TLR4-mutant Mice Research Articles

DNA attenuates enterocyte Toll-like receptor 4-mediated intestinal mucosal injury after remote trauma

Intestinal mucosal injury occurs after remote trauma although the mechanisms that sense remote injury and lead to intestinal epithelial disruption remain incompletely understood. We now hypothesize that Toll-like receptor 4 (TLR4) signaling on enterocytes after remote injury, potentially through the endogenous TLR4 ligand high-mobility group box-1 (HMGB1), could lead to intestinal dysfunction and bacterial translocation and that activation of TLR9 with DNA could reverse these effects. In support of this hypothesis, exposure of TLR4-expressing mice to bilateral femur fracture and systemic hypotension resulted in increased TLR4 expression and signaling and disruption of the ileal mucosa, leading to bacterial translocation, which was not observed in TLR4-mutant mice. TLR4 signaling in enterocytes, not immune cells, was required for this effect, as adenoviral-mediated inhibition of TLR4 in enterocytes prevented these findings. In seeking to identify the endogenous TLR4 ligands involved, the expression of HMGB1 was increased in the intestinal mucosa after injury in wild-type, but not TLR4-mutant, mice, and administration of anti-HMGB1 antibodies reduced both intestinal mucosal TLR4 signaling and bacterial translocation after remote trauma. Strikingly, mucosal injury was significantly increased in TLR9-mutant mice, whereas administration of exogenous DNA reduced the extent of TLR4-mediated enterocyte apoptosis, restored mucosal healing, and maintained the histological integrity of the intestinal barrier after remote injury. Taken together, these findings identify a novel link between remote injury and enterocyte TLR4 signaling leading to barrier injury, potentially through HMGB1 as a ligand, and demonstrate the reversal of these adverse effects through activation of TLR9.

Hypertonic Saline Enhances Host Defense and Reduces Apoptosis in Burn Mice by Increasing Toll-Like Receptors

Hypertonic saline (HTS) is useful in the management of intracranial hypertension and shock patients. The aim of this study was to investigate whether HTS enhances host defense in burn mice through the increase of Toll-like receptors (TLRs) and nuclear factor κB (NF-κB) activation. C57BL/6, TLR4, C3H/HeN, and C3H/HeJ (nonfunctional TLR4 mutant) mice underwent burn and were given 10 mL/kg HTS (7.5% NaCl, 1.28 mol/L), 10 mL/kg saline (154 mmol/L), or 80 mL/kg saline (154 mmol/L) at 8 h after burn. At 24 h after burn, mesenteric lymph nodes were harvested and assayed for bacterial translocation (BT). Next, animals received i.p. Escherichia coli challenge, and bacterial clearance was measured. Finally, peritoneal cells were isolated for assay of bacterial killing activity, phagocytic activity, apoptotic ratio, NF-κB DNA binding activity, and expression of TLR4, MyD88, p-Akt, pp38, macrophage inflammatory protein 2, and Bcl-xL. Hypertonic saline decreased BT in C57BL/6 and C3H/HeN mice but not in TLR4 mutant mice. Also, HTS increased bacterial clearance and bacterial killing activity and decreased apoptotic ratio of peritoneal cells from C57BL/6 and C3H/HeN mice but not TLR4 or C3H/HeJ mice. Finally, HTS increased NF-κB activity and expression of TLR4, MyD88, p-Akt, pp38, macrophage inflammatory protein 2, and Bcl-xL in C57BL/6 but not in TLR4 mice. Hypertonic saline increases bacterial clearance and bacterial killing activity and decreases thermal injury-induced BT in wild-type but not in TLR4 mutant mice. Given that HTS induces NF-κB activity and TLR4, MyD88, and pp38 expression but decreases the apoptosis of inflammatory cells, we conclude that HTS resuscitation enhances host defense against bacterial challenge and reduces apoptosis of inflammatory cells in burn mice by increasing TLR4 expression and NF-κB activation.

Potential Down-Regulation of Salivary Gland AQP5 by LPS via Cross-Coupling of NF-κB and p-c-Jun/c-Fos

The mRNA and protein levels of aquaporin (AQP)5 in the parotid gland were found to be potentially decreased by lipopolysaccharide (LPS) in vivo in C3H/HeN mice, but only weakly in C3H/HeJ, a TLR4 mutant mouse strain. In the LPS-injected mice, pilocarpine-stimulated saliva production was reduced by more than 50%. In a tissue culture system, the LPS-induced decrease in the AQP5 mRNA level was blocked completely by pyrrolidine dithiocarbamate, MG132, tyrphostin AG126, SP600125, and partially by SB203580, which are inhibitors for IkappaB kinase, 26S proteasome, ERK1/2, JNK, and p38 MAPK, respectively. In contrast, the expression of AQP1 mRNA was down-regulated by LPS and such down-regulation was blocked only by SP600125. The transcription factors NF-kappaB (p65 subunit), p-c-Jun, and c-Fos were increased by LPS given in vivo, whereas the protein-binding activities of the parotid gland extract toward the sequences for NF-kappaB but not AP-1-responsive elements present at the promoter region of the AQP5 gene were increased by LPS injection. Co-immunoprecipitation by using antibody columns suggested the physical association of the three transcription factors. These results suggest that LPS-induced potential down-regulation of expression of AQP5 mRNA in the parotid gland is mediated via a complex(es) of these two classes of transcription factors, NF-kappaB and p-c-Jun/c-Fos.

Protein Kinase C-η and Phospholipase D2 Pathway Regulates Foam Cell Formation via Regulator of G Protein Signaling 2

Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for Galpha(q), which is involved in regulating various vascular functions. To understand how RGS2 regulates foam cell formation, the present study identified signaling pathways controlled by lipopolysaccharide (LPS) and discovered new mechanisms whereby protein kinase C (PKC)-eta and phospholipase D (PLD) 2 regulate RGS2 expression. The toll-like receptor (TLR) 4 agonist LPS caused foam cell formation of Raw264.7 macrophages and dramatically decreased RGS2 mRNA expression. RGS2 down-regulation by LPS was partially recovered by TLR4 small interfering RNA (siRNA). Peritoneal macrophages were separated from wild-type and TLR4 mutant mice, and treatment with LPS showed RGS2 expression decrease in wild-type macrophages but no change in TLR4 mutant macrophages. RGS2 overexpression was suppressed, whereas RGS2 down-regulation accelerated foam cell formation by LPS. Treatment of PKC-eta pseudosubstrate weakened foam cell formation and recovered RGS2 down-regulation by LPS. In addition, LPS or phorbol 12-myristate 13-acetate stimulated PLD activity, and the pretreatment of PLD inhibitor weakened foam cell formation and recovered RGS2 down-regulation. Inhibition of PLD2 expression by siRNA also weakened foam cell formation and partially recovered LPS-mediated RGS2 down-regulation. On the other hand, PLD2 overexpression intensified RGS2 down-regulation and foam cell formation by LPS. These results suggest that LPS causes foam cell formation by increasing PKC-eta and PLD2 activity by down-regulating RGS2 expression via TLR4 dependently.

Dominant role of the MyD88-dependent signaling pathway in mediating early endotoxin-induced murine ileus

TLR4 ligation by pathogen-associated molecular patterns, such as Gram-negative bacteria-derived LPS, triggers a nonhematopoietic cell-mediated ileus during early endotoxemia. Our objective was to investigate the quantitative contributions of the two downstream signaling pathways of TLR4, namely the adapter proteins myeloid differentiation primary response gene 88 (MyD88) and Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF). Six hours after intraperitoneal injection of highly purified LPS (UP-LPS, 5 mg/kg), in vivo gastrointestinal transit and intestinal muscularis gene transcripts of inflammatory mediators chemokine (C-X-C motif) ligand 10, synonymous IP-10 (CXCL10), granulomonocyte colony stimulating factor (GM-CSF, synonymous CSF-2), IL-1beta, IL-6, IL-10, and inducible NO synthase (iNOS) were assessed in mice with transgenic loss-of-function for MyD88 or TRIF. LPS-induced MyD88 and TRIF mRNA upregulation was quantified within the intestinal muscularis of TLR4-competent and TLR4-mutant mice, and MyD88 mRNA levels were additionally measured in TLR4 bone marrow chimeras. MyD88 deficiency completely protected mice from early endotoxin-induced ileus, while TRIF deficiency partially ameliorated ileus severity. LPS induction of the primary downstream signaling element MyD88 was TLR4 dependent and was derived in equal amounts from both the hematopoietic and the nonhematopoietic cells. Conversely, no induction of TRIF mRNA was detectable. Significant gene induction of all inflammatory mediators was dependent on intracellular signal transduction by MyD88, while the TRIF MyD88-independent pathway predominantly regulated the molecular levels of CXCL10. In summary, MyD88 and TRIF are nonredundant signaling pathways in early endotoxin-induced rodent ileus, but MyD88 is the essential adaptor molecule for transduction of early TLR4-induced ileus and inflammatory signaling. The dependency of ileus on individual adaptor protein pathways is also reflected in the manifestation of specific molecular inflammatory events within the intestinal muscularis externa.

Central role of extracellular signal-regulated kinase and Toll-like receptor 4 in IL-10 production in regulatory dendritic cells induced by Trypanosoma cruzi

Several Trypanosoma cruzi molecules that stimulate macrophages activity were described as Toll-like receptor 2 (TLR2) ligands. Besides, the models of dendritic cells (DC) are poorly characterised. We have previously demonstrated that live-trypomastigotes (Tp) plus lipopolysaccharide (LPS) induce DC with tolerogenic properties that produce high levels of interleukin (IL)-10 and an impaired capacity to induce lymphoproliferation. Here, we show that the regulatory phenotype was observed with heat-killed trypomastigotes (Tphk) stimulation, ruling out DC infection. T. cruzi induced a particular DC activation state increasing LPS-activation of extracellular regulated kinase (ERK) 1/2 and signal transducer and activator of transcription (STAT) 3. Inhibition of ERK down-regulated IL-10 production and restored DC stimulatory capacity, showing the importance of this pathway in the DC modulation. A recent work shows that signalling via TLR4 and TLR2 induces a synergism in anti-inflammatory cytokine production in murine DC. Upon TLR2 and TLR4 stimulation using Pam 3Cys or LPS and Tphk in DC from TLR2 knock out (KO) or TLR4-mutant mice, we showed that high levels of IL-10 were independent of TLR2 but associated with TLR4 and NF-κB signallization. Although sialic acid has been described as a molecule responsible of DC inhibition, we determine that it is not associated with T. cruzi-IL-10 modulatory response. In conclusion, all these findings demonstrate a key role of ERK and TLR4 in association with NF-κB in IL-10 modulation induced by T. cruzi and suggest that this regulatory effect involves parasite–DC interactions not described yet.

PS204. Toll-like Receptors 2 and 4, but Not RAGE Deficiency Results in Greater Muscle Necrosis 2 Weeks After Hind-limb Ischemia: Implications for Muscle Recovery After Ischemic Injury

Innate immunity is the first line of defense against invading pathogens through receptors such as TLR2 and TLR4. These receptors also mediate responses to tissue injury through endogenous danger signals. We have shown that HMGB1, a danger signal, is released by hypoxic cells and promotes angiogenesis in vitro. Anti-HMGB1 antibody increases muscle necrosis in murine hind-limb ischemia but did not change endothelial density, supporting a role for HMGB1 and innate immunity in skeletal muscle recovery following ischemia. In this study, we examined the roles of the HMGB1 receptors TLR2, TLR4 and RAGE in mediating this protection. 10-12 wk old mice genetically deficient for TLR4 (C3H/HeJ, control HeOuJ; N = 4), TLR2 (N = 3), or RAGE (N = 4; control C57B/6, N = 4) underwent right femoral artery ligation. At 2wks, calf muscles were fixed, sectioned, and stained with H&E to quantify muscle necrosis (% area) and vWF for endothelial density. Statistical significance was determined by ANOVA. TLR4 mutant mice developed greater muscle necrosis following hind-limb ischemia than control HeOuJ mice (16.4% ± 4.6 vs 4.6 ± 2.1; p = 0.04). Muscle necrosis was also markedly increased in TLR2 KO (23.5 ± 17.3; P-0.04) while no necrosis was detected in the C57B/6 controls. RAGE KO mice also showed little necrosis. In contrast, the C57B/6 and RAGE KO mice exhibited significant muscle regeneration in the ischemic hind-limb. Endothelial density was similar between the knockout and control mice. The absence of TLR2 and TLR4 signaling is associated with increased skeletal muscle necrosis following ischemia and suggests that these receptors are necessary for muscle protection. In contrast, RAGE signaling is not involved. The protection offered by TLR2 and TLR4 did not involve endothelial proliferation but may involve regenerative processes. These data suggest that TLRs may be critical in tissue recovery from ischemia independent of angiogenesis.

10 Altered Gastrointestinal Motility and Reduced nNOS Neurons in TLR4 Mutant-Mice

10 Altered Gastrointestinal Motility and Reduced nNOS Neurons in TLR4 Mutant-Mice

M1675 Presence of Anti-Microbial Antibodies is Associated With Increased Risk for Systemic Infections in a Large Hungarian Cohort of Patients With Liver Cirrhosis

DNA fragmentation. Total RNA was isolated and IL-8, CXCL1, CXCL2, CCL2, ICAM, VCAM mRNA expression was determined by real time RT-PCR. NF-kB activation was determined by luciferase assay. Results: Exposure of LX2 cells to palmitate significantly induces IL-8 production and gene expression, as well as TLR4, CXCL1, CXCL2, CCL2, ICAM, VCAM mRNA expression. Palmitate induced IL-8 production can not be blocked by pan-caspase inhibitor. However, palmitate induced MIP-2 (rodent homolog of human IL-8) production was completely abrogated in HSCs from TLR4-mutant (C3H/HeJ) mice, but not from wildtype (C3H/HeOuJ) mice. Palmitate also induces NF-kB activation in a dose-dependent manner. Conclusions: Our data demonstrate that palmitate directly stimulates IL-8 production in HSCs via TLR4 signaling pathway, suggesting that HSCs play an important role in FFAs induced liver inflammation and injury.

Role of Toll‐like receptor signaling in hepatocytes in regulating the gene expression of drug metabolizing enzymes

In inflammation, drug metabolism and clearance is altered due to suppression of drug metabolizing enzymes (DMEs) and their regulatory nuclear receptors (NRs: PXR, CAR and RXRα). Inflammatory responses are mediated by cytokines produced by activation of Toll‐like receptors (TLRs) on liver macrophages or Kupffer cells (KCs). Cytokines act on hepatocytes to suppress gene expression. However, hepatocytes also express TLRs, and we hypothesize that TLRs on hepatocytes are directly involved in suppression of hepatic DMEs. Primary mouse hepatocytes from TLR4+/+ (C3HeB/FeJ) and TLR4‐mutant (C3H/HeJ) mice were treated with LPS (1 μg/ml) or saline. RNA and protein levels were analyzed by real‐time PCR and immunoblotting, respectively. The purity of the hepatocytes was assessed by CD68 staining for KC contamination. RNA levels of the major DMEs, Cyp3a11 and Ugt1a1 were reduced ~60% by LPS in TLR4+/+, but not TLR4‐mutant hepatocytes. The cell‐signaling components, JNK and NF‐κB, known regulators of NRs and DMEs were activated in TLR4+/+ and not TLR4‐mutant cells. Nuclear protein levels of RXRα were rapidly reduced after LPS treatment of TLR4+/+, but not TLR4‐mutant hepatocytes. Treatment of hepatocytes with the KC inhibitor, Gadolinium chloride did not attenuate the effect of LPS. Thus, both cytokine‐dependent and independent pathways are involved in the regulation of hepatic gene expression in response to LPS signals.

Hemorrhagic shock primes for IL-1β processing: role of upregulated TLR2 (136.1)

Abstract IL-1β contributes to post-trauma sepsis, and its release requires processing in inflammasomes. Hemorrhagic shock (HS) primes for IL-1β release in response to LPS via yet unclear mechanisms. We have reported that HS-activated neutrophils (PMN) augment LPS/TLR4 signaling upregulation of TLR2 in alveolar macrophages (AM), thereby sensitizing the AM to TLR2 ligands. Here, we addressed the role of upregulated TLR2 in the HS-primed IL-1β processing. WT, TLR4-mutant and TLR2 KO mice were subjected to HS, followed by intratracheal LPS (30 µg/kg B.W.) and PGN (30 µg/kg B.W.) at 2h and 4h after HS, respectively. Bronchoalveolar lavage fluids (BALF) were collected at 2 h after PGN for detection of IL-1β. In vitro, AM were co-cultured with PMN isolated from HS mice, and treated with LPS, PMN was then removed and PGN was added at 2h. IL-1β and caspase-1 maturation in the AM were assessed. In vivo treatments of LPS and PGN caused a marked increase in IL-1β in BALF in the WT/HS mice, but not in the WT/sham, TLR4-mutant/HS or TLR2 KO/HS mice. HS-activated PMN increased TLR2 expression in AM in response to LPS, and subsequently increased caspase-1 and IL-1β maturation in the AM as well as IL-1β release in response to PGN. Moreover increasing extracellular [K+] failed to prevent the TLR2 signal-induced IL-1β maturation. In HS, amplified TLR2 signaling acts as a second signal in stimulating caspase-1 and IL-1β maturation via mechanisms independent of K+ efflux.

Cooperative Interactions between TLR4 and TLR9 Regulate Interleukin 23 and 17 Production in a Murine Model of Gram Negative Bacterial Pneumonia

Toll like receptors play an important role in lung host defense against bacterial pathogens. In this study, we investigated independent and cooperative functions of TLR4 and TLR9 in microbial clearance and systemic dissemination during Gram-negative bacterial pneumonia. To access these responses, wildtype Balb/c mice, mice with defective TLR4 signaling (TLR4lps-d), mice deficient in TLR9 (TLR9−/−) and TLR4/9 double mutant mice (TLR4lps-d/TLR9−/−) were challenged with K. pneumoniae, then time-dependent lung bacterial clearance and systemic dissemination determined. We found impaired lung bacterial clearance in TLR4 and TLR9 single mutant mice, whereas the greatest impairment in clearance was observed in TLR4lps-d/TLR9−/− double mutant mice. Early lung expression of TNF-α, IL-12, and chemokines was TLR4 dependent, while IFN-γ production and the later expression of TNF-α and IL-12 was dependent on TLR9. Classical activation of lung macrophages and maximal induction of IL-23 and IL-17 required both TLR4 and TLR9. Finally, the i.t. instillation of IL-17 partially restored anti-bacterial immunity in TLR4lps-d/TLR9−/− double mutant mice. In conclusion, our studies indicate that TLR4 and TLR9 have both non-redundant and cooperative roles in lung innate responses during Gram-negative bacterial pneumonia and are both critical for IL-17 driven antibacterial host response.

Remote Ischemic Preconditioning by Hindlimb Occlusion Prevents Liver Ischemic/Reperfusion Injury

Hepatocellular injury caused by ischemia-reperfusion of the liver occurs in a number of clinical situations including major trauma, elective surgery of the liver, and liver transplantation. Several strategies have been used to prevent liver injury following ischemia-reperfusion (I/R). Among these, ischemic preconditioning has shown promise as a preventative approach. In this manuscript, we hypothesized that use of remote ischemic preconditioning by brief hindlimb ischemia might prevent liver dysfunction in a mouse model of liver I/R. C57/B mice were subjected to 60 minutes of partial liver ischemia with or without antecedent hindlimb vascular I/R. Blood was drawn for serum alanine aminotransferase levels at times following liver reperfusion. Liver inflammation was assessed by measuring serum and liver tumor necrosis factor (TNF)-alpha protein and mRNA. The role of toll-like receptor 4 (TLR4) in mediating protection was determined using the mouse strain HeJ, which has a mutated TLR4. Antecedent hindlimb ischemia (10 minutes) lessened I/R-induced elevation of serum alanine aminotransferase compared with untreated I/R animals. This protection correlated with a reduction in serum TNF-alpha protein levels as well as liver TNF-alpha mRNA and apoptosis. High Mobility Group-Box 1 (HMG-B1) levels in the blood were elevated after hindlimb ischemia and injection of HMG-B1 prior to liver recapitulated the protective effect of hindlimb occlusion. TLR4-mutant HeJ mice did not demonstrate protection with hindlimb preconditioning. Brief hindlimb occlusion prevents liver I/R injury. This effect appears to be related to release of HMG-B1 and is dependent on the presence of a functional TLR4. Remote ischemia preconditioning represents a novel approach to preventing distant organ injury.

Commensal microflora induce host defense and decrease bacterial translocation in burn mice through toll-like receptor 4

BackgroundMajor burn is associated with decreased gut barrier function and increased bacterial translocation (BT). This study is to investigate whether commensal microflora induce host defense and decrease BT in burn mice.MethodsFirst, we treated Wild type (WT) mice with antibiotics in drinking water for 4 weeks to deplete gut commensal microflora. At week 3, drinking water was supplemented with lipopolysaccharide (LPS); a ligand for TLR4, to trigger TLRs in gut. The intestinal permeability, glutathione level, NF-κB DNA-binding activity, TLR4 expression of intestinal mucosa, BT to mesenteric lymph nodes (MLNs), and bacterial killing activity of peritoneal cells were measured after thermal injury. Second, lung of animals were harvested for MPO activity and TNFα mRNA expression assay. Third, WT animals were treated with oral antibiotics with or without LPS supplement after burn. At 48 hr after burn, TLR4 expression of intestinal mucosa and bacterial killing activity of cells were examined. Finally, bacterial killing activity and BT to MLNs after thermal injury in C3H/HeJ (TLR4 mutant) mice were measured.ResultsBurn induced BT to MLNs in WT mice. Commensal depletion decreased TLR4 expression as well as NF-κB activation of intestine, myeloperoxidase (MPO) activity as well as TNFα expression of lung, and bacterial killing activity of peritoneal cells. Oral LPS supplement markedly reduced 81% of burn-induced BT and increased TLR4 expression, MPO activity of lung, as well as bacterial killing activity of peritoneal cells. LPS supplement did not change BT or bacterial killing activity in C3H/HeJ mice.ConclusionsCollectively, commensal microflora induce TLR4 expression of intestine and bacterial killing activity of inflammatory cells in burn. TLR4 ligand increases bacterial killing activity and decreases burn-induced BT. Taken together with the abolition of LPS effect in TLR4 mutant mice, we conclude that commensal microflora induce host defense and decrease bacterial translocation in burn mice through toll-like receptor 4.

The role of Toll-like receptor 4 in eliciting acquired immune responses against nontypeable Haemophilus influenzae following intranasal immunization with outer membrane protein

Objective Acute otitis media (AOM) is one of the most common infectious diseases in children. Nontypeable Haemophilus influenzae (NTHi) is considered a major pathogen in AOM . Current treatment options depend mainly on the use of antibiotics, thus developing vaccines to prevent this disease is an urgent goal for public health. Outer membrane proteins (OMPs) are promising candidate targets for vaccination against NTHi. Methods We used C3H/HeJ (Toll-like receptor 4 [TLR4]-mutant) mice, which arose spontaneously and have a non-functional TLR4 protein, and normal wild-type (WT) C3H/HeN mice. These mice were immunized intranasally with OMP from NTHi to investigate the mechanism of acquired immunity via TLR4. We examined the kinetics of mucosal and systemic antibody secretion and the migration of antibody producing lymphocytes to the mucosa in both strains during the course of intranasal immunization. Results The mucosal and systemic immune responses against OMP from NTHi were elicited in both TLR4-mutant and WT mice. However, the mucosal IgA, and systemic IgG, and Th1 immune responses in WT mice were stronger than those in TLR4-mutant mice. Conclusions TLR4 plays an important role in relation to Th1 function for optimal development of the acquired immune responses to OMP administered intranasally. The variety of immune responses via TLR4 expression needs to be taken into consideration of individual vaccinations to prevent AOM.

Cross-Talk between TLR4 and FcγReceptorIII (CD16) Pathways

Pathogen-pattern-recognition by Toll-like receptors (TLRs) and pathogen clearance after immune complex formation via engagement with Fc receptors (FcRs) represent central mechanisms that trigger the immune and inflammatory responses. In the present study, a linkage between TLR4 and FcγR was evaluated in vitro and in vivo. Most strikingly, in vitro activation of phagocytes by IgG immune complexes (IgGIC) resulted in an association of TLR4 with FcγRIII (CD16) based on co-immunoprecipitation analyses. Neutrophils and macrophages from TLR4 mutant (mut) mice were unresponsive to either lipopolysaccharide (LPS) or IgGIC in vitro, as determined by cytokine production. This phenomenon was accompanied by the inability to phosphorylate tyrosine residues within immunoreceptor tyrosine-based activation motifs (ITAMs) of the FcRγ-subunit. To transfer these findings in vivo, two different models of acute lung injury (ALI) induced by intratracheal administration of either LPS or IgGIC were employed. As expected, LPS-induced ALI was abolished in TLR4 mut and TLR4−/− mice. Unexpectedly, TLR4 mut and TLR4−/− mice were also resistant to development of ALI following IgGIC deposition in the lungs. In conclusion, our findings suggest that TLR4 and FcγRIII pathways are structurally and functionally connected at the receptor level and that TLR4 is indispensable for FcγRIII signaling via FcRγ-subunit activation.

Extracellular Hsp72, an endogenous DAMP, is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4

BackgroundNeutrophils play an important role in the pathophysiology of RSV, though RSV does not appear to directly activate neutrophils in the lower airways. Therefore locally produced cytokines or other molecules released by virally-infected airway epithelial cells are likely responsible for recruiting and activating neutrophils. Heat shock proteins (HSPs) are generally regarded as intracellular proteins acting as molecular chaperones; however, HSP72 can also be released from cells, and the implications of this release are not fully understood.MethodsHuman bronchial epithelial cells (16HBE14o-) were infected with RSV and Hsp72 levels were measured by Western blot and ELISA. Tracheal aspirates were obtained from critically ill children infected with RSV and analyzed for Hsp72 levels by ELISA. Primary human neutrophils and differentiated HL-60 cells were cultured with Hsp72 and supernatants analyzed for cytokine production. In some cases, cells were pretreated with polymyxin B prior to treatment with Hsp72. IκBα was assessed by Western blot and EMSA's were performed to determine NF-κB activation. HL-60 cells were pretreated with neutralizing antibody against TLR4 prior to Hsp72 treatment. Neutrophils were harvested from the bone marrow of wild type or TLR4-deficient mice prior to treatment with Hsp72.ResultsInfection of 16HBE14o- with RSV showed an induction of intracellular Hsp72 levels as well as extracellular release of Hsp72. Primary human neutrophils from normal donors and differentiated HL-60 cells treated with increasing concentrations of Hsp72 resulted in increased cytokine (IL-8 and TNFα) production. This effect was independent of the low levels of endotoxin in the Hsp72 preparation. Hsp72 mediated cytokine production via activation of NF-κB translocation and DNA binding. Using bone marrow-derived neutrophils from wild type and TLR4-mutant mice, we showed that Hsp72 directly activates neutrophil-derived cytokine production via the activation of TLR4.ConclusionCollectively these data suggest that extracellular Hsp72 is released from virally infected airway epithelial cells resulting in the recruitment and activation of neutrophils.

TheFasciola hepaticaTegumental Antigen Suppresses Dendritic Cell Maturation and Function

Parasitic worms and molecules derived from them have powerful anti-inflammatory properties and are shown to have therapeutic effects on inflammatory diseases. The helminth Fasciola hepatica has been reported to suppress antigen-specific Th1 responses in concurrent bacterial infections, thus demonstrating its anti-inflammatory ability in vivo. Here, F. hepatica tegumental antigen (Teg) was shown to significantly suppress serum levels of gamma interferon (IFN-gamma) and interleukin-12p70 (IL-12p70) in a model of septic shock. Since dendritic cells (DCs) are a good source of IL-12p70 and critical in driving adaptive immunity, we investigated the effects of F. hepatica Teg on the activation and function of murine DCs. While Teg alone did not induce cytokine production or cell surface marker expression on DCs, it significantly suppressed cytokine production (IL-12p70, IL-6, IL-10, tumor necrosis factor alpha, and nitrite) and cell surface marker expression (CD80, CD86, and CD40) in DCs matured with a range of Toll-like receptor (TLR) and non-TLR ligands. Teg works independently of the TLR4 pathway, since it still functioned in DCs generated from TLR4 mutant and knockout mice. It impaired DC function by inhibiting their phagocytic capacity and their ability to prime T cells. It does not appear to target the common components (extracellular signal-regulated kinase, Jun N-terminal protein kinase, or p38) of the TLR pathways; however, it suppressed the active p65 subunit of the transcription factor NF-kappaB in mature DCs, which could explain the impairment of proinflammatory cytokine production. Overall, our results demonstrate the potent anti-inflammatory properties of F. hepatica Teg and its therapeutic potential as an anti-inflammatory agent.

Hepatic Flavin-Containing Monooxygenase Gene Regulation in Different Mouse Inflammation Models

The objective of the study was to investigate the regulation of hepatic flavin-containing monooxygenases (Fmo) Fmo1, Fmo3, Fmo4, and Fmo5 in three different mouse models of inflammation, including treatment with Citrobacter rodentium, lipopolysaccharide (LPS), and dextran sulfate sodium (DSS). Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the steady-state mRNA levels for the various Fmo isoforms in these mouse models of inflammation during different treatment time courses. Fmo3 mRNA was most significantly down-regulated in C. rodentium-treated female mice. Fmo1, Fmo3, and Fmo5 mRNAs were also found to be down-regulated in LPS models of inflammation. The significant down-regulation of hepatic FMO3 protein during C. rodentium treatment was confirmed with Western blot analysis of liver microsomes from treated animals. Toll-like receptor (TLR) 4 is known to be responsible for LPS signaling in association with several proteins. To investigate whether TLR4 was responsible for regulation of Fmo genes in both LPS and C. rodentium animal models, Fmo mRNA levels in female wild-type (C3H/HeOuJ) and TLR4 mutant (C3H/HeJ) mice were compared in both inflammatory models by real-time RT-PCR. The results showed that Fmo3 down-regulation during C. rodentium infection is independent of TLR4. Whereas TLR4 is likely to play only a partial role in Fmo1 gene regulation in LPS-treated animals, our results show that the down-regulation of Fmo3 and Fmo5 in this model is TLR4-dependent. Unlike cytochrome P450 regulation measured in the same mouse strains, Fmo3 expression was largely refractory to down-regulation in the DSS model of inflammatory colitis.

Signaling through MyD88 Regulates Leukocyte Recruitment after Brain Injury

Injury to the CNS provokes an innate inflammatory reaction that engages infiltrating leukocytes with the capacity to repair and/or exacerbate tissue damage. The initial cues that orchestrate leukocyte entry remain poorly defined. We have used flow cytometry to investigate whether MyD88, an adaptor protein that transmits signals from TLRs and receptors for IL-1 and IL-18, regulates leukocyte infiltration into the stab-injured entorhinal cortex (EC) and into sites of axonal degeneration in the denervated hippocampus. We have previously established the kinetics of leukocyte entry into the denervated hippocampus. We now show that significant leukocyte entry into the EC occurs within 3-12 h of stab injury. Whereas T cells showed small, gradual increases over 8 days, macrophage infiltration was pronounced and peaked within 12-24 h. MyD88 deficiency significantly reduced macrophage and T cell recruitment to the stab-injured EC and the denervated hippocampus at 5 days post-injury. Whereas macrophage and T cell entry remained impaired into the denervated hippocampus of MyD88-deficient mice at 8 days, leukocyte infiltration into the stab-injured EC was restored to levels observed in wild-type mice. Transcripts for TNF-alpha, IL-1beta, and CCL2, which increased >50-fold after stab injury in C57BL/6 mice at the time of peak expression, were severely reduced in injured MyD88 knockout mice. Leukocyte recruitment and gene expression were unaffected in TLR2-deficient or TLR4 mutant mice. No significant differences in gene expression were observed in mice lacking IL-1R or IL-18R. These data show that MyD88-dependent signaling mediates proinflammatory gene expression and leukocyte recruitment after CNS injury.