Modulation of the Triggering Receptor Expressed on Myeloid Cells–1 Pathway during Pneumonia in Rats

Abstract

BackgroundTriggering receptor expressed on myeloid cells–1 (TREM-1) is a cell-surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. The aim of the present study was to investigate the effect of the modulation of the TREM-1 pathway during experimental pneumonia in rats

MethodsAdult male Wistar rats were intratracheally inoculated with Pseudomonas aeruginosa (PAO1 strain) and randomly treated or not treated with an analogue synthetic peptide derived from the extracellular moiety of TREM-1 (LP17)

ResultsP. aeruginosa induced a severe pneumonia associated with signs of severe sepsis within the first 24 h. In septic rats, LP17 improved hemodynamic status, attenuated the development of lactic acidosis and hypoxemia, modulated lung and systemic inflammatory responses and coagulation activation, reduced lung histological damage, and improved survival

ConclusionsThe modulation of the TREM-1 pathway by the use of such synthetic peptides as LP17 appears beneficial during P. aeruginosa pneumonia in rats in attenuating lung and systemic inflammatory responses

Sepsis constitutes a significant public health burden and remains an ever-present challenge in intensive care units. Its pathogenesis is now becoming better understood, and a greater comprehension of the complex network of immune, inflammatory, and hematological mediators involved in this disorder now allows for the development of rational and novel therapies

Stimulatory immunoreceptors play a central role in the recognition of foreign antigens or pathogens by the immune system [1]. Several immunoglobulin-like activating receptors have been characterized, including paired immunoglobulin receptors [2], NKp44 [3], and the Src homology 2 domain–containing protein tyrosine phosphatase substrate 1 family [4]. Recently, the triggering receptor expressed on myeloid cells (TREM) family, a new family of receptors that is distantly related to NKp44, has been described [5, 6]. All TREMs associate the adaptor DAP12 for signaling. Among the TREM family, TREM-1 has been identified both on human and murine polymorphonuclear cells and on mature monocytes [5]. Its expression by these effector cells is dramatically increased in skin, biological fluids, and tissues infected by gram-positive or gram-negative bacteria or fungi [7, 8]. By contrast, TREM-1 is not up-regulated in samples from patients with noninfectious inflammatory disorders such as psoriasis, ulcerative colitis, or vasculitis caused by immune complexes [7]. In mice, the engagement of TREM-1 with agonist monoclonal antibodies (MAbs) stimulates the production of several proinflammatory cytokines and chemokines [5, 9], along with rapid neutrophil degranulation and oxidative burst [10]. The activation of TREM-1 in the presence of Toll-like receptor (TLR)–2 or TLR-4 ligands amplifies the production of proinflammatory cytokines (tumor necrosis factor [TNF]–α, interleukin [IL]–1β, and granulocyte-macrophage colony-stimulating factor), together with the inhibition of IL-10 release [9]. Moreover, the activation of these TLRs up-regulates TREM-1 expression [5, 11]. Thus, TREM-1 and TLRs appear to cooperate in producing an inflammatory response. The role that TREM-1 plays as an amplifier of the inflammatory response has been confirmed in a mouse model of septic shock in which blocking signaling through TREM-1 partially protected mice from death [7, 12]. Both in vitro and in vivo, a synthetic peptide mimicking short, highly interspecies-conserved domains of TREM-1 attenuated the cytokine production of human monocytes and protected septic mice from hyperresponsiveness and death [12]. This peptide was efficient not only in preventing but also in down-modulating the deleterious effects of proinflammatory cytokines [12]

Considering the involvement of TREM-1 during pneumonia [13–15] and that nosocomial pneumonia is a major cause of sepsis in intensive care units, we sought to investigate the effect of such a peptide in a rat model of Pseudomonas aeruginosa pneumonia. Our hypothesis was that TREM-1 pathway modulation blunts the inflammatory response associated with pneumonia and provides protection from lung damage and death

Materials and Methods

RatsAdult male Wistar rats (350–380 g) were purchased from Centre d’élevage Depre. The experiments were performed in compliance with the National Institutes of Health Guidelines on the Use of Laboratory Animals and approved by our Institutional Animal Care and Use Committee

P. aeruginosa inoculum preparation and bacterial analysesA nonmucoid P. aeruginosa strain (serotype PAO1, ATCC BAA-47) was used for all studies. These bacteria were maintained in peptone broth containing 25% glycerol at −70°C. Before each experiment, the strain was propagated in tryptone soy agar plates for 24 h at 37°C. One colony was then transferred to tryptone soy broth for another 24 h at 37°C. On the day of the experiment, the bacteria were centrifuged at 3000 g for 15 min, and the pellet of bacteria was washed twice with PBS. P. aeruginosa solution was then resuspended in saline at a concentration of 7×10⁸–8×10⁸ cfu/mL, and a 0.5 mL/kg concentration of this solution was used for intratracheal inoculation as described below. Samples of bronchoalveolar lavage (BAL) fluid and lung homogenate were obtained aseptically for culture in rats with pneumonia 24 h after the bacterial instillation. The concentration of bacteria was then quantified by placing successive 10-fold dilutions of the bacterial suspension in tryptone soy agar plates and scoring visible colonies after 24 h incubation at 37°C

Bacterial pneumonia and LP17 treatmentLP17, spanning the complementary determining region loop 3 of TREM-1, was chemically synthesized (Pepscan Systems) as described elsewhere [12]. The correct peptide (LQVTDSGLYRCVIYHPP) was obtained in >99% yield; was homogeneous after preparative purification, as confirmed by mass spectrometry and analytic reversed phase high performance liquid chromatography; and was endotoxin free

Rats were anesthetized with ether for a brief period. A median incision was made in the anterior neck to expose the trachea, and 2 successive intratracheal instillations, using 25-gauge needles, were performed. Rats were allocated randomly to receive 0.1 mL of either saline or LP17 (1 mg in 0.1 mL of saline) solution (see below for details) and, 5 min later, a 0.5 mL/kg concentration of either saline or bacterial solution (P. aeruginosa). We therefore studied rats with pneumonia (the control group), rats with pneumonia treated with LP17 (the LP17 group), and normal rats (the sham group)

Preliminary studies established that a maximum severity of our model occurred between 18 and 24 h. We thus chose to start the mechanical ventilation 18 h after the intratracheal instillations, following a procedure described elsewhere [16]. The rats were anesthetized (intraperitoneally; 50 mg/kg pentobarbital sodium) and ventilated supine through an endotracheal tube (tidal volume, 7–8 mL/kg) (rodent ventilator no. 683; Harvard Apparatus) with a fraction of inspired oxygen of 1.0 and a respiratory rate of 60 breaths/min. Arterial pressure was continuously monitored through a carotid arterial line. In addition to the biological and histological analyses described below, a different set of rats was used to assess survival

BAL, differential cell count, and histological examinationBAL was performed 24 h after bacterial inoculation by flushing the lungs 9 times with 2.5 mL of 37°C sterile pyrogen-free physiological saline via the tracheal cannula. The first fraction was removed. The 8 other fractions of 2.5 mL were recovered and pooled. The total number of lung cells was counted using a standard hemocytometer, and cytospin preparations were made. The cells were air-dried and stained with May-Grünwald Giemsa. Differential cell counts on 200 cells were made using standard morphological criteria. To examine the CR3 expression (CD11b/CD18) on the surface of alveolar cells, flow-cytometric analysis (FACScalibur; Becton Dickinson) was performed after staining the cells with CD11b/CD18 MAbs (RnD Systems) or isotypic control antibodies [16]. Histopathological examinations were performed in a blinded fashion as described elsewhere [17]

Biological measurementsArterial blood gases and lactate concentrations were determined hourly 30 min after the initiation of mechanical ventilation on an automatic blood gas analyzer (ABL 735; Radiometer). Concentrations of TNF-α, IL-1β, and IL-6 in the plasma and the BAL fluid were determined by ELISA (Biosource), as were plasma and BAL fluid D-dimer (Asserachrome D-Di; Stago Diagnostica) and thrombin-antithrombin complex (TATc) concentrations (Enzygnost TAT micro; Dade Behring). All rats were administered a volume of saline corresponding to the volume of blood drawn after sampling

Statistical analysisComparisons between 2 groups were made using Student’s t test. Comparisons between >2 groups were made using a 1-way analysis of variance. Survival curves were compared using the log-rank test. A 2-tailed value of P<.05 was considered significant. The data are expressed as mean±SD. All analyses were performed with GraphPad Prism software (version 2.01; GraphPad)

Results

LP17-associated protection from hemodynamic deterioration, prevention of lactic acidosis, and improvement in oxygenationPneumonia induced a progressive mean arterial pressure (MAP) decrease in the control rats, from 129±8 to 102±4 mmHg between 18 and 22 h after P. aeruginosa instillation. The LP17 group showed a stable and constantly higher MAP than the control group (P<.0001) and no difference from that of the sham group (figure 1). The changes in heart rate, rectal temperature, lactic acidosis, and oxygenation are summarized in table 1. A protective effect of LP17 on the development of the lactic acidosis and the deterioration of oxygenation was observed

Figure 1

LP17-associated protection from hemodynamic deterioration. Mean arterial pressure (MAP) from LP17-treated rats (the LP17 group; n=8) was constantly higher than that of the control group (n=12) (P<.0001). Time 0 corresponds to the time of Pseudomonas aeruginosa instillation

Table 1

Systemic and selected biochemical parameters during Pseudomonas aeruginosa–induced pneumonia

No effect of LP17 on bacterial clearanceWe hypothesized that the improvement of hemodynamics and oxygenation observed with LP17 could be due to better bacterial clearance. To address this question, we measured bacterial load both in BAL fluid and lung homogenate 24 h after P. aeruginosa instillation. We were unable to show any effect that LP17 has on bacterial load, suggesting that LP17 has no effect on bacterial clearance. No P. aeruginosa could be found in the sham group

LP17-associated reductions in cellular infiltration and histological lung damageWe next investigated whether LP17 affects local cell recruitment. Pneumonia induced a massive cellular efflux as shown in figure 2A. LP17 reduced the alveolar cell number, compared with the control group (P=.01). The type of cells present in the BAL fluid was not affected by LP17, with 20%–25% macrophages and 70%–75% neutrophils (figure 2B) recruited, nor was the recruited neutrophils’ activation affected, as assessed by their CR3 (CD11b/CD18) expression (figure 2C). Histological study found severe lung injury; that is, intraalveolar hemorrhage, protein precipitation, leukocyte infiltration into the alveoli, and edematous thickening of perivascular space in the P. aeruginosa–instilled lungs (figure 3). These alterations were attenuated in the LP17 group. Therefore, LP17 prevents the massive cellular infiltration and histological damage induced by pneumonia

Figure 2

LP17-associated decreases in cellular infiltration, without influencing cell type or CR3 expression. A Reduced no. of alveolar cell in the LP17-treated rats (the LP group; n=8), compared with the control group (n=12). B No alteration in type of recruited cells by LP17 treatment. C No impairment of CR3 expression in alveolar cells by LP17 treatment. Measurements were performed 24 h after Pseudomonas aeruginosa instillation. MFI, mean fluorescence intensity. *P<.02, vs. sham group (n=6); ^#P=.03, vs. sham group; ^$P=.01, LP17 group vs. control group

Figure 3

Histopathological examination of the lungs 24 h after Pseudomonas aeruginosa instillation. Intraalveolar hemorrhage, protein precipitation, and leukocyte infiltration into the alveoli and edematous thickening of perivascular space were attenuated in the LP17-treated rats. A typical illustration (original magnification, ×200) is shown here. Top panel LP17-treated rat; bottom panel control rat

LP17-associated attenuation of local and systemic inflammatory response We next determined whether LP17 could alter the inflammatory cytokine response induced by pneumonia. We measured TNF-α, IL-1β, and IL-6, both in the BAL fluid 24 h after P. aeruginosa instillation and in the plasma 18, 20, and 22 h after instillation (figure 4). The concentrations of these 3 cytokines were elevated in the plasma 18 h after instillation and further increased during mechanical ventilation (i.e., 20 and 22 h after instillation). By contrast, they were barely detectable in the plasma from the LP17 group (P<.001 for each cytokine, compared with the control group). A similar increase was found in the BAL fluid, and once again the cytokine concentrations were decreased in the LP17 group, compared with the control group (figure 4). These data suggest that LP17 attenuates the pneumonia-induced inflammatory response both locally and systemically

Figure 4

Plasma and bronchoalveolar cytokine concentrations. Plasma (top) and bronchoalveolar lavage fluid (bottom) concentrations of tumor necrosis factor (TNF)–α (A) interleukin (IL)–1β (B) and IL-6 (C) were more elevated in the control group (n=12) than in LP17-treated rats (the LP17 group; n=8). Measurements were performed 24 h after Pseudomonas aeruginosa instillation. ND, not detected. *P<.001

LP17-associated decreases in local and systemic coagulation activationSepsis and pneumonia are associated with fibrin deposition in the pulmonary compartment and disturbances in hemostatic balance [18, 19]. We, therefore, investigated the effects that LP17 has on local and systemic coagulation activation. Both D-dimer and TATc concentrations were markedly elevated in plasma and BAL fluid during pneumonia (figure 5). LP17 treatment was associated with lower concentrations of these markers at all time points, except for plasma D-dimer 18 h after P. aeruginosa instillation. Thus, LP17 prevents pneumonia-induced coagulation activation

Figure 5

Plasma and bronchoalveolar D-dimer and thrombin-antithrombin complex (TATc) concentrations. Plasma and bronchoalveolar lavage fluid concentrations of D-dimer (A) and TATc (B) were more elevated in the control group (n=12) than in LP17-treated rats (the LP17 group; n=8). Measurements were performed 24 h after Pseudomonas aeruginosa instillation. *P<.001

LP17-associated improvements in survival rateWe investigated whether the clinical and biological effects of LP17 could confer a survival advantage in this experimental model of sepsis. Pneumonia was associated with a high death rate, with only 43% of the control rats surviving the first 24 h, and at day 3 the survival rate was only 14% (figure 6). By contrast, LP17 improved survival to 75% at day 1 and 43% at day 3 (P=.03, log-rank test). There were no late deaths (after day 3), indicating that LP17 did not merely delay the onset of lethality but conferred lasting protection. No deaths occurred in the sham group

Figure 6

LP17-associated protection of rats from death. Rats (n=17 in each group) were monitored during 1 week after Pseudomonas aeruginosa instillation. The survival rate was 14% in the control group, compared with 43% in LP17-treated rats (the LP17 group) (P=.03, log-rank test). No deaths were observed in the sham group

Discussion

In the present study, we report that the modulation of the TREM-1 pathway by means of treatment with LP17, a synthetic peptide, attenuates pulmonary and systemic inflammation and promotes survival during experimental bacterial pneumonia in rats. Overactivation of the DAP12/TREM-1 pathway appears to be deleterious, in that it promotes an excessive inflammatory response. Using a model of hepatic inflammation induced by Zymosan A (Nacaraitesque), Nochi et al. demonstrated an increase in granuloma formation in DAP12-overexpressing mice [20]. This was inhibited by hepatic transgenic expression of a soluble form of the extracellular domain of TREM-1, as an antagonist of DAP12 signaling. In a lipopolysaccharide-induced septic shock mouse model, administration of an agonist anti–TREM-1 MAb doubled the mortality rate from 50% to 100% [12]. By contrast, treatment with a TREM-1 fusion protein protected mice against shock induced by endotoxin or cecal ligation and puncture [7]. Monocytes and macrophages produce a soluble form of TREM-1 (sTREM-1), and the administration of LP17, a peptide mimicking this soluble receptor, reduced inflammatory hyperresponsiveness and lowered mortality in septic mice [12]

Studies using DAP12 knockout (DAP12^−/−) mice also provide indirect evidence of a role that the DAP12-associated receptor TREM-1 plays in the development of inflammation. Turnbull et al. recently observed that septic DAP12^−/− mice had reduced plasma cytokine concentrations, a decreased acute-phase response, and a lower mortality rate [21]. In cells isolated after sepsis and stimulated ex vivo, DAP12 signaling augmented lipopolysaccharide-mediated cytokine production. These data demonstrate that DAP12 signaling enhances the response to microbial products during sepsis, amplifying inflammation and contributing to mortality. Conflicting with these results, Hamerman et al. recently found that DAP12-deficient macrophages produced higher concentrations of inflammatory cytokines in response to TLR-2, TLR-4, and TLR-9 stimuli and that DAP12-deficient mice were more susceptible to endotoxic shock [22]. These findings suggest that 1 or more DAP12-pairing receptors negatively regulate signaling through TLRs. One of these could be a specific receptor for sTREM-1 (and then could recognize the TREM-1 peptide LP17); many DAP12-paired receptors have a related inhibitory receptor [22]

In the present study, we extend previous observations of a beneficial effect of LP17 [12] in another physiologically relevant model of infection. Our P. aeruginosa pneumonia model was severe enough to induce the development of a severe sepsis, as demonstrated by hemodynamic deterioration, the occurrence of a lactic acidosis, and hypoxemia. All these disorders were attenuated by LP17, and, importantly, survival was improved by this treatment

The mechanisms by which LP17 is beneficial during pneumonia could involve phagocytic cell recruitment or their killing capacity. In agreement with the results of Hamerman et al., we found no effect of LP17 in terms of bacterial colony count, in either BAL fluid or lung homogenate [22]. By contrast, Carpenter et al. recently reported that intrapulmonary delivery of an adenovirus vector expressing KARAP/DAP12 enhanced fungal clearance during experimental invasive pulmonary aspergillosis in neutropenic mice [23]. Nevertheless, this model is considerably different from ours, and this could largely explain the discrepancy

As expected, pneumonia induced massive cell recruitment within the lung and led to histological damage. LP17 prevented these alterations. Importantly, although the number of infiltrating cells was reduced, LP17 did not alter their nature (majority of polymorphonuclear neutrophils) or the level of expression of CR3, which is crucial to their phagocytic activity [24]

P. aeruginosa infection was also responsible for the development of an inflammatory response, both locally, as assessed by the BAL fluid cytokine concentrations, and systemically. LP17 blunted production of this cytokine within the lung but without completely inhibiting it. By contrast, the systemic inflammatory response was almost completely abolished and could explain the hemodynamic improvement. These findings are in agreement with those of previous studies investigating TREM-1 pathway modulation during other circumstances [7, 12]

Acute lung injury and pneumonia are associated with pulmonary activation of coagulation and impairment of fibrinolysis, resulting in fibrin deposition in the lung [18, 19]. We used D-dimer and TATc concentrations as markers for coagulation activation. We found that pneumonia elicited increases in the local and systemic concentrations of both markers and that these increases were markedly reduced by LP17 treatment. Because TREM-1 has no known action on coagulation, these data suggest that LP17 has an indirect effect on coagulation activation, probably by reducing the inflammatory response [25]

Although the effect of LP17 appears to be robust, some limitations deserve consideration. To obtain a severe model of sepsis, we chose to not administer antibiotics to the rats, and, therefore, mortality was high. Obviously, the relevance of this choice from a clinical point of view is questionable, because most of the patients who have pneumonia receive early and adequate antibiotherapy. Another limitation is that we administered LP17 at the time of P. aeruginosa instillation and did not investigate its effect when given as a delayed treatment. This was done to avoid repeated anesthesia that might have been difficult and deleterious in rats with already-developed pneumonia

In conclusion, the modulation of the TREM-1 pathway by the use of synthetic peptide, such as LP17, is beneficial during P. aeruginosa pneumonia in rats in attenuating lung and systemic inflammatory responses

Acknowledgments

We thank Chantal Montemont, Eliane Vautier, and Anne-Marie Carpentier for their valuable technical assistance

Potential conflicts of interest: none reported

Financial support: Fondation Recherche Médicale; Association des Chefs de Service du Centre Hospitalier Universitaire de Nancy, France