IL-10 Knockout Mice Research Articles

A Small Intestinal Helminth Infection Alters Colonic Mucus and Shapes the Colonic Mucus Microbiome

Infecting humans with controlled doses of small intestinal helminths, such as human hookworm, is proposed as a therapy for the colonic inflammatory disease ulcerative colitis. Strengthening the colonic mucus barrier is a potential mechanism by which small intestinal helminths could treat ulcerative colitis. In this study, we compare C57BL/6 mice infected with the small intestinal helminth Heligmosomoides polygyrus and uninfected controls to investigate changes in colonic mucus. Histology, gene expression, and immunofluorescent analysis demonstrate that this helminth induces goblet cell hyperplasia, and an upregulation of mucin sialylation, and goblet-cell-derived functional proteins resistin-like molecule-beta (RELM-β) and trefoil factors (TFFs), in the colon. Using IL-13 knockout mice, we reveal that these changes are predominantly IL-13-dependent. The assessment of the colonic mucus microbiome demonstrates that H. polygyrus infection increases the abundance of Ruminococcus gnavus, a commensal bacterium capable of utilising sialic acid as an energy source. This study also investigates a human cohort experimentally challenged with human hookworm. It demonstrates that TFF blood levels increase in individuals chronically infected with small intestinal helminths, highlighting a conserved mucus response between humans and mice. Overall, small intestinal helminths modify colonic mucus, highlighting this as a plausible mechanism by which human hookworm therapy could treat ulcerative colitis.

Interleukin-22 inhibits right ventricular remodelling by a unique mechanism in a pulmonary artery constriction model

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. Four weeks after PAB, RV function was measured by echocardiography and real-time PCR analysis was performed to measure Col1a1, Col3a1, BNP, and transcriptome analysis was performed. Survival confirmation of mice with moderate pulmonary artery stenosis was also performed up to 56 days. Results IL-22 KO mice had significantly higher 56-day mortality rate compared with WT mice after PAC (p<0.01). Four 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 4 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<0.001). The expressions of Col1a1 and Col3a1 were no difference between in IL22-KO mice and Wild mice. However, BNP, a cardioprotective marker, tended to be lower in IL-22KO mice. The transcriptome analysis also showed that the group of fibrosis-related genes that are elevated in PAC in WT mice were less elevated in IL-22 KO mice. Conclusion These results suggest that IL-22 is involved in RV remodelling during RV pressure loading without a general mechanism.

IL-6 deficiency accelerates cerebral cryptococcosis and alters glial cell responses

Cryptococcus neoformans (Cn) is an opportunistic encapsulated fungal pathogen that causes life-threatening meningoencephalitis in immunosuppressed individuals. Since IL-6 is important for blood-brain barrier support and its deficiency has been shown to facilitate Cn brain invasion, we investigated the impact of IL-6 on systemic Cn infection in vivo, focusing on central nervous system (CNS) colonization and glial responses, specifically microglia and astrocytes. IL-6 knock-out (IL-6−/−) mice showed faster mortality than C57BL/6 (Wild-type) and IL-6−/− supplemented with recombinant IL-6 (rIL-6; 40 pg/g/day) mice. Despite showing early lung inflammation but no major histological differences in pulmonary cryptococcosis progression among the experimental groups, IL-6−/− mice had significantly higher blood and brain tissue fungal burden at 7-days post infection. Exposure of cryptococci to rIL-6 in vitro increased capsule growth. In addition, IL-6−/− brains were characterized by an increased dystrophic microglia number during Cn infection, which are associated with neurodegeneration and senescence. In contrast, the brains of IL-6-producing or -supplemented mice displayed high numbers of activated and phagocytic microglia, which are related to a stronger anti-cryptococcal response or tissue repair. Likewise, culture of rIL-6 with microglia-like cells promoted high fungal phagocytosis and killing, whereas IL-6 silencing in microglia decreased fungal phagocytosis. Lastly, astrogliosis was high and moderate in infected brains removed from Wild-type and IL-6−/− supplemented with rIL-6 animals, respectively, while minimal astrogliosis was observed in IL-6−/− tissue, highlighting the potential of astrocytes in containing and combating cryptococcal infection. Our findings suggest a critical role for IL-6 in Cn CNS dissemination, neurocryptococcosis development, and host defense.

IL-10 deficiency aggravates cell senescence and accelerates BLM-induced pulmonary fibrosis in aged mice via PTEN/AKT/ERK pathway

BackgroundPulmonary fibrosis (PF) is an aging-related progressive lung disorder. The aged lung undergoes functional and structural changes termed immunosenescence and inflammaging, which facilitate the occurrence of fibrosis. Interleukin-10 (IL-10) is a potent anti-inflammatory and immunoregulatory cytokine, yet it remains unclear how IL-10 deficiency-induced immunosenescence participates in the development of PF.MethodsFirstly we evaluated the susceptibility to fibrosis and IL-10 expression in aged mice. Then 13-month-old wild-type (WT) and IL-10 knockout (KO) mice were subjected to bleomycin(BLM) and analyzed senescence-related markers by PCR, western blot and immunohistochemistry staining of p16, p21, p53, as well as DHE and SA-β-gal staining. We further compared 18-month-old WT mice with 13-month-old IL-10KO mice to assess aging-associated cell senescence and inflamation infiltration in both lung and BALF. Moreover, proliferation and apoptosis of alveolar type 2 cells(AT2) were evaluated by FCM, immunofluorescence, TUNEL staining, and TEM analysis. Recombinant IL-10 (rIL-10) was also administered intratracheally to evaluate its therapeutic potential and related mechanism. For the in vitro experiments, 10-week-old naïve pramily lung fibroblasts(PLFs) were treated with the culture medium of 13-month PLFs derived from WT, IL-10KO, or IL-10KO + rIL-10 respectively, and examined the secretion of senescence-associated secretory phenotype (SASP) factors and related pathways.ResultsThe aged mice displayed increased susceptibility to fibrosis and decreased IL-10 expression. The 13-month-old IL-10KO mice exhibited significant exacerbation of cell senescence compared to their contemporary WT mice, and even more severe epithelial-mesenchymal transition (EMT) than that of 18 month WT mice. These IL-10 deficient mice showed heightened inflammatory responses and accelerated PF progression. Intratracheal administration of rIL-10 reduced lung CD45 + cell infiltration by 15%, including a 6% reduction in granulocytes and a 10% reduction in macrophages, and increased the proportion of AT2 cells by approximately 8%. Additionally, rIL-10 significantly decreased α-SMA and collagen deposition, and reduced the expression of senescence proteins p16 and p21 by 50% in these mice. In vitro analysis revealed that conditioned media from IL-10 deficient mice promoted SASP secretion and upregulated senescence genes in naïve lung fibroblasts, which was mitigated by rIL-10 treatment. Mechanistically, rIL-10 inhibited TGF-β-Smad2/3 and PTEN/PI3K/AKT/ERK pathways, thereby suppressing senescence and fibrosis-related proteins.ConclusionsIL-10 deficiency in aged mice leads to accelerated cell senescence and exacerbated fibrosis, with IL-10KO-PLFs displaying increased SASP secretion. Recombinant IL-10 treatment effectively mitigates these effects, suggesting its potential as a therapeutic target for PF.Graphical

Monnieriside A from Evolvulus linarioides promotes antinociceptive effects in models of inflammatory and postoperative pain in male mice

Monnieriside A (MoA) is a chromone isolated from Evolvulus linarioides. This study investigated the antinociceptive potential of MoA in mice. MoA (0.01–100 mg/kg, i.p.) inhibited nociception in the inflammatory phase of the formalin test without causing motor impairment. MoA (0.1–100 mg/kg, i.p.) also reduced hindpaw mechanical allodynia caused by either intraplantar injection of Complete Freund’s Adjuvant (CFA) or surgical paw incision to simulate postoperative pain. Postoperative antinociception was accompanied by reduced IL-1β levels in the incised paw, assessed by ELISA. The antinociceptive action of MoA (100 mg/kg, i.p.) was preserved in IL-10 knockout mice submitted to paw incision, indicating that IL-10 is not essential to the antinociceptive effect. Interestingly, MoA (100 mg/kg, i.p.) increased the expression of TGF-β in IL-10 knockout mice, which could be a compensation mechanism leading to an antinociceptive effect in the absence of IL-10.

Positive Effect of Peptides Obtained from Nile Tilapia (Oreochromis niloticus) on Inflammation Regulation and Wound Healing

This study investigated the effect of natural compounds from Nile tilapia (Oreochromis niloticus) skin on wound healing in IL-10 knockout mice. The healing fraction, Fraction T19, was obtained through hydrolysis with trypsin. In vitro, T19 was not cytotoxic to RAW 264.7 macrophage cells, promoting increased cell proliferation and migration. In vivo, mice (n = 30) were divided into three groups with 12 mm wounds in the dorsal region: control (distilled water), T1 (T19 at 125 μg/mL), and T2 (T19 at 250 μg/mL). Daily applications were performed, with tissue removal after nine days. The results showed that T19 increased the production of nitric oxide (NO) and hydrogen peroxide (H2O2), preventing wound contamination. There was an increase in pro-inflammatory (IL-2 and IFNγ) and anti-inflammatory (IL-4) cytokines, as well as cell proliferation markers (PCNA and KI67). Antibodies CD31, CD163, and COX-2 indicated an increase in the formation of new vessels and a reduction in inflammation. Both groups treated with T19 showed better healing results, with better effects observed at higher doses. It was concluded that T19 can effectively modulate the skin repair process and represent an alternative therapeutic for improving the quality of wound skin, especially in the clinical context. Formulations using tilapia skin are safe and effective for accelerating wound healing.

Different mechanisms guide the antinociceptive effect of bone marrow-mononuclear cells and bone marrow-mesenchymal stem/stromal cells in trigeminal neuralgia

AimsTrigeminal neuralgia (TN) is a type of chronic orofacial pain evoked by trivial stimuli that manifests as episodes of excruciating and sudden, recurrent paroxysmal pain. Most patients are refractory to pharmacological therapy used for the treatment of TN. Mononuclear cells (MNC) and mesenchymal stem/stromal cells (MSC) have shown therapeutic potential in painful neuropathies, but their mechanism of action is not fully understood. The present work aimed to investigate the antinociceptive effect and mechanism of action of MNC and MSC in experimental TN. Materials and methodsMice submitted to the chronic constriction injury of the infraorbital nerve (CCI-ION) mouse model of TN received a single intravenous injection of saline, MNC, or MSC (1 × 106 cells/mouse). The effect of the treatments on the behavioral signs of painful neuropathy, morphological aspects of the infraorbital nerve, and inflammatory and oxidative stress markers in the infraorbital nerve were assessed. Key findingsMNC and MSC improved behavioral painful neuropathy, activated key cell signaling antioxidant pathways by increasing Nrf2 expression, and reduced the proinflammatory cytokines IL-1β and TNF-α. However, treatment with MSC, but not MNC, was associated with a sustained increase of IL-10 and with the re-establishment of the morphometric pattern of the infraorbital nerve, indicating a difference in the mechanism of action between MNC and MSC. In line with this result, in IL-10 knockout mice, MSC transplantation did not induce an antinociceptive effect. SignificanceImportantly, these data suggest an IL-10-induced disease-modifying profile related to MSC treatment and reinforce cell therapy's potential in treating trigeminal neuralgia.

Study on the protection of gingival epithelial barrier by interleukin-22 through regulating microbiota and E-cadherin expression

Objective: To investigate the regulatory effect and mechanism of interleukin-22 (IL-22) on the gingival epithelial barrier in the context of periodontal inflammation. Methods: IL-22 knockout (IL-22 KO) mice were constructed, and periodontitis mice models were established through oral gavage with polymicrobial inoculation. DNAs were extracted from the oral plaques of IL-22 KO periodontitis mice group (n=7) and their wild-type littermates periodontitis group (n=7) to establish a periodontitis-related oral microbiota database"PD-RiskMicroDB", determining the relationship between changes in oral microbiota and microbial function in two groups using 16S rRNA sequencing results. Gingival epithelial cells (GEC) were cultured by modified trypsinization method, and were stimulated with 100 μg/L IL-22, Porphyromonas gingivalis (Pg) (multiplicity of infection:100), separately or together for 3 and 12 hours. The experimental groups were as follows: control group (no stimulation), IL-22 group, Pg group and Pg+IL-22 group. The expression of barrier protein E-cadherin in each group at 3 h was detected by immunofluorescence, real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting. Fluorescein isothiocyanate-dextran-mediated epithelial cell permeability experiment was conducted to clarify the changes in permeability of GEC in each group at 3 and 12 h. The mRNA expressions of E-cadherin in the gingival epithelium of wild-type littermates periodontitis group and IL-22 KO periodontitis group were detected by RT-qPCR. Fifteen C57BL/6 wild-type mice were randomly divided into control group (n=5), periodontitis group (n=5) and periodontitis+IL-22 treatment group (n=5). RT-qPCR and immunohistochemistry (IHC) staining were used to detect the expression level of E-cadherin in the gingival epithelium of each group. Results: 16S rRNA sequencing results showed that the composition of oral microbiota changed in IL-22 KO periodontitis group, of which the abundance of bacterial genera related to periodontal tissue invasion was significantly increased (linear discriminant analysis score: 2.22, P=0.009), compared with wild-type littermates periodontitis group. In vitro cell experiments showed that after Pg infection for 3 hours, the cell connections of GEC in Pg group were interrupted, and the fluorescence intensity of E-cadherin was reduced in Pg group compared with the control group. Meanwhile, the mRNA and protein expression levels of E-cadherin (mRNA: 0.69±0.12; protein: 0.60±0.12) were downregulated compared with the control group [mRNA: 1.00±0.00 (P=0.043); protein: 1.04±0.08 (P=0.003)], respectively. The fluorescence intensity of E-cadherin in the Pg+IL-22 group was enhanced compared with Pg group, and expression levels of E-cadherin mRNA (1.16±0.10) and protein (0.98±0.07) in Pg+IL-22 group showed a significant increase compared with Pg group [mRNA: 0.69±0.12 (P=0.005); protein: 0.60±0.12 (P=0.007)]. The result of epithelial permeability test showed that there was no statistical difference in epithelial permeability among control group, Pg group, IL-22 group and Pg+IL-22 group with treatment for 3 hours (F=0.20, P=0.893). While when the treatment time turned to be 12 hours, the epithelial barrier permeability showed a significant increase in Pg group (1.39±0.15) compared with control group (1.00±0.00, P=0.027), and a decrease in Pg+IL-22 group (1.02±0.18) compared with Pg group (1.39±0.15, P=0.034). In vivo, the mRNA expression of E-cadherin in the gingival epithelium of IL-22 KO periodontitis group decreased significantly (0.32±0.21) compared with wild-type littermates periodontitis group (1.01±0.01) (t=5.70, P=0.005). Moreover, RT-qPCR and IHC staining results showed that the mRNA expression level of E-cadherin (0.40±0.07) and absorbance value of E-cadherin positive expression (0.02±0.00) in gingival epithelial tissue of periodontitis group were both significantly down-regulated compared with control group [mRNA: 1.00±0.00 (P=0.005); absorbance value of E-cadherin positive expression: 0.04±0.01 (P=0.006)]. Meanwhile, the mRNA expression level of E-cadherin (1.06±0.24) and the absorbance value of E-cadherin positive expression (0.03±0.01) were both observed increase in periodontitis+IL-22 treatment group compared with periodontitis group (P=0.003, P=0.039). Conclusions: IL-22 may exert a protective effect on the gingival epithelial barrier in an inflammatory environment by regulating the invasiveness of oral microbiota and the expression of host barrier protein.

IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells.

IL-2Rα knock out (KO) mice have been instrumental to discovering the immunoregulatory properties of IL-2Rα. While initially thought of only as a stimulatory cytokine, IL-2 and IL-2Rα KO mice revealed that this cytokine-receptor system controls immune responses through restimulation-induced cell death and by promoting the survival of T regulatory cells. Although described mostly in the context of lymphocytes, recent studies by our laboratory showed that IL-2R is expressed in smooth muscle cells. Given this finding, we sought to use IL-2Rα KO to determine the function of this receptor in vascular smooth muscle cells. Surprisingly, we found that IL-2Rα KO vascular smooth muscle cells had detectable IL-2Rα. We used multiple gene and protein-based methods to determine why IL-2Rα KO vascular smooth muscle cells exhibited IL-2Rα protein. These methods included: genomic sequencing, assessing cells and tissues for evidence of maternal microchimerism, and determining the half-life of IL-2Rα protein. Our studies demonstrated the following: (1) in addition to the cell surface, IL-2Rα is localized to the nucleus; (2) the genetic deletion of IL-2Rα is intact in IL-2Rα KO mice; (3) both IL-2Rα KO and WT tissues show evidence of maternal microchimerism, the likely source of IL-2Rα (4) IL-2Rα is transmitted between cells; (5) IL-2Rα has a long half-life; and (6) nuclear IL-2Rα contributes to the regulation of cell proliferation and size. Our findings suggest that the phenotype of complete IL-2Rα loss is more severe than demonstrated by IL-2Rα KO mice, and that IL-2Rα plays a here-to-fore unrecognized role in regulating cell proliferation in non-lymphoid cells.

The Role of Interleukin-6 in Regulating Glomerular Filtration Rate during Angiotensin II and High Salt Conditions

Previous results demonstrate that interleukin-6 (IL-6) increases mean arterial pressure (MAP) during Angiotensin II (Ang II)-salt hypertension. In addition, albumin excretion was lower in IL-6 knockout mice (KO) when compared to wild-type (WT). This study investigated the role of IL-6 on regulating glomerular filtration rate (GFR), renal plasma flow (RPF) and MAP in WT and IL-6 KO mice treated with a slow pressor dose of Ang II (200 ng/kg/min) +/- 6% high-salt (HS) diet. Male C57BL6 and IL-6 KO mice (12 weeks old) were treated with Ang II +/- 6% HS diet for 12 – 14 days. Mean arterial pressure, RPF and GFR were measured in anesthetized mice. MAP was 130 ± 7 mmHg and 91 ± 4 mmHg in control WT and IL-6 KO mice, respectively. Ang II treatment increased MAP in WT (153 ± 5 mmHg) and no change in IL-6 KO (83 ± 4 mmHg) mice. Ang II + 6% HS increased MAP to 150 ± 11 mmHg in WT and 93 ± 4 mmHg in IL-6 KO mice. Baseline RPF was 1822 ± 229 and 1912 ± 402 ml/min/g in control WT and IL-6 KO mice, respectively. Ang II treatment increased RPF in WT (3156 ± 753 ml/min/g), while lowering RPF in IL-6 KO (1648 ± 422 ml/min/g) mice. RPF during Ang II + 6% HS treatment was decreased in WT (1095 ± 305 ml/min/g) and IL-6 KO (1133 ml/min/g) mice. GFR was 756 ± ml/min/g and 788 ± ml/min/g in control WT and IL-6 KO mice, respectively. Ang II increased GFR in WT (1009 ± 63 ml/min/g) and no change in IL-6 KO (756 ± 23 ml/min/g). Ang II + 6% HS increased GFR in WT (1095 ± 146 ml/min/g) and no change in GFR of IL-6 KO (540 ± 207 ml/min/g) mice. Our results suggest that IL-6 reduces MAP during baseline, Ang II, and Ang II + 6% HS. The absence of IL-6 prevented increases in MAP, RPF and GFR during Ang II treatment. Our data also suggest that IL-6 contributes to increased MAP and GFR during Ang II + 6% HS treatment. K01HL092593-05. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Validation of a Microbiome Dependent Colitis Model via Fecal Microbial Transplant in Germ-Free IL-10 Deficient Mice

Abstract Inflammatory Bowel Diseases (IBD) are immune-mediated intestinal tract diseases with undefined etiologies. Proposed pathogenic mechanisms include abnormal inflammatory response to the intestinal microbiome. Mice deficient in IL-10, a critical cytokine for mucosal immune homeostasis, spontaneously develop enterocolitis; phenotypes are microbiome-sensitive. To establish a microbiome-dependent colitis model with improved construct validity and translational clinical relevance, we assessed colitis development in Germ-Free IL-10 knockout mice (GF IL-10 KO; Taconic Biosciences) following inoculation with fecal microbial transplant (FMT) from wild-type C57BL/6NTac mice at the Murine Pathogen Free™ (MPF™) health standard and assessed clinical responses to anti-IL-12/23p40 (anti-p40). Compared to control GF IL-10 KO mice, FMT-inoculated mice demonstrated reduced weight gain, elevated levels of fecal lipocalin 2 and calprotectin, and increased colon weight:length ratio at 8 weeks following FMT. FMT-inoculated mice also showed elevated histopathology scores, increased colonic CD3+ T cells and CD4+ TH cells, and epithelial hyperplasia by Ki-67 immunolabeling. Several colon cytokines were elevated in FMT-inoculated mice. Anti-p40 treatment significantly reduced all of these disease indicators with the exception of colon weight:length. These data provide a validated colitis model with relevant mechanisms for assessing the role of the microbiome and response to therapeutics in IBD.

Ginsenoside Re protects against kainate-induced neurotoxicity in mice by attenuating mitochondrial dysfunction through activation of the signal transducers and activators of transcription 3 signaling

It was demonstrated that ginsenosides exert anti-convulsive potentials and interleukin-6 (IL-6) is protective from excitotoxicity induced by kainate (KA), a model of temporal lobe epilepsy. Ginsenosides-mediated mitochondrial recovery is essential for attenuating KA-induced neurotoxicity, however, little is known about the effects of ginsenoside Re (GRe), one of the major ginsenosides. In this study, GRe significantly attenuated KA-induced seizures in mice. KA-induced redox changes were more evident in mitochondrial fraction than in cytosolic fraction in the hippocampus of mice. GRe significantly attenuated KA-induced mitochondrial oxidative stress (i.e. increases in reactive oxygen species, 4-hydroxynonenal, and protein carbonyl) and mitochondrial dysfunction (i.e. the increase in intra-mitochondrial Ca2+ and the decrease in mitochondrial membrane potential). GRe or mitochondrial protectant cyclosporin A restored phospho-signal transducers and activators of transcription 3 (STAT3) and IL-6 levels reduced by KA, and the effects of GRe were reversed by the JAK2 inhibitor AG490 and the mitochondrial toxin 3-nitropropionic acid (3-NP). Thus, we used IL-6 knockout (KO) mice to investigate whether the interaction between STAT3 and IL-6 is involved in the GRe effects. Importantly, KA-induced reduction of manganese superoxide dismutase (SOD-2) levels and neurodegeneration (i.e. astroglial inhibition, microglial activation, and neuronal loss) were more prominent in IL-6 KO than in wild-type (WT) mice. These KA-induced detrimental effects were attenuated by GRe in WT and, unexpectedly, IL-6 KO mice, which were counteracted by AG490 and 3-NP. Our results suggest that GRe attenuates KA-induced neurodegeneration via modulating mitochondrial oxidative burden, mitochondrial dysfunction, and STAT3 signaling in mice.

Effect of Dietary Supplementation with Omega-3 Fatty Acid on the Generation of Regulatory T Lymphocytes and on Antioxidant Parameters and Markers of Oxidative Stress in the Liver Tissue of IL-10 Knockout Mice.

chronic low-grade inflammation, or inflammaging, emerges as a crucial element in the aging process and is associated with cardiovascular and neurological diseases, sarcopenia, and malnutrition. Evidence suggests that omega-3 fatty acids present a potential therapeutic agent in the prevention and treatment of inflammatory diseases, mitigating oxidative stress, and improving muscle mass, attributes that are particularly relevant in the context of aging. The objective of the present study was to evaluate the effectiveness of supplementation with omega-3 fish oil in improving the immune response and oxidative stress in knockout mice for interleukin IL-10 (IL-10-/-). female C57BL/6 wild-type (WT) and interleukin IL-10 knockout (IL-10-/-) mice were fed during 90 days with a standard diet (control groups), or they were fed/supplemented with 10% of the omega-3 polyunsaturated fatty acid diet (omega-3 groups). Muscle, liver, intestinal, and mesenteric lymph node tissue were collected for analysis. the IL-10-/-+O3 group showed greater weight gain compared to the WT+O3 (p = 0.001) group. The IL-10-/-+O3 group exhibited a higher frequency of regulatory T cells than the IL-10-/- group (p = 0.001). It was found that animals in the IL-10-/-+O3 group had lower levels of steatosis when compared to the IL-10-/- group (p = 0.017). There was even greater vitamin E activity in the WT group compared to the IL-10-/-+O3 group (p = 0.001) and WT+O3 compared to IL-10-/-+O3 (p = 0.002), and when analyzing the marker of oxidative stress, MDA, an increase in lipid peroxidation was found in the IL-10-/-+O3 group when compared to the IL-10-/- group (p = 0.03). Muscle tissue histology showed decreased muscle fibers in the IL-10-/-+O3, IL-10-/-, and WT+O3 groups. the findings show a decrease in inflammation, an increase in oxidative stress markers, and a decrease in antioxidant markers in the IL-10-/-+O3 group, suggesting that supplementation with omega-3 fish oil might be a potential intervention for inflammaging that characterizes the aging process and age-related diseases.

Effects of interleukin-15 on autophagy regulation in the skeletal muscle of mice.

This study aimed to evaluate the role of skeletal muscle-derived interleukin (IL)-15 in the regulation of skeletal muscle autophagy using IL-15 knockout (KO) and transgenic (TG) mice. Male C57BL/6 wild-type (WT), IL-15 KO, and IL-15 TG mice were used in this study. Changes in muscle mass, forelimb grip strength, succinate dehydrogenase (SDH) activity, gene and protein expression levels of major regulators and indicators of autophagy, comprehensive gene expression, and DNA methylation in the gastrocnemius muscle were analyzed. Enrichment pathway analyses revealed that the pathology of IL-15 gene deficiency was related to the autophagosome pathway. Moreover, although IL-15 KO mice maintained gastrocnemius muscle mass, they exhibited a decrease in autophagy induction. IL-15 TG mice exhibited a decrease in gastrocnemius muscle mass and an increase in forelimb grip strength and SDH activity in skeletal muscle. In the gastrocnemius muscle, the ratio of phosphorylated adenosine monophosphate-activated protein kinase α (AMPKα) to total AMPKα and unc-51-like autophagy activating kinase 1 and Beclin1 protein expression were higher in the IL-15 TG group than in the WT group. IL-15 gene deficiency induces a decrease in autophagy induction. In contrast, IL-15 overexpression could improve muscle quality by activating autophagy induction while decreasing muscle mass. The regulation of IL-15 in autophagy in skeletal muscles may lead to the development of therapies for the autophagy-induced regulation of skeletal muscle mass and cellular quality control.NEW & NOTEWORTHY IL-15 gene deficiency can decrease autophagy induction. However, although IL-15 overexpression induced a decrease in muscle mass, it led to an improvement in muscle quality. Based on these results, understanding the role of IL-15 in regulating autophagy pathways within skeletal muscle may lead to the development of therapies for the autophagy-induced regulation of skeletal muscle mass and cellular quality control.

Abstract A023: Cytokine loss leads to decreased tumor differentiation and a more aggressive phenotype

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of only 11% and approximately half the patients present with distant metastasis at the time of diagnosis. PDAC is accompanied by pronounced alterations in stromal responses and immune surveillance programs, which are now recognized as some of the major drivers in PDAC tumor evolution. The role of immune modulators in driving aggressiveness of PDAC is poorly understood. We investigate how the loss of host-derived IL-12 and IL-23 in murine models of pancreatic cancer can lead to changes in changes in tumor morphology, cellular differentiation, and tumor immune microenvironment. In orthotopic tumors isolated from IL-12 and IL-23 knockout mice tumor morphology is altered and appears to be less differentiated. Noticeably, ductal area decreases in tumors from IL-23 and IL-12 knockout mice. Single cell RNA sequencing of tumors isolated from IL-12 and IL-23 knockout mice reveals a cluster of tumor cells with decreased gastric gene expression. Gastric genes such as TFF1, TFF2, MUC5AC, GKN1, and GKN2 have been implicated in pancreatic cancer development, progression, and tumor subtype. The classical pancreatic tumor subtype designation correlates with better differentiated pancreatic tumors as opposed to the more poorly differentiated tumors in the basal subtype. The classical expression signature is characterized by the expression of GATA6 and other epithelial cell terminal differentiation genes and includes several of these gastric genes. We hypothesize that loss of IL-12 and IL-23 leads to loss of tumor differentiation and to a more aggressive phenotype. Citation Format: Whitney J. Bell, Nancy Kren, Yan Wang, Jennifer Modliszewski, Yi Xu, Meijiang Guan, Kathleen DelGiorno, Jen Jen Yeh, Yuliya Pylayeva-Gupta. Cytokine loss leads to decreased tumor differentiation and a more aggressive phenotype [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A023.

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.

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.

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.