Acute respiratory infections are the cause of significant morbidity and mortality in patients with compromised immunity. Mortality that can be attributed to nosocomial pneumonia is as high as 50% (1). Hemorrhagic shock, such as that resulting from trauma, induces innate immune dysfunction and enhanced susceptibility to secondary infection (2). Ventilator-associated pneumonia is a frequent infectious complication of trauma and hemorrhagic shock (3). An analysis of the SENTRY Antimicrobial Surveillance Program, a recent multinational study, shows that the gram-negative bacterium Pseudomonas aeruginosa is the second most prevalent microbe causing hospital-acquired bacterial pneumonia (22.4% of all cases), and the most prevalent cause of ventilator-associated bacterial pneumonia (26.6% of all cases) (4). Multidrug resistance is of particular concern with P. aeruginosa, so understanding pathogenesis and developing therapies to promote host defense against secondary respiratory infection are especially important to curtail progression into sepsis and/or acute respiratory distress syndrome with high rates of mortality.
In the acute phase of the inflammatory response to infection, tissue-resident cells of the innate immune system first recognize the presence of a pathogen. In the lungs, alveolar macrophages and epithelial cells release pro-inflammatory cytokines, including tumor necrosis factor-α (TNFα) and interleukin 1β (IL-1β), and chemokines, including C-X-C motif ligand 1 (CXCL1), that initiate the recruitment of effector leukocytes. Cytokine storm, the exaggerated production and release of pro-inflammatory cytokines, is a pathological feature of infectious disease in the critically ill (5). In an experimental animal model of critical illness in which hemorrhagic shock precedes systemic infection via cecal ligation and puncture, prior hemorrhage amplified the levels of pro-inflammatory mediators IL-6 and macrophage inflammatory protein-2 in the lungs (6). Similarly, with a secondary pulmonary stimulus, hemorrhage primes for the enhanced generation of inflammatory mediators in the lungs of rats (7). However, in some experimental models of nosocomial pneumonia, prior insult led to a reduction in certain pro-inflammatory cytokines in response to subsequent respiratory infection (8, 9).
Neutrophils, by mobilizing from the circulation, are critical for host defense against bacterial infections of the lungs (10–14). Human patients (15, 16) and laboratory mice (10, 17, 18) with neutropenia have enhanced susceptibility and mortality to P. aeruginosa respiratory infection. In contrast to the multistep adhesion cascade that has been extensively characterized using animal imaging models and in-vitro systems, neutrophil recruitment from the capillary network of the lungs occurs through mechanisms distinct from those at play in postcapillary venules. In part, these mechanistic differences are due to higher relative rates of blood flow and wall shear stress in venules as compared to the pulmonary capillary bed (11). Neutrophils must deform and squeeze through the pulmonary capillaries, even during homeostasis (19). During inflammation, neutrophil stiffening occurs and results in their sequestration, retention and enrichment within the lungs (20). Presumably, pulmonary sequestration of neutrophils during respiratory infection provides a greater supply of neutrophils to engulf and kill bacteria. However, to complete their antimicrobial activities, neutrophils must emigrate from the capillary bed into the alveolar airspaces.
Neutrophil priming is a phenomenon in which neutrophils acquire a preactivated state that results in enhanced responsiveness to subsequent stimuli (21, 22). Studies in mice have described neutrophil priming that occurs following hemorrhagic shock (6, 7, 23). These studies suggest that prior hemorrhagic shock acts as a priming event for an exaggerated response by neutrophils, that is, triggered upon subsequent infectious challenge and results in injury to bystander tissues/organs, such as the lungs. For example, such damage is thought to be mediated by premature deployment of neutrophil effector functions such as reactive oxygen species generation, degranulation, and release of neutrophil extracellular traps (24, 25).
In addition to the direct tissue damage that accompanies a severe inflammatory response, an inability of the host to eliminate infection is thought to contribute to high rates of morbidity and mortality in the critically ill (5). Several experimental animal models have been developed to investigate the immune dysfunction that renders the severely injured host susceptible to lethal secondary infection (8, 9, 26, 27). Although the broader paradigm that injury/hemorrhage induces host immune suppression has been established, the specific mechanisms of immune dysfunction (e.g., impact on specific leukocyte subsets) and their prominence with respect to specific secondary infectious pathogens remain topics of active investigation. We thus set out to determine the impact of hemorrhagic shock on neutrophil recruitment in response to secondary respiratory infection by P. aeruginosa. While one might expect that hemorrhage, by priming an amplified inflammatory response to a second stimulus (28), would enhance the recruitment of neutrophils into the airspaces, our findings indicate that neutrophil recruitment is compromised in mice receiving controlled/fixed pressure, hypovolemic hemorrhage prior to respiratory infection. The hemorrhage-induced attenuation of neutrophil recruitment in response to P. aeruginosa is accompanied by a higher rate of mortality.
MATERIALS AND METHODS
All animal studies were approved by the Lifespan Animal Welfare Committee (Approval #0150-16, Office of Laboratory Animal Welfare Assurance #A3922-01) and were conducted in accordance with the Public Health Service guidelines for animal care and use. C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in a specific pathogen-free facility at Rhode Island Hospital with ambient temperature 22 ± 1°C and a 12-h light/dark cycle. Male C57BL/6 mice, 9 to 11 weeks old and weighing 20 g to 28 g that were used for this study, were provided ad libitum access to standard mouse chow and water. In each experiment, an equal number of mice for each experimental group from a given set of littermates were allocated prior to the hemorrhage/sham procedure. A total of 99 C57BL/6J mice were used in this study. In some cases, the same set of mice was used for multiple analyses. The following sets of mice were used: cytokine and bacterial burden analyses; neutrophil recruitment, priming, and blood count analyses; histological analyses; and survival analyses.
Hemorrhagic shock and resuscitation
The mouse model of nonlethal, fixed-pressure hemorrhagic shock has been described (6, 29). Throughout the procedure, animals were secured on a 37°C water-circulating heating pad (T/Pump, Stryker, Kalamazoo, MI). Dual femoral artery catheterization was performed on mice anesthetized with isoflurane. One catheter was used to monitor blood pressure (Blood Pressure Analyzer 400, Digi-Med, Louisville, KY) while the other catheter was used for controlled blood withdrawal. Blood was withdrawn until a mean arterial blood pressure of 35 ± 5 mmHg was reached and maintained for 90 min. Four times the shed blood volume of Ringer's lactate was infused as fluid resuscitation. At the conclusion of the hemorrhage procedure, catheters were removed, femoral vessels were ligated, and the incisions were sutured. Sham mice had their femoral arteries ligated, but without catheterization or blood withdrawal. Suture sites were bathed with lidocaine/bupivacaine as local analgesic. Immediately following the hemorrhage procedure, mice were monitored for 1 h until their return to normal locomotive/aroused state. For the 24-h period following hemorrhage, mice were monitored at 4 separate times to assess animal condition (activity level, respiratory signs, suture site integrity); all mice in this study recovered normally from hemorrhage/sham procedure without complication.
Acute respiratory infection
The cytotoxic P. aeruginosa strain PA103 (30) was grown on solid media before culturing to log phase in tryptic soy broth. Bacteria were suspended in sterile 0.9% saline and the number of colony-forming units (CFUs) was determined by a standard curve according to optical density at 540 nm. Intratracheal inhalation was performed under vaporized isoflurane anesthesia by pipetting a 50 μL bolus of either 0.9% saline alone or 3 × 105 CFU P. aeruginosa into the back of the oral cavity of mice secured on an intubation stand (Braintree Scientific, Braintree, MA). Intratracheal inhalation was performed at 24 h after hemorrhagic shock (timed from the end of fluid resuscitation) or sham procedure. For survival studies, mice were observed every 6 to 10 h for 4 days postinfection and then euthanized by carbon dioxide inhalation. For 24-h time point analyses of lungs, mice were euthanized by intraperitoneal injection of Fatal Plus euthanasia solution. For the survival study, a group size of 12 mice was determined by power analysis using Chi-square test of difference in proportion between sham/infection and hemorrhage/infection; fewer mice were used in the negative control group (hemorrhage/saline), but no statistical comparison was made to this group.
Quantification of lung cytokines/chemokines and bacterial burden
Twenty-four hours after receiving saline or P. aeruginosa, mice were euthanized and all lobes of the lung were harvested. Lung tissue was dissociated using an enzymatic digestion kit (Miltenyi, Auburn, CA). To quantify lung cytokines and chemokines, digested lung tissue was centrifuged to remove cells and supernatants were subjected to a cytometric bead array assay for multiplexed assessment of murine IL-6, CXCL1, TNFα, IL-1β, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), and CXCL5 (BioLegend, San Diego, CA). Group sizes for cytokine/chemokine analyses, as determined using power analyses based on expectations of response/variation (10-fold saline vs. PA; 3-fold sham–PA vs. Hem–PA), were: sham–saline, Hem–saline: n = 4; sham–PA: n = 10; and Hem–PA: n = 11. To quantify the burden of P. aeruginosa, serial dilutions of digested lung tissue were plated on tryptic soy agar plates. The total number of P. aeruginosa CFUs in the lungs were calculated by extrapolation of, typically, the number of colonies from 2 different dilutions. Counts of zero were imputed as 100 CFUs for the purpose of statistical analyses. This was further justified as an approximate limit of detection, as the smallest dilution factor plated for the assay was comprised of 10 μL of a 2.4 mL sample of the whole digested lung. Group sizes for bacterial burden analyses were: sham–PA: n = 14; Hem–PA: n = 15. Bacterial burden analyses required additional animals beyond those used in conjunction with quantifying cytokines due to the greater variability between individual mice.
Quantification of neutrophil recruitment
Twenty-four hours after receiving saline or P. aeruginosa, mice were euthanized and bronchoalveolar lavage (BAL) was obtained by repeated instillation/withdrawal of ice cold 1 mL phosphate buffered saline containing 5 mM ethylenediaminetetraacetic acid and 0.5% bovine serum albumin (total volume collected 4–5 mL per mouse). BAL samples were stained with the following antibodies to identify neutrophils: APC/Cy7 anti-Ly6G, Alexa Fluor 488 anti-CD11b, and PE anti-C-X-C motif chemokine receptor 2 (CXCR2) (all from BioLegend). Samples were analyzed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi, Bergisch Gladbach, Germany) and postacquisition analysis was performed using FlowJo software (Becton Dickinson, Franklin Lakes, NJ). Group sizes for analyses of BAL neutrophils were: Hem–saline: n = 4; sham–PA, Hem–PA: n = 7.
Twenty-four hours after receiving saline or P. aeruginosa, mice were euthanized and the lungs were fixed by gravity infusion of 10% buffered formalin. Sections from paraffin embedded tissue were stained with hematoxylin and eosin for histological examination on an Olympus BX60 microscope with a Chameleon3 color camera (FLIR Integrated Imaging Solutions, Wilsonville, Ore). Images of slides from n = 3 mice per condition were acquired by a blinded observer; representative images are shown.
Blood samples were collected by saphenous venipuncture, both at 24 h after sham or hemorrhage procedure (prior to infection) and at 24 h after intratracheal instillation of saline or P. aeruginosa. After erythrocyte lysis with ammonium chloride solution, samples were stained with the following antibodies: APC anti-CD54, Alexa Fluor 488 anti-CD11b, PE anti-CXCR2, and Brilliant Violet 421 anti-Ly6G (all from BioLegend). Samples were analyzed by flow cytometry as described above. Group sizes were n = 3 for all flow cytometry analyses of neutrophil priming (pre and postinfection), and were n = 10 for analyses of circulating neutrophil count.
Data are presented as mean ± SD. For comparison between 2 groups to assess bacterial burden and circulating neutrophil number, 2-tailed Student's t tests were performed. For all other experiments, comparison between more than 2 groups was performed by one-way analysis of variance and Tukey's post-hoc multiple comparisons test. Statistical analysis of Kaplan–Meier survival curves was performed by log-rank test. P values < 0.05 were considered significant, as indicated by * and † in figure legends. All statistical analyses were performed using Prism software (GraphPad, La Jolla, Calif).
Hemorrhage potentiates the inflammatory cytokine response to P. aeruginosa
To characterize the inflammatory environment of a “two hit” model of P. aeruginosa pneumonia secondary to hemorrhagic shock, we analyzed a panel of cytokines and chemokines in the lungs of mice. Groups of C57BL/6 mice received either a sham procedure or hemorrhagic shock by fixed-pressure blood removal and resuscitation. Twenty-four hours after hemorrhage, mice received an intratracheal bolus of 3 × 105 CFU P. aeruginosa or saline control. At 24 h postinfection, we digested lung tissue and quantified the concentration of IL-6, CXCL1, TNFα, IL-1β, IL-10, GM-CSF, and CXCL5. Of these, only CXCL5 did not exhibit a significant increase in the lungs of mice infected with P. aeruginosa compared to those given saline (Fig. 1; sham/Hem–saline, n = 4; sham–PA, n = 10; Hem–PA, n = 11). Prior hemorrhage significantly enhanced the levels of IL-6 and CXCL1 in lung tissue in response to infection (Fig. 1, sham–PA vs. Hem–PA). Hemorrhagic shock alone did not significantly impact inflammatory cytokine generation in the lungs (Fig. 1). On a technical note, the small discrepancy in group size between P. aeruginosa infected mice for this assay and for evaluating bacterial burden was the result of sample loss during tissue digestion. Taken together, these results suggest that hemorrhage serves as a priming event for an amplified inflammatory response to secondary respiratory infection with P. aeruginosa.
Neutrophil recruitment in response to hemorrhage and P. aeruginosa
Neutrophils play a critical role in the clearance of P. aeruginosa from infected airspaces (10, 17, 18). To successfully fulfill their host defense function, neutrophils must first traffic from the circulation into the lung parenchyma and alveolar airspaces. We therefore performed experiments to determine whether hemorrhagic shock impacts the recruitment of neutrophils in response to subsequent respiratory infection by P. aeruginosa. In this context, we define a recruited neutrophil as one that has emigrated from the vasculature and entered the bronchoalveolar airspaces.
Previous studies have demonstrated that hemorrhagic shock results in neutrophil priming with enhanced responsiveness to subsequent stimuli (6, 7, 23). At 24 h after hemorrhage, we analyzed circulating neutrophils for surface expression of CD54, a marker of neutrophil priming (22). As expected, peripheral blood neutrophils from hemorrhaged mice uniformly expressed higher levels of CD54 than neutrophils from mice that had received a sham procedure (Fig. 2 A and B; n = 3 per group). Hemorrhagic shock alone did not trigger neutrophil activation, as surface expression of CD11b, a constituent of secretory granules, was unchanged on circulating neutrophils both before and after secondary infection (Fig. 2 A and B). In addition, after sham or hemorrhage procedure, both before and after secondary infection, peripheral blood neutrophils from all groups expressed equivalent levels of CXCR2 (Fig. 2 A and B), the primary chemokine receptor for CXCL1 that drives neutrophil recruitment in a similar model (7).
Neutrophil recruitment into the alveolar airspaces occurs rapidly, with significant numbers entering within hours and continuing to accumulate for at least 24 h (31). In mice receiving either sham or hemorrhage procedure followed by intratracheal P. aeruginosa, we analyzed the number of circulating neutrophils, as release of neutrophils from bone marrow and other reservoirs is one mechanism for promoting recruitment to tissue sites. We observed that prior hemorrhage did not significantly impact circulating neutrophil numbers at 24 h postinfection (Fig. 2C; n = 10 per group), consistent with what has been observed in a similar model of pseudomonal pneumonia secondary to polytrauma (32). However, as shown in Figure 3A, hemorrhagic shock significantly attenuated the entry of neutrophils into the bronchoalveolar airspaces. While hemorrhage alone (n = 4) did not result in neutrophil appearance in the BAL, respiratory infection with P. aeruginosa induced the accumulation of neutrophils in the BAL that was reduced by about half in mice receiving prior hemorrhage (n = 7), as compared to sham animals (n = 7) (Fig. 3A).
Prior to entering the airspaces of the lung, neutrophils may accumulate in the lung vasculature and parenchyma (19, 31). To determine whether the hemorrhage-induced apparent reduction in neutrophil entry into the airspaces represented an overall attenuation of cellular infiltration of the lung tissue, we performed histological analyses at 24 h postinfection. In mice receiving P. aeruginosa, we observed robust cellular infiltrates in the context of either prior hemorrhagic shock or sham procedure (Fig. 3B). Taken together, these data suggest that prior hemorrhagic shock specifically compromises neutrophil recruitment into the airspaces in response to respiratory infection by P. aeruginosa.
Prior hemorrhage and the clearance of P. aeruginosa
Inhibition of neutrophil recruitment results in greater bacterial burden and mortality in a mouse model of P. aeruginosa pneumonia (13), demonstrating a prominent role for neutrophils in eradicating this pathogen from the lungs. As we observed that hemorrhagic shock reduced the subsequent recruitment of neutrophils by approximately 50%, we therefore sought to determine whether the clearance of P. aeruginosa from the lungs was also impacted by prior hemorrhage. At 24 h postinfection, we measured the number of P. aeruginosa CFUs in the lungs of mice receiving either sham procedure or hemorrhage the day prior to infection. Although a third of the mice that underwent hemorrhagic shock prior to infection (Hem–PA, n = 15) had more lung tissue P. aeruginosa CFUs than any of the mice that received sham procedure prior to infection (sham–PA, n = 14), the difference between these 2 groups was not statistically significant (Fig. 4). Thus, the attenuated recruitment of neutrophils into the bronchoalveolar airspaces of mice subjected to prior hemorrhagic shock may not significantly impact bacterial clearance at 24 h postinfection.
Impact of hemorrhagic shock on surviving secondary infection
While pseudomonal pneumonia in healthy individuals is rare, P. aeruginosa is a prevalent pathogen causing nosocomial pneumonia in the immunocompromised (15, 16). Hemorrhagic shock, a frequent result of traumatic injury, is followed by adaptive and maladaptive responses of the host, including systemic inflammatory response syndrome and concurrent compensatory anti-inflammatory response syndrome, that impact outcome to secondary infection (2, 5).
To assess whether prior hemorrhage alters mortality rate in response to secondary respiratory infection with P. aeruginosa, we monitored the survival of mice for up to 4 days postinfection. Controlled hemorrhagic shock and resuscitation without secondary infection did not cause mortality in C57BL/6 mice (data not shown), as expected based on previous studies (26). Respiratory infection following sham procedure (sham–PA, n = 12 mice) resulted in mortality in a small minority (2 out of 12) of mice (Fig. 5). By contrast, mice receiving hemorrhage/resuscitation 24 h prior to infection (Hem–PA, n = 13 mice) exhibited significantly lower survival at 4 days postinfection (Fig. 5). These data indicate that controlled/fixed-pressure, hypovolemic hemorrhagic shock predisposes mice to a lethal secondary respiratory infection by P. aeruginosa.
Secondary infections represent a major clinical challenge for individuals that survive an initial traumatic injury that is accompanied by severe blood loss and hemorrhagic shock. Animal models have been developed to further understand the impact of hemorrhagic shock on the immune system and how this relates to the pathogenesis of secondary infections in the hospital setting. Here, we have employed a mouse model in which controlled hemorrhage and resuscitation are followed by respiratory inoculation with the gram-negative P. aeruginosa, one of the most common bacterial pathogens causing nosocomial pneumonia (4). Focusing on the neutrophil recruitment response to P. aeruginosa, we observe that prior hemorrhage results in a marked decrease in the number of neutrophils entering the airspaces of the lungs and a higher mortality rate.
The mechanisms by which hemorrhagic shock attenuates neutrophil entry into P. aeruginosa-infected airspaces are likely to be complex. The recent work of our group (33) and of others (14) has demonstrated that the steps of recruitment in which a neutrophil emigrates from inside the pulmonary vasculature through the parenchyma and into the airspaces are highly regulated. Further, the sequestration and retention of neutrophils in pulmonary capillaries is augmented by hemorrhage prior to a second hit (6), but those activated intravascular neutrophils may emigrate into parenchyma with slower kinetics (33). Thus, while the dysfunction of neutrophil migration during critical illness is well documented (34), the impact of hemorrhagic shock on neutrophil recruitment to a localized secondary infection is less clear and, specifically within the lungs, there are potentially competing mechanisms at play. While hemorrhage-primed neutrophils would be expected to become more easily sequestered and retained within the pulmonary capillaries in response to a respiratory infection (7, 35), those retained and activated neutrophils may exhibit delayed or defective emigration into the interstitium and airspaces. We recently described how activated β2 integrins restrict neutrophil entry into the airspaces in response to P. aeruginosa(33), suggesting a potential mechanism by which hemorrhage could attenuate neutrophil recruitment. These competing mechanisms could explain the discrepancy between the hemorrhage-induced defects in neutrophil appearance in BAL that we observe with analogous studies in which prior hemorrhagic shock promoted neutrophil recruitment elicited by lipopolysaccharide (7). It also remains possible that the impact of hemorrhage on neutrophil recruitment depends upon the nature, identity, and/or magnitude of the second stimulus.
Our experiments were unable to detect a statistically significant difference in clearance of P. aeruginosa from lung tissue between mice receiving prior hemorrhage versus sham procedure. However, given the critical role of neutrophils in clearing P. aeruginosa, both in humans (15, 16) and in rodents (10, 17, 18), the potential relationship between changes in the number of recruited neutrophils and clearance of P. aeruginosa should continue to be investigated. Additional studies with adequate statistical power to detect differences in bacterial clearance in an outcome with high levels of variation would address some shortcomings of the current study. Operational efforts to achieve consistent planktonic P. aeruginosa virulence and the use of a randomization matrix for allocating group subjects would help minimize the heterogeneity in observed responses.
Whether the outcome of respiratory infection secondary to hemorrhagic shock is driven by tissue damage that is mediated by neutrophils, P. aeruginosa, or both, remains an open question that may be difficult to answer. While there are likely other factors involved in the negative impact of hemorrhage on animal survival, there is also evidence that hemorrhage-induced priming of leukocyte subsets enhance aspects of innate host defense. For example, alveolar macrophages are primed by hemorrhagic shock, via systemic oxidative stress, to produce and release higher levels of inflammatory cytokines (7, 28), and neutrophil priming enhances the phagocytosis of bacteria and generation of reactive oxygen species (22). It is possible that, in our study, hemorrhage-induced priming of neutrophil antimicrobial activity partially compensated for the defect in neutrophil recruitment, thereby balancing opposing effects on the clearance of P. aeruginosa from the lungs.
In conclusion, hemorrhagic shock leads to an attenuation of neutrophil entry into the airspaces in response to subsequent respiratory infection by P. aeruginosa. The rapid recruitment of neutrophils, which in the context of P. aeruginosa pneumonia is mediated by CXCL1/CXCR2 (13), was nevertheless reduced in hemorrhaged mice that had nearly 10 times as much CXCL1 in the lungs as control mice receiving the same bolus of P. aeruginosa. These findings suggest a biphasic neutrophil recruitment response with respect to CXCL1 in the lungs. Future studies that address the apparent competing forces of neutrophil sequestration within the pulmonary capillary network and chemotactic emigration into the airspaces will aid in reaching the balance between neutrophil-dependent host defense and neutrophil-mediated lung injury.
We thank Dr Jason Machan for help with statistical analyses.
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