IL-10 KO Mice Research Articles

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

O47 THE ROLE OF IL-33 AND ST2 IN THE MODULATION OF TARGET ORGAN DAMAGE IN ANGIOTENSIN II-DEPENDENT HYPERTENSION

Introduction: Hypertension is associated with a chronic inflammatory state and aberrant immune system activity. Interleukin-33/suppression of tumorigenicity 2 (IL-33/ST2) signalling axis is important in the modulation of atherosclerosis and cardiovascular fibrosis. We aimed to evaluate the modulation of IL-33 and ST2 in hypertensive mouse models and patients. Methods and Results: Hypertension was induced by a 14-day infusion of Ang-II (490ng/min/kg) in 12-week-old WT C57BL/6, ST2KO, IL33KO mice. IL-33 and ST2 mRNA and protein were evaluated using qPCR and Western blot in WT mice. Ang II-infusion significantly increased IL-33 and ST2 protein expression in target organs related to hypertension, particularly the aorta (1.0±0.1 vs 2.8±0.2, p<0.0001, and 0.963±0.068 vs 2.094±0.231, p=0.002, respectively). Further, mRNA expression of IL-33 and ST2 was elevated in hypertensive aortas when compared with control (1.0±0.053 vs 1.76±0.271, p=0.0185 and 1.0±0.039 vs 1.978±0.221, p=0.0039, respectively). IHC from WT mice illustrated the localisation of IL-33 and ST2 in Ang-II aortas in ECs and SMCs. Further in human hypertensive mammary arteries, the localisation was. In-vivo, IL33KO and ST2KO mice showed no altered BP at baseline or after 14 days of Ang-II compared to WT littermates. ST2KO Ang-II mice showed protection against endothelial dysfunction compared to WT Ang-II mice through isometric tension studies (p=0.0317). Additionally, Ang-II-induced IL33KO and ST2KO mice worsen cardiac fibrosis compared to WT littermates (p=0.0005,p<0.0001, respectively). Conclusion: Both IL-33 and its receptor, ST2 are increased in the vascular and perivascular tissues in hypertension. Additionally, the IL-33/ST2 axis has a role in regulating cardiac fibrosis.

Abstract 70: Cellular senescence as a driver of preeclampsia

Background: Preeclampsia (PE) is a severe hypertensive disorder of pregnancy of unknown pathophysiology. We previously have reported the presence of senescence in kidney and fat tissue of PE women, as well as accelerated epigenetic aging, which prompted us to postulate that cellular senescence may drive development of PE. Methods: We prospectively collected sera from pregnant women throughout pregnancy, and using multiplex Luminex analysis, measured 50 markers of the senescence-associated secretory phenotype (SASP). Comparisons were made between women who developed PE (n=10) vs. normotensive pregnancies (n=10). We then injected human PE serum into the IL-10KO mouse to induce PE and assess whether PE is associated with development of senescence. We measured plasma p16, a circulating indicator of cellular senescence, before and after inducing PE. We also compared plasma p16 and expressions of senescence-associated genes in the hearts and aortas of regular pregnant (PG, no PE serum injection) and PE mice (n=4). Results: There was a significant increase in several SASP markers in sera of women who developed PE, beginning in the first trimester, through delivery. Notably, eotaxin, MCP1, GROb, MIF, MMP2, TNFRII, IGFBP2 were significantly increased in PE women compared to their normotensive peers throughout all trimesters. Mice injected with PE serum exhibited all features of PE, as reported earlier. When measuring plasma p16 before and after PE serum injection, animals had higher values upon inducing PE. Plasma samples of PE mice demonstrated higher p16 concentrations compared to the PG group. The aortas of PE mice exhibited increased expressions of senescence associated genes p16, il6, and mcp1, whereas in the hearts, p16, Il1a and il8 were upregulated. Conclusion: The SASP is present very early in human pregnancy, even before PE has developed. Injection of SASP-enriched PE serum into IL-10KO mice induces cellular senescence in different tissues resulting in a PE phenotype. These results suggest that senescence may be an underlying mechanism of PE.

Klebsiella michiganasis as a tissue repairing commensal in gut inflammation

Abstract Commensal bacteria are recognized as contributors for both tissue inflammation and tissue homeostasis. Therefore, we tested their potential for gut inflammation and tissue repairing using Germ-Free (GF) mice using stools from healthy human donors. Although, DSS-treated WT GF mice all die within 10 days due to severe colonic epithelial damage, gnotobiotic WT mice colonized with a healthy microbiome survived and carried more IL-22 producing innate and adaptive immune cells. This suggests that healthy stools contain wound healing commensals, however, IL10 KO mice with the same stools developed colitis, indicating a colitogenic potential. To eliminate colitogenic from wound healing commensals we utilized various antibiotics in human stool inoculated mice. These treated mice showed significant reductions in their microbiomes. Importantly, IFNg/IL-17A double producing pathogenic CD4 T cells were decreased leading to no colitis in gavaged IL10 KO mice. Additionally, gnotobiotic WT mice colonized with cecum contents from treated human stool gavaged IL10 KO survived lethal DSS treatment. We isolated Klebsiella michiganasis (K. michiganasis) from their cecum and confirmed that monocolonization of K. michiganasis in GF WT mice reproduced the wound healing ability via both the IL-23 and IL1b pathways stimulating type 3 innate lymphoid cells to strongly produce IL-22. Our results suggest that non-pathogenic K. michiganasis is a promising commensal for combating gut inflammation.

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.

B cell-derived IL-10 is an autocrine survival factor and maintains immune homeostasis in the spleen

Abstract IL-10 is a key anti-inflammatory cytokine and IL-10 KO mice develop colitis. Mice with a global IL-10 KO or constitutive B cell-specific IL-10 KO (CD19-Cre.IL-10fl/fl) have otherwise normal B cells. Surprisingly, we found that within 14-21 days, mice with a tamoxifen (Tam) -induced B cell specific deletion of IL-10R (hCD20-ERT2-Cre.IL-10Rfl/fl) had a 50% decrease in splenic B cell number vs. Cre-controls. B cell frequency was unchanged due to a concomitant fall in both T cells and innate cells resulting in a 58% fall in splenocyte number. Plasma cells, CD4+ and CD8+ T cells, and neutrophils were disproportionately decreased, resulting in a 20-40% reduction in frequency. A similar but somewhat less severe loss of B cells/splenocytes was seen in Tam-treated hCD20-ERT2-Cre.IL-10fl/fl mice. Adoptive transfer competition studies in immunized µMT hosts treated with Tam revealed a selective loss of hCD20-ERT2-Cre.IL-10fl/fl vs. CD19-Cre.IL-10fl/fl B cells. Furthermore, by day 21, acute loss of B cell IL-10 or IL-10R resulted in ~20% weight loss and colon length, consistent with colitis (pathology pending). In conclusion, IL-10 is an autocrine B cell survival factor and acute loss of B cell IL-10 or IL-10R is associated with a marked contraction of B cells and associated collapse in T cells and innate cells in the spleen, resulting in immune dysregulation and colitis. This requirement for B cell IL-10 has not been previously recognized because it is compensated for developmentally.

A194 EARLY LIFE WESTERN-TYPE DIET ACCELERATES THE ONSET OF MURINE IL-10 KO COLITIS

Abstract Background Early life is a critical time for gut microbiome and immune development, including the establishment of proper host-microbe interactions. While exposures to western world environmental factors are associated with inflammatory bowel disease (IBD) susceptibility, the link between environment in early life and later disease onset is unclear. One of the most important western world environmental factors is diet, which can distinctly alter the gut microbiome. We believe an early life western-type diet (WD) can affect disease progression in a murine IL-10 KO colitis model through dysregulated T-cell responses against the microbiome. Aims We aimed to characterize how an early life WD given to IL-10 KO mice would affect colitis development and the gut microbiome. Methods IL-10 KO mice were housed in specific pathogen free conditions and fed normal chow (NC) or WD ad libitum, either between days 10 to 35 (early WD, eWD) or days 35 to 60 (adolescent WD, aWD). Stool was collected from mice at days 35, 56, and 84 for lipocalin-2 (LCN-2) and 16S rDNA sequencing. Mice were sacrificed at day 84, assessing mesenteric lymph node weight, gene expression, colon damage by histopathology, and colon lamina propria leukocyte (LPL) phenotype and cytokine expression by flow cytometry. Results Compared to NC and aWD, the eWD fed IL-10 KO mice displayed increased fecal LCN-2 at days 56 and 84, indicating greater inflammation. At sacrifice, eWD fed mice had larger mesenteric lymph nodes and more significant colon damage by histopathological scoring. Colon LPL preparations showed that eWD fed IL-10 KO mice had higher proportions and absolute numbers of effector and regulatory T-cells. Additionally, higher proportions and absolute numbers of those T-cells were IFNγ+, IL-17A+, and IL-22+. qPCR of distal colon tissue revealed increased gene expression of innate and type 3 proinflammatory cytokines such as Il1b, Tnfα, and Il23, while showing no change in Il6 and Il4. Taxa positively associated with WD were able to establish a persistent niche in eWD fed mice but not in aWD fed mice. An increase in only one specific taxon was associated with eWD at all timepoints while also most strongly correlating with inflammatory markers. Conclusions This data suggests that eWD feeding during gut microbiome and immune development is uniquely capable of increasing long-term susceptibility to intestinal inflammation in IL-10 KO mice. This appears to be associated with persistent colonization of potentially more inflammatory taxa. This work provides insight into the potential role of early life environmental risk factors, i.e. WD, in the later development of IBD. Funding Agencies CAG, CIHRUniversity of Toronto, Taconic Biosciences

P005 Exosome miR-132-3p derived from Adipose Stem Cells in Creeping fat Regulates RASA1/ERK1/2-dependant Lymphatic Functions and Improve Colitis and Mesenteritis

Abstract Background Creeping fat (CrF) is an important pathological characteristics of Crohn’s disease (CD) and characterized by lymphangiogenesis and abnormal lymphatic functions. Adipose tissue derived stem cells (ADSCs) can regulate surrounding cells function by delivering exosomes (exo). However, whether CrF-ADSC-exo could affect lymphatic endothelium to regulate lymphatic functions, and further influence procession of mesenteritis and colitis remains elusive. Methods Paired CrF and uninvolved mesenteric adipose tissue (uMAT) samples were collected from CD patients during operation, and exosomes were separated by ultra-centrifugation. Human lymphatic endothelial cells (HLECs) were cultured in vitro and evaluated the abilities in proliferation, migration and tube formation. IL-10 KO mice was used for assessing the treatment of CrF-ADSC-exo in experimental colitis and mesenteritis. Results ADSC-derived exosomes from paired CrF and uMAT samples were confirmed to transfer to LEC by immunofluorescence microscope. In vitro, CrF-ADSC-exo effectively stimulated HLECs in proliferation, migration and tube formation. In vivo, CrF-ADSC-exo injection increased lymphangiogenesis in MAT and improved lymphatic drainage, and further alleviated colitis and mesenteritis in IL-10 KO mice. We next adopted high-throughput sequencing of paired CrF-ADSC-exo and uMAT-ADSC-exo, and validated significantly high expression of miR-132-3p in CrF-ADSC-exo. In vitro, upregulation or downregulation of miR-132-3p in HLECs stimulated or inhibited the lymphatic abilities in proliferation, migration and tube formation, respectively. Mechanically, we demonstrated that CrF-ADSC-exo regulated lymphatic function through miR-132-3p-RASA1-ERK1/2 axis by western blot. CrF-ADSC-exo miR-132-3p improved lymphatic drainage function in MAT and reduced inflammatory colon and mesentery in IL-10 KO mice. The higher level of miR-132-3p in CrF from CD patients was tightly correlated with inflammatory mediators expressions when compared with uMAT and healthy MAT. Conclusion Our study discovers that CrF-ADSC-exo improves lymphatic functions and alleviates colitis and mesenteritis through miR-132-3p/RASA1/ERK1/2 axis, which discloses new role of CrF involved in CD procession and provides novel insight for CD treatment.

Role of maraviroc and/or rapamycin in the liver of IL10 KO mice with frailty syndrome.

Cellular senescence and low-grade inflammation favor the acceleration of aging. The liver is an essential metabolic organ because changes related to its function are related to age-related diseases. The objective of this study was to evaluate the effects of maraviroc (MVC) and/or rapamycin (RAPA) on liver tissue in an experimental model of frailty syndrome in mice, since MVC and RAPA are two molecules able to decrease CCR5 expression, which is overexpressed in patients with frailty. Methods: Eighty male homozygous IL10KO mice were randomly assigned to one of 4 groups (n = 20): i) IL10KO group; ii) MVC group, iii) RAPA group, and iv) MVC-RAPA group. Liver samples were analyzed. Gene expression quantification and western blotting were also performed. The proinflammatory cytokines IL-6 and IL-18 were decreased in MVC and MVC/RAPA groups, IL-12 was decreased in RAPA and MVC/RAPA groups and TNF-α was decreased in all therapeutic groups. P21 was decreased in RAPA and MVC/RAPA groups, Galactosidase beta-1, was also significantly reduced in all therapeutic groups, as were NF-kB1, NF-kB2 and STAT3. In all groups, mTOR and CCL5 were significantly reduced. CCR5 expression was decreased in the MVC and MVC/RAPA groups. Conclusion: MVC and RAPA may protect against some factors involved in liver aging. More studies will be necessary to verify their clinical applications.

Ileitis promotes MASLD progression via bile acid modulation and enhanced TGR5 signaling in ileal CD8+ T cells

Clinical evidence substantiates a link between inflammatory bowel disease, particularly Crohn's disease (CD), and metabolic dysfunction-associated steatotic liver disease (MASLD). This study aims to explore the underlying molecular mechanisms responsible for this association. MASLD was induced by administering high-fat and western diets, while inflammatory bowel disease was induced using DSS (dextran sulfate sodium) and the Il10 knockout (KO) mouse model. The investigation into the role of secondary bile acids (SBAs) in ileitis involved employing metagenomic sequencing, conducting metabolomics detection, performing fecal microbiota transplantation, and constructing CD8+ T cell-specific gene knockout mice. In MASLD+DSS and Il10 KO MASLD mice, we observed ileitis characterized by T-cell infiltration and activation in the terminal ileum. This condition resulted in decreased bile acid levels in the portal vein and liver, inhibited hepatic farnesoid X receptor (FXR) activation, and exacerbated MASLD. Metagenomic and metabolomic analysis of ileal contents revealed increased Clostridium proliferation and elevated SBA levels in MASLD-associated ileitis. Experiments using germ-free mice and fecal microbiota transplantation suggested an association between SBA and MASLD-related ileitis. Invitro, SBAs promoted CD8+ T-cell activation via the TGR5, mTOR, and oxidative phosphorylation pathways. Invivo, TGR5 KO in CD8+ T cells effectively alleviated ileitis and reversed the MASLD phenotype. Clinical data further supported these findings, demonstrating a positive correlation between ileitis and MASLD. MASLD-induced changes in intestinal flora result in elevated levels of SBAs in the ileum. In the presence of a compromised intestinal barrier, this leads to severe CD8+ T cell-mediated ileitis through the TGR5/mTOR/oxidative phosphorylation signaling pathway. Ileitis-induced tissue damage impairs enterohepatic circulation, inhibits hepatic FXR activation, and exacerbates the MASLD phenotype. Our study provides a comprehensive investigation of the interplay and underlying mechanisms connecting ileitis and metabolic dysfunction-associated steatotic liver disease (MASLD). Secondary bile acids produced by intestinal bacteria act as the critical link between MASLD and ileitis. Secondary bile acids exert their influence by disrupting liver lipid metabolism through the promotion of CD8+ T cell-mediated ileitis. In future endeavors to prevent and treat MASLD, it is essential to thoroughly account for the impact of the intestinal tract, especially the ileum, on liver function via the enterohepatic circulation.

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.

Genetic deficiencies of both IL-4 receptor alpha chain and IL-10 trigger early onset of severe colitis in mice

Inflammatory bowel diseases are associated with dysregulated inflammatory immune responses in the gastrointestinal tract. We found that deficiencies of both IL-4 receptor alpha chain (IL-4Rα) and IL-10 in BALB/c mice (IL-4Rα × IL-10 KO mice) highly induced spontaneous rectal prolapse and diarrhea. These mice also exhibited severe colitis in their cecum and colon and marked elevation of serum proinflammatory cytokines including TNFα and IFNγ. These pathologies were transmittable with their cecal contents containing Helicobacter spp. Their mesenteric LN cells produced TNFα and IFNγ in response to soluble H. hepaticus antigens and high titers of H. hepaticus-specific serum IgG were also detected. These results suggested the important function of IL-4Rα signaling in controlling the intestinal inflammation and the susceptibility to intestinal microbes including H. hepaticus. Therefore, these IL-4Rα × IL-10 KO mice potentially provide the significant murine model for clarifying the causes and control of spontaneous colitis and intestinal inflammation.

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.

Intestinal Tr1 Cells Confer Protection against Colitis in the Absence of Foxp3+ Regulatory T Cell-Derived IL-10.

The intestinal mucosa is continually exposed to diverse microbial and dietary Ags, requiring coordinated efforts by specialized populations of regulatory T cells (Tregs) to maintain homeostasis. Suppressive mechanisms used by intestinal Tregs include the secretion of anti-inflammatory cytokines such as IL-10 and TGF-β. Defects in IL-10 signaling are associated with severe infantile enterocolitis in humans, and mice deficient in IL-10 or its receptors develop spontaneous colitis. To determine the requirement of Foxp3+ Treg-specific IL-10 for protection against colitis, we generated Foxp3-specific IL-10 knockout (KO) mice (IL-10 conditional KO [cKO] mice). Colonic Foxp3+ Tregs isolated from IL-10cKO mice showed impaired ex vivo suppressive function, although IL-10cKO mice maintained normal body weights and developed only mild inflammation over 30 wk of age (in contrast to severe colitis in global IL-10KO mice). Protection from colitis in IL-10cKO mice was associated with an expanded population of IL-10-producing type 1 Tregs (Tr1, CD4+Foxp3-) in the colonic lamina propria that produced more IL-10 on a per-cell basis compared with wild-type intestinal Tr1 cells. Collectively, our findings reveal a role for Tr1 cells in the gut, as they expand to fill a tolerogenic niche in conditions of suboptimal Foxp3+ Treg-mediated suppression and provide functional protection against experimental colitis.

Wheat Germ Supplementation Attenuates Obesity-Induced Neuroinflammation in the RVLM of IL-10 KO mice

Obesity is known to be a major factor in the increase of cardiovascular risk and inflammatory responses in the brain. It has been found that high-fat diet (HFD)-induced obesity increases sympathetic nerve activity (SNA) through a cascade of chronic metabolic processes such as an increase in oxidative stress and inflammation. The rostral ventrolateral medulla (RVLM) is a specific part of the brainstem that regulates SNA. Wheat germ (WG), a rich source of bioactive compounds including polyunsaturated fatty acid, has been shown to have an anti-inflammatory effect on the gut environment of HFD-fed mice. A healthy gut microbiota has been shown to have a regulatory effect on the gut-brain axis, especially under stress. In this study, we investigated the effects of WG supplementation on RVLM inflammation in high-fat high cholesterol diet (HFHC)-fed IL-10 KO mice. Six-week-old female C57BL/6 (WT) and IL-10 KO mice ( n = 4-5/group) were randomly assigned to treatment groups: WT and IL-10 KO fed a control diet (10% fat kcal), IL-10 KO fed HFHC (43.4% fat kcal, 1% cholesterol) diet with or without 10% WG for 12 weeks. At the end of 12 wks of treatment, animals were sacrificed and RVLM mRNA expression of proinflammatory cytokines and chemokine were analyzed using qPCR. HFHC increased the expression of IL1b, TNFa, and MCP-1 in the RVLM. WG supplementation was able to reverse the mRNA expression of IL1b (p&lt;0.05), TNFa (p&lt;0.05), and MCP1 (p&lt;0.05). These results indicate that HFHC diet-induced obesity increases inflammation in the RVLM and WG supplementation could reverse these effects. Future studies would focus on understanding the mechanisms by which WG reverses the inflammation in the brain. NIH R15HL148844, AHA AIREA959725, and Oklahoma Agriculture Experiment Station 2153907 This is the full abstract presented at the American Physiology Summit 2023 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.

IL-4 role in the development of the lupus mouse model induced by non-bilayer phospholipids arrangements

Abstract Non-bilayer phospholipids arrangements (NPAs) are lipid molecular associations that are different from the bilayer and are transient. However, if they become stable, they can trigger a disease in mice that is similar to human systemic lupus erythematosus. NPAs can be stabilized in liposomes formed by cylindrical and conical lipids or in cell membranes by drugs such as procainamide or chlorpromazine. In wild-type BALB/c mice, this lupus model has been developed by administering liposomes with NPAs induced and stabilized with procainamide or chlorpromazine, by administering the drugs alone or by administering the monoclonal antibody H308; these last two procedures stabilize the NPAs in the membranes of mouse cells. Anti-NPAs, anti-cardiolipin, anti-histones and anti-coagulants auto-antibodies have been detected in the serum of these mice; in addition, the mice present facial lesions and alterations in the skin and kidney. In this work, we evaluated the participation of the T H2 response, through IL-4, which is its most characteristic cytokine, in the development of the NPAs-induced lupus model in mice. Wild type or IL-4 knockout BALB/c mice were administered with liposomes bearing drug-induced NPAs, the drugs alone, or anti-NPA H308 antibody to induce the lupus-like disease. IL-4 KO mice showed minor disease manifestations, compared to wild-type mice, with decreased production of anti-NPA IgG antibodies, no anti-cardiolipin, anti-histones and anticoagulant antibodies, and no kidney or skin lesions. In these mice, H308 was the only inducer of anti-NPA IgG antibodies. These findings suggest that IL-4 has a key role in the development of the murine lupus-like disease induced by NPA stabilization. This study was supported by grants from IPN (SIP20232128, SIP20232132 and SIP20232135).

Oral exposure to Escherichia colitoxin protects against lung allergen-induced inflammation

Abstract Recent studies suggest that the intestinal microenvironment regulate lung immune cells. Conversely, changes in the lung can modulate the gut immune system. Yet, mucosal-associated immune responses have been studied in a compartmentalized manner, as if there is no communication between different organs. Thus, the role of the gut-lung axis in mucosal immune responses is poorly understood. Using enterotoxigenic Escherichia coli (ETEC), we established a model of intestine-localized infection without translocation to any other tissue. In response to ETEC, type 2 innate lymphoid cells (ILC2s) and eosinophils accumulated in the intestinal lamina propria, even after bacterial clearance. In addition, oral ETEC transiently increased the numbers of ILC2 and eosinophils in the lung parenchyma. To test if oral ETEC promote lung immune responses to allergen, we exposed mice that recently cleared ETEC to intranasal papain/ovalbumin allergy induction model. Unexpectedly, ETEC-exposed mice were significantly resistant to allergen-induced lung inflammation, with reduced number of ILC2, Type 2 effector CD4 +T cells (Th2) and eosinophils. Paradoxically, oral ETEC-induced protection against lung allergen exposure was dependent on IL-33: IL-33KO mice developed partial lung inflammation in this model, and no protection induced by ETEC exposure was observed. Importantly, protection from allergy was achieved with ETEC heat-labile toxin (LT) oral exposure; we also found that the toxicity domain of LT is needed for this protection. Our results indicate that gastrointestinal bacterial infection mediators (toxins) negatively regulate the onset of allergic inflammation and may provide a novel immunological basis for the “hygiene hypothesis”. R01AI170649 FAPESP 2021/15185-2

Wheat Germ Supplementation Reduces Inflammation and Gut Epithelial Barrier Dysfunction in Female Interleukin-10 Knockout Mice Fed a Pro-Atherogenic Diet

BackgroundMice lacking IL-10 are prone to gut inflammation. Additionally, decreased production of short-chain fatty acids (SCFAs) plays a significant role in the high-fat (HF) diet-induced loss of gut epithelial integrity. We have previously shown that wheat germ (WG) supplementation increased ileal expression of IL-22, an important cytokine in maintaining gut epithelial homeostasis. ObjectivesThis study investigated the effects of WG supplementation on gut inflammation and epithelial integrity in IL-10 knockout mice fed a pro-atherogenic diet. MethodsEight-week-old female C57BL/6 wild type mice were fed a control diet (10% fat kcal), and age-matched knockout mice were randomly assigned to 1 of 3 diets (n = 10/group): control, high-fat high-cholesterol (HFHC) [(43.4% fat kcal (∼49% saturated fat, 1% cholesterol)], or HFHC + 10% WG (HFWG) for 12 wk. Fecal SCFAs and total indole, ileal, and serum proinflammatory cytokines, gene or protein expression of tight junctions, and immunomodulatory transcription factors were assessed. Data were analyzed by 1-way ANOVA, and P < 0.05 was considered statistically significant. ResultsFecal acetate, total SCFAs, and indole increased (P < 0.05) by at least 20% in HFWG compared with the other groups. WG increased (P < 0.0001, 2-fold) ileal Il22 (interleukin 22) to Il22ra2 (interleukin 22 receptor, alpha 2) mRNA ratio and prevented the HFHC diet-mediated increase in ileal protein expression of indoleamine 2,3 dioxygenase and pSTAT3 (phosphorylated signal transducer and activator of transcription 3). WG also prevented the HFHC diet-mediated reduction (P < 0.05) in ileal protein expression of the aryl hydrocarbon receptor and the tight junction protein, zonula occludens-1. Serum and ileal concentrations of the proinflammatory cytokine, IL-17, were lower (P < 0.05) by at least 30% in the HFWG group than in the HFHC group. ConclusionsOur findings demonstrate that the anti-inflammatory potential of WG in IL-10 KO mice consuming an atherogenic diet is partly attributable to its effects on the IL-22 signaling and pSTAT3-mediated production of T helper 17 proinflammatory cytokines.

Non-pathogenic microbiota accelerate age-related CpG Island methylation in colonic mucosa

ABSTRACT DNA methylation is an epigenetic process altered in cancer and ageing. Age-related methylation drift can be used to estimate lifespan and can be influenced by extrinsic factors such as diet. Here, we report that non-pathogenic microbiota accelerate age-related methylation drift in the colon when compared with germ-free mice. DNA methylation analyses showed that microbiota and IL10KO were associated with changes in 5% and 4.1% of CpG sites, while mice with both factors had 18% alterations. Microbiota, IL10KO, and their combination altered 0.4%, 0.4%, and 4% of CpG island methylation, respectively. These are comparable to what is seen in colon cancer. Ageing changes were accelerated in the IL10KO mice with microbiota, and the affected genes were more likely to be altered in colon cancer. Thus, the microbiota affect DNA methylation of the colon in patterns reminiscent of what is observed in ageing and colorectal cancer.

Abstract P3060: Circulating Gut Microbial Peptides Exacerbate Cardiac Dysfunction In IL10KO Mice Following Transaortic Constriction

Background: Heart failure is the leading cause of morbidity and mortality in the USA. Recently, impaired intestinal permeability is known to augment cardiac dysfunction. Though, the cardioprotective role of IL10 is well known, its role in reducing gut dysbiosis and maintaining intestinal permeability during hypertrophic heart failure is not known. We hypothesized that IL10 maintains intestinal permeability and inhibits the release of gut microbial peptides (GMPs) which ultimately improves cardiac function. Methods: WT &amp; IL10KO mice were subjected to TAC or sham surgery. Heart and intestine were collected to evaluate inflammation genes expression, immune cell infiltration (flow cytometry), and intestinal permeability (WB and immunohistochemistry). Blood was collected to quantify peptidoglycan (PGN) and other bacterial metabolites (TMAO) levels. The effect of PGN on mitochondrial functions was evaluated in neonatal cardiomyocytes. Results: Cardiac functions were significantly reduced in IL10KO mice following TAC. IL10 KO mice showed exacerbated inflammation (IL1β, IL6 and TNFα genes expression) following TAC. TAC induced leukocytes (monocyte/macrophage, T-cell and neutrophils) recruitments in the intestine in IL10KO mice. TAC mice showed gut dysbiosis (16S rRNA sequencing) in IL10KO mice. Further, IL10KO mice exhibited higher intestinal permeability as shown by increase in PV1 level and reduction in zonulin-1 (ZO-1) protein. Plasma PGN level was significantly increased in IL10KO mice following TAC. Pathway-based cardiotoxicity array showed a significant increase in uncoupler protein 2 (UCP2) and phospholamban (PLN; a calcium handling protein) genes expression in cardiomyocytes following PGN treatment. Finally, IL10 treatment restores PGN-induced decrease in mitochondrial membrane potential (TMRE assay) in cardiomyocytes. Conclusion: Taken together, data suggest that IL-10 helps to maintain gut wall permeability and reduced gut microbial peptide circulation to the heart and thus improves cardiac function by improving mitochondrial function. Ongoing investigations using molecular approaches will provide a better understanding of gut-heart signaling and its impact on the progress of heart failure.