HMGB‐1 induces c‐kit+ cell microvascular rolling and adhesion via both toll‐like receptor‐2 and toll‐like receptor‐4 of endothelial cells

High-mobility group box 1 (HMGB-1) is a strong chemo-attractive signal for both inflammatory and stem cells. The aim of this study is to evaluate the mechanisms regulating HMGB-1–mediated adhesion and rolling of c-kit⁺ cells and assess whether toll-like receptor-2 (TLR-2) and toll-like receptor-4 (TLR-4) of endothelial cells or c-kit⁺ cells are implicated in the activation of downstream migration signals to peripheral c-kit⁺ cells. Effects of HMGB-1 on the c-kit⁺ cells/endothelial interaction were evaluated by a cremaster muscle model in wild-type (WT), TLR-2 (−/−) and Tlr4 (LPS-del) mice. The mRNA and protein expression levels of endothelial nitric oxide synthase were determined by quantitative real-time PCR and immunofluorescence staining. Induction of crucial adhesion molecules for rolling and adhesion of stem cells and leukocytes were monitored in vivo and in vitro. Following local HMGB-1 administration, a significant increase in cell rolling was detected (32.4 ± 7.1% in ‘WT’ versus 9.9 ± 3.2% in ‘control’, P < 0.05). The number of firmly adherent c-kit⁺ cells was more than 13-fold higher than that of the control group (14.6 ± 5.1 cells/mm² in ‘WT’ versus 1.1 ± 1.0 cells/mm² in ‘control’, P < 0.05). In knockout animals, the fraction of rolling cells did not differ significantly from control levels. Firm endothelial adhesion was significantly reduced in TLR-2 (−/−) and Tlr4 (LPS-del) mice compared to WT mice (1.5 ± 1.4 cells/mm² in ‘TLR-2 (−/−)’ and 2.4 ± 1.4 cells/mm² in ‘Tlr4 (LPS-del)’ versus 14.6 ± 5.1 cells/mm² in ‘WT’, P < 0.05). TLR-2 (−/−) and Tlr4 (LPS-del) stem cells in WT mice did not show significant reduction in rolling and adhesion compared to WT cells. HMGB-1 mediates c-kit⁺ cell recruitment via endothelial TLR-2 and TLR-4.     

Ischemia-Reperfusion Lung Injury Is Attenuated in MyD88-Deficient Mice

Ischemia-reperfusion lung injury is a common cause of acute morbidity and mortality in lung transplant recipients and has been associated with subsequent development of bronchiolitis obliterans syndrome. Recognition of endogenous ligands released during cellular injury (damage-associated molecular patterns; DAMPs) by Toll-like receptors (TLRs), especially TLR4, has increasingly been recognized as a mechanism for inflammation resulting from tissue damage. TLR4 is implicated in the pathogenesis of ischemia-reperfusion injury of multiple organs including heart, liver, kidney and lung. Additionally, activation of TLRs other than TLR4 by DAMPs has been identified in tissues other than the lung. Because all known TLRs, with the exception of TLR3, signal via the MyD88 adapter protein, we hypothesized that lung ischemia-reperfusion injury was mediated by MyD88-dependent signaling. To test this hypothesis, we subjected C57BL/6 wildtype, Myd88 ^-/-, and Tlr4 ^-/- mice to 1 hr of left lung warm ischemia followed by 4 hr of reperfusion. We found that Myd88 ^-/- mice had significantly less MCP-1/CCL2 in the left lung following ischemia-reperfusion as compared with wildtype mice. This difference was associated with dramatically reduced lung permeability. Interestingly, Tlr4 ^-/- mice had only partial protection from ischemia-reperfusion as compared to Myd88 ^-/- mice, implicating other MyD88-dependent pathways in lung injury following ischemia-reperfusion. We also found that left lung ischemia-reperfusion caused remote inflammation in the right lung. Finally, using chimeric mice with MyD88 expression restricted to either myeloid or non-myeloid cells, we found that MyD88-dependent signaling in myeloid cells was necessary for ischemia-reperfusion induced lung permeability. We conclude that MyD88-dependent signaling through multiple receptors is important in the pathogenesis of acute lung inflammation and injury following ischemia and reperfusion.     

Toll-Like Receptor 4 Mediates the Response of Epithelial and Stromal Cells to Lipopolysaccharide in the Endometrium

Background

Ascending infections of the female genital tract with bacteria causes pelvic inflammatory disease (PID), preterm labour and infertility. Lipopolysaccharide (LPS) is the main component of the cell wall of Gram-negative bacteria. Innate immunity relies on the detection of LPS by Toll-like receptor 4 (TLR4) on host cells. Binding of LPS to TLR4 on immune cells stimulates secretion of pro-inflammatory cytokines such as IL-6, chemokines such as CXCL1 and CCL20, and prostaglandin E₂. The present study tested the hypothesis that TLR4 on endometrial epithelial and stromal cells is essential for the innate immune response to LPS in the female genital tract.

Methodology/Principal Findings

Wild type (WT) mice expressed TLR4 in the endometrium. Intrauterine infusion of purified LPS caused pelvic inflammatory disease, with accumulation of granulocytes throughout the endometrium of WT but not Tlr4^−/− mice. Intra-peritoneal infusion of LPS did not cause PID in WT or Tlr4^−/− mice, indicating the importance of TLR4 in the endometrium for the detection of LPS in the female genital tract. Stromal and epithelial cells isolated from the endometrium of WT but not Tlr4^−/− mice, secreted IL-6, CXCL1, CCL20 and prostaglandin E₂ in response to LPS, in a concentration and time dependent manner. Co-culture of combinations of stromal and epithelial cells from WT and Tlr4^−/− mice provided little evidence of stromal-epithelial interactions in the response to LPS.

Conclusions/Significance

The innate immune response to LPS in the female genital tract is dependent on TLR4 on the epithelial and stromal cells of the endometrium.     

De novo expression of circulating biglycan evokes an innate inflammatory tissue response via MyD88/TRIF pathways

Matrix-bound constituents, such as the small leucine-rich proteoglycan biglycan, can act as powerful signaling molecules when released by limited proteolysis of the extracellular matrix or de novo synthesized by macrophages in the circulation and body fluids. Specifically, biglycan acts as an endogenous ligand of innate immunity by directly engaging the Toll-like receptor (TLR)-2 and -4. In this study, we generated a transient transgenic mouse model where biglycan was de novo overproduced by hepatocytes driven by the albumin promoter. Transgenic biglycan was rapidly and abundantly synthesized by hepatocytes and released into the bloodstream. Notably, we found that circulating biglycan accumulated in the kidneys where it caused recruitment of leukocytes infiltrating the renal parenchyma concurrent with abnormal renal levels of chemoattractants CXCL1, CXCL2, CCL2 and CCL5. Using mice deficient in either TLR adapter proteins MyD88 or TRIF we discovered that MyD88 deficiency drastically reduced neutrophil and macrophage infiltration in the kidney, whereas TRIF deficiency decreased T cell infiltrates. Production of CXCL1, CXCL2 and CCL2 required MyD88, whereas the levels of T cell and macrophage attractant CCL5 required TRIF. Thus, we provide robust genetic evidence for circulating biglycan as a powerful pro-inflammatory mediator targeting the renal parenchyma. Furthermore, our results provide the first evidence that biglycan differentially triggers chemoattraction of leukocytes via two independent pathways, both under the control of TLR2/4, utilizing either MyD88 or TRIF adaptor proteins. As aberrant expression of biglycan occurs in several inflammatory diseases, this transient transgenic mouse model could serve as a valuable research tool in investigating the effects of increased biglycan expression in vivo and for the development of therapeutic strategies in the treatment of inflammatory diseases.     

Polysaccharides from Dicliptera chinensis ameliorate liver disturbance by regulating TLR-4/NF-κB and AMPK/Nrf2 signalling pathways

The purpose of this study was to alleviate liver disturbance by applying polysaccharides from Dicliptera chinensis (DCP) to act on the adenosine monophosphate–activated protein kinase/ nuclear factor erythroid 2-related factor 2 (AMPK/ Nrf2) oxidative stress pathway and the Toll-like receptor 4 (TLR-4)/ nuclear factor kappa-B (NF-κB) inflammatory pathway and to establish an in vivo liver disturbance model using male C57BL/6J and TLR-4 knockout (^−/−) mice. For this, we evaluated the expression levels of SREBP-1 and Nrf2 after silencing the expression of AMPK using siRNA technology. Our results show that with regard to the TLR-4/ NF-κB inflammatory pathway, DCP inhibits TLR-4, up-regulates the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), reduces the expression of phospho(p)-NF-κB and leads to the reduction of downstream inflammatory factors, such as tumour necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β, thereby inhibiting the inflammatory response. Regarding the AMPK/ Nrf2 oxidative stress pathway, DCP up-regulates the expression of p-AMPK and Nrf2, in addition to regulating glucose and lipid metabolism, oxidative stress and ameliorating liver disturbance symptoms. In summary, our study shows that DCP alleviates liver disturbances by inhibiting mechanisms used during liver inflammation and oxidative stress depression, which provides a new strategy for the clinical treatment of liver disturbance.     

Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4^−/−Abca4^−/−Rdh8^−/− mice displayed milder retinal degenerative phenotypes than Abca4^−/−Rdh8^−/− mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.     

Cinnamon extract reduces symptoms,inflammatory mediators and mast cell markers in murine IL-10−/− colitis

Inflammatory bowel disease (IBD) shows an increasing prevalence and harm in western countries. Conventional therapies are associated with bad compliance and adverse side effects. Natural substances like cinnamon extract (CE) could be an additional therapy. We found recently that CE acts anti-inflammatory on mast cells — discussed of being relevant in IBD. Here, we analysed the effects of CE on murine IL-10^−/− colitis as model for IBD. Mice were treated 12 weeks with or without CE in drinking water. Clinical scores and disease activity index were assessed. Colonic tissue samples were analysed for infiltration, tissue damage, bowel wall thickness, expression of pro-inflammatory mediators, mast cell proteases, tight junction proteins, and NF-κB signaling. Following treatment with CE, symptoms of murine colitis as well as increased infiltration of immune cells, tissue damage and bowel wall thickness in colon tissue of IL-10^−/− mice were diminished significantly. MIP-2, TNF, IFNγ, CCL2, CCL3, CCL4 and IL-1β as well as MC-CPA, MCP-1 and MCP-4 were strongly upregulated in IL-10^−/− mice compared to WT, but noteworthy not in CE group. Expression of tight junction proteins was not influenced by CE. Phosphorylation of IκB was slightly down-regulated in CE treated IL-10^−/− mice compared to IL-10^−/− controls. In summary, CE decreases inflammatory symptoms and expression of inflammatory markers in murine IL-10^−/− colitis. CE has no influence on tight junction proteins, but seems acting via reducing pro-inflammatory mediators and recruitment of neutrophil granulocytes probably by inhibiting NF-κB signaling.     

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

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

Prevention of renal apoB retention is protective against diabetic nephropathy: role of TGF-β inhibition

Animal studies demonstrate that hyperlipidemia and renal lipid accumulation contribute to the pathogenesis of diabetic nephropathy (DN). We previously demonstrated that renal lipoproteins colocalize with biglycan, a renal proteoglycan. The purpose of this study was to determine whether prevention of renal lipid (apoB) accumulation attenuates DN. Biglycan-deficient and biglycan wild-type Ldlr^−/− mice were made diabetic via streptozotocin and fed a high cholesterol diet. As biglycan deficiency is associated with elevated transforming growth factor-β (TGF-β), in some experiments mice were injected with either the TGF-β-neutralizing antibody, 1D11, or with 13C4, an irrelevant control antibody. Biglycan deficiency had no significant effect on renal apoB accumulation, but led to modest attenuation of DN with ∼30% reduction in albuminuria; however, biglycan deficiency caused a striking elevation in TGF-β. Use of 1D11 led to sustained suppression of TGF-β for approximately 8 weeks at a time. The 1D11 treatment caused decreased renal apoB accumulation, decreased albuminuria, decreased renal hypertrophy, and improved survival, compared with the 13C4 treatment. Thus, prevention of renal apoB accumulation is protective against development of DN. Furthermore, this study demonstrates that prevention of renal apoB accumulation is a mechanism by which TGF-β inhibition is nephroprotective.     

Toll-like receptor 3 plays a role in myocardial infarction and ischemia/reperfusion injury

Innate immune and inflammatory responses mediated by Toll like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. This study examined the role of TLR3 in myocardial injury induced by two models, namely, myocardial infarction (MI) and I/R. First, we examined the role of TLR3 in MI. TLR3 deficient (TLR3^−/−) and wild type (WT) mice were subjected to MI induced by permanent ligation of the left anterior descending (LAD) coronary artery for 21 days. Cardiac function was measured by echocardiography. Next, we examined whether TLR3 contributes to myocardial I/R injury. TLR3^−/− and WT mice were subjected to myocardial ischemia (45 min) followed by reperfusion for up to 3 days. Cardiac function and myocardial infarct size were examined. We also examined the effect of TLR3 deficiency on I/R-induced myocardial apoptosis and inflammatory cytokine production. TLR3^−/− mice showed significant attenuation of cardiac dysfunction after MI or I/R. Myocardial infarct size and myocardial apoptosis induced by I/R injury were significantly attenuated in TLR3^−/− mice. TLR3 deficiency increases B-cell lymphoma 2 (BCL2) levels and attenuates I/R-increased Fas, Fas ligand or CD95L (FasL), Fas-Associated protein with Death Domain (FADD), Bax and Bak levels in the myocardium. TLR3 deficiency also attenuates I/R-induced myocardial nuclear factor KappaB (NF-κB) binding activity, Tumor necrosis factor alpha (TNF-α) and Interleukin-1 beta (IL-1β) production as well as I/R-induced infiltration of neutrophils and macrophages into the myocardium. TLR3 plays an important role in myocardial injury induced by MI or I/R. The mechanisms involve activation of apoptotic signaling and NF-κB binding activity. Modulation of TLR3 may be an effective approach for ameliorating heart injury in heart attack patients.     

Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice

Background

Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model.

Results

The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 μg LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-μg LPS injection in Tlr4^-/- mice. When the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4^-/- mice was 30%. In the Tlr4^-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 μg LPS led to a significant induction in O₂^- detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4^-/- mice. Addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 only significantly increased the O₂^- level in the lung and liver of the Tlr4^-/- mice but not in the C57BL/6 mice following 500-μg LPS injection. In addition, the serum IL-1β and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4^-/- mice. Notably, IL-1β and IL-2 were significantly increased in Tlr4^-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 prior to LPS injection.

Conclusions

In this study, we demonstrate that innate resistance to LPS toxicity in Tlr4^-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O₂^- and inflammatory cytokines.     

Collagen receptors integrin alpha2beta1 and discoidin domain receptor 1 regulate maturation of the glomerular basement membrane and loss of integrin alpha2beta1 delays kidney fibrosis in COL4A3 knockout mice

Maturation of the glomerular basement membrane (GBM) is essential for maintaining the integrity of the renal filtration barrier. Impaired maturation causes proteinuria and renal fibrosis in the type IV collagen disease Alport syndrome. This study evaluates the role of collagen receptors in maturation of the GBM, matrix accumulation and renal fibrosis by using mice deficient for discoidin domain receptor 1 (DDR1), integrin subunit α2 (ITGA2), and type IV collagen α3 (COL4A3). Loss of both collagen receptors DDR1 and integrin α2β1 delays maturation of the GBM: due to a porous GBM filtration barrier high molecular weight proteinuria that more than doubles between day 60 and day 100. Thereafter, maturation of the GBM causes proteinuria to drop down to one tenth until day 200. Proteinuria and the porous GBM cause accumulation of glomerular and tubulointerstitial matrix, which both decrease significantly after GBM-maturation until day 250. In parallel, in a disease with impaired GBM-maturation such as Alport syndrome, loss of integrin α2β1 positively delays renal fibrosis: COL4A3^−/−/ITGA2^−/− double knockouts exhibited reduced proteinuria and urea nitrogen compared to COL4A3^−/−/ITGA2^+/− and COL4A3^−/−/ITGA2^+/+ mice. The double knockouts lived 20% longer and showed less glomerular and tubulointerstitial extracellular matrix deposition than the COL4A3^−/− Alport mice with normal integrin α2β1 expression. Electron microscopy illustrated improvements in the glomerular basement membrane structure. MMP2, MMP9, MMP12 and TIMP1 were expressed at significantly higher levels (compared to wild-type mice) in COL4A3^−/−/ITGA2^+/+ Alport mice, but not in COL4A3^+/+/ITGA2^−/− mice. In conclusion, the collagen receptors DDR1 and integrin α2β1 contribute to regulate GBM-maturation and to control matrix accumulation. As demonstrated in the type IV collagen disease Alport syndrome, glomerular cell–matrix interactions via collagen receptors play an important role in the progression of renal fibrosis.     

Biglycan and decorin differentially regulate signaling in the fetal membranes

Preterm birth is the leading cause of newborn mortality in the United States and about one third of cases are caused by preterm premature rupture of fetal membranes, a complication that is frequently observed in patients with Ehlers–Danlos Syndrome. Notably, a subtype of Ehlers–Danlos Syndrome is caused by expression of abnormal biglycan and decorin proteoglycans. As compound deficiency of these two small leucine-rich proteoglycans is a model of preterm birth, we investigated the fetal membranes of Bgn^−/−; Dcn^−/− double-null and single-null mice. Our results showed that biglycan signaling supported fetal membrane remodeling during early gestation in the absence of concomitant changes in TGFβ levels. In late gestation, biglycan signaling acted in a TGFβ-dependent manner to aid in membrane stabilization. In contrast, decorin signaling supported fetal membrane remodeling at early stages of gestation in a TGFβ-dependent manner, and fetal membrane stabilization at later stages of gestation without changes in TGFβ levels. Furthermore, exogenous soluble decorin was capable of rescuing the TGFβ signaling pathway in fetal membrane mesenchymal cells. Collectively, these findings provide novel targets for manipulation of fetal membrane extracellular matrix stability and could represent novel targets for research on preventive strategies for preterm premature rupture of fetal membranes.     

Age-Dependent TLR3 Expression of the Intestinal Epithelium Contributes to Rotavirus Susceptibility

Rotavirus is a major cause of diarrhea worldwide and exhibits a pronounced small intestinal epithelial cell (IEC) tropism. Both human infants and neonatal mice are highly susceptible, whereas adult individuals remain asymptomatic and shed only low numbers of viral particles. Here we investigated age-dependent mechanisms of the intestinal epithelial innate immune response to rotavirus infection in an oral mouse infection model. Expression of the innate immune receptor for viral dsRNA, Toll-like receptor (Tlr) 3 was low in the epithelium of suckling mice but strongly increased during the postnatal period inversely correlating with rotavirus susceptibility, viral shedding and histological damage. Adult mice deficient in Tlr3 (Tlr3^−/−) or the adaptor molecule Trif (Trif^Lps2/Lps2) exerted significantly higher viral shedding and decreased epithelial expression of proinflammatory and antiviral genes as compared to wild-type animals. In contrast, neonatal mice deficient in Tlr3 or Trif did not display impaired cell stimulation or enhanced rotavirus susceptibility. Using chimeric mice, a major contribution of the non-hematopoietic cell compartment in the Trif-mediated antiviral host response was detected in adult animals. Finally, a significant age-dependent increase of TLR3 expression was also detected in human small intestinal biopsies. Thus, upregulation of epithelial TLR3 expression during infancy might contribute to the age-dependent susceptibility to rotavirus infection.     

Burn-induced alterations in toll-like receptor-mediated responses by bronchoalveolar lavage cells

Burn is associated with profound inflammation and activation of the innate immune system in multiple organ beds, including the lung. Similarly, toll-like receptors (TLR) are associated with innate immune activation. Nonetheless, it is unclear what impact burn has on TLR-induced inflammatory responses in the lung.MethodsMale C57BL/6 mice were subjected to burn (3rd degree, 25% TBSA) or sham procedure and 1, 3 or 7 days thereafter, bronchoalveolar lavage (BAL) fluid was collected and cells were isolated and cultured in vitro with specific TLR agonists as follows: Zymosan (TLR-2), LPS (TLR-4) and CpG-ODN (TLR-9). Supernatants were collected 48 h later and assayed for inflammatory cytokine levels (IL-1β, IL-6, IL-10, IL-17, TNF-α, KC, MCP-1, MIP-1α, MIP-1β and RANTES) by Bioplex.ResultsBAL fluid from sham and burn mice did not contain detectable cytokine levels. BAL cells, irrespective of injury, were responsive to TLR-2 and TLR-4 activation. Seven days after burn, TLR-2 and TLR-4 mediated responses by BAL cells were enhanced as evidenced by increased production of IL-6, IL-17, TNF-α, MCP-1, MIP-1β and RANTES.ConclusionsBurn-induced changes in TLR-2 and TLR-4 reactivity may contribute to the development of post-burn complications, such as acute lung injury (ALI) and adult respiratory distress syndrome (ARDS).     

Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS^−/−) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS^−/− mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS^−/− mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS^−/− mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS^−/− mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-γ and TNF-α) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis.     

Tlr4 Deficiency Protects against Cardiac Pressure Overload Induced Hyperinflammation

Transverse aortic constriction provokes a pro-inflammatory reaction and results in cardiac hypertrophy. Endogenous ligands contribute to cardiac hypertrophy via toll-like receptor (TLR)-4 binding. A lack of TLR4 signaling diminishes hypertrophy and inflammation. Wild type mice undergoing aortic constriction respond to a lipopolysaccharide second-hit stimulus with hyperinflammation. The objective of this study was to assess whether other second-hit challenges utilizing TLR ligands provoke a comparable inflammatory reaction, and to find out whether this response is absent in TLR4 deficient mice. Assuming that cardiac stress alters the expression of pattern recognition receptors we analyzed the effects of transverse aortic constriction and second-hit virulence factor treatment on TLR expression, as well as cytokine regulation. Wild type and Tlr4 ^-/- mice were subjected to three days of TAC and subsequently confronted with gram-positive TLR2 ligand lipoteichoic acid (LTA, 15mg/g bodyweight) or synthetic CpG-oligodesoxynucleotide 1668 thioate (20 nmol/kg bodyweight, 30 min after D-galactosamin desensitization) signaling via TLR9. Hemodynamic measurements and organ preservation were performed 6 h after stimulation. Indeed, the study revealed a robust enhancement of LTA induced pattern recognition receptor and cytokine mRNA expression and a LTA-dependent reduction of hemodynamic pressure in TAC wild type mice. Second-Hit treatment with CpG-ODNs led to similar results. However, second-hit effects were abolished in Tlr4 ^-/- mice. In total, these data indicate for the first time that cardiac stress increases the inflammatory response towards both, gram-negative and gram-positive, TLR ligands as well as bacterial DNA. The decrease of the inflammatory response upon TLR2 and -9 ligand challenge in TAC Tlr4 ^-/- mice demonstrates that a lack of TLR4 signaling does not only prevent left ventricular hypertrophy but also protects the mice from a cardiac stress induced hyperinflammatory reaction.     

Interclass small leucine-rich repeat proteoglycan interactions regulate collagen fibrillogenesis and corneal stromal assembly

The corneal stroma is enriched in small leucine-rich proteoglycans (SLRPs), including both class I (decorin and biglycan) and class II (lumican, keratocan and fibromodulin). Transparency is dependent on the assembly and maintenance of a hierarchical stromal organization and SLRPs are critical regulatory molecules. We hypothesize that cooperative interclass SLRP interactions are involved in the regulation of stromal matrix assembly. We test this hypothesis using a compound Bgn^−/0/Lum^−/− mouse model and single Lum^−/− or Bgn^−/0 mouse models and wild type controls. SLRP expression was investigated using immuno-localization and immuno-blots. Structural relationships were defined using ultrastructural and morphometric approaches while transparency was analyzed using in vivo confocal microscopy. The compound Bgn^−/0/Lum^−/− corneas demonstrated gross opacity that was not seen in the Bgn^−/0 or wild type corneas and greater than that in the Lum^−/− mice. The Bgn^−/0/Lum^−/− corneas exhibited significantly increased opacity throughout the stroma compared to posterior opacity in the Lum^−/− and no opacity in Bgn^−/0 or wild type corneas. In the Bgn^−/0/Lum^−/− corneas there were abnormal lamellar and fibril structures consistent with the functional deficit in transparency. Lamellar structure was disrupted across the stroma with disorganized fibrils, and altered fibril packing. In addition, fibrils had larger and more heterogeneous diameters with an abnormal structure consistent with abnormal fibril growth. This was not observed in the Bgn^−/0 or wild type corneas and was restricted to the posterior stroma in Lum^−/− mice. The data demonstrate synergistic interclass regulatory interactions between lumican and biglycan. These interactions are involved in regulating both lamellar structure as well as collagen fibrillogenesis and therefore, corneal transparency.     

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

Background

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

Methodology/Principal Findings

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

Conclusions/Significance

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

Bmi1 regulate tooth and mandible development by inhibiting p16 signal pathway

To determine whether the deletion of p16 can correct tooth and mandible growth retardation caused by Bmi1 deficiency, we compared the tooth and mandible phenotypes of homozygous p16-deficient (p16^−/−) mice, homozygous Bmi1-deficient (Bmi1^−/−) mice, double homozygous Bmi1 and p16-deficient (Bmi1^−/−p16^−/−) mice to those of their wild-type littermates at 4 weeks of age by radiograph, histochemistry and immunohistochemistry. Results showed that compared to Bmi1^−/− mice, the dental mineral density, dental volume and dentin sialoprotein immunopositive areas were increased, whereas the ratio of the predentin area to total dentin area and that of biglycan immunopositive area to dentin area were decreased in Bmi1^−/−p16^−/− mice. These results indicate that the deletion of p16 can improve tooth development in Bmi1 knockout mice. Compared to Bmi1^−/− mice, the mandible mineral density, cortical thickness, alveolar bone volume, osteoblast number and activity, alkaline phosphatase positive area were all increased significantly in Bmi1^−/−p16^−/− mice. These results indicate that the deletion of p16 can improve mandible growth in Bmi1 knockout mice. Furthermore, the protein expression levels of cyclin D, CDK4 and p53 were increased significantly in p16^−/− mice compared with those from wild-type mice; the protein expression levels of cyclin D and CDK4 were decreased significantly, whereas those of p27 and p53 were increased significantly in Bmi1^−/− mice; these parameters were partly rescued in Bmi1^−/−p16^−/− mice compared with those from Bmi1^−/− mice. Therefore, our results indicate that Bmi1 plays roles in regulating tooth and mandible development by inhibiting p16 signal pathway which initiated entry into cell cycle.