MD2 contributes to the pathogenesis of perioperative neurocognitive disorder via the regulation of α5GABAA receptors in aged mice.

BACKGROUND: Perioperative neurocognitive disorder (PND) is a long-term postoperative complication in elderly surgical patients. The underlying mechanism of PND is unclear, and no effective therapies are currently available. It is believed that neuroinflammation plays an important role in triggering PND. The secreted glycoprotein myeloid differentiation factor 2 (MD2) functions as an activator of the Toll-like receptor 4 (TLR4) inflammatory pathway, and α5GABAA receptors (α5GABAARs) are known to play a key role in regulating inflammation-induced cognitive deficits. Thus, in this study, we aimed to investigate the role of MD2 in PND and determine whether α5GABAARs are involved in the function of MD2. METHODS: Eighteen-month-old C57BL/6J mice were subjected to laparotomy under isoflurane anesthesia to induce PND. The Barnes maze was used to assess spatial reference learning and memory, and the expression of hippocampal MD2 was assayed by western blotting. MD2 expression was downregulated by bilateral injection of AAV-shMD2 into the hippocampus or tail vein injection of the synthetic MD2 degrading peptide Tat-CIRP-CMA (TCM) to evaluate the effect of MD2. Primary cultured neurons from brain tissue block containing cortices and hippocampus were treated with Tat-CIRP-CMA to investigate whether downregulating MD2 expression affected the expression of α5GABAARs. Electrophysiology was employed to measure tonic currents. For α5GABAARs intervention experiments, L-655,708 and L-838,417 were used to inhibit or activate α5GABAARs, respectively. RESULTS: Surgery under inhaled isoflurane anesthesia induced cognitive impairments and elevated the expression of MD2 in the hippocampus. Downregulation of MD2 expression by AAV-shMD2 or Tat-CIRP-CMA improved the spatial reference learning and memory in animals subjected to anesthesia and surgery. Furthermore, Tat-CIRP-CMA treatment decreased the expression of membrane α5GABAARs and tonic currents in CA1 pyramidal neurons in the hippocampus. Inhibition of α5GABAARs by L-655,708 alleviated cognitive impairments after anesthesia and surgery. More importantly, activation of α5GABAARs by L-838,417 abrogated the protective effects of Tat-CIRP-CMA against anesthesia and surgery-induced spatial reference learning and memory deficits. CONCLUSIONS: MD2 contributes to the occurrence of PND by regulating α5GABAARs in aged mice, and Tat-CIRP-CMA is a promising neuroprotectant against PND.

Construction of a sensitive pyrogen-testing cell model by site-specific knock-in of multiple genes.

A pyrogen test is crucial for evaluating the safety of drugs and medical equipment, especially those involved in injections. As existing pyrogen tests, including the rabbit pyrogen test, the limulus amoebocyte lysate (LAL) test and the monocyte activation test have limitations, development of new models for pyrogen testing is necessary. Here we develop a sensitive cell model for pyrogen test based on the lipopolysaccharides (LPS) signal pathway. TLR4, MD2, and CD14 play key roles in the LPS-mediated pyrogen reaction. We established a new TLR4/MD2/CD14-specific overexpressing knock-in cell model using the CRISPR/CAS9 technology and homologous recombination to detect LPS. Stimulation of our TLR4/CD14/MD2 knock-in cell line model with LPS leads to the release of the cytokines IL-6 and TNF-alpha, with a detection limit of 0.005 EU/ml, which is greatly lower than the lower limit of 0.015 EU/ml detected by the Tachypleus amebocyte lysate (TAL) assay.

Carbon monoxide down-modulates Toll-like receptor 4/MD2 expression on innate immune cells and reduces endotoxic shock susceptibility.

Carbon monoxide (CO) has been recently reported as the main anti-inflammatory mediator of the haem-degrading enzyme haem-oxygenase 1 (HO-1). It has been shown that either HO-1 induction or CO treatment reduces the ability of monocytes to respond to inflammatory stimuli, such as lipopolysaccharide (LPS), due to an inhibition of the signalling pathways leading to nuclear factor-κB, mitogen-activated protein kinases and interferon regulatory factor 3 activation. Hence, it has been suggested that CO impairs the stimulation of the Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD2) complex located on the surface of immune cells. However, whether CO can negatively modulate the surface expression of the TLR4/MD2 complex in immune cells remains unknown. Here we report that either HO-1 induction or treatment with CO decreases the surface expression of TLR4/MD2 in dendritic cells (DC) and neutrophils. In addition, in a septic shock model of mice intraperitoneally injected with lipopolysaccharide (LPS), prophylactic treatment with CO protected animals from hypothermia, weight loss, mobility loss and death. Further, mice pre-treated with CO and challenged with LPS showed reduced recruitment of DC and neutrophils to peripheral blood, suggesting that this gas causes a systemic tolerance to endotoxin challenge. No differences in the amount of innate cells in lymphoid tissues were observed in CO-treated mice. Our results suggest that CO treatment reduces the expression of the TLR4/MD2 complex on the surface of myeloid cells, which renders them resistant to LPS priming in vitro, as well as in vivo in a model of endotoxic shock.

STAT1 regulates MD-2 expression in monocytes of sepsis via miR-30a.

Sepsis is a major cause of morbidity and mortality in critically ill patients. MD-2 is a 25-kDa lipopolysaccharide (LPS)-binding protein that forms a heterodimer with TLR42, but its regulation in sepsis is not clear. This study aims to investigate the molecular mechanism of regulation of MD-2. Inflammation cytokines in monocytes were analyzed by real-time RT-PCR and ELISA, and it was found that IL-10 was elevated significantly in the monocytes with LPS treatment. And then, when the cells were treated with IL-10, STAT1 was activated in the monocytes using Western blotting. It was also found that STAT1 could enhance MD-2 expression on transcriptional and posttranscriptional levels. Finally, miR-30a was predicted to the molecule that may regulate STAT1 expression. It was verified that STAT1 was a new target gene of miR-30a. miR-30a could inhibit IL-10-induced cytokine release by targeting STAT1-MD-2 in monocytes. In conclusion, this study for the first time demonstrated that miR-30a inhibits MD-2 expression by targeting of STAT1 in human monocytes.

LPS ligand and culture additives improve production of monomeric MD-1 and 2 in Pichia pastoris by decreasing aggregation and intermolecular disulfide bonding.

Myeloid differentiation proteins MD-1 and MD-2 have both been shown to form a heterogeneous collection of oligomers when expressed in absence of their respective receptor, RP105 and TLR4. The biological relevance of these oligomers is not clear. Only monomeric proteins have been found to be active and able to trigger an immune response to endotoxin by modulating the TLR4 pathway. In this study, we produced variants of MD-1 and MD-2 in Pichia pastoris. To minimize the time and expense of initial expression tests, small-scale cultures have been set up to allow the rapid identification of the highest expressing clone and the optimal expression conditions. The expression vectors used, the site of linearization and the locus of integration affected the yield of transformation. Next we screened culture additives and found that they significantly increased the fraction of monomeric proteins secreted in the culture medium (up to 15% of the total MD protein produced). We confirmed their presence by size-exclusion chromatography. Optimal anti-aggregation agents were protein-dependent except for LPS that presented stabilizing effects for all MD proteins. Contrary to previous reports, this study suggests that MD-1 can bind to LPS.

Expression of functional D299G.T399I polymorphic variant of TLR4 depends more on coexpression of MD-2 than does wild-type TLR4.

Two missense variants (D299G and T399I) of TLR4 are cosegregated in individuals of European descent and, in a number of test systems, result in reduced responsiveness to endotoxin. How these changes within the ectodomain (ecd) of TLR4 affect TLR4 function is unclear. For both wild-type and D299G.T399I TLR4, we used endotoxinCD14 and endotoxinMD-2 complexes of high specific radioactivity to measure: 1) interaction of recombinant MD-2TLR4 with endotoxinCD14 and TLR4 with endotoxinMD-2; 2) expression of functional MD-2TLR4 and TLR4; and 3) MD-2TLR4 and TLR4-dependent cellular endotoxin responsiveness. Both wild-type and D299G.T399I TLR4(ecd) demonstrated high affinity (K(d) approximately 200 pM) interaction of endotoxinCD14 with MD-2TLR4(ecd) and endotoxinMD-2 with TLR4(ecd). However, levels of functional TLR4 were reduced up to 2-fold when D299G.T399I TLR4 was coexpressed with MD-2 and >10-fold when expressed without MD-2, paralleling differences in cellular endotoxin responsiveness. The dramatic effect of the D299G.T399I haplotype on expression of functional TLR4 without MD-2 suggests that cells expressing TLR4 without MD-2 are most affected by these polymorphisms.

CD14-independent responses induced by a synthetic lipid A mimetic.

CRX-527 belongs to a new family of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates, which are considered as potential vaccine adjuvants or stand-alone immunotherapeutics to harness innate immune defenses. Since natural lipid A from bacterial LPS depends on membrane-bound (mCD14) or soluble CD14 for its TLR4 ligand activity, we investigated the involvement of both forms of CD14 in the responses elicited by CRX-527. First, we found that CRX-527 induces NF-kappaB and interferon regulatory factor-3 (IRF-3) activation in human embryonic kidney cells transfected with TLR4 and MD-2 genes alone, whereas the responses to LPS require either co-transfection of the gene encoding mCD14 or addition of soluble CD14. We then observed that monocyte-derived DC, which are devoid of mCD14 respond to CRX-527 but not to LPS in serum-free medium. Furthermore, we found that, in contrast to LPS, CRX-527 induces the production of cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which mCD14-dependent responses are defective. Finally, we demonstrated that splenocytes from CD14-deficient mice produce cytokines in response to CRX-527 but not to LPS. We conclude that the lipid A mimetic CRX-527 does not require the CD14 co-receptor to elicit TLR4-mediated responses.

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.

Beta2-integrin-induced p38 MAPK activation is a key mediator in the CD14/TLR4/MD2-dependent uptake of lipopolysaccharide by hepatocytes.

The liver is the main organ that clears circulating lipopolysaccharide (LPS), and hepatocytes are a major cell type involved in LPS uptake. Little is known about the mechanisms for LPS internalization in hepatocytes and what signaling pathways are involved. We show here that LPS uptake is initiated after formation of a multi-receptor complex within lipid rafts. We find that essential components for LPS uptake are CD14, TLR4, MD2, and the beta2-integrin CD11b/CD18. Activation of p38 MAPK is also essential for the initiation of LPS uptake, and interestingly, we show that this activation is not through TLR4 signaling by MyD88 but through activation of TIRAP via CD11b/CD18. However, TLR4/MD2 remain essential components at the cell surface as part of the LPS receptor complex. We therefore suggest novel roles for TLR4/MD2, CD11b/CD18, TIRAP, and p38 MAPK in LPS uptake by hepatocytes.

Preparation and characterization of agonistic monoclonal antibodies against Toll-like receptor 4-MD-2 complex.

Ligands for toll-like receptors (TLR) are known to induce a variety of immune responses. Selective induction of desirable responses would be important for the treatment of individual diseases with various pathogenesis. For this purpose, we established six MAbs against the TLR4/MD-2 complex (UT MAbs) from TLR4(-/-) mice or MD-2(-/-) mice. Three MAbs (UT12, 18, and 22) induced NF-kappaB activation and production of pro-inflammatory cytokines, but the other three (UT15, 41, and 49) did not induce such cell responses. Unlike lipopolysaccharide (LPS), agonistic UT MAbs did not require serum components for the functions. UT41 and UT49 recognized TLR4 in the absence of MD-2. On the other hand, the other four MAbs reacted to the TLR4/MD-2 complex, but not to solo TLR4. Agonistic UT MAbs shared the epitopes, but non-agonistic UT15 reacted to distinct epitope on the complex. UT MAbs appear to be useful analyzing the molecular mechanism of TLR signaling and will contribute to the development of novel immunotherapies.

Protein-energy malnutrition decreases the expression of TLR-4/MD-2 and CD14 receptors in peritoneal macrophages and reduces the synthesis of TNF-alpha in response to lipopolysaccharide (LPS) in mice.

Protein-energy malnutrition (PEM) modifies resistance to infection, impairing a number of physiological processes, including hematopoiesis. In this study, we examined a few aspects of the inflammatory response to LPS in a model of PEM. We evaluated the cellularity of the blood, bone marrow and spleen, as well as phagocytic, fungicidal and spreading activity, the production in vivo and in vitro of TNF-alpha, IL-1alpha and IL-6, and the expression of CD14 and TLR-4/MD-2 receptors in macrophages. Two-month-old male Swiss mice were submitted to PEM with a low-protein diet containing 4% protein as compared to 20% protein in the control diet. When the experimental group had attained about 20% loss of their original body weight, they were used in the experiments. Malnourished animals presented anemia, leucopenia and severe reduction in bone marrow, spleen and peritoneal cavity cellularity. The production of TNF-alpha, IL-1alpha and IL-6 stimulated in vivo with LPS and the production of IL-6 in bone marrow cells cultured with LPS and the production of TNF-alpha in bone marrow, spleen and peritoneal cells cultured with LPS were significantly lower in malnourished animals. The expression of CD14 and TLR-4/MD-2 receptors was found to be significantly lower in macrophages of malnourished animals. These findings suggest that malnourished animals present a deficient response to LPS. The lower expression of the CD14 and TLR-4/MD-2 receptors may be partly responsible for the immunodeficiency observed in the malnourished mice. These data lead us to infer that the nutritional state interferes with the activation of macrophages and with the capacity to mount an immune response.

Enhanced IL-10 production by TLR4- and TLR2-primed dendritic cells upon TLR restimulation.

LPS tolerance has been investigated extensively in monocytes/macrophages. However, the LPS restimulation studies are not well documented in dendritic cells (DCs). In the present study, we investigated influences of TLR restimulation using murine bone marrow-derived DCs. Purified bone marrow-derived DCs (>98% CD11c+ B220-) were stimulated with TLR4 and TLR2 ligands for 24 h and then cultured with medium alone for 48 h as a resting interval (TLR4,2-primed DCs). The TLR4-MD2 expression was markedly reduced immediately after the TLR stimulation, but was restored following the resting interval. The TLR4,2-primed DCs exhibited significantly enhanced IL-10 production, but markedly diminished IL-12p40 production upon TLR4 restimulation compared with naive (unprimed) DCs. TLR4-mediated activation of p38 MAPK was markedly suppressed, whereas that of ERK1/2 was enhanced in the TLR4,2-primed DCs compared with naive DCs. Blocking the activation of ERK1/2 with U0126 reduced the enhanced IL-10 production by the TLR4,2-primed DCs upon the TLR4 restimulation. The U0126 showed no significant effects on the IL-12p40 production. Thus, the enhanced ERK1/2 activation appears to be, at least in part, responsible for the enhanced IL-10 production in the TLR4,2-primed DCs. In addition, TNFR-associated factor 3 expression was significantly up-regulated in the TLR4,2-primed DCs compared with that in naive DCs. We demonstrated in this study that DCs primed with TLR4 and TLR2 ligands and rested for 48 h showed enhanced IL-10 production upon TLR4 restimulation. The enhanced IL-10 production by the TLR4,2-primed DCs may be attributed to the altered balance of intracellular signaling pathways via p38 MAPK, ERK1/2, and TNFR-associated factor 3 upon TLR restimulation.

Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance.

In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively active mouse DeltaTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, IkappaB-alpha degradation, and activation of NF-kappaB and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-kappaB activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes.

Functional TLRs and NODs in human gingival fibroblasts.

Since human gingival fibroblasts are the major cells in periodontal tissues, we hypothesized that gingival fibroblasts are endowed with receptors for bacterial components, which induce innate immune responses against invading bacteria. We found clear mRNA expression of Toll-like receptors (TLR)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, MD-2, MyD88, NOD1, and NOD2 in gingival fibroblasts. Gingival fibroblasts constitutively expressed these molecules. Upon stimulation with chemically synthesized ligands mimicking microbial products for these receptors, the production of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1, was markedly up-regulated. Furthermore, the production of pro-inflammatory cytokines induced by TLR and NOD ligands was significantly inhibited by an RNA interference assay targeted to NF-kappaB. These findings indicate that these innate immunity-related molecules in gingival fibroblasts are functional receptors involved in inflammatory reactions in periodontal tissues, which might be responsible for periodontal pathogenesis.

MD-2 expression is not required for cell surface targeting of Toll-like receptor 4 (TLR4).

The cell surface receptor complex formed by TLR4 and myeloid differentiation 2 (MD-2) is engaged when cells are exposed to LPS. Recent studies suggested that surface localization of functional mouse TLR4 (mTLR4) depends on the simultaneous expression of MD-2. As we did not observe a similar requirement, we conducted a comparative study of human TLR4 and mTLR4 surface expression in immune cells derived from the MD-2 knockout mouse and LPS-responsive cell lines and in cells that ectopically express TLR4. Our results indicate that in the human and mouse models, neither TLR4 function nor TLR4 surface targeting requires MD-2 coexpression. Accordingly, we report on one human cell line, which constitutively expresses functional TLR4 on the cell surface in the absence of MD-2 expression.

A protein associated with toll-like receptor 4 (PRAT4A) regulates cell surface expression of TLR4.

TLRs recognize microbial products. Their subcellular distribution is optimized for microbial recognition. Little is known, however, about mechanisms regulating the subcellular distribution of TLRs. LPS is recognized by the receptor complex consisting of TLR4 and MD-2. Although MD-2, a coreceptor for TLR4, enhances cell surface expression of TLR4, an additional mechanism regulating TLR4 distribution has been suggested. We show here that PRAT4A, a novel protein associated with TLR4, regulates cell surface expression of TLR4. PRAT4A is associated with the immature form of TLR4 but not with MD-2 or TLR2. PRAT4A knockdown abolished LPS responsiveness in a cell line expressing TLR4/MD-2, probably due to the lack of cell surface TLR4. PRAT4A knockdown down-regulated cell surface TLR4/MD-2 on dendritic cells. These results demonstrate a novel mechanism regulating TLR4/MD-2 expression on the cell surface.

Postnatal acquisition of endotoxin tolerance in intestinal epithelial cells.

The role of innate immune recognition by intestinal epithelial cells (IECs) in vivo is ill-defined. Here, we used highly enriched primary IECs to analyze Toll-like receptor (TLR) signaling and mechanisms that prevent inappropriate stimulation by the colonizing microflora. Although the lipopolysaccharide (LPS) receptor complex TLR4/MD-2 was present in fetal, neonatal, and adult IECs, LPS-induced nuclear factor kappaB (NF-kappaB) activation and chemokine (macrophage inflammatory protein 2 [MIP-2]) secretion was only detected in fetal IECs. Fetal intestinal macrophages, in contrast, were constitutively nonresponsive to LPS. Acquisition of LPS resistance was paralleled by a spontaneous activation of IECs shortly after birth as illustrated by phosphorylation of IkappaB-alpha and nuclear translocation of NF-kappaB p65 in situ as well as transcriptional activation of MIP-2. Importantly, the spontaneous IEC activation occurred in vaginally born mice but not in neonates delivered by Caesarean section or in TLR4-deficient mice, which together with local endotoxin measurements identified LPS as stimulatory agent. The postnatal loss of LPS responsiveness of IECs was associated with a posttranscriptional down-regulation of the interleukin 1 receptor-associated kinase 1, which was essential for epithelial TLR4 signaling in vitro. Thus, unlike intestinal macrophages, IECs acquire TLR tolerance immediately after birth by exposure to exogenous endotoxin to facilitate microbial colonization and the development of a stable intestinal host-microbe homeostasis.

Mechanism regulating cell surface expression and activation of Toll-like receptor 4.

The Toll family of receptors senses microbial invasion and activates defense responses. Toll-like receptor 4 (TLR4) is a member of the Toll family that senses lipopolysaccharide (LPS), a principal membrane component from Gram-negative bacteria. LPS is known as an endotoxin that strongly activates immune cells such as macrophages and dendritic cells. LPS recognition by TLR4 requires an additional accessory molecule, MD-2. MD-2 is associated with the extracellular portion of TLR4, directly binds to LPS, and regulates subsequent LPS-induced TLR4 clustering. LPS recognition occurs on the cell surface. The subcellular distribution of TLR was shown to influence TLR responses. An endoplasmic reticulum (ER) chaperone, glycoprotein 96, is required for the stability of TLR4 and the formation of a TLR4/MD-2 complex in ER. MD-2 facilitates TLR4 glycosylation and its trafficking to the cell surface. Recently, another molecule, a protein associated with Toll-like receptor 4 (PRAT4A), was shown to play a critical role in cell surface expression of TLR4. These molecules control LPS responsiveness by regulating the subcellular distribution of TLR4.