Toll-like receptor 3 expression in myeloid cells is essential for efficient regeneration after acute pancreatitis in mice

Stringent regulation of the inflammatory response is crucial for normal tissue regeneration. Here, we analyzed the role of Toll-like receptor 3 (TLR3) in pancreatic regeneration after acute pancreatitis (AP). AP was induced by caerulein treatment in mice with global TLR3 deficiency (TLR3^OFF) or in mice re-expressing TLR3 exclusively in the myeloid cell lineage (TLR3^Mye). Compared to WT mice, TLR3^OFF mice had a markedly increased formation of acinar-to-ductal metaplasia (ADM) that persisted until day 7 after initiation of AP. Pancreatic tissue of WT mice was completely regenerated after 5 days with no detectable ADM structures. The enhancing effect of TLR3-deficiency on ADM formation was closely linked with an increased and prolonged accumulation of macrophages in pancreata of TLR3^OFF mice. Importantly, the phenotype of TLR3^OFF mice was rescued in TLR3^Mye mice, demonstrating the causative role of myeloid cell selective TLR3 signaling. Moreover, in vitro stimulation of macrophages through TLR3 initiated cell death by a caspase-8-associated mechanism. Therefore, these findings provide evidence that TLR3 signaling in myeloid cells is sufficient to limit inflammation and ADM formation and to promote regeneration after AP. Notably, resolution of inflammation after AP was associated with macrophage sensitivity to TLR3-mediated cell death.     

DC‐SIGN reacts with TLR‐4 and regulates inflammatory cytokine expression via NF‐κB activation in renal tubular epithelial cells during acute renal injury

In the pathological process of acute kidney injury (AKI), innate immune receptors are essential in inflammatory response modulation; however, the precise molecular mechanisms are still unclear. Our study sought to demonstrate the inflammatory response mechanisms in renal tubular epithelial cells via Toll‐like receptor‐4 (TLR‐4) and dendritic cell‐specific intercellular adhesion molecule 3‐grabbing non‐integrin 1 (DC‐SIGN) signalling. We found that DC‐SIGN exhibited strong expression in renal tubular epithelial cells of human acute renal injury tissues. DC‐SIGN protein expression was increased significantly when renal tubular epithelial cells were exposed to lipopolysaccharide (LPS) for a short period. Furthermore, DC‐SIGN was involved in the activation of p65 by TLR‐4, which excluded p38 and c‐Jun N‐terminal kinases (JNK). Interleukin (IL)‐6 and tumour necrosis factor (TNF)‐α expression was decreased after DC‐SIGN knock‐down, and LPS induced endogenous interactions and plasma membrane co‐expression between TLR‐4 and DC‐SIGN. These results show that DC‐SIGN and TLR‐4 interactions regulate inflammatory responses in renal tubular epithelial cells and participate in AKI pathogenesis.     

T‐cell receptor activation of human CD4+ T cells shifts the innate TLR response from CXCL8hiIFN‐γnull to CXCL8loIFN‐γhi

Toll‐like receptors (TLRs) play a major part in providing innate immunity against pathogenic microorganisms. Recent studies show that these receptors are also expressed on T cells, which are the sentinels of adaptive immunity. Here, we have investigated the regulatory role of the T‐cell receptor in the functioning of these innate receptors in T cells. We show that freshly isolated human CD4⁺ T cells readily secrete the neutrophil chemoattractant CXCL8 upon activation with the TLR ligands Pam3CSK and flagellin. In contrast, TCR‐activated cells secrete considerably less CXCL8 but start producing IFN‐γ upon stimulation with TLR agonists in the absence of concomitant TCR engagement. These T cells show increased activation of p38 and JNK MAP‐kinases in response to TLR stimulation, and inhibition of p38 abrogates TLR‐induced IFN‐γ secretion. The shifting of the T‐cell innate immune response from CXCL8^hiIFN‐γ^null in freshly isolated to CXCL8^loIFN‐γ^hi in activated T cells is also observed in response to endogenous innate stimulus, IL‐1. These results suggest that the innate immune response of human CD4⁺ T cells switches from a proinflammatory to an effector type following activation of these cells through the antigen receptor.     

Role of Galectin‐3 in the pathophysiology underlying allergic lung inflammation in a tissue inhibitor of metalloproteinases 1 knockout model of murine asthma

Asthma is a chronic inflammatory respiratory disease characterized by airway inflammation, airway hyperresponsiveness and reversible airway obstruction. Understanding the mechanisms that underlie the various endotypes of asthma could lead to novel and more personalized therapies for individuals with asthma. Using a tissue inhibitor of metalloproteinases 1 (TIMP‐1) knockout murine allergic asthma model, we previously showed that TIMP‐1 deficiency results in an asthma phenotype, exhibiting airway hyperreactivity, enhanced eosinophilic inflammation and T helper type 2 cytokine gene and protein expression following sensitization with ovalbumin. In the current study, we compared the expression of Galectins and other key cytokines in a murine allergic asthma model using wild‐type and TIMP‐1 knockout mice. We also examined the effects of Galectin‐3 (Gal‐3) inhibition on a non‐T helper type 2 cytokine interleukin‐17 (IL‐17) to evaluate the relationship between Gal‐3 and the IL‐17 axis in allergic asthma. Our results showed a significant increase in Gal‐3, IL‐17 and transforming growth factor‐β₁ gene expression in lung tissue isolated from an allergic asthma murine model using TIMP‐1 knockout. Gal‐3 gene and protein expression levels were also significantly higher in lung tissue from an allergic asthma murine model using TIMP‐1 knockout. Our data show that Gal‐3 may regulate the IL‐17 axis and play a pivotal role in the modulation of inflammation during experimental allergic asthma.     

The role of inflammatory and parenchymal cells in acute pancreatitis

The infiltration of inflammatory cells into the pancreas is an early and central event in acute pancreatitis that promotes local injury and systemic complications of the disease. Recent research has yielded the important finding that resident cells of the pancreas (particularly acinar and pancreatic stellate cells) play a dynamic role in leukocyte attraction via secretion of chemokines and cytokines and expression of adhesion molecules. Significant progress has been made in recent years in our understanding of the role of leukocyte movement (adhesion to the blood vessel wall, transmigration through the blood vessel wall and infiltration into the parenchyma) in the pathophysiology of acute pancreatitis. This review discusses recent studies and describes the current state of knowledge in the field. It is clear that detailed elucidation of the numerous processes in the inflammatory cascade is an essential step towards the development of improved therapeutic strategies in acute pancreatitis. Studies to date suggest that combination therapy targeting different steps of the inflammatory cascade may be the treatment of choice for this disease.     

Cystathionine-gamma-lyase inhibitor attenuates acute lung injury induced by acute pancreatitis in rats

Introduction

Acute pancreatitis (AP) is known to induce injuries to extrapancreatic organs. Because respiratory dysfunction is the main cause of death in patients with severe AP, acute pancreatitis-associated lung injury (APALI) is a great challenge for clinicians. This study aimed to investigate the potential role of hydrogen sulfide (H₂S) in the pathogenesis of APALI.

Material and methods

Fifty-four SD rats were randomly divided into three groups: the AP group of rats that received injection of sodium deoxycholate into the common bile duct, the control group that underwent a sham operation, and the treatment group made by intraperitoneal injection of propargylglycine (PAG), an inhibitor of cystathionine-γ-lyase (CSE), into rats with AP. Histopathology of the lung was examined and the expression of CSE and TNF-α mRNA in lung tissue was detected by real-time polymerase chain reaction. The H₂S level in the serum was detected spectrophotometrically.

Results

The serum concentration of H2S and CSE and TNF-α expression in the lung were increased in AP rats modeled after 3 h and 6 h than in control rats (p < 0.05). Intraperitoneal injection of PAG could reduce the serum concentration of H₂S, reduce CSE and TNF-α expression, and alleviate the lung pathology (p < 0.05).

Conclusions

Taken together, our findings suggest that the H₂S/CSE system is crucially involved in the pathological process of APALI and represents a novel target for the therapy of APALI.     

Characterization of innate immune signalling receptors in virus‐induced acute asthma

Background The role of toll‐like receptors (TLRs) and innate immune activation in clinical asthma exacerbations and their relationship to virus infection are unclear. Objective This study aimed to characterize TLR expression and innate immune activity during virus infection in acute asthma. Methods Subjects with acute asthma, stable asthma and healthy controls were recruited and underwent spirometry and sputum induction with isotonic saline. Selected sputum was dispersed with dithiothreitol and total and differential leucocyte counts were performed. Selected sputum was also used for quantitative real‐time PCR for TLR2, TLR3, TLR4, IL‐10 and IP‐10mRNA expression. Sputum supernatant was used for the measurement of innate immune markers, including IL‐8, matrix metalloproteinase‐9 and neutrophil elastase activity. Viruses were detected using real‐time and gel‐based PCR. Results Sputum TLR2 mRNA expression was up‐regulated in both acute and stable asthma compared with healthy controls and decreased 4–6 weeks after acute exacerbation. Sputum TLR2 mRNA expression was elevated in viral, compared with non‐viral, acute asthma. Sputum TLR3 mRNA expression was similar in controls, stable and acute asthma. However, in acute asthma, subjects with virus‐induced acute asthma had significantly higher sputum TLR3 mRNA expression. Induced sputum gene expression for IP‐10 and IL‐10 were increased in viral, compared with non‐viral, acute asthma. In virus‐induced acute asthma, levels of IP‐10 and IL‐10 mRNA expression were correlated with the mRNA expression of TLR2 and TLR3. Conclusions and Clinical Relevance Virus‐induced acute asthma leads to specific induction of TLR2, TLR3, IP‐10 and IL‐10, suggesting that signalling via TLRs may play an important role in mediating airway inflammation, via both innate and adaptive pathways, in virus‐induced exacerbations. These mediators may provide potential treatment targets for virus‐induced asthma. They may also be useful in diagnosing the nature of acute asthma exacerbations and monitoring treatment responses, which would be useful in the clinical management of asthma exacerbations. Cite this as: L. G. Wood, J. L. Simpson, P. A. B. Wark, H. Powell and P. G. Gibson, Clinical & Experimental Allergy, 2011 (41) 640–648.     

Galectin‐3 is an amplifier of the interleukin‐1β‐mediated inflammatory response in corneal keratinocytes

Interleukin‐1β (IL‐1β) is a potent mediator of innate immunity commonly up‐regulated in a broad spectrum of inflammatory diseases. When bound to its cell surface receptor, IL‐1β initiates a signalling cascade that cooperatively induces the expression of canonical IL‐1 target genes such as IL‐8 and IL‐6. Here, we present galectin‐3 as a novel regulator of IL‐1β responses in corneal keratinocytes. Using the SNAP‐tag system and digitonin semi‐permeabilization, we show that recombinant exogenous galectin‐3 binds to the plasma membrane of keratinocytes and is internalized into cytoplasmic compartments. We find that exogenous galectin‐3, but not a dominant negative inhibitor of galectin‐3 polymerization lacking the N‐terminal domain, exacerbates the response to IL‐1β by stimulating the secretion of inflammatory cytokines. The activity of galectin‐3 could be reduced by a novel d ‐galactopyranoside derivative targeting the conserved galactoside‐binding site of galectins and did not involve interaction with IL‐1 receptor 1 or the induction of endogenous IL‐1β. Consistent with these observations, we demonstrate that small interfering RNA‐mediated suppression of endogenous galectin‐3 expression is sufficient to impair the IL‐1β‐induced secretion of IL‐8 and IL‐6 in a p38 mitogen‐activated protein kinase‐independent manner. Collectively, our findings provide a novel role for galectin‐3 as an amplifier of IL‐1β responses during epithelial inflammation through an as yet unidentified mechanism.     

Physiological aspects of Toll‐like receptor 4 activation in sepsis‐induced acute kidney injury

Sepsis‐induced acute kidney injury (SI‐AKI) is common and associated with high mortality. Survivors are at increased risk of chronic kidney disease. The precise mechanism underlying SI‐AKI is unknown, and no curative treatment exists. Toll‐like receptor 4 (TLR4) activates the innate immune system in response to exogenous microbial products. The result is an inflammatory reaction aimed at clearing a potential infection. However, the consequence may also be organ dysfunction as the immune response can cause collateral damage to host tissue. The purpose of this review is to describe the basis for how ligand binding to TLR4 has the potential to cause renal dysfunction and the mechanisms by which this may take place in gram‐negative sepsis. In addition, we highlight areas for future research that can further our knowledge of the pathogenesis of SI‐AKI in relation to TLR4 activation. TLR4 is expressed in the kidney. Activation of TLR4 causes cytokine and chemokine release as well as renal leucocyte infiltration. It also results in endothelial and tubular dysfunction in addition to altered renal metabolism and circulation. From a physiological standpoint, inhibiting TLR4 in large animal experimental SI‐AKI significantly improves renal function. Thus, current evidence indicates that TLR4 has the ability to mediate SI‐AKI by a number of mechanisms. The strong experimental evidence supporting a role of TLR4 in the pathogenesis of SI‐AKI in combination with the availability of pharmacological tools to target TLR4 warrants future human studies.     

Vitamin K3 attenuates lipopolysaccharide‐induced acute lung injury through inhibition of nuclear factor‐κB activation

Vitamin K is a family of fat‐soluble compounds including phylloquinone (vitamin K₁), menaquinone (vitamin K₂) and menadione (vitamin K₃). Recently, it was reported that vitamin K, especially vitamins K₁ and K₂, exerts a variety of biological effects, and these compounds are expected to be candidates for therapeutic agents against various diseases. In this study, we investigated the anti‐inflammatory effects of vitamin K₃ in in vitro cultured cell experiments and in vivo animal experiments. In human embryonic kidney (HEK)293 cells, vitamin K₃ inhibited the tumour necrosis factor (TNF)‐α‐evoked translocation of nuclear factor (NF)‐κB into the nucleus, although vitamins K₁ and K₂ did not. Vitamin K₃ also suppressed the lipopolysaccharide (LPS)‐induced nuclear translocation of NF‐κB and production of TNF‐α in mouse macrophage RAW264·7 cells. Moreover, the addition of vitamin K₃ before and after LPS administration attenuated the severity of lung injury in an animal model of acute lung injury/acute respiratory distress syndrome (ARDS), which occurs in the setting of acute severe illness complicated by systemic inflammation. In the ARDS model, vitamin K₃ also suppressed the LPS‐induced increase in the serum TNF‐α level and inhibited the LPS‐evoked nuclear translocation of NF‐κB in lung tissue. Despite marked efforts, little therapeutic progress has been made, and the mortality rate of ARDS remains high. Vitamin K₃ may be an effective therapeutic strategy against acute lung injury including ARDS.     

Attenuation of Th1 response through galectin‐9 and T‐cell Ig mucin 3 interaction inhibits autoimmune diabetes in NOD mice

Galectin‐9 (gal‐9), widely expressed in many tissues, regulates Th1 cells and induces their apoptosis through its receptor, T‐cell Ig mucin 3, which is mainly expressed on terminally differentiated Th1 cells. Type 1 diabetes is a Th1‐dominant autoimmune disease that specifically destroys insulin‐producing β cells. To suppress the Th1 immune response in the development of autoimmune diabetes, we overexpressed gal‐9 in NOD mice by injection of a plasmid encoding gal‐9. Mice treated with gal‐9 plasmid were significantly protected from diabetes and showed less severe insulitis compared with controls. Flow cytometric analyses in NOD‐T1/2 double transgenic mice showed that Th1‐cell population in spleen, pancreatic lymph node and pancreas was markedly decreased in gal‐9 plasmid‐treated mice, indicating a negative regulatory role of gal‐9 in the development of pathogenic Th1 cells. Splenocytes from gal‐9 plasmid‐treated mice were less responsive to mitogenic stimulation than splenocytes from the control group. However, adoptive transfer of splenocytes from gal‐9‐treated or control mice caused diabetes in NOD/SCID recipients with similar kinetics, suggesting that gal‐9 treatment does not induce active tolerance in NOD mice. We conclude that gal‐9 may downregulate Th1 immune response in NOD mice and could be used as a therapeutic target in autoimmune diabetes.     

Absence of Galectin‐1 accelerates CD8+ T cell‐mediated graft rejection

Galectin‐1 (Gal‐1) is a member of a family of endogenous β‐galactose‐binding proteins with a role in preventing autoimmune diseases and chronic inflammation. In this study, the involvement of Gal‐1 in graft rejection was investigated by using Gal‐1‐deficient mice (Gal‐1?/?). We demonstrate that in the absence of Gal‐1, skin grafts are rejected earlier compared with those of WT mice, and that this is due to the role played by CD8+ T cells in graft rejection. The difference in graft survival observed between Gal‐1?/? and WT mice was explained by both an increase in the percentage of antigen‐specific CD8+ T cells and by preferential secretion of IFN‐γ and IL‐17 by CD8+ T cells in Gal‐1?/? mice compared with WT mice. This study suggests that endogenous expression of Gal‐1 contributes to graft survival. The results obtained from the use of mice deficient in Gal‐1 also confirm a key role for CD8+ T cells in graft rejection.     

Galectin‐1 is essential for the induction of MOG35–55‐based intravenous tolerance in experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), intravenous (i.v.) injection of the antigen, myelin oligodendrocyte glycoprotein‐derived peptide, MOG_35–55, suppresses disease development, a phenomenon called i.v. tolerance. Galectin‐1, an endogenous glycan‐binding protein, is upregulated during autoimmune neuroinflammation and plays immunoregulatory roles by inducing tolerogenic dendritic cells (DCs) and IL‐10 producing regulatory type 1 T (Tr1) cells. To examine the role of galectin‐1 in i.v. tolerance, we administered MOG_35–55‐i.v. to wild‐type (WT) and galectin‐1 deficient (Lgals1^?/?) mice with ongoing EAE. MOG_35–55 suppressed disease in the WT, but not in the Lgals1^?/? mice. The numbers of Tr1 cells and Treg cells were increased in the CNS and periphery of tolerized WT mice. In contrast, Lgals1^?/? MOG‐i.v. mice had reduced numbers of Tr1 cells and Treg cells in the CNS and periphery, and reduced IL‐27, IL‐10, and TGF‐β1 expression in DCs in the periphery. DCs derived from i.v.‐tolerized WT mice suppressed disease when adoptively transferred into mice with ongoing EAE, whereas DCs from Lgals1^?/? MOG‐i.v. mice were not suppressive. These findings demonstrate that galectin‐1 is required for i.v. tolerance induction, likely via induction of tolerogenic DCs leading to enhanced development of Tr1 cells, Treg cells, and downregulation of proinflammatory responses.     

Release of inflammatory mediators by adipose tissue during acute pancreatitis

Obesity and lipid metabolism are associated with the severity of acute pancreatitis. Fat necrosis appears in the severe acute pancreatitis as a consequence of the release of lipolytic enzymes, but its potential role on the progression of the disease is unclear. In this study, we have examined the role of white adipose tissue as a source of inflammatory mediators that can promote systemic inflammation during experimental taurocholate‐induced acute pancreatitis in rats. The inflammatory status and the expression of TNFα, iNOS, adiponectin and IL‐10 were determined in necrotic and non‐necrotic areas of adipose tissue. Samples of adipose tissue were also used to induce the activation of macrophages in vitro. Finally, the release of TNFα to mesenterial vessels surrounded by necrotic or non‐necrotic fat was evaluated in ex vivo perfused mesenterium. A strong inflammatory infiltrate was observed in the border between necrotic and non‐necrotic areas of adipose tissue. In these areas, high expression of TNFα and iNOS and a reduced expression of IL‐10 were observed, while adiponectin showed only a moderate increase. Necrotic fat strongly activates peritoneal macrophages in vitro. Mesenterial areas with fat necrosis release to the vascular vessels significantly increased amounts of TNFα when compared to vessels without necrosis. Altogether, these results indicate that adipose tissue inflammation is a process secondary to acute pancreatitis but also contributes to the generation of mediators potentially involved in the induction of the systemic inflammatory response. In particular, the areas of fat necrosis are important sources of inflammatory mediators. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Triggering of TLR‐3, ‐4, NOD2, and DC‐SIGN reduces viral replication and increases T‐cell activation capacity of HIV‐infected human dendritic cells

A variety of signals influence the capacity of dendritic cells (DCs) to mount potent antiviral cytotoxic T‐cell (CTL) responses. In particular, innate immune sensing by pathogen recognition receptors, such as TLR and C‐type lectines, influences DC biology and affects their susceptibility to HIV infection. Yet, whether the combined effects of PPRs triggering and HIV infection influence HIV‐specific (HS) CTL responses remain enigmatic. Here, we dissect the impact of innate immune sensing by pathogen recognition receptors on DC maturation, HIV infection, and on the quality of HS CTL activation. Remarkably, ligand‐driven triggering of TLR‐3, ‐4, NOD2, and DC‐SIGN, despite reducing viral replication, markedly increased the capacity of infected DCs to stimulate HS CTLs. This was exemplified by the diversity and the quantity of cytokines produced by HS CTLs primed by these DCs. Infecting DCs with viruses harboring members of the APOBEC family of antiviral factors enhanced the antigen‐presenting skills of infected DCs. Our results highlight the tight interplay between innate and adaptive immunity and may help develop innovative immunotherapies against viral infections.     

Modulation of γδ T‐cell activation by neutrophil elastase

γδ T cells are non‐conventional, innate‐like T cells, characterized by a restricted T‐cell receptor repertoire. They participate in protective immunity responses against extracellular and intracellular pathogens, tumour surveillance, modulation of innate and adaptive immune responses, tissue healing, epithelial cell maintenance and regulation of physiological organ function. In this study, we investigated the role of neutrophils during the activation of human blood γδ T cells through CD3 molecules. We found that the up‐regulation of CD69 expression, and the production of interferon‐γ and tumour necrosis factor‐α induced by anti‐CD3 antibodies was potentiated by neutrophils. We found that inhibition of caspase‐1 and neutralization of interleukin‐18 did not affect neutrophil‐mediated modulation. By contrast, the treatment with serine protease inhibitors prevented the potentiation of γδ T‐cell activation induced by neutrophils. Moreover, the addition of elastase to γδ T‐cell culture increased their stimulation, and the treatment of neutrophils with elastase inhibitor prevented the effect of neutrophils on γδ T‐cell activation. Furthermore, we demonstrated that the effect of elastase on γδ T cells was mediated through the protease‐activated receptor, PAR1, because the inhibition of this receptor with a specific antagonist, RWJ56110, abrogated the effect of neutrophils on γδ T‐cell activation.     

High Mobility Group Box1 (HMGB1) released from cancer cells induces the expression of pro‐inflammatory cytokines in peritoneal fibroblasts

High Mobility Group Box1 protein (HMGB1), one of the mediators of inflammation, is associated with tumorigenesis. The HMGB1‐Receptor for advanced glycation end‐products (RAGE) in gastric adenocarcinoma tissues promoted gastric cancer growth, however, there are no reports concerning the relationship between the expression of HMGB1 in gastric cancer and cancer‐related inflammation. Fibroblasts exist most abundantly on cancer tissue where inflammation occurs. So, we studied the effects of HMGB1 released from cancer cells on the fibroblasts. The expression of HMGB1 in cancer cells and nuclear factor‐kappa B (NF‐kB) in fibroblasts were evaluated immunohistochemically in human gastric cancer specimens. Cytoplasmic HMGB1 expression in the cancer cells and nuclear translocation of NF‐kB in fibroblasts were detected at deeper invasion. To determine whether HMGB1 released from cancer cells induces the expression of pro‐inflammatory cytokines in fibroblasts, we analyzed the activation of Toll‐like receptor (TLR) signaling. Fibroblasts stimulated by recombinant HMGB1 and the HSC44PE‐conditioned medium showed the phosphorylation of Interleukin‐1 receptor associated‐kinase 4 (IRAK4), nuclear translocation of NF‐kB, and enhanced pro‐inflammatory cytokine expression. Treatment with HSC44PE‐conditioned‐medium transfected with siRNA‐HMGB1 reduced the expressions of pro‐inflammatory cytokines in the fibroblasts. We propose that HMGB1 released from cancer cells induces the expression of pro‐inflammatory cytokines in peritoneal fibroblasts through the HMGB1‐TLR2/4 pathway.