Intestine-Specific Mttp Deletion Increases the Severity of Experimental Colitis and Leads to Greater Tumor Burden in a Model of Colitis Associated Cancer

Background

Gut derived lipid factors have been implicated in systemic injury and inflammation but the precise pathways involved are unknown. In addition, dietary fat intake and obesity are independent risk factors for the development of colorectal cancer. Here we studied the severity of experimental colitis and the development of colitis associated cancer (CAC) in mice with an inducible block in chylomicron secretion and fat malabsorption, following intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO).

Methodology/Principal Findings

Mttp-IKO mice exhibited more severe injury with ∼90% mortality following dextran sodium sulfate (DSS) induced colitis, compared to <20% in controls. Intestinal permeability was increased in Mttp-IKO mice compared to controls, both at baseline and after DSS administration, in association with increased circulating levels of TNFα. DSS treatment increased colonic mRNA expression of IL-1β and IL-17A as well as inflammasome expression in both genotypes, but the abundance of TNFα was selectively increased in DSS treated Mttp-IKO mice. There was a 2-fold increase in colonic tumor burden in Mttp-IKO mice following azoxymethane/DSS treatment, which was associated with increased colonic inflammation as well as alterations in cytokine expression. To examine the pathways by which alterations in fatty acid abundance might interact with cytokine signaling to regulate colonic epithelial growth, we used primary murine myofibroblasts to demonstrate that palmitate induced expression of amphiregulin and epiregulin and augmented the increase in both of these growth mediators when added to IL-1βor to TNFα.

Conclusions

These studies demonstrate that Mttp-IKO mice, despite absorbing virtually no dietary fat, exhibit augmented fatty acid dependent signaling that in turn exacerbates colonic injury and increases tumor formation.     

Dynamics of pro-inflammatory and anti-inflammatory cytokine release during zcute inflammation in chronic obstructive pulmonary disease: an ex vivo study

Background

Exacerbations of Chronic obstructive pulmonary disease (COPD) are an important cause of the morbidity and mortality associated with the disease. Strategies to reduce exacerbation frequency are thus urgently required and depend on an understanding of the inflammatory milieu associated with exacerbation episodes. Bacterial colonisation has been shown to be related to the degree of airflow obstruction and increased exacerbation frequency. The aim of this study was to asses the kinetics of cytokine release from COPD parenchymal explants using an ex vivo model of lipopolysaccharide (LPS) induced acute inflammation.

Methods

Lung tissue from 24 patients classified by the GOLD guidelines (7F/17M, age 67.9 ± 2.0 yrs, FEV₁ 76.3 ± 3.5% of predicted) and 13 subjects with normal lung function (8F,5M, age 55.6 ± 4.1 yrs, FEV₁ 98.8 ± 4.1% of predicted) was stimulated with 100 ng/ml LPS alone or in combination with either neutralising TNFα or IL-10 antibodies and supernatant collected at 1,2,4,6,24, and 48 hr time points and analysed for IL-1β, IL-5, IL-6, CXCL8, IL-10 and TNFα using ELISA. Following culture, explants were embedded in glycol methacrylate and immunohistochemical staining was conducted to determine the cellular source of TNFα, and numbers of macrophages, neutrophils and mast cells.

Results

In our study TNFα was the initial and predictive cytokine released followed by IL-6, CXCL8 and IL-10 in the cytokine cascade following LPS exposure. The cytokine cascade was inhibited by the neutralisation of the TNFα released in response to LPS and augmented by the neutralisation of the anti-inflammatory cytokine IL-10. Immunohistochemical analysis indicated that TNFα was predominantly expressed in macrophages and mast cells. When patients were stratified by GOLD status, GOLD I (n = 11) and II (n = 13) individuals had an exaggerated TNFα responses but lacked a robust IL-10 response compared to patients with normal lung function (n = 13).

Conclusion

We report on a reliable ex vitro model for the investigation of acute lung inflammation and its resolution using lung parenchymal explants from COPD patients. We propose that differences in the production of both TNFα and IL-10 in COPD lung tissue following exposure to bacterial LPS may have important biological implications for both episodes of exacerbation, disease progression and amelioration.     

TLR4 Expression by Liver Resident Cells Mediates the Development of Glucose Intolerance and Insulin Resistance in Experimental Periodontitis

Background

Results from epidemiological studies indicate a close association between periodontitis and type 2 diabetes mellitus. However, the mechanism linking periodontitis to glucose intolerance (GI) and insulin resistance (IR) is unknown. We therefore tested the hypothesis that periodontitis induces the development of GI/IR through a liver Toll-like receptor 4 (TLR4) dependent mechanism.

Methods

TLR4 chimeric mice were developed by bone marrow transplantation using green fluorescent protein expressing TLR4WT mouse (GFPWT) as donor and TLR4 WT or TLR4-/- as recipient mice (GFPWT:WT and GFPWT:KO chimeras respectively). These chimeras were subjected to experimental chronic periodontitis induced by repeated applications of LPS to the gingival sulci for 18 weeks. The levels of GI/IR were monitored and plasma cytokines and LPS were determined at 18 weeks when differences in glucose tolerance were most apparent. Cytokine gene expression was measured in liver tissue by qPCR.

Results

Alveolar bone loss was significantly greater in GFPWT:WT chimeras treated with LPS compared with chimeras treated with PBS or GFPWT:KO chimeras. However, the degree of gingival inflammation was similar between GFPWT:WT and GFPWT:KO mice with LPS application. Severe GI/IR occurred in GFPWT:WT chimeras but not in the GFPWT:KO chimeras that were subjected to 18 weeks of LPS. Serum LPS was detected only in animals to which LPS was applied and the level was similar in GFPWT:WT and GFPWT:KO mice at the 18 week time point. Surprisingly, there was no significant difference in the plasma levels of IL1β, IL6 and TNFα at 18 weeks in spite of the severe GI/IR in the GFPWT:WT chimeras with LPS application. Also, no difference in the expression of TNFα or IL6 mRNA was detected in the liver of GFPWT:WT vs GFPWT:KO mice. In contrast, liver IL1β expression was significantly greater in GFPWT:WT chimeras compared to GFPWT:KO chimeras treated with LPS.

Conclusion

We observed that GFPWT:WT, but not GFPWT:KO chimeras, treated with LPS developed GI/IR despite similar degrees of gingival inflammation, circulating cytokine levels, and LPS concentrations. We conclude that LPS from periodontitis sites has a pivotal role in triggering the development of GI/IR through a mechanism that involves TLR4 expression by resident macrophages/Kupffer cells in the liver.     

Chronic Alcohol-Induced microRNA-155 Contributes to Neuroinflammation in a TLR4-Dependent Manner in Mice

Introduction

Alcohol-induced neuroinflammation is mediated by pro-inflammatory cytokines and chemokines including tumor necrosis factor-α (TNFα), monocyte chemotactic protein-1 (MCP1) and interleukin-1-beta (IL-1β). Toll-like receptor-4 (TLR4) pathway induced nuclear factor-κB (NF-κB) activation is involved in the pathogenesis of alcohol-induced neuroinflammation. Inflammation is a highly regulated process. Recent studies suggest that microRNAs (miRNAs) play crucial role in fine tuning gene expression and miR-155 is a major regulator of inflammation in immune cells after TLR stimulation.

Aim

To evaluate the role of miR-155 in the pathogenesis of alcohol-induced neuroinflammation.

Methods

Wild type (WT), miR-155- and TLR4-knockout (KO) mice received 5% ethanol-containing or isocaloric control diet for 5 weeks. Microglia markers were measured by q-RTPCR; inflammasome activation was measured by enzyme activity; TNFα, MCP1, IL-1β mRNA and protein were measured by q-RTPCR and ELISA; phospho-p65 protein and NF-κB were measured by Western-blotting and EMSA; miRNAs were measured by q-PCR in the cerebellum. MiR-155 was measured in immortalized and primary mouse microglia after lipopolysaccharide and ethanol stimulation.

Results

Chronic ethanol feeding up-regulated miR-155 and miR-132 expression in mouse cerebellum. Deficiency in miR-155 protected mice from alcohol-induced increase in inflammatory cytokines; TNFα, MCP1 protein and TNFα, MCP1, pro-IL-1β and pro-caspase-1 mRNA levels were reduced in miR-155 KO alcohol-fed mice. NF-κB was activated in WT but not in miR-155 KO alcohol-fed mice. However increases in cerebellar caspase-1 activity and IL-1β levels were similar in alcohol-fed miR-155-KO and WT mice. Alcohol-fed TLR4-KO mice were protected from the induction of miR-155. NF-κB activation measured by phosphorylation of p65 and neuroinflammation were reduced in alcohol-fed TLR4-KO compared to control mice. TLR4 stimulation with lipopolysaccharide in primary or immortalized mouse microglia resulted in increased miR-155.

Conclusion

Chronic alcohol induces miR-155 in the cerebellum in a TLR4-dependent manner. Alcohol-induced miR-155 regulates TNFα and MCP1 expression but not caspase-dependent IL-1β increase in neuroinflammation.     

Lipopolysaccharide Does Not Alter Small Airway Reactivity in Mouse Lung Slices

The bacterial endotoxin, lipopolysaccharide (LPS) has been associated with occupational airway diseases with asthma-like symptoms and in acute exacerbations of COPD. The direct and indirect effects of LPS on small airway reactivity have not been fully elucidated. We tested the hypothesis that both in vitro and in vivo LPS treatment would increase contraction and impair relaxation of mouse small airways. Lung slices were prepared from naïve Balb/C mice and cultured in the absence or presence of LPS (10 μg/ml) for up to 48 h for measurement of TNFα levels in conditioned media. Alternatively, mice were challenged with PBS or LPS in vivo once a day for 4 days for preparation of lung slices or for harvest of lungs for Q-PCR analysis of gene expression of pro-inflammatory cytokines and receptors involved in airway contraction. Reactivity of small airways to contractile agonists, methacholine and serotonin, and bronchodilator agents, salbutamol, isoprenaline and rosiglitazone, were assessed using phase-contrast microscopy. In vitro LPS treatment of slices increased TNFα release 6-fold but did not alter contraction or relaxation to any agonists tested. In vivo LPS treatment increased lung gene expression of TNFα, IL-1β and ryanodine receptor isoform 2 more than 5-fold. However there were no changes in reactivity in lung slices from these mice, even when also incubated with LPS ex vivo. Despite evidence of LPS-induced inflammation, neither airway hyperresponsiveness or impaired dilator reactivity were evident. The increase in ryanodine receptor isoform 2, known to regulate calcium signaling in vascular smooth muscle, warrants investigation. Since LPS failed to elicit changes in small airway reactivity in mouse lung slices following in vitro or in vivo treatment, alternative approaches are required to define the potential contribution of this endotoxin to altered small airway reactivity in human lung diseases.     

Deletion of Smad3 protects against C-reactive protein-induced renal fibrosis and inflammation in obstructive nephropathy

Introduction and Aims: Elevated plasma levels of C-reactive protein (CRP) are closely associated with progressive renal injury in patients with chronic kidney disease (CKD). Here, we tested a hypothesis that CRP may promote renal fibrosis and inflammation via a TGF-β/Smad3-dependent mechanism.Methods: Role and mechanisms of TGF-β/Smad3 in CRP-induced renal fibrosis and inflammation were examined in a mouse model of unilateral ureteral obstruction (UUO) induced in CRP Tg/Smad3 KO mice and in a rat tubular epithelial cell line in which Smad3 gene is stably knocked down (S3KD-NRK52E).Results: We found that mice overexpressing the human CRP gene were largely promoted renal inflammation and fibrosis as evidenced by increasing IL-1β, TNF-α, MCP-1 expression, F4/80⁺ macrophages infiltration, and marked accumulation of α-smooth muscle actin (α-SMA), collagen I and fibronectin in the UUO kidney, which were blunted when Smad3 gene was deleted in CRPtg-Smad3KO. Mechanistically, we found that the protection of renal inflammation and fibrosis in the UUO kidney of CRPtg-Smad3KO mice was associated with the inactivation of CD32-NF-κB and TGF-β/Smad3 signaling.Conclusion: In conclusion, Smad3 deficiency protects against CRP-mediated renal inflammation and fibrosis in the UUO kidney by inactivating CD32-NF-κB and TGF-β/Smad3 signaling.     

The Absence of Mrp4 Has No Effect on the Recruitment of Neutrophils and Eosinophils into the Lung after LPS,Cigarette Smoke or Allergen Challenge

The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4^−/− and Mrp4^+/+ mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4^−/− and Mrp4^+/+ mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4^+/+ or Mrp4^−/− mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.     

Hyaluronic Acid Oligosaccharides Suppress TLR3-Dependent Cytokine Expression in a TLR4-Dependent Manner

The release of endogenous molecules from the skin after injury has been proposed to influence inflammation. Recent studies have found that pro-inflammatory signals can be generated by damaged endogenous self-RNA, and this event is detected by TLR3. Conversely, release of endogenous fragments of hyaluronic acid (HA) after injury has been proposed to inhibit LPS induced inflammation driven by TLR4. In this study we investigated if HA oligomers could also influence inflammation mediated by TLR3. A tetramer form of HA (oligo-HA) was added to MH-S cells (mouse alveolar macrophage cell line) that were then activated by poly(I:C). ELISA analysis of culture supernatants showed that the presence of oligo-HA suppressed the poly(I:C) induced release of IL-6 and TNFα. IL-6 mRNA expression was also suppressed as measured by quantitative RT-PCR. To determine the mechanism of action for oligo-HA to inhibit poly(I:C), macrophages derived from wild-type (WT), Tlr2−/− or Tlr4−/− mice were treated with oligo-HA and poly(I:C). Similar to WT cells, Tlr2−/− macrophages were inhibited by oligo-HA and retained suppression of cytokine release. In contrast, Tlr4−/− macrophages lost the capacity to be suppressed by oligo-HA. An increase in Traf1 (TLR negative regulator) mRNA was observed after oligo-HA treatment of WT but not in Tlr4−/− macrophages, and oligo-HA did not suppress cytokine responsiveness in Traf1−/− macrophages. These results show that oligo-HA acts through TLR4 and TRAF1 to inhibit TLR3-dependent inflammation. This observation illustrates the complex immunomodulatory action of endogenous products released after injury.     

Antigen-Specific Interferon-Gamma Responses and Innate Cytokine Balance in TB-IRIS

Background

Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) remains a poorly understood complication in HIV-TB patients receiving antiretroviral therapy (ART). TB-IRIS could be associated with an exaggerated immune response to TB-antigens. We compared the recovery of IFNγ responses to recall and TB-antigens and explored in vitro innate cytokine production in TB-IRIS patients.

Methods

In a prospective cohort study of HIV-TB co-infected patients treated for TB before ART initiation, we compared 18 patients who developed TB-IRIS with 18 non-IRIS controls matched for age, sex and CD4 count. We analyzed IFNγ ELISpot responses to CMV, influenza, TB and LPS before ART and during TB-IRIS. CMV and LPS stimulated ELISpot supernatants were subsequently evaluated for production of IL-12p70, IL-6, TNFα and IL-10 by Luminex.

Results

Before ART, all responses were similar between TB-IRIS patients and non-IRIS controls. During TB-IRIS, IFNγ responses to TB and influenza antigens were comparable between TB-IRIS patients and non-IRIS controls, but responses to CMV and LPS remained significantly lower in TB-IRIS patients. Production of innate cytokines was similar between TB-IRIS patients and non-IRIS controls. However, upon LPS stimulation, IL-6/IL-10 and TNFα/IL-10 ratios were increased in TB-IRIS patients compared to non-IRIS controls.

Conclusion

TB-IRIS patients did not display excessive IFNγ responses to TB-antigens. In contrast, the reconstitution of CMV and LPS responses was delayed in the TB-IRIS group. For LPS, this was linked with a pro-inflammatory shift in the innate cytokine balance. These data are in support of a prominent role of the innate immune system in TB-IRIS.     

GSDME-mediated pyroptosis promotes inflammation and fibrosis in obstructive nephropathy

Renal tubular cell (RTC) death and inflammation contribute to the progression of obstructive nephropathy, but its underlying mechanisms have not been fully elucidated. Here, we showed that Gasdermin E (GSDME) expression level and GSDME-N domain generation determined the RTC fate response to TNFα under the condition of oxygen-glucose-serum deprivation. Deletion of Caspase-3 (Casp3) or Gsdme alleviated renal tubule damage and inflammation and finally prevented the development of hydronephrosis and kidney fibrosis after ureteral obstruction. Using bone marrow transplantation and cell type-specific Casp3 knockout mice, we demonstrated that Casp3/GSDME-mediated pyroptosis in renal parenchymal cells, but not in hematopoietic cells, played predominant roles in this process. We further showed that HMGB1 released from pyroptotic RTCs amplified inflammatory responses, which critically contributed to renal fibrogenesis. Specific deletion of Hmgb1 in RTCs alleviated caspase11 and IL-1β activation in macrophages. Collectively, our results uncovered that TNFα/Casp3/GSDME-mediated pyroptosis is responsible for the initiation of ureteral obstruction-induced renal tubule injury, which subsequentially contributes to the late-stage progression of hydronephrosis, inflammation, and fibrosis. This novel mechanism will provide valuable therapeutic insights for the treatment of obstructive nephropathy.Subject terms: Cell death and immune response, Kidney diseases     

Lack of the receptor for advanced glycation end-products attenuates E. coli pneumonia in mice

Background

The receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated.

Methodology/Principal Findings

C57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice.

Conclusions/Significance

Lack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation.     

Secreted Protein Acidic and Rich in Cysteine (SPARC) Exacerbates Colonic Inflammatory Symptoms in Dextran Sodium Sulphate-Induced Murine Colitis

Background

Secreted Protein Acidic and Rich in Cysteine (SPARC) is expressed during tissue repair and regulates cellular proliferation, migration and cytokine expression. The aim was to determine if SPARC modifies intestinal inflammation.

Methods

Wild-type (WT) and SPARC-null (KO) mice received 3% dextran sodium sulphate (DSS) for 7 days. Inflammation was assessed endoscopically, clinically and histologically. IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-12/IL23p40, TNF-α, IFN-γ, RANTES, MCP-1, MIP-1α, MIP-1β, MIG and TGF-β1 levels were measured by ELISA and cytometric bead array. Inflammatory cells were characterised by CD68, Ly6G, F4/80 and CD11b immunofluorescence staining and regulatory T cells from spleen and mesenteric lymph nodes were assessed by flow cytometry.

Results

KO mice had less weight loss and diarrhoea with less endoscopic and histological inflammation than WT animals. By day 35, all (n = 13) KO animals completely resolved the inflammation compared to 7 of 14 WT mice (p<0.01). Compared to WTs, KO animals at day 7 had less IL1β (p = 0.025) and MIG (p = 0.031) with higher TGFβ1 (p = 0.017) expression and a greater percentage of FoxP3+ regulatory T cells in the spleen and draining lymph nodes of KO animals (p<0.01). KO mice also had fewer CD68+ and F4/80+ macrophages, Ly6G+ neutrophils and CD11b+ cells infiltrating the inflamed colon.

Conclusions

Compared to WT, SPARC KO mice had less inflammation with fewer inflammatory cells and more regulatory T cells. Together, with increased TGF-β1 levels, this could aid in the more rapid resolution of inflammation and restoration of the intestinal mucosa suggesting that the presence of SPARC increases intestinal inflammation.     

Specific Inhibition of Histone Deacetylase 8 Reduces Gene Expression and Production of Proinflammatory Cytokines in Vitro and in Vivo

ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1β and other cytokines at 25–100 nm; at concentrations >200 nm, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC₅₀ of 285 nm). Here we compared the production of IL-1β, IL-1α, TNFα, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nm; at 1000 nm, the secretion of IL-1β was reduced by 76%, secretion of TNFα was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1α were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100–1000 nm. Gene expression of TNFα was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1β and TNFα was modestly reduced, IFNγ production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1β independent of inhibition of caspase-1 activity; however, synthesis of the IL-1β precursor was reduced by 40% without significant decrease in IL-1β mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNFα by 85% and reduced IL-1β by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity.     

Vascular Cellular Adhesion Molecule-1 (VCAM-1) Expression in Mice Retinal Vessels Is Affected by Both Hyperglycemia and Hyperlipidemia

Background

Inflammation has been proposed to be important in the pathogenesis of diabetic retinopathy. An early feature of inflammation is the release of cytokines leading to increased expression of endothelial activation markers such as vascular cellular adhesion molecule-1 (VCAM-1). Here we investigated the impact of diabetes and dyslipidemia on VCAM-1 expression in mouse retinal vessels, as well as the potential role of tumor necrosis factor-α (TNFα).

Methodology/Principal Findings

Expression of VCAM-1 was examined by confocal immunofluorescence microscopy in vessels of wild type (wt), hyperlipidemic (ApoE^−/−) and TNFα deficient (TNFα^−/−, ApoE^−/−/TNFα^−/−) mice. Eight weeks of streptozotocin-induced diabetes resulted in increased VCAM-1 in wt mice, predominantly in small vessels (<10 µm). Diabetic wt mice had higher total retinal TNFα, IL-6 and IL-1β mRNA than controls; as well as higher soluble VCAM-1 (sVCAM-1) in plasma. Lack of TNFα increased higher basal VCAM-1 protein and sVCAM-1, but failed to up-regulate IL-6 and IL-1β mRNA and VCAM-1 protein in response to diabetes. Basal VCAM-1 expression was higher in ApoE^−/− than in wt mice and both VCAM-1 mRNA and protein levels were further increased by high fat diet. These changes correlated to plasma cholesterol, LDL- and HDL-cholesterol, but not to triglycerides levels. Diabetes, despite further increasing plasma cholesterol in ApoE^−/− mice, had no effects on VCAM-1 protein expression or on sVCAM-1. However, it increased ICAM-1 mRNA expression in retinal vessels, which correlated to plasma triglycerides.

Conclusions/Significance

Hyperglycemia triggers an inflammatory response in the retina of normolipidemic mice and up-regulation of VCAM-1 in retinal vessels. Hypercholesterolemia effectively promotes VCAM-1 expression without evident stimulation of inflammation. Diabetes-induced endothelial activation in ApoE^−/− mice seems driven by elevated plasma triglycerides but not by cholesterol. Results also suggest a complex role for TNFα in the regulation of VCAM-1 expression, being protective under basal conditions but pro-inflammatory in response to diabetes.     

CPEB Regulation of TAK1 Synthesis Mediates Cytokine Production and the Inflammatory Immune Response

The cytoplasmic-element-binding (CPEB) protein is a sequence-specific RNA-binding protein that regulates cytoplasmic polyadenylation-induced translation. In mouse embryo fibroblasts (MEFs) lacking CPEB, many mRNAs encoding proteins involved in inflammation are misregulated. Correlated with this aberrant translation in MEFs, a macrophage cell line depleted of CPEB and treated with lipopolysaccharide (LPS) to stimulate the inflammatory immune response expresses high levels of interleukin-6 (IL-6), which is due to prolonged nuclear retention of NF-κB. Two proteins involved in NF-κB nuclear localization and IL-6 expression, IκBα and transforming growth factor beta-activated kinase 1 (TAK1), are present at excessively low and high steady-state levels, respectively, in LPS-treated CPEB-depleted macrophages. However, only TAK1 has an altered synthesis rate that is CPEB dependent and CPEB/TAK1 double depletion alleviates high IL-6 production. Peritoneal macrophages isolated from CPEB knockout (KO) mice treated with LPS in vitro also have prolonged NF-κB nuclear retention and produce high IL-6 levels. LPS-injected CPEB KO mice secrete prodigious amounts of IL-6 and other proinflammatory cytokines and exhibit hypersensitivity to endotoxic shock; these effects are mitigated when the animals are also injected with (5Z)-7-oxozeaenol, a potent and specific inhibitor of TAK1. These data show that CPEB control of TAK1 mRNA translation mediates the inflammatory immune response.     

Th17 Down-regulation Is Involved in Reduced Progression of Schistosomiasis Fibrosis in ICOSL KO Mice

Background

Granulomatous and fibrosing inflammation in response to parasite eggs is the main pathology that occurs during infection with Schistosoma spp. CD4+ T cells play critical roles in both host immune responses against parasitic infection and immunopathology in schistosomiasis,and coordinate many types of immune cells that contribute to fibrosis. ICOSL plays an important role in controlling specific aspects of T cell activation, differentiation, and function. Previous work has suggested that ICOS is essential for Th17 cell development. However, the immunopathogenesis of this pathway in schistosomiasis fibrosisis still unclear.

Methodology/Principal Findings

Using models of schistosomiasis in ICOSL KO and the C57BL/6 WT mice, we studied the role of the ICOSL/ICOS interaction in the mediation of the Th17 response in host granulomatous inflammation, particularly in liver fibrosis during S. japonicum infection, and investigated the immune responses and pathology of ICOSL KO mice in these models. The results showed that ICOSL KO mice exhibited improved survival, reduced liver granulomatous inflammation around parasite eggs, markedly inhibited hepatic fibrosis development, lower levels of Th17-related cytokines (IL-17/IL-21), Th2-related cytokines (IL-4/IL-6/IL-10), a pro-fibrotic cytokine (IL-13), and TGF-β1, but higher level of Th1-related cytokine (IFN-γ) compared to wild-type (WT) mice. The reduced progression of fibrogenesis was correlated with the down-regulation of Th17 and Th2 and the elimination of ICOSL/ICOS interactions.

Conclusions/Significance

Our findings suggest that IL-17-producing cells contribute to the hepatic granulomatous inflammation and subsequent fibrosis. Importantly, there was a clearly positive correlation between the presence of IL-17-producing cells and ICOS expression in ICOSL KO mice, and additional results indicated that Th17 was involved in the pathological tissue remodeling in liver fibrosis induced by schistosomiasis.     

Kuppfer Cells Trigger Nonalcoholic Steatohepatitis Development in Diet-induced Mouse Model through Tumor Necrosis Factor-α Production

Nonalcoholic steatohepatitis (NASH), characterized by lipid deposits within hepatocytes (steatosis), is associated with hepatic injury and inflammation and leads to the development of fibrosis, cirrhosis, and hepatocarcinoma. However, the pathogenic mechanism of NASH is not well understood. To determine the role of distinct innate myeloid subsets in the development of NASH, we examined the contribution of liver resident macrophages (i.e. Kupffer cells) and blood-derived monocytes in triggering liver inflammation and hepatic damage. Employing a murine model of NASH, we discovered a previously unappreciated role for TNFα and Kupffer cells in the initiation and progression of NASH. Sequential depletion of Kupffer cells reduced the incidence of liver injury, steatosis, and proinflammatory monocyte infiltration. Furthermore, our data show a differential contribution of Kupffer cells and blood monocytes during the development of NASH; Kupffer cells increased their production of TNFα, followed by infiltration of CD11b^intLy6C^hi monocytes, 2 and 10 days, respectively, after starting the methionine/choline-deficient (MCD) diet. Importantly, targeted knockdown of TNFα expression in myeloid cells decreased the incidence of NASH development by decreasing steatosis, liver damage, monocyte infiltration, and the production of inflammatory chemokines. Our findings suggest that the increase of TNFα-producing Kupffer cells in the liver is crucial for the early phase of NASH development by promoting blood monocyte infiltration through the production of IP-10 and MCP-1.     

Neurotropin Suppresses Inflammatory Cytokine Expression and Cell Death through Suppression of NF-κB and JNK in Hepatocytes

Inflammatory response and cell death in hepatocytes are hallmarks of chronic liver disease, and, therefore, can be effective therapeutic targets. Neurotropin® (NTP) is a drug widely used in Japan and China to treat chronic pain. Although NTP has been demonstrated to suppress chronic pain through the descending pain inhibitory system, the action mechanism of NTP remains elusive. We hypothesize that NTP functions to suppress inflammatory pathways, thereby attenuating disease progression. In the present study, we investigated whether NTP suppresses inflammatory signaling and cell death pathways induced by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) in hepatocytes. NTP suppressed nuclear factor-κB (NF-κB) activation induced by IL-1β and TNFα assessed by using hepatocytes isolated from NF-κB-green fluorescent protein (GFP) reporter mice and an NF-κB-luciferase reporter system. The expression of NF-κB target genes, Il6, Nos2, Cxcl1, ccl5 and Cxcl2 induced by IL-1β and TNFα was suppressed after NTP treatment. We also found that NTP suppressed the JNK phosphorylation induced by IL-1β and TNFα. Because JNK activation contributes to hepatocyte death, we determined that NTP treatment suppressed hepatocyte death induced by IL-1β and TNFα in combination with actinomycin D. Taken together, our data demonstrate that NTP attenuates IL-1β and TNFα-mediated inflammatory cytokine expression and cell death in hepatocytes through the suppression of NF-κB and JNK. The results from the present study suggest that NTP may become a preventive or therapeutic strategy for alcoholic and non-alcoholic fatty liver disease in which NF-κB and JNK are thought to take part.     

Peripheral TNFalpha, but not peripheral IL-1, requires endogenous IL-1 or TNFalpha induction in the brain for the febrile response

It is known that peripherally administered IL-1 and TNFα induce fever through mechanisms involving prostaglandin (PG)E₂. In this report, we compared the signaling cascade induced in the brain by TNFα and IL-1. Peripheral administration of TNFα-induced enhanced fever in IL-1 Receptor antagonist KO mice, suggesting that IL-1 is involved in the TNFα mediated fever. IL-1α, but not TNFα, induced fever in IL-1α/β/TNFα KO mice, although central administration of TNFα-induced fever. Only IL-1α, but not TNFα, induced IL-6 in the IL-1α/β/TNFα KO mouse brain, while both cytokines induced cyclooxygenase (Cox)-2. Icv administration of PGE₂ induced only transient fever in contrast to the TNFα- or IL-1α-induced fever that lasted longer. Taken together, either IL-1 or TNFα induction in the brain is required for the response induced by TNFα but not by IL-1α, and that both Cox-2 and IL-6 induction are required for prolonged febrile response against these cytokines.