Mechanical Ventilation Induces a Toll/Interleukin-1 Receptor Domain-containing Adapter-inducing Interferon β–dependent Inflammatory Response in Healthy Mice

All experiments were approved by the Regional Animal Ethics Committee (Nijmegen, The Netherlands) and performed under the guidelines of the Dutch Council for Animal Care and the National Institutes of Health.

To test the role of TRIF in our experimental model, studies were conducted by using TRIF mutant mice (C57BL6 background; 10–12 weeks; 25 ± 4 g; n = 31). Age-matched wild-type (WT) mice (C57BL6 background; 26 ± 3 g; n = 31) were used as controls. TRIF mutant mice were a kind gift from Bruce Beutler M.D., Ph.D. (Professor, Department of Immunology, The Scripps Research Institute, La Jolla, California), who identified and cloned the TRIF gene (called Lps2).13These TRIF mutant mice have a distal frameshift error in the Lps2 gene, which has an equivalent gene in humans.13WT mice were purchased from Charles River (Sulzfeld, Germany).

Mice were anesthetized with an intraperitoneal injection of a combination of ketamine, medetomidine, and atropine (KMA): 7.5 μl/g of body weight of induction KMA mix (consisting of 1.26 ml of ketamine, 100 mg/ml; 0.2 ml of medetomidine, 1 mg/ml; 1 ml of atropine, 0.5 mg/ml; and 5 ml of NaCl, 0.9%) was given just before intubation. Animals were orally intubated, mechanically ventilated (MiniVent®; Hugo Sachs Elektronik-Harvard apparatus, March-Hugstetten, Germany) for 4 h and killed immediately thereafter. The following settings were used during controlled MV: tidal volume 8 ml/kg body weight and frequency 150/min, which is well within the range of measured tidal volume and respiratory rate during spontaneous ventilation in C57BL6 mice.14All animals received 4 cm H²O positive end-expiratory pressure, and fraction of inspired oxygen was set to 0.4.

To maintain anesthesia, 5.0 μl/g of body weight boluses of maintenance KMA mix (consisting of 0.72 ml of ketamine, 100 mg/ml; 0.08 ml of medetomidine, 1 mg/ml; 0.3 ml of atropine, 0.5 mg/ml; and 18.9 ml of NaCl, 0.9%) were given, via  an intraperitoneally placed catheter, every 30 min. Rectal temperature was monitored continuously and maintained between 36.0°C and 37.5°C by using a heating pad.

The first set of experiments was performed to investigate the role of TRIF in MV-induced changes in cytokine profile, leukocyte influx, and NF-κB activity. Blood and lungs were harvested after 4 h of MV in WT mice (group V-WT, n = 8) and TRIF mutant mice (group V-TRIF, n = 8) or immediately after induction of anesthesia in WT (group C-WT, n = 8) and TRIF mutant mice (group C-TRIF, n = 8).

The second set of experiments was designed to assess changes in cardiopulmonary physiology. Continuous intraarterial carotid blood pressure was measured in mechanically ventilated WT mice (group V-WT, n = 15) and TRIF mutant mice (group V-TRIF, n = 15). Arterial blood gas analysis (iSTAT; Abbott, Birmingham, United Kingdom) was performed after 4 h of MV. We did not include these animals for the cytokine or histopathologic analysis to avoid possible interference with cytokine response resulting from instrumentation (i.e. , insertion of arterial catheter) and subsequent bacterial contamination. One TRIF mutant mouse died during instrumentation.

Lipopolysacharide was measured in experimental circuit by Limulus Amebocyte Lysate testing (Cambrex Bio Science, Walkersville, MD; detection limit 0.06 IU/ml) to rule out contamination with lipopolysacharide in our experimental setting. Indeed, no lipopolysacharide could be detected in air, tubing, or ventilator (data not shown).

Blood was collected by exsanguination, centrifuged at 14,000 rpm (13,000g ) (Eppendorf 5415 C; Nethler-Hinz GmbH, Hamburg, Germany) for 2 min, and plasma was stored at −80°C for later biochemical analysis. Immediately after exsanguination, heart and lungs were carefully removed en block via  midline sternotomy. The right upper and lower lobes were snap frozen in liquid nitrogen and stored at –80°C. The right middle lobe was fixed for light microscopy as described previously.7The left lung was homogenized for the measurement of cytokines.

Tumor necrosis factor-α, interleukin (IL)-6, IL-10, and keratinocyte-derived chemokine (KC) in the homogenized left lung and in plasma were analyzed by enzyme-linked-immunosorbent assay (ELISA) (for tumor necrosis factor-α, IL-6, and IL10: CytoSet, BioSource, CA; for KC: ELISA-Kit, R&D Systems, Minneapolis, MN). Because of insufficient amount of plasma, IL-1α and IL-1β could only be assessed in lung homogenate using specific radioimmunoassays, as described previously.15Lower detection limits: 40 pg/ml for IL-1α and IL-1β; 32 pg/ml for tumor necrosis factor-α; 160 pg/ml for IL-6; 80 pg/ml for IL-10; 160 pg/ml for KC.

NF-κB’s DNA-binding activity was determined by electrophoretic mobility shift assay. Nuclear proteins for electrophoretic mobility shift assay were isolated from liquid nitrogen frozen lungs. Lung tissue (20 mg) was homogenized in 5 ml of ice-cold buffer (HEPES 10 mm, 1.5 mm MgCl², 10 mm KCl and 0.6% Nonidet-P40, 0.5 mm dithiothreitol, and 0.2 mm phenylmethylsulphonylfluoride [Sigma-Aldrich, Zwijndrecht, The Netherlands]) and centrifuged for 30 s at 350g  (4°C). The supernatant was then incubated on ice for 5 min and centrifuged for 5 min at 6,000g  (4°C). The pellet was resuspended in 200 μl of buffer (10 mm HEPES, 1.5 mm MgCl², 10 mm KCL, 1.2 m sucrose, 0.5 mm dithiothreitol, and 0.2 mm phenylmethylsulphonylfluoride [Sigma-Aldrich]) and centrifuged for 30 min at 13,000g  (4°C). The pellet was then resuspended in 66 μl of buffer (HEPES 20 mm, 1.5 mm MgCl², 0.2 mm EDTA, 420 mm NaCl, 25% glycerol, 0.5 mm dithiothreitol, 0.2 mm phenylmethylsulphonylfluoride, 2.0 mm benzamidine, and 5.0 μg/ml leupeptine [Sigma-Aldrich]), incubated on ice for 20 min, and centrifuged for 2 min at 6000g  (4°C). The supernatants were used as nuclear extracts. Protein concentrations in these extracts were determined by using the Bio-Rad protein assay (Bio-Rad, Veenendaal, The Netherlands).

Double stranded oligonucleotides containing an NF-κb consensus binding site (5′-AGTTGAGGGGACTTTCCCAGGC-3′) were radiolabeled with 32[P]-adenosine triphosphate by using T4 polynucleotide kinase (Promega, Madison, WI). Labeled NF-κB oligonucleotides were mixed with nuclear extracts (10 μg) and incubated at room temperature for 20 min. Then, these samples were loaded on a 4% polyacrylamide gel. After electrophoresis for 45 min, the gel was dried and exposed for 24 h to an X-ray film. The bands on the film were quantified by using optical densitometry software (GeneTools, Syngene, Cambridge, United Kingdom).

For Polymerase Chain Reaction (PCR) analysis of messenger RNA, the right upper and lower lobes were homogenized with a micro-dismembrator II (Braun, Melsungen, Germany). Total RNA was extracted in 1 ml TRIzol reagent. Subsequently, 200 μl chloroform and 500 μl of 2-propanol (Merck) were used to separate the RNA from DNA and proteins. After a wash step with 75% ethanol (Merck, Darmstadt, Germany), the dry RNA was dissolved in 30 μl of water. To obtain double strain complementary DNA, DNase-treated total RNA 1 μg with oligo dT primers (0.01 μg/ml) was reverse transcribed in a real-time PCR with a total volume of 20 μl. Quantitative PCR was subsequently performed by using ABI/PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). PCRs of glyceraldehyde-3-phosphate dehydrogenase, IL-1β were performed with Sybr Green PCR Master Mix (Applied Biosystems), 5 μl of 1/20 diluted complementary DNA and primers in a final concentration of 300 nm in a total volume of 25 μl. The primers were developed by using Primer Express® software (Applied Biosystems). Quantification of the PCR signals of each sample was performed by comparing the cycle threshold values (Ct), in duplicate, of the gene of interest with the Ct values of the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene. IL-1β messenger RNA expression was expressed as relative expression to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. All primers were validated according to the protocol, and standard curves were all within the tolerable range.

The right middle lung lobe was fixed in 4% buffered formalin solution overnight at room temperature, dehydrated, and embedded in paraplast (Amstelstad, Amsterdam, The Netherlands). Sections of 4 μm-thicknesses were used. The enzyme activity of leukocytes was visualized by enzyme histochemistry by using chloracetatesterase staining (Leder staining). Leukocytes were counted manually (20 fields per mouse), and after automated correction for air/tissue ratio, the number of leukocytes/μm²was calculated.

Data are expressed as mean ± SD when distributed normally (leukocyte counts, relative messenger RNA expression) and expressed as Box (median, 25th, 75th percentile) and Whiskers (range) otherwise (cytokine concentrations). Statistical analysis was performed with SAS (SAS Institute Inc. Cary NC, version 8.02) statistical procedures. Cytokine concentrations are not normally distributed; therefore, Kruskal Wallis procedures were used with post hoc  comparisons of subgroups (Duncan). For the analysis of normally distributed data (leukocyte counts and relative messenger RNA expression), ANOVA was used with post hoc  comparison of group means (Duncan). Two-tailed P  values are reported, and the level of significance was set to P < 0.05.

In WT mice, MV increased levels of IL-1α, IL-1β, and KC (fig. 1), which is in line with previous data from our lab.7,8In contrast, MV in TRIF mutant mice elicited only a minor increase in IL-1β and KC. Levels of IL-1α after MV were not different between WT and TRIF mutant mice (fig. 1). In addition, MV increased the messenger RNA expression of IL-1β in WT mice, but not in TRIF mutant mice after MV, indicating that TRIF affects IL-1β transcription (fig. 2).

NF-κB activity after MV was significantly lower in TRIF mutant mice compared with WT mice (fig. 3). MV resulted in a pulmonary leukocyte influx in both WT and TRIF mutant mice (fig. 4).

In WT mice, MV increased levels of IL-6 and KC in plasma (fig. 5). In TRIF mutant mice, no increase of IL-6 was found after MV, and the increase in KC appeared less pronounced compared with WT mice.

The animals with an intraarterial cannula exhibited stable hemodynamic variables throughout the experiments. Mean arterial pressure was within normal limits and remained above 65 mmHg in all animals, which was in line with previous data from our lab.7Blood gas analysis showed pH 7.30 ± 0.07 in WT mice and pH 7.32 ± 0.08 in TRIF mutant mice, arterial oxygen tension of 146 ± 23 mmHg in WT mice and 157 ± 20 mmHg in TRIF mutant mice, arterial carbon dioxide tension of 41 ± 6 mmHg in WT mice and 39 ± 5 mmHg in TRIF mutant mice, bicarbonate of 17.4 ± 3 mmol/l in WT mice and 19.1 ± 3.1 mmol/l in TRIF mutant mice. Blood gas values, Pao²/Fio²ratios, and alveolar-arterial oxygen gradients were not different between WT and TRIF mutant mice after MV.

The current study confirms earlier observations from our laboratory7,8and others1,6,16that MV using clinical relevant ventilator settings results in a pulmonary and systemic inflammatory response. The current study extends these findings by showing that TRIF deficiency attenuates this inflammatory response after MV, by reducing NF-κB activation. TRIF deletion prevented pulmonary pro-IL-1β increase and systemic IL-6 increase after 4 h of MV. Also, pulmonary levels of IL-1β and KC were significantly lower in TRIF-deleted mice lungs compared with WT lungs after MV.

The TRIF pathway is a downstream pathway of TLR4 and TLR3,9,10that can cause delayed NF-κB activation.17Recently, we have shown the involvement of TLR4 in MV-induced inflammation.8The current study extends these findings by showing the involvement of TRIF signaling. We found that the MV-induced increase of KC and IL-1β was TRIF dependent. TRIF is also involved in the downstream signaling of TLR3; therefore, we cannot exclude the involvement of TLR3 in our model. However the results presented here closely resemble the results from our previous TLR4 experiment.8Therefore it is likely that in downstream signaling of TLR4 the TRIF pathway is involved in the inflammatory response after MV. Subsequent studies using TLR3 deleted mice are needed to confirm the importance of TLR4 in MV-induced inflammation.

Several studies have pointed out the involvement of the analyzed cytokines in lung injury. IL-1β has been shown to be among the most biologically active cytokines in the lungs;18,19it is therefore proposed to play an important role in the pathogenesis of lung injury.20In clinical studies, high tidal volume MV results in persistently high plasma levels of IL-1β, which is associated with distal organ failure and mortality.2,21In the current study, TRIF deletion prevented IL-1β increase at messenger RNA level and attenuated the increase of IL-1β at protein levels, indicating TRIF involvement at the level of transcription. This is interesting because it indicates major involvement of the TRIF pathway and may indicate only minor or no influence of the MyD88 pathway. However this hypothesis needs further investigation.

KC is a chemoattractant, but it also has a direct cytotoxic effect.22Jiang et al.  23found KC to be produced by pulmonary epithelial cells in a TLR4-dependent manner in direct response to bleomycin. More recently, functional TLR4 expression was found to be critical in the KC increase after hemorrhage.24The current study shows that the increase of KC after MV is at least partly TRIF-dependent.

Our data show that blocking TRIF-dependent pathway prevents the increase in plasma IL-6 after MV. Several studies suggest that IL-6 plays a role in the development of distal organ failure in ventilator-induced lung injury.2,25Accordingly, TRIF modulation may attenuate distal organ failure induced by MV.

It should be noticed that our model shows no evidence of either pulmonary dysfunction or distant organ failure, despite the development of an inflammatory response. Apparently, the trigger induced by the ventilator is relatively mild, and the lung is able to cope with the MV-induced inflammatory reaction. This has been demonstrated before in a study from our laboratory7and by a clinical study showing that 2 h of MV in healthy children resulted in enhanced cytokine concentrations without clinical signs of pulmonary dysfunction.16In acid-induced lung injury, TRIF deletion indeed diminished impairments in lung function.5Therefore, the inflammatory response after MV in healthy lungs may be too subtle to identify changes in cardiopulmonary physiology. However, the inflammatory response after MV is clinically relevant; this forms the basis for the two-hit hypothesis proposing that injury (e.g. , the critically ill patient) primes the immune system (first hit) for a lethal inflammatory reaction to a later, otherwise nonlethal, secondary insult (second hit), namely MV.26–28This enhanced host response can lead to distal organ failure and is previously linked to TLR4 reactivity,29which is interesting because we have shown that TLR4 is involved in the inflammatory response after MV in healthy lungs.8Downstream of TLR4, we identified the involvement of TRIF in this response. Therefore, inhibition of TRIF may be an effective strategy to prevent or attenuate MV-induced pulmonary and systemic inflammation. Figure 6shows a schematic overview of the downstream signaling pathways of TLR4.

KC is a major chemoattractant for leukocytes.30In a previous study, we have shown that 4 h of MV in healthy mice induces a TLR4-dependent pulmonary leukocyte influx after the increase of KC in the lung.7,8In the current study, this pulmonary leukocyte influx is confirmed. In TRIF mutant mice, leukocyte influx appeared less pronounced; however, this did not reach statistical significance. This might be explained by the fact that, the increase in KC in plasma in TRIF mutant mice also appeared to be reduced, without reaching statistical significance.

Our study has several limitations. First, all studies were performed in mice. It is unknown whether the response to MV in mice is similar to the response in humans. Second, only the effect of 4 h of MV with clinically relevant tidal volume was tested. In a previous study, we showed that the proinflammatory response is activated within 30 min after initiation of MV and intensifies up to 4 h after initiation of MV.7Preliminary observations show that the inflammatory response unaltered after 8 h of MV compared with 4 h of MV. We did not study the effects of TRIF deletion in lungs exposed to high-tidal volume MV, as the use of high-stretch MV is currently avoided in clinical practice. Third, the current study did not evaluate the effect of different ventilatory modes. Interestingly, recent studies indicate that spontaneous ventilatory efforts may improve respiratory function such as diaphragm function, pulmonary gas exchange, and hemodynamics after MV.31,32 

Factors other than MV possibly affecting TRIF were carefully avoided. Contamination with lipopolysacharide is suggested to be a confounding factor in many studies.33We therefore excluded lipopolysacharide contamination during the experiments. The possibility of triggering an inflammatory response by invasive procedures (i.e. , insertion of an intraarterial line)34was eliminated by performing experiments in noninvasively monitored animals. Previously, cardiopulmonary stability in invasively monitored animals has been documented.7In the current study, mice were slightly acidotic after MV. As hypercapnic acidosis attenuates MV-induced inflammation in healthy mice,35this could affect our results. However it is unlikely that the slight metabolic acidosis in the current study significantly affects our data. Indeed, correcting metabolic acidosis does not alter levels of cytokines after MV in healthy mice.36The possible immune modulating effects of anesthetics have been studied extensively.37Ketamine, for instance, is known to have an inhibitory effect on lipopolysacharide-induced cytokine production,38–41possibly by suppressing TLR4 expression.42Recently, it has been demonstrated that ketamine alone (without lipopolysacharide) also attenuated cytokine production in humans in the direct postoperative period after elective abdominal surgery.43In the current study, all animals received ketamine. Ideally, an additional control group of spontaneously breathing animals under ketamine, medetomidine and atropine anesthesia is needed. However, this will result in hypoventilation with severe respiratory acidosis and hemodynamic instability.

The current study supports a role for TRIF in the inflammatory reaction after MV in healthy lungs. Increasing the understanding of the innate immune response to MV and the contribution of MV to the “multiple hit” concept may lead to future treatment advances in ventilator-induced lung injury, in which TRIF may serve as a therapeutic target.

We are grateful for the help of Francien van de Pol, Ing., and Ilona van den Brink, Ing. (Lab technicians, Department of Anesthesiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands) for their expertise in performing the animal experiments. Ineke Verschueren, Ing. (Lab technician, Nijmegen Centre for Infectious Diseases, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands) for her help with cytokine assays, and Cindy Pigmans, Ing. (Lab technician, Department of Pulmonary Diseases, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands) for her help with analyzing NF-κB activity.