Murine Colitis Model Research Articles

Anthocyanin Extracted from Purple Sweet Potato Alleviates Dextran Sulfate Sodium-Induced Colitis in Mice by Suppressing Pyroptosis and Altering Intestinal Flora Structure.

The objective of this study was to examine the impact and underlying mechanisms of pelargonidin-3-galactoside (Pg3gal) produced from purple sweet potatoes on colonic inflammation induced by dextran sulfate sodium (DSS) in a murine model of ulcerative colitis (UC). C57BL/6J mice were categorized into four groups (n = 6 per group): DSS+Pg3gal, control, control+Pg3gal, and DSS. Colitis was induced by providing free access to 3% DSS for 10 days. The DSS+Pg3gal model mice received DSS concurrently with intragastric Pg3gal (25 mg/kg). The health of the mice was carefully monitored on a regular basis, and scores for the Disease Activity Index (DAI) were documented. A histological assessment was conducted using hematoxylin and eosin staining to evaluate the extent of mucosal injury present. The expression levels of IL-6, NLRP3, ASC, cleaved-Caspase-1, TNF-α, N-GSDMS, and cleaved-IL-1β proteins were evaluated by Western blot analysis. The process of 16S rRNA sequencing was carried out to examine the composition and relative abundance of gut microbiotas within the intestines of the mice. The DAI results revealed that Pg3gal significantly attenuated the DSS-induced UC in mice. In addition, it successfully alleviated the decline in colon size, improved the condition of colonic tissue, and significantly inhibited the production of proinflammatory cytokines, such as IL-6, IL-1β, and TNF-α, in the colon tissues. Additionally, Pg3gal modulated the DSS-induced imbalanced gut microbiota, as evidenced by decreased Proteobacteria and Deferribacteres and simultaneous elevation in Firmicutes, Bacteroidetes, and Verrucomicrobia. In summary, Pg3gal alleviated DSS-induced UC by inhibiting pyroptosis in intestinal epithelial cells and enhancing the structural integrity of the gut microbiota.

Comparison of a selective STAT3 inhibitor with a dual STAT3/STAT1 inhibitor using a dextran sulfate sodium murine colitis model: new insight into the debate on selectivity.

Recent advances in the treatment of inflammatory bowel disease include antitumor necrosis factor antibodies and the Janus kinase inhibitor tofacitinib, approved for ulcerative colitis. Janus kinase recruits signal transducers and activators of transcriptions (STAT), which are promising targets in inflammatory bowel diseases. However few inhibitors have been evaluated, and their selectivity with respect to STAT1 and STAT3 remains controversial. Here, we investigated the therapeutic potential of a selective inhibitor vs. a non-selective, closely related compound, in a dextran sulfate sodium (DSS) murine colitis model. Thirty Swiss/CD-1 male mice were used in this study. They were divided into a healthy control group, a colitis-DSS control group, a compound (cpd) 23-treated group, a cpd 46-treated group and an icariin-treated group. For the coadministration experiment with rutin, the cpd 46-treated group and the icariin-treated group were replaced by the oral rutin-treated group and the coadministration rutin/cpd 23-treated group. The effect of the tested inhibitors was also assessed by quantification of proinflammatory markers. The selective inhibitor had a significantly greater effect than the dual inhibitor on the disease activity index. We also noticed in curative treatment a significant decrease in the most abundant proinflammatory biomarker present in neutrophilic granulocytes, myeloperoxidase and on proinflammatory cytokines, including tumor necrosis factor-α, interferon-γ, interleukins -6 and -23, with a mild synergy with rutin, the glycoside of quercetin. The current study shows how STAT3 selective inhibitors can exert a significant therapeutic effect in the treatment of experimental DSS-colitis.

Active nanoparticle targeting of MUC5AC ameliorates therapeutic outcome in experimental colitis.

Inflammatory bowel diseases (IBDs), which include Crohn's disease (CD) and ulcerative colitis (UC), are chronic inflammatory diseases of the gastrointestinal tract and are characterized by chronic recurrent ulceration of the bowels. Colon-targeted drug delivery systems (DDS) have received significant attention for their potential to treat IBD by improving the inflamed tissue selectivity. Herein, antiMUC5AC-decorated drug loaded nanoparticles (NP) are suggested for active epithelial targeting and selective adhesion to the inflamed tissue in experimental colitis. NPs conjugated with antiMUC5AC (anti-MUC5) were tested for their degree of bioadhesion with HT29-MTX cells by comparison with non-targeted BSA-NP conjugates. In vivo, the selectivity of bioadhesion and the influence of ligand density in bioadhesion efficiency as well as the therapeutic benefit for glucocorticoid loaded anti-MUC5-NP were studied in a murine colitis model. Quantitative adhesion analyses showed that anti-MUC5-conjugated NP exhibited a much higher binding and selectivity to inflamed tissue compared to PNA-, IgG1- and BSA-NP conjugates used as controls. This bioadhesion efficiency was found to be dependent on the ligand density, present at the NP surface. The binding specificity between anti-MUC5 ligand and inflamed tissues was confirmed by fluorescence imaging. Both anti-MUC5-NP and all other glucocorticoid containing formulations led to a significant mitigation of the experimental colitis, as became evident from the substantial reduction of myeloperoxidase activity and pro-inflammatory cytokine concentrations (TNF-α, IL-1β). Targeted NP by using anti-MUC5 appears to be a very promising tool in future treatment of various types of local disorders affecting the gastro-intestinal tract but not limited to colitis.

Wuwei Kushen Changrong capsule alleviates DSS-induced colitis in mice via inhibition of NLRP3 inflammasome and STAT3 pathway.

Wuwei Kushen Changrong capsule (Composite Sophora Colon-soluble Capsule, CSCC) is a Chinese patent medicine developed to treat ulcerative colitis. Studies highlight CSCC potential efficacy for ulcerative colitis (UC) but unclear mechanism limits its widely treatment for patients. We aimed to investigate the anti-colitis efficacy of CSCC and explore the mechanism by which GPR43 inhibits the NLRP3/STAT3 signaling pathway, thereby mediating the protective effects of CSCC on the intestinal barrier. The protective effects of CSCC were evaluated in a murine ulcerative colitis model induced by 3% DSS. Assessments included body weight, Disease Activity Index (DAI) score, colon length, and histopathological score. Colon tissue, cell function, and immune-inflammatory status were evaluated using immunohistochemistry, immunofluorescence, ELISA, and real-time fluorescence quantitative PCR (RT-PCR). Protein expression levels of relevant pathways and receptors were measured using Western blot. All experiments were repeated. CSCC protected mice from DSS-induced colitis by upregulating Gpr43, promoting the expression of ZO-1 and Occludin tight junction proteins. Mechanistically, CSCC inhibits the MEK4/JNK1/STAT3 activation pathway, consequently suppressing the STAT3/NLRP3/IL-1β pathway and inhibiting the production of inflammatory factors such as IL-17A. The mechanisms through which CSCC protects against DSS-induced colitis may include upregulating Gpr43, inhibiting the STAT3/NLRP3 pathway, and suppressing inflammation factors like IL-17A. These findings highlight the mechanisms underlying CSCC's anti-colitis effects and suggest its potential as a therapeutic candidate for managing the progression of UC.

Kumujan B suppresses TNF-α-induced inflammatory response and alleviates experimental colitis in mice.

Treatments of inflammatory bowel disease (IBD) are diverse, but their efficacy is limited, and it is therefore urgent to find better therapies. Controlling mucosal inflammation is a must in IBD drug treatment. The occurrence of anti-tumor necrosis factor α (TNF-α) monoclonal antibodies has provided a safer and more efficacious therapy. However, this kind of treatment still faces failure in the form of loss of response. β-Carboline alkaloids own an anti-inflammatory pharmacological activity. While Kumujan B contains β-carboline, its biological activity remains unknown. In this study, we attempted to determine the anti-inflammatory effects of Kumujan B using both the TNF-α- induced in vitro inflammation and DSS-induced in vivo murine IBD models. Our data show that Kumujan B attenuated the expression of interleukin 1β (IL-1β) and interleukin 6 (IL-6) induced by TNF-α in mouse peritoneal macrophages. Kumujan B suppressed c-Jun N-terminal protein kinases (JNK) signaling, especially c-Jun, for anti-inflammatory response. Furthermore, Kumujan B promoted K11-linked ubiquitination and degradation of c-Jun through the proteasome pathway. In an in vivo study, Kumujan B inhibited the expression of IL-1β, IL-6, and TNF-α and improved the colon barrier function in dextran sulfate sodium salt (DSS)-induced experimental mice colitis. Kumujan B exhibited in vivo and in vitro anti-inflammatory effects, making it a potential therapeutic candidate for treating IBD.

Identification of highly selective SIK1/2 inhibitors that modulate innate immune activation and suppress intestinal inflammation.

The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.

Anion‐Dependent Layered Double Hydroxide Nanoparticles Regulate Differentiation of CD206+ CX3CR1+ Macrophages by Inhibiting the IL‐17 Signaling Pathway Contributing to Inflammatory Bowel Disease

AbstractInflammatory bowel disease (IBD) is a chronic and destructive autoimmune disease that has created a global burden. However, the pathogenesis and treatment strategies of IBD remain difficult problems to overcome. The anti‐inflammatory action of anion‐dependent layered double hydroxide (LDH) is evaluated in IBD. Raw264.7 macrophages induced by Lipopolysaccharide (LPS) produced low levels of pro‐inflammatory cytokines after LDH treatment. The results from dextran sulfate sodium (DSS)‐induced murine colitis models show that LDHs significantly reduce pro‐inflammatory cytokines in the colon tissue and inhibit colon atrophy. Most importantly, LDH with NO3− as the interlayer anion (LDH‐NO3−) demonstrates superior anti‐inflammatory ability compared to LDH with Cl− as the interlayer anion (LDH‐Cl−), both in vitro and in vivo. LDH‐NO3− promotes the differentiation of CD206+CX3CR1+ lamina propria macrophages, reduces the abundance of T helper 17 (Th17) cells, and inhibits the activation of the IL‐17 signaling pathway. LDH‐NO3− also limits the pro‐inflammatory effects of IL‐17A on macrophages, and the anti‐inflammatory effects of LDH‐NO3− are reversed by IL‐17RA‐siRNA. Suggesting that LDH effectively inhibits the inflammatory reaction induced by the interaction between macrophages and Th17 cells. This study demonstrates that LDH‐NO3− is a drug‐free nanomedicine that acts against IBD, providing application prospects for LDH‐NO3− in IBD treatment.

Structural characterization of a polysaccharide from Alhagi honey and its protective effect against inflammatory bowel disease by modulating gut microbiota dysbiosis

The Alhagi honey polysaccharide (AHP) exhibits notable anti-inflammatory, antioxidant, and immunomodulatory properties, positioning it as a promising candidate in traditional Chinese medicine. In this investigation, we successfully isolated and purified a neutral AHP, designated AHPN50-1a, subsequently elucidating its structural attributes. AHPN50-1a was found to have a molecular weight of 1.756 × 106 Da, featuring a structural motif characterized by a recurring (1→6)-α-GlcP linker. To comprehensively evaluate its therapeutic potential, we explored the protective effects of AHPN50-1 in a murine model of dextran sodium sulfate-induced colitis. Administration of AHPN50-1 at doses of 200 and 400 mg/kg/day resulted in improved food intake, increased body weight, and increased colon length in mice with acute colitis. Simultaneously, a reduction in the disease activity index and histological scores was observed. AHPN50-1 effectively mitigated colon tissue damage, down-regulated the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in colon tissue, restored intestinal microbiota diversity, and concentrations of short-chain fatty acids (SCFAs) of gut microbiota metabolites, thus alleviating intestinal inflammation in mice. In summary, our findings underscore the promise of AHPN50-1 as a valuable nutritional or dietary supplement for the treatment and prevention of inflammatory bowel disease.

ELMO1 ameliorates intestinal epithelial cellular senescence via SIRT1/p65 signaling in inflammatory bowel disease-related fibrosis.

Intestinal fibrosis is a common complication in inflammatory bowel disease (IBD), which still lacks of reliable markers and therapeutic options. Cellular senescence has been considered an important mechanism of intestinal fibrosis, but the underlying molecular link remains elusive. Tissues were stained using α-smooth muscle actin (α-SMA), fibronectin, and collagen I as markers of myofibroblastic differentiation. Cellular senescence was confirmed through Lamin B1 staining, senescence-associated β-galactosidase staining, and the expression of senescence-associated secretory phenotype (SASP) factors. We explored the relationship between senescence of intestinal epithelial cells (IECs) and intestinal fibrosis, as well as the molecular mechanism underlying this interaction. The effects of irisin on cellular senescence and fibrosis were determined. Here, we identify engulfment and cell motility protein 1 (ELMO1) as a novel biomarker for intestinal cellular senescence and fibrosis. In fibrostrictured tissues from patients and murine models with IBD, significantly high levels of cellular senescence score and factors were noted, which positively correlated with the fibrotic regulator fibronectin. Senescent IECs, not fibroblast itself, released SASP factors to regulate fibroblast activation. Prolonging exposure to severe and persistent injurious stimuli decreased ELMO1 expression, which dampened SIRT1 deacetylase activity, enhanced NF-κB (p65) acetylation, and thereby accelerated cellular senescence. Deletion of ELMO1 led to senescent IECs accumulation and triggered premature fibrosis in murine colitis. Furthermore, irisin, inhibiting the degradation of ELMO1, could downregulate p65 acetylation, reduce IECs senescence, and prevent incipient intestinal fibrosis in murine colitis models. This study reveals ELMO1 downregulation is an early symbol of intestinal senescence and fibrosis, and the altered ELMO1-SIRT1-p65 pathway plays an important role in intestinal cellular senescence and IBD-related fibrosis.

Depletion of muscularis macrophages ameliorates inflammation-driven dysmotility in murine colitis model

Previously, the presence of a blood-myenteric plexus barrier and its disruption was reported in experimentally induced colitis via a macrophage-dependent process. The aim of this study is to reveal how myenteric barrier disruption and subsequent neuronal injury affects gut motility in vivo in a murine colitis model. We induced colitis with dextran sulfate sodium (DSS), with the co-administration of liposome-encapsulated clodronate (l-clodronate) to simultaneously deplete blood monocytes contributing to macrophage infiltration in the inflamed muscularis of experimental mice. DSS-treated animals receiving concurrent l-clodronate injection showed significantly decreased blood monocyte numbers and colon muscularis macrophage (MM) density compared to DSS-treated control (DSS-vehicle). DSS-clodronate-treated mice exhibited significantly slower whole gut transit time than DSS-vehicle-treated animals and comparable to that of controls. Experiments with oral gavage-fed Evans-blue dye showed similar whole gut transit times in DSS-clodronate-treated mice as in control animals. Furthermore, qPCR-analysis and immunofluorescence on colon muscularis samples revealed that factors associated with neuroinflammation and neurodegeneration, including Bax1, Hdac4, IL-18, Casp8 and Hif1a are overexpressed after DSS-treatment, but not in the case of concurrent l-clodronate administration. Our findings highlight that MM-infiltration in the muscularis layer is responsible for colitis-associated dysmotility and enteric neuronal dysfunction along with the release of mediators associated with neurodegeneration in a murine experimental model.

Ligilactobacillus salivarius CNCM I-4866, a potential probiotic candidate, shows anti-inflammatory properties in vitro and in vivo

IntroductionThe aim of this work was to characterize a new strain of Ligilactobacillus salivarius (CNCM I-4866) (CNCM I-4866) to address its potential as probiotic with a special focus on intestinal inflammation. Potential anti-inflammatory abilities of this strain were evaluated through in vivo and in vitro experiments.MethodsFirstly, the strain was tested in a murine acute inflammation colitis model induced by DNBS. In vitro characterization was then performed with diverse tests: modulation capability of intestinal permeability; study of the impact on immunity profile through cytokines dosage; capacity to inhibit pathogens and adhere to intestinal cells lines. Production of metabolites, antibiotic resistance and survival to gastro-intestinal tract conditions were also tested.ResultsIn vitro assay has shown a reduction of colonic damage and markers of inflammation after treatment with CNCM I-4866. Transcriptomic analysis performed on colons showed the capacity of the strain to down-regulate pro-inflammatory cytokines. L. salivarius CNCM I-4866 exerted anti-inflammatory profile by reducing IL-8 production by TNF-α stimulated cell and modulated cytokines profile on peripheral blood mononuclear cells (PBMC). It protected intestinal integrity by increasing trans-epithelial electrical resistance (TEER) on Caco-2 TNF-α inflamed cells. Additionally, L. salivarius CNCM I-4866 displayed inhibition capacity on several intestinal pathogens and adhered to eukaryotic cells. Regarding safety and technical concerns, CNCM I-4866 was highly resistant to 0.3% of bile salts and produced mainly L-lactate. Finally, strain genomic characterization allowed us to confirm safety aspect of our strain, with no antibiotic gene resistance found.DiscussionTaken together, these results indicate that L. salivarius CNCM I-4866 could be a good probiotic candidate for intestinal inflammation, especially with its steady anti-inflammatory profile.

Enhancement of efferocytosis through biased FPR2 signaling attenuates intestinal inflammation

Efficient clearance of dying cells (efferocytosis) is an evolutionarily conserved process for tissue homeostasis. Genetic enhancement of efferocytosis exhibits therapeutic potential for inflammation resolution and tissue repair. However, pharmacological approaches to enhance efferocytosis remain sparse due to a lack of targets for modulation. Here, we report the identification of columbamine (COL) which enhances macrophage‐mediated efferocytosis and attenuates intestinal inflammation in a murine colitis model. COL enhances efferocytosis by promoting LC3‐associated phagocytosis (LAP), a non‐canonical form of autophagy. Transcriptome analysis and pharmacological characterization revealed that COL is a biased agonist that occupies a part of the ligand binding pocket of formyl peptide receptor 2 (FPR2), a G‐protein coupled receptor involved in inflammation regulation. Genetic ablation of the Fpr2 gene or treatment with an FPR2 antagonist abolishes COL‐induced efferocytosis, anti‐colitis activity and LAP. Taken together, our study identifies FPR2 as a potential target for modulating LC3‐associated efferocytosis to alleviate intestinal inflammation and highlights the therapeutic value of COL, a natural and biased agonist of FPR2, in the treatment of inflammatory bowel disease.

Acid-base transformative HADLA micelles alleviate colitis by restoring adaptive immunity and gut microbiome

Inflammatory bowel disease (IBD) is a worldwide public health issue with an increasing number of patients annually. However, there is no curative drug for IBD, and the present medication for IBD generally focuses on suppressing hyperactive immune responses, which can only delay disease progression but inevitably induce off-target side effects, including infections and cancers. Herein, late-model orally administered nanotherapeutic micelles (HADLA) were developed based on a conjugate of hyaluronic acid (HA) and dehydrolithocholic acid (DLA), which was simple to achieve and obtained satisfactory therapeutic efficacy in a murine colitis model with a full safety profile. HADLA is capable of targeting inflammatory colon tissues, restoring intestinal barrier function and reducing intestinal epithelial cell death. Moreover, it modulates the adaptive immune system by inhibiting the activation of pathogenic T helper 17 (Th17) cells, and it exhibits more remarkable effects in preventing colitis than DLA alone. Finally, HADLA exhibits a remarkable ability to modulate dysregulated gut microbiomes by increasing beneficial probiotics and decreasing pathogenic bacteria, such as Turicibacter. Compared with the current systemic or subcutaneous administration of biologics, this study opens new avenues in the oral delivery of immune-modulating nanomedicine and introduces DLA as a new medication for IBD treatment.

Orange juice containing Pediococcus acidilactici CE51 modulates the intestinal microbiota and reduces induced inflammation in a murine model of colitis

The management of inflammatory bowel diseases has been widely investigated, especially ulcerative colitis. Thus, studies with the application of new probiotic products are needed in the prevention/treatment of these clinical conditions. The objective of this work was to evaluate the effects of probiotic orange juice containing Pediococcus acidilactici CE51 in a murine model of colitis. 45 male Swiss lineage mice were used, divided into five groups (n = 9): control, colitis, colitis + probiotic (probiotic orange juice containing CE51), colitis + placebo (orange juice) and colitis + sulfasalazine (10 mg/kg/Weight). The induction of colitis was performed with dextran sodium sulfate (3%). The treatment time was 5 and 15 days after induction. Histopathological analysis, serum measurements of TNF-α and C-reactive protein and metagenomic analysis of feces were performed after euthanasia. Probiotic treatment reduced inflammation in the small intestine, large intestine and spleen. The probiotic did not alter the serum dosages of TNF-α and C-reactive protein. Their use maintained the quantitative ratio of the phylum Firmicutes/Bacteroidetes and increased Lactobacillus helveticus with 15 days of treatment (p < 0.05). The probiotic orange juice containing P. acidilactici CE51 positively modulated the gut microbiota composition and attenuated the inflammation induced in colitis.

Neutrophil extracellular trap induction through peptidylarginine deiminase 4 activity is involved in 2,4,6-trinitrobenzenesulfonic acid-induced colitis.

Neutrophil extracellular traps (NETs) are induced in the innate immune response against infectious agents and are also implicated in the pathogenesis of various cancers and autoimmune diseases. Peptidylarginine deiminase 4 (PAD4), an enzyme that converts arginine to citrulline, is also involved in NET formation. In this study, we investigated the pathogenic effect of PAD4 on NETs in inflammatory bowel disease using a trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. PAD4-deficient (PAD4KO) mice were generated by CRISPR-Cas9-mediated genomic editing. NETs were triggered in peritoneal neutrophils obtained from wild-type mice by A23187 (a calcium ionophore), but these responses were completely abolished in the PAD4KO mice. Experimental colitis was induced in wild-type and PAD4KO mice via an intrarectal injection of TNBS. TNBS injection resulted in body weight loss, extensive colonic erosion, and ulceration in wildtype mice. However, these responses were significantly attenuated following the administration of Cl-amidine (an inhibitor of pan-PADs) and DNase I (an inhibitor of NET formation), in combination with PAD4KO in mice. TNBS-induced increases in myeloperoxidase activity, inflammatory cytokine expression, and NET formation in the colon were significantly reduced following the administration of Cl-amidine, DNase I injection, and PAD4KO. These findings suggest that NET formation contributes to the pathogenesis of TNBS-induced colitis via PAD4. Thus, PAD4 is a promising target for the treatment of inflammatory bowel disease.

GPR4 Knockout Attenuates Intestinal Inflammation and Forestalls the Development of Colitis-Associated Colorectal Cancer in Murine Models.

GPR4 is a proton-sensing G protein-coupled receptor highly expressed in vascular endothelial cells and has been shown to potentiate intestinal inflammation in murine colitis models. Herein, we evaluated the proinflammatory role of GPR4 in the development of colitis-associated colorectal cancer (CAC) using the dextran sulfate sodium (DSS) and azoxymethane (AOM) mouse models in wild-type and GPR4 knockout mice. We found that GPR4 contributed to chronic intestinal inflammation and heightened DSS/AOM-induced intestinal tumor burden. Tumor blood vessel density was markedly reduced in mice deficient in GPR4, which correlated with increased tumor necrosis and reduced tumor cell proliferation. These data demonstrate that GPR4 ablation alleviates intestinal inflammation and reduces tumor angiogenesis, development, and progression in the AOM/DSS mouse model.

OGR1 (GPR68) and TDAG8 (GPR65) Have Antagonistic Effects in Models of Colonic Inflammation.

G-protein-coupled receptors (GPRs), including pro-inflammatory ovarian cancer GPR1 (OGR1/GPR68) and anti-inflammatory T cell death-associated gene 8 (TDAG8/GPR65), are involved in pH sensing and linked to inflammatory bowel disease (IBD). OGR1 and TDAG8 show opposite effects. To determine which effect is predominant or physiologically more relevant, we deleted both receptors in models of intestinal inflammation. Combined Ogr1 and Tdag8 deficiency was assessed in spontaneous and acute murine colitis models. Disease severity was assessed using clinical scores. Colon samples were analyzed using quantitative polymerase chain reaction (qPCR) and flow cytometry (FACS). In acute colitis, Ogr1-deficient mice showed significantly decreased clinical scores compared with wildtype (WT) mice, while Tdag8-deficient mice and double knockout (KO) mice presented similar scores to WT. In Il-10-spontaneous colitis, Ogr1-deficient mice presented significantly decreased, and Tdag8-deficient mice had increased inflammation. In the Il10-/- × Ogr1-/- × Tdag8-/- triple KO mice, inflammation was significantly decreased compared with Tdag8-/-. Absence of Ogr1 reduced pro-inflammatory cytokines in Tdag8-deficient mice. Tdag8-/- had significantly more IFNγ+ T-lymphocytes and IL-23 T-helper cells in the colon compared with WT. The absence of OGR1 significantly alleviates the intestinal damage mediated by the lack of functional TDAG8. Both OGR1 and TDAG8 represent potential new targets for therapeutic intervention.

Genetically Engineered Membrane‐Coated Nanoparticles as Versatile Platforms with Reduced Protein Corona for Targeted siRNA Delivery

AbstractLow uptake efficiency in vivo as well as systemic toxicities of nucleic‐acid nanovehicles substantially retard the clinical translation of gene therapy. Targeted gene delivery to specific cell populations with antibody display techniques and membrane‐coated nanoparticle (NP) approaches may solve these problems. Here, a new class of targeted membrane‐camouflaged nanosystem is successfully constructed, which is made of polydopamine (PDA) nanoparticles coated with biosynthetic antibody‐displaying membranes from stem cells. In murine models of rheumatoid arthritis and colitis, the membrane‐camouflaged nanocarriers displayed anti‐CD64 antibodies in a ligand‐oriented way via the biosynthetic method and has reduced protein corona due to the coating of the negatively charged cell membrane, thereby achieving remarkable therapeutic efficacy through silencing the TNF expression selectively in CD64‐positive immune cell subsets. By expressing a wide variety of functional protein ligands on the cellular membranes that are further coated onto PDA nanoparticles, the membrane‐camouflaged nanosystems can also serve as a versatile theranostic platform that enables antibody‐targeted gene/siRNA delivery to the cells of interest in vivo, especially immune cells.

Intestinal epithelial pH-sensing receptor GPR65 maintains mucosal homeostasis via regulating antimicrobial defense and restrains gut inflammation in inflammatory bowel disease

ABSTRACT Intestinal epithelial cell (IEC) regulation of barrier function and mucosal homeostasis enables the establishment of a harmonious gut microenvironment. However, host-derived regulatory networks that modulate intestinal antimicrobial defenses have not been fully defined. Herein we generated mice with IEC-specific deletion of Gpr65 (Gpr65 ΔIEC) and investigated the role of epithelial GPR65 using DSS- and C. rodentium-induced murine colitis models. RNA sequencing analysis was conducted on colonic IECs from Gpr65 fl/fl and Gpr65 ΔIEC mice, and colonoids and colonic epithelial cell lines were used to evaluate the pH-sensing effect of GPR65. The expression of GPR65 was determined in IECs from patients with inflammatory bowel disease (IBD) and DSS colitis mice by qRT-PCR, Western blot, and immunohistochemistry, respectively. We observed that the absence of GPR65 in IECs abrogated homeostatic antimicrobial programs, including the production of antimicrobial peptides (AMPs) and defense response-associated proteins. Gpr65 ΔIEC mice displayed dysbiosis of the gut microbiota and were prone to DSS- and C. rodentium-induced colitis, as characterized by significantly disrupted epithelial antimicrobial responses, pathogen invasion, and increased inflammatory infiltrates in the inflamed colon. RNA sequencing analysis revealed that deletion of GPR65 in IECs provoked dramatic transcriptome changes with respect to the downregulation of immune and defense responses to bacteria. Forced AMP induction assays conducted in vivo or in ex vivo colonoids revealed that IEC-intrinsic GPR65 signaling drove antimicrobial defense. Mechanistically, GPR65 signaling promoted STAT3 phosphorylation to optimize mucosal defense responses. Epithelial cell line and colonoid assays further confirmed that epithelial GPR65 sensing pH synergized with IL-22 to facilitate antimicrobial responses. Finally, the expression of GPR65 was markedly decreased in the inflamed epithelia of IBD patients and DSS colitis mice. Our findings define an important role of epithelial GPR65 in regulating intestinal homeostasis and mucosal inflammation and point toward a potential therapeutic approach by targeting GPR65 in the treatment of IBD.

Orally-Delivered, Cytokine-Engineered Extracellular Vesicles for Targeted Treatment of Inflammatory Bowel Disease.

The use of orally-administered therapeutic proteins for treatment of inflammatory bowel disease (IBD) has been limited due to the harsh gastrointestinal environment and low bioavailability that affects delivery to diseased sites. Here, a nested delivery system, termed Gal-IL10-EVs (C/A) that protects interleukin 10 (IL-10) from degradation in the stomach and enables targeted delivery of IL-10 to inflammatory macrophages infiltrating the colonic lamina propria, is reported. Extracellular vesicles (EVs) carrying IL-10 are designed to be secreted from genetically engineered mammalian cells by a plasmid system, and EVs are subsequently modified with galactose, endowing the targeted IL-10 delivery to inflammatory macrophages. Chitosan/alginate (C/A) hydrogel coating on Gal-IL10-EVs enables protection from harsh conditions in the gastrointestinal tract and favorable delivery to the colonic lumen, where the C/A hydrogel coating is removed at the diseased sites. Gal-IL10-EVs control the production of reactive oxygen species (ROS) and inhibit the expression of proinflammatory cytokines. In a murine model of colitis, Gal-IL10-EVs (C/A) alleviate IBD symptoms including inflammatory responses and disrupt colonic barriers. Taken together, Gal-IL10-EVs (C/A) features biocompatibility, pH-responsive drug release, and macrophage-targeting as a therapeutic platform for oral delivery of bioactive proteins for treating intestinal diseases.