Abstract Fibrogenesis is essential to wound healing, but aberrant fibrogenesis is a driver of many chronic diseases and cancers. Lysyl oxidases (LOX) play a pivotal role in fibrogenesis by catalyzing the oxidation of lysine residues to reactive aldehydes (allysine) in collagens and elastin, resulting in the crosslinking and excessive deposition of these extracellular matrix components. Currently, rapid and robust histological assays to visualize the spatial distribution of LOX activity are lacking, hindering the precise validation of anti-fibrotic therapies. Here, we present a histological fluorescent staining method to visualize fibrogenesis (active fibrosis) and LOX activity in tissue sections utilizing a bioorthogonal tag and a click reaction with a turn-on fluorophore. Notably, requiring only two commercial reagents, this protocol can be completed in under two hours and is compatible with other imaging modalities, including second-harmonic generation and immunofluorescence staining. We validated this method across various healthy and fibrotic mouse and human tissue specimens.

A diet low in fermentable oligo-, di-, monosaccharides and polyols (FODMAP) (LFD) is the most efficacious dietary therapy for irritable bowel syndrome (IBS). However, the mechanisms by which FODMAPs drive IBS pathophysiology are unclear. Patients with Rome IV diarrhea-predominant IBS (IBS-D) underwent barrier function evaluation pre- and post-LFD along with assessment of mast cell number and activation profile. Finally, fecal supernatants (FS) were administered intracolonically to wildtype mice with/without pharmacological inhibition, tlr4-/- mice and mast cell-deficient mice with/without mast cell reconstitution. Of 42 patients, 34 responded to LFD and 8 did not. IBS-D patients had a significant improvement in colonic barrier structure and function, mast cell number, and levels of mast cell mediators post-LFD. The magnitude of physiological changes did not correlate with the magnitude of clinical response. Humanization of germ-free mice with pre-LFD feces caused barrier dysfunction while post-LFD feces did not. Similarly, pre-LFD FS caused barrier dysfunction in wildtype mice, whereas post-LFD FS did not. LPS removal and TLR4 antagonism reversed pre-LFD IBS FS-induced barrier dysfunction. Barrier dysfunction was absent in tlr4-/- mice treated with pre-LFD FS. Similarly, pre-LFD IBS FS did not cause barrier dysfunction in wildtype mice treated with mast cell stabilizer, or in mast cell-deficient mice. However, barrier dysfunction was inducible in mast cell-deficient mice upon reconstitution with wildtype mast cells but not tlr4-/- mast cells. IBS-D patients exhibited improvement in colonic barrier dysfunction, mast cell recruitment, and activation post-LFD. FODMAP-mediated barrier dysfunction in IBS-D is mediated by direct activation of the TLR4 receptor on colonic mast cells by fecal LPS. gov (NCT04542018).

Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are characterized by intestinal inflammation and barrier dysfunction. While disruption of the intestinal barrier contributes to the pathogenesis of IBD, yet how colonic inflammation alters small intestinal homeostasis remains poorly defined. Here, we demonstrate that three models of colitis, including 2,4,6-Trinitrobenzenesulfonic acid (TNBS), Dextran Sulfate Sodium (DSS), and oxazolone, induce paracellular barrier dysfunction in the small intestine, with model-specific immune profiles. Th1/Th17-skewed responses (TNBS and DSS) were associated with microbiota-dependent upregulation of myosin light chain kinase (MLCK), RORγt⁺ CD4⁺ T cell expansion, and elevated expression of Nod2, which was not observed under Th2-dominant (oxazolone) conditions. Inhibition of MLCK restored barrier function and suppressed inflammation in a CD4⁺ T cell-dependent manner. Using Nod2-deficient and 2939insC mutant mice, we showed Nod2 as a critical regulator of small intestinal permeability during colitis. Bone marrow chimeras revealed compartment-specific roles of Nod2, with non-hematopoietic Nod2 sufficient to preserve epithelial integrity, while hematopoietic Nod2 expression is required to limit cytokine-mediated inflammation. Moreover, MLCK inhibition ameliorated intestinal lesions only in mice with Nod2 deficiency in the hematopoietic compartment. Finally, microbiota transfer experiments ruled out a causal role for dysbiosis in driving small intestinal permeability defects in Nod2-deficient mice. These findings uncover a Nod2-MLCK-CD4⁺ T cell axis linking colonic inflammation to small intestinal barrier dysfunction and highlight distinct immune-epithelial-microbial mechanisms shaping intestinal homeostasis during colitis.

The intestinal barrier is formed by epithelial cells and tight junctions. Rather than being impermeant, paracellular channels allow passive transport across the epithelial monolayer. The tight junction proteins claudins 2 and 15 permit selective cation, i.e., Na + and water flux, while excluding larger molecules, e.g., bacterial products. Despite forming functionally similar channels, distinct expression patterns, with claudin -2 or -15 predominating in infants or adults, respectively, suggest that these proteins have unique functions. Consistent with this, claudin-2, but not claudin-15, expression is increased in human and experimental mouse disease. This transcriptional upregulation is beneficial in enteric infection, where claudin-2 enhances fluid efflux and accelerates pathogen clearance. Conversely, claudin-2 upregulation exacerbates experimental sepsis and inflammatory bowel disease. The mechanisms that lead to these divergent effects of claudin-2 expression are unknown. Hypothesis: We hypothesize that claudin-2-dependent Na + and water permeability activate proinflammatory signaling within the intestinal mucosa. Methods: Ileal and colonic immune cells from wildtype (WT), intestinal epithelial-specific EGFP- Cldn2 transgenic ( Cldn2 Tg ), and intestinal epithelial-specific knockout ( Cldn2 ΔIEC ) mice were analyzed. Non-stressed mice were compared to mice with colonic mucosal damage-induced stress (intrarectal TNBS), systemic immune stress (intraperitoneal LPS), or psychological stress (restraint). Results: Weights and behaviors of WT, Cldn2 Tg , and Cldn2 ΔIEC mice were similar. As reported previously, Cldn2 Tg mice have increased fecal Na + and water. However, Cldn2 Tg mice also displayed ~2-fold expansion of CD4 + T cells, Th17 T cells, CD8 + T cells, and neutrophils as well as a ~2-fold reduction in Tregs relative to WT mice. Conversely, Tregs were increased ~2-fold, but Th17 cell numbers were reduced by half in Cldn2 ΔIEC mice relative to WT mice. Therefore, claudin-2 expression increases the number of pro-inflammatory cells, with Cldn2 Tg > WT > Cldn2 ΔIEC , while decreasing Tregs. Relative differences in inflammatory and immunoregulatory cells across the three genotypes were maintained after exposure to each stressor, but absolute numbers of proinflammatory cells increased, and Tregs decreased. Specific stressor-induced changes included increased Th1 cell, ILC1, and ILC2 numbers in Cldn2 Tg mice and reduced ILC3 and NK cell numbers in Cldn2 ΔIEC mice relative to WT mice after damage. In contrast, systemic immune activation increased mucosal Th2 cell and eosinophil numbers in Cldn2 Tg mice but reduced ILC3 ~4-fold in Cldn2 ΔIEC mice relative to WT mice. Psychological stress increased Th2 (ileum), Th1 (colon), NK, and ILC2 cells but dramatically reduced Treg numbers in Cldn2 Tg mice relative to WT mice. Thus, claudin-2 expression primes the mucosa for proinflammatory stress responses, while claudin-2 deletion amplifies immunoregulation. Conclusions: Claudin-2 upregulation and the following immune activation may represent a primitive host-defense response. This is consistent with the beneficial effects of claudin-2 upregulation during infection. However, this host-defense response appears maladaptive in inflammatory disorders, where mucosal immune activation in the absence of a pathogen exacerbates disease. Although further study is needed to determine the mechanisms by which paracellular Na + and water permeability have such profound effects on mucosal immune status, these data highlight the therapeutic potential of claudin-2-modifying agents in a wide range of intestinal and systemic disorders. Funding: NIDDK R01DK061931, R01DK068271, and P30 DK034854. Disclosures: None. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

BACKGROUND & AIMS: A diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (LFD) is the most efficacious dietary therapy for irritable bowel syndrome (IBS). However, the mechanisms by which fermentable oligosaccharides, disaccharides, monosaccharides, and polyols drive IBS pathophysiology are unclear. METHODS: Patients with Rome IV diarrhea-predominant IBS (IBS-D) underwent barrier function evaluation preand post-LFD along with assessment of mast cell number and activation profile. Finally, fecal supernatants (FS) were administered intracolonically to wild-type mice with and without pharmacologic inhibition, toll-like receptor 4 (tlr4)-/- mice, and mast cell-deficient mice with/without mast cell reconstitution. RESULTS: Of 42 patients, 34 responded to the LFD and 8 did not. Patients with IBS-D had significant improvement in colonic barrier structure and function, mast cell number, and levels of mast cell mediators post-LFD. The magnitude of physiological changes did not correlate with the magnitude of clinical response. Humanization of germ-free mice with pre-LFD feces caused barrier dysfunction and post-LFD feces did not. Similarly, pre-LFD FS caused barrier dysfunction in wild-type mice, whereas post-LFD FS did not. Lipopolysaccharide removal and TLR4 antagonism reversed pre-LFD IBS FS-induced barrier dysfunction. Barrier dysfunction was absent in tlr4-/- mice treated with pre-LFD FS. Similarly, pre-LFD IBS FS did not cause barrier dysfunction in wild-type mice treated with mast cell stabilizer or in mast cell-deficient mice. However, barrier dysfunction was inducible in mast cell-deficient mice upon reconstitution with wild-type mast cells, but not tlr4-/- mast cells. CONCLUSIONS: Patients with IBS-D exhibited improvement in colonic barrier dysfunction, mast cell recruitment, and activation post LFD. Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols-mediated barrier dysfunction in IBS-D is mediated by direct activation of the TLR4 receptor on colonic mast cells by fecal lipopolysaccharide. Clinicaltrials.gov, Number: NCT04542018.

Intestinal epithelial barrier loss has been suggested as a pathogenic factor in Hirschsprung-associated enterocolitis and intestinal dysfunction in children with Hirschsprung disease (HD), but there is a paucity of comprehensive studies on the tight junction proteins that regulate paracellular permeability in this population. This case-control study aimed to determine if colonic epithelial tight junction protein expression is altered in children with HD. We use quantitative immunofluorescence microscopy to assess the expression of tight junction proteins (claudin-1, claudin-2, claudin-3, claudin-4, claudin-7, claudin-15, ZO-1, ZO-2, and occludin) in 29 children with HD who underwent surgical reconstruction and 16 controls who underwent transmural colorectal surgical resection for other etiologies between January 2015 and December 2021. We found that the expression of claudin-2, claudin-15, and occludin was reduced in both ganglionic and aganglionic colon specimens from children with HD compared with controls. Expression of other tight junction proteins did not differ between the groups. Together with previous studies, these data suggest that decreased expression of paracellular Na+ and water channel-forming claudin-2 and claudin-15 may limit luminal hydration, enhance fecal stasis, and promote dysbiosis. Conversely, occludin downregulation can not only increase paracellular macromolecular flux but also limit epithelial sensitivity to apoptotic stimuli. Together, these changes in expression of claudin-2 and claudin-15, as well as occludin, may promote intestinal dysfunction and contribute to Hirschsprung-associated enterocolitis pathogenesis following surgical reconstruction.

Epithelial homeostasis and the formation of selectively permeable tissue barriers depend on claudins. In the intestine, claudin-2 and claudin-15 both form paracellular water and cation, i.e., Na + , selective channels and are differentially development and in disease. The observation that knockout of both claudins results in perinatal death but that deletion of only one are viable demonstrates functional redundancy of claudin-2 and claudin-15. HYPOTHESIS: We hypothesized that differences in the molecular interactions underlying claudin-2 and claudin-15 polymerization may explain the distinct patterns of their expression in development and disease. METHODS: We used sequence alignments, structural predictions, and molecular dynamics simulations to compare claudins 2 and 15. Polymerization, i.e., strand formation, was imaged by super-resolution (STED) microscopy. Transepithelial electrical resistance was measured to assess channel conductivity. RESULTS: Sequence analysis showed that glutamine-81 (Q81) within the second transmembrane helix is unique: other claudins have arginine at the corresponding site, e.g., claudin-15 R79 . Evolutionary conservation of claudin-2 Q81 suggests that it may confer unique structural or functional properties. Consistent with this, molecular dynamics simulations showed that claudin-2 Q81 mutation altered the numbers and sites of potential homomeric interactions. Comprehensive screening of claudin-2 Q81 and claudin-15 R79 mutants confirmed the essential contributions of these residues to assembly and architecture of polymeric strand networks in nonepithelial cells, indicating distinct claudin-2 and claudin-15 polymerization interfaces. When expressed in epithelial cells, claudin-2 Q81 or claudin-15 R79 mutagenesis altered strand network complexity and paracellular channel function. Complementary mutations of non-conserved residues within the third transmembrane helix rescued claudin function, indicating that a novel intramembranous interaction modifies claudin helix bundling, polymerization, and function. CONCLUSION: These data define a previously unrecognized interface between the second and third transmembrane helices that stabilizes claudin structure to support polymerization and channel function. Moreover, the distinct impact of corresponding mutations on claudin-2 and claudin-15 structure and function suggest that the role and importance of this intramembranous binding interface differs within the claudin protein family. We speculate that this interface may modify strand dynamics that dictate unique functional or regulatory aspects of otherwise similar claudins. DFG Walter Benjamin Fellowship (531212047), HDDC Pilot and Feasibility Grant (2024A004855), NIH individual grants (R01DK061931, R01DK068271, R21GM144801, R01DK048106) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Epithelial tissue barriers are composed of claudin-based polymeric strands. In addition to creating barriers, some claudins assemble into charge-selective channels that accommodate small molecules up to ~5Å diameter, e.g., ions, but not larger molecules, to create the pore pathway. Although tight junction associated Marvel proteins (TAMPs; occludin, tricellulin, and MARVELD3) do not self-assemble into polymers, it is clear that they can regulate the leak pathway that allows macromolecules up to ~125Å diameter to cross tight junctions. In vivo studies have shown that mice with global knockout of either occludin or tricellulin are viable and have only subtle phenotypes. Similarly, only subtle changes are detected in epithelial cells (MDCK or Caco-2) lacking one TAMP. In contrast, marked delays in barrier development and increased leak pathway permeability are apparent when any two or all three TAMPs are eliminated. We sought to understand the impact of TAMPs on claudin meshwork architecture and function. HYPOTHESIS: We hypothesized that each TAMP would have distinct effects on claudin polymerization and tight junction strand architecture. METHODS: Claudin proteins were expressed individually or in combination with a TAMP. After fixation, claudins or TAMPs were immmunolabelled with STAR RED or STAR ORANGE, respectively. Claudin strand structures and localization of each protein within meshworks were visualized using super-resolution STED microscopy. RESULTS: Consistent with prior studies, meshworks formed by individual claudins differed when expressed in U2OS osteosarcoma cells. For example, claudin-1 was concentrated in punctae along strands within a dense meshwork. In contrast, claudin-3 formed continuous strands that defined a meshwork with relatively small spaces. When co-expressed with individual claudins, each TAMP had distinct effects on meshwork architectures. Tricellulin increased mesh density, i.e., reduced the area of enclosed spaces. Consistent with this, the density of nodes, i.e., sites at which two or more strands intersect, were more frequent in the presence of tricellulin and tended to form right angles. In contrast, occludin induced intracellular claudin aggregates, interfered with strand assembly, and elongated both internode distances and the spaces defined by the meshwork. CONCLUSION: These data show that TAMPs modify polymeric claudin meshwork architecture. Although TAMPs had distinct effects on meshwork architecture of individual claudins, common themes characterized the effect of each TAMP across multiple claudins. In molecular terms, these data are difficult to understand in the absence of defined sites of TAMP-claudin interactions. However, our previous work has shown that, at least for claudins 1 and 2, ZO-1 can mediate indirect interactions with occludin. Such scaffolding functions of ZO-1 or ZO-2 or interactions with other proteins may also underlie the distinct enrichment of tricellulin at tricellular tight junctions, versus occludin and MARVELD3 localization within both bicellular and tricellular tight junction regions, as well as the unique anchoring and exchange properties of each TAMP. Thus, future studies will focus on defining the potential contributions of ZO-1/ZO-2 and F-actin TAMP effects on claudin architecture. NIDDK grants R01DK061931, R01DK068271, R21GM144801, and P30DK034854 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Gut barrier dysfunction is a hallmark of inflammatory bowel disease (IBD), characterized by increased intestinal permeability, epithelial disruption, and chronic inflammation. Despite growing insights into the gut microbiota’s role, the mechanisms underlying probiotic-driven metabolic reprogramming of gut barrier function remain elusive. This study hypothesizes that Lacticaseibacillus rhamnosus GG (LGG), in synergy with dietary tryptophan, reprograms host metabolism to restore intestinal homeostasis and enhance barrier integrity. Using germ-free mice colonized with LGG and fed tryptophan-deficient or -sufficient diets, we employed untargeted metabolomics, transcriptomics, and a novel metabolome-transcriptome correlation analysis (METRCA). Functional validation was performed via in vitro assays, ex vivo organoids, and DSS-induced colitis mouse models. LGG robustly enhanced gut barrier function through two complementary mechanisms. First, LGG significantly upregulated argininosuccinate lyase (ASL), a critical enzyme in arginine biosynthesis, alleviating the accumulation of argininosuccinate (ASA)—a barrier-disrupting metabolite that perturbs tight junction proteins such as Ocln and Tjp1. These effects were evident in both experimental colitis and human IBD, where reduced ASL expression correlated with disease severity. Second, LGG stimulated the production of methylnicotinamide (MNA), a vitamin B 3 derivative that exhibited potent barrier-protective effects by enhancing epithelial tight junctions and promoting healing in DSS-induced colitis. METRCA analyses revealed strong correlations between LGG-regulated metabolites, including indole derivatives and MNA, and genes associated with arginine metabolism and epithelial barrier integrity. Notably, MNA reduced intestinal permeability and inflammatory cytokine production, positioning it as a key effector in LGG’s mechanism of action. These findings were further validated by in silico analyses of human IBD datasets, demonstrating that LGG and dietary tryptophan synergistically modulate arginine biosynthesis and reduce ASA levels, ultimately mitigating gut barrier dysfunction. This study highlights LGG’s dual capacity to enhance gut barrier function by leveraging dietary tryptophan to drive arginine metabolism and vitamin B3 synthesis. By targeting the metabolic disruptions underlying IBD, LGG emerges as a transformative precision probiotic. These exciting findings establish a framework for microbiome-based therapeutic strategies, emphasizing the potential of dietary and probiotic interventions to restore intestinal barrier integrity in IBD and related disorders. (NIH R01-AT010243, R01-DK102934, R01-DK119198 and NSF 1754783) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background & aims: Humans with WNT2B deficiency have severe intestinal disease, including significant inflammatory injury, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. Methods: We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the baseline histology and health of the small intestine and colon, and the impact of inflammatory challenge using dextran sodium sulfate (DSS). We also evaluated human intestinal tissue. Results: Mice with WNT2B deficiency had normal baseline histology but enhanced susceptibility to DSS colitis because of an increased early injury response. Although intestinal stem cells markers were decreased, epithelial proliferation was similar to control subjects. Wnt2b KO mice showed an enhanced inflammatory signature after DSS treatment. Wnt2b KO colon and human WNT2B-deficient organoids had increased levels of CXCR4 and IL6, and biopsy tissue from humans showed increased neutrophils. Conclusions: WNT2B is important for regulation of inflammation in the intestine. Absence of WNT2B leads to increased expression of inflammatory cytokines and increased susceptibility to gastrointestinal inflammation, particularly in the colon.

ABSTRACT:Sepsis induces intestinal hyperpermeability, which is associated with higher mortality. Occludin is a tight junction protein that plays a critical role in regulating disease-associated intestinal barrier loss. This study examined the role of intestinal occludin on gut barrier function and survival in a preclinical model of sepsis. Intestinal epithelial-specific occludin knockout (occludin KO IEC ) mice and wild type controls were subjected to intra-abdominal sepsis and sacrificed at predetermined endpoints for mechanistic studies or followed for survival. Occludin KO IEC mice had a significant increase in intestinal permeability, which was induced only in the setting of sepsis as knockout mice and control mice had similar baseline permeability. The worsened barrier was specific to the leak pathway of permeability, without changes in either the pore or unrestricted pathways. Increased sepsis-induced permeability was associated with increased levels of the tight junction ZO-1 in occludin KO IEC mice. Occludin KO IEC mice also had significant increases in systemic cytokines IL-6 and MCP-1 and increased bacteremia. Furthermore, occludin KO IEC mice had higher levels of jejunal IL-1β and MCP-1 as well as increased MCP-1 and IL-17A in the peritoneal fluid although peritoneal bacteria levels were unchanged. Notably, 7-day mortality was significantly higher in occludin KO IEC mice following sepsis. Occludin thus plays a critical role in preserving gut barrier function and mediating survival during sepsis, associated with alterations in inflammation and bacteremia. Agents that preserve occludin function may represent a new therapeutic strategy in the treatment of sepsis.

Macrophage-mediated inflammation has been implicated in the pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH); however, the immunometabolic program underlying the regulation of macrophage activation remains unclear. Beta-arrestin 2, a multifunctional adaptor protein, is highly expressed in bone marrow tissues and macrophages and is involved in metabolism disorders. Here, we observed that β-arrestin 2 expression was significantly increased in the liver macrophages and circulating monocytes of patients with MASH compared with healthy controls and positively correlated with the severity of metabolic dysfunction-associated steatotic liver disease (MASLD). Global or myeloid Arrb2 deficiency prevented the development of MASH in mice. Further study showed that β-arrestin 2 acted as an adaptor protein and promoted ubiquitination of immune responsive gene 1 (IRG1) to prevent increased itaconate production in macrophages, which resulted in enhanced succinate dehydrogenase activity, thereby promoting the release of mitochondrial reactive oxygen species and M1 polarization. Myeloid β-arrestin 2 depletion may be a potential approach for MASH.

Background & Aims: Lacticaseibacillus rhamnosus GG (LGG) is the world’s most consumed probiotic but its mechanism of action on intestinal permeability and differentiation along with its interactions with an essential source of signaling metabolites, dietary tryptophan (trp), are unclear. Methods: Untargeted metabolomic and transcriptomic analyses were performed in LGG monocolonized germ-free mice fed trp-free or -sufficient diets. LGG-derived metabolites were profiled in vitro under anaerobic and aerobic conditions. Multiomic correlations using a newly developed algorithm discovered novel metabolites tightly linked to tight junction and cell differentiation genes whose abundances were regulated by LGG and dietary trp. Barrier-modulation by these metabolites were functionally tested in Caco2 cells, mouse enteroids, and dextran sulfate sodium experimental colitis. The contribution of these metabolites to barrier protection is delineated at specific tight junction proteins and enterocyte-promoting factors with gain and loss of function approaches. Results: LGG, strictly with dietary trp, promotes the enterocyte program and expression of tight junction genes, particularly Ocln. Functional evaluations of fecal and serum metabolites synergistically stimulated by LGG and trp revealed a novel vitamin B3 metabolism pathway, with methylnicotinamide (MNA) unexpectedly being the most robust barrier-protective metabolite in vitro and in vivo. Reduced serum MNA is significantly associated with increased disease activity in patients with inflammatory bowel disease. Exogenous MNA enhances gut barrier in homeostasis and robustly promotes colonic healing in dextran sulfate sodium colitis. MNA is sufficient to promote intestinal epithelial Ocln and RNF43, a master inhibitor of Wnt. Blocking trp or vitamin B3 absorption abolishes barrier recovery in vivo. Conclusions: Our study uncovers a novel LGG-regulated dietary trp-dependent production of MNA that protects the gut barrier against colitis.

Intestinal epithelial expression of the tight junction protein claudin- 2, which forms paracellular cation and water channels, is precisely regulated during development and in disease. Here, we show that small intestinal epithelial claudin- 2 expression is selectively upregulated in septic patients. Similar changes occurred in septic mice, where claudin- 2 upregulation coincided with increased flux across the paracellular pore pathway. In order to define the significance of these changes, sepsis was induced in claudin- 2 knockout (KO) and wild- type (WT) mice. Sepsis- induced increases in pore pathway permeability were prevented by claudin- 2 KO. Moreover, claudin- 2 deletion reduced interleukin- 17 production and T cell activation and limited intestinal damage. These effects were associated with reduced numbers of neutrophils, macrophages, dendritic cells, and bacteria within the peritoneal fluid of septic claudin- 2 KO mice. Most strikingly, claudin- 2 deletion dramatically enhanced survival in sepsis. Finally, the microbial changes induced by sepsis were less pathogenic in claudin- 2 KO mice as survival of healthy WT mice injected with cecal slurry collected from WT mice 24 h after sepsis was far worse than that of healthy WT mice injected with cecal slurry collected from claudin- 2 KO mice 24 h after sepsis. Claudin- 2 upregulation and increased pore pathway permeability are, therefore, key intermediates that contribute to development of dysbiosis, intestinal damage, inflammation, ineffective pathogen control, and increased mortality in sepsis. The striking impact of claudin- 2 deletion on progression of the lethal cascade activated during sepsis suggests that claudin- 2 may be an attractive therapeutic target in septic patients.

Intestinal epithelia express two long myosin light chain kinase (MLCK) splice variants, MLCK1 and MLCK2 that differ by the absence of a complete immunoglobulin-like (Ig) domain 3 within MLCK2. Only MLCK1 is associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn’s disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key MLCK1 domains to prevent recruitment, restore barrier function, and limit inflammatory bowel disease progression.