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Published in final edited form as: Prostaglandins Other Lipid Mediat. 2022 Jan 8;159:106617. doi: 10.1016/j.prostaglandins.2022.106617

Resolvin D2 promotes host defense in a 2 - hit model of sepsis with secondary lung infection

JM Walker 1, PY Kadiyam Sundarasivarao 1, JM Thornton 1, K Sochacki 1, A Rodriguez 1, BW Spur 1, NK Acharya 1,2, K Yin 1,*
PMCID: PMC8920764  NIHMSID: NIHMS1771460  PMID: 35007703

Abstract

In the development of sepsis, there is early, massive inflammation which can lead to multiple organ failure. Later there is an immunosuppressed phase where the host is susceptible to secondary infections or is unable to clear existing infection. Specialized Pro-resolving Mediators (SPMs) are endogenously produced lipids which resolve infection by decreasing bacteria load and reducing systemic inflammatory response. There has been little work studying if SPMs given late, can promote host defense. We examined if an SPM, Resolvin D2 (RvD2) could promote host defense in a 2-hit mouse model of cecal ligation and puncture (CLP) sepsis and secondary Pseudomonas aeruginosa lung infection. RvD2 given 48h after mild CLP (1st hit), increased gene expression of Toll-like receptor-2 (TLR-2) and alveolar macrophage/monocyte phagocytic ability compared to CLP mice given saline vehicle. In this model, RvD2 did not affect plasma IL-6 or IL-10. These effects induced by RvD2, lowered lung bacterial load and decreased mortality after the secondary infection of Pseudomonas aeruginosa (2nd hit). Splenic T-cell numbers were also increased in RvD2 treated mice compared to saline vehicle treated animals. The results suggest that RvD2 promoted mechanisms of host defense in a 2-hit model sepsis and secondary lung infection.

Keywords: alveolar macrophage, phagocytosis, Pseudomonas aeruginosa, Toll-like receptors, cytokines

INTRODUCTION

In sepsis, the host responds to severe bacterial infections by producing an inflammatory response to clear the invading pathogen [1]. This host response is activated by binding of Pathogen Associated Molecular Patterns (PAMPs) to pathogen receptors such as Toll-like receptors (TLRs) found on immune cells [2]. The binding to these receptors initiates an upregulation of a variety of inflammatory genes which result in leukocyte migration to the site of infection and the release of large amounts of inflammatory mediators. This inflammation signaling is important for the host to clear pathogen but if excessive can cause tissue damage and organ injury. Later in sepsis this response becomes dysregulated, and the immune system is suppressed [3 - 7]. The suppression is characterized by non-functional macrophages or lymphocytes as well as lymphocyte death [3 - 7]. In this condition the host is susceptible to secondary opportunistic infections. Therefore, infection resolution is vital as continued bacterial load will cause excessive pro-inflammatory or dysregulated inflammatory responses without returning to homeostasis. At the cellular level, infection resolution involves reducing inflammatory mediator release to prevent excessive inflammation, maintaining signaling pathways and maintaining/increasing leukocyte phagocytic ability to ensure clearance of a secondary infection.

Recently, a large body of work has focused on endogenously produced lipids, the Specialized Pro-resolving Mediators (SPMs) which induce inflammation resolution but are not immunosuppressive [8 - 16]. Lipoxin A4 (LxA4) is an arachidonic acid metabolite while Resolvins (Rvs) are metabolites of the omega-3 fatty acids. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) give rise to the D and E series Rvs respectively. RvD2 has been shown to increase survival, reduce systemic inflammation and attenuate bacterial load in murine cecal ligation and puncture (CLP) sepsis [10, 11]. RvD1 enhanced host defense by increasing macrophage phagocytic ability and lowered antibiotic requirements in a model of E. coli infection [12] suggesting that RvD1 enhances host defense to clear bacteria. Similarly, RvE1 and RvD4 have been shown to augment macrophage phagocytic ability and reduce bacterial load (14, 15). LxA4 has been reported to reduce LPS-induced lung injury or bacterial load [17, 18] and directly stimulate macrophage phagocytic ability [19]. Most studies however, have only examined the actions of SPMs relatively early in the development of sepsis. There are no studies which have investigated the possible beneficial actions of an SPM given later in sepsis to reduce infection/injury from a 2nd hit. We used this model because (i) it enables us to examine the effects of RvD2 on host defense mechanisms when given late in disease development (ii) allows us to investigate tissue and cell host defense mechanisms by which RvD2 may be beneficial during primary infection and before a secondary lung bacterial infection. Therefore, using a 2-hit model allows for a clearer understanding of the in vivo mechanism by which RvD2 can promote host defense in sepsis.

The 2-hit model of CLP and pulmonary Pseudomonas aeruginosa (P. aeruginosa) inoculation has been reported as a clinically relevant sepsis model in which the host becomes susceptible to a secondary lung infection after an initial primary infectious peritonitis [20, 21]. One mechanism is believed to be loss of T-cell lymphocytes [20, 21], which then decreases host defense against a secondary infection. The secondary infection increases the likelihood of lung injury as well as multiple organ failure. As RvD2 has been reported to promote macrophage activity and augment host defense to reduce antibiotic concentrations for bacterial clearance [9, 11], it is plausible that RvD2 can promote host defense and attenuate consequences of a secondary infection.

P. aeruginosa is an opportunistic pathogen commonly found to infect immunocompromised patients such as those with cystic fibrosis [22]. In such immunocompromised patients, P. aeruginosa infections are particularly virulent as the bacteria is resistant to many antibiotics due partially to its ability to form biofilms which encapsulates the bacteria [23] and protects it from antibiotic attack and partially from development of antibiotic resistance [24]. In addition, P. aeruginosa virulence is enhanced by the release of the exotoxin, pyocyanin which has been shown to be particularly injurious in the lungs [25].

In this study we first examined the effect of RvD2, systemic inflammatory response, TLR expression in spleen and lung macrophage phagocytic ability in a less severe model of CLP induced sepsis. Next, we investigated the effects of RvD2 on lung bacteria load and survival after secondary pulmonary inoculation of P. aeruginosa (2nd hit).

METHODS

All experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Rowan University, and the study strictly complied with the U.S. National Institutes of Health guidelines on the use of experimental animals.

RvD2 synthesis

RvD2 was prepared by total organic synthesis as previously reported [26]. The purity of the compound was measured by HPLC-Mass Spectrometry and determined to be >98% purity. 1ug of RvD2 was dissolved in 500 μl sterile saline which had been bubbled with argon, on days of experiments.

Surgery

CLP was performed on male CD1 mice (11-14 weeks old) (Envigo, Somerset, NJ USA) using modified methods as previously published [20]. On the day of surgery, mice were anesthetized with 2.5 % isoflurane (+ O2). Prior to surgery, Buprenorphine SR (1 mg/kg) (ZooPharm, Laramie, WY, USA) was given s.c. A 1 cm-long midline incision was made in the abdomen to expose the cecum. The cecum was removed, and the distal third of the cecum was ligated with 4.0 surgical silk. Using a 27-gauge needle, the cecum was punctured twice, through and through. A small drop of feces was extruded through the holes to ensure patency of the punctures. The cecum was placed back into the abdomen and the musculature was closed with sutures, followed by the closure of the skin with wound clips. For sham controls, ceca were removed but not ligated, or punctured. Sterile saline (2 mL/100 g) was given s.c. immediately after surgery. Mice were monitored every 8 hours. CLP mice were anesthetized and injected with either RvD2 (100 ng/mouse) or saline vehicle via the tail vein. 24h later, mice were anesthetized with ketamine/xylazine (100/10 mg/kg, i.p.). Blood was obtained by intracardiac puncture into 1 ml syringes containing 50 mM EDTA. The blood was centrifuged at 1000 x g for 10 min. Plasma was removed and stored at −70°C until analysis. Spleens and the left lobe of the lung were flash frozen in liquid nitrogen before being stored at −70°C. In separate studies, tracheas were cannulated using 23G tubing adaptors inserted into PE50 tubing. Lungs were then lavaged with 1.4 ml PBS (GrowCells.com, Irvine, CA) pH 7.4 containing sodium citrate (0.38% final concentration). Cells obtained from bronchoalveolar lavage (BAL) were used for the macrophage phagocytosis assay.

2-hit model of CLP + Pseudomonas aeruginosa

For the 2-hit model (Figure 1), mice were anesthetized with isoflurane 72h after initial surgery and P. aeruginosa (A27853)(~5X109) or saline vehicle was inoculated intranasally. Mice continued to be monitored every 8h. At 24h post inoculation, mice were sacrificed to obtain, spleens and bronchoalveolar lavage for flow cytometric and lung bacteria load measurements respectively. In separate experiments, lung sections were taken for histology. In other studies, mice were sacrificed at 3days post P/ aeruginosa instillation for survival studies.

Figure 1.

Figure 1

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Experimental design of 2-hit model of CLP-sepsis + P. aeruginosa.

Survival studies

For survival studies, the protocol as written was employed with 2 exceptions. We did not use Buprenorphine-SR and ketamine/xylazine (100/10 mg/kg, i.p.) was used instead of isoflurane for inoculation of P. aeruginosa.

Plasma and lung cytokine concentrations

Plasma IL-6 , IL-10 and Macrophage Inflammatory Protein (MIP-2) concentrations were measured by using ELISA (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions. Frozen lung tissues were thawed at room temperature and transferred to a tube containing 2 ml of Tissue Protein Extraction Reagent (TPER) (Thermo Fisher Scientific, Waltham, MA) and Pierce Protease inhibitor cocktail tablets (1 tablet per 10 ml of TPER)(Thermo Fisher Scientific, Waltham, MA). Tissues were homogenized for 30 seconds. Homogenized samples were immediately centrifuged at 9000 X g for 10 min at 4°C. 50 μl of sample was then used in ELISA assays. Protein levels are quantified by using the Bradford assay. Tissue levels were standardized to mg total protein.

Bacterial load

1:10 serial dilutions of blood were made and aseptically spread onto tryptic soy agar plates (Sigma-Aldrich, St. Louis, MO, USA) to measure aerobic bacteria growth. The plates were then incubated overnight at 37°C. The number of colony forming units (CFU) was assessed 24h later by observers blinded to the treatment groups.

Bronchoalveolar macrophage/monocyte phagocytosis

pHrodo Red E. coli BioParticles™ conjugated for phagocytosis (Thermo Fisher Scientific, Waltham, MA, USA) were reconstituted according to manufacturer’s instructions at 20 mg/ml in a glass vial, using PBS (Molecular Biologicals International, Irvine, CA). Bioparticles were sonicated in an ultrasonic bath until particles were dispersed. Opsonizing reagent for E. coli (Thermo Fisher Scientific, Waltham, MA, USA) was dissolved in 0.5 ml water. Equal volumes of E. coli BioParticles™ and opsonizing reagent were incubated together for 1 h at 37°C on the day of the lavage. Bioparticles were then washed three times with PBS, centrifuging at 1500 x g, 15 min, 4°C between each wash. BAL cells were centrifuged at 800 x g and resuspended in RPMI 1640 with 10% Fetal Bovine Serum and L-glutamine (Corning). Cells were counted in a Countess II FL (Thermo Fisher Scientific, Waltham, MA, USA). 250,000 BAL cells were pipetted into each well of a 4 well chamber slide (Nunc Lab-Tek, Thermo Fisher Scientific, Waltham, MA, USA). Chamber slides were placed in a 5% CO2 incubator and allowed to incubate for 2 h to allow for macrophage adherence. This method is modified from previously published methods (6) in our lab where more than 95% of adherent cells were macrophages. The medium was removed and 0.5 ml of opsonized pHrodo Red E. Coli Bioparticles was added to each chamber. Slides were incubated for 45 min at 37°C with an identical control slide incubated at 0°C. The ratio of cells to particles was 20:1. After incubation with the pHrodo particles, they were removed and cold 0.5 ml RPMI 1640 was added to each slide. Images were obtained by a digital camera (AxioCam MRm) attached to a Zeiss Axiovert 40 CFL Microscope using Texas Red filter. The percentage of cells containing E. coli boparticles was calculated by an observer blinded to the treatment groups.

RNA isolation from spleen tissue

Frozen spleen samples (30 mg) were transferred into RNAlater™-ICE Frozen Tissue Transition Solution (Thermo Fisher Scientific, Waltham MA, USA) and stored overnight at 4°C to prevent mRNA degradation while thawing. Next day, spleen tissue was ground in liquid nitrogen using a mortar and pestle, then homogenized using shredder columns (Qiagen, Germantown, MD, USA). RNA was purified using RNeasy Protect kit (Qiagen, Germantown, MD, USA). Purity and concentration of RNA was analyzed in a nanophotometer (IMPLEN P330) at absorbances A260/280 (protein contamination) and A260/230 (buffer contamination) before proceeding to cDNA preparation.

Synthesis of cDNA

Approximately 2 μg of total RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham MA, USA) with RNAse inhibitor (Thermo Fisher Scientific, Waltham, MA). Concentration of cDNA was measured using a nanophotometer after cDNA synthesis.

Quantitative real-time PCR

q-PCR was performed using Taqman probes (Thermo Fisher Scientific, Waltham MA, USA). (Table 1). 10 ng of cDNA together with primers was used for the Taqman assay (Taqman Fast Advanced Mastermix; Thermo Fisher Scientific, Waltham, MA, USA). Primer sequences (Table 1) for TLRs and β-actin were purchased from Thermo Fisher (Thermo Fisher, Scientific, Waltham, MA, USA). The q-PCR reactions were carried out in an Eppendorf Realplex Mastercycler (Eppendorf, Westbury, NY, USA). Fold change was calculated using double-delta CT. Relative quantitation was performed against tissue taken from sham mice (animals which undergo laparotomy only) with β- actin as reference gene.

Table 1:

Primer sequences for TLRs and β-actin

GENE Probe Name Amplicon length
(nucleotides)
TLR2 Mm00442346_m1 69
TLR3 Mm01207404_m1 121
TLR4 Mm00445273_m1 87
TLR5 Mm07297422_m1 86
TLR7 Mm00446590_m1 125
TLR9 Mm00446193_m1 60
β-Actin Mm00607939_s1 115
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Isolation of murine splenocytes

The spleen was removed from each mouse using forceps and placed in a small petri dish containing 10 ml of PBS pH 7.4 with 0.38% sodium citrate. Spleens were mashed through a 70 μm Nylon cell strainer (VWR, Swedesboro, NJ, USA) with the blunt end of a 3 ml syringe. The cell strainer was rinsed with 2.5 ml of PBS pH 7.4 containing 0.38% sodium citrate. The mashed spleen was centrifuged at 4°C, 800 x g for 8 minutes. The supernatant was discarded and 5 ml of Gibco ACK Lysis Buffer (Thermo Fisher Scientific, Waltham, MA, USA) was added to the pellet, resuspended, and incubated for 5 min. 30 ml of cold PBS was added to the resuspended cells, mixed gently, and centrifuged at 4°C, 800 x g for 8 min. The supernatant was removed, and pellet resuspended in 2% FCS in PBS pH 7.4 and used for flow cytometric analysis.

Flow cytometry

All antibodies were purchased from BioLegend (San Diego, CA, USA) and titrated to determine appropriate concentration for labeling. Flow cytometry was performed using the Attune Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed with FlowJo v10.2-Software (FlowJo LLC, OR, USA). Unstained controls and single-color controls were utilized to establish base line gate settings for each antibody-fluorophore combination. Cells were counted using a Coulter Counter (Beckman Counter, Indianapolis, IN, USA). Dead cells were stained using Live Dead Fixable Near-IR Dead cell stain kit (Thermo Fisher Scientific, Waltham, MA, USA) and excluded from analysis. Splenic B-cells were stained with anti-mouse/human CD45R/B220, clone RA3-6B2. Splenic T-cells were stained using anti-mouse CD3 antibody, clone 17A2, anti-mouse CD4 antibody, clone GK1.5, and CD8a antibody, clone 53-6.7 before being analyzed by flow cytometry.

Lung histology

The left lobe of lungs of mice 24h after being instilled intranasally with P. aeruginosa, were taken and placed in 10% neutral buffered formalin. After processing and embedment in paraffin wax, 6 μm thick sections were cut and mounted on slides. Sections were then stained with hematoxylin and eosin. Sections were examined by operators who were blinded to the different groups.

Statistical analyses

A t-test was used for comparison of differences in cell differential and TLR expression data. Data for other measurements were analyzed using one-way analysis of variance. Differences between groups were then determined using the Bonferroni’s test. A Mann-Whitney U test was used for comparison of bacterial load. Difference in survival was analyzed by log rank test. For all analyses, P < 0.05 was taken as significant. All data are expressed as mean ± S.E.M.

RESULTS

Sham or mild CLP surgery was performed on CD1 mice as described in the Methods section. 48h after surgery, the CLP mice were given either RvD2 or vehicle saline (i.v). 24h later, blood and spleens were taken from all mice for analyses.

Plasma cytokine:

RvD2 or vehicle saline was administered 48 h after CLP surgery. Blood was collected by intracardiac puncture 24 h after RvD2 or vehicle administration. Plasma cytokine levels were measured by ELISA. Plasma pro-inflammatory IL-6 levels (Figure 2a) were significantly raised in CLP animals compared to sham controls but there was no difference between CLP and CLP + RvD2 groups. Overall, plasma IL-6 levels were relatively low at 48h after mild CLP. There was no difference in plasma IL-10 levels (anti-inflammatory cytokine) or MIP-2 (chemoattractant cytokine) between any of the groups (Figures 2b and 2c). Lung tissue cytokine levels showed no difference between any of the groups (Figures 2d, 2e and 2f).These results suggest that RvD2 did not significantly affect the overall systemic and lung tissue levels of inflammatory mediators when administered late in this sepsis model.

Figure 2.

Figure 2

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Plasma cytokine levels of sham, CLP given vehicle saline (CLP) and CLP mice given Resolvin D2 (RvD2; 100 ng/mouse, i.v.). Treatments were administered 48 h after surgery and mice sacrificed 24 h later to obtain plasma and lungs. (a) CLP surgery increased plasma IL-6 levels compared to sham controls but RvD2 did not have a significant effect. There were no significant differences in plasma levels of (b) IL-10 or (c) MIP-2 between sham, CLP or CLP + RvD2. Results are mean ± s.e.m. P < 0.05 compared to sham, n = 6 for sham, n = 7 for CLP, n = 7 for CLP + RvD2. There were no differences in lung tissue levels of (a) IL-6, (b) IL-10 or (c) MIP-2. N = 3 for sham, n = 7 for CLP and n = 7 for CLP + RvD2.

TLR signaling:

TLR signaling is an important early step to promote the innate immune response. To investigate if RvD2 altered TLR gene expression, we performed qRT-PCR on splenic cells isolated from the mice in each different group. Gene expression of TLRs 2 and 4 were increased in both groups of CLP mice compared to sham controls (fold change above 1). RvD2 increased TLR-2 expression even more compared to CLP + vehicle (Figure 3a). RvD2 did not affect splenic cell TLR-4 gene expression or expression of any the other TLRs tested (Figure 3). These results suggest that RvD2 selectively increased TLR-2 gene expression in splenocytes when given late in CLP sepsis.

Figure 3.

Figure 3

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Gene expression of Toll-like receptors (TLRs) in spleens. 48h after CLP surgery was performed, CLP mice were given either vehicle saline or RvD2 (100 ng/mouse; i.v.). 24h after treatment, mice were sacrificed and spleens removed for qPCR of TLRs. All TLR expression is fold change compared to sham control not given any treatment. RvD2 expression increased TLR-2 expression but not other TLRs. * P < 0.05, n = 9 for all groups.

Alveolar macrophage/monocyte phagocytic ability:

We reasoned that with increased TLR signaling in the spleen there could be increased macrophage activity distal to the site of infection. Therefore, we next examined the effects of late RvD2 administration (48h after surgery) on phagocytic ability in alveolar macrophages/monocytes. 24h after RvD2 or vehicle administration, BAL was performed. The phagocytosis assay was performed as detailed in Methods section. RvD2 increased alveolar macrophage/monocyte phagocytic ability (Figure 4) suggesting that RvD2 increased macrophage/monocyte phagocytic ability in an organ distal to the site of infection.

Figure 4.

Figure 4

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Alveolar macrophage/monocyte phagocytosis after CLP. At 48h after CLP, mice were given either vehicle saline or RvD2 (100 ng/mouse, i.v.). 24h after these injections, bronchoalveolar lavage was performed and alveolar macrophages/monocytes isolated. The adherent cells were opsonized and incubated with pHRodo E.coli (50:1) for 45 min before images were taken and counted by observers blinded to the different groups. RvD2 increased the phagocytic ability of alveolar macrophage/monocytes. * P< 0.05, n = 4 for all groups.

Alveolar bacteria load after 2nd hit:

Next, in separate studies, we examined if the increase in TLR signaling and alveolar macrophage/monocyte phagocytic ability translates to decreased bacteria load in the lung. To assess this, P. aeruginosa (2nd hit) was administered intranasally 24 h after RvD2 or vehicle saline administration. 24h after P. aeruginosa administration, lungs were lavaged, serial dilutions made and plated on tryptic soy agar (TSA) plates. Figure 5 shows that RvD2 administration reduced lung bacteria load. Taken together with the previous data, it is plausible that RvD2 administration given late in this CLP model was able to increase lung macrophage phagocytic ability to attenuate lung bacteria load after secondary infection.

Figure 5.

Figure 5

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Bacteria load of bronchoalveolar lavage taken from lungs of CLP mice 24h after Pseudomonas aeruginosa intranasal instillation. RvD2 treatment significantly reduced bacteria load. * P < 0.05, when compared to CLP + Pseudomonas, n = 10 for each group.

Splenic T-cells:

24 h after P. aeruginosa administration (2nd hit) spleens were obtained and splenocytes were analyzed by flow cytometry. Splenic T-cell numbers were increased in RvD2 treated mice 24h after P. aeruginosa administration, compared to vehicle treated mice (Figure 6). There was no significant change in splenic B-cell numbers. These results support the hypothesis that RvD2 promotes or preserves immune cell signaling to increase bacterial clearance after a 2nd hit.

Figure 6.

Figure 6

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Splenic cell numbers 24h after Pseudomonas inoculation. 24 after Pseudomonas inoculation spleens were harvested from CLP mice given either saline or RvD2. Spleens were stained for CD4, CD8 T-cells as well as for B-cells. RvD2 treatment increased CD4 and CD8 T-cells compared to CLP mice given vehicle saline. There was no significant difference in B-cells between groups. Data are expressed as mean ± s.e.m.* P < 0.05, n = 6 for both groups.

Lung histology:

Histological sections of lungs taken from surviving CLP mice 24h after being given P. aeruginosa showed that there was infiltration of inflammatory cells (neutrophils and macrophages) which could fill individual alveoli. Prior administration of RvD2 significantly reduced this inflammatory response (Figures 7a and 7b). At this time point there was no overt lung injury such as alveolar wall thickening or edema that was observed.

Figure 7:

Figure 7:

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(a) Representative photographs of lung sections stained with H & E, taken 24h after intranasal Pseudomonas aeruginosa inoculation. After Pseudomonas instillation there was neutrophil and monocyte/macrophage infiltration (circles) into the alveoli airspace which could be observed on occasion to block or plug the alveolar airspace. In RvD2 treated mice there was less infiltration of these inflammatory cells (circles) and significantly more clear airspaces. No overt lung injury alveolar wall thickening, edema, could be observed. All photos taken at 40X magnification. (b) Sections were scored for inflammation/injury by an observer who was blind to the treatment groups. There was significantly less inflammation in the lung sections taken from RvD2 treated mice. *P < 0.05, n = 4 for all groups.

Survival studies:

In survival studies, CLP mice were observed for 3 days after P. aeruginosa (2nd hit) inoculation. RvD2 administered 24 h before P. aeruginosa inoculation, increased survival compared to mice given vehicle saline (Figure 8).

Figure 8.

Figure 8

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Sham or CLP was performed on CD1 mice. 48h after surgery, either RvD2 (100 ng/mouse, i.v.) or saline was injected into CLP mice. Pseudomonas aeruginosa was inoculated into the mice intranasally. Mice were monitored for 3 days after inoculation. After Pseudomonas inoculation, CLP mice given RvD2 had greater survival compared to CLP mice given saline. * P < 0.05 for n = 16 for CLP controls, n = 16 for CLP = RvD2, n = 9 for sham.

DISCUSSION

Host defense is vital for clearance of bacterial infection, regulation of the innate immune system and return of the host to homeostasis. Overly active host defense can lead to chronic inflammation and tissue injury while inadequate or dysregulated host defense may lead to chronic infection or secondary infection(s). Dysregulation of host defense and increased susceptibility to secondary infections is a late hallmark of sepsis. The inability to clear a secondary infection is thought to be due to decreased immune signaling and subsequent inadequate macrophage function [3 - 7]. It is important to note that our model of mild sepsis allows us to interrogate suppressed host defense which is unable to clear a “2nd hit” to an organ distal to the primary site of infection.

Previous work by us and other groups have suggested that various SPMs can reduce inflammation without causing immunosuppression [8 - 16]. In these studies, the SPMs were administered early in animal models when there was hyperinflammation. The SPMs used were beneficial because they “resolved” the profound acute inflammatory response and decreased bacteria load. Here, we first characterized the effects of an SPM, RvD2, given a full 48h after initial insult. We measured systemic inflammatory response, TLR expression and alveolar macrophage phagocytic ability in a model of mild sepsis (1st hit). In this setting, RvD2 did not affect plasma IL-6, IL-10 or MIP-2 but increased splenic TLR-2 gene expression and alveolar macrophage phagocytic ability. We then investigated if RvD2 affected secondary P. aeruginosa lung infection (2nd hit). Our results show that RvD2 decreased P. aeruginosa lung injury and increased survival.

An early signaling mechanism for the innate immune system is the TLR system. With respect to bacterial infections, TLRs 2 and 4 are the most important signaling receptors [27 - 30]. TLR-2 ligands include LPS, lipoteichoic acid, peptidoglycans from gram negative and gram positive bacterial cell wall components. The major TLR-4 ligand is LPS, a core component of the gram negative bacterial cell wall. Stimulation of TLRs 2 and 4 leads to inflammatory cytokine production through activation of nuclear factor kappa-B (NF-κB). Our results showed that splenic cells from RvD2 treated CLP mice, had increased TLR-2 gene expression compared to vehicle treated CLP mice without significantly affecting TLR-4 expression. These results are different from an in vitro study showing that RvD2 decreased LPS induced TLR-4 expression [31]. The difference is probably due to a combination of complex cellular signaling between different cell types within the spleen and a milder sepsis model. Work by other investigators is inconsistent where increased TLR-2 expression in human monocytes correlated with increased sepsis mortality and TLR-2 ligands attenuated cardiac dysfunction in CLP-sepsis [32, 33]. On the other hand, early blockade of TLR-2 decreased mortality in models of severe CLP-sepsis [34, 35]. Taken together with previous work, our results suggest that (i) RvD2 was not immunosuppressive and (ii) increased TLR-2 expression can lead to greater bacteria clearance late in mild CLP-sepsis.

This small but significant increased expression of TLR-2 was not accompanied with any increase in circulating IL-6, IL-10 or MIP-2 levels. Similarly, lung tissue levels of these cytokines were not affected by RvD2 administration. A previous report provided evidence that 17(R)-RvD1 increased TNFα production and macrophage phagocytic ability when stimulated with live E. coli in vitro but not exogenous LPS [36]. This finding is consistent with our results using RvD2 in vivo with the exception that RvD2 did not increase inflammatory response in vivo. A possible explanation to this difference is that in vivo, macrophages are in contact with many different cell-types which could modulate their response. Our results are not consistent with previous results showing that RvD2 reduces plasma inflammatory cytokine levels [9]. This difference is probably due to the difference in the severity of the sepsis model and the timing of the administration. In our mild sepsis model, the plasma levels of IL-6 are relatively low and RvD2 administration did not further affect its levels. Furthermore, we administered the RvD2 48h after mild CLP surgery when plasma cytokine levels had already plateaued and fallen while in the studies mentioned above, RvD2 was given early (1h after surgery), well before systemic inflammatory response had plateaued.

In other published work, RvD2 reduced the level of antibiotics necessary to clear bacterial infection [11, 13] and is consistent with our studies. The mechanism postulated there was that RvD2 upregulated host defense to clear a primary pathogen. Our work has advanced this concept to show that RvD2 can increase host defense by increasing expression of splenic TLR-2 and increasing phagocytic ability of macrophage/monocytes at a site distal to the primary site of infection.

Alveolar macrophage activity is essential for bacterial clearance in the lung during infection [37 - 39]. Use of resolvins has been reported to promote pulmonary clearance of bacteria [12, 15] by increasing macrophage activity. In these reports however, the resolvins were administered to immune stable mice which did not have any prior infection or inflammatory stimuli. Our data showing that there was increased mortality when P. aeruginosa was introduced as a secondary infection compared to sham mice given P. aeruginosa, provides evidence that in these mice, pulmonary bacteria clearance was not adequate. Along these lines, our results show that giving RvD2 intravenously 48 h after CLP surgery and 24 h before P. aeruginosa infection promoted pulmonary bacterial clearance, reduced lung inflammation and increased survival. This beneficial effect may be at least partially due to increased alveolar macrophage/monocyte phagocytic activity and is consistent with other reports showing that RvD1 enhanced P. aeruginosa clearance in long-term infection and a murine cystic fibrosis model [39, 40].

There are several studies which have reported that splenic lymphocytes are reduced in the 2-hit model of CLP-sepsis and secondary pulmonary infection [20, 21]. It is postulated that the decrease in T-cell numbers plays a major part in the host’s decreased ability to clear the secondary infection. This is due primarily to T-cell signaling of macrophage activity and other T-cell signaling functions [41 - 43]. Our results showed that splenic T-cell numbers (both CD4 and CD8) were greater in mice given RvD2 than mice given vehicle 24h after secondary infection. This result suggests that RvD2 can preserve immune cell signaling during this infection and increase macrophage/monocyte function to clear infection. The mechanism for this increase in splenic T-cell number is unclear but may be a result of reduced infection setting up a feedback loop where initial increased alveolar macrophage phagocytic ability reduces infection/injury which preserves T-cell number. The mechanism and significance of this increase in splenic T-cell number is currently a subject of investigation in the lab.

Results from our study suggest that in less lethal, less inflammatory bacterial sepsis, RvD2 promotes host defense by increasing TLR-2 signaling and macrophage/monocyte phagocytic ability when given 48 h after sepsis onset. These actions occur without any effects on systemic inflammation. Acting through these mechanisms, RvD2 provides better clearance and increases survival after secondary lung infection. Taken together, these studies provide evidence that an SPM such as RvD2 is beneficial in a 2-hit rodent model where the host’s ability to clear infection is compromised.

HIGHLIGHTS.

  • The development of sepsis is often biphasic where there is an early inflammatory phase followed by a later immunosuppressed phase characterized by the host’s inability to clear a secondary infection.

  • RvD2 given late, at 48h after mild cecal ligation and puncture (CLP) sepsis, increased Toll-like Receptor 2 (TLR-2) expression in the spleen but did not affect plasma cytokine levels compared to CLP mice given saline vehicle.

  • RvD2 given at 48h after CLP increased alveolar adherent macrophage/monocyte cells’ phagocytic ability compared to mice given saline vehicle.

  • Treatment with RvD2 promoted lung clearance of Pseudomonas aeruginosa (P. aeruginosa) secondary infection in CLP mice.

  • RvD2 increased survival in the 2-hit model of CLP with P. aeruginosa secondary infection.

Acknowledgments

We thank Rachael Wilson for helping in caring for the animals during these studies. This work was supported by NIH (RO1 AI128202).

Footnotes

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