TLR4 Molecules Research Articles

A homogalacturonan from Hippophae rhamnoides L. Berries enhance immunomodulatory activity through TLR4/MyD88 pathway mediated activation of macrophages

Our previous study isolated a natural high-methoxyl homogalacturonan (HRWP-A) from Hippophae rhamnoides and showed antitumor activity in vivo. In this study, the immunomodulatory activity and mechanisms of action of HRWP-A were further investigated. Results showed that HRWP-A could recover the body condition and activated macrophage in Cyclophosphamide (CTX)-induced immunosuppressed mice. Further, we investigated the possible mechanism underlying the effects of HRWP-A on mouse peritoneal macrophages. qPCR and western blot revealed that HRWP-A upregulated the expression of TLR4 mRNA in vitro. This process was accompanied by a clear increase in MyD88 expression and p-IκB-α, but these effects were largely abrogated by pretreatment with anti-TLR4 antibodies. The effects of HRWP-A on macrophage NO, IL-1β and IL-6 production were also inhibited by anti-TLR4 antibodies and were greatly influenced by the NF-κB inhibitor PDTC. Moreover, HRWP-A failed to induce the production of NO, IL-1β and IL-6 in peritoneal macrophages prepared from C3H/HeJ mice, which have a point mutation in the Tlr4 gene, suggesting the involvement of the TLR4 molecule in HRWP-A-mediated macrophage activation. These results may have important implications for our understanding of the structure-activity relationship of immunopotentiating polysaccharides from medicinal herbs.

Structural characterization of TLRs with novel agonists

Abstract Small molecule TLR4 and TLR2 agonists have been reported, but no structural data reveals their activation mechanism and detailed interactions with the TLRs. We have developed two small molecule agonists, Neoseptin-3 and Diprovocim, that activate TLR4/MD-2 and TLR1/TLR2 complexes, respectively, with exquisitely specific structure activity relationships. These two molecules bear no structural similarity to the natural ligands, lipopolysaccharide (LPS) and tri-acylated lipopeptide (Pam3CSK4). The crystal structures of Neoseptin-3 in complex with mouse TLR4/MD-2 and Diprovocim in complex with human TLR2 provide the first glimpse of how these TLRs bind to unconventional agonists, revealing unique and unexpected binding modes. Neoseptin-3 binds as an asymmetrical dimer within the hydrophobic pocket of MD-2, and induces an active receptor complex (a dimer of TLR4/MD-2) similar to that induced by lipid A. However, Neoseptin-3 and lipid A form different molecular contacts with TLR4/MD-2 to achieve receptor activation. Diprovocim forms a symmetrical dimer and interacts with the same hydrophobic pocket of TLR2 as Pam3CSK4, inducing homodimerization of TLR2 that has a different conformation than the active TLR1/TLR2 heterodimer. Diprovocim binds to TLR2 through an extensive intermolecular hydrogen bonding network that is not observed in the Pam3CSK4/TLR2/TLR1 structure. These two structures are now guiding us in optimization of TLR4/MD-2 and TLR1/TLR2 agonists and antagonists for clinical applications.

MP26-09 INVOLVEMENT OF TOLL-LIKE RECEPTOR 4 IN NONBACTERIAL BLADDER INFLAMMATION AND FREQUENT URINATION VIA INFLAMMASOME PATHWAYS IN SPONTANEOUSLY HYPERTENSIVE RATS

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology I1 Apr 2017MP26-09 INVOLVEMENT OF TOLL-LIKE RECEPTOR 4 IN NONBACTERIAL BLADDER INFLAMMATION AND FREQUENT URINATION VIA INFLAMMASOME PATHWAYS IN SPONTANEOUSLY HYPERTENSIVE RATS Shinsuke Mizoguchi, Kenichi Mori, Naoyuki Yamanaka, Fuminori Sato, Naoki Yoshimura, and Hiromitsu Mimata Shinsuke MizoguchiShinsuke Mizoguchi More articles by this author , Kenichi MoriKenichi Mori More articles by this author , Naoyuki YamanakaNaoyuki Yamanaka More articles by this author , Fuminori SatoFuminori Sato More articles by this author , Naoki YoshimuraNaoki Yoshimura More articles by this author , and Hiromitsu MimataHiromitsu Mimata More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.780AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Although several studies have suggested that chronic bladder inflammation associated with oxidative stress contribute to overactive bladder (OAB), the underlying pathophysiological mechanism is unclear. Recently, the toll-like receptor (TLR) 4 has been reported to play a role in triggering chronic inflammatory conditions through activation of NLRP3 inflammasome pathways followed by production of IL1β and IL18. Although oxidative stress is one of factors that activate TLR4, it is not known whether changes in the expression of TLR4 are involved in the development of OAB caused by chronic inflammation. We therefore investigated an alteration of histopathology and expression of TLR4 and NLRP3 inflammasome-related molecules in the bladder using spontaneously hypertensive rats (SHRs) as an OAB model. METHODS Twenty-weeks-old male SHRs and Wistar Kyoto rats (control) were used. After voiding function was analyzed by using metabolic cages, the bladder was excised for analysis of histopathology and mRNA expression. Hematoxylin eosin and Masson′s trichrome stain were performed to analyze bladder inflammatory condition and fibrosis. Immunohistostaining for NLRP3, TLR4 was also performed. Expression levels of NLRP3, IL1β, IL-18, IL6, IL8 and TGFβ mRNA in the bladder were investigated by real-time PCR. Statistical analysis was performed using Mann-Whitney U test. P value less than 0.05 was considered statistically significant. RESULTS In voiding function analyses, single urine volume was significantly decreased and voiding frequency was significantly increased in SHRs compared to control rats. In histological evaluation, suburothelial fibrosis was shown in SHRs compared to controls. Furthermore, immunohistostaining showed localized expression of NLRP3 and TLR4 in the bladder urothelium in both groups. In RT-qPCR analyses, mRNA expression levels of NLRP3, TLR4, IL1β, IL-18, IL6, IL8 and TGFβ were significantly increased in SHRs in the bladder compared to controls. CONCLUSIONS These results suggest that activation of TLR4 associated with oxidative stress is implicated in bladder chronic inflammation, which leads to frequent urination through NLRP3 inflammasome pathways. Therefore, further clarification of interactions between TLR4 and inflammasome pathways may offer new therapeutic targets for OAB associated with chronic inflammation. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e322-e323 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Shinsuke Mizoguchi More articles by this author Kenichi Mori More articles by this author Naoyuki Yamanaka More articles by this author Fuminori Sato More articles by this author Naoki Yoshimura More articles by this author Hiromitsu Mimata More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

TLR4 gene polymorphism associated with lifetime cigarette smoking in bipolar disorder

Bipolar disorder (BD) is associated with an increased risk of tobacco dependence, the leading addictive substance worldwide. Toll-like receptor 4, a sensor of pathogens and of products of cellular stress and damage, is a major innate immune molecule and logical candidate gene for tobacco dependence in BD because (i) the involvement of TLR4 molecules in several substance use disorders has been suggested, (ii) and the association between the TLR4 gene and BD. We analysed herein the potential association between six TLR4 polymorphisms and lifetime tobacco smoking in 514 BD patients. Significant association between tobacco smoking and rs10759932 was found (genotype, Cochrane Armitage trend test, p=0.008, pcorrected=0.040 and alleles, p=0.008; pcorrected=0.040), where the C minor allele is associated with a protective effect, even after adjusting for confounding factors (OR=1.54 [1.04–2.30], p=0.03). Our results suggest that TLR4 gene polymorphism may act as an intermediate factor for the association between tobacco smoking addiction and BD.

Better Management of Alcohol Liver Disease Using a 'Microstructured Synbox' System Comprising L. plantarum and EGCG.

Synergistic combination of probiotics with carbohydrate based prebiotics is widely employed for the treatment of various gut related disorders. However, such carbohydrate based prebiotics encourage the growth of pathogens and probiotics, equally. Aim of the study was (i) to explore the possibility of using epigallocatechin gallate (EGCG) a phenolic compound, as a prebiotic for L.plantarum; (ii) to develop and evaluate a microstructured synbox (microencapsulating both probiotic and EGCG together) in rat model of alcohol liver disease (ALD); and, (iii) to confirm whether the combination can address issues of EGCG bioavailability and probiotic survivability in adverse gut conditions. Growth enhancing effect of EGCG on L. plantarum (12.8±0.5 log 10 units) was significantly (p≤0.05) better than inulin (11.4±0.38 log 10 units), a natural storage carbohydrate. The formulated synbox significantly modulated the levels of alcohol, endotoxin, hepatic enzymes and restored the hepatoarchitecture in comparison to simultaneous administration of free agents. Additionally, using a battery of techniques, levels of various cellular and molecular markers viz. NF-kB/p50, TNF-α, IL12/p40, and signalling molecules TLR4, CD14, MD2, MyD88 and COX-2 were observed to be suppressed. Developed microbead synbox, as a single delivery system for both the agents showed synergism and hence, holds promise as a therapeutic option for ALD management.

Frontline Science: HIV infection of Kupffer cells results in an amplified proinflammatory response to LPS.

End-stage liver disease is a common cause of non-AIDS-related mortality in HIV+ patients, despite effective anti-retroviral therapies (ARTs). HIV-1 infection causes gut CD4 depletion and is thought to contribute to increased gut permeability, bacterial translocation, and immune activation. Microbial products drain from the gut into the liver via the portal vein where Kupffer cells (KCs), the resident liver macrophage, clear translocated microbial products. As bacterial translocation is implicated in fibrogenesis in HIV patients through unclear mechanisms, we tested the hypothesis that HIV infection of KCs alters their response to LPS in a TLR4-dependent manner. We showed that HIV-1 productively infected KCs, enhanced cell-surface TLR4 and CD14 expression, and increased IL-6 and TNF-α expression, which was blocked by a small molecule TLR4 inhibitor. Our study demonstrated that HIV infection sensitizes KCs to the proinflammatory effects of LPS in a TLR4-dependent manner. These findings suggest that HIV-1-infected KCs and their dysregulated innate immune response to LPS may play a role in hepatic inflammation and fibrosis and represent a novel target for therapy.

Novel Toll-like receptor-4 antagonist (+)-naloxone protects mice from inflammation-induced preterm birth.

Toll-like receptor 4 (TLR4) activation by bacterial infection, or by sterile inflammatory insult is a primary trigger of spontaneous preterm birth. Here we utilize mouse models to investigate the efficacy of a novel small molecule TLR4 antagonist, (+)-naloxone, the non-opioid isomer of the opioid receptor antagonist (−)-naloxone, in infection-associated preterm birth. Treatment with (+)-naloxone prevented preterm delivery and alleviated fetal demise in utero elicited by i.p. LPS administration in late gestation. A similar effect with protection from preterm birth and perinatal death, and partial correction of reduced birth weight and postnatal mortality, was conferred by (+)-naloxone administration after intrauterine administration of heat-killed E. coli. Local induction by E. coli of inflammatory cytokine genes Il1b, Il6, Tnf and Il10 in fetal membranes was suppressed by (+)-naloxone, and cytokine expression in the placenta, and uterine myometrium and decidua, was also attenuated. These data demonstrate that inhibition of TLR4 signaling with the novel TLR4 antagonist (+)-naloxone can suppress the inflammatory cascade of preterm parturition, to prevent preterm birth and perinatal death. Further studies are warranted to investigate the utility of small molecule inhibition of TLR-driven inflammation as a component of strategies for fetal protection and delaying preterm birth in the clinical setting.

Toll-like receptor 4 signalling mediates inflammation in skeletal muscle of patients with chronic kidney disease.

BackgroundInflammation in skeletal muscle is implicated in the pathogenesis of insulin resistance and cachexia but why uremia up‐regulates pro‐inflammatory cytokines is unknown. Toll‐like receptors (TLRs) regulate locally the innate immune responses, but it is unknown whether in chronic kidney disease (CKD) TLR4 muscle signalling is altered. The aim of the study is to investigate whether in CKD muscle, TLRs had abnormal function and may be involved in transcription of pro‐inflammatory cytokine.MethodsTLR4, phospho‐p65, phospho‐ikBα, tumour necrosis factor (TNF)‐α, phospho p38, Murf 1, and atrogin were studied in skeletal muscle from nondiabetic CKD stage 5 patients (n = 29) and controls (n = 14) by immunohistochemistry, western blot, and RT–PCR. Muscle cell cultures (C2C12) exposed to uremic serum were employed to study TLR4 expression (western blot and RT–PCR) and TLR‐driven signalling. TLR4 signalling was abrogated by a small molecule chemical inhibitor or TLR4 siRNA. Phospho AKT and phospho p38 were evaluated by western blot.ResultsCKD subjects had elevated TLR4 gene and protein expression. Also expression of NFkB, p38 MAPK and the NFkB‐regulated gene TNF‐α was increased. At multivariate analysis, TLR4 protein content was predicted by eGFR and Subjective Global Assessment, suggesting that the progressive decline in renal function and wasting mediate TLR4 activation. In C2C12, uremic serum increased TLR4 as well as TNF‐α and down‐regulated pAkt. These effects were prevented by blockade of TLR4.ConclusionsCKD promotes muscle inflammation through an up‐regulation of TLR4, which may activate downward inflammatory signals such as TNF‐α and NFkB‐regulated genes.

Stimulation through CD40 and TLR-4 Is an Effective Host Directed Therapy against Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading cause of morbidity and mortality among all infectious diseases. Failure of Bacillus Calmette Guerin as a vaccine and serious side-effects and toxicity due to long-term TB drug regime are the major hurdles associated with TB control. The problem is further compounded by the emergence of drug-resistance strains of Mycobacterium tuberculosis (Mtb). Consequently, it demands a serious attempt to explore safer and superior treatment approaches. Recently, an improved understanding of host–pathogen interaction has opened up new avenues for immunotherapy for treating TB. Although, dendritic cells (DCs) show a profound role in generating immunity against Mtb, their immunotherapeutic potential needs to be precisely investigated in controlling TB. Here, we have devised an approach of bolstering DCs efficacy against Mtb by delivering signals through CD40 and TLR-4 molecules. We found that DCs triggered through CD40 and TLR-4 showed increased secretion of IL-12, IL-6, and TNF-α. It also augmented autophagy. Interestingly, CD40 and TLR-4 stimulation along with the suboptimal dose of anti-TB drugs significantly fortified their efficacy to kill Mtb. Importantly, animals treated with the agonists of CD40 and TLR-4 boosted Th1 and Th17 immunity. Furthermore, it amplified the pool of memory CD4 T cells as well as CD8 T cells. Furthermore, substantial reduction in the bacterial burden in the lungs was observed. Notably, this adjunct therapy employing immunomodulators and chemotherapy can reinvigorate host immunity suppressed due to drugs and Mtb. Moreover, it would strengthen the potency of drugs in curing TB.

HBD-3 regulation of the immune response and the LPS/TLR4-mediated signaling pathway.

The aim of the study was to investigate the mechanisms of human β-defensin 3 (HBD-3) regulation of the immune response and the lipopolysaccharide/Toll-like receptor-4 (LPS/TLR4)-mediated signaling pathway. A TLR4 extracellular gene fragment was cloned into the pET32a plasmid to determine its expression in Escherichia coli (E. coli) and purification. A dialysis labeling method was used to stain HBD-3 with fluorescein isothiocyanate (FITC). FITC-HBD-3 was used to induce the differentiation of human peripheral blood mononuclear cells (MNC) into immature dendritic cells (imDC) in vitro. Binding reactions were established using FITC-HBD-3 and sTLR4 into cell suspensions. Flow cytometry (FCM) was used to analyze the results. Western blot analysis confirmed the identity of nuclear factor-κB (NF-κB) and was used to quantify its nuclear translocation. The results showed that, HBD-3 bound to imDC in a Ca2+-dependent manner, and sTLR4 and LPS competitively inhibited the binding. HBD-3 competitively blocked the binding of LPS and imDC by binding to imDC. HBD-3 significantly decreased the translocation of LPS-induced NF-κB into the nucleus. In conclusion, HBD-3 can competitively inhibit the binding of LPS and imDC through its binding to TLR4 molecules, which are expressed in imDC, thereby preventing LPS from inducing the maturity of the imDCs.

Thrombomodulin Protects Against Bacterial Keratitis, Is Anti-Inflammatory, but Not Angiogenic.

Thrombomodulin (TM) is a multidomain, transmembrane protein with anti-inflammatory properties. Thrombomodulin domain (D) 1 is lectin-like, interacting with Lewis Y antigen on lipopolysaccharide, and with HMGB1, while TMD23 is associated with angiogenic and anti-inflammatory functions. Thus, we tested if TM is protective against Pseudomonas aeruginosa keratitis and whether it enhanced corneal vascularity. Eyes of C57BL/6 (B6) mice were injected with recombinant TM (rTM), rTMD1, or PBS subconjunctivally before and intraperitoneally after infection with P. aeruginosa. Clinical scores, photography with a slit lamp, RT-PCR, ELISA, myeloperoxidase (MPO) assay, viable bacterial plate counts, and India ink perfusion were used to assess the disease response and corneal vascularity (rTM only). Recombinant TM versus PBS treatment reduced clinical scores and corneal opacity. Corneal mRNA levels for HMGB1 were unchanged, but proinflammatory molecules IL-1β, CXCL2, NF-κB, TLR4, and RAGE were decreased; anti-inflammatory molecules SIGIRR and ST2 were increased. ELISA confirmed the mRNA data for HMGB1, IL-1β, and CXCL2 proteins. Both neutrophil influx and viable bacterial plate counts also were decreased after rTM treatment. Protein levels for angiogenic molecules VEGF, VEGFR-1, and VEGFR-2 were measured at 5 days post infection and were not different or reduced significantly after rTM treatment. Further, perfusion with India ink revealed similar vessel ingrowth between the two groups. Similar studies were performed with rTMD1, but disease severity, mRNA, proteins, MPO, and plate counts were not changed from controls. These data provide evidence that rTM treatment is protective against bacterial keratitis, does not reduce HMGB1, and is not angiogenic.

Synthetic and natural small molecule TLR4 antagonists inhibit motoneuron death in cultures from ALS mouse model

Increasing evidence indicates that inflammatory responses could play a critical role in the pathogenesis of motor neuron injury in amyotrophic lateral sclerosis (ALS). Recent findings have underlined the role of Toll-like receptors (TLRs) and the involvement of both the innate and adaptive immune responses in ALS pathogenesis. In particular, abnormal TLR4 signaling in pro-inflammatory microglia cells has been related to motoneuron degeneration leading to ALS. In this study the effect of small molecule TLR4 antagonists on in vitro ALS models has been investigated. Two different types of synthetic glycolipids and the phenol fraction extracted from commercial extra-virgin olive oil (EVOO) were selected since they efficiently inhibit TLR4 stimulus in HEK cells by interacting with the TLR4·MD-2 complex and CD14 co-receptor. Here, TLR4 antagonists efficiently protected motoneurons from LPS-induced lethality in spinal cord cultures, and inhibited the interleukine-1β production by LPS-stimulated microglia. In motoneurons/glia cocultures obtained from wild type or SOD1 G93A mice, motoneuron death induced by SOD1mut glia was counteracted by TLR4 antagonists. The release of nitric oxide by LPS treatment or SOD1mut glia was also inhibited by EVOO, suggesting that the action of this natural extract could be mainly related to the modulation of this inflammatory mediator.

A novel small molecule TLR4 antagonist (IAXO-102) negatively regulates non-hematopoietic toll like receptor 4 signalling and inhibits aortic aneurysms development

ObjectivesThe toll-like receptors (TLRs), including TLR4, have been shown to play a crucial role in vascular inflammatory diseases, such as atherosclerosis and aneurysm. The main goal of this study was to determine the potential of IAXO-102 (Innaxon, Tewkesbury), a novel small molecule TLR4 antagonist, to modulate non-hematopoietic TLR4 proinflammatory signalling and inhibit experimental abdominal aortic aneurysm (AAA) development. MethodsHuman umbilical vein endothelial cells (HUVEC) and Angiotensin II-induced experimental AAA development were our in vitro and in vivo models respectively. Western blotting, antibody array and ELISA approaches were used to explore the effect of IAXO-102 on TLR4 functional activity on two levels: modulation of TLR4-induced mitogen activated protein kinases (MAPK) and p65 NF-kB phosphorylation and expression of TLR4 dependent proinflammatory proteins. ResultsFollowing activation of TLR4, in vitro/in vivo data revealed that IAXO-102 inhibited MAPK and p65 NF-kB phosphorylation associated with down regulation of the expression of TLR4 and TLR4 dependent proinflammatory proteins. Furthermore, IAXO-102 decreased Angiotensin II-induced aortic expansion, rupture and incidence of AAA. ConclusionsThese results demonstrate the ability of IAXO-102 to negatively regulate TLR4 signalling and to inhibit experimental AAA development, suggesting the potential therapeutic use of this TLR4 antagonist for pharmacological intervention of AAA.

Abstract LB-230: SOCS5 mediates defective function of monocyte-derived dendritic cells in patients with chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western countries. This clinically heterogeneous disease is characterized by the progressive accumulation of monoclonal B cells co-expressing CD5, CD19, CD20 and CD23 in the peripheral blood and in the primary and secondary lymphoid organs. Additionally, a range of immune cells are altered in patients with CLL. The resulting immunologic defects predispose patients to severe recurrent infections, which is the major life-threatening complication associated with CLL. Functional impairment of dendritic cells (DCs) in CLL not only diminishes the response to microorganisms, but it also allows for tumor escape from immune control. Understanding the mechanism for this effect can provide insight into the molecular regulation of DC function and may also suggest therapeutic strategies to reverse the immunosuppressive effect of tumors on DCs. To elucidate the mechanism for DC dysfunction in patients with CLL, we focused on signal transduction pathways that regulate the expression of genes necessary for the immune response. We found that monocytes from CLL patients have a decrease in IL-4-induced activation of the transcription factor STAT6, which prevents the phenotypic and functional maturation of DCs. This does not reflect a generalized defect in cytokine-induced signal transduction, as the activation of the related transcription factor STAT5 in response to GM-CSF is unaffected. Monocyte-derived DCs from CLL patients display low levels of HLA-DR, costimulatory molecules and CD83, and reduced secretion of pro-inflammatory cytokines. These changes are associated with low expression of TLR4 and related molecules, which decrease the ability of these cells to respond to stimulus afforded by LPS, impairing their complete maturation. Consequently, monocyte-derived DCs from CLL patients have decreased ability to induce proliferation of T-cells and display an increased induction of immune-suppressive regulatory T cell. Although monocytes from CLL patients exhibit high IL-4R expression, activation of the downstream transcription factor STAT6 is inhibited because of increased expression of the negative regulator SOCS5. It is known that CLL cells produce IL-10, leading to elevated serum levels of this cytokine. IL-10-treatment of monocytes from healthy donors mimics the alteration in signaling observed in patients, through enhanced STAT3-dependent expression of SOCS5, which inhibits STAT6 activation and leads to defective DC differentiation. These findings suggest that SOCS5 can mediate the impaired function of DCs in CLL patients, and may be a new potential therapeutic target for reversing cancer-associated immune suppression. Note: This abstract was not presented at the meeting. Citation Format: Patricia A. Toniolo, Suhu Liu, Sarah R. Walker, Jose Alexandre M. Barbuto, David A. Frank. SOCS5 mediates defective function of monocyte-derived dendritic cells in patients with chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-230. doi:10.1158/1538-7445.AM2015-LB-230

A novel small mimetic molecule TLR4 antagonist (IAXO-102) modulates TLR4 proinflammatory signalling and inhibits aortic aneurysms development

A novel small mimetic molecule TLR4 antagonist (IAXO-102) modulates TLR4 proinflammatory signalling and inhibits aortic aneurysms development

Alkaline phosphatase protects lipopolysaccharide-induced early pregnancy defects in mice.

Excessive cytokine inflammatory response due to chronic or superphysiological level of microbial infection during pregnancy leads to pregnancy complications such as early pregnancy defects/loss and preterm birth. Bacterial toxin lipopolysaccharide (LPS), long recognized as a potent proinflammatory mediator, has been identified as a risk factor for pregnancy complications. Alkaline phosphatase (AP) isozymes have been shown to detoxify LPS by dephosphorylation. In this study, we examined the role of alkaline phosphatase (AP) in mitigating LPS-induced early pregnancy complications in mice. We found that 1) the uterus prior to implantation and implantation sites following embryo implantation produce LPS recognition and dephosphorylation molecules TLR4 and tissue non-specific AP (TNAP) isozyme, respectively; 2) uterine TNAP isozyme dephosphorylates LPS at its sites of production; 3) while LPS administration following embryo implantation elicits proinflammatory cytokine mRNA levels at the embryo implantation sites (EISs) and causes early pregnancy loss, dephosphorylated LPS neither triggers proinflammatory cytokine mRNA levels at the EISs nor induces pregnancy complications; 4) AP isozyme supplementation to accelerate LPS detoxification attenuates LPS-induced pregnancy complications following embryo implantation. These findings suggest that a LPS dephosphorylation strategy using AP isozyme may have a unique therapeutic potential to mitigate LPS- or Gram-negative bacteria-induced pregnancy complications in at-risk women.

Angiotensin‐II Induced Hypertensive Renal Injury is Mediated through HMGB1‐TLR4 Signaling in Rat Tubulo‐Epithelial Cells

Angiotensin II is a vaso-constrictive peptide that regulates blood pressure homeostasis. Even though the inflammatory effects of AngII in renal pathophysiology have been studied, there still exists a paucity of data with regard to the mechanism of action of AngII-mediated kidney injury. The objective of this study was to elucidate the mechanistic role of HMGB1-TLR4 signaling in AngII-induced inflammation in the kidney. Rat tubular epithelial cells (NRK52E) were treated with AngII over a preset time-course. In another set of experiments, HMGB1 was neutralized and TLR4 was knocked down using small interfering RNA targeting TLR4. Cell extracts were subjected to RT-PCR, immunoblotting, flow cytometry, ELISA. AngII induced inflammation in NRK52E cells increased gene and protein expression of TLR4, HMGB1 and proinflammatory cytokines. Pretreatment with Losartan attenuated the AngII-induced expression of TLR4 and inflammatory molecules. TLR4 silencing was used to elucidate the specific role played by TLR4 in Ang...

Angiotensin II‐Induced TLR4 Mediated Abdominal Aortic Aneurysm formation in ApoE‐/‐ mice is Dependent on STAT3

Abdominal Aortic Aneurysm (AAA) is a major cause of mortality and morbidity in older men. Angiotensin II (ANGII) treatment of ApoE-/-mice is a model for the study of the pathogenesis and treatment of AAA. Macrophage infiltration has been shown to play a role in the AAA formation. Here we demonstrate that ANGII mediates an increase in the phosphorylation of STAT3 in the suprarenal aorta from ApoE-/- mice.The pSTAT3 inhibitor S3I-201 decreased the incidence of AAA from 80% to 40% in these mice. A time course of the development of AAAs demonstrated that 7-10 days after initiation of ANG II infusion, macrophage infiltration increased 3 fold compared to control and the number of macrophages with the M1 proinflammatory phenotype determined by FACS analysis of CD11c+ cells had increased 3 fold, with no change in the M2 CD206+ “healing” macrophages. Interestingly only 2 of 16 mice had developed AAAs at this time point. At day 28 of ANG II infusion 80% of 19 mice had developed AAAs, 67% of which demonstrated multiple clots and complex aneurysms. In these mice macrophages had increased 6 fold over control in extracts of the suprarenal aorta and the number of M1 macrophages had increased 6 fold by FACS and qPCR, while the number of M2 macrophages had decreased by 50% (N=6,n p<0.008). Treatment of mice with S3I-201 reversed the ratio of M1 to M2 macrophages by 50%. In ApoE-/- TLR4-/- or in ApoE-/- mice treated with ANGII and/or the small molecule TLR4 inhibitor Eritoran, levels of AAA formation, M1 macrophages and pSTAT3 levels decreased by 50%. These data support a role of pSTAT3 in TLR4 dependent AAA formation and a possible therapeutic role for TLR4 inhibition in AAA.

Decreased expression of heat shock proteins may lead to compromised wound healing in type 2 diabetes mellitus patients

BackgroundHeat shock proteins (HSPs) are inducible stress proteins expressed in cells exposed to stress. HSPs promote wound healing by recruitment of dermal fibroblasts to the site of injury and bring about protein homeostasis. Diabetic wounds are hard to heal and inadequate HSPs may be important contributors in the etiology of diabetic foot ulcers (DFU). ObjectiveTo analyze the differential expression of HSPs and their downstream molecules in human diabetic wounds compared to control wounds. MethodsExpressional levels of HSP27, HSP47 and HSP70 and their downstream molecules like TLR4, p38-MAPK were seen in biopsies from 101 human diabetic wounds compared to 8 control subjects without diabetes using RT-PCR, western blot and immunohistochemistry. ResultsOur study suggested a significant down regulation of HSP70, HSP47 and HSP27 (p value=<0.001 for HSP70; p value=0.007 for HSP47; p value=0.007 for HSP27) in DFU along with their downstream molecules TLR4 and p38-MAPK (p value=0.006 for p38-MAPK; p value=0.02 for TLR4). HSP70 levels were significantly lower in male subjects and their levels increased significantly with the grades of wound on Wagner’s scale. Infection status of the wounds was found to be significantly associated with the increased levels of HSP70 and HSP27 in infected diabetic wounds. ConclusionsOur study demonstrates that the down regulation of HSPs in diabetic wounds is associated with wound healing impairment in T2DM subjects.

Expression profiling and functional analysis of Toll-like receptors in primary healthy human nasal epithelial cells shows no correlation and a refractory LPS response.

BackgroundInnate immune recognition via Toll-like receptors (TLRs) on barrier cells like epithelial cells has been shown to influence the regulation of local immune responses. Here we determine expression level variations and functionality of TLRs in nasal epithelial cells from healthy donors.MethodsExpression levels of the different TLRs on primary nasal epithelial cells from healthy donors derived from inferior turbinates was determined by RT-PCR. Functionality of the TLRs was determined by stimulation with the respective ligand and evaluation of released mediators by Luminex ELISA.ResultsPrimary nasal epithelial cells express different levels of TLR1-6 and TLR9. We were unable to detect mRNA of TLR7, TLR8 and TLR10. Stimulation with Poly(I:C) resulted in a significant increased secretion of IL-4, IL-6, RANTES, IP-10, MIP-1β, VEGF, FGF, IL-1RA, IL-2R and G-CSF. Stimulation with PGN only resulted in significant increased production of IL-6, VEGF and IL-1RA. Although the expression of TLR4 and co-stimulatory molecules could be confirmed, primary nasal epithelial cells appeared to be unresponsive to stimulation with LPS. Furthermore, we observed huge individual differences in TLR agonist-induced mediator release, which did not correlate with the respective expression of TLRs.ConclusionOur data suggest that nasal epithelium seems to have developed a delicate system of discrimination and recognition of microbial patterns. Hypo-responsiveness to LPS could provide a mechanism to dampen the inflammatory response in the nasal mucosa in order to avoid a chronic inflammatory response. Individual, differential expression of TLRs on epithelial cells and functionality in terms of released mediators might be a crucial factor in explaining why some people develop allergies to common inhaled antigens, and others do not.