IL-10 Knockout Research Articles

Reduced estrogen signaling contributes to bone loss and cardiac dysfunction in interleukin-10 knockout mice.

Characterization of the interleukin (IL)-10 knockout (KO) mouse with chronic gut inflammation, cardiovascular dysfunction, and bone loss suggests a critical role for this cytokine in interorgan communication within the gut, bone, and cardiovascular axis. We sought to understand the role of IL-10 in the cross-talk between these systems. Six-week-old IL-10 KO mice and their wild type (WT) counterparts were maintained on a standard rodent diet for 3 or 6 months. Gene expression of proinflammatory markers and Fgf23, serum 17β-estradiol (E2), and cardiac protein expression were assessed. Ileal Il17a and Tnf mRNA increased while Il6 mRNA increased in the bone and heart by at least 2-fold in IL-10 KO mice. Bone Dmp1 and Phex mRNA were repressed at 6 months in IL-10 KO mice, resulting in increased Fgf23 mRNA (~4-fold) that contributed to increased fibrosis. In the IL-10 KO mice, gut bacterial β-glucuronidase activity and ovarian Cyp19a1 mRNA were lower (p < 0.05), consistent with reduced serum E2 and reduced cardiac pNOS3 (Ser1119 ) in these mice. Treatment of ileal lymphocytes with E2 reduced gut inflammation in WT but not IL-10 KO mice. In conclusion, our data suggest that diminished estrogen and defective bone mineralization increased FGF23 which contributed to cardiac fibrosis in the IL-10 KO mouse.

Acute exercise modulates Trim63 and Bmal1 in the skeletal muscle of IL-10 knockout mice

The anti-inflammatory role of physical exercise is mediated by interleukin 10 (IL-10), and their release is possibly upregulated in response to IL-6. Previous studies demonstrated that mice lacking IL-6 (IL-6 KO mice) exhibited diminished exercise tolerance, and reduced strength. Rev-erbα, a transcriptional suppressor involved in circadian rhythm, has been discovered to inhibit the expression of genes linked to bodily functions, encompassing inflammation and metabolism. It also plays a significant role in skeletal muscle and exercise performance capacity. Given the potential association between Rev-erbα and the immune system and the fact that both pathways are modulated following acute aerobic exercise, we examined the physical performance of IL-10 KO mice and analyzed the modulation of the atrophy and Rev-erbα pathways in the muscle of wild type (WT) and IL-10 KO mice following one session of acute exercise. For each phenotype, WT and IL-10 KO were divided into two subgroups (Control and Exercise). The acute exercise session started at 6 m/min, followed by 3 m/min increments every 3 min until animal exhaustion. Two hours after the end of the exercise protocol, the gastrocnemius muscle was removed and prepared for the reverse transcription-quantitative polymerase chain reaction (RT-q-PCR) and immunoblotting technique. In summary, compared to WT, the IL-10 KO animals showed lower body weight and grip strength in the baseline. The IL-10 control group presented a lower protein content of BMAL1. After the exercise protocol, the IL-10 KO group had higher mRNA levels of Trim63 (atrophy signaling pathway) and lower mRNA levels of Clock and Bmal1 (Rev-erbα signaling pathway). This is the first study showing the relationship between Rev-erbα and atrophy in IL-10 KO mice. Also, we accessed a public database that analyzed the gastrocnemius of MuRF KO mice submitted to two processes of muscle atrophy, a denervation surgery and dexamethasone (Dexa) injections. Independently of knockout, the denervation demonstrated lower Nr1d1 levels. In conclusion, IL-10 seems to be a determinant in the Rev-erbα pathway and atrophy after acute exercise, with no modulation in the baseline state.

The Roles of IL-18 in a Realistic Partial Body Irradiation with 5% Bone Marrow Sparing (PBI/BM5) Model.

IL-18 has been shown to play important roles in response to total body irradiation. However, homogenous total body irradiation is not a realistic model to reflect the radiation exposure in a real nuclear event. To further study the roles of IL-18 in a real nuclear scenario, we developed a mouse partial body irradiation with 5% bone marrow sparing (PBI/BM5) model to mimic the inhomogeneous radiation exposure. We established the dose response curves of PBI/BM5 using different radiation doses ranging from 12 to 16 Gy. Using the PBI/BM5 model, we showed that IL-18 knockout mice were significantly more radiation resistant than the wild-type mice at 14.73 Gy. We further studied the hematopoietic changes using a complete blood count, bone marrow colony-forming assays, and serum cytokine assays on the mice exposed to PBI/BM5 with IL-18BP treatment and wild-type/IL-18 knockout mice. In conclusion, our data suggest that IL-18 plays important roles in mouse survival in a realistic nuclear exposure model, potentially through the IL-18/IFNγ pathway.

IL-6 Mutation Attenuates Liver Injury Caused by Aeromonas hydrophila Infection by Reducing Oxidative Stress in Zebrafish.

Interleukin-6 (IL-6), a pleiotropic cytokine, plays a crucial role in acute stress induced by bacterial infection and is strongly associated with reactive oxygen species (ROS) production. However, the role of IL-6 in the liver of fish after Aeromonas hydrophila infection remains unclear. Therefore, this study constructed a zebrafish (Danio rerio) il-6 knockout line by CRISPR/Cas9 to investigate the function of IL-6 in the liver post bacterial infection. After infection with A. hydrophila, pathological observation showed that il-6-/- zebrafish exhibited milder liver damage than wild-type (WT) zebrafish. Moreover, liver transcriptome sequencing revealed that 2432 genes were significantly up-regulated and 1706 genes were significantly down-regulated in il-6-/- fish compared with WT fish after A. hydrophila infection. Further, gene ontology (GO) analysis showed that differentially expressed genes (DEGs) were significantly enriched in redox-related terms, including oxidoreductase activity, copper ion transport, etc. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were significantly enriched in pathways such as the PPAR signaling pathway, suggesting that il-6 mutation has a significant effect on redox processes in the liver after A. hydrophila infection. Additionally, il-6-/- zebrafish exhibited lower malondialdehyde (MDA) levels and higher superoxide dismutase (SOD) activities in the liver compared with WT zebrafish following A. hydrophila infection, indicating that IL-6 deficiency mitigates oxidative stress induced by A. hydrophila infection in the liver. These findings provide a basis for further studies on the role of IL-6 in regulating oxidative stress in response to bacterial infections.

Abstract B014: Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity

Abstract Ovarian cancer (OvCa) is among the most aggressive and lethal cancers with a poor response to immune checkpoint blockade (ICB) despite its modest tumor mutational burden and expression of known cancer antigens. Consequently, it is critical to understand the molecular mechanisms of tumor immune escape and resistance to immunotherapies. Intercellular signaling between cancer cells and the tumor microenvironment (TME) is a crucial determinant of tumor immunity and given the immunosuppressive nature of OvCa TME, it is of utmost importance to dissect how this immunosuppressed TME is established. To begin to determine which cancer-cell genes may function cell externally in controlling of OvCa immunity, we employed a first-of-its-kind spatial functional genomics approach we developed, called Perturb-map, which enables dozens of CRISPR gene knock-out (KO) cells to be resolved in a tissue/tumor by imaging, along with cells in the local TME. We used Perturb-map to determine how 35 different genes, identified through ligand-receptor analysis of ovarian tumors, influenced tumor response to anti-PD-1 treatment in an OvCa mouse model. Strikingly, Il4 KO tumors were unique amongst the 35 gene KOs making the tumors significantly more responsive to anti-PD-1 treatment despite not impacting tumor growth in the absence of immunotherapy. Further experiments with single CRISPR KO tumors validated the Perturb-map finding, where Il4 KO combined with anti-PD-1 treatment resulted in a major reduction in tumor growth and a significant increase in survival. Cyclic immunofluorescence (CyCIF) multiplex imaging revealed that loss of cancer cell-derived IL-4 led to a significant reduction in pro-tumor macrophages and an increase in dendritic cell (DC) subsets as well as both progenitor and terminally exhausted CD8 T cells. Upon anti-PD-1 treatment, Il4 KO tumors had significant enrichment for activated CD8 T cells and B cells. Importantly, spatial analysis of the tumor samples identified enrichment of T follicular helper cells, B cells, and DCs in tertiary lymphoid structures in tumors with combined IL-4 loss and PD-1 inhibition. Further, we analyzed a human OvCa single-cell transcriptomics dataset and demonstrated that an IL-4 response signature is expressed in tumor-associated macrophages, indicating active IL-4 signaling in human ovarian tumors. Importantly, we employed a CRISPR-knock-in expression reporter in mouse tumors as well as immunohistochemistry of both human and mouse tumor samples to confirm that IL-4 is expressed by cancer cells. Thus, we identified cancer cell-secreted IL-4 as a potent regulator of OvCa TME and response to ICB. Given that an anti-IL-4-receptor antibody is already in clinical use to treat immune diseases, our findings have the potential to be translated to the clinical management of this dreadful disease. Citation Format: Gurkan Mollaoglu, Alexander Tepper, Hunter Potak, Luisanna Pia, Angelo Amabile, Chiara Falcomata, Jaime Mateus-Tique, Noam Rabinovich, Rachel Brody, Lindsay Browning, Jia-Ren Lin, Peter Sorger, Sandro Santagata, Miriam Merad, Alessia Baccarini, Brian Brown. Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B014.

The critical role of interleukin-6 in protection against neurotropic flavivirus infection.

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are emerging mosquito-borne flaviviruses causing encephalitis globally. No specific drug or therapy exists to treat flavivirus-induced neurological diseases. The lack of specific therapeutics underscores an urgent need to determine the function of important host factors involved in flavivirus replication and disease progression. Interleukin-6 (IL-6) upregulation has been observed during viral infections in both mice and humans, implying that it may influence the disease outcome significantly. Herein, we investigated the function of IL-6 in the pathogenesis of neurotropic flavivirus infections. First, we examined the role of IL-6 in flavivirus-infected human neuroblastoma cells, SK-N-SH, and found that IL-6 neutralization increased the WNV or JEV replication and inhibited the expression of key cytokines. We further evaluated the role of IL-6 by infecting primary mouse cells derived from IL-6 knockout (IL-6-/-) mice and wild-type (WT) mice with WNV or JEV. The results exhibited increased virus yields in the cells lacking the IL-6 gene. Next, our in vivo approach revealed that IL-6-/- mice had significantly higher morbidity and mortality after subcutaneous infection with the pathogenic WNV NY99 or JEV Nakayama strain compared to WT mice. The non-pathogenic WNV Eg101 strain did not cause mortality in WT mice but resulted in 60% mortality in IL-6-/- mice, indicating that IL-6 is required for the survival of mice after the peripheral inoculation of WNV or JEV. We also observed significantly higher viremia and brain viral load in IL-6-/- mice than in WT mice. Subsequently, we explored innate immune responses in WT and IL-6-/- mice after WNV NY99 infection. Our data demonstrated that the IL-6-/- mice had reduced levels of key cytokines in the serum during early infection but elevated levels of proinflammatory cytokines in the brain later, along with suppressed anti-inflammatory cytokines. In addition, mRNA expression of IFN-α and IFN-β was significantly lower in the infected IL-6-/- mice. In conclusion, these data suggest that the lack of IL-6 exacerbates WNV or JEV infection in vitro and in vivo by causing an increase in virus replication and dysregulating host immune response.

Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease.

To our knowledge, IL-10-KO mice have not previously been used to investigate the interactions of host, microbiota, and broccoli, broccoli sprouts, or broccoli bioactives in resolving symptoms of CD. We showed that a diet containing 10% raw broccoli sprouts increased the plasma concentration of the anti-inflammatory compound sulforaphane and protected mice to varying degrees against disease symptoms, including weight loss or stagnation, fecal blood, and diarrhea. Younger mice responded more strongly to the diet, further reducing symptoms, as well as increased gut bacterial richness, increased bacterial community similarity to each other, and more location-specific communities than older mice on the diet intervention. Crohn's disease disrupts the lives of patients and requires people to alter dietary and lifestyle habits to manage symptoms. The current medical treatment is expensive with significant side effects, and a dietary intervention represents an affordable, accessible, and simple strategy to reduce the burden of symptoms.

Interleukin-22 deficiency exacerbates right ventricular remodeling during pressure overload

Abstract Background Fibrosis and remodeling of right ventricle (RV) are associated with prognosis in patients with RV failure. Interleukin-22 (IL-22) is a member of the IL-10 cytokine family, play roles in tissue protection and wound repair. We have previously shown that IL-22 plays an important role in cardiac remodeling after acute myocardial infarction; however, the role of IL-22 in RV fibrosis and remodeling remains elusive. Purpose We investigated the role of IL-22 in the pathogenesis of RV remodeling during pressure overload. Methods Pulmonary artery banding (PAB) is performed by placing a 6-0 suture around the pulmonary artery over a 24 G needle in wild type mice (C57BL/6J) and IL-22 knockout mice (IL-22 KO). Only mice with moderate pulmonary artery stenosis (peak pressure gradient across the pulmonary band: 25–40 mmHg at 1 week after surgery by echo Doppler) were included in the study protocol. Two weeks after PAB, RV function was measured by echocardiography and real-time PCR analysis was performed to measure IL-22, IL-22 receptor subunit alpha 1 (IL-22RA1), and IL-22 signaling inhibitor IL-22 binding protein (IL-22BP). Results 2 weeks after PAB, IL-22 KO mice showed markedly increased RV weight (IL-22 KO mice vs. wild-type mice; RV/LV+IVS: 0.48±0.05 vs 0.40±0.02, P=0.002). IL-22 KO mice exhibited significant RV enlargement and RV dysfunction 2 weeks after PAB. (IL-22 KO mice vs wild type mice; RVEDV: 10±2.3mm2 vs 12.9±3.1mm2, P=0.005, TAPSE: 10.8±1.8mm vs 9.5±1.7mm, P=0.04, FAC: 35.2±7.8% vs 23±6.0%, P&amp;lt;0.001). The expression of IL-22, IL-22RA1 and IL-22BP in the RV tended to increase 2 weeks after PAB (wild-type mice with PAB (n=3) vs. wild-type mice with sham; IL-22: 0.0023 vs 0.0028, P=0.18 IL-22RA1: 1.489 vs 1.773, P=0.175, IL-22BP: 0.025 vs 0.067, p=0.005). Conclusion These results suggest that IL-22 is involved in RV remodeling during RV pressure overload.

Limitation of amino acid availability by bacterial populations during enhanced colitis in IBD mouse model.

Inflammatory bowel disease is associated with an increase in Enterobacteriaceae and Enterococcus species; however, the specific mechanisms are unclear. Previous research has reported the associations between microbiota and inflammation, here we investigate potential pathways that specific bacteria populations use to drive gut inflammation. Richie et al. show that these bacterial populations utilize an alternate sulfur metabolism and are tolerant of host-derived immune-response products. These metabolic pathways drive host gut inflammation and fuel over colonization of these pathobionts in the dysbiotic colon. Cultured isolates from dysbiotic mice indicated faster growth supplemented with L-cysteine, showing these microbes can utilize essential host nutrients.

Native lamin A/C proteomes and novel partners from heart and skeletal muscle in a mouse chronic inflammation model of human frailty.

Clinical frailty affects ∼10% of people over age 65 and is studied in a chronically inflamed (Interleukin-10 knockout; "IL10-KO") mouse model. Frailty phenotypes overlap the spectrum of diseases ("laminopathies") caused by mutations in LMNA. LMNA encodes nuclear intermediate filament proteins lamin A and lamin C ("lamin A/C"), important for tissue-specific signaling, metabolism and chromatin regulation. We hypothesized that wildtype lamin A/C associations with tissue-specific partners are perturbed by chronic inflammation, potentially contributing to dysfunction in frailty. To test this idea we immunoprecipitated native lamin A/C and associated proteins from skeletal muscle, hearts and brains of old (21-22months) IL10-KO versus control C57Bl/6 female mice, and labeled with Tandem Mass Tags for identification and quantitation by mass spectrometry. We identified 502 candidate lamin-binding proteins from skeletal muscle, and 340 from heart, including 62 proteins identified in both tissues. Candidates included frailty phenotype-relevant proteins Perm1 and Fam210a, and nuclear membrane protein Tmem38a, required for muscle-specific genome organization. These and most other candidates were unaffected by IL10-KO, but still important as potential lamin A/C-binding proteins in native heart or muscle. A subset of candidates (21 in skeletal muscle, 30 in heart) showed significantly different lamin A/C-association in an IL10-KO tissue (p < 0.05), including AldoA and Gins3 affected in heart, and Lmcd1 and Fabp4 affected in skeletal muscle. To screen for binding, eleven candidates plus prelamin A and emerin controls were arrayed as synthetic 20-mer peptides (7-residue stagger) and incubated with recombinant purified lamin A "tail" residues 385-646 under relatively stringent conditions. We detected strong lamin A binding to peptides solvent exposed in Lmcd1, AldoA, Perm1, and Tmem38a, and plausible binding to Csrp3 (muscle LIM protein). These results validated both proteomes as sources for native lamin A/C-binding proteins in heart and muscle, identified four candidate genes for Emery-Dreifuss muscular dystrophy (CSRP3, LMCD1, ALDOA, and PERM1), support a lamin A-interactive molecular role for Tmem38A, and supported the hypothesis that lamin A/C interactions with at least two partners (AldoA in heart, transcription factor Lmcd1 in muscle) are altered in the IL10-KO model of frailty.

Protective actions of a luminally acting 5-HT4 receptor agonist in mouse models of colitis.

5-hydroxytryptamine 4 receptors (5-HT4 Rs) are expressed in the colonic epithelium, and previous studies have demonstrated that luminal administration of agonists enhances motility, suppresses nociception, and is protective in models of inflammation. We investigated whether stimulation with a luminally acting 5-HT4 R agonist is comparable to previously tested absorbable compounds. The dextran sodium sulfate (DSS), trinitrobenzene sulfonic acid (TNBS), and interleukin 10 knockout (IL-10KO) models of colitis were used to test the protective effects of the luminally acting 5-HT4 R agonist, 5HT4-LA1, in the absence and presence of a 5-HT4 R antagonist. The compounds were delivered by enema to mice either before (prevention) or after (recovery) the onset of active colitis. Outcome measure included disease activity index (DAI) and histological evaluation of colon tissue, and effects on wound healing and fecal water content were also assessed. Daily enema of 5HT4-LA1 attenuated the development of, and accelerated recovery from, active colitis. Enema administration of 5HT4-LA1 did not attenuate the development of colitis in 5-HT4 R knockout mice. Stimulation of 5-HT4 Rs with 5HT4-LA1 increased Caco-2 cell migration (accelerated wound healing). Daily administration of 5HT4-LA1 did not increase fecal water content in active colitis. Luminally restricted 5-HT4 R agonists are comparable to absorbable compounds in attenuating and accelerating recovery from active colitis. Luminally acting 5-HT4 R agonists may be useful as an adjuvant to current inflammatory bowel disease (IBD) treatments to enhance epithelial healing.

The Role of the IL-33/ST2 Axis in CpG-Induced Macrophage Activation Syndrome.

Macrophage activation syndrome (MAS) is a fatal inflammatory condition, which is often associated with the elevation of multiple proinflammatory cytokines and multiple organ dysfunction. Previous studies have shown that ST2 contributes to T cell overactivation and plays a detrimental role in mouse models of primary hemophagocytic lymphohistiocytosis. The purpose of this study was to investigate the role of the IL-33/ST2 axis in a mouse model of MAS induced by repeated injections of cytosine-phosphate-guanine (CpG). Serum cytokines were determined using the cytometric bead array by flow cytometry. IL-33 and ST2 were detected by immunohistochemistry and real-time quantitative PCR in the liver and spleen of mice. CD3 and F4/80 in the liver were detected by immunohistochemistry. Inflammatory macrophages and effector memory T lymphocytes were detected by flow cytometry. The CpG-induced MAS model was successfully induced after repeated CpG injections, presenting with hypercytokinemia and hepatosplenomegaly. The numbers of IL-33 positive cells in the liver and spleen decreased significantly, while the expression of ST2 in the liver tended to increase in the mice with MAS. IL-33 and St2 knockout mice showed similar levels of hepatosplenomegaly, peripheral blood count, and cytokine storm when compared with wild-type (WT) mice after induction of MAS. There were also no significant differences in liver pathology (including inflammatory cell infiltration of CD3 and F4/80) and levels of splenic inflammatory macrophages and effector memory T cells between the WT and knockout mice. These results suggested that IL-33 decreased in the liver and spleen tissues of MAS mice. Further results suggest that IL-33 and St2 knockout mice have no treatment potential in CpG-induced MAS. Thus, the IL-33/ST2 axis has little effect on the prognosis of CpG-induced MAS.

The gut microbiome changes in wild type and IL-18 knockout mice after 9.0 Gy total body irradiation

BackgroundRecent studies have shown that gut microbiome plays important roles in response to radiation exposure. IL-18, an inflammatory cytokine, is highly elevated in mice, mini-pigs and nonhuman primates after radiation exposure. Blocking IL-18 using its endogenous binding protein (IL-18BP) increases mice survival after radiation exposure by decreasing bone marrow interferon-gamma levels.MethodsTo further characterize the roles of IL-18 in response to radiation, both wild type and IL-18 knockout (IL-18 KO) mice were exposed to 9.0 Gy total body irradiation (TBI). The 30-day survival result demonstrated that IL-18 KO mice were significantly more resistant to radiation compared to the wild type mice (p < 0.0001). Mouse faecal samples were collected at pre-radiation (d0), d1, d3, d7, d14, d21 and d29 after radiation exposure. Microbiome profiling was performed on the faecal samples using 16S and ITS sequencing technology.ResultsData analysis showed that there was significant difference in the bacterial microbiome between wild type and IL-18 KO mice. Cohousing of wild type and IL-18 KO mice decreased the bacterial microbiome difference between the two genotypes. Much fewer bacterial genera were significantly changed in wild type mice than the IL-18 KO mice after radiation exposure. The different composition of the IL-18 KO mice and wild type mice persisted even after radiation exposure. Bacterial genera that significantly correlated with other genera were identified in the IL-18 KO and wild type mice. The metabolic pathways that differentially expressed in both genotypes were identified. The animal bacterial microbiome data could be used to predict the animal’s radiation status. The fungal microbiome had no significant difference regarding genotype or time after radiation exposure.ConclusionThe current study helps understand the gut microbiome in different genetic backgrounds and its temporal changes after radiation exposure. Our data provide insight into the mechanisms underlying radiation-induced toxicity and help identify bacteria important in response to radiation.

CSF-1R+ Macrophages Control the Gut Microbiome-Enhanced Liver Invariant NKT Function through IL-18.

The gut microbiome is an important modulator of the host immune system. In this study, we found that altering the gut microbiome by oral vancomycin increases liver invariant NKT (iNKT) cell function. Enhanced iNKT cytokine production and activation marker expression were observed in vancomycin-treated mice following both Ag-specific and Ag-independent invivo iNKT stimulations, with a more prominent effect in the liver than in the spleen. Fecal transplantation studies demonstrated that the iNKT functional regulation is mediated by altering the gut microbiome but uncoupled from the modulation of iNKT cell population size. Interestingly, when stimulated invitro, iNKT cells from vancomycin-treated mice did not show increased activation, suggesting an indirect regulation. iNKT cells expressed high levels of IL-18 receptor, and vancomycin increased the expression of IL-18 in the liver. Blocking IL-18 by neutralizing Ab or using genetically deficient mice attenuated the enhanced iNKT activation. Liver macrophages were identified as a major source of IL-18. General macrophage depletion by clodronate abolished this iNKT activation. Using anti-CSF-1R depletion or LyzCrexCSF-1RLsL-DTR mice identified CSF-1R+ macrophages as a critical modulator of iNKT function. Vancomycin treatment had no effect on iNKT cell function invivo in IL-18 knockout macrophage reconstituted mice. Together, our results demonstrate that the gut microbiome controls liver iNKT function via regulating CSF-1R+ macrophages to produce IL-18.

A gut-restricted glutamate carboxypeptidase II inhibitor reduces monocytic inflammation and improves preclinical colitis.

There is an urgent need to develop therapeutics for inflammatory bowel disease (IBD) because up to 40% of patients with moderate-to-severe IBD are not adequately controlled with existing drugs. Glutamate carboxypeptidase II (GCPII) has emerged as a promising therapeutic target. This enzyme is minimally expressed in normal ileum and colon, but it is markedly up-regulated in biopsies from patients with IBD and preclinical colitis models. Here, we generated a class of GCPII inhibitors designed to be gut-restricted for oral administration, and we interrogated efficacy and mechanism using in vitro and in vivo models. The lead inhibitor, (S)-IBD3540, was potent (half maximal inhibitory concentration = 4 nanomolar), selective, gut-restricted (AUCcolon/plasma > 50 in mice with colitis), and efficacious in acute and chronic rodent colitis models. In dextran sulfate sodium-induced colitis, oral (S)-IBD3540 inhibited >75% of colon GCPII activity, dose-dependently improved gross and histologic disease, and markedly attenuated monocytic inflammation. In spontaneous colitis in interleukin-10 (IL-10) knockout mice, once-daily oral (S)-IBD3540 initiated after disease onset improved disease, normalized colon histology, and attenuated inflammation as evidenced by reduced fecal lipocalin 2 and colon pro-inflammatory cytokines/chemokines, including tumor necrosis factor-α and IL-17. Using primary human colon epithelial air-liquid interface monolayers to interrogate the mechanism, we further found that (S)-IBD3540 protected against submersion-induced oxidative stress injury by decreasing barrier permeability, normalizing tight junction protein expression, and reducing procaspase-3 activation. Together, this work demonstrated that local inhibition of dysregulated gastrointestinal GCPII using the gut-restricted, orally active, small-molecule (S)-IBD3540 is a promising approach for IBD treatment.

Diet high in linoleic acid dysregulates the intestinal endocannabinoid system and increases susceptibility to colitis in Mice

ABSTRACT Inflammatory bowel disease (IBD) is a multifactorial disease with increasing incidence in the U.S. suggesting that environmental factors, including diet, are involved. It has been suggested that excessive consumption of linoleic acid (LA, C18:2 omega-6), which must be obtained from the diet, may promote the development of IBD in humans. To demonstrate a causal link between LA and IBD, we show that a high fat diet (HFD) based on soybean oil (SO), which is comprised of ~55% LA, increases susceptibility to colitis in several models, including IBD-susceptible IL10 knockout mice. This effect was not observed with low-LA HFDs derived from genetically modified soybean oil or olive oil. The conventional SO HFD causes classical IBD symptoms including immune dysfunction, increased intestinal epithelial barrier permeability, and disruption of the balance of isoforms from the IBD susceptibility gene Hepatocyte Nuclear Factor 4α (HNF4α). The SO HFD causes gut dysbiosis, including increased abundance of an endogenous adherent invasive Escherichia coli (AIEC), which can use LA as a carbon source. Metabolomic analysis shows that in the mouse gut, even in the absence of bacteria, the presence of soybean oil increases levels of LA, oxylipins and prostaglandins. Many compounds in the endocannabinoid system, which are protective against IBD, are decreased by SO both in vivo and in vitro. These results indicate that a high LA diet increases susceptibility to colitis via microbial and host-initiated pathways involving alterations in the balance of bioactive metabolites of omega-6 and omega-3 polyunsaturated fatty acids, as well as HNF4α isoforms.

The role of interleukin-18 and interleukin-18 binding protein in K/BxN serum transfer-induced arthritis.

Interleukin-18 is a proinflammatory cytokine, the activity of which is regulated by its natural inhibitor, IL-18 binding protein (IL-18BP). Elevated circulating levels of IL-18 have been observed in patients with systemic juvenile idiopathic arthritis (sJIA) and adult-onset Still's disease (AOSD), two conditions associated with dysregulated innate immune responses. This study examines the expression and function of IL-18 and IL-18BP in K/BxN serum transfer arthritis (STA), a model that is uniquely dependent on innate immune responses. Naïve and serum transfer-induced arthritis (STA) wild-type (WT) mice were used to examine the articular levels of IL-18 and IL-18BP mRNA by RT-qPCR. The cellular sources of IL-18BP in the joints were determined by using Il18bp-tdTomato reporter knock-in mice. The incidence and severity of arthritis, including mRNA levels of different cytokines, were compared in IL-18BP or IL-18 knock-out (KO) mice and their WT littermates. IL-18 and IL-18BP mRNA levels were significantly increased in arthritic as compared to normal joints. Synovial neutrophils, macrophages, and endothelial cells represented the cellular sources of IL-18BP in arthritic joints, whereas IL-18BP production was limited to endothelial cells in non-inflamed joints. The incidence and severity of arthritis were similar in IL-18BP KO and IL-18 KO compared to their WT littermates. Transcript levels of different inflammatory cytokines were not different in the two KO mouse lines compared to WT mice. Although IL-18 and IL-18BP levels were increased in arthritic joints, our results show that the IL-18/IL-18BP balance is not involved in the regulation of STA.

Endogenous interleukin-22 prevents cardiac rupture after myocardial infarction in mice.

Myocardial infarction (MI) can result in fatal myocardial rupture or heart failure due to adverse remodeling and dysfunction of the left ventricle. Although recent studies have shown that exogenous interleukin (IL)-22 shows cardioprotective effect after MI, the pathophysiological significance of endogenous IL-22 is unknown. In this study, we investigated the role of endogenous IL-22 in a mouse model of MI. We produced MI model by permanent ligation of the left coronary artery in wild-type (WT) and IL-22 knock-out (KO) mice. The post-MI survival rate was significantly worse in IL-22KO mice than in WT mice due to a higher rate of cardiac rupture. Although IL-22KO mice exhibited a significantly greater infarct size than WT mice, there was no significant difference in left ventricular geometry or function between WT and IL-22KO mice. IL-22KO mice showed increase in infiltrating macrophages and myofibroblasts, and altered expression pattern of inflammation- and extracellular matrix (ECM)-related genes after MI. While IL-22KO mice showed no obvious changes in cardiac morphology or function before MI, expressions of matrix metalloproteinase (MMP)-2 and MMP-9 were increased, whereas that of tissue inhibitor of MMPs (TIMP)-3 was decreased in cardiac tissue. Protein expression of IL-22 receptor complex, IL-22 receptor alpha 1 (IL-22R1) and IL-10 receptor beta (IL-10RB), were increased in cardiac tissue 3 days after MI, regardless of the genotype. We propose that endogenous IL-22 plays an important role in preventing cardiac rupture after MI, possibly by regulating inflammation and ECM metabolism.

Maresin 1 activates brown adipose tissue and promotes browning of white adipose tissue in mice

ObjectiveMaresin 1 (MaR1) is a docosahexaenoic acid-derived proresolving lipid mediator with insulin-sensitizing and anti-steatosis properties. Here, we aim to unravel MaR1 actions on brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning. MethodsMaR1 actions were tested in cultured murine brown adipocytes and in human mesenchymal stem cells (hMSC)-derived adipocytes. In vivo effects of MaR1 were tested in diet-induced obese (DIO) mice and lean WT and Il6 knockout (Il6−/−) mice. ResultsIn cultured differentiated murine brown adipocytes, MaR1 reduces the expression of inflammatory genes, while stimulates glucose uptake, fatty acid utilization and oxygen consumption rate, along with the upregulation of mitochondrial mass and genes involved in mitochondrial biogenesis and function and the thermogenic program. In Leucine Rich Repeat Containing G Protein-Coupled Receptor 6 (LGR6)-depleted brown adipocytes using siRNA, the stimulatory effect of MaR1 on thermogenic genes was abrogated. In DIO mice, MaR1 promotes BAT remodeling, characterized by higher expression of genes encoding for master regulators of mitochondrial biogenesis and function and iBAT thermogenic activation, together with increased M2 macrophage markers. In addition, MaR1-treated DIO mice exhibit a better response to cold-induced BAT activation. Moreover, MaR1 induces a beige adipocyte signature in inguinal WAT of DIO mice and in hMSC-derived adipocytes. MaR1 potentiates Il6 expression in brown adipocytes and BAT of cold exposed lean WT mice. Interestingly, the thermogenic properties of MaR1 were abrogated in Il6−/− mice. ConclusionsThese data reveal MaR1 as a novel agent that promotes BAT activation and WAT browning by regulating thermogenic program in adipocytes and M2 polarization of macrophages. Moreover, our data suggest that LGR6 receptor is mediating MaR1 actions on brown adipocytes, and that IL-6 is required for the thermogenic effects of MaR1.

Magnesium increases numbers of Foxp3+ Treg cells and reduces arthritis severity and joint damage in an IL-10-dependent manner mediated by the intestinal microbiome.

Rheumatoid arthritis (RA) is a common autoimmune disease with emerging environmental and microbiome risk factors. The western diet is typically deficient in magnesium (Mg), and there is some evidence suggesting that Mg may have anti-inflammatory properties. But the actual role of Mg supplementation in arthritis or in T cell subsets has not been explored. We investigated the role of a high Mg diet in two different mouse models of RA induced with the KRN serum, and collagen-induced arthritis. We also characterized the phenotypes of splenocytes, gene expression, and an extensive intestinal microbiome analyses including fecal material transplantation (FMT). The high Mg diet group was significantly protected with reduced arthritis severity and joint damage, and reduced expression of IL-1β, IL-6, and TNFα. The high Mg group also had increased numbers of Foxp3+ Treg cells and IL-10-producing T cells. The high Mg protective effect disappeared in IL-10 knockout mice. FMT from the high Mg diet mice recreated the phenotypes seen in the diet-treated mice, with reduced arthritis severity, increased Foxp3+ Treg, and increased IL-10-producing T cells. Intestinal microbiome analyses using 16S rDNA sequencing revealed diet-specific changes, including reduced levels of RA-associated Prevotella in the high Mg group, while increasing levels of Bacteroides and other bacteria associated with increased production of short-chain fatty acids. Metagenomic analyses implicated additional pathways including L-tryptophan biosynthesis and arginine deiminase. We describe a new role for Mg in suppressing arthritis, in expanding Foxp3+ T reg cells and in the production of IL-10, and show that these effects are mediated by the intestinal microbiome. Our discoveries suggest a novel strategy for modifying the intestinal microbiome to treat RA and other autoimmune and inflammatory diseases. None.