Toll-like Receptor 2 Antibodies Research Articles

Loss of Toll-like Receptor 2 Results in Accelerated Leukemogenesis in the NUP98-HOXD13 Mouse Model of MDS

The myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis and a high risk of leukemic transformation. Stem cell transplantation is the only curative treatment available to a subset of patients and is associated with significant morbidity and mortality; thus, new therapies are needed. Multiple studies have shown a correlation between MDS and enhanced toll-like receptor 2 (TLR2) signaling, suggesting this might contribute to disease pathogenesis. In fact, an inhibitory TLR2 antibody is currently in clinical trial for use in MDS (OPN-305, Opsona Therapeutics). Despite enthusiasm for TLR2 as a potential drug target, the role of TLR2 in MDS remains unclear. Increased TLR2 signaling has been associated with low-risk disease, higher rates of apoptosis associated with improved prognosis, and longer survival (Zeng et al ., Exp Cell Res 2016; Wei et al., Leukemia 2013), suggesting TLR2 may, in fact, have a protective role in MDS. Thus, we are exploring how TLR2 signaling regulates cell fate in MDS to shape efforts to target this pathway in patients.To elucidate the contribution of TLR2 signaling to MDS pathogenesis, we determined the effects of TLR2 loss in a mouse model of MDS (expressing the NUP98-HOXD13 fusion from the hematopoietic Vav-1 promoter). These “ NHD13” mice recapitulate many features of human MDS and die of leukemia or cytopenias (Lin et al ., Blood 2005). Similar to CD34+ cells in humans, we found a nearly two-fold increase of TLR2 on NHD13 HSCs as compared to controls. NHD13 mice were crossed to Tlr2-/- mice to generate NHD13;Tlr2-/-, NHD13;Tlr2+/+, Tlr2-/-, and wild-type (WT) groups. Interestingly, loss of TLR2 in NHD13 mice did not confer an improvement in peripheral cytopenias, but was associated with accelerated leukemogenesis and significantly worse survival (p=0.016, with a median survival of 335 days in NHD13;Tlr2-/- mice vs. 370 days for NHD13;Tlr2+/+ mice; in both cohorts, leukemia was the primary cause of death- 12/20 for NHD13;Tlr2-/- and 10/13 for NHD13;Tlr2+/+). An even more significant acceleration of leukemogenesis and death was observed in NHD13 mice crossed to mice lacking myeloid differentiation primary response gene 88 (MyD88), an adaptor required for most TLR signaling, including TLR2 (p<0.0001, with a median survival of 218 days in NHD13;MyD88-/- mice vs. 370 days in NHD13 mice).To investigate the potential mechanism by which TLR2 signaling loss accelerates leukemogenesis in NHD13 mice, hematopoietic stem and progenitor cells (c-Kit+, Sca-1+, Lin-; HSPCs) of premalignant NHD13;Tlr2+/+ and NHD13;Tlr2-/- mice were evaluated for cell cycling, cell death, and DNA damage. While loss of TLR2 in the NHD13 mice did not confer a significant difference in HSPC cycling, it did reduce cell death (HSPCs of NHD13+;Tlr2+/+ mice display 18.4% Annexin V+ cells, while those of NHD13+;Tlr2-/- mice display only 6.2%, p=0.0008). An assessment of caspase-1 and caspase-3/7 activities showed a significant reduction in activated caspase-1 in the HSPCs of NHD13;Tlr2-/- mice compared to NHD13; Tlr2+/+ mice (average luminescence units: 7310 vs. 10164), suggesting that TLR2 specifically promotes inflammasome-dependent cell death in the NHD13 mice. RNA sequencing of sorted HSPCs also demonstrated an enrichment of apoptosis-related gene pathways in the NHD13 compared to the NHD13;Tlr2-/- cells. Moreover, the accumulated HSPCs in the NHD13;Tlr2-/- mice showed elevated levels of γH2AX staining as compared to NHD13; Tlr2+/+ mice and WT controls, indicating TLR2 loss promotes the survival of damaged HSPCs.As apoptosis of HSPCs is characteristic of low-risk MDS and is lost as the disease progresses to leukemia, these data suggest that some TLR2 signaling may be protective against leukemic transformation via promotion of apoptosis of damaged premalignant HSCs. Further supporting this idea, we found that in 38% of cases evaluated, TLR2 surface expression was lost from the malignant cells upon transformation to acute leukemia in the NHD13 mice. Thus, down-regulation of TLR2 may be a later event in the progression of MDS to acute leukemia, and complete inhibition of this pathway may, in fact, facilitate the survival of pre-leukemic HSPCs. Ongoing experiments are aimed at elucidating the mechanism by which TLR2 signaling promotes premalignant cell death in MDS and understanding how TLR2 is down-regulated upon transformation to leukemia. DisclosuresNo relevant conflicts of interest to declare.

Porphyromonas gingivalis lipopolysaccharide promotes T- helper 17 cell differentiation from human CD4+ naïve T cells via toll-like receptor-2 in vitro

ObjectivesThe persistence of T-helper 17 (Th17) cells has been shown to support chronic inflammation and mediate tissue destruction in periodontitis. However, little is known regarding the underlying mechanisms that regulate Th17 cell differentiation in the periodontal inflammatory context. The objective of this study was to explore the possible effect and mechanism of lipopolysaccharide (LPS) from Porphyromonas gingivalis on Th17 cell differentiation. MethodsActivated human CD4+CD45RA+ naïve T cells were stimulated with different doses of LPS from virulent and avirulent P. gingivalis strains combined with Th17 driven cytokines in vitro. Flow cytometry was used to analyze the differentiation ratio of Th17 cells. IL-17A protein expression and IL-17, retinoid-related orphan receptor C (RORC) and toll-like receptor 2 (TLR2) mRNA transcription were analysed by ELISA and real-time qPCR, respectively. The role of TLR2 in Th17 cell differentiation was further confirmed by TLR2 blocking assay. ResultsLPS from P. gingivalis (Pg-LPS) up-regulated Th17 cell differentiation ratios, expression of IL-17 and RORC mRNA, and IL-17 concentration in culture supernatant, with 0.1 μg/mL LPS from the virulent P. gingivalis strain being the most effectively. Furthermore, Pg-LPS also up-regulated expression of TLR2 on T cells during Th17 differentiation, and the differentiation was attenuated by treatment with TLR2 antibody. ConclusionsThese results suggest that Pg-LPS promotes Th17 cell differentiation in vitro, and TLR2 signalling may be involved in this process. LPS from the virulent P. gingivalis strain up-regulated Th17 cell differentiation more effectively, which may be associated with the pathogenicity of different P. gingivalis strains.

Crosstalk between TLR2 and Sphk1 in microglia in the cerebral ischemia/reperfusion-induced inflammatory response.

Stroke is associated with high morbidity and mortality, and much remains unknown about the injury-related mechanisms that occur following reperfusion. This study aimed to explore the roles of Toll-like receptor 2 (TLR2) and sphingosine kinase 1 (Sphk1) in microglial cells in inflammatory responses induced by cerebral ischemia/reperfusion (I/R). For this purpose, C57BL/6 mice were randomly divided into 4 groups as follows: the sham-operated group, the I/R group, the I/R group treated with TLR2 antibody, and the I/R group treated with N,N-dimethylsphingosine. Focal cerebral I/R was induced by middle cerebral artery occlusion. Double-labeling immunofluorescence was used to observe the protein expression of TLR2 and Sphk1 in the ischemic brain tissue. Quantitative polymerase chain reaction was performed to determine the mRNA levels of TLR2 and Sphkl in ischemic brain tissue. Enzyme-linked immunosorbent assay was carried out to detect the protein contents of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-17 and IL-23 in ischemic brain tissue. The results revealed that I/R upregulated TLR2 and Sphk1 expression in microglial cells, and the inhibition of either TLR2 or Sphk1 inhibited the expression of the pro-inflammatory cytokines, IL-1β, TNF-α, IL-17 and IL-23. Notably, the inhibition of TLR2 activity also decreased Sphk1 expression. These results thus indicate that the activation of microglial cells, via a TLR2→Sphk1→pro-inflammatory cytokine (IL-1β, TNF-α, IL-17 and IL-23) pathway, may participate in I/R injury.

Systemic TLR2 Antibody Application in Renal Ischaemia and Reperfusion Injury Decreases AKT Phosphorylation and Increases Apoptosis in the Mouse Kidney.

Acute kidney injury remains an important cause of renal dysfunction. In this context, Toll-like receptors have been demonstrated to play a critical role in the induction of innate and inflammatory responses. Among these, Toll-like receptor 2 (TLR2) is constitutively expressed in tubular epithelial cells (TECs) of the kidney and is also known to mediate ischaemia reperfusion (IR) injury. Adult male C57BL/6JRj mice were randomized into seven groups (n = 8): a non-operative control group (CTRL) and six interventional groups in which mice were subjected to a 30 min. bilateral renal ischaemia. Immediately before reperfusion, mice were treated either with saline or with TLR2 antibody (clone T2.5) and harvested after ischaemia and reperfusion for 3, 24 and 48 hr. Analysed kidney homogenates of TLR2 antibody-treated mice displayed significantly decreased levels of TLR2 protein after 3 hr of IR compared to saline-treated mice. Accordingly, the degree of AKT phosphorylation was significantly decreased after 3 hr of IR compared to saline-treated animals. TUNEL staining revealed significantly higher apoptosis rates in TLR2 antibody-treated animals compared to saline-treated mice after 3 and 24 hr of IR. Further, a positive correlation between TLR2 protein expression and phosphorylation of AKT as well as a negative correlation with the number of TUNEL-positive cells could be observed. Inhibition of TLR2 and its signalling pathway by a single application of TLR2 antibody results in reduced phosphorylation of AKT and consecutively increased apoptosis.

Silver nanoparticles induce apoptosis through the Toll-like receptor 2 pathway

The objective of this study was to evaluate the effects of silver nanoparticles (AgNPs) on the Toll-like receptor 2 (TLR-2) pathway in cultured cells. Human chondrocytes, periodontal ligament (PDL) cells, and SCC-9 cells (squamous cell carcinoma from the tongue) were cultured and subjected to cytotoxicity assays. To evaluate the effects of AgNPs on the TLR-2 pathway, TLR-2 small interfering (si) RNA or TLR-2 antibodies were applied to the chondrocytes, followed by the application of AgNPs. AgNPs induced dose-dependent effects on the examined cell types in terms of both cytotoxicity and TLR-2 expression levels. AgNP-mediated apoptosis was reduced after treatment with TLR-2 siRNA in both PDL cells and chondrocytes. Furthermore, functional blocking of TLR-2 with anti-TLR2 antibodies inhibited AgNP-mediated cytotoxicity. AgNPs increased c-Jun phosphorylation, an effect that was reversed after treatment with TLR-2 siRNA. The results indicate that AgNP-mediated apoptosis most likely occurs via the TLR-2 pathway.

PM10‐biogenic fraction drives the seasonal variation of proinflammatory response in A549 cells

PM10 was collected in a Milan urban site, representative of the city air quality, during winter and summer 2006. Mean daily PM10 concentration was 48 μg m(-3) during summer and 148 μg m(-3) during winter. Particles collected on Teflon filters were chemically characterized and the endotoxin content determined by the LAL test. PM10-induced cell toxicity, assessed with MTT and LDH methods, and proinflammatory potential, monitored by IL-6 and IL-8 cytokines release, were investigated on the human alveolar epithelial cell line A549 exposed to increasing doses of PM. Besides untreated cells, exposure to inert carbon particles (2-12 μm) was also used as additional control. Both cell toxicity and proinflammatory potency resulted to be higher for summer PM10 with respect of winter PM10, with IL-6 showing the highest dose-dependent release. The relevance of biogenic components adsorbed onto PM10 in eliciting the proinflammatory mediators release was investigated by inhibition experiments. Polymixin B (Poly) was used to inhibit particle-bind LPS while Toll-like receptor-2 antibody (a-TLR2) to specifically block the activation of this receptor. While cell viability was not modulated in cells coexposed to PM10 and Poly or a-TLR2 or both, inflammatory response did it, with IL-6 release being the most inhibited. In conclusion, Milan PM10-induced seasonal-dependent biological effects, with summer particles showing higher cytotoxic and proinflammatory potential. Cytotoxicity seemed to be unaffected by the PM biogenic components, while inflammation was significantly reduced after the inhibition of some biogenic activated pathways. Besides, the PM-associated biogenic activity does not entirely justify the PM-induced inflammatory effects. © 2010 Wiley Periodicals, Inc. Environ Toxicol 2012.

Toll‐like receptor 2 monoclonal antibody or/and Toll‐like receptor 4 monoclonal antibody increase counts of Lactobacilli and Bifidobacteria in dextran sulfate sodium‐induced colitis in mice

The accurate pathogenesis of ulcerative colitis (UC) is not yet well understood. Recently, Toll-like receptor 2 (TLR2), TLR4 and gut microbial flora have been proved as playing important roles in the process of UC. This study was to evaluate the effect of TLR2 and TLR4 monoclonal antibodies on gut microbial flora in dextran sulfate sodium (DSS)-induced colitis in a mouse model. We evaluated the effects of the TLR2 and TLR4 monoclonal antibodies on the development of DSS-induced colitis. Clinical symptoms were evaluated by the disease activity index (DAI), while tissue samples were evaluated by histological scoring (HS). Meanwhile, the mucosal mRNA expressions of TLR2, TLR4, interferon-γ (IFN-γ), interleukin-4 (IL-4) and IL-17 were analyzed by Realtime polymerase chain reaction (PCR). The mucosal protein TRAF6, TAB1, P-IKK, P-P38α mitogen-activated protein kinase (MAPL) and c-jun expressions of the TLR2 and TLR4 signaling pathways were analyzed using Western blot. The mucosal nuclear factor kappa B (NF-κB) was analyzed using electrophoretic mobility shift assay. Fecal samples were obtained directly from the cecum for microbiological studies. Expressions of TLR2 and TLR4 in colonic epithelial cells on DSS-induced colitis were much higher than normal ones. After the treatment with TLR2mAb and TLR4mAb, DAI and HS were decreased significantly. The UC model group showed a conspicuous increase of Escherichia coli and decreases of Lactobacillus spp. and Bifidobacterium spp. After being treated with TLR2mAb or/and TLR4mAb, Lactobacillus spp. and Bifidobacterium spp. increased to the normal level. TLR2mAb and TLR4mAb can suppress the development of DSS-induced colitis and increase counts of Lactobacilli and Bifidobacteria.

Extracellular HMGB1 Enhances Proliferation, Differentiation and Migration of Human CD34+ Hematopoietic Stem Cells in Vitro

High-mobility group box 1 protein (HMGB1) is a chromatin protein and acts as a cytokine involved in inflammation, cell proliferation, differentiation, migration and stem cell recruitment. So far, little is known about its effect on hematopoietic stem cells (HSCs). In this study, we investigated whether receptors for HMGB1 are expressed on human CD34+ HSCs, and whether HMGB1 could affect HSCs proliferation, differentiation and migration in vitro. As examined by FACS analysis and RT-PCR, cord blood CD34+ HSCs express the HMGB1 receptors RAGE (receptor for advanced glycation end products), TLR2 (Toll-like receptor 2) and TLR4. To study the effects of HMGB1 on CD34+ HSCs proliferation and differentiation, freshly isolated cord blood CD34+ HSCs were cultured for 7 days in medium alone or in the presence of HMGB1. Flow cytometric analysis showed that HMGB1 (50ng/ml) can induce the differentiation of CD34+ HSCs along the granulo-monocytic (CD14+, CD13+) lineage and erythropoiesis (CD71+). In contrast, HMGB1 did not induce the expression of CD41, a marker for megakaryocyte lineage. The numbers of cells cultured in the presence of HMGB1 were always increased in comparison with controls. Furthermore, higher numbers of granulomonocytic progenitors (CFU-GM), erythroid progenitors (BFU-E), and CFU-MIX were confirmed by CFC assays in a HMGB1 dose dependent manner after 14-day culture. The results suggest that HMGB1 enhances CD34+ HSCs proliferation and differentiation. We next assessed the effects of HMGB1 on HSCs migration by chemotaxis assay using Boyden chambers. HMGB1 dose-dependently increased the chemotactic migration of CD34+ HSCs. A neutralizing anti-RAGE antibody significantly blocked the HMGB1-induced migration of HSCs, whereas neutralizing TLR2 and TLR4 antibodies did not significantly influence HMGB1-stimulated HSCs migration, suggesting that the migratory effect of HMGB1 on human HSCs is predominantly mediated by RAGE. In summary, our results provide the first report of HMGB1 receptors expression profile of human cord blood CD34+ HSCs and demonstrate that HMGB1 can increase the proliferation and migration of HSCs and directly induce of HSCs along myeloid differentiation and erythropoiesis in vitro. Further studies will be needed to clarify the mechanism of HMGB1 activation and the physiological function of HMGB1 in HSCs in vivo.

Ｈｉｇｈ Ｍｏｂｉｌｉｔｙ Ｇｒｏｕｐ Ｂｏｘ １（ＨＭＧＢ１）が培養ヒト歯根膜細胞に与える影響

The periodontal ligament (PDL) is a fibrous connective tissue that exists in the cementum and the alveolar bone. Periodontitis is a chronic inflammatory disease that is caused by a bacterial infection in the periodontal region. This infection increases the production of inflammatory cytokines and causes the destruction of periodontal tissue. High mobility group box 1 (HMGB1) is a nuclear nonhiston DNA-binding protein that is present in many eukaryotic cells. HMGB1 is released actively from macrophages and monocytes stimulated by lipopolysaccharide or tumor necrosis factor-alpha and passively from damaged cells and necrotic cells. Extracellular HMGB1 signals through a specialized receptor for advanced glycation end products (RAGE), toll-like receptor 2 (TLR2), and toll-like receptor 4 (TLR4). According to a recent report, HMGB1 is of concern in periodontitis. The purpose of this study was to examine the effect of HMGB1 in PDL cells. To investigate RAGE, TLR2 and TLR4 mRNA in PDL cells, reverse transcript-polymerase chain reaction experiments were performed. PDL cells were stimulated with HMGB1, with or without anti-RAGE, TLR2 and TLR4 antibodies. IL-6 and IL-11 production was measured using an enzyme-linked immunosorbent assay, and mRNA expression was quantified by real-time PCR. PDL cells expressed RAGE, TLR2 and TLR4 mRNA. Production and mRNA expression of IL-6 and IL-11 were augmented in PDL cells stimulated with HMGB1. In addition, they were also suppressed by anti-RAGE, TLR2 and TLR4 antibodies. In conclusion, PDL cells produce IL-6 and IL-11 in response to HMGB1 via RAGE, TLR2 and TLR4.

Expression of TLR-2, TLR-4, NOD2 and pNF-κB in a Neonatal Rat Model of Necrotizing Enterocolitis

BackgroundThe etiology of necrotizing enterocolitis (NEC) results from a combination of several risk factors that act synergistically and occurs in the same circumstances as those which lead to innate immunity activation. Pattern recognition molecules could be an important player in the initiation of an exaggerated inflammatory response leading to intestinal injury in NEC.Methodology/Principal FindingsWe specifically evaluated intestinal epithelial cell (IEC) expression of Toll-like receptor 2 (TLR-2), TLR-4, NOD2 and phosphorylated NF-κB (pNF-κB) after mucosal injury in a rat model of NEC induced by prematurity, systemic hypoxia, and a rich protein formula. In the control group (group 1), neonatal rats were full-term and breast-fed; in the experimental groups, rat pups were preterm at day 21 of gestation and rat-milk fed (group 2) or hand-gavaged with a protein rich formula after a hypoxia–reoxygenation procedure (group 3). Morphological mucosal changes in the small bowel were scored on hematoxylin- and eosin-stained sections. Immunohistochemistry was performed on frozen tissue sections using anti TLR-2 and active pNF-κB p65 antibodies. Real-time RT-PCR was performed to assess mRNA expression of NOD2, TLR-2 and TLR-4. Proliferation and apoptosis were studied in paraffin sections using anti Ki-67 and caspase-3 antibodies, respectively. The combination of immaturity, protein rich formula and a hypoxia–reoxygenation procedure induces pathological mucosal damage consistent with NEC. There was an overexpression of TLR-2, and pNF-κB in IECs that was correlated with the severity of mucosal damage, together with an increase of apoptotic IECs and markedly impaired proliferation. In addition, these immunological alterations appeared before severe mucosal damage. TLR-2 mRNA were also increased in NEC together with TLR-4 mRNA using real-time RT-PCR whereas NOD2 expression was unchanged.Conclusions/SignificanceThese results show that this rat model of NEC induced mucosal injury, leading to a highly responsive IEC phenotype and suggesting that alterations in the innate immune system participates in the pathogenesis of NEC and are enhanced by prematurity.

Toll-like Receptor-2 Is Essential for the Development of Palmitate-induced Insulin Resistance in Myotubes

Fatty acids can activate proinflammatory pathways leading to the development of insulin resistance, but the mechanism is undiscovered. Toll like receptor 2 (TLR2) recognizes lipids, activates proinflammatory pathways, and is genetically associated with inflammatory diseases. This study aimed to examine the role of TLR2 in palmitate-induced insulin resistance in C2C12 myotubes. Treatment with palmitate rapidly induced the association of myeloid differentiation factor 88 (MyD88) with the TLR2 receptor, activated the stress-linked kinases p38, JNK, and protein kinase C, induced degradation of IkappaBalpha, and increased NF-kappaB DNA binding. The activation of these pathways by palmitate was sensitive and temporally regulated and occurred within the upper physiologic range of saturated fatty acid concentrations in vivo, suggesting a receptor-mediated event and not simple lipotoxicity. When compared with an equimolar concentration of palmitate, fibroblast-stimulating lipopeptide-1, a known TLR2 ligand, was a slightly more potent activator of signal transduction and interleukin (IL)-6 production. Palmitate inhibited insulin signal transduction in C2C12 cells beginning 1-2 h after exposure and reached a maximum at 12-16 h. An antagonist TLR2 antibody, mAb 2.5, led to a 50-60% decrease in palmitate-induced IL-6 production and partially restored insulin signal transduction, whereas an isotype-matched control antibody had no effect. RNA interference-mediated inhibition of TLR2 and MyD88 expression in C2C12 muscle cells resulted in a near complete inhibition of palmitate-induced insulin resistance and IL-6 production. This study provides strong evidence that TLR2 mediates the initial events of fatty acid-induced insulin resistance in muscle.

Lipopolysaccharide and Double-stranded RNA Up-regulate Toll-like Receptor 2 Independently of Myeloid Differentiation Factor 88

Toll-like receptor 2 (TLR2) is a signaling receptor for a variety of microbial products, including bacterial lipoproteins and peptidoglycan, and is central in initiating immune responses toward Gram-positive bacteria, spirochetes, and mycobacteria. The mechanisms behind regulation of TLR2 protein expression are still not well understood. By using a newly developed monoclonal antibody against mouse TLR2, we detected TLR2 protein expression on macrophages, neutrophils, and dendritic cells. Endogenous macrophage TLR2 localized mostly to the cell membrane, with particular accumulation around phagosomes containing zymosan. Treatment of macrophages with the TLR2 antibody diminished cellular response to lipoproteins and down-regulated membrane TLR2. Marked up-regulation of surface TLR2 was observed on macrophages in response to whole bacteria, lipoproteins, lipopolysaccharide, poly(I-C) (double-stranded RNA), R848, and CpG DNA, and this up-regulation appeared to be a very sensitive marker for the presence of microbial products. Up-regulation of TLR2 in response to stimuli correlated with an increased response to secondary lipoprotein exposure following a low concentration of primary lipoprotein challenge. By comparison, exposure to a larger primary challenge induced a hyporeactive state. Most interestingly, lipopolysaccharide- and double-stranded RNA-induced up-regulation of surface TLR2 in macrophages was found to be MyD88-independent, whereas the up-regulation in response to lipoproteins, R848, and CpG DNA was absent in MyD88-deficient cells. We conclude that complex mechanisms regulate expression and signaling via TLR2. Up-regulation of TLR2 in the presence of low, yet clinically relevant amounts of microbial products may be an important mechanism by which the immune system boosts its response to a beginning infection.

Differential expression of inducible nitric oxide synthase is associated with differential Toll-like receptor-4 expression in chicken macrophages from different genetic backgrounds

The purpose of this study was to examine iNOS gene expression and activity in macrophages from different chicken genetic lines against various bacterial LPS. Furthermore, the possible involvement of surface LPS receptors as candidates for differential iNOS gene induction in these genetic lines of chicken was also examined. Sephadex-elicited abdominal macrophages (1×10 6) as well as iNOS hyper-responder macrophages from a transformed chicken macrophage cell line, MQ-NCSU, were exposed to 5 μg/ml LPS from E. coli, Shigella flexneri, Serratia marcensces, and Salmonella typhimurium. Nitrite levels were quantitated in the culture supernatant fractions of macrophages after 24 h by the Griess method. The results showed that macrophages from K-strain (B 15B 15) (range from two separate trials: 31–89 μM) and MQ-NCSU (22–81 μM) were high responders whereas macrophages from both GB1 (B 13B 13) (15–38 μM) and GB2 (B 6B 6) (7–15 μM) chickens were low responders against all LPSs used. Northern blot analysis revealed that K-strain macrophages expressed higher intensity of 4.5 Kb iNOS mRNA (iNOS/β-actin ratio) than macrophages from GB2 regardless of the LPS source. To elucidate possible molecular mechanism(s) involved in iNOS gene expression in these two strains of chickens, the constitutive expression of LPS-related macrophage cell surface receptors, CD14, Toll-like receptor-2 (TLR2), and Toll-like receptor-4 (TLR4), was examined via flow cytometry using anti-human CD14, TLR2 and TLR4 antibodies. CD14 surface expression and intensity was not different between macrophages from K-strain or GB2 chickens. In contrast, while the overall percentage of TLR4-positive macrophages was the same (K-strain, trial 1=92%, trial 2=62%; GB2, trial 1=91%, trial 2=64%), the mean fluorescence intensity (MFI), an indicator of receptor number, was significantly higher ( P=0.05) in K-strain macrophages (MFI: trial 1=145; trial 2=131) than GB2 macrophages (MFI: trial 1=101; trial 2=98). Furthermore, TLR2 (a previously thought candidate as LPS signaling molecule) positive cell numbers were higher in K-strain than the GB2 macrophages in one of the two trials with no difference in the intensity of TLR2 expression in either trial. These findings suggest that the observed differences in iNOS expression and activity among the K-strain (hyper-responder) and GB2 (hypo-responder) chickens are, at least in part, due to differential expression of TLR4 (an LPS signaling molecule), leading to more intense LPS-mediated activation of K-macrophages.