cytotoxic T-lymphocyte antigen-4 by ipilimumab results in dysregulation of gastrointestinal immunity in patients with advanced melanoma.
      ,
      • Luoma A.M.
      • Suo S.
      • Williams H.L.
      • Sharova T.
      • Sullivan K.
      • Manos M.
      • et al.
      Molecular pathways of colon inflammation induced by cancer immunotherapy.
      ,
      • Ouaknine Krief J.
      • Helly de Tauriers P.
      • Dumenil C.
      • Neveux N.
      • Dumoulin J.
      • Giraud V.
      • et al.
      Role of antibiotic use, plasma citrulline and blood microbiome in advanced non-small cell lung cancer patients treated with nivolumab.
      ] Interventions that could modulate the integrity, permeability, and immune activity of the gut wall into a more favorable state for response to ICI have the potential to improve disease outcomes in patients. To develop such interventions, we need to understand how gut wall characteristics can impact the strength and timing of the anticancer immune response to ICI. Furthermore, this understanding might also provide us with tools to better predict response to ICI.
      Here, we provide an overview of markers that can be used to characterize the integrity, permeability, and immune activity of the gut wall. We also review interventions that can modulate these gut wall characteristics. Finally, we present a future perspective on how these gut wall characterization markers and interventions might improve ICI treatment strategies.

      Search strategy and selection criteria

      We retrieved articles from PubMed published up to February 2022 reporting results for relevant markers and interventions. Search terms are provided in Suppl. Table 1. Only articles published in English, Q1, and Q2 journals, with full-text availability, and concerning human subjects were considered eligible. Next, titles, abstracts, and full text were sequentially assessed to remove non-relevant articles. Articles reporting gastrointestinal malignancy markers or disease-specific markers, conference abstracts, commentaries, editorial correspondence, or pre-clinical studies were excluded. If multiple studies address the same marker, we selected the article(s) that reported results with the highest level of evidence as defined by the Oxford 2011 Levels of Evidence, v2.1. To identify articles missed by our search strategy, we screened the references of the remaining articles for additional relevant studies. The final selection of references included for each marker and intervention are provided in Suppl. Tables 2–5.

      Characterizing gut wall integrity, permeability, and immune activity

      The gut, formed by the small and large intestine, is selectively permeable. While protection from potentially noxious luminal contents by the gut wall is essential, low-grade exposure of the body to the gut's contents builds and maintains an adequate immune defense. [
      • Agace W.W.
      • McCoy K.D.
      Regionalized development and maintenance of the intestinal adaptive immune landscape.
      ] Two components of the gut wall are vital to preserving intestinal homeostasis: the physical gut barrier and the intestinal immune system.
      We identified markers that characterize the integrity and permeability of the physical gut barrier (n = 11, Table 1) and the activity of the intestinal immune system (n = 10, Table 2).
      Table 1Physical gut wall markers.
      MarkerSpecimenCut-offSens/Spec (%)ContextLoERef.
      Lactulose:

      Mannitol

      Urine> 0.03NRAsymptomatic first-degree relatives of individuals with Crohn’s disease2[
      • Turpin W.
      • Lee S.-H.
      • Raygoza Garay J.A.
      • Madsen K.L.
      • Meddings J.B.
      • Bedrani L.
      • et al.
      Increased intestinal permeability is associated with later development of Crohn’s disease.
      ]
      i-FABPPlasma & Serum382 pg/mL80/87Celiac disease vs normal2[
      • Adriaanse M.P.M.
      • Tack G.J.
      • Passos V.L.
      • Damoiseaux J.G.M.C.
      • Schreurs M.W.J.
      • van Wijck K.
      • et al.
      Serum I-FABP as marker for enterocyte damage in coeliac disease and its relation to villous atrophy and circulating autoantibodies.
      ]
      CitrullinePlasma & Serum20 µmol/L80/84Enteropathies* vs normal2[
      • Fragkos K.C.
      • Forbes A.
      Citrulline as a marker of intestinal function and absorption in clinical settings: A systematic review and meta-analysis.
      ]
      TJ proteinsBiopsyNRNRIntestinal ischemia–reperfusion3[
      • Schellekens D.H.
      • Hundscheid I.HR.
      • Leenarts C.A.
      • Grootjans J.
      • Lenaerts K.
      • Buurman W.A.
      • et al.
      Human small intestine is capable of restoring barrier function after short ischemic periods.
      ]
      Transepithelial electrical resistanceBiopsyNRNRFunctional dyspepsia vs normal3[
      • Nojkov B.
      • Zhou S.-Y.
      • Dolan R.D.
      • Davis E.M.
      • Appelman H.D.
      • Guo X.
      • et al.
      Evidence of duodenal epithelial barrier impairment and increased pyroptosis in patients with functional dyspepsia on confocal laser endomicroscopy and “ex vivo” mucosa analysis.
      ]
      TJ and ZA function with lanthanum nitrate electron microscopyBiopsyNRNRIntestinal ischemia–reperfusion3[
      • Schellekens D.H.
      • Hundscheid I.HR.
      • Leenarts C.A.
      • Grootjans J.
      • Lenaerts K.
      • Buurman W.A.
      • et al.
      Human small intestine is capable of restoring barrier function after short ischemic periods.
      ]
      Confocal leak scoreeCLE ileal imagesConfocal leak score

      >13
      95/98Symptomatic IBD vs asymptomatic IBD vs normal2[
      • Chang J.
      • Leong R.W.
      • Wasinger V.C.
      • Ip M.
      • Yang M.
      • Phan T.G.
      Impaired intestinal permeability contributes to ongoing bowel symptoms in patients with inflammatory bowel disease and mucosal healing.
      ]
      Epithelial gap densitypCLE duodenal images> 15 gaps / 1000 cells79/88Functional dyspepsia vs normal2[
      • Nojkov B.
      • Zhou S.-Y.
      • Dolan R.D.
      • Davis E.M.
      • Appelman H.D.
      • Guo X.
      • et al.
      Evidence of duodenal epithelial barrier impairment and increased pyroptosis in patients with functional dyspepsia on confocal laser endomicroscopy and “ex vivo” mucosa analysis.
      ]
      51Cr-EDTAUrineNRNRIBS vs normal3[
      • Dunlop S.P.
      • Hebden J.
      • Campbell E.
      • Naesdal J.
      • Olbe L.
      • Perkins A.C.
      • et al.
      Abnormal intestinal permeability in subgroups of diarrhea-predominant irritable bowel syndromes.
      ]
      D-lactatePlasma & UrineNRNRComparison of patients with acute gastrointestinal injury grade I-IV3[
      • Li H.
      • Chen Y.
      • Huo F.
      • Wang Y.
      • Zhang D.
      Association between acute gastrointestinal injury and biomarkers of intestinal barrier function in critically ill patients.
      ]
      CarotenoidsSerumNRNRChildren with no, mild, moderate, or severe malnutrition3[
      • Vieira M.M.
      • Paik J.
      • Blaner W.S.
      • Soares A.M.
      • Mota R.MS.
      • Guerrant R.L.
      • et al.
      Carotenoids, retinol, and intestinal barrier function in children from northeastern Brazil.
      ]
      *Celiac disease, tropical enteropathy, Crohn’s disease, mucositis, acute rejection in intestinal transplantation.
      51Cr-EDTA, Chromium-51 ethylenediaminetetraacetic acid; D-lactate, D-lactate dehydrogenase; eCLE, endoscope-based confocal laser endomicroscopy; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; i-FABP, intestinal fatty acid-binding protein; LoE, Level of Evidence (as defined by Oxford 2011 Levels of Evidence, v2.1.); NR, not reported; pCLE, probe-based confocal laser endomicroscopy; Sens, sensitivity; Spec, specificity; TJ, tight junction; ZA, zonula adherens.
      Table 2Source articles of intestinal immune system markers.
      MarkerSpecimenCut-offSens/Spec (%)ContextLoERef.
      LactoferrinFeces>7.25 µg/g82/79IBD vs controls (IBS and normal)1[
      • Mosli M.H.
      • Zou G.
      • Garg S.K.
      • Feagan S.G.
      • MacDonald J.K.
      • Chande N.
      • et al.
      C-reactive protein, fecal calprotectin, and stool lactoferrin for detection of endoscopic activity in symptomatic inflammatory bowel disease patients: a systematic review and meta-analysis.
      ]
      CalprotectinFeces>50 µg/g88/73IBD vs controls (IBS and normal)1[
      • Mosli M.H.
      • Zou G.
      • Garg S.K.
      • Feagan S.G.
      • MacDonald J.K.
      • Chande N.
      • et al.
      C-reactive protein, fecal calprotectin, and stool lactoferrin for detection of endoscopic activity in symptomatic inflammatory bowel disease patients: a systematic review and meta-analysis.
      ]
      S100A12Feces>0.8 µg/g



      100/81



      100/91



      86/96
      Crohn's disease vs normal

      Ulcerative colitis vs normal

      IBS vs normal
      2[
      • Kaiser T.
      • Langhorst J.
      • Wittkowski H.
      • Becker K.
      • Friedrich A.W.
      • Rueffer A.
      • et al.
      Faecal S100A12 as a non-invasive marker distinguishing inflammatory bowel disease from irritable bowel syndrome.
      ]
      Lipocalin-2Feces0.81 µg/g95//96IBD vs controls (IEC, IBS or normal)2[
      • Thorsvik S.
      • Damås J.K.
      • Granlund A.vB.
      • Flo T.H.
      • Bergh K.
      • Østvik A.E.
      • et al.
      Fecal neutrophil gelatinase-associated lipocalin as a biomarker for inflammatory bowel disease.
      ]
      CHI3L1Feces>13.7 ng/g85/89IBD vs normal2[
      • Aomatsu T.
      • Imaeda H.
      • Matsumoto K.
      • Kimura E.
      • Yoden A.
      • Tamai H.
      • et al.
      Faecal chitinase 3-like-1: a novel biomarker of disease activity in paediatric inflammatory bowel disease.
      ]
      Immune cellsBiopsyNRNR
      IE CD3 + T cells, LP CD45 + cells and eosinophils



      NR




      NR
      Celiac disease vs NCGWS vs non-NCGWS3[
      • Carroccio A.
      • Giannone G.
      • Mansueto P.
      • Soresi M.
      • La Blasca F.
      • Fayer F.
      • et al.
      Duodenal and rectal mucosa inflammation in patients with non-celiac wheat sensitivity.
      ]
      MacrophagesNRNRIBD vs normal3[
      • Perminow G.
      • Reikvam D.H.
      • Lyckander L.G.
      • Brandtzaeg P.
      • Vatn M.H.
      • Carlsen H.S.
      Increased number and activation of colonic macrophages in pediatric patients with untreated Crohn's disease.
      ]
      MAIT cells

      NR


      NR
      Ulcerative colitis vs normal3[
      • Haga K.
      • Chiba A.
      • Shibuya T.
      • Osada T.
      • Ishikawa D.
      • Kodani T.
      • et al.
      MAIT cells are activated and accumulated in the inflamed mucosa of ulcerative colitis.
      ]
      CD4 + CD25+

      FoxP3 + cells
      Ulcerative colitis vs normal3[
      • Holmén N.
      • Lundgren A.
      • Lundin S.
      • Bergin A.-M.
      • Rudin A.
      • Sjövall H.
      • et al.
      Functional CD4+CD25high regulatory T cells are enriched in the colonic mucosa of patients with active ulcerative colitis and increase with disease activity.
      ]
      PMN-elastaseFeces>19 ng/g84/87IBD vs IBS2[
      • Langhorst J.
      • Elsenbruch S.
      • Koelzer J.
      • Rueffer A.
      • Michalsen A.
      • Dobos J.B.
      Noninvasive markers in the assessment of intestinal inflammation in inflammatory bowel diseases: performance of fecal lactoferrin, calprotectin, and PMN-Elastase, CRP, and clinical indices.
      ]
      DPP4FecesNRNRIBD2[
      • Pinto-Lopes P.
      • Melo F.
      • Afonso J.
      • Pinto-Lopes R.
      • Rocha C.
      • Melo D.
      • et al.
      Fecal Dipeptidyl Peptidase-4: An emergent biomarker in inflammatory bowel disease.
      ]
      HMGB1FecesNRNRIBD vs normal3[
      • Palone F.
      • Vitali R.
      • Cucchiara S.
      • Mennini M.
      • Armuzzi A.
      • Pugliese D.
      • et al.
      Fecal HMGB1 Reveals microscopic inflammation in adult and pediatric patients with inflammatory bowel disease in clinical and endoscopic remission.
      ]
      UMHUrineNRNRIBD vs normal3[
      • Winterkamp S.
      • Weidenhiller M.
      • Otte P.
      • Stolper J.
      • Schwab D.
      • Hahn E.G.
      • et al.
      Urinary excretion of N-methylhistamine as a marker of disease activity in inflammatory bowel disease.
      ]
      CHI3L1, Chitinase 3-like-1; DPP4, Dipeptidyl Peptidase-4; HMGB1, high mobility group box 1; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; IE, intra-epithelial; IEC, Infectious enterocolitis; LoE, Level of Evidence (evidence as defined by the Oxford 2011 Levels of Evidence, v2.1.); LP, lamina propria; MAIT, mucosal-associated invariant T cells; NCGWS; non-coeliac gluten/wheat sensitivity; NR, not reported; PMN, polymorphonuclear neutrophil; S100A12, S100 calcium-binding protein A12; Sens, sensitivity; Spec, specificity; UMH, urinary excretion of n-methylhistamine.

      Characterizing the integrity and permeability of the physical gut barrier

      Physical gut barrier integrity and permeability can be measured non-invasively using sugar absorption tests, the most common of which is the lactulose-to-mannitol (L:M) ratio. Under physiological conditions, an intact physical gut barrier preferentially facilitates absorption of mannitol over lactulose, resulting in a low L:M urinary ratio. When the physical gut barrier becomes disrupted and more permeable, absorption of lactulose increases, resulting in an increased L:M ratio. Differences in methodologies across laboratories hamper the standardization and comparison of studies using L:M ratios. [
      • Turpin W.
      • Lee S.-H.
      • Raygoza Garay J.A.
      • Madsen K.L.
      • Meddings J.B.
      • Bedrani L.
      • et al.
      Increased intestinal permeability is associated with later development of Crohn’s disease.
      ,
      • Camilleri M.
      Leaky gut: mechanisms, measurement and clinical implications in humans.
      ,
      • Seethaler B.
      • Basrai M.
      • Neyrinck A.M.
      • Nazare J.-A.
      • Walter J.
      • Delzenne N.M.
      • et al.
      Biomarkers for assessment of intestinal permeability in clinical practice.
      ]
      Blood-based alternatives such as intestinal fatty acid-binding protein (i-FABP) and citrulline can also be used to characterize physical gut barrier integrity and permeability. i-FABP is a water-soluble cytosolic protein expressed by mature enterocytes. When intestinal mucosal damage occurs, i-FABP is released into the circulation. [
      • Adriaanse M.P.M.
      • Tack G.J.
      • Passos V.L.
      • Damoiseaux J.G.M.C.
      • Schreurs M.W.J.
      • van Wijck K.
      • et al.
      Serum I-FABP as marker for enterocyte damage in coeliac disease and its relation to villous atrophy and circulating autoantibodies.
      ,
      • Bischoff S.C.
      • Barbara G.
      • Buurman W.
      • Ockhuizen T.
      • Schulzke J.-D.
      • Serino M.
      • et al.
      Intestinal permeability–a new target for disease prevention and therapy.
      ,
      • Derikx J.P.M.
      • Vreugdenhil A.C.E.
      • Van den Neucker A.M.
      • Grootjans J.
      • van Bijnen A.A.
      • Damoiseaux J.G.M.C.
      • et al.
      A pilot study on the noninvasive evaluation of intestinal damage in celiac disease using I-FABP and L-FABP.
      ] Citrulline is an amino acid produced by small bowel enterocytes from glutamine and derived amino acids Decreased plasma citrulline reflects reduced enterocyte mass and function. [
      • Fragkos K.C.
      • Forbes A.
      Citrulline as a marker of intestinal function and absorption in clinical settings: A systematic review and meta-analysis.
      ,
      • Hueso T.
      • Gauthier J.
      • Joncquel Chevalier-Curt M.
      • Magro L.
      • Coiteux V.
      • Dulery R.
      • et al.
      Association between low plasma level of citrulline before allogeneic hematopoietic cell transplantation and severe gastrointestinal graft vs host disease.
      ]
      Ex-vivo assessment of physical gut barrier integrity and permeability is possible through immunohistochemical staining of tight junctions and zonula adherens components. [
      • Schellekens D.H.
      • Hundscheid I.HR.
      • Leenarts C.A.
      • Grootjans J.
      • Lenaerts K.
      • Buurman W.A.
      • et al.
      Human small intestine is capable of restoring barrier function after short ischemic periods.
      ] Physical gut barrier integrity can also be assessed ex-vivo by measuring transepithelial electrical resistance with Ussing chambers. [
      • Nojkov B.
      • Zhou S.-Y.
      • Dolan R.D.
      • Davis E.M.
      • Appelman H.D.
      • Guo X.
      • et al.
      Evidence of duodenal epithelial barrier impairment and increased pyroptosis in patients with functional dyspepsia on confocal laser endomicroscopy and “ex vivo” mucosa analysis.
      ]
      In-vivo physical gut barrier integrity assessment is possible using probe- or endoscope-based confocal laser endomicroscopy. Confocal laser endomicroscopy can visualize leakage of lanthanum nitrate into the paracellular space. This leakage is, which is associated with tight junction and zonula adherens loss. [
      • Schellekens D.H.
      • Hundscheid I.HR.
      • Leenarts C.A.
      • Grootjans J.
      • Lenaerts K.
      • Buurman W.A.
      • et al.
      Human small intestine is capable of restoring barrier function after short ischemic periods.
      ] Confocal laser endomicroscopy, combined with intravenous fluorescein administration, allows grading of physical barrier integrity and permeability using the Confocal Leak Score. The Confocal Leak Score measures fluorescein leakage from the submucosa into the gut lumen through epithelial breaks. The Confocal Leak Score has been validated and is predictive for diarrhea motions per day in inflammatory bowel disease (IBD). [
      • Chang J.
      • Leong R.W.
      • Wasinger V.C.
      • Ip M.
      • Yang M.
      • Phan T.G.
      Impaired intestinal permeability contributes to ongoing bowel symptoms in patients with inflammatory bowel disease and mucosal healing.
      ] In-vivo and ex-vivo tissue analyses may be cumbersome for patients. Still, in the case of in-vivo analysis, they offer a level of real-time diagnostic accuracy that is not obtainable otherwise.

      Characterizing the activity of the intestinal immune system

      Calprotectin and lactoferrin are the most commonly used markers of intestinal immune activation. Calprotectin is the predominant cytosolic protein in neutrophils, while lactoferrin is a protein in neutrophil secondary granules. Their elevation in feces can reflect microscopic and macroscopic intestinal inflammation. Calprotectin can serve to predict and monitor IBD flare-ups and distinguish IBD from non-inflammatory intestinal conditions. [
      • Mosli M.H.
      • Zou G.
      • Garg S.K.
      • Feagan S.G.
      • MacDonald J.K.
      • Chande N.
      • et al.
      C-reactive protein, fecal calprotectin, and stool lactoferrin for detection of endoscopic activity in symptomatic inflammatory bowel disease patients: a systematic review and meta-analysis.
      ,
      • Jukic A.
      • Bakiri L.
      • Wagner E.F.
      • Tilg H.
      • Adolph T.E.
      Calprotectin: from biomarker to biological function.
      ,

      Zollner A, Schmiderer A, Reider SJ, et al. Faecal biomarkers in inflammatory bowel diseases: calprotectin versus lipocalin-2—a comparative study. J Crohn Colitis. 2021;15;43-35. doi: 10.1093/ecco-jcc/jjaa124.

      ] Variations in calprotectin and lactoferrin levels across age groups have been reported, therefore age-adjusted cut-off values for both markers have been proposed. [
      • Joshi S.
      • Lewis S.J.
      • Creanor S.
      • Ayling R.M.
      Age-related faecal calprotectin, lactoferrin and tumour M2-PK concentrations in healthy volunteers.
      ]
      Fecal markers S100 calcium-binding protein A12 (S100A12), lipocalin-2, and Chitinase 3-like-1 (CHI3L) are not yet clinically validated alternatives to detect bowel inflammation. S100A12, like calprotectin, is a cytoplasmic neutrophil-derived protein released upon neutrophil activation. [
      • Kaiser T.
      • Langhorst J.
      • Wittkowski H.
      • Becker K.
      • Friedrich A.W.
      • Rueffer A.
      • et al.
      Faecal S100A12 as a non-invasive marker distinguishing inflammatory bowel disease from irritable bowel syndrome.
      ] Lipocalin-2 is a glycoprotein with immunomodulatory and antimicrobial effects secreted into the gut lumen primarily by enterocytes but also by macrophages, monocytes, and granulocytes in response to proinflammatory stimuli. Lipocalin-2 can detect low-grade inflammation and is equivalent to fecal calprotectin in IBD. [
      • Thorsvik S.
      • Damås J.K.
      • Granlund A.vB.
      • Flo T.H.
      • Bergh K.
      • Østvik A.E.
      • et al.
      Fecal neutrophil gelatinase-associated lipocalin as a biomarker for inflammatory bowel disease.
      ,
      • Jukic A.
      • Bakiri L.
      • Wagner E.F.
      • Tilg H.
      • Adolph T.E.
      Calprotectin: from biomarker to biological function.
      ] Glycoprotein CHI3L is highly expressed in colon epithelial cells and lamina propria macrophages at sites of mucosal inflammation. Fecal CHI3L levels correlate with clinical and endoscopic IBD activity. [
      • Aomatsu T.
      • Imaeda H.
      • Matsumoto K.
      • Kimura E.
      • Yoden A.
      • Tamai H.
      • et al.
      Faecal chitinase 3-like-1: a novel biomarker of disease activity in paediatric inflammatory bowel disease.
      ]
      Detailed characterization of immune cell infiltrates is possible through ex-vivo assessment of biopsies using immunohistochemical stainings, flow cytometry, or quantitative real-time polymerase chain reaction. Thus, immune cell populations can be quantified and characterized, and their infiltration patterns analyzed [
      • Dunlop S.P.
      • Hebden J.
      • Campbell E.
      • Naesdal J.
      • Olbe L.
      • Perkins A.C.
      • et al.
      Abnormal intestinal permeability in subgroups of diarrhea-predominant irritable bowel syndromes.
      ,
      • Li H.
      • Chen Y.
      • Huo F.
      • Wang Y.
      • Zhang D.
      Association between acute gastrointestinal injury and biomarkers of intestinal barrier function in critically ill patients.
      ,
      • Vieira M.M.
      • Paik J.
      • Blaner W.S.
      • Soares A.M.
      • Mota R.MS.
      • Guerrant R.L.
      • et al.
      Carotenoids, retinol, and intestinal barrier function in children from northeastern Brazil.
      ,
      • Mosli M.H.
      • Zou G.
      • Garg S.K.
      • Feagan S.G.
      • MacDonald J.K.
      • Chande N.
      • et al.
      C-reactive protein, fecal calprotectin, and stool lactoferrin for detection of endoscopic activity in symptomatic inflammatory bowel disease patients: a systematic review and meta-analysis.
      ,
      • Kaiser T.
      • Langhorst J.
      • Wittkowski H.
      • Becker K.
      • Friedrich A.W.
      • Rueffer A.
      • et al.
      Faecal S100A12 as a non-invasive marker distinguishing inflammatory bowel disease from irritable bowel syndrome.
      ,
      • Thorsvik S.
      • Damås J.K.
      • Granlund A.vB.
      • Flo T.H.
      • Bergh K.
      • Østvik A.E.
      • et al.
      Fecal neutrophil gelatinase-associated lipocalin as a biomarker for inflammatory bowel disease.
      ,
      • Aomatsu T.
      • Imaeda H.
      • Matsumoto K.
      • Kimura E.
      • Yoden A.
      • Tamai H.
      • et al.
      Faecal chitinase 3-like-1: a novel biomarker of disease activity in paediatric inflammatory bowel disease.
      ,
      • Carroccio A.
      • Giannone G.
      • Mansueto P.
      • Soresi M.
      • La Blasca F.
      • Fayer F.
      • et al.
      Duodenal and rectal mucosa inflammation in patients with non-celiac wheat sensitivity.
      ,
      • Perminow G.
      • Reikvam D.H.
      • Lyckander L.G.
      • Brandtzaeg P.
      • Vatn M.H.
      • Carlsen H.S.
      Increased number and activation of colonic macrophages in pediatric patients with untreated Crohn's disease.
      ,
      • Haga K.
      • Chiba A.
      • Shibuya T.
      • Osada T.
      • Ishikawa D.
      • Kodani T.
      • et al.
      MAIT cells are activated and accumulated in the inflamed mucosa of ulcerative colitis.
      ,
      • Holmén N.
      • Lundgren A.
      • Lundin S.
      • Bergin A.-M.
      • Rudin A.
      • Sjövall H.
      • et al.
      Functional CD4+CD25high regulatory T cells are enriched in the colonic mucosa of patients with active ulcerative colitis and increase with disease activity.
      ,
      • Jukic A.
      • Bakiri L.
      • Wagner E.F.
      • Tilg H.
      • Adolph T.E.
      Calprotectin: from biomarker to biological function.
      ,

      Zollner A, Schmiderer A, Reider SJ, et al. Faecal biomarkers in inflammatory bowel diseases: calprotectin versus lipocalin-2—a comparative study. J Crohn Colitis. 2021;15;43-35. doi: 10.1093/ecco-jcc/jjaa124.

      ,
      • Joshi S.
      • Lewis S.J.
      • Creanor S.
      • Ayling R.M.
      Age-related faecal calprotectin, lactoferrin and tumour M2-PK concentrations in healthy volunteers.
      ]. Biopsies are considered the gold standard to diagnose and monitor intestinal inflammation. However, biopsies they face the limitation that they represent a small tissue area and therefore may not be representative.

      Interventions modulating the integrity, permeability, and immune activity of the gut wall

      Modulation of the integrity, permeability, and immune activity of the gut wall is possible through pharmaceutical intervention, diet and supplements, pre- and pro-biotics, and FMT. We identified 11 pharmaceutical interventions, one dietary intervention, and three microbiota-directed interventions (Table 3).
      Table 3Source articles of gut wall directed interventions.
      InterventionMechanism of actionGut-selective?EffectContextLoERef.
      CorticosteroidsInteract with glucocorticoid receptors in cell nuclei

      Promote tolerogenic over immunogenic phenotype, neutralize proinflammatory cytokines
      NoAnti-inflammatory and mucosal healing

      IBD1[
      • Pola S.
      • Patel D.
      • Ramamoorthy S.
      • McLemore E.
      • Fahmy M.
      • Rivera–Nieves J.
      • et al.
      Strategies for the care of adults hospitalized for active ulcerative colitis.
      ]
      Integrin inhibitor

      Not fully understood

      Vedolizumab blocks α4β7 integrin on T and B cells
      YesAnti-inflammatory and mucosal healingIBD

      1[
      • Schreiber S.
      • Dignass A.
      • Peyrin-Biroulet L.
      • Hather G.
      • Demuth D.
      • Mosli M.
      • et al.
      Systematic review with meta-analysis: real-world effectiveness and safety of vedolizumab in patients with inflammatory bowel disease.
      ]

      ThiopurinesInhibit nucleotide and purine synthesis, reducing the proliferation of rapidly dividing cells

      Block gene activation of effector T cells, downregulate their cytotoxic activity and reduce B cell infiltration in the gut mucosa
      NoAnti-inflammatory, mucosal healingCrohn’s disease1[
      • Chatu S.
      • Subramanian V.
      • Saxena S.
      • et al.
      The role of thiopurines in reducing the need for surgical resection in Crohn’s disease: a systematic review and meta-analysis.
      ]
      CyclosporineCalcineurin inhibitorNoAnti-inflammatory and mucosal healingUlcerative colitis1[
      • Van Assche G.
      • D'Haens G.
      • Noman M.
      • et al.
      Randomized, double-blind comparison of 4 mg/kg versus 2 mg/kg intravenous cyclosporine in severe ulcerative colitis.
      ]
      JAK-inhibitorsSelectively inhibit JAK-1 and JAK-2, thus downmodulating signaling of pro-inflammatory cytokines of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21NoAnti-inflammatory and mucosal healingIBD1[
      • Salas A.
      • Hernandez-Rocha C.
      • Duijvestein M.
      • Faubion W.
      • McGovern D.
      • Vermeire S.
      • et al.
      JAK-STAT pathway targeting for the treatment of inflammatory bowel disease.
      ]
      5-ASANot fully understood

      Metabolized by IECs, local effect
      YesAnti-inflammatory and mucosal healingIBD2[
      • Ogata H.
      • Yokoyama T.
      • Mizushima S.
      • Hagino A.
      • Hibi T.
      Comparison of efficacy of once daily multimatrix mesalazine 2.4 g/day and 4.8 g/day with other 5-aminosalicylic acid preparation in active ulcerative colitis: a randomized, double-blind study.
      ]
      UstekinumabInhibits the T-helper 1 and 17 pathways by blocking the p40 subunit of IL-12 & IL-23NoAnti-inflammatory and mucosal healingCrohn’s disease2[

      Straatmijer T, Biemans VBC, Hoentjen F, et al. Ustekinumab for Crohn's Disease: Two-Year Results of the Initiative on Crohn and Colitis (ICC) Registry, a Nationwide Prospective Observational Cohort Study. J Crohns Colitis. 2021;15:1920-1930. doi: 10.1093/ecco-jcc/jjab081.

      ]
      TNF-α inhibitorsInhibit the proinflammatory activity of the cytokine TNF-αNoAnti-inflammatory, mucosal healingUlcerative colitis2[
      • Hämäläinen A.
      • Sipponen T.
      • Kolho K.L.
      Infliximab in pediatric inflammatory bowel disease rapidly decreases fecal calprotectin levels.
      ]

      Improve physical gut barrier function and decrease permeabilityCrohn’s disease2[
      • Noth R.
      • Stüber E.
      • Häsler R.
      • Nikolaus S.
      • Kühbacher T.
      • Hampe J.
      • et al.
      Anti-TNF-α antibodies improve intestinal barrier function in Crohn’s disease.
      ]
      Dersalazine sodiumDecreases expression of inflammatory genes and proinflammatory cytokines in colonic tissue through anti-platelet activating factor activityYesAnti-inflammatory and mucosal healing

      Ulcerative colitis.

      2[
      • Noth R.
      • Stüber E.
      • Häsler R.
      • Nikolaus S.
      • Kühbacher T.
      • Hampe J.
      • et al.
      Anti-TNF-α antibodies improve intestinal barrier function in Crohn’s disease.
      ,
      • Pontes C.
      • Vives R.
      • Torres F.
      • Panés J.
      Safety and activity of dersalazine sodium in patients with mild-to-moderate active colitis: double-blind randomised proof of concept study.
      ]
      Propionyl-L-carnitineSource of l-carnitine and propionyl-coenzyme-A for colonocytes, proposed to in this way facilitate energy releaseYesMucosal healingUlcerative colitis, as co-treatment2[
      • Mikhailova T.L.
      • Sishkova E.
      • Poniewierka E.
      • Zhidkov K.P.
      • Bakulin I.G.
      • Kupcinskas L.
      • et al.
      Randomised clinical trial: the efficacy and safety of propionyl-L-carnitine therapy in patients with ulcerative colitis receiving stable oral treatment.
      ]
      Lazarotide acetatePeptide derived from zonula occludens toxin, modulator of tight-junctions and inhibitor of paracellular permeabilityYesIncreases physical gut barrier integrity and decreases intestinal permeabilityCoeliac’s disease2[
      • Leffler D.A.
      • Kelly C.P.
      • Green P.H.
      • et al.
      Larazotide acetate for persistent symptoms of celiac disease despite a gluten-free diet: a randomised controlled trial.
      ]

      LMWHNot fully understood

      Thought to modulate complement pathways and proinflammatory cytokines
      NoAnti-inflammatoryUlcerative colitis2[
      • Celasco G.
      • Papa A.
      • Jones R.
      • et al.
      Clinical trial: oral colon-release parnaparin sodium tablets (CB-01-05 MMX) for active left-sided ulcerative colitis.
      ]

      Oral glutamineNot fully understood

      Glutamine is a major source of energy for enterocytes
      NoDecreases intestinal permeabilityPost-infectious IBS-D3[
      • Zhou QiQi
      • Verne M.L.
      • Fields J.Z.
      • Lefante J.J.
      • Basra S.
      • Salameh H.
      • et al.
      Randomised placebo-controlled trial of dietary glutamine supplements for postinfectious irritable bowel syndrome.
      ]
      Prebiotics

      Not fully understoodNoAnti-inflammatory, functional changes gut microbiotaIBD1[
      • Bindels L.B.
      • Delzenne N.M.
      • Cani P.D.
      • Walter J.
      Towards a more comprehensive concept for prebiotics.
      ]
      ProbioticsNot fully understoodNoMucosal healing, improve physical gut barrier integrity, decrease intestinal permeability, anti-inflammatoryIBD, IBS, C. difficile, SIBO1[
      • Valcheva R.
      • Koleva P.
      • Martínez I.
      • Walter J.
      • Gänzle M.G.
      • Dieleman L.A.
      Inulin- type fructans improve active ulcerative colitis associated with microbiota changes and increased short-chain fatty acids levels.
      ]
      FMTNot fully understoodNoMucosal healing, anti-inflammatory, eubiosis?Ulcerative colitis1[
      • Narula N.
      • Kassam Z.
      • Yuan Y.
      • Colombel J.F.
      • Ponsioen C.
      • Reinisch W.
      • et al.
      Systematic review and meta-analysis: fecal microbiota transplantation for treatment of active ulcerative colitis.
      ]
      5-ASAs, mesalamine or 5-aminosalicylic acid; FMT, fecal microbial transplant; IBD, inflammatory bowel disease; IBS-D, diarrhea-predominant irritable bowel syndrome; IL, interleukin; JAK, Janus kinase; LMWH, low-molecular weight heparin; LoE, Level of Evidence (evidence as defined by the Oxford 2011 Levels of Evidence, v2.1.); SIBO, small intestinal bacterial overgrowth; TNF, tumor necrosis factor alpha.

      Pharmaceutical interventions

      Clinically approved treatments that non-selectively promote gut mucosal healing through immunomodulation are Janus kinase (JAK) inhibitors, tumor necrosis factor-α (TNF-α) inhibitors, corticosteroids, cyclosporine, ustekinumab, and thiopurines. In addition, selective modulation of the intestinal immune system is possible with 5-aminosalicylic acid and vedolizumab. [
      • Pola S.
      • Patel D.
      • Ramamoorthy S.
      • McLemore E.
      • Fahmy M.
      • Rivera–Nieves J.
      • et al.
      Strategies for the care of adults hospitalized for active ulcerative colitis.
      ,
      • Schreiber S.
      • Dignass A.
      • Peyrin-Biroulet L.
      • Hather G.
      • Demuth D.
      • Mosli M.
      • et al.
      Systematic review with meta-analysis: real-world effectiveness and safety of vedolizumab in patients with inflammatory bowel disease.
      ,
      • Chatu S.
      • Subramanian V.
      • Saxena S.
      • et al.
      The role of thiopurines in reducing the need for surgical resection in Crohn’s disease: a systematic review and meta-analysis.
      ,
      • Van Assche G.
      • D'Haens G.
      • Noman M.
      • et al.
      Randomized, double-blind comparison of 4 mg/kg versus 2 mg/kg intravenous cyclosporine in severe ulcerative colitis.
      ,
      • Salas A.
      • Hernandez-Rocha C.
      • Duijvestein M.
      • Faubion W.
      • McGovern D.
      • Vermeire S.
      • et al.
      JAK-STAT pathway targeting for the treatment of inflammatory bowel disease.
      ,
      • Ogata H.
      • Yokoyama T.
      • Mizushima S.
      • Hagino A.
      • Hibi T.
      Comparison of efficacy of once daily multimatrix mesalazine 2.4 g/day and 4.8 g/day with other 5-aminosalicylic acid preparation in active ulcerative colitis: a randomized, double-blind study.
      ,

      Straatmijer T, Biemans VBC, Hoentjen F, et al. Ustekinumab for Crohn's Disease: Two-Year Results of the Initiative on Crohn and Colitis (ICC) Registry, a Nationwide Prospective Observational Cohort Study. J Crohns Colitis. 2021;15:1920-1930. doi: 10.1093/ecco-jcc/jjab081.

      ,
      • Hämäläinen A.
      • Sipponen T.
      • Kolho K.L.
      Infliximab in pediatric inflammatory bowel disease rapidly decreases fecal calprotectin levels.
      ,
      • Noth R.
      • Stüber E.
      • Häsler R.
      • Nikolaus S.
      • Kühbacher T.
      • Hampe J.
      • et al.
      Anti-TNF-α antibodies improve intestinal barrier function in Crohn’s disease.
      ,
      • Pantavou K.
      • Yiallourou A.I.
      • Piovani D.
      • Evripidou D.
      • Danese S.
      • Peyrin-Biroulet L.
      • et al.
      Efficacy and safety of biologic agents and tofacitinib in moderate-to-severe ulcerative colitis: A systematic overview of meta-analyses.
      ,
      • Dubois-Camacho K.
      • Ottum P.A.
      • Franco-Muñoz D.
      • De la Fuente M.
      • Torres-Riquelme A.
      • Díaz-Jiménez D.
      • et al.
      Glucocorticosteroid therapy in inflammatory bowel diseases: From clinical practice to molecular biology.
      ,
      • D'Haens G.
      Systematic review: second-generation vs. conventional corticosteroids for induction of remission in ulcerative colitis.
      ,
      • Zeissig S.
      • Rosati E.
      • Dowds C.M.
      • Aden K.
      • Bethge J.
      • Schulte B.
      • et al.
      Vedolizumab is associated with changes in innate rather than adaptive immunity in patients with inflammatory bowel disease.
      ,
      • Hanauer S.B.
      New lessons: classic treatments, expanding options in ulcerative colitis.
      ]
      Next to these clinically used gut wall modulators, larazotide acetate and propionyl-L-carnitine increase physical gut wall integrity and decrease intestinal permeability, colonic-release low-molecular-weight heparin promotes mucosal healing and reduces inflammation, and dersalazine sodium modulates intestinal immune system activity. [
      • Pontes C.
      • Vives R.
      • Torres F.
      • Panés J.
      Safety and activity of dersalazine sodium in patients with mild-to-moderate active colitis: double-blind randomised proof of concept study.
      ,
      • Mikhailova T.L.
      • Sishkova E.
      • Poniewierka E.
      • Zhidkov K.P.
      • Bakulin I.G.
      • Kupcinskas L.
      • et al.
      Randomised clinical trial: the efficacy and safety of propionyl-L-carnitine therapy in patients with ulcerative colitis receiving stable oral treatment.
      ,
      • Leffler D.A.
      • Kelly C.P.
      • Green P.H.
      • et al.
      Larazotide acetate for persistent symptoms of celiac disease despite a gluten-free diet: a randomised controlled trial.
      ,
      • Celasco G.
      • Papa A.
      • Jones R.
      • et al.
      Clinical trial: oral colon-release parnaparin sodium tablets (CB-01-05 MMX) for active left-sided ulcerative colitis.
      ]

      Dietary interventions

      Improvement of integrity, permeability, and immune activity of the gut wall with diets has been explored in a broad range of gastrointestinal conditions. While consensus on dietary interventions targeting gut wall function remains to be reached, certain dietary patterns are considered to be noxious or protective. High consumption of sugar, animal protein, and saturated fats is associated with gut microbiota dysbiosis, intestinal inflammation, and increased risk for IBD. High fiber consumption has been associated with more favorable and varied gut microbiota composition and increased short-chain fatty acid (SCFA) production, thus promoting gut wall homeostasis. [
      • Sasson A.N.
      • Ingram R.J.M.
      • Zhang Z.
      • Taylor L.M.
      • Ananthakrishnan A.N.
      • Kaplan G.G.
      • et al.
      The role of precision nutrition in the modulation of microbial composition and function in people with inflammatory bowel disease.
      ,
      • Fritsch J.
      • Garces L.
      • Quintero M.A.
      • Pignac-Kobinger J.
      • Santander A.M.
      • Fernández I.
      • et al.
      Low-fat, high-fiber diet reduces markers of inflammation and dysbiosis and improves quality of life in patients with ulcerative colitis.
      ,
      • Spencer C.N.
      • McQuade J.L.
      • Gopalakrishnan V.
      • McCulloch J.A.
      • Vetizou M.
      • Cogdill A.P.
      • et al.
      Dietary fiber and probiotics influence the gut microbiome and melanoma immunotherapy response.
      ]
      We identified one dietary intervention directed at the gut wall: oral glutamine supplementation. Glutamine is a major energy source for enterocytes. Without glutamine, enterocytes decay, causing physical gut barrier disruption and increased permeability. Oral supplementation of glutamine normalizes intestinal permeability in patients with postinfectious diarrhea-predominant IBS. [
      • Zhou QiQi
      • Verne M.L.
      • Fields J.Z.
      • Lefante J.J.
      • Basra S.
      • Salameh H.
      • et al.
      Randomised placebo-controlled trial of dietary glutamine supplements for postinfectious irritable bowel syndrome.
      ]

      Interventions with prebiotics, probiotics, and gut microbiota

      The use of pre- and probiotics to reinstate gut wall homeostasis shows promising results in experimental settings. Prebiotics are non-digestible compounds that are metabolized by gut microbiota. Prebiotics promote favorable gut microbiota composition and/or activity, resulting in beneficial physiological effects on the host. [
      • Bindels L.B.
      • Delzenne N.M.
      • Cani P.D.
      • Walter J.
      Towards a more comprehensive concept for prebiotics.
      ] Suppletion with prebiotic inulin-type-ϐ fructans suppletion is beneficial for some patients with ulcerative colitis. It reduces inflammation, promotes mucosal healing, and induces functional and compositional microbiota changes. [
      • Valcheva R.
      • Koleva P.
      • Martínez I.
      • Walter J.
      • Gänzle M.G.
      • Dieleman L.A.
      Inulin- type fructans improve active ulcerative colitis associated with microbiota changes and increased short-chain fatty acids levels.
      ] Probiotics are live microorganisms that can be beneficial to the host's health. Probiotics exert their effects by promoting a favorable gut microbiota composition and functionality, improving physical gut barrier function, immunomodulation and modulating physiological processes on the host. [
      • Judkins T.C.
      • Archer D.L.
      • Kramer D.C.
      • Solch R.J.
      Probiotics, nutrition, and the small intestine.
      ] In diarrhea-predominant IBS, lactic acid bacteria supplementation improves mucosal barrier function. [
      • Zeng J.
      • Li Y.Q.
      • Zuo X.L.
      • Zhen Y.B.
      • Yang J.
      • Liu C.H.
      Clinical trial: effect of active lactic acid bacteria on mucosal barrier function in patients with diarrhoea-predominant irritable bowel syndrome.
      ] When added to the conventional treatment of patients with mild to moderate ulcerative colitis, probiotic mix VSL#3 can boost mucosal healing and increase endoscopic remission rates. [
      • Mardini H.E.
      • Grigorian A.Y.
      Probiotic mix VSL#3 is effective adjunctive therapy for mild to moderately active ulcerative colitis: a meta-analysis.
      ] While studies suggest that use of prebiotic and probiotics may be beneficial in certain clinical settings like IBD, the variation in formulations and dosages used, as well as the limited data available from controlled trials, preclude deriving firm conclusions on their effects and efficacy. [
      • Bindels L.B.
      • Delzenne N.M.
      • Cani P.D.
      • Walter J.
      Towards a more comprehensive concept for prebiotics.
      ] Importantly, data suggest that probiotic supplementation may also enhance the therapeutic effects of ICI therapy. Supplementation with CMBM588, a probiotic containing Clostridium butyricum which produces the SCFA butyrate appears to improve progression-free survival in patients with metastatic renal cell cancer receiving dual ICI therapy. Patients receiving CBM588 showed changes in gut microbiota functionality, like upregulated rhamnose synthesis and an increase of the SCFA propionate production. [
      • Dizman N.
      • Meza L.
      • Bergerot P.
      • Alcantara M.
      • Dorff T.
      • Lyou Y.
      • et al.
      Nivolumab plus ipilimumab with or without live bacterial supplementation in metastatic renal cell carcinoma: a randomized phase 1 trial.
      ] Manipulation of the gut microbiota through FMT has been explored in the context of severe reduction in microbial diversity caused by pathogens like C. difficile and as an experimental treatment in IBD. In patients with C. difficile, FMT induced remission and symptomatic relief. [
      • Cammarota G.
      • Ianiro G.
      • Gasbarrini A.
      Fecal microbiota transplantation for the treatment of Clostridium difficile infection: a systematic review.
      ] In patients with ulcerative colitis, FMT increased diversity, induced remission, and mucosal healing. FMT did not lead to objective changes in inflammatory markers like calprotectin. [
      • Narula N.
      • Kassam Z.
      • Yuan Y.
      • Colombel J.F.
      • Ponsioen C.
      • Reinisch W.
      • et al.
      Systematic review and meta-analysis: fecal microbiota transplantation for treatment of active ulcerative colitis.
      ,
      • Colman R.J.
      • Rubin D.T.
      Fecal microbiota transplantation as therapy for inflammatory bowel disease: a systematic review and meta-analysis.
      ] The exact effects of FMT are unknown. It is hypothesized that FMT promotes recolonization of bacteria with properties beneficial for health, and thus an optimal balance of microbiota in the gastrointestinal tract. While the short-term effects of FMT are promising, several aspects remain unclear. Ongoing studies will provide more data on the effect of FMT on objective endpoints such as mucosal healing or disease extent. Further research is required to address the impact of bowel preparation on FMT effectiveness, to determine the best administration route and dosage of FMT as well as to define the long-term effects of FMT on the recipient. [
      • Narula N.
      • Kassam Z.
      • Yuan Y.
      • Colombel J.F.
      • Ponsioen C.
      • Reinisch W.
      • et al.
      Systematic review and meta-analysis: fecal microbiota transplantation for treatment of active ulcerative colitis.
      ,
      • Cammarota G.
      • Ianiro G.
      • Gasbarrini A.
      Fecal microbiota transplantation for the treatment of Clostridium difficile infection: a systematic review.
      ]

      Gut wall integrity, permeability, and immune activity in the context of ICI treatment

      To date, efforts have focused on the relationship between gut lumen microbiota composition and the strength of the anticancer immune response induced by ICI. In addition, an association between gut mucosa microbial composition and response to ICI and to gastrointestinal immune-related adverse events was recently shown. [
      • Sakurai T.
      • De Velasco M.A.
      • Sakai K.
      • Nagai T.
      • Nishiyama H.
      • Hashimoto K.
      • et al.
      Integrative analysis of gut microbiome and host transcriptomes reveals associations between treatment outcomes and immunotherapy-induced colitis.
      ] In initial ICI non-responders, FMT, and re-introduction of ICI can lead to tumor response. [
      • Baruch E.N.
      • Youngster I.
      • Ben-Betzalel G.
      • Ortenberg R.
      • Lahat A.
      • Katz L.
      • et al.
      Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients.
      ] However, there is no consensus on which bacterial species are favorable. [
      • Baruch E.N.
      • Youngster I.
      • Ben-Betzalel G.
      • Ortenberg R.
      • Lahat A.
      • Katz L.
      • et al.
      Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients.
      ,
      • Davar D.
      • Dzutsev A.K.
      • McCulloch J.A.
      • Rodrigues R.R.
      • Chauvin J.-M.
      • Morrison R.M.
      • et al.
      Fecal microbiota transplant overcomes resistance to anti-PD-1 therapy in melanoma patients.
      ] Promising data suggest that supplementation with probiotics may improve clinical outcomes of patients receiving dual ICI therapy. [
      • Dizman N.
      • Meza L.
      • Bergerot P.
      • Alcantara M.
      • Dorff T.
      • Lyou Y.
      • et al.
      Nivolumab plus ipilimumab with or without live bacterial supplementation in metastatic renal cell carcinoma: a randomized phase 1 trial.
      ]
      It is becoming increasingly evident that microbial functionality may be equally relevant or even more relevant than gut microbiota composition itself. Shotgun metagenomic sequencing was performed on pre-ICI-treatment stool samples collected in 5 observational cohorts recruiting ICI-naive patients with advanced cutaneous melanoma (n = 165). It showed that the gut microbiome has a relevant but cohort-dependent association with response to ICI. [
      • Lee K.A.
      • Thomas A.M.
      • Bolte L.A.
      • Björk J.R.
      • de Ruijter L.K.
      • Armanini F.
      • et al.
      Cross-cohort gut microbiome associations with immune checkpoint inhibitor response in advanced melanoma.
      ] We are currently generating metabolomics data and will measure markers discussed in this review to characterize the gut wall in the abovementioned cohorts of ICI-naïve patients.
      SCFAs have been associated with response to ICI. SCFAs are microbial metabolites produced by microbial fermentation of dietary fibers. SCFAs interact with the gut wall, contribute to intestinal immune homeostasis and intestinal epithelial barrier integrity. Patients with non-small cell lung cancer (NSCLC) responding to programmed cell death protein 1 (PD-1) antibody therapy had a higher baseline fecal SCFAs concentration. [
      • Zizzari I.
      • Di Filippo A.
      • Scirocchi F.
      • Di Pietro F.
      • Rahimi H.
      • Ugolini A.
      • et al.
      Soluble immune checkpoints, gut metabolites and performance status as parameters of response to nivolumab treatment in NSCLC patients.
      ] In line with this, in patients with a wide range of malignancies treated with PD-1 antibodies, high baseline fecal concentrations of SCFAs propionic, butyric, valeric, and acetic acid and high plasma levels of isovaleric acid were associated with longer progression-free survival. [
      • Nomura M.
      • Nagatomo R.
      • Doi K.
      • Shimizu J.
      • Baba K.
      • Saito T.
      • et al.
      Association of short-chain fatty acids in the gut microbiome with clinical response to treatment with nivolumab or pembrolizumab in patients with solid cancer tumors.
      ] Furthermore, in patients with NSCLC, SCFAs were shown to be the main metabolites produced by the gut microbiota of long-term responders to PD-1 antibody therapy. [
      • Botticelli A.
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      Gut metabolomics profiling of non-small cell lung cancer (NSCLC) patients under immunotherapy treatment.
      ] In contrast, low baseline serum propionic and butyric acid were associated with longer progression-free survival in patients with metastatic melanoma and metastatic prostate cancer receiving cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibodies. [
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      ] Due to differences in the methodologies applied and modest size of patient populations included, no clear conclusions can be drawn from the divergent results across the abovementioned studies. Adequate enterocyte function, reflected by high plasma citrulline levels (≥20 μM) at baseline, correlated with favorable response to ICI, longer progression-free, and overall survival in patients with advanced NSCLC receiving anti-PD-1 therapy. [
      • Ouaknine Krief J.
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      • Dumenil C.
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      • Dumoulin J.
      • Giraud V.
      • et al.
      Role of antibiotic use, plasma citrulline and blood microbiome in advanced non-small cell lung cancer patients treated with nivolumab.
      ] The effects of ICI therapy on the intestinal immune system have been investigated in more detail in the context of ICI-induced colitis. In ICI-induced colitis, fecal calprotectin and lactoferrin levels are elevated. [
      • Berman D.
      • Parker S.M.
      • Siegel J.
      • et al.
      Blockade of cytotoxic T-lymphocyte antigen-4 by ipilimumab results in dysregulation of gastrointestinal immunity in patients with advanced melanoma.
      ,
      • Abu-Sbeih H.
      • Ali F.S.
      • Luo W.
      • Qiao W.
      • Raju G.S.
      • Wang Y.
      Importance of endoscopic and histological evaluation in the management of immune checkpoint inhibitor-induced colitis.
      ,
      • Abu-Sbeih H.
      • Ali F.S.
      • Wang X.
      • et al.
      Early introduction of selective immunosuppressive therapy associated with favourable clinical outcomes in patients with immune checkpoint inhibitor-induced colitis.
      ,

      Gambichler T, Brown V, Steuke AK, Schmitz L, Stockfleth E, Susok. Baseline laboratory parameters predicting clinical outcome in melanoma patients treated with ipilimumab: a single-centre analysis. J Eur Acad Dermatology Venereol. 2018;32:972–977. doi: 10.1111/jdv.14629.

      ] ICI-induced colitis is characterized by a heavy CD8 + T cell mucosal infiltrate. [
      • Luoma A.M.
      • Suo S.
      • Williams H.L.
      • Sharova T.
      • Sullivan K.
      • Manos M.
      • et al.
      Molecular pathways of colon inflammation induced by cancer immunotherapy.
      ,
      • Hone Lopez S.
      • Kats-Ugurlu G.
      • Renken R.J.
      • Buikema H.J.
      • de Groot M.R.
      • Visschedijk M.C.
      • et al.
      Immune checkpoint inhibitor treatment induces colitis with heavy infiltration of CD8 + T cells and an infiltration pattern that resembles ulcerative colitis.
      ] In the ongoing DEFENCE study, colon biopsies and blood samples are obtained at baseline and during ICI treatment to determine the effects of ICI on the physical gut wall and the intestinal immune system activation in relation to ICI tumor response. [

      National Library of Medicine (U.S.). (2021, October -). Deep Phenotyping of the Gut Immune System During Immune Checkpoint Inhibitor Therapy – DEFENCE. Identifier NCT04600180. https://clinicaltrials.gov/ct2/show/NCT04600180.

      ]

      Future perspective: Exploiting the gut wall to predict and potentiate tumor response to ICI

      Compelling data suggest that gut microbiota composition and functionality are relevant to tumor response to ICI. This raises the question of whether this may also be the case for the gut wall. To study this hypothesis, the gut wall phenotypes of responders and non-responders to ICI should be defined and compared. This can be done by measuring physical gut barrier integrity, intestinal permeability, and intestinal inflammation markers at baseline and during ICI treatment. In addition, the interplay between gut wall immune composition and activity status and gut microbiota, as well as tumor and patient characteristics should be taken into account. Determining PD-1 and CTLA-4 expression of cells in the gut wall may also be of interest, since these ICI targets have a role in maintaining intestinal immune system homeostasis. [
      • Chulkina M.
      • Beswick E.J.
      • Pinchuk I.V.
      Role of PD-L1 in Gut Mucosa Tolerance and Chronic Inflammation.
      ,
      • Barnes M.J.
      • Griseri T.
      • Johnson A.M.
      • Young W.
      • Powrie F.
      • Izcue A.
      CTLA-4 promotes Foxp3 induction and regulatory T cell accumulation in the intestinal lamina propria.
      ] If data suggest a role for the gut wall in the response to ICI, and specific gut wall phenotypes are associated with response to ICI, interventions to promote such beneficial phenotypes should be considered.
      Together, the data obtained from exploring the role of the gut wall in tumor response to ICI might lead to novel markers and therapeutic targets with which to predict and potentiate the response to ICI.

      Funding

      This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

      Author contribution

      All authors contributed to manuscript conception, drafting of the manuscript, provided critical review and revisions, and approved the final version of the manuscript.

      CRediT authorship contribution statement

      Sara Hone Lopez: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Mathilde Jalving: Conceptualization, Methodology, Writing – review & editing, Supervision. Rudolf S.N. Fehrmann: Conceptualization, Methodology, Writing – review & editing. Wouter B. Nagengast: Conceptualization, Writing – review & editing. Elisabeth G.E. de Vries: Conceptualization, Methodology, Writing – review & editing. Jacco J. de Haan: Conceptualization, Methodology, Writing – original draft, Writing – review & editing, Supervision.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Appendix A. Supplementary material

      The following are the Supplementary data to this article:

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