Activation of Toll-like receptor 4 is associated with insulin resistance in adipocytes

Chronic inflammation is closely associated with metabolic disorders such as obesity and type 2 diabetes, however, the underlying mechanism is unclear. Toll-like receptors (TLRs) play a key role in innate immune response as well as inflammatory signals. Here, we observed that mRNA level of TLR4 was induced during adipocyte differentiation and remarkably enhanced in fat tissues of obese db/db mice. In addition, activation of TLR4 with either LPS or free fatty acids stimulated NFkappaB signaling and expression of inflammatory cytokine genes, such as TNFalpha and IL-6 in 3T3-L1 adipocytes. Furthermore, we discovered that TLR4 activation in 3T3-L1 adipocytes provoked insulin resistance. Taken together, these results suggest that activation of TLR4 in adipocyte might be implicated in the onset of insulin resistance in obesity and type 2 diabetes.     

TLR-3 is Present in Human Adipocytes,but Its Signalling is Not Required for Obesity-Induced Inflammation in Adipose Tissue In Vivo

Innate immunity plays a pivotal role in obesity-induced low-grade inflammation originating from adipose tissue. Key receptors of the innate immune system including Toll-like receptors-2 and -4 (TLRs) are triggered by nutrient excess to promote inflammation. The role of other TLRs in this process is largely unknown. In addition to double-stranded viral mRNA, TLR-3 can also recognize mRNA from dying endogenous cells, a process that is frequently observed within obese adipose tissue. Here, we identified profound expression of TLR-3 in adipocytes and investigated its role during diet-induced obesity. Human adipose tissue biopsies (n=80) and an adipocyte cell-line were used to study TLR-3 expression and function. TLR-3-/- and WT animals were exposed to a high-fat diet (HFD) for 16 weeks to induce obesity. Expression of TLR-3 was significantly higher in human adipocytes compared to the non-adipocyte cells part of the adipose tissue. In vitro, TLR-3 expression was induced during differentiation of adipocytes and stimulation of the receptor led to elevated expression of pro-inflammatory cytokines. In vivo, TLR-3 deficiency did not significantly influence HFD-induced obesity, insulin sensitivity or inflammation. In humans, TLR-3 expression in adipose tissue did not correlate with BMI or insulin sensitivity (HOMA-IR). Together, our results demonstrate that TLR-3 is highly expressed in adipocytes and functionally active. However, TLR-3 appears to play a redundant role in obesity-induced inflammation and insulin resistance.     

Lipopolysaccharide activates an innate immune system response in human adipose tissue in obesity and type 2 diabetes

Type 2 diabetes (T2DM) is associated with chronic low-grade inflammation. Adipose tissue (AT) may represent an important site of inflammation. 3T3-L1 studies have demonstrated that lipopolysaccharide (LPS) activates toll-like receptors (TLRs) to cause inflammation. For this study, we 1) examined activation of TLRs and adipocytokines by LPS in human abdominal subcutaneous (AbdSc) adipocytes, 2) examined blockade of NF-kappaB in human AbdSc adipocytes, 3) examined the innate immune pathway in AbdSc AT from lean, obese, and T2DM subjects, and 4) examined the association of circulating LPS in T2DM subjects. The findings showed that LPS increased TLR-2 protein expression twofold (P<0.05). Treatment of AbdSc adipocytes with LPS caused a significant increase in TNF-alpha and IL-6 secretion (IL-6, Control: 2.7+/-0.5 vs. LPS: 4.8+/-0.3 ng/ml; P<0.001; TNF-alpha, Control: 1.0+/-0.83 vs. LPS: 32.8+/-6.23 pg/ml; P<0.001). NF-kappaB inhibitor reduced IL-6 in AbdSc adipocytes (Control: 2.7+/-0.5 vs. NF-kappaB inhibitor: 2.1+/-0.4 ng/ml; P<0.001). AbdSc AT protein expression for TLR-2, MyD88, TRAF6, and NF-kappaB was increased in T2DM patients (P<0.05), and TLR-2, TRAF-6, and NF-kappaB were increased in LPS-treated adipocytes (P<0.05). Circulating LPS was 76% higher in T2DM subjects compared with matched controls. LPS correlated with insulin in controls (r=0.678, P<0.0001). Rosiglitazone (RSG) significantly reduced both fasting serum insulin levels (reduced by 51%, P=0.0395) and serum LPS (reduced by 35%, P=0.0139) in a subgroup of previously untreated T2DM patients. In summary, our results suggest that T2DM is associated with increased endotoxemia, with AT able to initiate an innate immune response. Thus, increased adiposity may increase proinflammatory cytokines and therefore contribute to the pathogenic risk of T2DM.     

Bacterial lipopolysaccharide activates NF-kappaB through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells. Differential expression of TLR-4 and TLR-2 in endothelial cells

A missense mutation in the cytoplasmic domain of the Toll-like receptor-4 (TLR-4) has been identified as the defect responsible for lipopolysaccharide (LPS) hyporesponsiveness in C3H/HeJ mice. TLR-4 and TLR-2 have recently been implicated in LPS signaling in studies where these receptors were overexpressed in LPS non-responsive 293 human embryonic kidney cells. However, the signaling role of TLR-4 or TLR-2 in human cells with natural LPS response remains largely undefined. Here we show that human dermal microvessel endothelial cells (HMEC) and human umbilical vein endothelial cells express predominantly TLR-4 but very weak TLR-2 and respond vigorously to LPS but not to Mycobacterium tuberculosis 19-kDa lipoprotein. Transient transfection of non-signaling mutant forms of TLR-4 and anti-TLR-4 monoclonal antibody inhibited LPS-induced NF-kappaB activation in HMEC, while a monoclonal antibody against TLR-2 was ineffective. In contrast to LPS responsiveness, the ability of HMEC to respond to 19-kDa lipoprotein correlated with the expression of TLR-2. Transfection of TLR-2 into HMEC conferred responsiveness to 19-kDa lipoprotein. These data indicate that TLR-4 is the LPS signaling receptor in HMEC and that human endothelial cells (EC) express predominantly TLR-4 and weak TLR-2, which may explain why they do not respond to 19-kDa lipoprotein. The differential expression of TLRs on human EC may have important implications in the participation of vascular EC in innate immune defense mechanisms against various infectious pathogens, which may use different TLRs to signal.     

Protein kinase C epsilon: a new target to control inflammation and immune-mediated disorders

Recent advances in understanding the molecular basis for mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns by a set of germline-encoded receptors: the Toll-like receptors (TLRs). TLRs have been identified as being part of a large family of pathogen-recognition receptors that play a decisive role in the induction of both innate and adaptive immunity. Indeed, activation of T lymphocytes depends on their interaction with dendritic cells previously stimulated by TLR agonists such as bacterial lipopolysaccharide (LPS), a TLR-4 ligand. A novel PKC epsilon (epsilon) was recently found to be a critical component of TLR-4 signaling pathway and thereby to play a key role in macrophage and dendritic cell (DC) activation in response to LPS. Thus, controlling the kinase activity of PKC epsilon might represent an efficient strategy to prevent or treat certain inflammatory disorders of microbial origin.     

Interleukin-6 and interleukin-15 are selectively regulated by lipopolysaccharide and interferon-gamma in primary pig adipocytes

3T3-L1 adipocytes express the lipopolysaccharide (LPS) receptor and respond to direct stimulation with the antigen by increasing the expression of inflammatory mediators. Activation of this receptor by its ligand in the macrophage causes the activation and translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus where it regulates the expression of proinflammatory cytokines and other target genes. We investigated whether LPS could stimulate NF-kappaB translocation in primary pig adipocytes and regulate the expression and secretion of TNF-alpha and IL-6. LPS clearly induced the nuclear translocation of NF-kappaB and also upregulated (P < 0.05) the mRNA expression and secretion of IL-6 into the culture medium. An induction of TNF-alpha expression by LPS was not detected, but with extended incubation (8 h), there was a modest increase (P < 0.09) in the media concentration of this cytokine. Inhibition of either ERK1/2, PKC, or the inhibitory G protein (Gi) with U-0126, bisindolylmaleimide HCl, and pertussis toxin, respectively, blocked (P < 0.05) the increase in IL-6 expression caused by LPS. Because LPS administration in vivo increases circulating concentrations of IFN-gamma, and because this cytokine also regulates multiple immune modulators in the adipocyte, we also determined whether IFN-gamma regulates cytokine expression in primary adipocytes. Although the expression of IL-6 and TNF-alpha was unresponsive to IFN-gamma, the expression of IL-15 was markedly upregulated (P < 0.01). Furthermore, the induction of IL-15 expression by IFN-gamma was blocked by inhibition of PKC. These data indicate that NF-kappaB is responsive to LPS in the adipocyte and also identify key mediators of LPS-induced IL-6 expression. In addition, we provide novel evidence that IFN-gamma targets the adipocyte to induce IL-15 expression, thus indicating a possible role for the adipocyte in the regulation of T-cell function and muscle metabolism during the innate immune response.     

Inducible Toll-like receptor and NF-kappaB regulatory pathway expression in human adipose tissue

Objective: Inflammatory activity in fat tissue has recently been implicated in mechanisms of insulin resistance and obesity‐related metabolic dysfunction. Toll‐like receptors (TLRs) play a key role in innate immune responses and recent studies implicate the TLR pathway in mechanisms of inflammation and atherosclerosis. The aim of this study was to examine differential TLR expression and function in human adipose tissue. Methods and Procedures: We biopsied subcutaneous abdominal fat from 16 obese subjects (age 39 ± 11 years, BMI 49 ± 14 kg/m²) and characterized TLR expression using quantitative real‐time PCR and confocal immunofluorescence imaging. In tissue culture, we stimulated isolated human adipocytes with Pam3CSK4 and lipopolysaccharide (LPS) (TLR2 and TLR4 agonists, respectively) and quantified TLR activity, interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α) production, and nuclear factor‐κB (NF‐κB) p65 nuclear activation using real‐time PCR, enzyme‐linked immunosorbent assay (ELISA), and immunofluorescence. Results: TLR1, 2, and 4 protein colocalized with adiponectin in human adipocytes with TLR4 exhibiting the highest immunohistochemical expression. Using real‐time PCR, we confirmed higher level of gene expression for TLR4 as compared to other members of the TLR family (TLR1, 2, 7, 8) in human adipose depots (P < 0.001). In tissue culture, adipocyte TLR2/TLR4 mRNA expression and protein increased significantly following Pam3CSK4 and LPS (P < 0.001). TLR2/TLR4 stimulation was associated with NF‐κB p65 nuclear translocation and proinflammatory cytokine production. Discussion: The findings demonstrate that TLRs are inducible in adipose tissue and linked with downstream NF‐κB activation and cytokine release. Adipose stores may play a dynamic role in the regulation of inflammation and innate immunity in human subjects via modulation of the TLR/NF‐κB regulatory pathway.     

Implication of CpG-ODN and reactive oxygen species in the inhibition of intracellular growth of Salmonella typhimurium in hepatocytes

Bacterial DNA acts as an alert signal for eukaryotic cells through immunostimulatory CpG motifs. These sequences have therapeutic properties promoting protective immune TH1 responses and are recognized by a membrane protein belonging to the Toll-like receptor (TLR) family, named TLR-9. The aim of this study was to test the capability of murine hepatocytes to sense bacterial DNA and to develop antibacterial mechanisms against Salmonella typhimurium. We show that hepatocyte cell lines and mRNA extracts from murine liver constitutively express TLR-9, which is down-regulated by LPS and the mix of IFNgamma, IL-1beta and LPS. Also, we have found that hepatocyte cell lines can sense the presence of bacterial DNA and respond to it by increasing the pool of intracellular peroxides. This results in inhibition of intracellular growth of S. typhimurium when infected cells were incubated in the presence of CpG synthetic oligonucleotides (CpG-ODN). Expression of hepatocyte Mn-SOD is also induced by stimulation with CpG-oligodeoxynucleotides, LPS, and the mix of IFNgamma, IL-1beta and LPS. These results reinforce the prominent role of hepatocytes as a microbial product-responsive cell and the capabilities of CpG-ODN sequences as potent inducers of the innate immune response through the activation of a broad range of cell types.     

NOD1 activation induces proinflammatory gene expression and insulin resistance in 3T3-L1 adipocytes

Chronic inflammation is associated with obesity and insulin resistance; however, the underlying mechanisms are not fully understood. Pattern recognition receptors Toll-like receptors and nucleotide-oligomerization domain-containing proteins play critical roles in innate immune response. Here, we report that activation of nucleotide binding oligomerization domain-containing protein-1 (NOD1) in adipocytes induces proinflammatory response and impairs insulin signaling and insulin-induced glucose uptake. NOD1 and NOD2 mRNA are markedly increased in differentiated murine 3T3-L1 adipocytes and human primary adipocyte culture upon adipocyte conversion. Moreover, NOD1 mRNA is markedly increased only in the fat tissues in diet-induced obese mice, but not in genetically obese ob/ob mice. Stimulation of NOD1 with a synthetic ligand Tri-DAP induces proinflammatory chemokine MCP-1, RANTES, and cytokine TNF-α and MIP-2 (human IL-8 homolog) and IL-6 mRNA expression in 3T3-L1 adipocytes in a time- and dose-dependent manner. Similar proinflammatory profiles are observed in human primary adipocyte culture stimulated with Tri-DAP. Furthermore, NOD1 activation suppresses insulin signaling, as revealed by attenuated tyrosine phosphorylation and increased inhibitory serine phosphorylation, of IRS-1 and attenuated phosphorylation of Akt and downstream target GSK3α/3β, resulting in decreased insulin-induced glucose uptake in 3T3-L1 adipocytes. Together, our results suggest that NOD1 may play an important role in adipose inflammation and insulin resistance in diet-induced obesity.     

TLR-2 and TLR-9 are sensors of apoptosis in a mouse model of doxorubicin-induced acute inflammation

Anthracycline antibiotics are inducers of an immunogenic form of apoptosis that has immunostimulatory properties because of the release of damage-associated molecular patterns. To study the mechanisms used by the innate immune system to sense this immunogenic form of cell death, we established an in vivo model of cell death induced by intraperitoneal injection of doxorubicin, a prototype of anthracyclines. The acute sterile inflammation in this model is characterized by rapid influx of neutrophils and increased levels of IL-6 and monocyte chemotactic protein-1. We demonstrate that acute inflammation induced by doxorubicin is associated with apoptosis of monocytes/macrophages and that it is specific for doxorubicin, an immunogenic chemotherapeutic. Further, the inflammatory response is significantly reduced in mice deficient in myeloid differentiation primary response gene 88 (MyD88), TLR-2 or TLR-9. Importantly, a TLR-9 antagonist reduces the recruitment of neutrophils induced by doxorubicin. By contrast, the acute inflammatory response is not affected in TRIF(Lps2) mutant mice and in TLR-3, TLR-4 and caspase-1 knockout mice, which shows that the inflammasome does not have a major role in doxorubicin-induced acute inflammation. Our findings provide important new insights into how the innate immune system senses immunogenic apoptotic cells and clearly demonstrate that the TLR-2/TLR-9-MyD88 signaling pathways have a central role in initiating the acute inflammatory response to this immunogenic form of apoptosis.     

Identification of a TLR-independent pathway for Borrelia burgdorferi-induced expression of matrix metalloproteinases and inflammatory mediators through binding to integrin alpha 3 beta 1

Borrelia burgdorferi stimulates a robust inflammatory response at sites of localization. Binding of borrelial lipoproteins to TLR-2 is one pathway important in the host response to B. burgdorferi. However, while TLR-2 is clearly important in control of infection, inflammation is actually worsened in the absence of TLR-2 or the shared TLR adapter molecule, MyD88, suggesting that there are alternative pathways regulating inflammation. Integrins are cell surface receptors that play an important role in cell to cell communications and that can activate inflammatory signaling pathways. In this study, we report for the first time that B. burgdorferi binds to integrin alpha(3)beta(1) and that binding of B. burgdorferi to this integrin results in induction of proinflammatory cytokines, chemokines, and end-effector molecules such as matrix metalloproteinases in primary human chondrocyte cells. Expression of these same molecules is not affected by the absence of MyD88 in murine articular cartilage, suggesting that the two pathways act independently in activating host inflammatory responses to B. burgdorferi. B. burgdorferi-induced alpha(3) signaling is mediated by JNK, but not p38 MAPK. In summary, we have identified a new host receptor for B. burgdorferi, integrin alpha(3)beta(1); binding of B. burgdorferi to integrin alpha(3)beta(1) results in the release of inflammatory mediators and is proposed as a TLR-independent pathway for activation of the innate immune response by the organism.     

Association of insulin receptor substrate 1 (IRS-1) y895 with Grb-2 mediates the insulin signaling involved in IRS-1-deficient brown adipocyte mitogenesis

We have recently generated immortalized fetal brown adipocyte cell lines from insulin receptor substrate 1 (IRS-1) knockout mice and demonstrated an impairment in insulin-induced lipid synthesis as compared to wild-type cell lines. In this study, we investigated the consequences of IRS-1 deficiency on mitogenesis in response to insulin. The lack of IRS-1 resulted in the inability of insulin-stimulated IRS-1-deficient brown adipocytes to increase DNA synthesis and enter into S/G2/M phases of the cell cycle. These cells showed a severe impairment in activating mitogen-activated protein kinase kinase (MEK1/2) and p42-p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. IRS-1-deficient cells also lacked tyrosine phosphorylation of SHC and showed no SHC-Grb-2 association in response to insulin. The mitogenic response to insulin could be partially restored by enhancing IRS-2 tyrosine phosphorylation and its association with Grb-2 by inhibition of phosphatidylinositol 3-kinase activity through a feedback mechanism. Reconstitution of IRS-1-deficient brown adipocytes with wild-type IRS-1 restored insulin-induced IRS-1 and SHC tyrosine phosphorylation and IRS-1-Grb-2, IRS-1-SHC, and SHC-Grb-2 associations, leading to the activation of MAPK and enhancement of DNA synthesis. Reconstitution of IRS-1-deficient brown adipocytes with the IRS-1 mutant Tyr895Phe, which lacks IRS-1-Grb-2 binding, restored SHC-IRS-1 association and SHC-Grb-2 association. However, the lack of IRS-1-Grb-2 association impaired MAPK activation and DNA synthesis in insulin-stimulated mutant cells. These data provide strong evidence for an essential role of IRS-1 and its direct association with Grb-2 in the insulin signaling pathway leading to MAPK activation and mitogenesis in brown adipocytes.     

Innate recognition of lipopolysaccharide by Toll-like receptor 4-MD-2

Toll-like receptors (TLRs) are pathogen recognition molecules that activate the immune system as part of the innate immune response. Microbial recognition by TLRs plays a crucial role in the host immune system's decision to respond or not to a particular microbial infection. Lipopolysaccharide (LPS), a membrane glycolipid of Gram-negative bacteria, exhibits strong immunostimulating activity among TLR ligands and has been studied in great detail. Recent studies have shown that cell surface TLR4-MD-2 physically interacts with LPS and triggers the release of an LPS signal, revealing a host-pathogen interaction mediated by TLR.     

Synthetic Toll like receptor-4 (TLR-4) agonist peptides as a novel class of adjuvants

Background

Adjuvants serve as catalysts of the innate immune response by initiating a localized site of inflammation that is mitigated by the interactions between antigens and toll like receptor (TLR) proteins. Currently, the majority of vaccines are formulated with aluminum based adjuvants, which are associated with various side effects. In an effort to develop a new class of adjuvants, agonists of TLR proteins, such as bacterial products, would be natural candidates. Lipopolysaccharide (LPS), a major structural component of gram negative bacteria cell walls, induces the systemic inflammation observed in septic shock by interacting with TLR-4. The use of synthetic peptides of LPS or TLR-4 agonists, which mimic the interaction between TLR-4 and LPS, can potentially regulate cellular signal transduction pathways such that a localized inflammatory response is achieved similar to that generated by adjuvants.

Methodology/Principal Findings

We report the identification and activity of several peptides isolated using phage display combinatorial peptide technology, which functionally mimicked LPS. The activity of the LPS-TLR-4 interaction was assessed by NF-κB nuclear translocation analyses in HEK-BLUE™-4 cells, a cell culture model that expresses only TLR-4, and the murine macrophage cell line, RAW264.7. Furthermore, the LPS peptide mimics were capable of inducing inflammatory cytokine secretion from RAW264.7 cells. Lastly, ELISA analysis of serum from vaccinated BALB/c mice revealed that the LPS peptide mimics act as a functional adjuvant.

Conclusions/Significance

Our data demonstrate the identification of synthetic peptides that mimic LPS by interacting with TLR-4. This LPS mimotope-TLR-4 interaction will allow for the development and use of these peptides as a new class of adjuvants, namely TLR-4 agonists.     

Toll-like receptor 4, but not toll-like receptor 2, is a signaling receptor for Escherichia and Salmonella lipopolysaccharides

Two members of the mammalian Toll-like receptor (TLR) family, TLR2 and TLR4, have been implicated as receptors mediating cellular activation in response to bacterial LPS. Through the use of mAbs raised against human TLR2 and TLR4, we have conducted studies in human cell lines and whole blood to ascertain the relative contribution of these receptors to LPS induced cytokine release. We show that the contribution of TLR2 and TLR4 to LPS-induced cellular activation correlates with the relative expression levels of these two TLRs in a given cell type. In addition, we have found that significant differences in cell stimulatory activity exist between various smooth and rough LPS types that cannot be ascribed to known LPS structural features. These results suggest that impurities in the LPS may be responsible for some of the activity and this would be in agreement with recently published results of others. Upon repurification, none of the commercial LPS preparations activate cells through TLR2, but continue to stimulate cells with comparable activity through TLR4. Our results confirm recent findings that TLR4, but not TLR2, mediates cellular activation in response to LPS derived from both Escherichia coli and Salmonella minnesota. Additionally, we show that TLR4 is the predominant signaling receptor for LPS in human whole blood.     

The effects of NOD activation on adipocyte differentiation

Objective:

Obesity is associated with chronic inflammation. Toll‐like receptors (TLR) and NOD‐like receptors (NLR) are two families of pattern recognition receptors that play important roles in immune response and inflammation in adipocytes. It has been reported that TLR4 and TLR2 activation induce proinflammatory changes that impair adipocyte differentiation. However, the effects of activation of NOD1 and NOD2, the two prominent members of NLR, on adipocyte differentiation have not been studied.

Design and Methods:

3T3‐L1 and human adipose‐derived stem cells were tested for adipocyte differentiation in the presence or absence of NOD ligand. Adipocyte differentiation was evaluated by the adipocyte markers gene expression and Oil Red O staining for lipid accumulation.

Results:

Activation of NOD1, but not NOD2, by a synthetic ligand dose‐dependently suppressed 3T3‐L1 adipocyte differentiation as revealed by Oil Red O stained cell morphology, lipid accumulation, and attenuated gene expression of adipocyte markers (PPARγ, C/EBPα, SCD, FABP4, Adiponectin). Activation of NOD1, but not NOD2, induced NF‐κB activation, which correlated with their abilities to suppress ligand‐induced PPARγ transaction. Moreover, the suppressive effect by NOD1 activation was reversed by IκB super‐repressor which blocks NF‐κB activation. The suppression by NOD1 ligand C12‐iEDAP on adipocyte differentiation was reversed by small RNA interference targeting NOD1, demonstrating the specificity of NOD1 activation. In contrast, activation of NOD1 and NOD2 both significantly suppressed adipocyte differentiation of human adipose‐derived adult stem cells, demonstrating the species specific effects of NOD activation. In contrast to enhanced leptin mRNA by LPS and TNFα, NOD1 activation suppressed leptin mRNA in adipocytes, suggesting the differential effects of NOD1 activation in adipocytes.

Conclusions:

Overall, our results suggest that NOD1 represents a novel target for adipose inflammation in obesity.     

Effects of Separate and Concomitant TLR-2 and TLR-4 Activation in Peripheral Blood Mononuclear Cells of Newborn and Adult Horses

Deficient innate and adaptive immune responses cause newborn mammals to be more susceptible to bacterial infections than adult individuals. Toll-like receptors (TLRs) are known to play a pivotal role in bacterial recognition and subsequent immune responses. Several studies have indicated that activation of certain TLRs, in particular TLR-2, can result in suppression of inflammatory pathology. In this study, we isolated peripheral blood mononuclear cells (PBMCs) from adult and newborn horses to investigate the influence of TLR-2 activation on the inflammatory response mediated by TLR-4. Data were analysed in a Bayesian hierarchical linear regression model, accounting for variation between horses. In general, cytokine responses were lower in PBMCs derived from foals compared with PBMCs from adult horses. Whereas in foal PBMCs expression of TLR-2, TLR-4, and TLR-9 was not influenced by separate and concomitant TLR-2 and TLR-4 activation, in adult horse PBMCs, both TLR ligands caused significant up-regulation of TLR-2 and down-regulation of TLR-9. Moreover, in adult horse PBMCs, interleukin-10 protein production and mRNA expression increased significantly following concomitant TLR-2 and TLR-4 activation (compared with sole TLR-4 activation). In foal PBMCs, this effect was not observed. In both adult and foal PBMCs, the lipopolysaccharide-induced pro-inflammatory response was not influenced by pre-incubation and co-stimulation with the specific TLR-2 ligand Pam3-Cys-Ser-Lys4. This indicates that the published data on other species cannot be translated directly to the horse, and stresses the necessity to confirm results obtained in other species in target animals. Future research should aim to identify other methods or substances that enhance TLR functionality and bacterial defence in foals, thereby lowering susceptibility to life-threatening infections during the first period of life.     

Expression of the human apoE2 isoform in adipocytes: altered cellular processing and impaired adipocyte lipogenesis

Expression of apoE in adipocytes has been shown to have an important role in modulating adipocyte triglyceride (TG) metabolism and gene expression that is independent of circulating and extracellular apoE. The impact of adipocyte expression of common human apoE isoforms was evaluated using adipocytes harvested from human apoE2, -3, and -4 knock-in mice. Expression of the apoE2 isoform was associated with an increase in adipocyte apoE gene expression and apoE synthesis. Newly synthesized apoE2 was unstable in adipocytes and demonstrated increased degradation and decreased secretion. ApoE2-expressing mice were hyperlipidemic, and had increased size of gonadal fat pads and of adipocytes, compared with apoE3 mice. In isolated cells, however, expression of the apoE2 isoform produced defective lipogenesis and increased TG hydrolysis. Incubation of adipose tissue with apoE3-containing TG-rich lipoproteins resulted in a significant increase in TG in adipose tissue from apoE3 and -E4 mice, but not apoE2 mice. Reduced capacity to internalize FFA as lipogenic substrate contributed to defective lipogenesis. Newly synthesized apoE2 is unstable in adipocytes and results in decreased adipocyte TG synthesis and defective FA uptake. These changes recapitulate those observed in apoE knockout adipocytes and have implications for understanding metabolic disturbances in humans expressing the E2 isoform.