A Toll-Like Receptor-Responsive Kinase, Protein Kinase R, Is Inactivated in Endotoxin Tolerance through Differential K63/K48 Ubiquitination

ABSTRACTOverwhelming 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.IMPORTANCE“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.

Download Full-text

Role of Toll-like Receptor 4 Mutations in Gram-Negative Septic Shock

Archives of Internal Medicine ◽

10.1001/archinte.162.21.2496 ◽

2002 ◽

Vol 162 (21) ◽

pp. 2496-2496 ◽

Cited By ~ 2

Author(s):

S. Yende

Keyword(s):

Septic Shock ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Gram Negative

Download Full-text

Central Role of Toll-Like Receptor 4 Signaling and Host Defense in Experimental Pneumonia Caused by Gram-Negative Bacteria

Infection and Immunity ◽

10.1128/iai.73.1.532-545.2005 ◽

2005 ◽

Vol 73 (1) ◽

pp. 532-545 ◽

Cited By ~ 90

Author(s):

Jill R. Schurr ◽

Erana Young ◽

Pat Byrne ◽

Chad Steele ◽

Judd E. Shellito ◽

...

Keyword(s):

Gene Expression ◽

Adaptor Protein ◽

Mouse Strains ◽

Gram Negative Bacteria ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling ◽

Gram Negative ◽

Experimental Pneumonia

ABSTRACT Toll-like receptor 4 (TLR4) has been identified as a receptor for lipopolysaccharide. However, the precise role of TLR4 in regulating gene expression in response to an infection caused by gram-negative bacteria has not been fully elucidated. The role of TLR4 signaling in coordinating gene expression was assessed by gene expression profiling in lung tissue in a mouse model of experimental pneumonia with a low-dose infection of Klebsiella pneumoniae. We analyzed four mouse strains: C57BL/6 mice, which are resistant to bacterial dissemination; 129/SvJ mice, which are susceptible; C3H/HeJ mice, which are susceptible and have defective TLR4 signaling; and their respective control strain, C3H/HeN (intermediate resistance). At 4 h after infection, C57BL/6 and C3H/HeN mice demonstrated the greatest number of genes, with 67 shared induced genes which were TLR4 dependent and highly associated with the resistance phenotype. These genes included cytokine and chemokine genes required for neutrophil activation or recruitment, growth factor receptors, MyD88 (a critical adaptor protein for TLR signaling), and adhesion molecules. TLR4 signaling accounted for over 74% of the gene expression in the C3H background. These data suggest that early TLR4 signaling controls the vast majority of gene expression in the lung in response to an infection caused by gram-negative bacteria and that this subsequent gene expression determines survival of the host.

Download Full-text

Role ofToll-Like Receptor 4 in Gram-Positive and Gram-Negative Pneumonia inMice

Infection and Immunity ◽

10.1128/iai.72.2.788-794.2004 ◽

2004 ◽

Vol 72 (2) ◽

pp. 788-794 ◽

Cited By ~ 171

Author(s):

Judith Branger ◽

Sylvia Knapp ◽

Sebastiaan Weijer ◽

Jaklien C. Leemans ◽

Jennie M. Pater ◽

...

Keyword(s):

Immune Response ◽

Mutant Mice ◽

Respiratory Pathogen ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Gram Positive ◽

Gram Negative ◽

High Level ◽

Bacterial Outgrowth

ABSTRACT To determine the role of Toll-like receptor 4 (TLR4) in the immune response to pneumonia, C3H/HeJ mice (which display a mutant nonfunctional TLR4) and C3H/HeN wild-type mice were intranasally infected with either Streptococcus pneumoniae (a common gram-positive respiratory pathogen) or Klebsiella pneumoniae (a common gram-negative respiratory pathogen). In cases of pneumococcal pneumonia, TLR4 mutant mice showed a reduced survival only after infection with low-level bacterial doses, which was associated with a higher bacterial burden in their lungs 48 h postinfection. In Klebsiella pneumonia, TLR4 mutant mice demonstrated a shortened survival after infection with either a low- or a high-level bacterial dose together with an enhanced bacterial outgrowth in their lungs. These data suggest that TLR4 contributes to a protective immune response in both pneumococcal and Klebsiella pneumonia and that its role is more important in respiratory tract infection caused by the latter (gram-negative) pathogen.

Download Full-text

MyD88, but Not Toll-Like Receptors 4 and 2, Is Required for Efficient Clearance of Brucella abortus

Infection and Immunity ◽

10.1128/iai.73.8.5137-5143.2005 ◽

2005 ◽

Vol 73 (8) ◽

pp. 5137-5143 ◽

Cited By ~ 67

Author(s):

David S. Weiss ◽

Kiyoshi Takeda ◽

Shizuo Akira ◽

Arturo Zychlinsky ◽

Edgardo Moreno

Keyword(s):

Myeloid Differentiation ◽

Toll Like Receptor ◽

Immunostimulatory Activity ◽

Toll Like Receptor 4 ◽

Gram Negative ◽

Macrophage Response ◽

Tlr4 Activation ◽

Myeloid Differentiation Factor ◽

Kinetics Of

ABSTRACT It is not clear how the host initially recognizes and responds to infection by gram-negative pathogenic Brucella spp. It was previously shown (D. S. Weiss, B. Raupach, K. Takeda, S. Akira, and A. Zychlinsky, J. Immunol. 172:4463-4469, 2004) that the early macrophage response against gram-negative bacteria is mediated by Toll-like receptor 4 (TLR4), which signals in response to lipopolysaccharide (LPS). Brucella, however, has a noncanonical LPS which does not have potent immunostimulatory activity. We evaluated the kinetics of TLR4 activation and the cytokine response in murine macrophages after Brucella infection. We found that during infection of macrophages, Brucella avoids activation of TLR4 at 6 h but activates TLR4, TLR2, and myeloid differentiation factor 88 (MyD88) at 24 h postinfection. Interestingly, even though its activation is delayed, MyD88 is important for host defense against Brucella infection in vivo, since MyD88−/− mice do not clear the bacteria as efficiently as wild-type, TLR4−/−, TLR2−/−, or TLR4/TLR2−/− mice.

Download Full-text

Platelet-bound lipopolysaccharide enhances Fc receptor–mediated phagocytosis of IgG-opsonized platelets

Blood ◽

10.1182/blood-2006-12-062695 ◽

2007 ◽

Vol 109 (11) ◽

pp. 4803-4805 ◽

Cited By ~ 91

Author(s):

John W. Semple ◽

Rukhsana Aslam ◽

Michael Kim ◽

Edwin R. Speck ◽

John Freedman

Keyword(s):

Flow Cytometry ◽

Toll Like Receptor ◽

Autoimmune Thrombocytopenia ◽

Toll Like Receptor 4 ◽

Platelet Destruction ◽

Gram Negative ◽

Synergistic Increase

Abstract Platelets express Toll-like receptor 4 (TLR4), and this has been shown to be responsible for the thrombocytopenia induced by lipopolysaccharide (LPS) administration in vivo. We studied the role of LPS in mediating platelet phagocytosis by THP-1 cells in vitro by flow cytometry. Opsonization of platelets with an IgG monoclonal (W6/32) antibody or with IgG autoantibody-positive sera from patients with autoimmune thrombocytopenia (AITP) significantly enhanced platelet phagocytosis (P < .001). In contrast, platelet phagocytosis did not occur if platelets were bound with only LPS. If, however, the LPS-bound platelets were also opsonized with either W6/32 or autoantibody-positive sera with titers greater than 4, there was a significant and synergistic increase in Fc-dependent platelet phagocytosis (P < .001, P = .003, P = .048, and P = .047). These results suggest that, in the presence of antiplatelet antibodies, bacterial products can significantly alter platelet phagocytosis, and this may have relevance to how Gram-negative infections enhance platelet destruction in some patients with AITP.

Download Full-text

Electric Stimulation of Ear Reduces the Effect of Toll-Like Receptor 4 Signaling Pathway on Kainic Acid-Induced Epileptic Seizures in Rats

BioMed Research International ◽

10.1155/2018/5407256 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 6

Author(s):

En-Tzu Liao ◽

Yi-Wen Lin ◽

Chun-Ping Huang ◽

Nou-Ying Tang ◽

Ching-Liang Hsieh

Keyword(s):

Protein Kinase ◽

Signaling Pathway ◽

Kainic Acid ◽

Epileptic Seizure ◽

Electric Stimulation ◽

Epileptic Seizures ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Brain Areas

Epilepsy is a common clinical syndrome with recurrent neuronal discharges in the temporal lobe, cerebral cortex, and hippocampus. Clinical antiepileptic medicines are often ineffective or of little benefit in 30% of epileptic patients and usually cause severe side effects. Emerging evidence indicates the crucial role of inflammatory mediators in epilepsy. The current study investigates the role of toll-like receptor 4 (TLR4) and its underlying mechanisms in kainic acid- (KA-) induced epileptic seizures in rats. Experimental KA injection successfully initiated an epileptic seizure accompanied by increased expression of TLR4 in the prefrontal cortex, hippocampus, and somatosensory cortex. In addition, calcium-sensitive phosphorylated Ca2+/calmodulin-dependent protein kinase II (pCaMKIIα) increased after the initiation of the epileptic seizure. Furthermore, downstream-phosphorylated signal-regulated kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 kinase simultaneously increased in these brain areas. Moreover, the transcriptional factor phosphorylated nuclear factor-κB (pNF-κB) increased, suggesting that nucleus transcription was affected. Furthermore, the aforementioned molecules decreased by an electric stimulation (ES) of either 2 Hz or 15 Hz of the ear in the three brain areas. Accordingly, we suggest that ES of the ear can successfully control epileptic seizures by regulating the TLR4 signaling pathway and has a therapeutic benefit in reducing epileptic seizures.

Download Full-text

Pretreatment with Propofol Reduces Pulmonary Injury in a Pig Model of Intestinal Ischemia-Reperfusion via Suppressing the High-Mobility Group Box 1 Protein (HMGB1)/Toll-Like Receptor 4 (TLR4)/Protein Kinase R (PKR) Signaling Pathway

Medical Science Monitor ◽

10.12659/msm.930478 ◽

2021 ◽

Vol 27 ◽

Author(s):

Wen-yu Bian ◽

Ya-ping Chen ◽

Bo Xu ◽

Jun Tang

Keyword(s):

Protein Kinase ◽

Signaling Pathway ◽

Intestinal Ischemia ◽

Ischemia Reperfusion ◽

High Mobility ◽

Pulmonary Injury ◽

Toll Like Receptor ◽

Protein Kinase R ◽

Toll Like Receptor 4 ◽

Intestinal Ischemia Reperfusion

Download Full-text

Toll-Like Receptor 4 Protects Against Clostridium perfringens Infection in Mice

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.633440 ◽

2021 ◽

Vol 11 ◽

Author(s):

Masaya Takehara ◽

Keiko Kobayashi ◽

Masahiro Nagahama

Keyword(s):

Clostridium Perfringens ◽

Gram Negative Bacteria ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Gram Positive ◽

Gram Negative ◽

Tlr4 Agonist ◽

Innate Ability ◽

Recognition Receptor

Toll-like receptor 4 (TLR4) has been reported to protect against Gram-negative bacteria by acting as a pathogen recognition receptor that senses mainly lipopolysaccharide (LPS) from Gram-negative bacteria. However, the role of TLR4 in Gram-positive bacterial infection is less well understood. Clostridium perfringens type A is a Gram-positive bacterium that causes gas gangrene characterized by severe myonecrosis. It was previously demonstrated that C. perfringens θ-toxin is a TLR4 agonist, but the role of TLR4 in C. perfringens infection is unclear. Here, TLR4-defective C3H/HeJ mice infected with C. perfringens showed a remarkable decrease in survival rate, an increase in viable bacterial counts, and accelerated destruction of myofibrils at the infection site compared with wild-type C3H/HeN mice. These results demonstrate that TLR4 plays an important role in the elimination of C. perfringens. Remarkable increases in levels of inflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF), were observed in C. perfringens-infected C3H/HeN mice, whereas the increases were limited in C3H/HeJ mice. Generally, increased G-CSF accelerates granulopoiesis in the bone marrow and the spleen to exacerbate neutrophil production, resulting in elimination of bacteria. The number of neutrophils in the spleen was increased in C. perfringens-infected C3H/HeN mice compared with non-infected mice, while the increase was lower in C. perfringens-infected C3H/HeJ mice. Furthermore, DNA microarray analysis revealed that the mutation in TLR4 partially affects host gene expression during C. perfringens infection. Together, our results illustrate that TLR4 is crucial for the innate ability to eliminate C. perfringens.

Download Full-text

Mitochondrial dsRNAs activate PKR and TLR3 to promote chondrocyte degeneration in osteoarthritis

10.1101/2020.06.17.156323 ◽

2020 ◽

Author(s):

Sujin Kim ◽

Keonyong Lee ◽

Yong Seok Choi ◽

Jayoung Ku ◽

Yun Jong Lee ◽

...

Keyword(s):

Immune Response ◽

Protein Kinase ◽

Inflammatory Cytokines ◽

Degenerative Diseases ◽

Toll Like Receptor ◽

Protein Kinase R ◽

Molecular Identity ◽

Synovial Fluids ◽

Elevated Expression

ABSTRACTProtein kinase R (PKR) is an immune response protein that becomes activated by long double-stranded RNAs (dsRNAs). Several studies reported the misactivation of PKR in patients of degenerative diseases including primary osteoarthritis (OA). However, the molecular identity of PKR-activating dsRNAs remains unknown. Here, we investigate the role of mitochondrial dsRNAs (mt-dsRNAs) in the development of OA. We find that in response to OA-mimicking stressors, cytosolic efflux of mt-dsRNAs is increased, leading to PKR activation and subsequent induction of inflammatory cytokines and apoptosis. Moreover, mt-dsRNAs are exported to the extracellular space where they activate toll-like receptor 3. Elevated expression of mt-dsRNAs in the synovial fluids of OA patients further supports our data. Lastly, we show that autophagy protects chondrocytes from mitochondrial dysfunction partly by removing cytosolic mt-dsRNAs. Together, these findings establish the PKR-mt-dsRNA as a critical regulatory axis in OA development and suggest mt-dsRNAs as a potential target in fighting OA.

Download Full-text