Hyperresponsivity to low-dose endotoxin during progression to nonalcoholic steatohepatitis is regulated by leptin-mediated signaling

Although bacterial endotoxin, such as lipopolysaccharide (LPS), plays a key role in the pathogenesis of nonalcoholic steatohepatitis (NASH), detailed mechanisms of this pathogenesis remain unclear. Here, we demonstrate that upregulation of CD14 by leptin-mediated signaling is critical to hyperreactivity against endotoxin during NASH progression. Upregulation of CD14 in Kupffer cells and hyperreactivity against low-dose LPS were observed in high-fat diet (HFD)-induced steatosis mice, but not chow-fed-control mice. Hyperresponsivity against low-dose LPS led to accelerated NASH progression, including liver inflammation and fibrosis. Administering leptin in chow-fed mice caused increased hepatic expression of CD14 via STAT3 signaling, resulting in hyperreactivity against low-dose LPS without steatosis. In contrast, a marked decrease in hepatic CD14 expression was observed in leptin-deficient ob/ob mice, despite severe steatosis. Our results indicate that obesity-induced leptin plays a crucial role in NASH progression via enhanced responsivity to endotoxin, and we propose a mechanism of bacteria-mediated progression of NASH.     

Oil composition of high-fat diet affects metabolic inflammation differently in connection with endotoxin receptors in mice

Low-grade inflammation observed in obesity is a risk factor for cardiovascular disease. Recent studies revealed that this would be linked to gut-derived endotoxemia during fat digestion in high-fat diets, but nothing is known about the effect of lipid composition. The study was designed to test the impact of oil composition of high-fat diets on endotoxin metabolism and inflammation in mice. C57/Bl6 mice were fed for 8 wk with chow or isocaloric isolipidic diets enriched with oils differing in fatty acid composition: milk fat, palm oil, rapeseed oil, or sunflower oil. In vitro, adipocytes (3T3-L1) were stimulated or not with lipopolysaccharide (LPS; endotoxin) and incubated with different fatty acids. In mice, the palm group presented the highest level of IL-6 in plasma (P < 0.01) together with the highest expression in adipose tissue of IL-1β and of LPS-sensing TLR4 and CD14 (P < 0.05). The higher inflammation in the palm group was correlated with a greater ratio of LPS-binding protein (LBP)/sCD14 in plasma (P < 0.05). The rapeseed group resulted in higher sCD14 than the palm group, which was associated with lower inflammation in both plasma and adipose tissue despite higher plasma endotoxemia. Taken together, our results reveal that the palm oil-based diet resulted in the most active transport of LPS toward tissues via high LBP and low sCD14 and the greatest inflammatory outcomes. In contrast, a rapeseed oil-based diet seemed to result in an endotoxin metabolism driven toward less inflammatory pathways. This shows that dietary fat composition can contribute to modulate the onset of low-grade inflammation through the quality of endotoxin receptors.     

Mechanisms of endotoxin tolerance in human intestinal microvascular endothelial cells

Lipopolysaccharide (endotoxin) tolerance is well described in monocytes and macrophages, but is less well characterized in endothelial cells. Because intestinal microvascular endothelial cells exhibit a strong immune response to LPS challenge and play a critical regulatory role in gut inflammation, we sought to characterize the activation response of these cells to repeated LPS exposure. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were stimulated with LPS over 6-60 h and activation was assessed using U937 leukocyte adhesion, expression of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, manganese superoxide dismutase, HLA-DR, and CD86. Effect of repeat LPS stimulation on HIMEC NF-kappaB and mitogen-activated protein kinase (MAPK) activation, generation of superoxide anion, and Toll-like receptor 4 expression was characterized. LPS pretreatment of HIMEC for 24-48 h significantly decreased leukocyte adhesion after subsequent LPS stimulation. LPS pretreatment inhibited expression of E-selectin, VCAM-1, IL-6, and CD86, while ICAM-1, IL-8, and HLA-DR were not altered. Manganese superoxide dismutase expression increased with repeated LPS stimulation, with a reduction in intracellular superoxide. NF-kappaB activation was transiently inhibited by LPS pretreatment for 6 h, but not at later time points. In contrast, p44/42 MAPK, p38 MAPK, and c-Jun N-terminal kinase activation demonstrated inhibition by LPS pretreatment 24 or 48 h prior. Toll-like receptor 4 expression on HIMEC was not altered by LPS. HIMEC exhibit endotoxin tolerance after repeat LPS exposure in vitro, characterized by diminished activation and intracellular superoxide anion concentration, and reduced leukocyte adhesion. HIMEC possess specific mechanisms of immunoregulatory hyporesponsiveness to repeated LPS exposure.     

Expression of CD14 corrects the slow response to lipopolysaccharide in the 1B8 mutant of the B cell lymphoma 70Z/3

 B cells and macrophages both activate NF-κB/Rel in response to lipopolysaccharide (LPS), but differ in sensitivity to LPS and in downstream genes that are activated. CD14 is a high-affinity receptor for LPS found on macrophages, but not B cells. We expressed human CD14 (hCD14) in the mouse B lymphoma, 70Z/3, and a mutant, 1B8, which responds slowly to LPS, to test whether expression of hCD14 could correct or bypass the defect in 1B8 cells. We compared the timing and extent of known responses to LPS in 70Z/3 cells and the 1B8 mutants. The hCD14⁺ 1B8 and 70Z/3 cells responded more rapidly and were sensitive to 100-fold lower levels of LPS than their untransfected counterparts. Degradation of the IκB-α and -β molecules and translocation of the NF-κB/Rel complexes into the nucleus were more rapid and the steady-state levels of Igk mRNA and mIgM on the cell surface were markedly increased in cells that expressed hCD14. The LPS response of the hCD14⁺ 1B8 and 70Z/3 cells showed subtle differences. In the 1B8 hCD14 cells, the p50/p50 complexes were never abundant in nuclear extracts, and degradation of IκB-β was slower than in hCD14 70Z/3 cells. This partial correction of the 1B8 phenotype suggests that the defective component in 1B8 participates in the CD14 signaling pathway and could include the B-cell LPS receptor itself. Received: 3 June 1997 / Revised: 26 June 1997     

Neurogenic peptides and the cardiomyopathy of magnesium-deficiency: Effects of substance P-receptor inhibition

Dietary deficiency of magnesium (Mg) in rodents results in cardiomyopathic lesion formation. In our rat model, these lesions develop after 3 weeks on the Mg-deficient diet; significant elevation of several cytokines, IL-1, IL-6 and TNF also occurs. In probing the mechanisms of lesion formation, we obtained data supporting the participation of free radicals (Freedman AMet al.: Bioch Biophys Res Commun 1990; 170: 1102). Recently, we identified an early elevation of circulating substance P and proposed a role of neurogenic peptides during Mg-deficiency (Weglicki WB, Phillips TM: Am J Phys 1992; 262: R734). The present study was designed to evaluate the contribution of neurogenic peptides to the pathogenesis of Mg-deficiency. In the blood, substance-P and calcitonin gene related peptide (CGRP) are elevated during the first week on the diet. During the second week, circulating histamine, PGE₂ and TBAR-materials were elevated and red cell glutathione was reduced, all prior to the elevation of the inflammatory cytokines during the third week. When the rats were treated with the substance P-receptor blocker [CP-96,345], the levels of substance P and CGRP remained elevated; however, increases in histamine, PGE₂, TBAR-materials, and the decrease in red cell glutathione were inhibited; also, the development of cardiac lesions was inhibited significantly. These data support a central role for neurogenic peptides, especially substance P, in the development of cardiomyopathic lesions during Mg-deficiency.     

Residual Endotoxin Contaminations in Recombinant Proteins Are Sufficient to Activate Human CD1c+ Dendritic Cells

Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c⁺ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002–2 ng/ml). We show that CD1c⁺ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c⁺ DCs was closely correlated with high CD14 expression levels observed in CD1c⁺ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.     

Peyer’s Patches and Mesenteric Lymph Nodes Cooperatively Promote Enteropathy in a Mouse Model of Food Allergy

Background and Objective

To improve the efficacy and safety of tolerance induction for food allergies, identifying the tissues responsible for inducing intestinal inflammation and subsequent oral tolerance is important. We used OVA23-3 mice, which express an ovalbumin-specific T-cell receptor, to elucidate the roles of local and systemic immune tissues in intestinal inflammation.

Methods and Results

OVA23-3 mice developed marked enteropathy after consuming a diet containing egg white (EW diet) for 10 days but overcame the enteropathy (despite continued moderate inflammation) after receiving EW diet for a total of 28 days. Injecting mice with anti-IL-4 antibody or cyclosporine A confirmed the involvement of Th2 cells in the development of the enteropathy. To assess the individual contributions of Peyer’s patches (PPs), mesenteric lymph nodes (MLNs), and the spleen to the generation of effector CD4⁺ T-cells, we analyzed the IL-4 production, proliferation in response to ovalbumin, and CD4⁺ T-cell numbers of these tissues. EW feeding for 10 days induced significant IL-4 production in PPs, the infiltration of numerous CD4⁺ T-cells into MLNs, and a decrease in CD4⁺ T-cell numbers in spleen. On day 28, CD4⁺ T-cells from all tissues had attenuated responses to ovalbumin, suggesting tolerance acquisition, although MLN CD4⁺ T-cells still maintained IL-4 production with proliferation. In addition, removal of MLNs but not the spleen decreased the severity of enteropathy and PP-disrupted mice showed delayed onset of EW-induced inflammatory responses. Disruption of peripheral lymphoid tissues or of both PPs and MLNs almost completely prevented the enteropathy.

Conclusions

PPs and MLNs coordinately promote enteropathy by generating effector T-cells during the initial and exacerbated phases, respectively; the spleen is dispensable for enteropathy and shows tolerogenic responses throughout EW-feeding. The regulation of PPs may suppress the initiation of intestinal inflammation, subsequently restricting MLNs and inhibiting the progression of food-allergic enteropathy.     

TLR4 is the signaling but not the lipopolysaccharide uptake receptor

TLR4 is the primary recognition molecule for inflammatory responses initiated by bacterial LPS (endotoxin). Internalization of endotoxin by various cell types is an important step for its removal and detoxification. Because of its role as an LPS-signaling receptor, TLR4 has been suggested to be involved in cellular LPS uptake as well. LPS uptake was investigated in primary monocytes and endothelial cells derived from TLR4 and CD14 knockout C57BL/6 mice using tritiated and fluorescein-labeled LPS. Intracellular LPS distribution was investigated by deconvolution confocal microscopy. We could not observe any difference in LPS uptake and intracellular LPS distribution in either monocytes or endothelial cells between TLR4(-/-) and wild-type cells. As expected, CD14(-/-) monocytes showed a highly impaired LPS uptake, confirming CD14-dependent uptake in monocytes. Upon longer incubation periods, the CD14-deficient monocytes mimicked the LPS uptake pattern of endothelial cells. Endothelial cell LPS uptake is slower than monocyte uptake, LBP rather than CD14 dependent, and sensitive to polyanionic polymers, which have been shown to block scavenger receptor-dependent uptake mechanisms. We conclude that TLR4 is not involved in cellular LPS uptake mechanisms. In membrane CD14-positive cells, LPS is predominantly taken up via CD14-mediated pathways, whereas in the CD14-negative endothelial cells, there is a role for scavenger receptor-dependent pathways.     

N-acetylcysteine reduces inflammation in the small intestine by regulating redox, EGF and TLR4 signaling

This study determined whether N-acetylcysteine (NAC) could affect intestinal redox status, proinflammatory cytokines, epidermal growth factor (EGF), EGF receptor (EGFR), Toll-like receptor-4 (TLR4), and aquaporin-8 in a lipopolysaccharide (LPS)-challenged piglet model. Eighteen piglets (35-day-old) were randomly allocated into one of the three treatments (control, LPS and NAC). The control and LPS groups were fed a basal diet, and the NAC group received the basal diet +500 mg/kg NAC. On days 10, 13, and 20 of the trial, the LPS- and NAC-treated piglets received intraperitoneal administration of LPS (100 μg/kg BW), whereas the control group received the same volume of saline. On days 10 and 20, venous blood samples were obtained at 3 h post LPS or saline injection. On day 21 of the trial, piglets were killed to obtain the intestinal mucosa for analysis. Compared with the control group, LPS challenge reduced (P < 0.05) the activities of superoxide dismutase, catalase, and glutathione peroxidase in jejunal mucosae, while increasing (P < 0.05) the concentrations of malondialdehyde, H₂O₂, O₂ ^·? and the ratio of oxidized to reduced glutathione in jejunal mucosae, and concentrations of TNF-α, cortisol, interleukin-6, and prostaglandin E₂ in both plasma and intestinal mucosae. These adverse effects of LPS were attenuated (P < 0.05) by NAC supplementation. Moreover, NAC prevented LPS-induced increases in abundances of intestinal HSP70 and NF-κB p65 proteins and TLR4 mRNA. NAC supplementation enhanced plasma EGF concentration and intestinal EGFR mRNA levels. Collectively, these results indicate that dietary NAC supplementation alleviates LPS-induced intestinal inflammation via regulating redox, EGF, and TLR4 signaling.     

Modification of Cytokine Milieu by A2A Adenosine Receptor Signaling–Possible Application for Inflammatory Diseases

Pro‐inflammatory cytokine TNF‐alpha (TNF) production from in vitro lipopolysaccharide (LPS)‐stimulated human peripheral blood CD14⁺ cells (PB‐CD14) was inhibited by A_2A adenosine receptor (AdoR) (A_2AR) or ß₂ adrenergic receptor (ADR) (ß₂R) signaling in a concentration‐dependent manner. These inhibitory effects were presumably mediated by the increase in intracellular cAMP. Furthermore A_2AR agonist and ß₂R agonist synergistically inhibited the TNF production of LPS‐stimulated PB‐CD14 cells. These results suggest that the anti‐inflammatory effect of extracellular adenosine is, at least in part, due to the modification of the cytokine milieu via A_2A signaling, and that the targeting of both A_2AR and ß₂R may have strong therapeutic potential for the inflammatory diseases.     

Spirulina platensis alleviates chronic inflammation with modulation of gut microbiota and intestinal permeability in rats fed a high‐fat diet

Recent research suggested that taking a high‐fat diet (HFD) may lead to a gut microbiota imbalance and colon tissue damage. This would lead to increased intestinal permeability and consequent constant circulation of low‐grade inflammatory cytokines. Spirulina platensis can protect against HFD‐induced metabolic inflammation and can stimulate the growth of beneficial bacteria in in vitro stool cultures. However, it is unknown whether this beneficial effect acts on intestinal tissues. In this study, rats were fed a high‐fat diet fed with 3% S platensis for 14 weeks. We analysed endotoxin, the composition of the microbiota, inflammation and gut permeability. We found that S platensis decreased the bodyweight and visceral fat pads weight of the HFD‐fed rats. In addition, it lowered the levels of lipopolysaccharide and pro‐inflammatory cytokines in serum. Our results showed that S platensis could largely reduce the relative amount of Proteobacteria and the Firmicutes/Bacteroidetes ratio in faecal samples from HFD‐fed rats. S platensis significantly reduced intestinal inflammation, as shown by decreased expression of myeloid differentiation factor 88 (MyD88), toll‐like receptor 4 (TLR4), NF‐κB (p65) and inflammatory cytokines. S platensis also ameliorated the increased permeability and decreased expression of tight junction proteins in the intestinal mucosa, such as ZO‐1, Occludin and Claudin‐1. Therefore, in HFD‐induced gut dysbiosis rats, S platensis benefits health by inhibiting chronic inflammation and gut dysbiosis, and modulating gut permeability.     

Antagonism by ethanol of endotoxin-induced tissue factor activation in relation to the depressed endotoxin binding to monocyte-like U937 cells

Our previous study has reported that ethanol (ETOH) partially inhibited the endotoxin (LPS)-induced tissue factor (TF)-activation in monocytes including blood peripheral monocytes as well as cultured leukemic U937 and THP-1 cells. The present study shows a strong correlation (r=0·92; p<0·01) between TF-activation and depression in LPS binding blocked by ETOH in U937 cells. The antagonism by ETOH of LPS binding was not due to a direct extracellular blockade, since ETOH did not affect the affinity of fluorescein isothiocyanate (FITC)-LPS or -anti CD14 mAb on U937 cells. After U937 cells were treated with 2 per cent (v/v) ETOH for 3 h, LPS binding was however drastically inhibited as shown by immunostaining with FITC-LPS which was viewed on a confocal laser scanning microscope. The results imply that cellular events of the ETOH effect mediate this inhibition of LPS binding. Anti-CD14 mAb (UCHM-1) inhibited LPS binding in a dose-dependent fashion, revealing a competitive specific binding to the LPS receptor. The results suggest that CD14 plays an important role in the recognition of LPS. FITC-UCHM-1 binding was significantly reduced in the cells pretreated with 2 per cent (v/v) ETOH for 3 h, indicating that ETOH modulates the ability to express CD14. CD14 expression was upregulated by priming with LPS which was offset by ETOH. Acetaldehyde, a possible metabolite of ETOH, was tested with no effect on CD14 expression. Taken together, our results show that ETOH downregulates the recognition of LPS, and suggest that the inhibitory action is likely to be mediated by the depression in CD14 expression which was also accompanied by a significantly altered membrane fluidity. Thus, the antagonism by ETOH of the binding of LPS results in a depression in the LPS-induced TF-activation. © 1997 John Wiley & Sons, Ltd.     

Interleukin-4 inhibits lipopolysaccharide-induced expression of prostaglandin H synthase-2 in human alveolar macrophages

Several studies have shown that interleukin-4 (IL-4) down-regulates synthesis of prostaglandin E₂ (PGE₂). We evaluated the mechanisms for this suppression in human alveolar macrophages (HAMs). Normal HAMs were obtained from healthy nonsmoking volunteers. The cells either remained unstimulated, or were exposed to 10 μg/ml of lipopolysaccharide (LPS) and/or various amounts of IL-4. LPS alone induced the synthesis of large amounts of PGE₂ and prostaglandin H synthase-2 (PGHS-2) protein. This effect of LPS was suppressed by increasing amounts of IL-4. Expression of LPS-induced PGHS-2 mRNA was also inhibited by IL-4. In addition, IL-4 inhibited expression of CD14, which is a receptor for LPS bound to the LPS-binding protein (LBP). We conclude that IL-4 down-regulates LPS-induced release of PGE₂, by reducing expression of the enzyme, PGHS-2. One potential mechanism for this effect of IL-4 is a reduced expression of CD14, which is the LPS-LBP receptor. © 1995 Wiley-Liss Inc.     

Cutting edge: cells that carry A null allele for toll-like receptor 2 are capable of responding to endotoxin.

Toll-like receptor (TLR) 2 and TLR4 have been implicated in the responses of cells to LPS (endotoxin). CD14-transfected Chinese hamster ovary (CHO)-K1 fibroblasts (CHO/CD14) are exquisitely sensitive to endotoxin. Sequence analysis of CHO-TLR2, compared with human and mouse TLR2, revealed a single base pair deletion. This frameshift mutation resulted in an alternative stop codon, encoding a protein devoid of transmembrane and intracellular domains. CHO-TLR2 cDNA failed to enable LPS signaling upon transient transfection into human epithelial kidney 293 cells. Site-directed mutagenesis of CHO-TLR2 enabled expression of a presumed full-length hamster TLR2 that conferred LPS responsiveness in human epithelial kidney 293 cells. Genomic TLR2 DNA from primary hamster macrophages also contained the frameshift mutation found in CHO fibroblasts. Nevertheless, hamster peritoneal macrophages were found to respond normally to LPS, as evidenced by the induction of cytokines. These results imply that expression of TLR2 is sufficient but not essential for mammalian responses to endotoxin.     

Rough and smooth forms of fluorescein-labelled bacterial endotoxin exhibit CD14/LBP dependent and independent binding that is influencedby endotoxin concentration.

Lipopolysaccharide (LPS, or endotoxin), is a major constituent of the outer membrane of Gram-negative bacteria. Bacteria express either smooth LPS, which is composed of O-antigen (O-Ag), complete core oligosaccharides, and the lipid A, or rough LPS which lack O-Ag but possess lipid A and progressively shorter core oligosaccharides. CD14 has been described as the receptor for complexes of LPS with LPS-binding protein (LBP). Using flow cytometry we have compared the binding of Salmonella minnesota rough LPS (ReLPS) and Escherichia coli smooth LPS labelled with fluorescein isothiocyanate (FITC-LPS) to Chinese hamster ovary (CHO) cells transfected with human CD14 gene (hCD14-CHO), to MonoMac 6 cells and to endothelial cells. Our results showed that both forms of LPS display the same binding characteristics, and that the binding of FITC-LPS to cells was both CD14- and LBP-dependent for LPS concentrations up to 100 ng.mL-1. At LPS concentrations higher than 100 ng.mL-1 we observed CD14/LBP-independent binding. CD14/LBP-dependent binding was dose dependent, saturable, and enhanced in the presence of human pooled serum (HPS), and the monoclonal anti-CD14 antibody (MY4) or unlabelled LPS could outcompete it.     

Losartan, an antagonist of AT1 receptor for angiotensin II, attenuates lipopolysaccharide-induced acute lung injury in rat

Angiotensin II (Ang II) plays an important role in inflammatory process. Acute lung injury (ALI), an inflammatory disorder of the lung, is commonly associated with endotoxemia; however, the mechanism that endotoxin (lipopolysaccharide, LPS) induces the inflammatory response in ALI is not well defined. Here, we showed, in LPS-induced ALI rat model, that Ang II and Ang II type 1 (AT₁) receptor were significantly increased in lung tissues, compared with those in controls. Meanwhile, nuclear factor (NF)-κB-DNA-binding activity, tumor necrosis factor (TNF)-α mRNA, and pneumocytic apoptosis were significantly increased. Moreover, pretreatment of rats with losartan, an antagonist of AT₁ receptor for Ang II, improved the inflammation, reduced the elevation of Ang II and AT₁ receptor, and inhibited NF-κB-DNA-binding activity, expression of TNF-α mRNA, and pneumocytic apoptosis. The data indicate that Ang II may mediate the inflammatory process in LPS-induced ALI through AT₁ receptor, which can be blocked by losartan.     

The pro-inflammatory signature of lipopolysaccharide in spontaneous contracting embryoid bodies differentiated from mouse embryonic stem cells

Embryonic stem (ES) cells differentiate towards all three germ layers, including cardiac cells and leukocytes, and may be therefore suitable to model inflammatory reactions in vitro. In the present study, embryoid bodies differentiated from mouse ES cells were treated with increasing doses of lipopolysaccharide (LPS) to mimic infection with gram-negative bacteria. LPS treatment dose-dependent increased contraction frequency of cardiac cell areas and calcium spikes and increased protein expression of α-actinin. LPS treatment increased the expression of the macrophage marker CD68 and CD69, which is upregulated after activation on T cells, B cells and NK cells. LPS dose-dependent increased protein expression of toll-like receptor 4 (TLR4). Moreover, upregulation of NLR family pyrin domain containing 3 (NLRP3), IL-1ß and cleaved caspase 1 was observed, indicating activation of inflammasome. In parallel, generation of reactive oxygen species (ROS), nitric oxide (NO), and expression of NOX1, NOX2, NOX4 and eNOS occurred. ROS generation, NOX2 expression and NO generation were downregulated by the TLR4 receptor antagonist TAK-242 which abolished the LPS-induced positive chronotropic effect of LPS. In conclusion, our data demonstrate that LPS induced a pro-inflammatory cellular immune response in tissues derived from ES cells, recommending the in vitro model of embryoid bodies for inflammation research.