The Mutyh Base Excision Repair Gene Influences the Inflammatory Response in a Mouse Model of Ulcerative Colitis

Background

The Mutyh DNA glycosylase is involved in the repair of oxidized DNA bases. Mutations in the human MUTYH gene are responsible for colorectal cancer in familial adenomatous polyposis. Since defective DNA repair genes might contribute to the increased cancer risk associated with inflammatory bowel diseases, we compared the inflammatory response of wild-type and Mutyh^−/− mice to oxidative stress.

Methodology/Principal Findings

The severity of colitis, changes in expression of genes involved in DNA repair and inflammation, DNA 8-oxoguanine levels and microsatellite instability were analysed in colon of mice treated with dextran sulfate sodium (DSS). The Mutyh^−/− phenotpe was associated with a significant accumulation of 8-oxoguanine in colon DNA of treated mice. A single DSS cycle induced severe acute ulcerative colitis in wild-type mice, whereas lesions were modest in Mutyh^−/− mice, and this was associated with moderate variations in the expression of several cytokines. Eight DSS cycles caused chronic colitis in both wild-type and Mutyh^−/− mice. Lymphoid hyperplasia and a significant reduction in Foxp3⁺ regulatory T cells were observed only in Mutyh^−/− mice.

Conclusions

The findings indicate that, in this model of ulcerative colitis, Mutyh plays a major role in maintaining intestinal integrity by affecting the inflammatory response.     

Interleukin 13 and Serotonin: Linking the Immune and Endocrine Systems in Murine Models of Intestinal Inflammation

Objective

Infiltration of activated immune cells and increased cytokine production define the immunophenotype of gastrointestinal (GI) inflammation. In addition, intestinal inflammation is accompanied by alteration in the numbers of serotonin (5-hydroxytryptamine; 5-HT) synthesizing enterochromaffin (EC) cells and in 5-HT amount. It has been established that EC cells express interleukin (IL)-13 receptor, additionally IL-13 has been implicated in the pathogenesis of ulcerative colitis. In this study, we investigated the role of IL-13 mediated 5-HT signaling in pathogenesis of colitis.

Methodology

Colitis was induced in IL-13 deficient (IL-13−/−) and wild-type (WT) mice with dextran sulfate sodium (DSS) and dinitrobenzene sulfonic acid (DNBS), as well as in IL-13−/− mice given recombinant mouse IL-13 (rmIL-13) and 5-hydroxytryptamine (5-HTP), the direct precursor of 5-HT.

Principal Findings and Conclusion

Elevated colonic IL-13 levels were observed in WT mice receiving DSS in comparison to control. IL-13−/− mice administered DSS exhibited significantly reduced severity of colitis compared to WT mice as reflected by macroscopic and histological damage assessments. Following DSS administration, significantly lower pro-inflammatory cytokine production and fewer infiltrating macrophages were observed in IL-13−/− mice compared to WT. The reduced severity of colitis observed in IL-13−/− mice was also accompanied by down-regulation of EC cell numbers and colonic 5-HT content. In addition, increasing colonic 5-HT content by administration of rmIL-13 or 5-HTP exacerbated severity of DSS colitis in IL-13−/− mice. IL-13−/− mice also exhibited reduced severity of DNBS-induced colitis. These results demonstrate that IL-13 plays a critical role in the pathogenesis of experimental colitis and 5-HT is an important mediator of IL-13 driven intestinal inflammation. This study revealed important information on immune-endocrine axis in gut in relation to inflammation which may ultimately lead to better strategy in managing various intestinal inflammatory conditions including inflammatory bowel disease.     

High Beta-Palmitate Fat Controls the Intestinal Inflammatory Response and Limits Intestinal Damage in Mucin Muc2 Deficient Mice

Background

Palmitic-acid esterified to the sn-1,3 positions of the glycerol backbone (alpha, alpha’-palmitate), the predominant palmitate conformation in regular infant formula fat, is poorly absorbed and might cause abdominal discomfort. In contrast, palmitic-acid esterified to the sn-2 position (beta-palmitate), the main palmitate conformation in human milk fat, is well absorbed. The aim of the present study was to examine the influence of high alpha, alpha’-palmitate fat (HAPF) diet and high beta-palmitate fat (HBPF) diet on colitis development in Muc2 deficient (Muc2^−/−) mice, a well-described animal model for spontaneous enterocolitis due to the lack of a protective mucus layer.

Methods

Muc2^−/− mice received AIN-93G reference diet, HAPF diet or HBPF diet for 5 weeks after weaning. Clinical symptoms, intestinal morphology and inflammation in the distal colon were analyzed.

Results

Both HBPF diet and AIN-93G diet limited the extent of intestinal erosions and morphological damage in Muc2^−/− mice compared with HAPF diet. In addition, the immunosuppressive regulatory T (Treg) cell response as demonstrated by the up-regulation of Foxp3, Tgfb1 and Ebi3 gene expression levels was enhanced by HBPF diet compared with AIN-93G and HAPF diets. HBPF diet also increased the gene expression of Pparg and enzymatic antioxidants (Sod1, Sod3 and Gpx1), genes all reported to be involved in promoting an immunosuppressive Treg cell response and to protect against colitis.

Conclusions

This study shows for the first time that HBPF diet limits the intestinal mucosal damage and controls the inflammatory response in Muc2^−/− mice by inducing an immunosuppressive Treg cell response.     

Sirtuin 3, a New Target of PGC-1α, Plays an Important Role in the Suppression of ROS and Mitochondrial Biogenesis

Background

Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1α induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.

Results

Here we show that PGC-1α strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1α led to decreased Sirt3 gene expression. PGC-1α activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR) binding element (ERRE) (−407/−399) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRα bound to the identified ERRE and PGC-1α co-localized with ERRα in the mSirt3 promoter. Knockdown of ERRα reduced the induction of Sirt3 by PGC-1α in C₂C₁₂ myotubes. Furthermore, Sirt3 was essential for PGC-1α-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1α in C₂C₁₂ myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1α on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1α on mitochondrial biogenesis in C₂C₁₂ myotubes.

Conclusion

Our results indicate that Sirt3 functions as a downstream target gene of PGC-1α and mediates the PGC-1α effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation. The elucidation of the molecular mechanisms of SIRT3 regulation and its physiological functions may provide a novel target for treating ROS-related disease.     

Dexamethasone induces apoptosis in pulmonary arterial smooth muscle cells

Background

Dexamethasone suppressed inflammation and haemodynamic changes in an animal model of pulmonary arterial hypertension (PAH). A major target for dexamethasone actions is NF-κB, which is activated in pulmonary vascular cells and perivascular inflammatory cells in PAH. Reverse remodelling is an important concept in PAH disease therapy, and further to its anti-proliferative effects, we sought to explore whether dexamethasone augments pulmonary arterial smooth muscle cell (PASMC) apoptosis.

Methods

Analysis of apoptosis markers (caspase 3, in-situ DNA fragmentation) and NF-κB (p65 and phospho-IKK-α/β) activation was performed on lung tissue from rats with monocrotaline (MCT)-induced pulmonary hypertension (PH), before and after day 14–28 treatment with dexamethasone (5 mg/kg/day). PASMC were cultured from this rat PH model and from normal human lung following lung cancer surgery. Following stimulation with TNF-α (10 ng/ml), the effects of dexamethasone (10⁻⁸–10⁻⁶ M) and IKK2 (NF-κB) inhibition (AS602868, 0–3 μM (0-3×10⁻⁶ M) on IL-6 and CXCL8 release and apoptosis was determined by ELISA and by Hoechst staining. NF-κB activation was measured by TransAm assay.

Results

Dexamethasone treatment of rats with MCT-induced PH in vivo led to PASMC apoptosis as displayed by increased caspase 3 expression and DNA fragmentation. A similar effect was seen in vitro using TNF-α-simulated human and rat PASMC following both dexamethasone and IKK2 inhibition. Increased apoptosis was associated with a reduction in NF-κB activation and in IL-6 and CXCL8 release from PASMC.

Conclusions

Dexamethasone exerted reverse-remodelling effects by augmenting apoptosis and reversing inflammation in PASMC possibly via inhibition of NF-κB. Future PAH therapies may involve targeting these important inflammatory pathways.     

Lipid Alterations in Experimental Murine Colitis: Role of Ceramide and Imipramine for Matrix Metalloproteinase-1 Expression

Background

Dietary lipids or pharmacologic modulation of lipid metabolism are potential therapeutic strategies in inflammatory bowel disease (IBD). Therefore, we analysed alterations of bioactive lipids in experimental models of colitis and examined the functional consequence of the second messenger ceramide in inflammatory pathways leading to tissue destruction.

Methodology/Principal Findings

Chronic colitis was induced by dextran-sulphate-sodium (DSS) or transfer of CD4⁺CD62L⁺ cells into RAG1^−/−-mice. Lipid content of isolated murine intestinal epithelial cells (IEC) was analysed by tandem mass spectrometry. Concentrations of MMP-1 in supernatants of Caco-2-IEC and human intestinal fibroblasts from patients with ulcerative colitis were determined by ELISA. Imipramine was used for pharmacologic inhibition of acid sphingomyelinase (ASM). Ceramide increased by 71% in chronic DSS–induced colitis and by 159% in the transfer model of colitis. Lysophosphatidylcholine (LPC) decreased by 22% in both models. No changes were detected for phosphatidylcholine. Generation of ceramide by exogenous SMase increased MMP-1-protein production of Caco-2-IEC up to 7-fold. Inhibition of ASM completely abolished the induction of MMP-1 by TNF or IL-1β in Caco-2-IEC and human intestinal fibroblasts.

Conclusions/Significance

Mucosal inflammation leads to accumulation of ceramide and decrease of LPC in the intestinal epithelium. One aspect of ceramide generation is an increase of MMP-1. Induction of MMP-1 by TNF or IL-1β is completely blocked by inhibition of ASM with imipramine. Therefore, inhibition of ASM may offer a treatment strategy to reduce MMP-1 expression and tissue destruction in inflammatory conditions.     

CCL25/CCR9 interactions regulate large intestinal inflammation in a murine model of acute colitis

Background & Aims

CCL25/CCR9 is a non-promiscuous chemokine/receptor pair and a key regulator of leukocyte migration to the small intestine. We investigated here whether CCL25/CCR9 interactions also play a role in the regulation of inflammatory responses in the large intestine.

Methods

Acute inflammation and recovery in wild-type (WT) and CCR9^−/− mice was studied in a model of dextran sulfate sodium (DSS)-induced colitis. Distribution studies and phenotypic characterization of dendritic cell subsets and macrophage were performed by flow cytometry. Inflammatory bowel disease (IBD) scores were assessed and expression of inflammatory cytokines was studied at the mRNA and the protein level.

Results

CCL25 and CCR9 are both expressed in the large intestine and are upregulated during DSS colitis. CCR9^−/− mice are more susceptible to DSS colitis than WT littermate controls as shown by higher mortality, increased IBD score and delayed recovery. During recovery, the CCR9^−/− colonic mucosa is characterized by the accumulation of activated macrophages and elevated levels of Th1/Th17 inflammatory cytokines. Activated plasmacytoid dendritic cells (DCs) accumulate in mesenteric lymph nodes (MLNs) of CCR9^−/− animals, altering the local ratio of DC subsets. Upon re-stimulation, T cells isolated from these MLNs secrete significantly higher levels of TNFα, IFNγ, IL2, IL-6 and IL-17A while down modulating IL-10 production.

Conclusions

Our results demonstrate that CCL25/CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa by balancing different subsets of dendritic cells. These findings have important implications for the use of CCR9-inhibitors in therapy of human IBD as they indicate a potential risk for patients with large intestinal inflammation.     

iNOS-Dependent Increase in Colonic Mucus Thickness in DSS-Colitic Rats

Aim

To investigate colonic mucus thickness in vivo in health and during experimental inflammatory bowel disease.

Methods

Colitis was induced with 5% DSS in drinking water for 8 days prior to experiment, when the descending colonic mucosa of anesthetized rats was studied using intravital microscopy. Mucus thickness was measured with micropipettes attached to a micromanipulator. To assess the contributions of NOS and prostaglandins in the regulation of colonic mucus thickness, the non-selective NOS-inhibitor L-NNA (10 mg/kg bolus followed by 3 mg/kg/h), the selective iNOS-inhibitor L-NIL (10 mg/kg bolus followed by 3 mg/kg/h) and the non-selective COX-inhibitor diclofenac (5 mg/kg) were administered intravenously prior to experiment. To further investigate the role of iNOS in the regulation of colonic mucus thickness, iNOS −/− mice were used.

Results

Colitic rats had a thicker firmly adherent mucus layer following 8 days of DSS treatment than untreated rats (88±2 µm vs 76±1 µm). During induction of colitis, the thickness of the colonic mucus layer initially decreased but was from day 3 significantly thicker than in untreated rats. Diclofenac reduced the mucus thickness similarly in colitic and untreated rats (−16±5 µm vs −14±2 µm). While L-NNA had no effect on colonic mucus thickness in DSS or untreated controls (+3±2 µm vs +3±1 µm), L-NIL reduced the mucus thickness significantly more in colitic rats than in controls (−33±4 µm vs −10±3 µm). The importance of iNOS in regulating the colonic mucus thickness was confirmed in iNOS−/− mice, which had thinner colonic mucus than wild-type mice (35±3 µm vs 50±2 µm, respectively). Furthermore, immunohistochemistry revealed increased levels of iNOS in the colonic surface epithelium following DSS treatment.

Conclusion

Both prostaglandins and nitric oxide regulate basal colonic mucus thickness. During onset of colitis, the thickness of the mucus layer is initially reduced followed by an iNOS mediated increase.     

The bile acid receptor GPBAR-1 (TGR5) modulates integrity of intestinal barrier and immune response to experimental colitis

Background

GP-BAR1, a member G protein coupled receptor superfamily, is a cell surface bile acid-activated receptor highly expressed in the ileum and colon. In monocytes, ligation of GP-BAR1 by secondary bile acids results in a cAMP-dependent attenuation of cytokine generation.

Aims

To investigate the role GP-BAR1 in regulating intestinal homeostasis and inflammation-driven immune dysfunction in rodent models of colitis.

Methods

Colitis was induced in wild type and GP-BAR1^−/− mice by DSS and TNBS administration. Potential GP-BAR1 agonists were identified by in silico screening and computational docking studies.

Results

GP-BAR1^−/− mice develop an abnormal morphology of colonic mucous cells and an altered molecular architecture of epithelial tight junctions with increased expression and abnormal subcellular distribution of zonulin 1 resulting in increased intestinal permeability and susceptibility to develop severe colitis in response to DSS at early stage of life. By in silico screening and docking studies we identified ciprofloxacin as a GP-BAR1 ligand. In monocytes, ciprofloxacin increases cAMP concentrations and attenuates TNFα release induced by TLR4 ligation in a GP-BAR1 dependent manner. Treating mice rendered colitic by TNBS with ciprofloxacin and oleanolic acid, a well characterized GP-BAR1 ligand, abrogates signs and symptoms of colitis. Colonic expression of GP-BAR1 mRNA increases in rodent models of colitis and tissues from Crohn''s disease patients. Flow cytometry analysis demonstrates that ≈90% of CD14+ cells isolated from the lamina propria of TNBS-treated mice stained positively for GP-BAR1.

Conclusions

GP-BAR1 regulates intestinal barrier structure. Its expression increases in rodent models of colitis and Crohn''s disease. Ciprofloxacin is a GP-BAR1 ligand.     

VSL#3 Resets Insulin Signaling and Protects against NASH and Atherosclerosis in a Model of Genetic Dyslipidemia and Intestinal Inflammation

Background

Signals generated by the inflammed intestine are thought to contribute to metabolic derangement. The intestinal microbiota contributes to instructing the immune system beyond the intestinal wall and its modulation is a potential target for treating systemic disorders.

Aims

To investigate the pathogenetic role of low grade intestinal inflammation in the development of steatohepatitis and atherosclerosis in a model of genetic dyslipidemia and to test the therapeutic potential of a probiotics intervention in protecting against development of these disorders.

Results

ApoE^−/− mice were randomized to receive vehicle or VSL#3, a mixture of eight probiotics, at the dose of 20×10⁹ colony-forming units/kg/day for three months alone or in combination with 0.2% of dextran sulfate sodium (DSS) in drinking water. Administering DSS to ApoE^−/− mice failed to induce signs and symptoms of colitis but increased intestinal permeability to dextran FITC and, while had no effect on serum lipids, increased the blood levels of markers of liver injury and insulin resistance. DSS administration associated with low level inflammation of intestinal and mesenteric adipose tissues, caused liver histopathology features of steatohepatitis and severe atherosclerotic lesions in the aorta. These changes were prevented by VSL#3 intervention. Specifically, VSL#3 reversed insulin resistance, prevented development of histologic features of mesenteric adipose tissue inflammation, steatohepatitis and reduced the extent of aortic plaques. Conditioned media obtained from cultured probiotics caused the direct transactivation of peroxisome proliferator-activated receptor-γ, Farnesoid-X-receptors and vitamin D receptor.

Conclusions

Low grade intestinal inflammation drives a transition from steatosis to steatohepatitis and worsens the severity of atherosclerosis in a genetic model of dyslipidemia. VSL#3 intervention modulates the expression of nuclear receptors, corrects for insulin resistance in liver and adipose tissues and protects against development of steatohepatitis and atherosclerosis.     

NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

Background

Chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood.

Methodology/Principal Findings

We found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47^phox−/− mice and gp91^phox-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-κB activation, and elevated downstream pro-inflammatory cytokines (TNF-α, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-κB activation.

Conclusions/Significance

These studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD.     

Telomere-Mediated Chromosomal Instability Triggers TLR4 Induced Inflammation and Death in Mice

Background

Telomeres are essential to maintain chromosomal stability. Cells derived from mice lacking telomerase RNA component (mTERC^−/− mice) display elevated telomere-mediated chromosome instability. Age-dependent telomere shortening and associated chromosome instability reduce the capacity to respond to cellular stress occurring during inflammation and cancer. Inflammation is one of the important risk factors in cancer progression. Controlled innate immune responses mediated by Toll-like receptors (TLR) are required for host defense against infection. Our aim was to understand the role of chromosome/genome instability in the initiation and maintenance of inflammation.

Methodology/Principal Findings

We examined the function of TLR4 in telomerase deficient mTERC^−/− mice harbouring chromosome instability which did not develop any overt immunological disorder in pathogen-free condition or any form of cancers at this stage. Chromosome instability was measured in metaphase spreads prepared from wildtype (mTERC^+/+), mTERC^+/− and mTERC^−/− mouse splenocytes. Peritoneal and/or bone marrow-derived macrophages were used to examine the responses of TLR4 by their ability to produce inflammatory mediators TNFα and IL6. Our results demonstrate that TLR4 is highly up-regulated in the immune cells derived from telomerase-null (mTERC^−/−) mice and lipopolysaccharide, a natural ligand for TLR4 stabilises NF-κB binding to its promoter by down-regulating ATF-3 in mTERC^−/− macrophages.

Conclusions/Significance

Our findings implied that background chromosome instability in the cellular level stabilises the action of TLR4-induced NF-κB action and sensitises cells to produce excess pro-inflammatory mediators. Chromosome/genomic instability data raises optimism for controlling inflammation by non-toxic TLR antagonists among high-risk groups.     

Roles of Apolipoprotein E (ApoE) and Inducible Nitric Oxide Synthase (iNOS) in Inflammation and Apoptosis in Preeclampsia Pathogenesis and Progression

Objectives

To investigate potential roles of inducible nitric oxide synthase (iNOS) and apolipoprotein (apoE) in inflammation and apoptosis promoting pathological changes in preeclampsia in pregnant mice with apoE and/or iNOS knock out.

Methods

B6.129 mice were crossed to produce WT, apoE^−/−, apoE^+/−, iNOS^−/−, iNOS^+/− and apoE^−/−iNOS^−/− groups. Variants were confirmed by PCR. Serum lipid parameters (triglycerides, TG; total cholesterol, TC; high density lipoprotein, HDL; and low density lipoprotein, LDL), NO levels and placental electronic microscopic ultrastructures were evaluated, and blood pressure (BP), 24-hour urine protein and pregnancy outcomes were recorded for pregnant F1 generation mice. Placental expressions of inflammatory (tumor necrosis factor-α, TNF-α; interleukin-6, IL-6; nuclear factor-κB, NF-κb) and apoptotic markers (Bcl-2 associated X protein, Bax, B-cell lymphoma/leukemia-2, Bcl-2, and Caspase-3) were evaluated via Western blot.

Results

Serum lipids, BP and 24-hour urine protein levels were shown to be significantly higher and parturition and placenta weights were lower in apoE^−/− and apoE^−/−iNOS^−/− groups (p<0.05). NO levels were lower in the apoE^−/−iNOS^−/− group. In addition, inflammatory/apoptosis parameters, including TNF-α, IL-6, NF-κb, Bax, Bcl-2 and Caspase-3 in the apoE^−/−iNOS^−/− group (p<0.01), as well as in the apoE^−/− group (p<0.05), and NF-κB, Bax in iNOS^−/− group (p<0.05) were higher compared with WT group. However, most of the inflammatory/apoptosis parameters in the iNOS^+/− and the apoE^+/− groups (p>0.05) showed no differences. In addition, placenta vascular endothelial and trophoblast cell morphological changes were demonstrated in both the apoE^−/−iNOS^−/− and apoE^−/− groups.

Conclusion

Elevated lipid metabolism and inflammatory/apoptosis parameters suggest a potentially significant role of apoE in preeclampsia pathology, as well as a relationship between iNOS and preeclampsia progression.     

Altered exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury

Background

The NF-κB signaling pathway plays a role in local and remote tissue damage following ischemia-reperfusion (I/R) injury to skeletal muscles. Evidence suggests that exosomes can act as intercellular communicators by transporting active proteins to remote cells and may play a role in regulating inflammatory processes. This study aimed to profile the exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury.

Results

To investigate the potential changes in protein expression mediated by NF-κB in secreted exosomes in the serum following I/R injury, the levels of circulating exosomal proteomes in C57BL/6 and NF-κB^−/− mice were compared using two dimensional differential in-gel electrophoresis (2-DE), liquid chromatography tandem mass spectrometry (LC-MS/MS), and proteomic analysis. In C57BL/6 mice, the levels of circulating exosomal proteins, including complement component C3 prepropeptide, PK-120 precursor, alpha-amylase one precursor, beta-enolase isoform 1, and adenylosuccinate synthetase isozyme 1, increased following I/R injury. However, in the NF-κB^−/− mice, the expression of the following was upregulated in the exosomes: protease, serine 1; glyceraldehyde-3-phosphate dehydrogenase-like isoform 1; glyceraldehyde-3-phosphate dehydrogenase; and pregnancy zone protein. In contrast, the expression of apolipoprotein B, complement component C3 prepropeptide, and immunoglobulin kappa light chain variable region was downregulated in NF-κB^−/− mice. Bioinformatic annotation using the Protein Analysis Through Evolutionary Relationships (PANTHER) database revealed that the expression of the exosomal proteins that participate in metabolic processes and in biological regulation was lower in NF-κB^−/− mice than in C57BL/6 mice, whereas the expression of proteins that participate in the response to stimuli, in cellular processes, and in the immune system was higher.

Conclusions

The data presented in this study suggest that NF-κB might regulate exosomal protein expression at a remote site via circulation following I/R injury.     

Targeted deletion of neuropeptide Y (NPY) modulates experimental colitis

Background

Neurogenic inflammation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). We examined the role of neuropeptide Y (NPY) and neuronal nitric oxide synthase (nNOS) in modulating colitis.

Methods

Colitis was induced by administration of dextran sodium sulphate (3% DSS) or streptomycin pre-treated Salmonella typhimurium (S.T.) in wild type (WT) and NPY (NPY^−/−) knockout mice. Colitis was assessed by clinical score, histological score and myeloperoxidase activity. NPY and nNOS expression was assessed by immunostaining. Oxidative stress was assessed by measuring catalase activity, glutathione and nitrite levels. Colonic motility was assessed by isometric muscle recording in WT and DSS-treated mice.

Results

DSS/S.T. induced an increase in enteric neuronal NPY and nNOS expression in WT mice. WT mice were more susceptible to inflammation compared to NPY^−/− as indicated by higher clinical & histological scores, and myeloperoxidase (MPO) activity (p<0.01). DSS-WT mice had increased nitrite, decreased glutathione (GSH) levels and increased catalase activity indicating more oxidative stress. The lower histological scores, MPO and chemokine KC in S.T.-treated nNOS^−/− and NPY^−/−/nNOS^−/− mice supported the finding that loss of NPY-induced nNOS attenuated inflammation. The inflammation resulted in chronic impairment of colonic motility in DSS-WT mice. NPY –treated rat enteric neurons in vitro exhibited increased nitrite and TNF-α production.

Conclusions

NPY mediated increase in nNOS is a determinant of oxidative stress and subsequent inflammation. Our study highlights the role of neuronal NPY and nNOS as mediators of inflammatory processes in IBD.     

Differential Role for CD80 and CD86 in the Regulation of the Innate Immune Response in Murine Polymicrobial Sepsis

Background

Inflammation in the early stages of sepsis is governed by the innate immune response. Costimulatory molecules are a receptor/ligand class of molecules capable of regulation of inflammation in innate immunity via macrophage/neutrophil contact. We recently described that CD80/86 ligation is required for maximal macrophage activation and CD80/86^−/− mice display reduced mortality and inflammatory cytokine production after cecal ligation and puncture (CLP). However, these data also demonstrate differential regulation of CD80 and CD86 expression in sepsis, suggesting a divergent role for these receptors. Therefore, the goal of this study was to determine the individual contribution of CD80/86 family members in regulating inflammation in sepsis.

Methodology/Principal Findings

CD80^−/− mice had improved survival after CLP when compared to WT or CD86^−/− mice. This was associated with preferential attenuation of inflammatory cytokine production in CD80^−/− mice. Results were confirmed with pharmacologic blockade, with anti-CD80 mAb rescuing mice when administered before or after CLP. In vitro, activation of macrophages with neutrophil lipid rafts caused selective disassociation of IRAK-M, a negative regulator of NF-κB signaling from CD80; providing a mechanism for preferential regulation of cytokine production by CD80. Finally, in humans, upregulation of CD80 and loss of constitutive CD86 expression on monocytes was associated with higher severity of illness and inflammation confirming the findings in our mouse model.

Conclusions

In conclusion, our data describe a differential role for CD80 and CD86 in regulation of inflammation in the innate immune response to sepsis. Future therapeutic strategies for blockade of the CD80/86 system in sepsis should focus on direct inhibition of CD80.     

Guanylate Cyclase C Deficiency Causes Severe Inflammation in a Murine Model of Spontaneous Colitis

Background

Guanylate Cyclase C (GC-C; Gucy2c) is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses.

Methods

We utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C^+/+ and GC-C^−/− mice were bred with interleukin (IL)-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10^−/−, GC-C^+/+IL-10^−/− and GC-C^−/−IL-10^−/− mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line.

Results

Relative to GC-C^+/+ animals, intraperitoneal lipopolysaccharide injection into GC-C^−/− mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C^−/−IL-10^−/− animals was significantly more severe relative to GC-C^+/+IL-10^−/− mice. Unlike GC-C^+/+IL-10^−/− controls, colon pathology in GC-C^−/−IL-10^−/− animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C^−/−IL-10^−/− mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells.

Conclusions

The GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by systemic cytokine burst or loss of mucosal immune cell immunosuppression. These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.     

Deficient Production of Reactive Oxygen Species Leads to Severe Chronic DSS-Induced Colitis in Ncf1/p47phox-Mutant Mice

Background

Colitis is a common clinical complication in chronic granulomatous disease (CGD), a primary immunodeficiency caused by impaired oxidative burst. Existing experimental data from NADPH-oxidase knockout mice propose contradictory roles for the involvement of reactive oxygen species in colitis chronicity and severity. Since genetically controlled mice with a point-mutation in the Ncf1 gene are susceptible to chronic inflammation and autoimmunity, we tested whether they presented increased predisposition to develop chronic colitis.

Methods

Colitis was induced in Ncf1-mutant and wild-_type mice by a 1^st 7-days cycle of dextran sulfate sodium (DSS), intercalated by a 7-days resting period followed by a 2^nd 7-days DSS-cycle. Cytokines were quantified locally in the colon inflammatory infiltrates and in the serum. Leukocyte infiltration and morphological alterations of the colon mucosa were assessed by immunohistochemistry.

Results

Clinical scores demonstrated a more severe colitis in Ncf1-mutant mice than controls, with no recovery during the resting period and a severe chronic colitis after the 2^nd cycle, confirmed by histopathology and presence of infiltrating neutrophils, macrophages, plasmocytes and lymphocytes in the colon. Severe colitis was mediated by increased local expression of cytokines (IL-6, IL-10, TNF-α, IFN-γ and IL-17A) and phosphorylation of Leucine-rich repeat kinase 2 (LRRK2). Serological cytokine titers of those inflammatory cytokines were more elevated in Ncf1-mutant than control mice, and were accompanied by systemic changes in functional subsets of monocytes, CD4⁺T and B cells.

Conclusion

This suggests that an ineffective oxidative burst leads to severe chronic colitis through local accumulation of peroxynitrites, pro-inflammatory cytokines and lymphocytes and systemic immune deregulation similar to CGD.