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(TRIF)-Mediated Signaling Contributes to Innate Immune Responses in the Lung during Escherichia coli Pneumonia1
* Division of Respiratory Infections, Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206;
Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, CO 80262; and
Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
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Abstract |
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(TRIF) is an adaptor for TLR3 and TLR4, and is associated with the MyD88-independent cascade. However, the importance of TRIF in immune responses against pulmonary bacterial pathogens is not well understood. We investigated the involvement of TRIF in a murine model of Escherichia coli pneumonia. TRIF–/– mice infected with E. coli display attenuated neutrophil migration; NF-
B activation; and TNF-
, IL-6, and LPS-induced C-X-C chemokine production in the lungs. In addition, E. coli-induced phosphorylation of JNK, ERK, and p38 MAPK was detected in bone marrow-derived macrophages (BMMs) of TRIF+/+ mice, but attenuated in BMMs of TRIF–/– mice. Furthermore, E. coli-induced TNF- and IL-6 production was attenuated in BMMs of TRIF–/– mice. E. coli LPS-induced late MAPK activation, and TNF- and IL-6 production were abolished in BMMs of TRIF–/– mice. Moreover, TRIF is not required for LPS-induced neutrophil influx, and keratinocyte cell-derived chemokine, MIP-2, and LPS-induced C-X-C chemokine production in the lungs. Using TLR3–/– mice, we ruled out the role of TLR3-mediated TRIF-dependent neutrophil influx during E. coli pneumonia. A TLR4-blocking Ab inhibited E. coli-induced TNF- and IL-6 in BMMs of both TRIF–/– and TRIF+/+ mice, suggesting that TRIF-mediated signaling involves TLR4. We also found that TRIF is critical to control E. coli burden in the lungs and E. coli dissemination. Thus, rapid activation of TRIF-dependent TLR4-mediated signaling cascade serves to augment pulmonary host defense against a Gram-negative pathogen. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Pathogen recognition is mediated by a set of germline-encoded type I transmembrane proteins termed TLRs (6, 7, 8, 9). These receptors rapidly detect microbial infections based on their unique microbial structures or pathogen-associated molecular patterns. Of 11 characterized TLRs, TLR2, 4, and 5 recognize bacterial peptidoglycan, endotoxin (LPS), and flagellin, respectively (6, 7, 8, 9). A remarkable feature of TLR signaling is that it is analogous to that of IL-1R cascade (10). The cytoplasmic region of TLRs contain Toll/IL-1R (TIR)3 domain, which is critical for interaction between TLRs and TIR-containing adaptors. TLRs recruit adaptor molecules to subsequently activate downstream signaling cascades. Upon ligand binding to TLR4, the cytoplasmic adaptor proteins TIR domain-containing adaptor protein (TIRAP) and MyD88 are recruited to the TLR signaling complex, which results in the early activation of NF-B and subsequent production of cytokines and chemokines, and this cascade is called the MyD88-dependent pathway (11, 12, 13). Activation of TLR also recruits other adaptor proteins TIR domain-containing adaptor inducing IFN- (TRIF) and TRIF-related adaptor molecule (TRAM), and this pathway activates late NF-B and type I IFN response and is called the MyD88-independent pathway (14, 15, 16). Whereas all TLRs (except TLR3) seem to be dependent on MyD88 for their downstream signaling, TLR3 and TLR4 seem to be dependent on TRIF for their downstream cascades (14).
Previous investigations have demonstrated the important roles of adaptor proteins in the MyD88-dependent cascade of TLR4 signaling, such as TIRAP and MyD88, in host defense in the lung. For example, MyD88 is shown to be important for host response in the lung against Pseudomonas aeruginosa (17, 18), nontypeable Haemophilus influenzae (19), and Klebsiella pneumoniae (20, 21). We recently reported that TIRAP plays a critical role in host response in the lung during pneumonia induced by Escherichia coli (22) and K. pneumoniae (23), but not Pseudomonas aeruginosa (23). In contrast, the role of adaptors in the MyD88-independent cascade, such as TRIF and TRAM against pulmonary bacterial pathogens, is not well understood.
In this study, we sought to define the role of TRIF in pulmonary host defense against a Gram-negative pathogen, E. coli. We also used a bacterial product from E. coli, endotoxin (LPS), a canonical TLR4 agonist, to demonstrate whether TRIF is important to induce lung inflammation. We observed that activation of the TLR4-mediated TRIF-dependent signaling cascade seems to be important for an effective innate immune response in the lung against E. coli despite the fact that TLR4-TRIF signaling does not seem to be required to induce innate pulmonary immune response against E. coli LPS. Our results provide new insights into the pulmonary innate immune mechanisms by which Gram-negative pathogens induce severe pneumonia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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TRIF gene-disrupted mice (TRIF–/–) were on a C57BL/6 x 129/Sv random hybrid background (14), and wild-type controls (TRIF+/+) were on a background matched to TRIF–/– mice. TLR4 (24), MyD88 (12, 13), and TLR3 gene-deficient mice (14) were on a C57BL/6 background and, therefore, C57BL/6 were used as controls. Mouse infection protocols were approved by the Animal Care and Use Committee of the National Jewish Medical and Research Center. Pathogen-free, 8- to 10-wk-old female mice, ranging from 18 to 24 g in weight, were used in all of our in vivo and in vitro experiments.
Mouse infection protocol
Bacteria were prepared for mouse inoculation, as described in previous studies (22). E. coli (American Type Culture Collection 25922) were grown in trypticase soy broth at 37°C overnight under constant agitation. Bacteria were harvested by centrifugation, washed twice in sterile isotonic saline, and resuspended in sterile 0.9% saline at a concentration of 20 x 106 CFU/ml. Mouse strains were anesthetized with i.p. avertin (250 mg/kg), followed by intratracheal (i.t.) inoculation of 50 µl of bacteria (106 CFU/mouse). The neck incision was closed with sterile staples under aseptic conditions. Control mice were i.t. inoculated in a similar manner with 50 µl of saline. The initial mouse inoculums were confirmed by plating serial 10-fold dilutions on MacConkey and tryptic soy agar (TSA) plates.
Mouse LPS challenge protocol
The induction of lung inflammation in a murine model by LPS aerosolization has been described in our previous studies (25, 26, 27, 28). Briefly, mice were exposed to 0.3 mg of LPS/ml in 0.9% saline by aerosolization for 20 min under a laminar flow hood by using a flow rate of 2 L/min. Control mice were exposed to 0.9% saline in a similar fashion.
Bronchoalveolar lavage fluid (BALF) collection
At the indicated time points after E. coli or LPS challenge, mice were euthanized by CO2 asphyxia and exsanguinated by cardiac puncture. A midventral incision was used to open the thoracic cavity, and the trachea was isolated and cannulated with a 20-gauge catheter. BALF was obtained from the whole lung to collect cells in the airspace and to obtain proteins for cytokine and chemokine detection, as described previously (25, 26, 27, 28). A total of 3.0 ml of BALF was retrieved from each mouse, and 0.5 ml of BALF was centrifuged and placed on glass cytospin slides, which were then stained by modified Wright-Giemsa staining (Diff-Quick; Fisher Scientific) to determine leukocyte subtypes based on their cellular and nuclear morphology. A total of 500 cells was counted in this respect. Leukocytes in BALF were determined using a hemocytometer. For determination of keratinocyte cell-derived chemokine (KC), MIP-2, LPS-induced C-X-C chemokine (LIX), TNF-, and IL-6 by ELISA, 2 ml of the undiluted cell-free BALF was centrifuged, passed via a 0.22-µm filter, and used immediately or kept at –20°C.
NF-B activation
Translocation of the p65 subunit of NF-B into the nucleus of lung cells was detected using an ELISA-based assay using oligonucleotides to specifically recognize the 5'-GGGACTTTCC-3' nucleotide sequence of Rel/NF-B family (Active Motif), as described in our previous publications (22, 29). A total of 20 µg of nuclear extract obtained from each lung sample after 2 h post-E. coli or post-LPS challenge was added to the NF-B-specific oligonucleotide-coated 96-well plate and incubated for 1 h at room temperature. After washing the wells three times, a primary Ab specific for p65 subunit of Rel/NF-B was added and incubated for 1 h at room temperature. The wells were washed three times to remove excess primary Ab, an anti-HRP conjugate was added, and the color development was monitored continuously. Color development was measured at the OD of 450 nM. This assay system has no cross-reactivity with any other transcription factors because of the immobilized specific nucleotide sequence and the specific Ab for Rel/NF-B.
Cytokine and chemokine determination in BALF
Cytokine and chemokine levels were assessed in BALF or culture supernatants of bone marrow-derived macrophage (BMM) cultures using a cytokine- or chemokine-specific sandwich ELISA, as described in our earlier publications (25, 26, 27, 28). The minimum detection limit is 2 pg/ml cytokine or chemokine protein.
BMM culture
BMMs were differentiated using DMEM containing 10% FBS and L-929 cell supernatant, as described (30). BMMs were grown in 12-well plates for 6 days, and cells were then stimulated for the indicated times with E. coli LPS (100 ng/ml), flagellin (100 µg/ml), or viable E. coli (105 or 106/ml), and processed in SDS-loading buffer before being separated on SDS polyacrylamide gels. Gels were transferred onto nitrocellulose membranes, and proteins were detected using the indicated Ab specific for phosphorylated or total MAPKs, such as JNK, ERK, and p38. The Western blots were quantitated and expressed as fold increase in response to E. coli after background (unstimulated) subtraction. In another set of experiments, BMMs were pretreated with a TLR4-blocking Ab (27) or isotype Ab. Supernatants were harvested from BMMs at 18 h after stimulation with the same agonists for TNF- and IL-6 detection by ELISA.
Bacterial enumeration in the lungs and spleens
Whole lungs were homogenized in 10 ml of sterile saline for 30 s, and resulting 20 µl of homogenates was plated in serial 10-fold dilutions on MacConkey and TSA plates. Bacterial colonies were counted after incubation at 37°C in 2 ml of saline for 24 h. To demonstrate E. coli dissemination, spleens were homogenized for 15 s in 2 ml of sterile saline for bacterial culture.
Uptake/killing assay
To assess whether neutrophils or alveolar macrophages (AMs) from TRIF–/– mice have impaired ability to uptake and/or kill E. coli, an uptake/bactericidal assay was performed, as described (22, 28). E. coli obtained after two washings with sterile 0.9% saline was used at a concentration of 106 CFU/ml, was mixed with 106/ml murine bone marrow-derived neutrophils or AMs in RPMI 1640 medium containing 10% FBS in a microfuge tube, and rotated at 50 rpm at 37°C for 2 h. The bacterial colonies were enumerated after serially diluting the supernatant at several 10-fold dilutions and culturing 20 µl of culture on MacConkey and TSA plates.
Actin polymerization measurement
To determine whether neutrophils from TRIF–/– mice have impaired actin assembly in response to inflammatory mediators, cells were exposed to KC, MIP-2, and TNF-, as described previously (31, 32). Briefly, isolated mouse band 3 neutrophils from the bone marrow were incubated with 5 ng/ml MIP-2 or KC for 15 min, 1 µg/ml TNF- for 1 h, or kept unstimulated for 1 h at 37°C. At the end of incubation, neutrophils were fixed and actin cytoskeleton changes were recorded using a flow cytometer.
Data analysis
All data are expressed as means ± SE. Data were analyzed with Student’s t test (between two groups) or with the one-way ANOVA (>2 groups). Survival curves were compared by Wilcoxon rank sign test. Differences in data values were defined significant at a p value of <0.05 using Kaleidagraph (Synergy Software).
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Results |
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First, we examined the role of MyD88 in Gram-negative bacterial pneumonia. Mice deficient in MyD88 (MyD88–/–) or its littermate controls (MyD88+/+) were challenged with E. coli through the i.t. route, and neutrophils were counted as an index of lung inflammation. In MyD88–/– mice, neutrophil counts in the BALF were substantially reduced in response to 106 CFU/mouse E. coli at 24 h postinfection (Fig. 1A), demonstrating that the MyD88-dependent cascade is critical to induce lung defense against E. coli. Although reduced, MyD88–/– mice still had significant neutrophil influx in response to 106 CFU/mouse E. coli (Fig. 1A), indicating that the MyD88-independent cascade also plays a role. We next sought to ascertain the role of TLR4 in Gram-negative pneumonia. We found that TLR4–/– mice had attenuated neutrophil influx in response to the same dose of E. coli (Fig. 1A), demonstrating the involvement of TLR4-dependent and TLR4-independent cascades. In sharp contrast with the E. coli data, MyD88- and TLR4-dependent cascade, but not TLR4-independent cascade, is required for E. coli LPS-induced inflammation in the lung (Fig. 1B).
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Because neutrophil recruitment in MyD88–/– mice was attenuated, but not abolished, in response to E. coli (Fig. 1A), we investigated whether TRIF in the MyD88-independent signaling cascade is required for E. coli-induced lung inflammation. When TRIF–/– and TRIF+/+ mice were i.t. inoculated with E. coli, reduced neutrophil migration was observed in BALF of TRIF–/– mice as compared with their wild-type controls at both 6 and 24 h after postinfection (Fig. 2A). In contrast, no apparent difference in neutrophil influx in response to E. coli LPS was noted between TRIF–/– and TRIF+/+ mice at 8 and 24 h after postchallenge (Fig. 2B). These findings conclude that TRIF contributes to E. coli-, but not LPS-induced lung inflammation, including neutrophil recruitment.
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TLR3 deficiency does not influence lung inflammation after E. coli challenge
Because the TLR3 signaling cascade is also associated with TRIF (14), we next examined the role of TLR3-mediated TRIF signaling in E. coli-induced neutrophil accumulation in the lungs in response to E. coli challenge. As shown in Fig. 3, there were no differences in neutrophil influx in the lungs between the TLR3–/– and TLR3+/+ mice observed after E. coli challenge (Fig. 3). These data demonstrate that TLR3-mediated TRIF-dependent cascade does not play a significant role in the induction of pulmonary inflammation following E. coli infection. Because TRIF is an adaptor molecule for both TLR3 and TLR4, these observations support that TRIF-dependent signaling triggered by E. coli is initiated by TLR4.
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B activation in the lung against E. coli
We next sought to determine whether the absence of functional TRIF influenced NF-B translocation in the lung in response to E. coli infection. We measured active NF-B in the nuclear fraction of whole lung homogenates after E. coli and E. coli LPS challenge. Our observations demonstrate that TRIF-dependent signaling mediates NF-B activation in lung cells following E. coli challenge (Fig. 4). Although NF-B translocated in the lungs of TRIF–/– and TRIF+/+ after LPS challenge, a modest, but significant reduction in NF-B translocation was observed in the lungs of TRIF–/– mice as compared with their littermate controls (TRIF+/+) after LPS challenge (Fig. 4).
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BALF studies were performed following challenge with E. coli or E. coli LPS. Although production of KC and MIP-2 was not decreased in response to E. coli (Fig. 5, B and C), TNF-, IL-6, and LIX production was reduced in TRIF–/– mice (Fig. 5, A, D, and E). In contrast, only TNF- was decreased in TRIF–/– mice in response to E. coli LPS as compared with their wild-type controls (Fig. 5F). No significant production of cytokines/chemokines was observed in BALF of TRIF–/– and TRIF+/+ mice after saline challenge (data not shown). These observations indicate that TRIF plays an important role in the differential expression of cytokines and chemokines in the lungs after E. coli and E. coli LPS challenge. These findings also suggest that TRIF-mediated production of LIX contributes to neutrophil influx in the lungs in response to E. coli.
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and IL-6 production in response to E. coli and E. coli LPSNext, we determined the effects of TRIF deficiency on MAPK signaling. When TRIF–/– macrophages were stimulated with E. coli, reduced activation of JNK, ERK, and p38 was observed only at late time point, i.e., at 60 min (Fig. 6, A and B). In addition, late MAPK activation was abolished in macrophages obtained from TRIF–/– mice in response to E. coli LPS (Fig. 6A). No differences were observed between macrophages obtained from TRIF–/– and TRIF+/+ mice in response to flagellin, a canonical TLR5 agonist (Fig. 6A). These data suggest that TRIF is important for E. coli and E. coli LPS-induced late activation of MAPKs in BMMs.
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and IL-6 by TRIF–/– BMMs to E. coli (Fig. 6C). In a similar manner, TNF- and IL-6 production by TRIF–/– BMMs was abolished after E. coli LPS stimulation (Fig. 6D). To demonstrate whether TRIF-dependent cascade involves TLR4 in E. coli-induced effects in BMMs of TRIF–/– and TRIF+/+ mice, we used a TLR4-blocking Ab. Our results demonstrate that TLR4 Ab blocks E. coli-induced TNF- and IL-6 responses in BMMs obtained from both TRIF–/– and TRIF+/+ mice (Fig. 6E), suggesting that TLR4-dependent TRIF-mediated (MyD88-independent) signaling cascade is essential for cytokine responses against E. coli. TRIF contributes to E. coli clearance in the lung and bacterial dissemination
Having established that TRIF contributes to efficient innate immune response in the lungs against E. coli, we next investigated whether TRIF is important for host defense against this pathogen. The lungs of TRIF–/– mice had more E. coli burden in the lungs as compared with its littermate controls at 24 h postinfection (Fig. 7A). More bacterial dissemination was noted in TRIF–/– mice at 6 and 24 h post-E. coli challenge (Fig. 7B). Furthermore, survival did not differ between TRIF–/– and TRIF+/+ mice after E. coli challenge (106/mouse) (Fig. 7C). Taken together, these findings demonstrate that TRIF is essential in limiting bacterial colonization in the lungs and bacterial dissemination in the bloodstream despite the fact that TRIF is not important for survival.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Innate immune response plays a critical role in bacterial clearance both locally and systematically. TLRs are important in the induction of innate immunity to bacterial pathogens (6). TLRs, particularly TLR4, induce at least two divergent signaling cascades that involve at least four adaptor molecules. The MyD88-dependent cascade involves TIRAP and MyD88 (11, 12, 13), whereas the MyD88-independent cascades involve TRIF and TRAM (14, 15). Although there is abundant evidence from in vivo studies that MyD88-dependent signaling cascades contribute to lung defense against bacterial pathogens (17, 18, 19, 20, 21, 22, 23), the role of the MyD88-independent cascade in the induction of host defense in the lung has not been well studied. We attempted to define the role of TRIF, an adaptor in the MyD88-independent cascade, in host defense in the lung against a Gram-negative bacterium E. coli. The present investigation clearly established that TRIF contributes to lung immune response and limits bacterial growth in the lungs and bacterial dissemination during E. coli pneumonia. Furthermore, our results demonstrate that genetic deletion of TRIF did not affect LPS-induced pulmonary inflammation.
Neutrophil accumulation in the lungs is a pathological hallmark of bacterial pneumonia (33, 34, 35). In our earlier studies, we have established that TIRAP is important for neutrophil recruitment into the lungs during E. coli pneumonia (22). In those investigations, the importance of MyD88 or TLR4 in neutrophil recruitment was not examined. In the current investigation, we have observed that MyD88 and TLR4 are required for neutrophil accumulation in the lungs during E. coli pneumonia. Our findings are consistent with a previous report, indicating that TLR4 is important for neutrophil influx during E. coli pneumonia (36). Our observations also demonstrate that TRIF is important for neutrophil recruitment during E. coli pneumonia. It has been well documented that TRIF is an adaptor molecule for TLR3- and TLR4-mediated signaling cascades (14, 15). The observation of unimpaired neutrophil accumulation in TLR3–/– mice during E. coli pneumonia (Fig. 3), together with the finding that anti-TLR4 Ab blocks E. coli-induced TNF- and IL-6 expression in BMMs of TRIF–/– and TRIF+/+ mice (Fig. 6), point toward the involvement of TRIF in the TLR4 signaling cascade. In this regard, the sole previous investigation determined the role of TRIF to a bacterial pathogen and concluded that TLR4-mediated TRIF-dependent cascade does not play a role in neutrophil recruitment in the lungs after nontypeable H. influenzae infection. However, two earlier investigations (19, 37) determined the role of TLR4 in pulmonary defense against H. influenzae in vivo using TLR4-mutant mice and reported an enhanced susceptibility in mutant mice. Therefore, our study is the first one demonstrating the role of TRIF associated with TLR4 in the induction of neutrophil recruitment in the lung against a bacterial pathogen. Because we have established a critical role of TIRAP in E. coli-induced lung infection (22), these data (22) together with our current findings demonstrate that both MyD88-dependent and MyD88-independent cascades of TLR4 are required for the induction of host defense against pulmonary E. coli infection.
In the current investigation, we revealed that TRIF in the MyD88-independent cascade does contribute to lung inflammation after E. coli challenge. Given the fact that TRIF–/– neutrophils have an unimpaired actin assembly (Fig. 2) and TRIF–/– neutrophils and AMs have an intact bactericidal capability (Fig. 7), the defective influx of neutrophils into the airways is most likely the result of attenuated LIX, TNF-, and IL-6 production in the lungs of TRIF–/– mice. However, our findings rule out the role of TRIF in E. coli LPS-induced neutrophil accumulation. In this context, E. coli LPS is known to activate TLR4 signaling cascade to induce lung inflammation. Using C3H/HeJ mice, which have mutated/nonfunctional TLR4, we have established that E. coli LPS lung inflammation requires TLR4 (27). We have also illustrated that the TIRAP adaptor molecule in the MyD88-dependent cascade is critical to induce lung inflammation after viable E. coli and E. coli LPS challenge (22). The differential requirement of TLR4-TRIF signaling axis in the induction of lung inflammation between E. coli and E. coli LPS is unexpected, and it brings up several possibilities, as follows: 1) the affinity and/or avidity of E. coli binding to TLR4 is different than E. coli LPS; 2) the presence of another receptor in addition to TLR4 is responsible for E. coli-induced effects; and 3) E. coli, but not LPS, binding to TLR4 or other cell surface receptor recruits one or more intracellular signaling molecules that signal via TRIF. A proposed scheme for this is presented in Fig. 8. Future studies are required to examine these possibilities.
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B is one of the well-studied transcription factors that mediate the activation of numerous inflammatory cytokines and chemokines (33, 34, 35, 36, 38, 39). Elegant studies have demonstrated that NF-B activation is a pivotal event in the pathogenesis of bacterial pneumonia (33, 34, 35). It has been established that adaptors transduce cell surface TLR signals to NF-B, which results in NF-B-dependent transcription (6, 7, 8). The MyD88-dependent cascade induces early NF-B translocation, whereas the MyD88-independent cascade causes late NF-B activation (6, 7, 8). Our findings suggest that TRIF-dependent NF-B activation is a critical mediator to induce LIX, TNF-, and IL-6 expression in the lungs in response to E. coli (Figs. 4 and 5). Although our investigation demonstrates a role for TRIF in E. coli-induced lung inflammation, a recent study using H. influenzae indicates that TRIF does not play a role in cytokine and chemokine expression in the lungs (19). The discrepancy between our findings and their observations (19) could be explained by the nature of the pathogens and time points used to measure chemokines/cytokines in the lungs.
Our results demonstrate that TRIF is an important molecule for MAPK activation in BMMs (Fig. 6). A remarkable feature of MAPK activation is that only the late MAPK activation was attenuated or abolished in BMMs obtained from TRIF–/– mice after E. coli and E. coli LPS challenge. These results are consistent with previous reports stating that activation of TLR4 leads to different phase of MAPK activation (11, 12, 13, 14, 15). Whereas activation of the TLR4-mediated MyD88-dependent signaling leads to an immediate-phase activation of MAPKs, TLR4-mediated MyD88-independent signaling causes a late-phase activation of MAPKs (11, 12, 13, 14, 15). Our findings also suggest that MAPK signaling is important for TNF- and IL-6 production in BMMs in response to E. coli and E. coli LPS. Although IL-6 production in BMMs obtained from TRIF–/– mice in response to LPS was abolished, we did not observe a difference of IL-6 levels in BALF obtained from TRIF–/– and TRIF+/+ mice. Although several reasons could have contributed to this discrepancy of IL-6 expression in BMMs vs BALF, we speculate that IL-6 production caused by LPS in nonmyeloid (resident) cells is not TRIF dependent. This explanation is supported by the fact that LIX production by viable E. coli is TRIF dependent, whereas LIX induction by E. coli LPS is TRIF independent (Fig. 5, E and J). In this context, we have demonstrated that LIX is predominantly produced by resident cells, i.e., alveolar type II cells after LPS challenge (25, 26).
The sentinel event in lung defense is the clearance of bacterial pathogens from the lungs and limitation of bacterial dissemination in the bloodstream. In our model of murine pneumonia, we have highlighted the importance of TRIF for antibacterial defense in the lung against E. coli. Although a previous study investigated the role of TRIF in H. influenzae infection, TRIF does not contribute to antibacterial defense in the lung in their model (19). To our knowledge, we have demonstrated for the first time that TRIF is capable of limiting bacterial growth in the lungs. The main reason underlying discrepancy between Wieland et al. (19) and ours are the time points used to measure bacterial CFUs in the lungs. The higher bacterial growth in the lungs of TRIF–/– mice and its dissemination may be due to attenuated neutrophil accumulation in the lungs (Fig. 2A) due to reduced LIX production (Fig. 5). Another potential mechanism for higher bacterial growth in the lungs and more bacterial dissemination in TRIF–/– mice may be decreased activation of recruited neutrophils by TNF- and IL-6. Thus, the role of TRIF in pulmonary host defense against E. coli infection is most likely dependent on the interplay among E. coli-induced inflammatory mediators in the lung.
We conclude that the TLR4-mediated TRIF-dependent pathway is important to initiate a prompt and effective host response against E. coli, which is essential for bacterial clearance in the lung and bloodstream. Specific targeting of TRIF may prove to be a feasible option to attenuate excessive neutrophil recruitment in the lung during Gram-negative bacterial pneumonia.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by a grant from the University of Colorado Health Sciences Center (to S.J.), a biomedical research grant from the American Lung Association (RG-22442-N; to S.J.), a grant from the American Heart Association (0275035N; to M.B.F.), and a grant from the National Institutes of Health (HL-068876; to G.S.W.). 
2 Address correspondence and reprint requests to Dr. Samithamby Jeyaseelan, Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson Street Neustadt D-403, Denver, CO 80206. E-mail address: JeyaseelanS{at}njc.org
3 Abbreviations used in this paper: TIR, Toll/IL-1R; AM, alveolar macrophage; BALF, bronchoalveolar lavage fluid; BMM, bone marrow-derived macrophage; i.t., intratracheal; KC, keratinocyte cell-derived chemokine; LIX, LPS-induced C-X-C chemokine; TIRAP, TIR domain-containing adaptor protein; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain-containing adaptor inducing IFN-; TSA, tryptic soy agar.
Received for publication June 21, 2006. Accepted for publication December 15, 2006.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B activation and neutrophil recruitment require IL-1 and TNF receptor signaling during pneumococcal pneumonia. J. Immunol. 175: 7530-7535. This article has been cited by other articles:
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 M. Leendertse, R. J. L. Willems, I. A. J. Giebelen, P. S. van den Pangaart, W. J. Wiersinga, A. F. de Vos, S. Florquin, M. J. M. Bonten, and T. van der Poll TLR2-Dependent MyD88 Signaling Contributes to Early Host Defense in Murine Enterococcus faecium Peritonitis J. Immunol., April 1, 2008; 180(7): 4865 - 4874. [Abstract] [Full Text] [PDF]
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