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1.  Toll-Like Receptor 2 Impairs Host Defense in Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis) 
PLoS Medicine  2007;4(7):e248.
Toll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis.
Methods and Findings
Patient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury.
Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis.
Willem Wiersinga and colleagues find up-regulation of multiple Toll-like receptors (TLRs) in peripheral blood cells of patients with melioidosis. However, only TLR2 had an effect on the immune response in a mouse model.
Editors' Summary
Melioidosis is a severe tropical infection caused by the bacterium Burkholderia pseudomallei. This soil-dwelling pathogen (disease-causing organism) enters the body through cuts, by swallowed contaminated water, or by inhaled contaminated dust. Here, it can cause a severe lung infection or spread into the blood stream and around the body, where it causes widespread inflammation (sepsis) and organ failure. Untreated septic melioidosis is usually fatal. Even with antibiotic therapy, half the people who develop it in Thailand (a hot spot for melioidosis) die. B. pseudomallei is a “gram-negative” bacterium. That is, it is surrounded by a membrane that stops it taking up a stain used to detect bacteria. This membrane contains a molecule called lipopolysaccharide (LPS). Proteins on immune system cells called Toll-like receptors (TLRs), of which there are many, recognize LPS and other surface molecules common to different pathogens and tell the cells to make cytokines. These cytokines stimulate the immune system to kill the pathogen but also cause inflammation, the underlying problem in septic melioidosis and other forms of sepsis. In other words, TLRs are two-edged swords—they provide an essential first-line defense against pathogens, but cause life-threatening inflammation if overstimulated.
Why Was This Study Done?
It isn't known which TLRs are involved in melioidosis. TLR4 normally detects LPS, but the surface of B. pseudomallei also carries molecules that interact with TLR2. Understanding how B. pseudomallei interacts with TLRs might suggest new, more effective ways to treat septic melioidosis. Better remedies for this disease are badly needed because, as well as the infections it causes in the community, the US Centers for Disease Control and Prevention has identified B. pseudomallei as a potential bioterrorism agent. In this study, the researchers have characterized the expression and function of TLRs in septic melioidosis using human, in vitro (test tube), and animal approaches.
What Did the Researchers Do and Find?
The researchers isolated monocytes and granulocytes (immune system cells involved in first-line defenses against pathogens) from patients with melioidosis and from healthy people. The patients' cells made more TLR1, TLR2, TLR4, and CD14 (a protein that enhances the activation of immune system cells by LPS) than those of the healthy controls and more of the mRNAs encoding several other TLRs. Next, the researchers tested the ability of heat-killed B. pseudomallei to induce the release of TNFα (a cytokine produced in response to TLR signaling) from macrophages (immune system cells that swallow up pathogens) isolated from wild-type mice and from mice lacking TLR2 or TLR4. Macrophages isolated from wild-type mice made more TNFα than those from TLR2- or TLR4-deficient mice. In addition, a human kidney cell line engineered to express CD14/TLR2 or CD14/TLR4 but not the parent cell line released IL8 (another cytokine) when stimulated with heat-killed B. pseudomallei. Other experiments in these human cell lines showed that LPS purified from B. pseudomallei signals through TLR2 but not through TLR4. Finally, the researchers tested the ability of TLR2- and TLR4-deficient mice to survive after infection with live B. pseudomallei. Compared with TLR4-deficient or wild-type mice, the TLR2-deficient mice had a strong survival advantage, a lower bacterial load, reduced lung inflammation, and less organ damage.
What Do These Findings Mean?
These findings show that people with melioidosis have increased expression of several TLRs, any one of which might cause the sepsis associated with B. pseudomallei infection. The in vitro findings indicate that TLR2 and TLR4 both contribute to the responsiveness of immune cells to B. pseudomallei in test tubes, but that only TLR2 detects the LPS of this bacterium. This unexpected result—TLR4 normally responds to LPS—might indicate that there is something unique about the LPS of B. pseudomallei. Finally, the survival of TLR2-deficient mice after infection with B. pseudomallei suggests that TLR2-mediated dysregulation of the immune system in response to invasive B. pseudomallei might cause septic melioidosis. Although these results need confirming in people, they suggest that inhibition of TLR2 in combination with antibiotic therapy might improve outcomes for people with melioidosis.
Additional Information.
Please access these Web sites via the online version of this summary at
Information is available from the US Centers for Disease Control and Prevention on melioidosis (in English and Spanish)
The UK Health Protection Agency provides information for the public and health professionals on melioidosis
Wikipedia has pages on melioidosis and on Toll-like receptors (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The MedlinePlus encyclopedia contains a page on sepsis (in English and Spanish)
PMCID: PMC1950213  PMID: 17676990
2.  Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and inflammatory response 
Bacterial and viral exacerbations play a crucial role in a variety of lung diseases including COPD or asthma. Since the lung epithelium is a major source of various inflammatory mediators that affect the immune response, we analyzed the inflammatory reaction of primary lung epithelial cells to different microbial molecules that are recognized by Toll-like receptors (TLR).
The effects of TLR ligands on primary small airway epithelial cells were analyzed in detail with respect to cytokine, chemokine and matrix metalloproteinase secretion. In addition, the regulation of the expression of TLRs and their adaptor proteins in small airway epithelial cells was investigated.
Our data demonstrate that poly(I:C), a synthetic analog of viral dsRNA, mediated the strongest proinflammatory effects among the tested ligands, including an increased secretion of IL-6, IL-8, TNF-α, GM-CSF, GRO-α, TARC, MCP-1, MIP-3α, RANTES, IFN-β, IP-10 and ITAC as well as an increased release of MMP-1, MMP-8, MMP-9, MMP-10 and MMP-13. Furthermore, our data show that poly(I:C) as well as type-1 and type-2 cytokines have a pronounced effect on the expression of TLRs and molecules involved in TLR signaling in small airway epithelial cells. Poly(I:C) induced an elevated expression of TLR1, TLR2 and TLR3 and increased the gene expression of the general TLR adaptor MyD88 and IRAK-2. Simultaneously, poly(I:C) decreased the expression of TLR5, TLR6 and TOLLIP.
Poly(I:C), an analog of viral dsRNA and a TLR3 ligand, triggers a strong inflammatory response in small airway epithelial cells that is likely to contribute to viral exacerbations of pulmonary diseases like asthma or COPD. The pronounced effects of poly(I:C) on the expression of Toll-like receptors and molecules involved in TLR signaling is assumed to influence the immune response of the lung epithelium to viral and bacterial infections. Likewise, the regulation of TLR expression by type-1 and type-2 cytokines is important considering the impact of exogenous and endogenous TLR ligands on Th1 or Th2 driven pulmonary inflammations like COPD or asthma, respectively.
PMCID: PMC1315317  PMID: 16316467
3.  Endotoxin Is Not Essential for the Development of Cockroach Induced Allergic Airway Inflammation 
Yonsei Medical Journal  2012;53(3):593-602.
Cockroach (CR) is an important inhalant allergen and can induce allergic asthma. However, the mechanism by which CR induces airway allergic inflammation and the role of endotoxin in CR extract are not clearly understood in regards to the development of airway inflammation. In this study, we evaluated whether endotoxin is essential to the development of CR induced airway allergic inflammation in mice.
Materials and Methods
Airway allergic inflammation was induced by intranasal administration of either CR extract, CR with additional endotoxin, or endotoxin depleted CR extract, respectively, in BALB/c wild type mice. CR induced inflammation was also evaluated with toll like receptor-4 (TLR-4) mutant (C3H/HeJ) and wild type (C3H/HeN) mice.
Intranasal administration of CR extracts significantly induced airway hyperresponsiveness (AHR), eosinophilic and neutrophilic airway inflammation, as well as goblet cell hyperplasia in a dose-dependent manner. The addition of endotoxin along with CR allergen attenuated eosinophilic inflammation, interleukin (IL)-13 level, and goblet cell hyperplasia of respiratory epithelium; however, it did not affect the development of AHR. Endotoxin depletion in CR extract did not attenuate eosinophilic inflammation and lymphocytosis in BAL fluid, AHR and IL-13 expression in the lungs compared to CR alone. The attenuation of AHR, eosinophilic inflammation, and goblet cell hyperplasia induced by CR extract alone was not different between TLR-4 mutant and the wild type mice. In addition, heat inactivated CR extract administration induced attenuated AHR and eosinophilic inflammation.
Endotoxin in CR extracts may not be essential to the development of airway inflammation.
PMCID: PMC3343443  PMID: 22477005
Cockroach; endotoxin; toll like receptor-4
4.  IL-13 dampens human airway epithelial innate immunity through induction of IL-1 receptor–associated kinase M 
Impaired airway mucosal immunity can contribute to increased respiratory tract infections in asthmatic patients, but the involved molecular mechanisms have not been fully clarified. Airway epithelial cells serve as the first line of respiratory mucosal defense to eliminate inhaled pathogens through various mechanisms, including Toll-like receptor (TLR) pathways. Our previous studies suggest that impaired TLR2 function in TH2 cytokine–exposed airways might decrease immune responses to pathogens and subsequently exacerbate allergic inflammation. IL-1 receptor–associated kinase M (IRAK-M) negatively regulates TLR signaling. However, IRAK-M expression in airway epithelium from asthmatic patients and its functions under a TH2 cytokine milieu remain unclear.
We sought to evaluate the role of IRAK-M in IL-13–inhibited TLR2 signaling in human airway epithelial cells. Methods: We examined IRAK-M protein expression in epithelia from asthmatic patients versus that in normal airway epithelia. Moreover, IRAK-M regulation and function in modulating innate immunity (eg, TLR2 signaling) were investigated in cultured human airway epithelial cells with or without IL-13 stimulation.
IRAK-M protein levels were increased in asthmatic airway epithelium. Furthermore, in primary human airway epithelial cells, IL-13 consistently upregulated IRAK-M expression, largely through activation of phosphoinositide 3-kinase pathway. Specifically, phosphoinositide 3-kinase activation led to c-Jun binding to human IRAK-M gene promoter and IRAK-M upregulation. Functionally, IL-13–induced IRAK-M suppressed airway epithelial TLR2 signaling activation (eg, TLR2 and human β-defensin 2), partly through inhibiting activation of nuclear factor κB.
Our data indicate that epithelial IRAK-M overexpression in TH2 cytokine–exposed airways inhibits TLR2 signaling, providing a novel mechanism for the increased susceptibility of infections in asthmatic patients.
PMCID: PMC3348857  PMID: 22154382
IL-13; IL-1 receptor–associated kinase M; Toll-like receptor 2; airway epithelial cells
5.  Asparagine Endopeptidase Controls Anti-Influenza Virus Immune Responses through TLR7 Activation 
PLoS Pathogens  2012;8(8):e1002841.
Intracellular Toll-like receptors (TLRs) expressed by dendritic cells recognize nucleic acids derived from pathogens and play an important role in the immune responses against the influenza virus (IAV), a single-stranded RNA sensed by different receptors including TLR7. However, the importance of TLR7 processing in the development of anti-viral immune responses is not known. Here we report that asparagine endopeptidase (AEP) deficient mice are unable to generate a strong anti-IAV response, as demonstrated by reduced inflammation, cross presentation of cell-associated antigens and priming of CD8+ T cells following TLR7-dependent pulmonary infection induced by IAV. Moreover, AEP deficient lung epithelial- or myeloid-cells exhibit impaired TLR7 signaling due to defective processing of this receptor. Indeed, TLR7 requires a proteolytic cleavage by AEP to generate a C-terminal fragment competent for signaling. Thus, AEP activity is critical for TLR7 processing, opening new possibilities for the treatment of influenza and TLR7-dependent inflammatory diseases.
Author Summary
Influenza A virus, a negative stranded RNA, can cause severe illness in humans and animals and stimulates many receptors including Toll like receptors 7 (TLR7). TLR signaling induces maturation of dendritic cells and the production of a variety of inflammatory cytokines that are crucial for both innate and adaptive immunity. TLR7 is an intracellular receptor, which resides in endosomes and senses viruses to trigger host defence. Previous data have shown that TLR9 requires proteolysis to be functional but it is unclear whether other intracellular TLRs (TLR3 and TLR7) are also subject to degradation. Here, we used a protease deficient mouse model to show the in vivo importance of TLR7 processing in influenza infection. Inflammation monitored by cytokine release and adaptive immunity measured by cross priming of CD8+ T cells was significantly reduced in infected protease-deficient animals in comparison to control animals. We showed that TLR7 requires a proteolytic cleavage by a cysteine endopeptidase in order to be functional. Our findings indicate that TLR7 processing mediated by a protease, asparagynil endopeptidase, is critical for inducing robust anti-influenza immune responses. Given our results, targeting TLR7 response in the lungs through proteases may offer new therapeutic potential in pulmonary infection.
PMCID: PMC3420946  PMID: 22916010
6.  Toll-like receptors 2, 3 and 4 and thymic stromal lymphopoietin expression in fatal asthma 
Airway inflammation in asthma involves innate immune responses. Toll-like receptors (TLRs) and thymic stromal lymphopoietin (TSLP) are thought to be involved in airway inflammation, but their expression in asthmatics’ both large and small airways has not been investigated.
To analyze the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics and compare their expression in smoking and nonsmoking asthmatics; to investigate whether TLR expression is associated with eosinophilic or neutrophilic airway inflammation and with Mycoplasma pneumoniae and Chlamydophila pneumoniae infection.
Using immunohistochemistry and image analysis, we investigated TLR2, TLR3, TLR4 and TSLP expression in large and small airways of 24 victims of fatal asthma, FA, (13 nonsmokers, 11 smokers) and 9 deceased control subjects (DCtrl). TLRs were also measured in 18 mild asthmatics (MA) and 12 healthy controls (HCtrl). Mycoplasma pneumoniae and Chlamydophila pneumoniae in autopsy lung tissue was analyzed using real-time polymerase chain reaction. Airway eosinophils and neutrophils were measured in all subjects.
Fatal asthma patients had higher TLR2 in the epithelial and outer layers of large and small airways compared with DCtrls. Smoking asthmatics had lower TLR2 levels in the inner and outer layers of the small airways than nonsmoking asthmatics. TSLP was increased in the epithelial and outer layers of the large airways of FA. FA patients had greater TLR3 expression in the outer layer of large airways and greater TLR4 expression in the outer layer of small airways. Eosinophilic airway inflammation was associated with TLR expression in the epithelium of FA. No bacterial DNA was detected in FA or DCtrls. MA and HCtrls had only a small difference in TLR3 expression.
Conclusions and Clinical Relevance
Increased expression of TLR 2, 3 and 4 and TSLP in fatal asthma may contribute to the acute inflammation surrounding asthma deaths.
PMCID: PMC3459227  PMID: 22994343
lung; innate immunity; immunohistochemistry
7.  House dust mite allergen induces asthma via TLR4 triggering of airway structural cells 
Nature medicine  2009;15(4):410-416.
Barrier epithelial cells and airway dendritic cells (DC) make up the first line of defence against inhaled substances like house dust mite (HDM) allergen and endotoxin. We hypothesized that these cells need to communicate to cause allergic disease. Using irradiated chimeric mice, we demonstrate that TLR4 expression on radioresistant lung structural cells is required and sufficient for DC activation in the lung and for priming of effector T helper responses to HDM. TLR4 triggering on structural cells caused production of the innate proallergic cytokines thymic stromal lymphopoietin, granulocyte-macrophage colony stimulating factor, interleukin-25 and IL-33. The absence of TLR4 on structural cells, but not on hematopoietic cells, abolished HDM driven allergic airway inflammation. Finally, inhalation of a TLR4 antagonist to target exposed epithelial cells suppressed the salient features of asthma including bronchial hyperreactivity. Our data identify an innate immune function of airway epithelial cells that drives allergic inflammation via activation of mucosal DCs.
PMCID: PMC2789255  PMID: 19330007
8.  PAI-1 is an essential component of the pulmonary host response during Pseudomonas aeruginosa pneumonia in mice 
Thorax  2011;66(9):788-796.
Elevated plasma and bronchoalveolar lavage fluid plasminogen activator inhibitor 1 (PAI-1) levels are associated with adverse clinical outcome in patients with pneumonia caused by Pseudomonas aeruginosa. However, whether PAI-1 plays a pathogenic role in the breakdown of the alveolar–capillary barrier caused by P aeruginosa is unknown.
The role of PAI-1 in pulmonary host defence and survival during P aeruginosa pneumonia in mice was tested. The in vitro mechanisms by which P aeruginosa causes PAI-1 gene and protein expression in lung endothelial and epithelial cells were also examined.
Methods and results
PAI-1 null and wild-type mice that were pretreated with the PAI-1 inhibitor Tiplaxtinin had a significantly lower increase in lung vascular permeability than wild-type littermates after the airspace instillation of 1 × 107 colony-forming units (CFU) of P aeruginosa bacteria. Furthermore, P aeruginosa in vitro induced the expression of the PAI-1 gene and protein in a TLR4/p38/RhoA/NF-κB (Toll-like receptor 4/p38/RhoA/nuclear factor-κB) manner in lung endothelial and alveolar epithelial cells. However, in vivo disruption of PAI-1 signalling was associated with higher mortality at 24 h (p<0.03) and higher bacterial burden in the lungs secondary to decreased neutrophil migration into the distal airspace in response to P aeruginosa.
The results indicate that PAI-1 is a critical mediator that controls the development of the early lung inflammation that is required for the activation of the later innate immune response necessary for the eradication of P aeruginosa from the distal airspaces of the lung.
PMCID: PMC3282176  PMID: 21768189
9.  The Therapeutic Potential of Toll-like Receptor 7 Stimulation in Asthma 
Asthma is an inflammatory disorder of the airways frequently characterized by an excessive Th2 adaptive immune response. Activation of Toll-like receptor (TLR)-7, a single-stranded viral RNA receptor that is highly expressed in the airways, triggers a rapid innate immune response and favors a subsequent Th1 response. Because of this role in pulmonary immunoregulation, TLR7 has gained considerable interest as a therapeutic target in asthma. Synthetic TLR7 ligands, including the imidazoquinolines imiquimod (R837) and resiquimod (R848), and 8-hydroxyadenine derivatives have been developed for other clinical indications. TLR7 activation prevents ovalbumin-induced airway hyperreactivity, eosinophilic inflammation, goblet cell hyperplasia and airway remodeling in murine models of asthma. TLR7 activation also inhibits viral replication in the lung and prevents virus-induced airway hyperreactivity. Furthermore, it has recently been shown that stimulating TLR7 rapidly relaxes airway smooth muscle, dilating the airways. This bronchodilating effect, which occurs in seconds to minutes and depends on rapid production of nitric oxide, indicates that TLR7 can signal via previously unrecognized pathways. The effects of decreasing the allergic Th2 response, acting as an immediate bronchodilator, and promoting an antiviral immune environment, make TLR7 an attractive drug target. We examine the current understanding of TLR7 as a therapeutic target and its translation to asthma treatment in humans.
PMCID: PMC4049154  PMID: 23078048
asthma; Toll-like Receptor 7; inflammation; bronchodilator
10.  Differential expression of Toll-like receptors on human alveolar macrophages and autologous peripheral monocytes 
Respiratory Research  2010;11(1):2.
Toll-like receptors (TLRs) are critical components in the regulation of pulmonary immune responses and the recognition of respiratory pathogens such as Mycobacterium Tuberculosis (M.tb). Through examination of human alveolar macrophages this study attempts to better define the expression profiles of TLR2, TLR4 and TLR9 in the human lung compartment which are as yet still poorly defined.
Sixteen healthy subjects underwent venipuncture, and eleven subjects underwent additional bronchoalveolar lavage to obtain peripheral blood mononuclear and bronchoalveolar cells, respectively. Surface and intracellular expression of TLRs was assessed by fluorescence-activated cell sorting and qRT-PCR. Cells were stimulated with TLR-specific ligands and cytokine production assessed by ELISA and cytokine bead array.
Surface expression of TLR2 was significantly lower on alveolar macrophages than on blood monocytes (1.2 ± 0.4% vs. 57 ± 11.1%, relative mean fluorescence intensity [rMFI]: 0.9 ± 0.1 vs. 3.2 ± 0.1, p < 0.05). The proportion of TLR4 and TLR9-expressing cells and the rMFIs of TLR4 were comparable between alveolar macrophages and monocytes. The surface expression of TLR9 however, was higher on alveolar macrophages than on monocytes (rMFI, 218.4 ± 187.3 vs. 4.4 ± 1.4, p < 0.05) while the intracellular expression of the receptor and the proportion of TLR9 positive cells were similar in both cell types. TLR2, TLR4 and TLR9 mRNA expression was lower in bronchoalveolar cells than in monocytes.
Pam3Cys, LPS, and M.tb DNA upregulated TLR2, TLR4 and TLR9 mRNA in both, bronchoalveolar cells and monocytes. Corresponding with the reduced surface and mRNA expression of TLR2, Pam3Cys induced lower production of TNF-α, IL-1β and IL-6 in bronchoalveolar cells than in monocytes. Despite comparable expression of TLR4 on both cell types, LPS induced higher levels of IL-10 in monocytes than in alveolar macrophages. M.tb DNA, the ligand for TLR9, induced similar levels of cytokines in both cell types.
The TLR expression profile of autologous human alveolar macrophages and monocytes is not identical, therefore perhaps contributing to compartmentalized immune responses in the lungs and systemically. These dissimilarities may have important implications for the design and efficacy evaluation of vaccines with TLR-stimulating adjuvants that target the respiratory tract.
PMCID: PMC2817655  PMID: 20051129
11.  Regulation of IL-4 Receptor Signaling by STUB1 in Lung Inflammation 
Rationale: IL-4Rα, the common receptor component for IL-4 and IL-13, plays a critical role in IL-4– and IL-13–mediated signaling pathways that regulate airway inflammation and remodeling. However, the regulatory mechanisms underlying IL-4Rα turnover and its signal termination remain elusive.
Objectives: To evaluate the role of STUB1 (STIP1 homology and U-Box containing protein 1) in regulating IL-4R signaling in airway inflammation.
Methods: The roles of STUB1 in IL-4Rα degradation and its signaling were investigated by immunoblot, immunoprecipitation, and flow cytometry. The involvement of STUB1 in airway inflammation was determined in vivo by measuring lung inflammatory cells infiltration, mucus production, serum lgE levels, and alveolar macrophage M2 activation in STUB1−/− mice. STUB1 expression was evaluated in airway epithelium of patients with asthma and lung tissues of subjects with chronic obstructive pulmonary disease.
Measurements and Main Results: STUB1 interacted with IL-4Rα and targeted it for ubiquitination-mediated proteasomal degradation, terminating IL-4 or IL-13 signaling. STUB1 knockout cells showed increased levels of IL-4Rα and sustained STAT6 activation, whereas STUB1 overexpression reduced IL-4Rα levels. Mice deficient in STUB1 had spontaneous airway inflammation, alternative M2 activation of alveolar macrophage, and increased serum IgE. STUB1 levels were increased in airways of subjects with asthma or chronic obstructive pulmonary disease, suggesting that up-regulation of STUB1 might be an important feedback mechanism to dampen IL-4R signaling in airway inflammation.
Conclusions: Our study identified a previously uncharacterized role for STUB1 in regulating IL-4R signaling, which might provide a new strategy for attenuating airway inflammation.
PMCID: PMC3919125  PMID: 24251647
IL-4R signaling; STUB1; airway inflammation
12.  Toll-Like Receptor 4-Myeloid Differentiation Factor 88 Signaling Contributes to Ventilator-induced Lung Injury in Mice 
Anesthesiology  2010;113(3):619-629.
The mechanisms of ventilator-induced lung injury, an iatrogenic inflammatory condition induced by mechanical ventilation, are not completely understood. Toll-like receptor 4 (TLR4) signaling via the adaptor protein, myeloid differentiation factor 88 (MyD88) is proinflammatory and plays a critical role in host immune response to invading pathogen and noninfectious tissue injury. The role of TLR4-MyD88 signaling in ventilator-induced lung injury remains incompletely understood.
Mice were ventilated with low or high tidal volume (HTV), 7 or 20 ml/kg, after tracheotomy for 4 h. Control mice were tracheotomized without ventilation. Lung injury was assessed by: alveolar capillary permeability to Evans blue albumin, wet/dry ratio, bronchoalveolar lavage analysis for cell counts, total proteins and cytokines, lung histopathology, and plasma cytokine levels.
Wildtype mice subjected to HTV had increased: pulmonary permeability; inflammatory cell infiltration/lung edema; and interleukin-6/macrophage-inflammatory protein-2 in the lavage compared to control. In HTV, inhibitor of κB alpha decreased whereas phosphorylated extracellular signal-regulated kinases increased. TLR4 mutant and MyD88−/− mice showed markedly attenuated response to HTV, including less lung inflammation; pulmonary edema; and cell number, protein content, and the cytokines in the lavage. Furthermore, compared to wildtype, both TLR4 mutant and MyD88−/− mice had significantly higher inhibitor of κB alpha and reduced extracellular signal-regulated kinases phosphorylation following HTV.
TLR4-MyD88 signaling plays an important role in the development of ventilator-induced lung injury in mice, possibly through mechanisms involving nuclear factor-κB and mitogen-activated protein kinase pathways.
PMCID: PMC3726314  PMID: 20683250
13.  Hyaluronan fragments contribute to the ozone-primed immune response to LPS 
Hyaluronan is a high molecular weight component of pulmonary extracelluar matrix and lung injury can generate low molecular weight hyaluronan fragment (HA) that functions as endogenous ligand to cell surface receptors CD44 and toll like receptor 4 (TLR4). This leads to activation of intracellular NFκB signaling and pro-inflammatory cytokine production. Based on previous information that ozone exposure causes increased HA in bronchial alveolar lavage fluid (BALF) and ozone pre-exposure primes immune response to inhaled LPS, we hypothesized that HA production during ozone exposure augments the inflammatory response to LPS. We demonstrate that acute ozone exposure at 1ppm for 3 hours primes the immune response to low dose aerosolized LPS in C57BL/6J mice, resulting in increased neutrophil recruitment into the airspaces, increased levels of protein and pro-inflammatory cytokines in the BALF, and increased airway hyperresponsiveness (AHR). Intratracheal instillation of endotoxin-free HA (25 μg) enhances the biological response to inhaled LPS in a manner similar to ozone pre-exposure. In vitro studies using bone marrow-derived macrophages indicate that HA enhances LPS responses measured by TNFα production while immunofluorescence staining of murine alveolar macrophages demonstrates that HA induces TLR4 peripheralization and lipid raft co-localization. Collectively, our observations support that ozone primes macrophage responsiveness to low dose LPS, in part, due to hyaluronan-induced TLR4 peripheralization in lung macrophages.
PMCID: PMC3691842  PMID: 21037098
environmental; ozone; lipopolysaccharide; hyaluronan; TLR4; toll-like receptor; innate immunity; macrophage; lung injury
14.  Legionella pneumophila induces human beta Defensin-3 in pulmonary cells 
Respiratory Research  2010;11(1):93.
Legionella pneumophila is an important causative agent of severe pneumonia in humans. Human alveolar epithelium and macrophages are effective barriers for inhaled microorganisms and actively participate in the initiation of innate host defense. The beta defensin-3 (hBD-3), an antimicrobial peptide is an important component of the innate immune response of the human lung. Therefore we hypothesize that hBD-3 might be important for immune defense towards L. pneumophila.
We investigated the effects of L. pneumophila and different TLR agonists on pulmonary cells in regard to hBD-3 expression by ELISA. Furthermore, siRNA-mediated inhibition of TLRs as well as chemical inhibition of potential downstream signaling molecules was used for functional analysis.
L. pneumophila induced release of hBD-3 in pulmonary epithelium and alveolar macrophages. A similar response was observed when epithelial cells were treated with different TLR agonists. Inhibition of TLR2, TLR5, and TLR9 expression led to a decreased hBD-3 expression. Furthermore expression of hBD-3 was mediated through a JNK dependent activation of AP-1 (c-Jun) but appeared to be independent of NF-κB. Additionally, we demonstrate that hBD-3 elicited a strong antimicrobial effect on L. pneumophila replication.
Taken together, human pulmonary cells produce hBD-3 upon L. pneumophila infection via a TLR-JNK-AP-1-dependent pathway which may contribute to an efficient innate immune defense.
PMCID: PMC2910005  PMID: 20615218
15.  Scavenger Receptor Class A Plays a Central Role in Mediating Mortality and the Development of the Pro-Inflammatory Phenotype in Polymicrobial Sepsis 
PLoS Pathogens  2012;8(10):e1002967.
Sepsis is a frequent complication in critical illness. The mechanisms that are involved in initiation and propagation of the disease are not well understood. Scavenger receptor A (SRA) is a membrane receptor that binds multiple polyanions such as oxidized LDL and endotoxin. Recent studies suggest that SRA acts as a pattern recognition receptor in the innate immune response. The goal of the present study was to determine the role of SRA in polymicrobial sepsis. SRA deficient (SRA−/−) and C57BL/6JB/6J (WT) male mice were subjected to cecal ligation and puncture (CLP) to induce polymicrobial sepsis. NFκB activity, myeloperoxidase activity, and co-association of SRA with toll like receptor (TLR) 4 and TLR2 was analyzed in the lungs. Spleens were analyzed for apoptosis. Serum cytokines and chemokines were assayed. Blood and peritoneal fluid were cultured for aerobic and anaerobic bacterial burdens. Long term survival was significantly increased in SRA−/− septic mice (53.6% vs. 3.6%, p<0.05) when compared to WT mice. NFκB activity was 45.5% lower in the lungs of SRA−/− septic mice versus WT septic mice (p<0.05). Serum levels of interleukin (IL)-5, IL-6, IL-10 and monocyte chemoattractant protein −1 were significantly lower in septic SRA−/− mice when compared to septic WT mice (p<0.05). We found that SRA immuno-precipitated with TLR4, but not TLR2, in the lungs of WT septic mice. We also found that septic SRA−/− mice had lower bacterial burdens than WT septic mice. SRA deficiency had no effect on pulmonary neutrophil infiltration or splenocyte apoptosis during sepsis. We conclude that SRA plays a pivotal, and previously unknown, role in mediating the pathophysiology of sepsis/septic shock in a murine model of polymicrobial sepsis. Mechanistically, SRA interacts with TLR4 to enhance the development of the pro-inflammatory phenotype and mediate the morbidity and mortality of sepsis/septic shock.
Author Summary
Trauma and other critical illnesses can progress to septic shock. The mechanisms that result in this progression are not understood. For this reason, there are no proven treatments available, and the mortality rate from sepsis remains quite high. We have found that mice that lack a certain cell surface protein, scavenger receptor A, have a higher rate of survival from a surgically induced sepsis than those that have the receptor. Previously, this receptor has been found to play a role in atherosclerosis, and more recently, to play a role in the immune response to infection. In this study we have found that in addition to improved survival, mice without scavenger receptor A have fewer bacteria in their abdominal cavities and in their blood. They also have lower levels of inflammation. We demonstrated that scavenger receptor A interacts with another protein involved in inflammation and infection, toll like receptor 4. This interaction might be one mechanism for the effects seen in mice without scavenger receptor A. These studies provide a better understanding of the underlying mechanisms of sepsis. Drugs that target scavenger receptor A could result in better therapies for sepsis.
PMCID: PMC3469655  PMID: 23071440
16.  Toll-Like Receptor 2 Regulates Organic Dust–Induced Airway Inflammation 
Organic dust exposure in agricultural environments results in significant airway inflammatory diseases. Gram-positive cell wall components are present in high concentrations in animal farming dusts, but their role in mediating dust-induced airway inflammation is not clear. This study investigated the role of Toll-like receptor (TLR) 2, a pattern recognition receptor for gram-positive cell wall products, in regulating swine facility organic dust extract (DE)–induced airway inflammation in mice. Isolated lung macrophages from TLR2 knockout mice demonstrated reduced TNF-α, IL-6, keratinocyte chemoattractant/CXCL1, but not macrophage inflammatory protein-2/CXCL2 expression, after DE stimulation ex vivo. Next, using an established mouse model of intranasal inhalation challenge, we analyzed bronchoalveolar lavage fluid and lung tissue in TLR2-deficient and wild-type (WT) mice after single and repetitive DE challenge. Neutrophil influx and select cytokines/chemokines were significantly lower in TLR2-deficient mice at 5 and 24 hours after single DE challenge. After daily exposure to DE for 2 weeks, there were significant reductions in total cellularity, neutrophil influx, and TNF-α, IL-6, CXCL1, but not CXCL2 expression, in TLR2-deficient mice as compared with WT animals. Lung pathology revealed that bronchiolar inflammation, but not alveolar inflammation, was reduced in TLR2-deficient mice after repetitive exposure. Airway hyperresponsiveness to methacholine after dust exposure was similar in both groups. Finally, airway inflammatory responses in WT mice after challenge with a TLR2 agonist, peptidoglycan, resembled DE-induced responses. Collectively, these results demonstrate that the TLR2 pathway is important in regulating swine facility organic dust–induced airway inflammation, which suggests the importance of TLR2 agonists in mediating large animal farming–induced airway inflammatory responses.
PMCID: PMC3208620  PMID: 21278324
Toll-like receptor 2; swine/pig facility; peptidoglycan; organic dust; lung pathology
17.  Early Life Co-Administration of Cockroach Allergen and Endotoxin Augments Pulmonary and Systemic Responses 
Environmental exposures to cockroach allergen and endotoxin are recognized epidemiological risk factors for the early development of allergies and asthma in children. Because of this, it is important to examine the role of early life concurrent inhalation exposures to cockroach allergen and endotoxin in the pathogenesis of allergic airways disease.
We examined the effects of repeated concomitant endotoxin and cockroach allergen inhalation on the pulmonary and systemic immune responses of newborn and juvenile mice.
C3H/HeBFeJ mice were exposed to inhaled endotoxin and cockroach allergen via intranasal instillation from day 2 to 21 after birth, and systemic and pulmonary responses were examined in serum, bronchoalveolar lavage fluid, and lung tissue.
Cockroach allergen exposures induced pulmonary eosinophilic inflammation, total and allergen specific IgE, IgG1, and IgG2a production, and alveolar remodeling. Co-exposures with endotoxin and cockroach allergen significantly increased serum IgE and IgG1, lung inflammation, and alveolar wall thickness, and decreased airspace volume density. Importantly, compared to exposures with individual substances, the responses to co-exposures were more than additive.
Repeated inhalation exposures of neonatal and juvenile mice to endotoxin and cockroach allergen increased the pulmonary inflammatory and systemic immune responses in a synergistic manner and enhanced alveolar remodeling in the developing lung. These data underscore the importance of evaluating the effect of multiple, concurrent environmental exposures, and of using an experimental model that incorporates clinically relevant timing and route of exposures.
PMCID: PMC3408223  PMID: 19438589
endotoxin; cockroach allergen; lung inflammation; allergy; enzyme hypothesis; synergistic effect
18.  Functional Effects of Toll-Like Receptor (TLR)3, 7, 9, RIG-I and MDA-5 Stimulation in Nasal Epithelial Cells 
PLoS ONE  2014;9(6):e98239.
The human nasal epithelium is an important physical barrier, and a part of the innate immune defense that protect against pathogens. The epithelial cells recognize microbial components by pattern-recognition receptors (PRRs), and thereby trigger an immune response. Even though TLR3, TLR7, TLR9, RIG-I and MDA-5 are all known to respond to viral stimulation, their potential role in chronic airway inflammation triggered by local cytokine release remains to be established.
mRNA and corresponding protein expression of TLR3, TLR7, TLR9, RIG-I and MDA-5 were analyzed in nasal biopsies and various upper airway epithelial cell lines using real-time reverse transcription PCR, immunohistochemistry and flow cytometry. Ligand induced, cytokine release, was evaluated with ELISA.
Nasal biopsies were found to express TLR3, TLR7, TLR9, RIG-I and MDA-5, with the most abundant expression in the surface epithelium. These receptors were verified in primary human nasal epithelial cell (HNEC) as well as in the airway epithelial cell lines Detroit-562 and FaDu. Poly(I:C) (TLR3) and R-837 (TLR7) stimulation increased secretion of IL-6 and GM-CSF from the nasal mucosa and the epithelial cell lines. CpG (TLR9) stimulation caused release of IL-8 in the nasal mucosa and in FaDu. Poly(I:C)/LyoVec (RIG-I/MDA-5) stimulation activated the secretion of IFN-β in the nasal mucosa. A corresponding release was also detected from HNEC and Detroit-562.
The nasal epithelium has the ability to recognize viral intrusion through TLR and RLR receptors, and the subsequent response might have a role in exacerbation of inflammatory diseases like allergic rhinitis and chronic rhinosinusitis.
PMCID: PMC4041746  PMID: 24886842
19.  Sessile alveolar macrophages modulate immunity through connexin 43-based epithelial communication 
Nature  2014;506(7489):503-506.
Tissue-resident macrophages of barrier organs constitute the first line of defense against pathogens at the systemic interface with the ambient environment. In lung, resident alveolar macrophages (AMs) provide sentinel function against inhaled pathogens1. Bacterial constituents ligate toll-like receptors (TLRs) on AMs2, causing AMs to secrete proinflammatory cytokines3 that activate alveolar epithelial receptors4, leading to recruitment of neutrophils that engulf pathogens5,6. However, since the AM-induced immune response could itself cause tissue injury, it is unclear how AMs modulate the response to prevent injury. Here, through real-time alveolar imaging in situ, we show that a subset of AMs attached to the alveolar wall, formed connexin 43 (Cx43)-containing gap junctional channels (GJCs) with the epithelium. During lipopolysaccharide (LPS)-induced inflammation, the AMs remained alveolus-attached and sessile, and they established intercommunication through synchronized Ca2+ waves, using the epithelium as the conducting pathway. The intercommunication was immunosuppressive, involving Ca2+ dependent activation of Akt, since AM-specific knockout of Cx43 enhanced alveolar neutrophil recruitment and secretion of proinflammatory cytokines in the bronchoalveolar lavage (BAL). The picture emerges of a novel immunomodulatory process in which a subset of alveolus-attached AMs intercommunicates immunosuppressive signals to reduce endotoxin-induced lung inflammation.
PMCID: PMC4117212  PMID: 24463523
20.  The innate immune response of equine bronchial epithelial cells is altered by training 
Veterinary Research  2015;46(1):3.
Respiratory diseases, including inflammatory airway disease (IAD), viral and bacterial infections, are common problems in exercising horses. The airway epithelium constitutes a major physical barrier against airborne infections and plays an essential role in the lung innate immune response mainly through toll-like receptor (TLR) activation. The aim of this study was to develop a model for the culture of equine bronchial epithelial cells (EBEC) in vitro and to explore EBEC innate immune responses in trained horses. Bronchial epithelial biopsies were taken from 6 adult horses during lower airway endoscopy. EBEC were grown in vitro by an explant method. The innate immune response of EBEC was evaluated in vitro by treatment with TLR ligands. TLR3 is the most strongly expressed TLR at the mRNA level in EBEC and stimulation of EBEC with Poly(I:C), an analog of viral dsRNA, triggers a strong secretion of IFN-β, TNF-α, IL-6 and CXCL8. We further evaluated the EBEC innate immune response in horses that underwent a 4-month-training program. While training had no effect on TLR mRNA expression in EBEC as well as in bronchial biopsies, it increased the production of IFN-β after stimulation with a TLR3 ligand and decreased the secretion of TNF-α and IL-6 after stimulation with a TLR2 and TLR3 ligand. These findings may be implicated in the increased risk for viral and bacterial infections observed in sport horses. Altogether, we report a successful model for the culture of EBEC that can be applied to the investigation of pathophysiologic conditions in longitudinal studies.
PMCID: PMC4297379  PMID: 25595212
21.  Airway Epithelial Expression of Toll-like Receptor 5 is Down-regulated in Healthy Smokers and Smokers with Chronic Obstructive Pulmonary Disease 
The toll-like receptors (TLRs) are important components of the respiratory epithelium host innate defense, enabling the airway surface to recognize and respond to a variety of insults in inhaled air. Based on the knowledge that smokers are more susceptible to pulmonary infection and that the airway epithelium of smokers with chronic obstructive pulmonary disease (COPD) is characterized by bacterial colonization and acute exacerbation of airway infections, we assessed whether smoking alters expression of TLRs in human small airway epithelium, the primary site of smoking-induced disease. Microarrays were used to survey the TLR family gene expression in small airway (10th–12th order) epithelium from healthy nonsmokers (n=60), healthy smokers (n=73) and smokers with COPD (n=36). Using the criteria of detection call of present in ≥50%, 6 of 10 TLRs (1, 2, 3, 4, 5 and 8) were expressed. Compared to nonsmokers, the most striking change was for TLR5, which was down-regulated in healthy smokers (1.4-fold, p<10−10) and smokers with COPD (1.6-fold, p<10−11). TaqMan RT-PCR confirmed these observations. Bronchial biopsy immunofluorescence studies showed that TLR5 was expressed mainly on the apical side of the epithelium and was decreased in healthy smokers and smokers with COPD. In vitro, the level of TLR5 downstream genes, IL-6 and IL-8, were highly induced by flagellin in TLR5 high-expressing cells compared to TLR5 low-expressing cells. In the context that TLR5 functions to recognize pathogens and activate innate immune responses, the smoking-induced down-regulation of TLR5 may contribute to smoking-related susceptibility to airway infection, at least for flagellated bacteria.
PMCID: PMC3579667  PMID: 22855713
22.  Human Airway Epithelial Cells Sense Pseudomonas aeruginosa Infection via Recognition of Flagellin by Toll-Like Receptor 5  
Infection and Immunity  2005;73(11):7151-7160.
Pseudomonas aeruginosa, an opportunistic respiratory pathogen that infects the majority of patients with cystic fibrosis, initiates host inflammatory responses through interaction with airway epithelial cells. The Toll-like receptors (TLRs) are a family of pathogen pattern recognition receptors that play key roles in host innate immunity. In this study we aimed to determine whether TLRs mediate the interaction between P. aeruginosa and airway epithelial cells. Individual murine TLRs (TLR1 to TLR9) and dual combinations of these TLRs that activate an NF-κB-driven luciferase reporter in response to PAO1 were screened in HEK 293 cells. TLR5, TLR2, a combination of TLR1 and TLR2, or a combination of TLR2 and TLR6 responded to PAO1. Another P. aeruginosa strain, strain PAK, activated TLR5 similarly, while the isogenic flagellin-deficient strain PAK/fliC and the flagellum-free bacterium Haemophilus influenzae failed to activate TLR5. Reverse transcription-PCR was used to probe the presence of multiple TLRs (including TLR5) in primary human airway epithelial cells (HAECs). Immunostaining with TLR5 antibodies showed that TLR5 was expressed in HAECs and on the apical surface of the human trachea epithelium. In HAECs, PAO1, PAK, and Burkholderia cepacia, but not flagellin-deficient strain PAK/fliC or a B. cepacia fliC mutant, activated the NF-κB reporter. Dominant negative TLR5 specifically blocked the response to P. aeruginosa but not to the response to lipoteichoic acid, a specific ligand of TLR2. We also determined that MyD88, IRAK, TRAF6, and Toll-interacting protein (Tollip), but not TIRAP, were involved in the TLR-mediated response to P. aeruginosa in HAECs. These findings demonstrate that the airway epithelial receptor TLR5 senses P. aeruginosa through its flagellin protein, which may have an important role in the initiation of the host inflammatory reaction to clear the invading pathogen.
PMCID: PMC1273914  PMID: 16239509
23.  Silencing airway epithelial cell-derived hepcidin exacerbates sepsis-induced acute lung injury 
Critical Care  2014;18(4):470.
The production of antimicrobial peptides by airway epithelial cells is an important component of the innate immune response to pulmonary infection and inflammation. Hepcidin is a β-defensin-like antimicrobial peptide and acts as a principal iron regulatory hormone. Hepcidin is mostly produced by hepatocytes, but is also expressed by other cells, such as airway epithelial cells. However, nothing is known about its function in lung infections and inflammatory diseases. We therefore sought to investigate the role of airway epithelial cell-derived hepcidin in sepsis-induced acute lung injury.
Acute lung injury was induced by polymicrobial sepsis via cecal ligation and puncture (CLP) surgery. Adenovirus-mediated short hairpin RNA specific for the mouse hepcidin gene hepc1 and control adenovirus were intratracheally injected into mice. The adenovirus-mediated knockdown of hepcidin in airway epithelial cells was evaluated in vivo. Lung injury and the seven-day survival rate were assessed. The levels of hepcidin-related iron export protein ferroportin were measured, and the iron content and function of alveolar macrophages were evaluated.
The hepcidin level in airway epithelial cells was upregulated during polymicrobial sepsis. The knockdown of airway epithelial cell-derived hepcidin aggravated the polymicrobial sepsis-induced lung injury and pulmonary bacterial infection and increased mortality (53.33% in Ad-shHepc1-treated mice versus 12.5% in Ad-shNeg-treated mice, P <0.05). The knockdown of hepcidin in airway epithelial cells also led to reduced ferroportin degradation and a low intracellular iron content in alveolar macrophages. Moreover, alveolar macrophages form the airway epithelial cell-derived hepcidin knockdown mice showed impaired phagocytic ability than those from the control mice.
Airway epithelial cell-derived hepcidin plays an important role in CLP-induced acute lung injury. The severe lung injury in the airway epithelial cell-derived hepcidin knockdown mice is at least partially related to the altered intracellular iron level and function of alveolar macrophages.
PMCID: PMC4243715  PMID: 25096529
24.  Chlamydia pneumoniae Infection Induced Allergic Airway Sensitization Is Controlled by Regulatory T-Cells and Plasmacytoid Dendritic Cells 
PLoS ONE  2011;6(6):e20784.
Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2−/−, and TLR4−/− mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2−/− mice, but not in TLR4−/− mice, due to differential Treg responses in these genotypes. TLR2−/− mice had reduced numbers of Tregs in the lung during CP infection while TLR4−/− mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs.
PMCID: PMC3112152  PMID: 21695198
25.  A Toll-Like Receptor-Responsive Kinase, Protein Kinase R, Is Inactivated in Endotoxin Tolerance through Differential K63/K48 Ubiquitination 
mBio  2010;1(5):e00239-10.
Overwhelming inflammation triggered by systemic infection in bacterial sepsis contributes to the pathology of this condition. Toll-like receptors (TLRs) are important in early septic inflammation. As a safeguard, the innate immune system has evolved to counter excessive inflammation through the induction of “tolerance.” In endotoxin tolerance, TLR signaling is inhibited and/or attenuated by multiple mechanisms that mitigate the ability of lipopolysaccharide (LPS) to activate critical kinases through TLR4. Here, we describe a novel mechanism. Protein kinase R (PKR), a kinase normally activated by a subset of TLRs, is rendered unresponsive to LPS in endotoxin-tolerized cells. In its naive state, PKR is subject to K63-linked ubiquitination (Ub), followed by K48-linked Ub, in response to LPS. In tolerance, the kinetics of this differential Ub is altered, resulting in a predominance of K48-linked chains, concomitant with a loss of PKR activation. These findings provide a novel mechanism by which a TLR-responsive kinase may be rendered inactive in tolerance.
“Endotoxin tolerance” is a period of transient unresponsiveness to the lipopolysaccharide (LPS) outer membrane component of Gram-negative bacteria that is induced by prior exposure to LPS through Toll-like receptor 4 (TLR4). The loss of LPS-inducible cytokine production by macrophages from patients who have experienced Gram-negative sepsis is well documented, and the increased susceptibility of such patients to reinfection has been attributed to the development of endotoxin tolerance. Multiple mechanisms have been proffered to account for this attenuated response. Using the LPS-responsive kinase protein kinase R (PKR), we have identified differential K48 versus K63 ubiquitination as an additional molecular mechanism by which signal-transducing elements may be inactivated in a state of endotoxin tolerance. This work is highly significant because it links recent discoveries concerning the important role of ubiquitination of signaling molecules in regulating TLR signaling with the loss of LPS responsiveness in tolerance.
PMCID: PMC2962435  PMID: 20978539

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