Resolvin D2 restores neutrophil directionality and improves survival after burns

Abstract

Following severe burns and trauma injuries, the changes of neutrophil migratory phenotype are a double-edged sword. Activated neutrophils migrate into injured tissues and help contain microbial infections, but they can also enter normal tissues and damage vital organs. Depleting the neutrophils from circulation protects vital organs against neutrophil-induced damage but leaves the body exposed to infectious complications. Here we show that restoring normal neutrophil migratory phenotype in rats with burn injuries correlates with improved survival in a classical double-injury model of sequential burn and septic insults. We uncovered that the directionality of neutrophils from burned rats can be restored both in vitro by 1 nM resolvin D2 (RvD2) and in vivo by RvD2 for 7 d, 25 ng/kg body mass (8–10 ng/rat). Restoring neutrophil directionality dramatically increases survival after a second septic insult at d 9 postburn. Survival of RvD2-treated animals increases from 0 to 100% after lipopolysaccharide injection and is extended by 1 wk after cecal ligation. Survival does not significantly increase when the restoration of neutrophil directionality is incomplete, following shorter regimens of RvD2. We conclude that restoring neutrophil directionality using RvD2 could have prophylactic value and delay lethal complications after burn injuries.—Kurihara T., Jones, C. N., Yu, Y.-M., Fischman, A. J., Watada, S., Tompkins, R. G., Fagan, S. P., Irimia, D. Resolvin D2 restores neutrophil directionality and improves survival after burns.

Traumatic injuries, including automobile and industrial accidents, gunshot injuries, and burns, remain the leading cause of death in working-age Americans under the age of 45 (1). Increasingly sophisticated medical and surgical treatments are available, and many victims who would not have survived a decade ago are now often saved (2). As the emergency medical and surgical treatment of patients with traumatic injuries improves, new problems emerge. The survival of the severely injured patients after the first week in the hospital is now limited by infections and multiple organ failure complications (3). These emerging problems are most daunting in the context of burn injuries, when such complications are now responsible for more than two-thirds of patient deaths in the hospital (4). Addressing infectious and inflammatory complications early and effectively in the patients with burns and traumatic injuries remains a significant unmet clinical need, augmented by the difficulties of diagnosing infections early (5, 6) and the increasing frequency of multidrug resistant bacteria (7).

Toward the ultimate goal of preventing and treating septic complications in patients with burn and traumatic injuries, we focused our attention on neutrophils. Neutrophils are part of the innate immune system, the first line of defense against various microbes, and are promptly activated after burn injury (8). The role of this activation is to contain infections through damaged skin and tissues. However, the prolonged activation of the innate immune system also appears to have significant deleterious effects on recovery after injury (9). Neutrophils appear to be most severely affected within days after burn injury (10), and multiple functional alterations have been documented. Such functions include the neutrophils' ability to kill microbes, production of cytokines (11, 12), and chemotaxis in response to chemical gradients (13).

Chemotaxis is a sine qua non condition for proper employment of all the other neutrophil functions. Among the neutrophil functions impaired after burn injury, a defective ability for chemotaxis will have the largest effect. If neutrophils do not follow the guidance of chemical signal gradients into the target tissues, they cannot reach their destination, and their ability to kill microbes and limit infections becomes useless. At the same time, neutrophils that veer away from guiding chemical gradients may end up inside various healthy tissues. When activated by a second stimulus, neutrophils could release their lytic enzyme inside healthy tissues and initiate tissue damage that could lead to organ dysfunction (14). In various animal models of burn injury, interventions aimed at depleting neutrophils (15) or blocking various chemokine receptors on neutrophils and inhibiting neutrophil activities (16, 17) have demonstrated protective effects on vital organs. A drawback of all these interventions was, however, the concomitant increase in the rate of infections in the treated animals (17, 18). In this context, restoring normal neutrophil chemotaxis after burn injury could simultaneously address the ability of neutrophils to fight against local infections and reduce the unnecessary inflammation in healthy tissues.

To measure the changes in neutrophil chemotaxis function after burn injury, we employed a rat model of burn injury (19) and microfluidic devices that enabled us to decouple the speed and directionality during chemotaxis (20). We found that neutrophil directionality deteriorates progressively for days after burn injury. To probe whether restoring the impaired neutrophil activity could improve the outcome of septic complications after burn injury, we measured the direct effects of recently discovered resolvins D1 and D2 (RvD1 and RvD2) on neutrophil chemotaxis (21, 22). We found that resolvins can quickly restore the defective directional motility of neutrophils from burned rats in vitro. We also found that the defects of neutrophil motility speed and directionality after burns can be rescued in vivo after 7 d of systemic administration of RvD2. Notably, 2 d after the end of the 7-d RvD2 treatment, the restored neutrophil motility function correlated with dramatically improved survival of the burn injured rats after second septic insults. Our results suggest that restoring normal neutrophil motility using RvD2 could have prophylactic value after severe burns, ultimately decreasing morbidity and mortality from infectious and inflammatory complications.

MATERIALS AND METHODS

Rat model of burn injury

Wistar male rats were purchased from Charles River Laboratories (Raleigh, NC, USA). All rats were housed in air-conditioned rooms with 12-h light-dark cycle and provided with free access to commercial laboratory food and water ad libitum. Rats weighing 300–400 g were used in the experiments after a 4- to 7-d acclimatization period. Before the burn procedure, cecal ligation, or cardiac puncture, rats were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital sodium. All animal protocols were approved by Subcommittee on Research Animal Care of the Massachusetts General Hospital. Thermal injury was produced using a protocol described previously (19), with minor modifications. Briefly, after the back fur of the trunk was clipped, the rats were placed in a mold exposing 30% of total body surface area (TBSA), and the exposed area was immersed in boiling water for 12 s, producing a full thickness burn. Sham-burned animals were similarly treated, with the exception that they were immersed in the room temperature water. Immediately after burn or sham-burn procedures, all animals received fluid resuscitation with 40 ml/kg i.p. of normal saline.

Second septic insult

For the lipopolysaccharide (LPS)-injection experiments, rats were injected with 2 mg/kg of LPS derived from Escherichia coli 0111:B4 (Sigma-Aldrich, St. Louis, MO, USA) via tail vein at 9 d after burn or sham-burn treatment. Vehicle solution was injected into the other 9 d postburn rats. The survival rates were evaluated at 7 d after the second insult. For the cecal ligation experiments, the rats abdominal region was shaved at d 9 postburn or sham-burn treatment. The abdomen was opened with a 2-cm midline incision. The cecum was ligated just distal to the ileocecal valve so that bowel continuity was preserved. No puncture was performed. The midline incision was then closed. After surgery, the animals were allowed to recover in their cages with free access to food and water. Sham operation rats also underwent surgery 9 d after burn with an abdominal incision; the cecum was exposed without ligation, and the abdominal incision was closed. These rats were surveyed for 23 d postburn.

Neutrophil isolation

Blood was collected every 3 d postburn (d 0, 3, 6, and 9). For each time point, whole blood was collected by cardiac puncture, using EDTA as anticoagulant. Rats were euthanized soon after the blood collection. Neutrophils were isolated from whole blood by means of a 2-step discontinuous Histopaque (Sigma-Aldrich) gradient centrifugation method. The densities of the Histopaque gradient were 1.083 and 1.119 g/ml. Whole blood (3 ml) was gently layered on the gradient (3 ml of each density) and centrifuged at 400 g for 45 min at room temperature. The polymorphonuclear (PMN) leukocyte band and all volume between the band and the red blood cell layer were collected. To further purify neutrophils, the EasySep negative selection kit for rat neutrophils (Stemcell Technologies, Vancouver, BC, Canada) was used.

Microfluidic devices for measuring neutrophil directional motility

Microfluidic devices used to evaluate the directional migration of neutrophils were fabricated using standard microfabrication technologies. Two layers (3 and 50 μm thick) of SU8 photoresist (Microchem, Newton, MA, USA) were sequentially patterned on a silicon wafer using photolithography masks and standard photoresist processing cycles (according to the instructions from the manufacturer). The wafer with patterned photoresist was used as a mold to produce pieces of polydimethylsiloxane (PDMS, Fisher Scientific, Fair Lawn, NJ, USA), which were subsequently bonded irreversibly to standard glass slides (1×3 inches, Fisher). Three devices for neutrophil motility were bonded together on one glass slide. Devices were fabricated in batches, stored dry at room temperature, and used as needed. The microfluidic devices were primed with a mixed solution of chemoattractant 100 nM N-formyl-methionylleucyl-phenylalanine (fMLP; Sigma-Aldrich) and extracellular matrix protein (25 nM rat fibronectin; Sigma-Aldrich). The role of fibronectin was to promote neutrophil adhesion inside the channels. The mixed solution was prepared in Hanks' buffered salt solution (HBSS; Sigma-Aldrich) with 0.2% rat serum albumin (Sigma-Aldrich). To prime the devices, a 1-ml syringe filled with the solution of chemoattractant and extracellular matrix was connected to one port of the device. By applying pressure to the syringe, the device was filled with the mixed solution, and after the exit port was clamped, the additional pressure helped displace air, which diffused out through PDMS. At 15 min after priming the devices, neutrophils were loaded into the device at the same time washing the chemoattractant out from the main channel. A chemoattractant gradient was soon established in the side channels by diffusion of the chemoattractant between the end of side channels filled with chemoattractant (acting as a source) and the main channel was filled with buffer (acting as a sink). Neutrophil migration started immediately and was recorded at multiple locations inside the device using a fully automated, time-lapse imaging system, Zeiss Axiovert 200M inverted microscope (Carl Zeiss, Oberkochen, Germany).

Neutrophil motility after incubation with resolvins in vitro

Neutrophils from healthy (control) and 9 d postburn rats were incubated in vitro with resolvins at various concentrations for 5 min at 37C. Resolvins were stored at −80°C in ethanol and were thawed and diluted in HBSS to the target concentration right before the incubation with neutrophils. Neutrophils were then washed twice with HBSS by repeated centrifugation and resuspension to remove excess resolvins. After resuspension in HBSS with 0.2% rat serum albumin, neutrophils were loaded in the microfluidic devices, and their directional migration was evaluated.

Neutrophil motility after incubation with serum from burned rats and resolvin D2 in vitro

Neutrophils from healthy rats were separated using the Histopaque gradient method and incubated with serum separated from blood of burned rats (d 9) for 3 h at 37°C. Neutrophils were then washed twice with HBSS by repeated centrifugation and resuspension to remove excess serum. After resuspension in HBSS with 0.2% rat serum albumin, neutrophils were loaded in the microfluidic devices, and their directional migration was evaluated. To probe the effect of RvD2 on neutrophils exposed to serum from burned rats, neutrophils from normal rats were incubated with serum from burned rats for 30 min, washed twice with HBSS, incubated with RvD2 at various concentrations for 5 min, washed twice, and resuspended in HBSS with 0.2% rat serum albumin.

Image analysis

To calculate average neutrophil velocity, all neutrophils entering the straight channels were tracked manually for the first 10 min, using ImageJ open-source software (U.S. National Institutes of Health, Bethesda, MD, USA). To evaluate neutrophil directionality, we counted the number of neutrophils that stopped at the posts and then calculated the fraction of “trapped” neutrophils relative to the total number of neutrophils that entered the channels with 3 posts. Also, to measure neutrophil directionality, we counted the neutrophils in the channels with symmetric and asymmetric bifurcations that turned back toward the main channel when they reached the second bifurcation (also defined as “lost” neutrophils) and reported this number to the total number of neutrophils entered the channels with bifurcations. To further quantify the cumulative effect of speed and directionality changes in rat neutrophils after burn injury, we counted the number of neutrophils in 5 distinct regions along the channels, at several time points, during the first 60 min after each neutrophil entered the side channels of the devices. The 5 regions were defined as 100-μm sections along the channels' length, from zone I close to the entrance to the side channels to zone V, starting at 400 μm away from the entrance.

Resolvin treatment after burn injury

Burned rats were injected daily with 25 ng/kg RvD1 or RvD2 (Cayman Chemical, Ann Arbor, MI, USA) via tail vein (usually 8–10 ng/animal). The first injection was at 2 h after burn injury and was followed by daily injections until ∼48 h before the motility assay or cecal ligation or LPS injection; that is, 8 administrations were performed over 7 d.

Normal neutrophil injection at d 9 postburn

Neutrophils from healthy rats were separated as described before, washed twice with HBSS with 0.2% rat serum albumin by repeated centrifugation, and resuspended in normal serum. All neutrophils from 1 healthy rat were injected in the circulation of 1 burned rat, with 30% TBSA, at d 9 postburn. Control rats (sham burned) also received the injection of neutrophils. After 6 h, LPS (2 mg/kg) was injected, and rats were surveyed for up to 2 d.

Cytokine measurements

Blood was drawn from the tail vein every 3 d for the first 9 d postburn. At 3 and 6 d postburn, blood was collected before RvD2 or vehicle injection. Blood samples were collected every day after cecal ligation (d 9 to 13) and at 0, 1, 3, 6, 12, and 24 h after the LPS challenge. Serum was separated by centrifugation at 1200 g for 10 min at 4°C and stored at −80°C until analyzed. Cytokines were determined using custom-designed electrochemiluminescence immunoassay plates run on a Sector Imager 2400 (Mesoscale Discovery, Gaithersburg, MD, USA) according to the manufacturer's protocols.

Statistics

The rate of trapped neutrophils and turned-back neutrophils was analyzed with the χ² test. Cytokine levels were analyzed using Student's t test. The survival rate of rats was analyzed with the log-rank test. The other data were analyzed with 1-way ANOVA. A value of P < 0.05 was regarded as significant.

RESULTS

Neutrophil speed and directionality decrease after burn injury

We measured the average speed and directionality of neutrophils isolated from whole blood in rats with 30% TBSA scald burn, at a 3 d interval, for 9 d postburn, using microfluidic devices (Fig. 1A, B). The average velocity of neutrophils moving through straight channels in response to a gradient of fMLP decreased from 21.9 ± 7.8 μm/min (average±sd; n=206 total neutrophils measured; n=3 rats) in control group to 19.3 ± 6.6 (n=197; n=3), 14.2 ± 5.2 (n=113; n=3), and 13.5 ± 5.9 (n=147; n=3) μm/min at 3, 6, and 9 d postburn, respectively (Fig. 1D). The average neutrophil velocity at 6 and 9 d postburn was significantly slower than that in the control group (P<0.05).

Figure 1.

Neutrophil speed and directionality are impaired after burn injury. A) Three-dimensional schematics of the microfluidic device. Device consists of a main channel connected to hundreds of side channels having 3- × 6-μm cross section and 1000-μm length. Three designs for the side channels were implemented: straight channels, channels with 3 posts, and bifurcated channels with symmetric or asymmetric design. B) Chemoattractant gradients are established by diffusion along the side channels between the side channels primed with chemoattractant and the main channel filled with buffer. In this image, the chemoattractant was replaced by a red dye of comparable molecular weight to visualize the formation of the gradient. C) Neutrophils introduced into the main channel follow the chemoattractant gradients, enter into the side channels, and move along the channels. To measure the spatial distribution of neutrophils migrating through the straight channels, we defined 5 parallel zones, 100 μm broad, from zone I at the entrance to the side channels, to zone V, extending >400 μm away from the entrance. D) Distribution and mean velocities of neutrophils entering into the straight side channels were calculated for the first 10 min of migration. Mean velocities at 6 and 9 d postburn were significantly slower than control (n=3). *P < 0.05. E) Spatial distribution of neutrophils for the first 1 h after each neutrophil entered into the straight channel. Asterisk indicates that a significantly lower percentage of neutrophils reached zone V at 6 and 9 d postburn compared with control.

In addition to the decrease in speed, neutrophils from burned rats also displayed defects of directionality during migration toward fMLP. Inside channels with 3 posts, the fraction of the neutrophils trapped after encountering the posts increased from 4.9% in the control group (18 of 364 neutrophils that entered the channels with posts) to 11.2% (38 of 339), 23.6% (38 of 161), and 27.7% (57 of 206) at 3, 6, and 9 d postburn, respectively. Moreover, inside the symmetric and asymmetric bifurcating channels, the fraction of lost neutrophils that turned back at the second bifurcation toward the loading channel increased from 4.8% in the control group (27 of 557 that entered the channels with bifurcations; n=3), to 5.1% (24 of 472; n=3), 8.0% (29 of 364; n=3), 8.6% (35 of 408; n=3) at 3, 6, and 9 d postburn, respectively.

To quantify the cumulative effect of speed and directionality changes in rat neutrophils after the burn injury, we defined 5 zones along the channels' length, each zone 100 μm wide, from zone I at the entrance to the side channels, to zone V at 400 μm away from the entrance (Fig. 1C). We followed each neutrophil for 30 min after entering the channels and then calculated the changes in the overall distribution of neutrophils in the 5 zones on this relative time scale. The fraction of neutrophils to reach zone V at 30 min decreased dramatically from 61.7% in the control group to 10.7, 6.2, and 5.4% at 3, 6, 9 d postburn group, respectively (Fig. 1E). This decrease was larger than expected based on the changes in speed or directionality alone, suggestive for a cumulative effect of the 2 parameters on migration in a complex environment.

Resolvins in vitro restore the motility of neutrophils from burned rats while stopping the migration of neutrophils from normal rats

To probe the effect of resolvins on neutrophils from burned animals, we incubated neutrophils with RvD1 and RvD2 for 5 min. We found that the migration of neutrophils from normal rats (controls) was completely blocked after exposure to 1 nM of higher concentrations of RvD2. Surprisingly, the motility of neutrophils from burned rats toward fMLP was restored close to normal values after preincubation with 1 nM RvD1 or RvD2 (Fig. 2A).

Figure 2.

Resolvins in vitro have distinct effects on neutrophils from rats with burn injury compared with healthy controls. A) Divergent effect of resolvin exposure on the average migration speed of neutrophils from healthy control and burn-injury rats (n=3). Migration of neutrophils from healthy rats is blocked by 1 nM RvD2, while the migration of neutrophils from rats with burn injury is enhanced by 1 nM RvD2 and RvD1. Stereochemical conformations of RvD1 (7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) and RvD2 (7S,16R,17S-trihydroxy-4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid) are included. *P < 0.05. B) Divergent dose-response effects of RvD2 on the average migration speed, trapping at post, orientation at bifurcations, and position in the channels for neutrophils from healthy and burn-injury rats. C) Montage of microscopy images of neutrophils trapped and lost at bifurcations. Scale bar = 100 μm; time interval between successive frames is 1 min.

We measured the dose response of normal neutrophils to RvD2 and found that the migration speed was progressively reduced with increasing concentration from 0.01 to 1 nM, and neutrophils were completely immotile at concentrations >1 nM (Fig. 2B). The rate of neutrophil trapping at posts was close to normal values for all concentrations < 1 nM, and neutrophil directionality at bifurcations was better after incubation with either 0.1 or 0.5 nM RvD2. The cumulative effect of RvD2 on normal neutrophils was overall inhibitory, reducing the fraction of neutrophils to reach zone V at 30 min from 61.7 to ∼20% at 0.1 and 0.5 nM concentrations.

In contrast, the migration of migration-defective neutrophils from burned rats was improved after short incubation with RvD2 at concentrations as low as 0.01 nM (Fig. 2B, C). The directionality of the moving cells was dramatically restored to values comparable to those in healthy animals, while the migration speed was only slightly improved. The fraction of burn neutrophils trapped at the posts was decreased by >3 times, and the orientation at bifurcations improved 4-fold after exposure to 1 nM RvD2. The cumulative effect of RvD2 on burn neutrophils was maximal at 1 nM concentration, when 45.7% of the neutrophils entering the side channels reached zone V in 30 min. This represents 1 order of magnitude more neutrophils reaching zone V compared with 5.4% of the neutrophils from burn animals in the absence of RvD2. Interestingly, a beneficial effect was present at concentrations as high as 100 nM, 2 orders of magnitude higher than the inhibitory concentrations for normal neutrophils.

When normal neutrophils were incubated with serum from burned rats for 3 h, their velocity decreased significantly from 21 to 13 μm/min. The penetration ratio was also decreased to 3% at 30 min, the rate of neutrophil trapping at posts was up to 16%, and the rate of lost neutrophils was up to 7%. When judged by these 4 metrics, the motility of normal neutrophils incubated with burn serum appeared to be slightly worse than that of neutrophils from burned rats at d 9. Interestingly, the incubation with RvD2 did not restore the migration speed or the directionality of burn-serum-incubated neutrophils. The average migration speed toward fMLP remained <15 μm/min for all RvD2 doses and penetration ratio to zone V remained <10%. Neutrophil migration toward fMLP was completely blocked at 10 nM RvD2, a dose ≥1 order of magnitude higher than that required for correcting the neutrophils from burned rats (Supplemental Fig. S1). Overall, the migration of burn-serum-incubated neutrophils was closer to that of neutrophils from burned animals, while the effect of RvD2 on migrating burn-serum-incubated neutrophils was closer to that of neutrophils from healthy donors than burn neutrophils.

RvD2 but not RvD1 restores neutrophil motility phenotype in vivo

To test whether the defects in neutrophil motility after burn injury can also be reversed in vivo, we administered RvD1 and RvD2 to burned rats. Resolvins were administered intravenously, 25 ng/kg daily, starting at 2 h after the injury at d 0 and the last dose was administered at d 7 (Fig. 3A). The neutrophil motility assay was performed ∼48 h after the end of the resolvin treatment at d 9 postburn. The average velocity of neutrophils from RvD2-treated animals toward fMLP was 20.9 ± 7.6 μm/min (n=173 total neutrophils measured; n=3 blood samples), comparable to the velocity of neutrophils from the control rats (21.9±7.8 μm/min; n=203; n=3). The directionality of neutrophils after RvD2 treatment was also improved, measured by a decrease in the numbers of trapped neutrophils at the posts (8.5%, 22 of 258 after burn and RvD2 treatment vs. 4.9% control) and a decrease in the numbers of lost neutrophils (4.9%). These directionality values were comparable to those of neutrophils from the rats in the control group (Fig. 3C, D, and Supplemental Movie S1). The cumulative measure of neutrophil motility phenotype at d 9 postburn also improved significantly after RvD2 treatment for 7 d. The percentage of neutrophils reaching zone V was 27.8% after 30 min (Fig. 3B). Shorter regimens of RvD2, of 3 administrations during first 2 d at the same dosage of 25 ng/kg daily, failed to restore neutrophil motility phenotype, and velocity at d 9 was comparable to that of neutrophils from untreated rats (15.3±5.4 μm/min; n=152; n=3). RvD1 treatment for 7 d had only partial effect on restoring velocity at d 9 postburn (13.0±5.1 μm/min: n=162, n=3) and had a small effect on directionality (Fig. 3B). After RvD1 treatment, 15.5% of neutrophils in channels with posts were trapped, and 8.6% of neutrophils were lost at the second bifurcation. The percentage of neutrophils that reached zone V after 30 min was 3.7%, comparable to untreated rats at d 9 postburn. These results also indicate that the measured effects are the result of these two resolvins, which were different in the two treatments, rather than their formulation or administration, which was the same for both treatments.

Figure 3.

Neutrophil motility is restored after RvD2 treatment for 7 d. A) In burned rats, neutrophil motility was measured at 0, 3, 6, and 9 d. In rats treated with RvD1 and RvD2, neutrophil motility was measured at d 9. RvD1 and RvD2 were administered first at 2 h after burn injury (d 0) and continued daily until d 7 postburn. B) RvD2 but not RvD1 restored the neutrophil average velocity at d 9 postburn to values close to control (n=3). A larger fraction of neutrophils was able to reach zone V in rats that received RvD2 compared with RvD1. C) Fraction of neutrophils trapped in the channels with posts increases progressively after the burn injury. Trapping at the first, second, and third post is presented as different shades of gray. The cumulative fraction of trapped neutrophils at all 3 posts decreased significantly after RvD2 treatment. *P < 0.05 vs. control group. D) Proportion of neutrophils turning back at the second bifurcation, away from the chemoattractant, also increased with time after burn. Fraction of turned-back neutrophils was restored to values comparable to controls in rats that received RvD2 treatment but not in RvD1-treated rats. *P < 0.05 vs. control group.

Survival improves significantly after second septic insult in RvD2-treated rats

To further probe the implications of functional neutrophils after RvD2 pretreatment, we examined the survival rate of rats after a second insult, either LPS injection or cecal ligation. In control experiments, all immunocompetent, sham-burned, control rats survived after the LPS injection (n=5). All sham-sepsis rats, which underwent 30% TBSA burn and vehicle injection at d 9 postburn, also survived for 1 wk after the vehicle injection. In the absence of treatment, all burned rats died in the first day after LPS injection (n=5; P<0.01; Fig. 4A). However, when burned rats that previously received RvD2 pretreatment (d 0 to 7) were exposed to similar second insults (d 9), the rate of survival increased dramatically. All rats (n=5) that received the RvD2 pretreatment survived the LPS challenge. Injecting normal neutrophils into burned rats at d 9 postburn did not improve survival after LPS injection. All rats receiving normal neutrophils (n=3) died within 12 h after LPS injection, whereas all sham-burn rats survived.

Figure 4.

Survival of burned rats after second septic insult. A, B) Rats treated with RvD2 after burn injury survived in larger numbers (burn+sepsis+RvD2; red line) after the second insult, either LPS (A) or cecal ligation (B), compared with untreated animals (burn+sepsis; blue line), as well as RvD1-pretreated animals (green line). No rats died within observation period in the sham-burned group (black dashed line) and sham-sepsis group (black solid line). *P < 0.05 for burn + sepsis vs. burn + sepsis + RvD2 groups (n=5). C) Survival rate after septic insult [either LPS (open squares) or cecal ligation (solid circles)] increased with the increasing the rate of penetration to zone V at 30 min for the different options of resolvin treatment. D) Increase in survival for burned rats after LPS insult was proportional to the dose of RvD2 (open triangles), with EC₅₀ between 0.25 and 2.5 ng/kg. Only minor increase in survival was noted for RvD1 (solid squares) for doses up to 750 ng/kg (n=4).

We also examined the survival of rats with burn injuries after cecal ligation at d 9 postburn. In the absence of treatment, all rats (n=5) died between d 11 and 15 postburn (Fig. 4B). With RvD2 treatment, the rats lived on average 1 wk longer than the untreated ones and died between d 10 and 22 postburn. In control experiments, all sham-burned rats with cecal ligation (n=5) were alive at d 23 postsham burn.

Only 1 of 5 burned rats pretreated with the same amount of RvD1 survived after the second insult (LPS; Fig. 4A). Shorter regimens of resolvins, 3 d at 25 ng/kg dose (d 4–7 postburn), also did not offer protection after the second insult. Overall, the survival after burn injury and resolvin-treated rats appears to be proportional to the cumulative index of penetration to zone V at 30 min, suggestive for a role for neutrophil motility in protection from sepsis after burn injuries (Fig. 4C). Lower daily doses of RvD2 at 0.25 and 2.5 ng/kg for 7 d were less effective in protecting the burned rats after LPS injection (2 of 4 rats died in both cases). Furthermore, higher daily doses of RvD1 at 750 ng/kg for 7 d had increased survival only up to 50% (2 of 4 rats; Fig. 4D).

Changes in cytokines levels after burn and second septic insult in untreated rats

To further understand the effects of resolvins on the innate immune system after burn injuries, we measured the levels of IL-1β, IL-6, TNF-α, IL-10, CINC-1, and CINC-3 cytokines level after burn and following septic insult, with and without RvD2 pretreatment (Fig. 5 and Supplemental Table S1). The levels of IL-1β, IL-6, and CINC-3 increased by 1 order of magnitude at d 9 compared with before the burn injury. The levels of TNF-α, IL-10, and CINC-1 increased slightly and stayed at the same order of magnitude. At d 4 after the cecal ligation (d 13 postburn), the levels of all cytokines increased compared with before ligation (d 9 postburn), 3-fold for IL-10, 6-fold for CINC-1, 30-fold for IL-1β, 60-fold for CINC-3, and >100-fold for IL-6 and TNF-α. Following the LPS insult, the levels for all 6 cytokines increased quickly, between 1 and 3 orders of magnitude at 3 h, after which they returned progressively down to almost preinjury levels at 24 h.

Figure 5.

Cytokine levels in burn-injured rats with and without RvD2 pretreatment. Levels of IL-1β, IL-6, TNF-α, IL-10, CINC-1, and CINC-3 cytokines were measured during the first 9 d postburn, with and without RvD2 pretreatment (green dashed lines and green solid lines, respectively). Cytokine levels after cecal ligation were recorded for 4 d after the second insult with and without RvD2 pretreatment (red dashed lines and red solid lines, respectively; n=10). Cytokine levels after LPS were recorded for 24 h after the second insult with and without RvD2 pretreatment (blue dashed lines and blue solid lines, respectively). With the exception of IL-10, the levels of all other cytokines before the second insult were lower in RvD2-treated rats compared with untreated ones. One order of magnitude lower cytokine levels of IL-1β, IL-6, TNF-α, and CINC-3 were recorded after cecal ligation in RvD2-pretreated rats. However, ≥2 order of magnitude surges in all cytokines were recorded within hours after the LPS challenge. The surges in IL-6 and CINC-3 were significantly smaller in rats that pretreated with RvD2 before the second insult. Results are presented as means ± se (n≥3). Note the logarithmic scale of cytokine levels. *P < 0.05.

Changes in cytokine levels after burn and second septic insult in RvD2-pretreated rats

At d 6 and 9 postburn in burned rats that received RvD2 pretreatment, the levels for IL-1β, and IL-6 were lower by >50% compared with untreated rats (Fig. 5). The levels of CINC-1, TNF-α, and CINC-3 were lower by ∼30%, and the level of IL-10 was 30% higher in treated vs. untreated rats. At 4 d after the cecal ligation (d 13 postburn), the levels for TNF-α and CINC-3 cytokines were 1 order of magnitude lower in rats that previously received RvD2 pretreatment compared with the untreated rats. The levels of IL-1β and IL-6 in RvD2-pretreated rats were half the values in untreated ones, while the levels of IL-10 and CINC-1 were comparable. However, it is important to note that despite RvD2 pretreatment, the levels of TNF-α, IL-1β, IL-6, and CINC-1 were all significantly higher (between 10- and 30-fold) at d 13 than they were at d 9 before the cecal ligation. The fold changes of all cytokines during the 4 d after cecal ligation relative to preinsult levels (d 9) were smaller in RvD2 pretreated vs. untreated rats, with the exception of IL-10 and CINC-1, which were comparable.

In burned rats with LPS challenge (2 mg/kg), cytokine levels followed the same dynamics of fast increase followed by slower decrease in both RvD2-pretreated and untreated rats. The absolute cytokine levels were roughly the same (50 to 150%) for treated and untreated animals, with the exception of IL-10 and CINC-3, which were lower by more than half in treated vs. untreated. Surprisingly, when comparing the fold changes post- and pre-LPS values (d 9 postburn), IL-1β, IL-6, and CINC-1 increased up to 3 times more in relative terms in RvD2-pretreated compared with untreated animals. Fold changes of TNF-α were only 10–20% higher in treated than untreated animals for the first 6 h after LPS, while the fold changes of IL-10 were all less than half the values in pretreated compared with untreated rats.

DISCUSSION

Here we report that the RvD2 treatment for 7 d after burn injury in rats restores the defective neutrophil motility and dramatically increases survival after a second septic insult. We also observed that resolvins have the previously unknown ability to correct the speed and directionality defects of neutrophils from burned rats, restoring their normal motility. These findings were enabled by advances in microfluidic assays for precise measurements of neutrophil directionality and speed during chemotaxis and the use of proresolving lipid mediators to control systemic inflammation in the context of severe burn injuries.

To measure the chemotaxis characteristics of rat neutrophils, we employed microfluidic devices with microchannels. The size of the channels was comparable to the size of the pores in connective tissues (23). Using these channels, we found that the average speed of neutrophils from healthy rats was comparable to that of neutrophils from healthy humans, migrating through channels of comparable size (13) After the burn injury, the rat neutrophil migration speed decreased continuously from 1 to 9 d postburn. This decrease had a different profile compared with that measured in human patients with burn injuries for which fast decrease followed by partial recovery at 3–5 d postburn was documented (13). This difference could be explained in part by the effects of immediate treatment of patients with burn injuries arriving to the hospital and the lack of significant therapeutic intervention in the rat burn model. For example, it has been suggested before that early burn wound excision and grafting can partially restore neutrophil function and delivery to inflamed tissues (24).

To measure the directionality of neutrophils, we incorporated posts and bifurcations in the new design of the channels. These features replicate in a controlled manner some of the obstacles that neutrophils moving through heterogeneous tissues could encounter, e.g., larger fibers, cells, and connective tissue. The design of these features is such that the neutrophils are forced to make directional decisions and are constrained to binary choices during chemotaxis. The orientation of the neutrophils from healthy rats was comparable to that previously reported for neutrophils from healthy humans. Neutrophils from healthy rats would pick the shortest route toward the chemoattractant in 95% of the encounters at bifurcations and pass by posts without changing their speed (20). In bifurcating channels, neutrophils with defects of directionality are identified when they turn back toward the starting point, against the direction of the guiding gradients, and could be counted to quantify the alterations of directionality. When confronted with posts, neutrophils with defective directionality do not maintain a stable leading edge and cannot deform in the smaller spaces. It is likely that neutrophils that turn back at bifurcations or are trapped at posts would have not been able to effectively migrate, when for example tracking microbes inside tissues. Moreover, relatively small changes of directionality could have significant effects in conditions where several decisions have to be made in a sequence. When neutrophils migrating through tissues may have to steer around multiple obstacles to reach their target, the effect of errors in directionality will accumulate and grow in exponential fashion with the increasing number of obstacles. For example, one could estimate for a trajectory with 10 obstacles that an increased the rate of directionality errors from 5 to 10% will result in a decrease of the total fraction of cells correctly navigating toward a target from 60% to ∼35% (Supplemental Fig. S2). The consequences of directionality errors in vivo are more complex. First, less neutrophils will arrive to the sites of infection in burned animals compared with normal animals. Second, a large number of cells will get lost in the perivascular space and release their enzymes at improper locations, where they could produce unnecessary damage of healthy tissues when stimulated again (14).

The precision in measuring the directionality of neutrophil migration enabled by the microfluidic devices described in this study cannot be accomplished using any of the existing cell migration assays. For example, the most widely used cell migration assay, the transwell assay (Boyden chamber), is an endpoint assay that does not allow the observations of cells during migration, and measurements of cell directionality are not possible. In other assays in which the migration of neutrophils on flat surfaces can be observed (including the traditional Dunn chamber, the micropipette experiments, and the majority of the modern microfluidic assays; ref. 25), directionality is indirectly estimated using statistical methods from the trajectories of all moving cells. In contrast with existing methods that are inherently imprecise in estimating neutrophil directionality, the current technique that forces moving cells to make binary decisions is easy to quantify and is intrinsically insensitive to noise, resulting in very high accuracy of neutrophil directionality measurements.

The precision of neutrophil motility measurements enabled us to refine the length and dose of an efficient protocol for restoring neutrophil chemotaxis ability in burned rats. Two resolvins in particular were key for such restoration, RvD1 and RvD2, endogenous lipid mediators derived from docosahexaenoic acid (DHA) and generated during the resolution phase of acute inflammation (21, 22). Previous studies found RvD1 to be very effective in stopping neutrophil migration, stimulating proresolving actions in murine air pouch and peritonitis (26, 27), and enhancing the containment of bacterial infections while lowering antibiotic requirements (28). RvD2 was found among many other actions, to increase neutrophil ability for phagocytosis and killing of E. coli, improving the survival rate during cecal ligation and puncture insult in a mouse model for sepsis (29) and to inhibit inflammatory pain (30). However, it is important to note that resolvins are not immune suppressive (28) and unlike steroidal anti-inflammatory drugs, they do not impair further immune responses.

When we quantified the action of resolvins on neutrophils in vitro, we found, surprisingly, that this action is dependent on the status of the donor animals. When acting on neutrophils from normal rats, resolvins inhibit neutrophil migration only slightly at concentrations <1 nM, and stop neutrophil migration completely at 1 nM and higher concentrations, consistent with previous data in mice and humans (31). In contrast, when acting on neutrophils from burned rats, resolvins have the ability to restore the directionality and speed of neutrophil migration toward fMLP. RvD2 at increasing concentrations from 0.01 to 1 nM can progressively restore the speed and improve directionality of neutrophils from burned rats. Even more unexpected, the motility of neutrophils from burned animals is not stopped even when exposed to 10 or 100 nM RvD2, which would have completely stopped normal neutrophils. Acute exposure of normal neutrophils to burn serum changes their dose response to RvD2, increasing the concentration at which neutrophils stop migrating toward fMLP chemoattractant. However this change appears to be incomplete, considering that neither the decrease in speed nor the defective directionality as measured after exposure to burn serum are corrected by RvD2. In agreement with the rapid loss of normal migratory phenotype by normal neutrophils in burn serum, normal neutrophils did not offer any protective benefit when injected into burn animals exposed to a second insult. It is possible that the presence of dysfunctional neutrophils in large numbers in the circulation also counters any beneficial effect from the smaller numbers of normal neutrophils injected. Emerging studies on roles of resolvin receptor-mediated effects on target cells (32) could help better understand the differences of resolvin activity on normal and defective neutrophils. For example, recently, the activity of resolvins D1 on neutrophils has been determined to be mediated by two G-protein-coupled receptors, ALX/FPR2 and GPR32, expressed on neutrophils (33, 34). The specific receptors for RvD2 remain to be described. It will also be interesting to test whether the changes in neutrophil responsiveness to RvD2 correlate with other neutrophil phenotype changes previously reported in neutrophils in the context of burn injuries (35).

We identified two important consequences of neutrophil directionality restoration by resolvins in vivo. First, RvD2 at 25 ng/kg (8–10 ng/animal) for 7 d has the ability to restore the directionality and speed of neutrophils in rats with burn injury. The restored motility of neutrophils was maintained for ≥2 d after the RvD2 treatment ended, when most of the RvD2 was likely already cleared from the circulation (36). Interestingly, although the in vitro effect of RvD1 on neutrophil speed was comparable to that of RvD2, the effect of RvD1 in vivo was much weaker. The remarkable potency of RvD2 is further demonstrated from the calculation of the effective dose to save 50% of animals (ED₅₀) to be between 0.25 and 2.5 ng/kg body mass (0.08–1 ng/animal). One possible explanation for the differences in vivo between RvD1 and RvD2 could be the different rates of metabolism and inactivation at the timescale of immediate action after each administration (27). These differences might have not been obvious in the in vitro assay due to the shorter duration of neutrophil exposure to the resolvins. Differences between RvD1 and RvD2 have been reported before. In a mouse model of colitis, it was found that RvD1 has higher potency compared with RvD2 (37), a reversed order of potency compared with our current study. In a more recent study comparing the roles of RvD1 and RvD2 during inflammation in obese mice, it was found that RvD2 is metabolically more stable in adipose tissue (33).

A second, more dramatic consequence of the neutrophil-restoring activity of resolvins in vivo was the improved survival of the burned rats in which neutrophil directional motility was restored by RvD2 treatment. Whereas none of the untreated rats with burn injury and abnormal neutrophil motility survived after the LPS injection, all burned rats treated with RvD2 survived the LPS injection. Survival after cecal ligation in RvD2-treated rats was also improved. Treated rats survived on average 7 d longer than the untreated counterparts. One could speculate that the differences in survival after LPS and cecal ligation are due to the differences in the nature of the septic insult. Whereas the LPS effects are transient, those of cecal ligation are prolonged due to the continuous presence and growth of bacteria in the ligated cecum. As the restoration of neutrophil function after RvD2 treatment washes off, the continuous presence of the untreated burn wound and significant inflammation in the cecum could result in significant release of in alterations of the neutrophil phenotype again 1 wk later. Further studies using longer regimens of RvD2 could clarify the origin of these differences.

To explain the relationship among neutrophil directionality, its restoration by resolvins, and its survival after second septic insult, we propose a hypothesis centered on the known interactions between neutrophils and monocytes. In healthy animals, when normal neutrophils follow gradients into tissues toward a site of inflammation, they end up in the same locations where monocytes will also arrive with a 6- to 24-h delay, following specific chemotactic signals. These monocytes have the ability to inhibit neutrophil activity, phagocyte apoptotic neutrophils, and terminate inflammation. However, when neutrophils do not have good directionality and scatter through tissues, they end up at random locations, away from the locations where monocytes are supposed to find and inhibit them. These unchecked neutrophils could be activated by subsequent inflammatory stimuli, damaging tissues and triggering complications that could lead to the death of untreated animals. By restoring neutrophil directionality, resolvins could effectively be bringing the neutrophils again under monocyte control. The precise details of the relationship between neutrophil directionality, its restoration by resolvins, and itssurvival after second septic insult remain to be clarified in future studies.

Other actions of resolvins, in addition to restoring neutrophil motility, could have contributed to these differences as well. One such example could be the significant changes in the level of several circulating cytokines known not only to be produced by neutrophils and monocytes but also important in modulating the activity of neutrophils and monocytes after burns as well (38). The levels of the cytokines were comparable in treated and untreated rats at 9 d postburn. However, in untreated rats, the levels of 4 of 6 cytokines after the cecal ligation or the LPS insult at 9 d after the burn injury were ∼1 order of magnitude higher than in RvD2-treated rats. The dynamics of cytokine level changes for IL-1β, IL-6, TNF-α, CINC-1, and CINC-3 were considerably different after the second insult in RvD2 pretreated vs. untreated animals. The faster dynamics of cytokine levels in animals pretreated with RvD2 may be more important for survival, including a more substantial surge of cytokines released during the early time after stimulation and a more effective suppression of the cytokine levels after the acute-phase. Further studies should investigate whether a self-sustaining stimulation loop is possible between the cytokines produced by the innate immune cells under conditions of stimulation and the subsequent response of the immune cells to these cytokine. For example, the levels of IL-10, known to be produced in significant amounts by neutrophils after burn injuries (39), decrease twice as fast in RvD2-treated rats compared with untreated rats after LPS insult, suggesting that the normalizing effect of RvD2 pretreatment is not limited to the motility of neutrophil but could include other neutrophil functions as well. The length of RvD2 treatment necessary to restore normal neutrophil motility in vivo is suggestive for complex interactions may take place between neutrophils and cytokines. In evaluating the complexity of resolvin actions, new data support a life span for neutrophils at >5 d (40) together with data that show the life span of the neutrophils increases even further in the presence of postburn inflammation (41). Other actions of resolvins will have to be taken into account as well e.g., the reported preventive action against deep dermal thrombosis (42), the modulation of neutrophil and monocyte trafficking and interactions (43), or the recently discovered inhibitory effect on inflammatory pain (30), a known contributor to complications after burn injuries (44).

In summary, our study in a rat double injury model (burn and sepsis) shows a dramatic improvement of survival after restoring neutrophil motility function by resolvin D2 treatment. Restored neutrophil motility correlated with protection, 2 d after the end of treatment, against lethal complications of septic insults. Our study also validates the measures of the neutrophil motility performed in vitro using the microfluidic devices and their relevance to the overall activity of neutrophils in vivo. While neutrophil motility is a sine qua non condition for bringing the neutrophils to their target locations (14), neutrophil directionality and speed are quantifiable prerequisites for the success of any other of their immune related functions. If similar correlations are uncovered in humans, measuring neutrophil motility could be used as a biomarker for the risk of complications after sepsis. Such correlations could eventually have important implications for the monitoring of the innate immune responses in patients with burn injuries and the prophylactic efforts for protecting patients against infections and sepsis.