Myeloid differentiation-2 is a potential biomarker for the amplification process of allergic airway sensitization in mice.

BACKGROUND: Allergic sensitization is a key step in the pathogenesis of asthma. However, little is known about the molecules that are critical regulators for establishing allergic sensitization of the airway. Thus, we conducted global gene expression profiling to identify candidate genes and signaling pathways involved in house dust mite (HDM)-induced allergic sensitization in the murine airway. METHODS: We sensitized and challenged mice with HDM or saline as a control through the airway on days 1 and 8. We evaluated eosinophilia in bronchoalveolar lavage fluid (BALF), airway inflammation, and mucus production on days 7 and 14. We extracted total RNA from lung tissues of HDM- and saline-sensitized mice on days 7 and 14. Microarray analyses were performed to identify up-regulated genes in the lungs of HDM-sensitized mice compared to the control mice. Data analyses were performed using GeneSpring software and gene networks were generated using Ingenuity Pathways Analysis (IPA). RESULTS: We identified 50 HDM-mediated, stepwise up-regulated genes in response to allergic sensitization and amplification of allergic airway inflammation. The highest expressed gene was myeloid differentiation-2 (MD-2), a lipopolysaccharide (LPS)-binding component of Toll-like receptor (TLR) 4 signaling complex. MD-2 protein was expressed in lung vascular endothelial cells and was increased in the serum of HDM-sensitized mice, but not in the control mice. CONCLUSIONS: Our data suggest MD-2 is a critical regulator of the establishment of allergic airway sensitization to HDM in mice. Serum MD-2 may represent a potential biomarker for the amplification of allergic sensitization and allergic inflammation.

Lymphocyte antigen 96: A new potential biomarker and immune target in Parkinson's disease.

BACKGROUND: Lymphocyte antigen 96 (LY96) plays an important role in innate immunity and has been reported to be associated with various neurological diseases. However, its role in Parkinson's disease (PD) remains unclear. METHODS: Transcriptome data from a total of 49 patients with PD and 34 healthy controls were downloaded from the Gene Expression Omnibus (GEO) database to analyse the expression pattern of LY96 and its relationship with gene function and immune-related markers. In addition, peripheral blood samples were collected from clinical patients to validate LY96 mRNA expression levels. Finally, an in vitro cell model of PD based on highly differentiated SH-SY5Y cells was constructed, with small interfering RNA-silenced LY96 expression, and LY96 mRNA level, cell viability, flow cytometry, and mitochondrial membrane potential assays were performed. RESULTS: The results of the analyses of the GEO database and clinical samples revealed significantly abnormally high LY96 expression in patients with PD compared with healthy controls. The results of cell experiments showed that inhibiting LY96 expression alleviated adverse cellular effects by increasing cell viability, reducing apoptosis, and reducing oxidative stress. Gene set enrichment analysis showed that LY96 was positively correlated with T1 helper cells, T2 helper cells, neutrophils, natural killer T cells, myeloid-derived suppressor cells, macrophages, and activated CD4 cells, and may participate in PD through natural killer cell-mediated cytotoxicity pathways and extracellular matrix receptor interaction pathways. CONCLUSION: These findings suggested that LY96 might be a novel potential biomarker for PD, and offer insights into its immunoregulatory role.

IL-10 enhances MD-2 and CD14 expression in monocytes and the proteins are increased and correlated in HIV-infected patients.

Soluble proteins that bind LPS, like myeloid differentiation-2 (MD-2) and CD14, have essential roles in regulating LPS signaling through TLR4. During a gram-negative bacterial infection, the host may control the response by adjusting the levels of soluble MD-2 and CD14. To address the surface expression of MD-2 on human leukocytes, we developed a mAb, IIC1, that recognized MD-2 both free and when bound to TLR4. MD-2 was found on the surface of freshly isolated monocytes, on a subpopulation of CD19(+) B-cells and on CD15(+) neutrophils. LPS transiently reduced the MD-2 levels on monocytes, which is most likely due to endocytosis of the LPS receptor complex since MD-2 colocalized with TLR4 in early endosomes after LPS stimulation. In the absence of LPS, MD-2 partly colocalized with TLR4 in Golgi trans and medial compartments. Cultivating monocytes for 18-20 h resulted in loss of MD-2 expression on the surface, which was reversed either by LPS or IL-10. Furthermore, addition of IL-10, but not LPS, resulted in a considerable increase in mRNA for both MD-2 and CD14. Using ELISA, we demonstrated that IL-10 had a profound dose- and time-related effect on the release of soluble MD-2 and soluble CD14 from monocytes. In HIV-infected patients, the amounts of MD-2, CD14, and IL-10 increased significantly in the patient group with AIDS. Of interest, we found that IL-10, CD14, and MD-2 levels were positively correlated, suggesting that IL-10 may be a driving force for increased release of MD-2 and CD14 during systemic inflammation.

Soluble MD-2 and Heme in Sickle Cell Disease Plasma Promote Pro-Inflammatory Signaling in Endothelial Cells.

Recent evidence indicates that hemolysis in sickle cell disease (SCD) promotes inflammation via innate immune signaling through toll-like receptor 4 (TLR4). Free heme released by hemolyzed red blood cells can bind to myeloid differentiation factor-2 (MD-2) and activate TLR4 pro-inflammatory signaling on endothelium to promote vaso-occlusion and acute chest syndrome in murine models of SCD. MD-2 is co-expressed with TLR4 on cell membranes, but in inflammatory conditions, soluble MD-2 (sMD-2) is elevated in plasma. sMD-2 levels were significantly increased in human and murine sickle (SS) plasma as compared to normal (AA) plasma. Human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells incubated with human SS plasma had significant increases in pro-inflammatory IL-8, IL-6, and soluble VCAM-1 secretion compared to endothelial cells incubated with AA plasma. The increase in HUVEC IL-8 secretion was blocked by depletion of sMD-2 from SS plasma and enhanced by the addition of sMD-2 to AA plasma. The TLR4 signaling inhibitor, TAK-242, inhibited HUVEC IL-8 secretion in response to SS plasma by 85%. Heme-agarose pull-down assays and UV/Vis spectroscopy demonstrated that heme binds to sMD-2. Hemopexin, a high affinity heme-binding protein, inhibited HUVEC IL-8 secretion induced by SS plasma or SS and AA plasma supplemented with sMD-2. These data suggest that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in SCD.

Biomarkers of treatment success in fully sensitive pulmonary tuberculosis patients: a multicenter longitudinal study.

Aim: Novel biomarkers that are able to accurately monitor tuberculosis (TB) treatment effectiveness are needed to adjust therapy and identify a need for a regimen change. Materials & methods: In our study, conducted on a cohort comprising 100 pulmonary TB patients, we analyzed the role of plasma cytokines and Toll-like receptors expression as biomarkers of treatment response. Results: Changes in toll-interacting protein (TOLLIP) and lymphocyte antigen 96 (LY96) gene expression as well as nine cytokine levels over the first 2 months were significantly associated with successful treatment outcome. Successful treatment was associated with higher serum concentration of Toll-like receptor-2. Conclusion: Our results suggest that differential expression of specific effector molecules and dynamics of selected cytokines may help to identify those responding to TB treatment early.

Increased release of sMD-2 during human endotoxemia and sepsis: a role for endothelial cells.

MD-2 is the crucial cofactor of TLR4 in the detection of LPS. Here, we show that soluble MD-2 (sMD-2) circulates in plasma of healthy individuals as a polymeric protein. The total amount of sMD-2 in septic plasma was strongly elevated and contained both sMD-2 polymers and monomers, the latter representing the putative biologically active form of MD-2. Moreover, during experimental human endotoxemia, the monomeric and total sMD-2 content in plasma increased with the kinetics of an acute phase protein. The increase in sMD-2 monomers was paralleled by enhanced TLR4 costimulatory activity. The presence of functional sMD-2 during endotoxemia and sepsis was confirmed by immunodepletion. Immunohistochemistry revealed that MD-2 expression in septic patients was strongly enhanced on endothelium and multiple inflammatory cells in lung and liver. In vitro studies showed that sMD-2 release appears to be restricted to endothelial cells and dendritic cells. Release of sMD-2 by endothelial cells was strongly enhanced by LPS and TNF-alpha stimulation. Taken together, this study demonstrates the increase of both circulating polymeric and functional monomeric sMD-2 during endotoxemia and sepsis, and evidence is provided that the endothelium is involved in this process.

Immune biomarkers for the diagnosis of mild traumatic brain injury.

BACKGROUND: In 2010, there were approximately 2.2 million emergency room visits associated with traumatic brain injury (TBI), with 80 percent diagnosed as mild TBI or concussion. In addition, there are a large number of TBIs, especially mild TBIs, which go either unreported by patients or initially undiagnosed by clinicians. Our team has previously identified a panel of immune-related genes that can diagnose ischemic stroke at triage, and due to shared pathophysiological mechanisms of TBI and stroke, we hypothesized that this panel of genes may also be utilized for the diagnosis of TBI. OBJECTIVES: The primary aims of this pilot study were to: (1) characterize changes in a panel of immune-related genes in TBI; (2) identify immune-related biomarkers that may be used to diagnose TBI and (3) describe the peripheral immune response following TBI. METHODS: Blood was drawn from TBI patients no later than 24 h of injury onset and matched control subjects. Real-time PCR was used to measure gene expression, and a white blood cell differential was performed to obtain neutrophil and lymphocyte percentages. RESULTS: Relative mRNA expression of ARG1, LY96, MMP9, s100a12 was significantly increased and CCR7 was significantly decreased in peripheral blood of TBI patients within 24 hours of injury compared to control subjects. We also observed a different pattern of leukocyte dynamics following TBI between mild and severe TBI. CONCLUSIONS: We have described a panel of immune-related genes that can accurately predict/diagnose TBI with higher sensitivity and specificity of other biomarkers to date.

Soluble markers of the Toll-like receptor 4 pathway differentiate between active and latent tuberculosis and are associated with treatment responses.

BACKGROUND: Biomarkers to differentiate between active tuberculosis (TB) and latent TB infection (LTBI) and to monitor treatment responses are requested to complement TB diagnostics and control, particularly in patients with multi-drug resistant TB. We have studied soluble markers of the Toll-like-receptor 4 (TLR-4) pathway in various stages of TB disease and during anti-TB treatment. METHODS: Plasma samples from patients with culture confirmed drug-sensitive TB (n = 19) were collected before and after 2, 8 and 24 weeks of efficient anti-TB treatment and in a LTBI group (n = 6). Soluble (s) CD14 and myeloid differentiation-2 (MD-2) were analyzed by the Enzyme-linked immunosorbent assay (ELISA). Lipopolysaccharide (LPS) was analyzed by the Limulus Amebocyte Lysate colorimetric assay. Nonparametric statistics were applied. RESULTS: Plasma levels of sCD14 (p<0.001), MD-2 (p = 0.036) and LPS (p = 0.069) were elevated at baseline in patients with untreated active TB compared to the LTBI group. MD-2 concentrations decreased after 2 weeks of treatment (p = 0.011), while LPS levels decreased after 8 weeks (p = 0.005). In contrast, sCD14 levels increased after 2 weeks (p = 0.047) with a subsequent modest decrease throughout the treatment period. There was no significant difference in concentrations of any of these markers between patients with pulmonary and extrapulmonary TB or between patients with or without symptoms. CONCLUSION: Our data suggest that plasma levels of LPS, MD-2 and sCD14 can discriminate between active TB and LTBI. A decline in LPS and MD-2 concentrations was associated with response to anti-TB treatment. The clinical potential of these soluble TLR-4 pathway proteins needs to be further explored.

Circulating levels of HMGB1 are correlated strongly with MD2 in HIV-infection: possible implication for TLR4-signalling and chronic immune activation.

Progressive HIV infection is characterized by profound enterocyte damage, microbial translocation and chronic immune activation. We aimed to test whether High Mobility Group Box protein 1(HMGB1), a marker of cell death, alone, or in combination with LPS, might contribute to HIV-associated immune activation and progression. Altogether, 29 untreated HIV-infected individuals, 25 inflammatory bowel disease (IBD) patients and 30 controls were included. HIV-infected patients had lower plasma LPS levels than IBD patients, but higher levels of soluble CD14 and Myeloid Differentiation (MD) 2, which interacts with TLR4 to initiate LPS-signalling. Furthermore, plasma levels of HMGB1 and MD2 were correlated directly within the HIV-infected cohort (r = 0.89, P < 0.001) and the IBD-cohort (r = 0.85, P < 0.001), implying HMGB1 signalling through the MD2/TLR4-pathway. HMGB1 and LPS, although not inter-correlated, were both moderately (r = 0.4) correlated with CD38 density on CD8+ T cells in HIV progressors. The highest levels of CD38 density and MD2 were found in progressors with plasma levels of both LPS and HMGB1 above the fiftieth percentile. Our results could imply that, in some patients, immune activation is triggered by microbial translocation, in some by cell death and in some by HMGB1 in complex with bacterial products through activation of the MD2/TLR4-pathway.

Sera from patients with Crohn's disease break bacterial lipopolysaccharide tolerance of human intestinal epithelial cells via MD-2 activity.

Myeloid differentiation (MD)-2 is linked to the cell surface as a Toll-like receptor (TLR) 4-bound protein though may also function as a soluble receptor to enable the lipopolysaccharide (LPS)-driven response. We recently demonstrated the importance of MD-2 either as a cell-associated or as a soluble receptor in the control of intestinal epithelial cell response toward LPS. High levels of circulating MD-2 were recently proposed as a risk factor for infectious/ inflammatory diseases as septic shock. We hypothesized that MD-2 might be present in sera from patients with inflammatory bowel disease and have pathogenic consequences. We analysed MD-2 activity in sera from patients with inflammatory bowel disease or from healthy subjects. We measured MD-2 activity as the capacity to mediate LPS-driven stimulation of intestinal epithelial cells (HT29). We found that sera from patients with inflammatory bowel disease, particularly Crohn's disease, endowed HT29 cells with a markedly higher LPS-dependent stimulating capacity as compared to sera from healthy subjects. The effect of sera was specific for LPS activation and was reduced in the presence of anti-MD-2, and anti-TLR4 antibodies. We conclude that sera from patients with inflammatory bowel disease might contain increased MD-2. This might result in higher local availability of the protein leading to a loss of tolerance toward gut microbiota.

Effect of plasma phospholipid transfer protein deficiency on lethal endotoxemia in mice.

Lipopolysaccharides (LPS) are components of Gram-negative bacteria. The cellular response from the host to LPS is mediated through stepwise interactions involving the lipopolysaccharide-binding protein (LBP), CD14, and MD-2, which produces the rearrangement of TLR4. In addition to LBP, the lipid transfer/lipopolysaccharide-binding protein gene family includes the phospholipid transfer protein (PLTP). Here we show that the intravascular redistribution of LPS from the plasma lipoprotein-free fraction toward circulating lipoproteins is delayed in PLTP-deficient mice. In agreement with earlier in vitro studies, which predicted the neutralization of the endotoxic properties of LPS when associated with lipoproteins, significant increases in the plasma concentration of proinflammatory cytokines were found in PLTP-deficient as compared with wild type mice. Similar inflammatory damage occurred in tissues from wild type and PLTP-deficient mice 24 h after one single intraperitoneal injection of LPS but with a more severe accumulation of red blood cells in glomeruli of LPS-injected PLTP-deficient mice. Complementary ex vivo experiments on isolated splenocytes from wild type and PLTP-deficient mice further supported the ability of cell-derived PLTP to prevent LPS-mediated inflammation and cytotoxicity when combined with lipoprotein acceptors. Finally, PLTP deficiency in mice led to a significant increase in LPS-induced mortality. It is concluded that increasing circulating levels of PLTP may constitute a new and promising strategy in preventing endotoxic shock.

Characterization of monoclonal antibodies to human soluble MD-2 protein.

Toll-like receptors (TLRs) are mammalian innate immune recognition receptors that are activated by pathogen associated molecular patterns (PAMPs). TLR4 is the signaling molecule of the lipopolysaccharide (LPS) receptor complex. TLR4 associates with its adapter molecule, MD-2, which is absolutely required for LPS-induced activation of TLR4. MD-2 exists as a cell surface protein in association with TLR4 and as secreted forms consisting of MD-2 monomers and multimers. To facilitate the studies of MD-2 distribution, abundance, and function, we produced monoclonal antibodies (MAbs) to baculovirally expressed soluble MD-2 (sMD-2). Eleven MAbs were characterized by enzyme-linked immunosorbent assay (ELISA) with soluble TLR4/MD-2 complex (sTLR4/MD-2) and sMD-2, Western blotting against sMD-2 monomer and multimers, and inhibition of direct LPS binding to sMD-2. Four MAbs preferentially recognized mainly MD-2 oligomers, not monomers, as judged by Western blotting and ELISA. Anti-MD-2 MAbs useful for indirect immunofluorescent staining of cells expressing TLR4 and MD-2 were identified. One MAb that recognized all forms of MD-2 was used in an ELISA to measure sMD-2 in normal human sera as well as sera from intensive care patients with and without sepsis. Serum levels of sMD-2 were undetectable or very low in normal and in nonsepsis patients but significantly (p < 0.05) increased in sepsis patients. These MAbs should therefore be very useful new tools for studies of MD-2 expression and function in health and disease.

Pharmacological inhibition of endotoxin responses is achieved by targeting the TLR4 coreceptor, MD-2.

The detection of Gram-negative LPS depends upon the proper function of the TLR4-MD-2 receptor complex in immune cells. TLR4 is the signal transduction component of the LPS receptor, whereas MD-2 is the endotoxin-binding unit. MD-2 appears to activate TLR4 when bound to TLR4 and ligated by LPS. Only the monomeric form of MD-2 was found to bind LPS and only monomeric MD-2 interacts with TLR4. Monomeric MD-2 binds TLR4 with an apparent Kd of 12 nM; this binding avidity was unaltered in the presence of endotoxin. E5564, an LPS antagonist, appears to inhibit cellular activation by competitively preventing the binding of LPS to MD-2. Depletion of endogenous soluble MD-2 from human serum, with an immobilized TLR4 fusion protein, abrogated TLR4-mediated LPS responses. By determining the concentration of added-back MD-2 that restored normal LPS responsiveness, the concentration of MD-2 was estimated to be approximately 50 nM. Similarly, purified TLR4-Fc fusion protein, when added to the supernatants of TLR4-expressing cells in culture, inhibited the interaction of MD-2 with TLR4, thus preventing LPS stimulation. The ability to inhibit the effects of LPS as a result of the binding of TLR4-Fc or E5564 to MD-2 highlights MD-2 as the logical target for drug therapies designed to pharmacologically intervene against endotoxin-induced disease.