IL-10 Mice Research Articles

Mast cell-derived IL-10 protects intestinal barrier integrity during malaria in mice and regulates parasite transmission to Anopheles stephensi with a female-biased immune response.

Malaria is strongly predisposed to bacteremia, which is associated with increased gastrointestinal permeability and a poor clinical prognosis. We previously identified mast cells (MCs) as mediators of intestinal permeability in malaria and described multiple cytokines that rise with parasitemia, including interleukin (IL)-10, which could protect the host from an inflammatory response and alter parasite transmission to Anopheles mosquitoes. Here, we used the Cre-loxP system and non-lethal Plasmodium yoelii yoelii 17XNL to study the roles of MC-derived IL-10 in malaria immunity and transmission. Our data suggest a sex-biased and local inflammatory response mediated by MC-derived IL-10, supported by early increased number and activation of MCs in females relative to males. Increased parasitemia in female MC IL-10 (-) mice was associated with increased ileal levels of chemokines and plasma myeloperoxidase (MPO). We also observed increased intestinal permeability in female and male MC IL-10 (-) mice relative to MC IL-10 (+) mice but no differences in blood bacterial 16S DNA levels. Transmission success of P. yoelii to A. stephensi was higher in female relative to male mice and from female and male MC IL-10 (-) mice relative to MC IL-10 (+) mice. These patterns were associated with increased plasma levels of pro-inflammatory cytokines in female MC IL-10 (-) mice and increased plasma levels of chemokines and markers of neutrophil activation in male MC IL-10 (-) mice. Overall, these data suggest that MC-derived IL-10 protects intestinal barrier integrity, regulates parasite transmission, and controls local and systemic host immune responses during malaria, with a female bias.

OR27-02 The Bone Anabolic Effect Of An Antibody Blocking Oxidized Phospholipids Is Associated With An Increase In Wnt10b In Osteoblasts.

Abstract Disclosure: M. Palmieri: None. I. Nookaew: None. M.J. Schuller-Almeida: None. H. Kim: None. T.E. Jospeh: None. S.C. Manolagas: None. C.A. O'Brien: None. E. Ambrogini: None. Oxidized phospholipids containing phosphatidylcholine (PC-OxPLs) are toxic molecules present on oxidized low-density lipoproteins (OxLDL) and apoptotic cells; and are pathogenic in many diseases, including osteoporosis. OxPLs decrease Wnt and BMP2 signaling, induce ferroptosis and stimulate pro-inflammatory cytokine production, in atherosclerosis and nonalcoholic steatohepatitis. The natural antibody E06 blocks PC-OxPLs. Transgenic expression of a single chain (scFv) form of the antigen-binding domain of E06 IgM (E06-scFv) increases bone mass in 6-month-old mice and attenuates high fat diet- and age-induced bone loss by increasing osteoblast number and bone formation rate and reducing osteoblast apoptosis. To elucidate the mechanism(s) mediating the bone anabolic effect of E06-scFv we performed bulk-RNA sequencing (bulk-RNA-seq) of vertebral bone and single cell RNA-sequencing (scRNA-seq) of mesenchymal and myeloid cells from long bones of WT and E06-scFv transgenic mice. Earlier bulk-RNA-seq performed in vertebral bone of 22- month-old females had shown that E06-scFv upregulates Wnt10b, but not other Wnt ligands, and the Wnt signaling pathway. We report now that scRNA-seq of mesenchymal cells isolated from 7-month-old female mice revealed that Wnt10b expression is increased by 3-fold (adj-P=3.04E-11) in pre-osteoblasts and osteoblasts from E06-scFv transgenic mice as compared to cells from WT mice. Wnt target genes were also upregulated. An integrated analysis of 9 published scRNA-seq datasets from bone resident mesenchymal and hematopoietic cell types revealed that the majority of Wnt10b in the bone microenvironment originates from cells of the osteoblast lineage. Further, OxLDL decreased Wnt10b and Wnt target genes in calvaria- or bone marrow-derived osteoblastic cell cultures. In contrast, OxLDL did not cause the ferroptosis associated upregulation of Chac1, Ptgs2, Slc7A11, and Acsl4, and the downregulation of Rgs4 in calvaria cells. In addition, no changes in the expression of these genes were detected in the scRNA-seq of mesenchymal cells from E06-scFv compared to WT mice. BMP signaling pathways were also unchanged in this scRNA-seq analysis. Finally, using bulk-RNA-seq of vertebral bone and scRNA-seq of myeloid cells, we could not detect changes in the expression of pro-inflammatory cytokines, such as Tnf-α, Il-6, Il1-α, Il-β, Il-4, Il-12, Lif, Il-23, interferon-α, and interferon-β, between WT and E06-scFv mice. Together, these findings indicate the PC-OxPLs decrease Wnt10b and E06-scFv increases Wnt10b in bone but has no effect on other pathways shown to be affected by OxPLs in other tissues. Thus, Wnt10b may be a critical mediator of the bone anabolic effects of E06-scFv. Presentation: Saturday, June 17, 2023

Microenvironment-dependent growth of Sezary cells in humanized IL-15 mice.

Sezary syndrome (SS) is a rare, aggressive leukemic variant of cutaneous T-cell lymphoma (CTCL) that lacks adequate therapeutic options and representative small-animal models. Here, we demonstrate that IL-15 is a critical CTCL growth factor. Importantly, an immunodeficient knock-in mouse model genetically engineered to express human IL-15 uniquely supported the growth of SS patient samples relative to conventional immunodeficient mouse strains. SS patient-derived xenograft (PDX) models recapacitated key pathological features of the human disease, including skin infiltration and spread of leukemic cells to the periphery, and maintained the dependence on human IL-15 upon serial in vivo passaging. Detailed molecular characterization of the engrafted cells by single-cell transcriptomic analysis revealed congruent neoplastic gene expression signatures but distinct clonal engraftment patterns. Overall, we document an important dependence of Sezary cell survival and proliferation on IL-15 signaling and the utility of immunodeficient humanized IL-15 mice as hosts for SS - and potentially other T and NK cell-derived hematologic malignancies - PDX model generation. Furthermore, these studies advocate the thorough molecular understanding of the resultant PDX models to maximize their translational impact.

CCL22 mutations drive natural killer cell lymphoproliferative disease by deregulating microenvironmental crosstalk.

Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is characterized by clonal expansion of natural killer (NK) cells where the underlying genetic mechanisms are incompletely understood. In the present study, we report somatic mutations in the chemokine gene CCL22 as the hallmark of a distinct subset of CLPD-NK. CCL22 mutations were enriched at highly conserved residues, mutually exclusive of STAT3 mutations and associated with gene expression programs that resembled normal CD16dim/CD56bright NK cells. Mechanistically, the mutations resulted in ligand-biased chemokine receptor signaling, with decreased internalization of the G-protein-coupled receptor (GPCR) for CCL22, CCR4, via impaired β-arrestin recruitment. This resulted in increased cell chemotaxis in vitro, bidirectional crosstalk with the hematopoietic microenvironment and enhanced NK cell proliferation in vivo in transgenic human IL-15 mice. Somatic CCL22 mutations illustrate a unique mechanism of tumor formation in which gain-of-function chemokine mutations promote tumorigenesis by biased GPCR signaling and dysregulation of microenvironmental crosstalk.

Losartan Mitigates Oxidative Stress in the Brains of Aged IL10-/- Mice

Abstract Chronic inflammation has been linked to frailty and declined cognition in older adults. Activation of the renin-angiotensin system (RAS) through the angiotensin Type1 receptor (AT1R) has been suggested as a contributory factor that links both inflammation and aging. Here we examined the impact of 4 weeks of oral Losartan treatment on IL10-/- mice brains, a mouse model of chronic inflammation and frailty. Frontal cortex, cerebellar, and hippocampal tissue of aged (100 weeks old) male IL10-/- mice were studied. Western blot techniques were employed to quantify changes in brain AT1R, nitrotyrosine (NT) as an oxidative stress marker, and Tau proteins. Our data show that aged IL-10 mice have significantly higher levels of AT1R in the cortex tissue but not in cerebellar or hippocampal tissue compared to age and sex-matched WT mice (0.63 + 0.35 vs 1.5 + 0.54, WT vs IL10, respectively, P<0.004). When treated with LOS, brain cortical tissue of IL10 -/- mice showed significant decreases in levels of AT1R (1.5 + 0.54 vs 0.98 + 0.50, IL10 vs LOS treated IL10, respectively, P<0.04), NT (0.72 + 0.12 vs 0.42 + 0.10, IL10 vs LOS treated IL10, respectively, P<0.009), and Tau protein (1.3 + 0.31 vs 0.15 + 0.08, IL10 vs LOS treated IL10, respectively, P<0.004) as compared to control IL10-/- mice. Losartan treatment had no significant effect on hippocampal AT1R or NT levels. Our results highlight the impact of Losartan, a drug commonly prescribed for the treatment of high blood pressure, on the brain-specific angiotensin system and its downstream effects on brain oxidative stress and Tau pathology.

Upregulation of TRPC5 in hippocampal excitatory synapses improves memory impairment associated with neuroinflammation in microglia knockout IL-10 mice

BackgroundMembers of the transient receptor potential canonical (TRPC) protein family are widely distributed in the hippocampus of mammals and exert respective and cooperative influences on the functions of neurons. The relationship between specific TRPC subtypes and neuroinflammation is receiving increasing attention.MethodsUsing Cx3cr1CreERIL-10−/− transgenic mice and their littermates to study the relationship between TRPC channels and memory impairment.ResultsWe demonstrated that Cx3cr1CreERIL-10−/− mice displayed spatial memory deficits in object location recognition (OLR) and Morris water maze (MWM) tasks. The decreased levels of TRPC4 and TRPC5 in the hippocampal regions were verified via reverse transcription polymerase chain reaction, western blotting, and immunofluorescence tests. The expression of postsynaptic density protein 95 (PSD95) and synaptophysin in the hippocampus decreased with an imbalance in the local inflammatory environment in the hippocampus. The number of cells positive for ionized calcium-binding adaptor molecule 1 (Iba1), a glial fibrillary acidic protein (GFAP), increased with the high expression of interleukin 6 (IL-6) in Cx3cr1CreERIL-10−/− mice. The nod-like receptor protein 3 (NLRP3) inflammasome was also involved in this process, and the cytokines IL-1β and IL-18 activated by NLRP3 were also elevated by western blotting. The co-localization of TRPC5 and calmodulin-dependent protein kinase IIα (CaMKIIα) significantly decreased TRPC5 expression in excitatory neurons. AAV9-CaMKIIα-TRPC5 was used to upregulate TRPC5 in excitatory neurons in the hippocampus.ConclusionsThe results showed that the upregulation of TRPC5 improved the memory performance of Cx3cr1CreERIL-10−/− mice related to inhibiting NLRP3 inflammasome-associated neuroinflammation.Graphical

Dysregulated microRNA expression in IL-4 transgenic mice, an animal model of atopic dermatitis.

IL-4 plays an important role in the pathogenesis of atopic dermatitis (AD). Previously we showed that the expression of genes in chemotaxis, angiogenesis, inflammation and barrier functions is dysregulated in IL-4 transgenic (Tg) mice, a well-characterized AD mouse model. In this study, we aim to study differential expression of microRNAs in IL-4 Tg mice. As compared with wild-type mice, we found that 10 and79 microRNAs are dysregulated in the skin of IL-4 mice before and after the onset of skin lesions, respectively. Bioinformatic analysis and previous reports show that these dysregulated microRNAs may be involved in the NF-κB, TLRs, IL-4/IL-13, MAPK and other pathways. We also found that miR-139-5p and miR-196b-3p are significantly up-regulated in the peripheral blood of IL-4 Tg mice. Taken together, our data have identified many dysregulated microRNAs in IL-4 Tg mice, which may play important roles in AD pathogenesis and pathophysiology.

Croton urucurana Baillon stem bark ointment accelerates the closure of cutaneous wounds in knockout IL-10 mice

Ethnopharmacological relevanceCroton urucurana Baill. (Euphorbiaceae) is a plant used in Brazilian popular medicine for the treatment of wound healing, inflammatory diseases, gastritis, infections, and hemorrhoids. AimThe present study aimed to evaluate the in vivo wound healing activity of an ointment based on ethanolic extract of C. urucurana stem bark, at concentrations of 5% and 10%, and to relate it with compounds that could be associated with this activity. Materials and methodsAnalyses by FIA-ESI-IT-MSn were carried out to investigate the chemical composition of C. urucurana. Knockout IL-10 (n = 60) mice and wild type C57 (n = 12) mice were separated into 6 groups to evaluate the wound healing activity. Knockout IL-10 mice: SAL (0.9% saline); BAS (ointment base); SS (1% silver sulfadiazine); CR1 (ointment with extract of C. urucurana 5%); CR2 (ointment with extract of C. urucurana 10%); and wild mice C57: SALC57 (Saline 0.9%). A circular wound with 10 mm in diameter was generated on the dorsal of the animals. Tissue specimen of the wounds were removed on days 7 and 14 of the treatment for histopathological, oxidative status and analyses of pro-and anti-inflammatory cytokines in scar tissue. ResultsIn the phytochemical profile, twelve proanthocyanidins were identified (in the form of monomers, dimers, trimers, and tetramers), based on (epi)catechin and (epi)gallocatechin. Furthermore, two quercetin derivatives and two alkaloids were detected. The groups treated with CR1 and CR2 ointments presented higher rate of wound closure, increased total number of cells, mast cells, blood vessels and higher deposition of type III and I collagen. In addition, they showed increased amount of pro-inflammatory cytokines (IL- 2 and IFN-γ), and anti-inflmatory cytokines (IL-4), on the 7th day of treatment. ConclusionThe results presented support the popular use of preparations based on the bark of C. urucurana as a healing compound.

Interleukin-10 resolves pain hypersensitivity induced by cisplatin by reversing sensory neuron hyperexcitability.

Understanding the mechanisms that drive transition from acute to chronic pain is essential to identify new therapeutic targets. The importance of endogenous resolution pathways acting as a "brake" to prevent development of chronic pain has been largely ignored. We examined the role of interleukin-10 (IL-10) in resolution of neuropathic pain induced by cisplatin. In search of an underlying mechanism, we studied the effect of cisplatin and IL-10 on spontaneous activity (SA) in dorsal root ganglia neurons. Cisplatin (2 mg/kg daily for 3 days) induced mechanical hypersensitivity that resolved within 3 weeks. In both sexes, resolution of mechanical hypersensitivity was delayed in Il10 mice, in WT mice treated intrathecally with neutralizing anti-IL-10 antibody, and in mice with cell-targeted deletion of IL-10R1 on advillin-positive sensory neurons. Electrophysiologically, small- to medium-sized dorsal root ganglia neurons from cisplatin-treated mice displayed an increase in the incidence of SA. Cisplatin treatment also depolarized the resting membrane potential, and decreased action potential voltage threshold and rheobase, while increasing ongoing activity at -45 mV and the amplitude of depolarizing spontaneous fluctuations. In vitro addition of IL-10 (10 ng/mL) reversed the effect of cisplatin on SA and on the depolarizing spontaneous fluctuation amplitudes, but unexpectedly had little effect on the other electrophysiological parameters affected by cisplatin. Collectively, our findings challenge the prevailing concept that IL-10 resolves pain solely by dampening neuroinflammation and demonstrate in a model of chemotherapy-induced neuropathic pain that endogenous IL-10 prevents transition to chronic pain by binding to IL-10 receptors on sensory neurons to regulate their activity.

IL-4 controls activated neutrophil FcγR2b expression and migration into inflamed joints

Neutrophils are the most abundant immune cells found in actively inflamed joints of patients with rheumatoid arthritis (RA), and most animal models for RA depend on neutrophils for the induction of joint inflammation. Exogenous IL-4 and IL-13 protect mice from antibody-mediated joint inflammation, although the mechanism is not understood. Neutrophils display a very strong basal expression of STAT6, which is responsible for signaling following exposure to IL-4 and IL-13. Still, the role of IL-4 and IL-13 in neutrophil biology has not been well studied. This can be explained by the low neutrophil surface expression of the IL-4 receptor α-chain (IL-4Rα), essential for IL-4- and IL-13-induced STAT6 signaling. Here we identify that colony stimulating factor 3 (CSF3), released during acute inflammation, mediates potent STAT3-dependent neutrophil IL-4Rα up-regulation during sterile inflammatory conditions. We further demonstrate that IL-4 limits neutrophil migration to inflamed joints, and that CSF3 combined with IL-4 or IL-13 results in a prominent neutrophil up-regulation of the inhibitory Fcγ receptor (FcγR2b). Taking these data together, we demonstrate that the IL-4 and CSF3 pathways are linked and play important roles in regulating proinflammatory neutrophil behavior.

The development of colitis in Il10-/- mice is dependent on IL-22.

Mice deficient in the IL-10 pathway are the most widely-used models of intestinal immunopathology.IL-17A is strongly implicated in gut disease in mice and humans, but conflicting evidence has drawn IL-17’s role in the gut into question.IL-22 regulates antimicrobial and repair activities of intestinal epithelial cells (IECs) and is closely associated with IL-17A responses but it’s role in chronic disease is uncertain. We report that IL-22, like IL-17A, is aberrantly expressed in colitic Il10−/− mice. While IL-22+Th17 cells were elevated in the colon, IL-22-producing ILC3s were highly enriched in the small intestines of Il10−/− mice. Remarkably, Il10−/−Il22−/− mice did not develop colitis despite retaining high levels of Th17 cells and remaining colonized with colitogenic Helicobacter spp.. Accordant with IL-22-induced IEC proliferation, the epithelia hyperplasia observed in Il10−/− animals was reversed in Il10−/−Il22−/− mice. Also, the high levels of antimicrobial IL-22-target genes, including Reg3g, were normalized in Il10−/−Il22−/− mice. Consistent with a heightened antimicrobial environment, Il10−/− mice had reduced diversity of the fecal microbiome that was reestablished in Il10−/−Il22−/− animals. These data suggest that spontaneous colitis in Il10−/− mice is driven by IL-22 and implicates an underappreciated IL-10-IL-22 axis in regulating intestinal homeostasis.

Antioxidant and Anti-Inflammatory Effects of Anacardium occidentale L. and Anacardium microcarpum D. Extracts on the Liver of IL-10 Knockout Mice.

Background The Anacardium occidentale L. (cashew) and Anacardium microcarpum D. (cajuí) are plants commonly found in Brazil. They present phytochemical compounds with antioxidant and anti-inflammatory action. Therefore, the objective of this study was to analyze the antioxidant and anti-inflammatory activities of ethanolic extracts from leaves of A. occidentale and A. microcarpum and its effect on the hepatic tissue in experimental knockout models after they received Paracetamol®. Methods Ethanol extracts from A. occidentale and A. microcarpum leaves were prepared. Total phenolics were determined by Folin–Ciocalteau reagent, and flavonoids are based on the complexation reaction with the aluminum metal, forming a colored complex. Fingerprint HPLC was performed to detect phenolic compounds. Knockout IL-10 mice randomly divided into six groups were used and received the following treatments: G1, only water; G2, A. occidentale extract; G3, A. microcarpum extract; G4, Paracetamol®; G5, Paracetamol® + A. occidentale extract (400 mg/kg); G6, Paracetamol® + A. microcarpum extract (400 mg/kg). Biochemical parameters of the blood and differential count of leukocytes were done. Oxidative markers and histopathological analyses were performed on their liver tissue. Results Phenolic compounds and total flavonoids were detected in both two extracts analyzed. The HPLC fingerprint detected phenolic acid, gallic acid, and catechin flavonoid in the two extracts. Histopathological analyses of the hepatic tissue permitted evaluation of nuclear increase, sinusoid congestion, and inflammatory infiltrate. A. microcarpum presented more antioxidant activity increasing antioxidant enzyme levels and reducing TBARS and carbonyl protein when compared to the other treatments after exposure to Paracetamol®. Histopathological analyses showed a decrease in the inflammatory infiltrate after treatment with extracts. Conclusion Our findings indicate that both extracts, especially A. microcarpum, can reduce hepatic damage in knockout mice exposed to paracetamol, indicating the curative power of these extracts reducing lipid peroxidation and in the morphofunctional damage to the liver parenchyma.

Single-cell RNA sequencing unveils an IL-10-producing helper subset that sustains humoral immunity during persistent infection

During chronic viral infection, the inflammatory function of CD4 T-cells becomes gradually attenuated. Concurrently, Th1 cells progressively acquire the capacity to secrete the cytokine IL-10, a potent suppressor of antiviral T cell responses. To determine the transcriptional changes that underlie this adaption process, we applied a single-cell RNA-sequencing approach and assessed the heterogeneity of IL-10-expressing CD4 T-cells during chronic infection. Here we show an IL-10-producing population with a robust Tfh-signature. Using IL-10 and IL-21 double-reporter mice, we further demonstrate that IL-10+IL-21+co-producing Tfh cells arise predominantly during chronic but not acute LCMV infection. Importantly, depletion of IL-10+IL-21+co-producing CD4 T-cells or deletion of Il10 specifically in Tfh cells results in impaired humoral immunity and viral control. Mechanistically, B cell-intrinsic IL-10 signaling is required for sustaining germinal center reactions. Thus, our findings elucidate a critical role for Tfh-derived IL-10 in promoting humoral immunity during persistent viral infection.

Mast Cells Play a Protective Role in Spontaneous Colitis

Abstract Mast cells are modulators of inflammation and are activated during inflammatory bowel disease (IBD). Previous work has demonstrated that mast cells are proinflammatory in acute models of chemical colitis. However, the role of mast cells in chronic, spontaneous IBD models remains to be fully defined. We hypothesized that mast cells play a protective role in chronic, spontaneous colitis. We compared colitis and intestinal barrier function in IL10−/− mice to mast cell-deficient, colitis prone mice (double knockout (DKO): IL-10−/− x KitWsh/Wsh). Systemic engraftment of DKO mice with mast cells was performed. Mast cell-deficient DKO mice exhibited more severe colitis compared to mice that lacked IL-10 alone, indicated by increased colitis scores, mucosal hypertrophy, increased intestinal permeability, and spontaneous colonic cytokine production. Reconstitution of DKO mice with mast cells prevented colitis (reduced colitis scores, mucosal hypertrophy, TNF, and intestinal permeability). PCR array analyses demonstrated the influence of mast cells on cytokine expression. Analysis of the microbiota revealed sex-specific differences between IL10 and DKO mice, in particular, the abundance of Akkermansia muciniphila, a mucus degrader associated with colitis. Derivation of DKO mice into a germ-free environment revealed that the microbiota is required for disease development. RNA-seq on colon samples revealed candidate genes for the protective effects of mast cells. These results demonstrate that mast cell deficiency exacerbates disease in a chronic, spontaneous IBD model, indicating that mast cells may have protective functions in IBD, and potentially other chronic inflammatory disorders.

Intestinal overexpression of interleukin (IL)-15 promotes tissue eosinophilia and goblet cell hyperplasia.

Interleukin (IL)-15 overexpression in eosinophilic gastrointestinal disorders is reported, but IL-15's role in promoting eosinophilic gastroenteritis is largely unknown. Therefore, we generated enterocyte-overexpressed IL-15 transgenic mice using Fabpi promoter. The Fabpi-IL-15 (iIL-15) transgenic mice showed induced IL-15 levels in the jejunum with a marked increase in jejunum eosinophils. However, no induction of eosinophilia in the blood or any other gastrointestinal segment was observed. Eosinophilia in the jejunum villus was substantially higher in iIL-15 mice compared to wild-type mice. In addition, goblet cell hyperplasia was also observed in the jejunum of iIL-15 mice. Furthermore, a significant correlation between induced IL-15 transcript and the IL-18 transcripts was observed. Therefore, to further understand the role of IL-18 in IL-15 mice associated gastrointestinal disorders, we generated iIL-15/IL-18Rα-/- mice. Using these mice, we found that IL-18 has an important role in promoting IL-15-induced eosinophilia. As intestinal IL-15 overexpression is reported in food intolerance, we examined OVA intolerance in iIL-15 mice. The OVA-sensitized and challenged iIL-15 mice experienced weight loss, diarrhea and eosinophilia in the jejunum. Taken together, our findings demonstrate that intestinal IL-15 overexpression induces IL-18-dependent eosinophilia and immunoglobulins in the intestine that promotes food allergic responses.

Interleukin-4 mediates the analgesia produced by low-intensity exercise in mice with neuropathic pain.

Peripheral nerve injury (PNI) activates the immune system, resulting in increased proinflammatory cytokines at the site of injury and in the spinal cord dorsal horn. Exercise modulates the immune system promoting an anti-inflammatory phenotype of macrophages in uninjured muscle, and increases in anti-inflammatory cytokines can promote healing and analgesia. We proposed that PNI will decrease, and treadmill exercise will increase, release of anti-inflammatory cytokines at the site of injury and in the spinal cord. We show that 2 weeks of treadmill exercise improves neuropathic pain behaviors in mice: mechanical hyperalgesia, escape and avoidance behavior, and spontaneous locomotor activity. Peripheral nerve injury reduced anti-inflammatory cytokines (interleukin-4 [IL-4], IL-1ra, and IL-5) at the site of nerve injury and in the spinal dorsal horn, whereas exercise restored IL-4, IL-1ra, and IL-5 concentrations to preinjury levels. IL4 mice and mice treated with IL-4 antibody did not develop analgesia to treadmill exercise. Using immunohistochemical staining of the sciatic nerve, treadmill exercise increased the percentage of M2 macrophages (secretes anti-inflammatory cytokines) and decreased M1 macrophages (secretes proinflammatory cytokines) when compared with sedentary mice. The increased M2 and decreased M1 macrophages in exercised mice did not occur in IL-4 mice. In the spinal cord, PNI increased glial cell activation, brain-derived neurotrophic factor and β-nerve growth factor levels, and decreased IL-4 and IL-1ra levels, whereas treadmill exercise suppressed glial cells activation (Glial Fibrillary Acidic Protein and Iba1 immunoreactivity), reduced brain-derived neurotrophic factor and β-nerve growth factor, and increased IL-4, IL-1ra, and IL-5 concentrations. Our results suggest that IL-4 mediates the analgesia produced by low-intensity exercise by modulating peripheral and central neuroimmune responses in mice with neuropathic pain.

Intestinal type 1 regulatory T cells migrate to periphery to suppress diabetogenic T cells and prevent diabetes development

Growing insight into the pathogenesis of autoimmune diseases and numerous studies in preclinical models highlights the potential of regulatory T cells to restore tolerance. By using non-obese diabetic (NOD) BDC2.5 TCR-transgenic (Tg), and IL-10 and Foxp3 double-reporter mice, we demonstrate that alteration of gut microbiota during cohousing experiments or treatment with anti-CD3 mAb significantly increase intestinal IL-10-producing type 1 regulatory T (Tr1) cells and decrease diabetes incidence. These intestinal antigen-specific Tr1 cells have the ability to migrate to the periphery via a variety of chemokine receptors such as CCR4, CCR5, and CCR7 and to suppress proliferation of Th1 cells in the pancreas. The ability of Tr1 cells to cure diabetes in NOD mice required IL-10 signaling, as Tr1 cells could not suppress CD4+ T cells with a dominant-negative IL-10R. Taken together, our data show a key role of intestinal Tr1 cells in the control of effector T cells and development of diabetes. Therefore, modulating gut-associated lymphoid tissue to boost Tr1 cells may be important in type 1 diabetes management.

T follicular helper cell-derived IL-10 sustains humoral immunity during chronic viral infection

Abstract Although it is well-appreciated that Tfh cells play a critical role in regulating antiviral humoral immunity, the detailed cellular and molecular pathways that govern their differentiation and function during chronic viral infection remains incompletely understood. In this study, we generated IL-10 and IL-21 double-reporter mice to study the dynamic expression of the immunoregulatory cytokines IL-10 and IL-21, which are known to differentially regulate T cell-mediated control over viral replication. Infection of these double-reporter mice with either an acute or chronic strain of LCMV, revealed a unique subset of IL-10+IL-21+co-producing Tfh cells that arise only during chronic viral infection. Notably, compared to IL-21-single producing Tfh cells, IL-10+IL-21+co-producing Tfh cells exhibited an enhanced germinal center (GC) Tfh-like profile, as exemplified by their increased per cell expression of PD-1, CXCR5, and Bcl-6. Additional analyses identified that IL-10-producing Tfh cells have an increased capacity to form stable Tfh-B cell conjugates compared to their IL-10− Tfh counterparts, suggesting that IL-10+ Tfh cells may specialize in providing help signals to B cells during chronic infection. Importantly, depletion of IL-10+IL-21+ co-producing CD4 T cells or deletion of Il10 specifically from Tfh cells resulted in impaired GC B cell responses, LCMV-specific antibody production and viral control. Mechanistically, we show that B cell intrinsic IL-10 signaling is indispensable for GC B cell differentiation and function. Collectively, our findings delineate a heterogeneous population of Tfh cells and elucidate a critical role for Tfh-derived IL-10 in promoting humoral immunity during persistent viral infection.

Alterations in Enterohepatic Fgf15 Signaling and Changes in Bile Acid Composition Depend on Localization of Murine Intestinal Inflammation.

Fibroblast growth factor (FGF) 15/19 is part of the gut-liver crosstalk accounting for bile acid (BA) metabolism regulation. Dysregulation of fibroblast growth factor 15/19 signaling is observed in different pathological conditions, for example, in gastrointestinal diseases such as inflammatory bowel disease (IBD). To understand the molecular bases, we analyzed the enterohepatic regulation of Fgf15-mediated pathway in 2 different inflammatory bowel disease mouse models. Target genes of the BA-farnesoid-X-receptor (Fxr)-Ffg15 axis were quantified by RT-PCR or western blotting in gut and liver of dextran sulfate sodium (DSS)-treated and IL10 mice. Serum Fgf15 levels were analyzed by ELISA. Biliary and fecal BA composition was differentiated by HPLC-MS/MS. Dextran sulfate sodium-treated mice with ileum-sparing colitis showed higher Fgf15 serum levels. In contrast, IL10 mice with ileitis had a trend toward decreased Fgf15 serum levels compared with controls and increased expression of Asbt as a negative Fxr-target gene. In hepatic tissue of both models, no histological changes, but higher interleukin 6 (IL-6) mRNA expression and down-regulation of Fxr and Cytochrom P450 7a1 mRNA expression were observed. Fibroblast growth factor receptor 4 up-regulation was in line with higher Fgf15 serum levels in dextran sulfate sodium-treated mice. A distinct fecal BA profile was observed in both models with significantly higher levels of taurine-conjugated BA in particular tauro-β-muricholic acid in IL10 mice. Ileum-sparing colitis is characterized by activation of Fxr-Fgf15 signaling with higher expression of Fxr-target gene Fgf15, whereas ileal inflammation showed no signs of Fxr-Fgf15 activation. Abundance of BA such as T-β-MCA may be important for intestinal Fxr activation in mice.

MicroRNA-320a Strengthens Intestinal Barrier Function and Follows the Course of Experimental Colitis.

Inflammatory bowel disease is a chronic-remittent disorder with the risk of disabling complications due to uncontrolled inflammation. Accurate biomarkers are needed to noninvasively monitor the disease course to tailor therapy. We evaluated the potential of the specific microRNA (miR)-320a to monitor disease activity in experimental colitis or patients with Crohn's disease and investigated its functional role in intestinal epithelial barrier formation. The impact of miR-320a on intestinal barrier function was tested in vitro in T84 epithelial cells by transepithelial resistance measurement and quantitative real-time polymerase chain reaction analysis on inflammatory and microbial stimulation. Experimental colitis was studied in dextran sodium sulfate colitis, T-cell transfer colitis, and IL-10 mice. Disease course was monitored by body weight measurement, colonoscopy, and histological examination. MiR-320a expression during inflammation was assessed in T84 cells, murine blood, and colonic tissue and in peripheral blood from patients with Crohn's disease with active or quiescent disease. MiR-320a transfection of T84 cells reinforced barrier integrity reflected by increased transepithelial resistance (P < 0.01) and inhibited barrier-destructive enteropathogenic Escherichia coli effects resulting in increased tight junction protein JAM-A expression (P = 0.02) and decrease of barrier integrity-destabilizing miR-320a target PPP2R5B (P < 0.001). Tumor necrosis factor-α and interleukin-1β stimulation increased a miR-320a epxression in T84 cells. MiR-320a level was increased in blood samples from colitic mice and patients with Crohn's disease showing a strong correlation with disease activity (r = 0.67). MiR-320a strengthens intestinal barrier function in vitro and has the potential to monitor disease activity of colitic mice. Future studies are needed to further evaluate the potential of miR-320a in patients with inflammatory bowel disease.