Gasdermin D (GSDMD)-mediated interleukin (IL)-33 secretion by lung epithelial cells initiates airway inflammation upon allergen challenge. How environmental allergens activate GSDMD remains elusive. Here, we demonstrate that exposing epithelial cells to allergens triggers protease-activated receptor 1 (PAR1)-dependent ferritinophagy, elevating intracellular labile iron. This iron pool is essential for noncanonical, protease-independent GSDMD activation. The iron chaperone poly(rC)-binding protein 2 (PCBP2) delivers iron directly to GSDMD, initiating a highly localized Fenton reaction. This generates constrained hydroxyl radicals that cleave GSDMD, releasing the active N-terminal p40 fragment to form pores for IL-33 release. Blocking any step of this iron-GSDMD pathway, via iron chelation or genetic ablation, abolishes IL-33 secretion, prevents group 2 innate lymphoid cell (ILC2) activation, and mitigates allergic airway inflammation and tissue damage in mice. Our findings reveal an unconventional, iron-catalyzed, and protease-independent mechanism for GSDMD activation, offering potential new therapeutic targets for allergic inflammatory diseases.

Abstract Anemia is one of the most debilitating and frequent complication of inflammatory bowel diseases (IBD) and is often treated with iron supplementation, which has limited efficacy and causes additional co-morbidities. Damaged intestinal barrier function is a hallmark of IBD and causes the translocation of endotoxins from gut bacteria into the bloodstream. A previous study in mice reported that endotoxins suppress erythropoiesis by reprogramming erythroblastic island macrophages (EBI Mφ). Here, we show that IBD patients and mice with acute colitis developed endotoxemia associated with anemia. Endotoxemia in IBD patients was negatively correlated with blood erythrocyte counts. In line with this, mice with colitis caused by drinking water containing dextrin sodium sulphate (DSS) had endotoxemia together with anemia characterized by reduced red blood cell counts, hemoglobin content and hematocrit, and reduced medullary erythropoiesis which was in part compensated by increased extramedullary erythropoiesis. As the endotoxin receptor TLR4 is expressed by CD169 + gut-resident macrophages as well as erythroid island macrophages in the bone marrow, we tested the hypothesis that TLR4 expressed by these CD169 + macrophages mediate both inflammatory colitis and anemia. Indeed, mice with a conditional deletion of the Tlr4 gene specifically in CD169 + tissue-resident macrophages were protected from DSS-induced anemia, inflammation and colitis. In addition, treatment with the TLR4 inhibitor C34 abated inflammation and anemia in DSS mice. These results suggest that endotoxins leaking from the inflamed gut may play a crucial role in IBD and associated anemia via CD169 + tissue-resident macrophages and that drugs targeting TLR4 may protect against IBD-associated anemia.

Cluster of differentiation 47 (CD47) is a widely expressed transmembrane protein that plays a crucial role in immune self-recognition. Cancer cells upregulate CD47 expression to promote immune escape through activating the "don't eat me" signal via interactions with signal regulatory protein α (SIRPα) on macrophages. The effectiveness of anti-CD47 antibodies has been demonstrated in multiple tumor models. However, since CD47 is also expressed in human red blood cells (RBCs) and platelets, the clinical application of anti-CD47 antibodies requires careful consideration of blood toxicity. One major obstacle to the clinical application of CD47 antibodies is the hemagglutination caused by RBCs cross-linking. In this study, we generated Hu1C8, a humanized anti-CD47 monoclonal antibody that demonstrated increased selectivity for binding to CD47 on cancer cells and lacked hemagglutination activity. Epitope mapping and the crystal structure of the Hu1C8 Fab-CD47 extracellular domain (ECD) complex revealed that Hu1C8 binds to a distinct epitope of CD47 in a Ca2+-dependent manner. The unique recognition and binding mode allowed Hu1C8 to bind CD47 on RBCs with reduced hemagglutination activity while still maintaining effective antitumor activity. These findings demonstrate a feasible strategy for developing CD47 antibodies with high antitumor activity but low RBC hemagglutination activity. Our study elucidates how epitope-specific antibody influences antibody-induced cell cross-linking, offering innovative strategies for antibody design to either leverage or avoid cell cross-linking effects.

Immune checkpoint therapy has transformed cancer treatment, yet efficacy and safety challenges persist. Selectively inhibiting tumor-infiltrating regulatory T cells (Ti-Tregs) while enhancing CD8+ T cell function are complementary strategies in cancer immunotherapy. Here, we engineered a bispecific antibody, FRP303, targeting 4-1BB and CCR8, which are co-expressed on a highly immunosuppressive subset of Ti-Tregs. In vivo, FRP303 outperformed monoclonal antibodies in CT26 and MC38 colorectal tumors and poorly immunogenic B16F10 melanoma. Treatment with FRP303 reduced Ti-Treg frequency, increased CD8+ T cell infiltration, and elevated antitumor cytokines IFN-γ and TNF-α. Safety assessments showed FRP303 does not disrupt immune homeostasis in peripheral tissues or induce significant hepatotoxicity. Moreover, FRP303 demonstrated strong synergistic effects when combined with a PD-1 antibody. In summary, FRP303 mediated anti-tumor activity through a dual mechanism involving the selective depletion of Ti-Tregs and the enhancement of CD8+ T cell function, offering a promising strategy for cancer immunotherapy.

Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions of the gastrointestinal tract. Anemia is the most common and debilitating comorbidity in IBD and leads to chronic fatigue, poor quality of life and increased rates of hospitalization. We investigated the mechanisms of anemia and drugs targeting these mechanisms to reduce anemia associated with IBD. Our study discovered that both patients and mice with IBD and ulcerative colitis (induced by 3% dextran sodium sulfate [DSS] in drinking water) displayed endotoxemia. We also found a positive correlation between anemia and endotoxemia in patients with IBD. Mice with colitis were anemic with reduced quantities of red blood cells, hemoglobin and hematocrit. Medullary erythropoiesis was reduced and partially compensated by extramedullary erythropoiesis in the spleen in mice with colitis.We have previously shown that endotoxins inhibit medullary erythropoiesis indirectly via erythroblastic islands macrophages (EBI Mφ), which express CD169 antigen and endotoxin receptor TLR4. To investigate the role of EBI Mφ on colitis-induced anemia, we specifically deleted TLR4 gene in CD169+ Mφ. Cd169Cre:Tlr4fl/fl mice did not develop anemia and showed reduced colitis severity in response to DSS suggesting that endotoxemia may contribute to both anemia and gut inflammation in IBD. We then investigated the effect of inhibitors targeting endotoxin (colistin sulfate/polymyxin E) or its receptor TLR4 (C34), we found that colistin sulfate reduced the severity of colitis with reduced gut inflammation and reduced anemia. In conclusion, we show for the first time that endotoxins leaking from the inflamed gut may play a crucial role in IBD and associated anemia and that drugs targeting endotoxins protect against IBD-associated anemia.

Modulation or depletion of tumor-infiltrating Tregs (Ti-Tregs) is a promising strategy in the field of antitumor immunotherapy. However, this approach poses challenges due to the diversity within the Treg population and the lack of precision in targeting Ti-Tregs. To selectively and efficiently eliminate Ti-Tregs while sparing other immune cells, we developed a bispecific antibody, FT10-Fab, targeting TNFR2 and CCR8, which are highly expressed on Ti-Tregs. Our results showed that FT10-Fab outperformed the monotherapies in several tumor models by significantly reducing the proportion of Ti-Tregs while increasing the proportion of CD8+ T cells. FT10-Fab was able to target and eliminate Ti-Tregs expressing TNFR2 or CCR8 (TNFR2+or CCR8+ Tregs), particularly TNFR2+ CCR8+ Tregs, which are the most important proliferative and protumorigenic Tregs. In addition, FT10-Fab relies on CD8+ T cells for its antitumor function and induces robust immune memory. Furthermore, the combination of FT10-Fab with PD-1 blockade showed synergistic therapeutic efficacy against tumors by significantly suppressing Tregs and enhancing effector CD8+ T cell function. Taken together, our findings suggest that precision depletion of Ti-Tregs via the bispecific TNFR2/CCR8 antibody is a potential therapeutic for cancer immunotherapy, while combination with anti-PD1 amplifies the antitumor effect.

Glucocorticoid drugs (GCs), while effective in systemic lupus erythematosus (SLE), cause severe systemic side effects due to lack of tissue-specificity. To overcome this bottleneck, we developed a CD74-directed antibody-drug conjugate (Bud-ADC) to deliver budesonide, a potent GC drug, selectively to target CD74-expressing immune cells (e.g., B cells, dendritic cells), which play an important role in SLE pathogenesis. Bud-ADC combines a cross-species anti-CD74 antibody with budesonide via a cleavable linker, enabling immunosuppression on targeted cells. In vitro, Bud-ADC selectively inhibited CD74-high immune cell activation and cytokine production. In two SLE mouse models, Bud-ADC significantly alleviated disease hallmarks-reducing autoantibodies, splenomegaly, and kidney damage-while showing superior efficacy to free budesonide at equivalent doses. The therapeutic effects involved both direct targeting of CD74-high immune cells and indirect modulation of T cell responses despite low CD74 expression. This study establishes CD74-targeted ADC as a novel strategy to enhance GC efficacy in SLE, aiming at minimizing off-target toxicity while maintaining broad immunosuppressive activity. The translatable design supports further preclinical and clinical development for autoimmune diseases.

Current treatments for ulcerative colitis (UC) remain limited, highlighting the need for novel therapeutic strategies. Trilobatin (TLB), a naturally derived food additive, exhibits potential anti-inflammatory properties. In this study, a dextran sulfate sodium (DSS)-induced animal model is used to investigate the effects of TLB on UC. It is found TLB significantly alleviates DSS-induced UC in mice, as evidenced by a reduction in the disease activity index, an increase in colon length, improvement in histopathological lesions. Furthermore, TLB treatment results in a decrease in proinflammatory cytokines and an increase in anti-inflammatory cytokines. TLB mitigates UC by modulating the intestinal microbiota, particularly Akkermansia, which enhances tryptophan metabolism and upregulates the production of xanthurenic acid (XANA). To confirm the role of TLB-induced microbiota changes, experiments are performed with pseudogerm-free mice and fecal transplantation. It is also identified XANA as a key metabolite that mediates TLB's protective effects. Both TLB and XANA markedly activate the aromatic hydrocarbon receptor (AhR). Administration of an AhR antagonist abrogates their protective effects, thereby confirming the involvement of AhR in the underlying mechanism. In conclusion, the study reveals a novel mechanism through which TLB alleviates UC by correcting microbiota imbalances, regulating tryptophan metabolism, enhancing XANA production, and activating AhR.

The bacterial strain is key to fermentation, and the intestinal tract in livestock and poultry is a resource bank of good natural strains. The objective of this study was to evaluate the effect of soybean meal fermented using Lactobacillus casei, isolated from healthy broiler intestines with excellent organic acid production, on the intestinal health and growth performance of broilers. A total of 120 Arbor Acre male broiler chickens aged 21 days were fed until 42 days of age. These chickens were randomly divided into four groups with five replicates per group. Each replicate contained six broiler chickens. The specific groups were the control group (basal diet), the low-dose fermented soybean meal (FSBM) additive group (FSBML, basal diet + 0.2 kg/t FSBM), the middle-dose FSBM additive group (FSBMM, basal diet + 2 kg/t FSBM), and the high-dose FSBM additive group (FSBMH, basal diet + 5 kg/t FSBM). The results demonstrated a significant increase in the average daily feed intake (ADFI) and average daily gain (ADG) of the FSBMH group (p < 0.05). The FSBMH group displayed a significantly increased villus height (VH) to crypt depth (CD) ratio (VH/CD) for the duodenum (p < 0.05) and rectum (p < 0.05). The examination of the ileal mucosa showed that the FSBMH group (p < 0.05) had significantly higher levels of glutathione (GSH) activity, as well as higher relative mRNA expression of ZO-1, ZO-2, Occludin, IL-4, IL-6, MCP-1, TNF-α, IFN-α, IFN-β, and IFN-γ. However, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were significantly lower in the FSBMH group (p < 0.05). The FSBMH group also showed higher levels of Nitriliruptoraceae and Ruminococcaceae. In conclusion, the addition of 5 kg/t FSBM to diets had an ameliorative effect on broiler growth performance and intestinal health.

Metabolic dysfunction-associated steatohepatitis (MASH) is the most prevalent cause of liver disease worldwide, with a single approved therapeutic. Previous research has shown that interleukin-22 (IL-22) can suppress β-cell stress, reduce local islet inflammation, restore appropriate insulin production, reverse hyperglycemia, and ameliorate insulin resistance in preclinical models of diabetes. In clinical trials long-acting forms of IL-22 have led to increased proliferation in the skin and intestine, where the IL-22RA1 receptor is highly expressed. To maximise beneficial effects whilst reducing the risk of epithelial proliferation and cancer, we designed short-acting IL-22-bispecific biologic drugs that successfully targeted the liver and pancreas. Here we show 10-fold lower doses of these bispecific biologics exceed the beneficial effects of native IL-22 in multiple preclinical models of MASH, without off-target effects. Treatment restores glycemic control, markedly reduces hepatic steatosis, inflammation, and fibrogenesis. These short-acting IL-22-bispecific targeted biologics are a promising new therapeutic approach for MASH.

Encapsulation of drugs into nanocarriers is proven to be highly promising approach in reducing drug toxicity and enhancing therapeutic efficacy. However, controlling the loading efficiency and capacity, and release of therapeutics at specific disease site has remained a key challenge, particularly for toxic chemotherapeutic drugs. This work explored the effect of treatment with empty silica nanoparticles (SNPs) and a chemotherapeutic drug either together (i.e. co-treatment) or in tandem (i.e. temporally spaced) on the cell ablation ability of the drug. The study also investigated whether the efficacy of the drug in response to these treatments was dependent on the morphology of particles. SNPs of four different morphologies (solid: SSNPs, dendritic: DSNPs, mesoporous: MSNPs, and rod: RSNP) were used, while cisplatin (CisPt) served as model chemotherapeutic. The efficacy of CisPt as a function of SNPs morphology and temporal treatment strategy was tested in HeLa cells. The results indicated that the morphology of particles as well as treatment strategy (i.e. co-incubation and post treatment) had an impact on not only the cell viability but also the cell death pathways, as evidenced by varying IC50 values and the flow cytometry analysis. Interestingly, co-treatment of SNPs with CisPt resulted in an across-the-board lower IC50 value compared to when the cells were first treated with SNPs for 24 h followed by CisPt treatment and even when CisPt was loaded into the particles for most of the SNPs.

Introduction: MDM2 is known as the primary negative regulator of p53, MDM2 promotes lung cancer fibrosis and lung injury through p53-dependent and p53-independent pathways. However, the mechanism by which MDM2 influences the pathogenesis of asthma is unknown. In this study, we investigated the function of MDM2 in lung epithelial cells in type 2 lung inflammation. Methods: We used type II alveolar epithelial cell-specific heterozygous knockout of Mdm2 mice to validate its function. Then papain-induced asthma model was established, and changes in inflammation were observed by measuring immunohistochemistry and flow cytometry analysis. Results: In this study, we knockdown the mouse Mdm2 gene in type 2 alveolar epithelial cells. We demonstrated that heterozygous Mdm2 gene-deleted mice were highly susceptible to protease allergen papain-induced pulmonary inflammation characterized by increased ILC2 numbers, IL-5 and IL-13 cytokine levels, and lung pathology. A mechanistic study showed that following the decreased expression of Mdm2 in lung epithelial cells and A549 cell line, p53 was overactivated, and the expression of its downstream genes p21, Puma, and Noxa was elevated, which resulted in apoptosis. After Mdm2 knockdown, the mRNA expression of inflammation-related gene IL-25, HMGB1 and TNF-α were increased, which further amplified the downstream ILC2 response and lung inflammation. Conclusion: These results indicate that Mdm2 maintains the homeostasis of lung epithelial cells by targeting P53, and regulate the function of lung epithelial cells under type 2 lung inflammation.

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by exacerbated synovial inflammation and joint destruction. Recent studies suggest toll-like receptor 4 (TLR4) internalization facilitate inflammatory response of macrophage. The role of TLR4 internalization in the pathogenesis of RA is unknown. Purpose: To investigate the role and mechanism of TLR4 internalization in macrophage inflammatory response of RA and explore whether TLR4 internalization mediates the anti-arthritic effect of Xiaowugui (XWG) decoction, a patented herbal formula used in China. Methods: The co-expression of TLR4 and the internalization marker, early endosome antigen 1 (EEA1), in the synovial samples of RA patients and joint tissue of collagen-induced arthritis (CIA) mice, were evaluated using immunofluorescence. The effect of Rab5a-mediated early internalization of TLR4 on the activation induced by lipopolysaccharide (LPS) in RAW264.7 cells was investigated using small interfering RNAs that act against Rab5a. CIA was induced in Rab5a-/- mice to evaluate the role of Rab5a in vivo. The disease progression and expression of Rab5a and TLR4 in the joint tissue were evaluated in CIA mice treated with XWG. Inflammatory factors production, TLR4 internalization, and activation of downstream signaling pathways were examined in RAW264.7 cells treated with XWG in vitro. Results: The co-expression and co-localization of TLR4 and EEA1 were elevated in the synovial samples of RA patients and joint tissue of CIA mice. Pharmaceutical inhibition of TLR4 internalization reduced macrophages inflammatory responses induced by LPS. The co-expression and co-localization of Rab5a and TLR4 were significantly increased in macrophages treated with LPS. Silencing Rab5a reduced LPS-induced TLR4 internalization, inflammatory factors production, and phosphorylation of Jun N-terminal kinases (JNK) and p65. Genetic deletion of Rab5a inhibited TLR4 internalization and the development of arthritis in vivo. The co-expression of TLR4 and Rab5a was also elevated in the synovial samples of RA patients. XWG treatment of mice with CIA alleviated arthritis and reduced the co-expression of Rab5a and TLR4 in the joint tissue. XWG treatment of macrophage inhibited LPS-induced IL-6 and TNF-alpha production, co-expression of Rab5a and TLR4, and phosphorylation of JNK and p65. Conclusions: Our findings highlight the pathogenic role of TLR4 internalization in patients with RA and identify a novel Rab5a-dependent internalization pathway that promotes macrophage inflammatory response. XWG treatment demonstrated outstanding therapeutic effects in experimental arthritis, and targeting the Rab5amediated internalization of TLR4 may be the main underlying mechanism.

Cardiovascular diseases (CVD) are the leading cause of mortality worldwide despite an aggressive reduction of traditional cardiovascular risk factors. Underlying inflammatory conditions such as inflammatory bowel disease (IBD) increase the risk of developing CVD. A broad understanding of the underlying pathophysiological processes between IBD and CVD is required to treat and prevent cardiovascular events in patients with IBD. This review highlights the commonality between IBD and CVD, including dysregulated immune response, genetics, environmental risk factors, altered gut microbiome, stress, endothelial dysfunction and abnormalities, to shed light on an essential area of modern medicine.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease accompanied by both local and systemic comorbidities. Genetic factors play a role in the development of IPF and certain associated comorbidities. Nevertheless, it is uncertain whether there are shared genetic factors underlying IPF and these comorbidities. To bridge this knowledge gap, we conducted a systematic investigation into the shared genetic architecture between IPF and ten prevalent heritable comorbidities (i.e., body mass index [BMI], coronary artery disease [CAD], chronic obstructive pulmonary disease [COPD], gastroesophageal reflux disease, lung cancer, major depressive disorder [MDD], obstructive sleep apnoea, pulmonary hypertension [PH], stroke, and type 2 diabetes), by utilizing large-scale summary data from their respective genome-wide association studies and multi-omics studies. We revealed significant (false discovery rate [FDR] < 0.05) and moderate genetic correlations between IPF and seven comorbidities, excluding lung cancer, MDD and PH. Evidence suggested a partially putative causal effect of IPF on CAD. Notably, we observed FDR-significant genetic enrichments in lung for the cross-trait between IPF and CAD and in liver for the cross-trait between IPF and COPD. Additionally, we identified 65 FDR-significant genes over-represented in 20 biological pathways related to the etiology of IPF, BMI, and COPD, including inflammation-related mucin gene clusters. Several of these genes were associated with clinically relevant drugs for the treatment of IPF, CAD, and/or COPD. Our results underscore the pervasive shared genetic basis between IPF and its common comorbidities and hold future implications for early diagnosis of IPF-related comorbidities, drug repurposing, and the development of novel therapies for IPF.

Inflammatory bowel diseases (IBD), which include Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory conditions of the gastrointestinal tract and highly prevalent in Australia and USA. Anemia is debilitating and a common complication of IBD associated with chronic fatigue, poor quality of life and increased rate of hospitalization. However, current treatments for anemia in IBD are limited to iron supplementation which has limited efficacy. In this study, we investigated the cause of anemia in IBD patients and in a mouse colitis model. We have previously shown that endotoxins/lipopolysaccharides (LPS) inhibit bone marrow (BM) erythropoiesis in a TLR4- and MyD88-dependent manner in mice. Therefore, we first measured blood plasma concentrations of endotoxins in IBD patients and mouse model of acute colitis. Blood endotoxin concentrations were significantly increased relative to healthy controls in IBD patients (~4-fold in UC and ~2.2-fold in CD patients) and in mice with colitis induced by sodium dextran sulphate (DSS) (~4-fold). This is consistent with disruption of the intestinal barrier observed in IBD patients and DSS-treated mice allowing translocation of endotoxins produced by the gut bacterial flora into the circulation. A positive correlation between anemia and endotoxemia was found in IBD patients. To investigate the effect of gut inflammation on erythropoiesis, we measured the markers of erythropoiesis in mice with acute colitis induced by 3% DSS in drinking water. Mice with acute colitis had significant anemia with reduced number of red blood cells (RBCs; controls mean 9.0 x 1012 ± 0.7 /L vs DSS-treated 7.5 x 1012± 1.1 /L), hemoglobin (HgB; mean 140 ± 9.1 g/L vs 117 g/L ± 15 g/L) and hematocrit (HCT; mean 0.41 ± 0.02 vs 0.34 ± 0.05) compared to control mice. In line with this, we found that acute colitis in mice caused a marked whitening of the bone marrow (BM) with reduced number of erythrocytes (mean 4.3 x 106 ± 1.8 erythrocytes/femur vs 2.3 x 106 ± 1.4 erythrocytes/femur), accompanied by shortened RBC half-life (t1/2 = 19.7 ± 2.9 days in controls vs t1/2 = 12.8 ± 4.4 days in DSS treated mice) in the blood and increased blood concentration of interferon-γ (mean 4.7 pg/mL ± 0.8 vs 7.6 pg/mL ± 2.7). Interferon- γ is known to reduce RBC lifespan by increasing their clearance in the spleen. Acute colitis increased RBC size (~1.15-fold increase in red cell width and ~ 3.5-fold increase in blood reticulocytes) consistent with colitis induced anemia. This reduced medullary erythropoiesis was compensated in part by extramedullary erythropoiesis in the spleen. Patients with IBD had increased levels of hepcidin (~4-fold increase) and interleukin -6 (IL-6; ~1.3-fold increase) in their blood. However, mice with acute colitis only showed an increase in blood IL-6 concentration (~9-fold increase) but no change in hepcidin, suggesting that there is a combination of both iron-deficiency anemia and anemia of inflammation in these settings. We have previously shown that endotoxin inhibits erythropoiesis indirectly by deregulating erythroblastic islands macrophages (EBI Mφ) which express the CD169/Siglec1 antigen and endotoxin receptor TLR4. To investigate the role of EBI Mφ on colitis-induced anemia, we specifically deleted TLR-4 in CD169+ Mφ by crossing Tlr4flox/flox mice with Siglec1Cre mice (expressing Cre recombinase in CD169+ tissue Mφ). Mice lacking Tlr4 gene specifically in CD169+ Mφ did not develop anemia in response to DSS with normal blood RBC, HgB, HCT, reticulocytes numbers, BM erythrocyte numbers and no extramedullary erythropoiesis in the spleen compared to mice with Tlr4 gene in CD169+ macrophages. In addition to the protective effect of Tlr4 deletion in CD169+ Mφ on anemia, we found that these mice were also protected from colitis with improved body weight, colon length and histological scores suggesting that endotoxemia may contribute to both anemia and colitis in IBD. In conclusion, we show for the first time that inflammation via endotoxins may play a crucial role in anemia in IBD and there is a correlation between anemia and endotoxemia in IBD.

Cell adhesion molecule close homolog of L1 (CHL1) is implicated in tumorigenesis of various malignancies. However, its role and underlying molecular mechanisms in colorectal cancer (CRC) remain unclear. The present study aimed to evaluate the specific biological functions and mechanisms of CHL1, in order to provide a theoretical basis for the use of CHL1 as a biological target in CRC. CHL1 expression was originally determined in CRC cell lines. Subsequently, CHL1 overexpression was induced by plasmid transfection in HT29 and SW480 cells, and cell proliferation, migration and invasion were evaluated using the Cell Counting Kit-8, clone formation, organoids formation and Transwell assays. Immunofluorescence and western blotting were performed to assess the protein expression of E-cadherin or N-cadherin. Differentially expressed genes (DEGs) were further evaluated using RNA-sequencing (RNA-seq) in HT29 and SW480 cells following CHL1 overexpression and functional enrichment analysis. Western blotting was performed to validate the expression of proteins related to the nuclear factor kappa B (NF-kappa B) signaling pathway. The TNMplot online database revealed the significant downregulation of CHL1 in CRC tissues. The results indicated that exogenous CHL1 overexpression significantly inhibited the proliferative, organoid-forming, migratory and invasive abilities of HT29 and SW480 cells, and increased E-cadherin protein expression. Additionally, CHL1 overexpression reduced xenograft tumor growth in vivo. RNA-seq and functional analysis revealed that DEGs in CHL1 overexpressing cells were mainly enriched in the NF-kappa B signaling pathway. The expression of p-p65 and p-p65/p65 ratio were significantly reduced in HT29 and SW480 cells, following CHL1 overexpression. Additionally, the inhibitory effects of CHL1 overexpression on CRC cell proliferation, organoid formation, migration and invasion were partially counteracted following the overexpression of p65 expression. Overall, the present study demonstrates that CHL1 inhibits CRC cell growth, migration and invasion through the inactivation of the NF-kappa B signaling pathway.

The cytokine Interleukin (IL)-20 belongs to the IL-10 superfamily. IL-20 levels are reported to increase in the intestines of Ulcerative Colitis (UC) patients, however not much is known about its effects on intestinal epithelial cells. Here, we investigated the influence of IL-20 on intestinal epithelial cell lines and primary intestinal organoid cultures. By using chemical-induced (dextran sodium sulphate; DSS) colitis and a spontaneous model of colitis (Winnie mice), we assess whether recombinant IL-20 treatment is beneficial in reducing/improving pathology. Following stimulation with IL-20, intestinal primary organoids from wild-type and Winnie mice increased the expression of ERK1/2. However, this was lost when cells were differentiated into secretory goblet cells. Importantly, IL-20 treatment significantly reduced endoplasmic reticulum (ER) stress, as measured by spliced-XBP1 in epithelial cells, and this effect was lost in the goblet cells. IL-20 treatment in vivo in the DSS and Winnie models had minimal effects on pathology, but a decrease in macrophage activation was noted. Taken together, these data suggest a possible, but subtle role of IL-20 on epithelial cells in vivo. The therapeutic potential of IL-20 could be harnessed by the development of a targeted therapy or combination therapy to improve the healing of the mucosal barrier.