Resolvin D1 enhances the resolution of lung inflammation caused by long-term Pseudomonas aeruginosa infection

Abstract

Pseudomonas aeruginosa lung infection is a main cause of disability and mortality worldwide. Acute inflammation and its timely resolution are crucial for ensuring bacterial clearance and limiting tissue damage. Here, we investigated protective actions of resolvin (Rv) D1 in lung infection induced by the RP73 clinical strain of P. aeruginosa. RvD1 significantly diminished bacterial growth and neutrophil infiltration during acute pneumonia caused by RP73. Inoculum of RP73, immobilized in agar beads, resulted in persistent lung infection up to 21 days, leading to a non resolving inflammation reminiscent of human pathology. RvD1 significantly reduced bacterial titer, leukocyte infiltration, and lung tissue damage. In murine lung macrophages sorted during P. aeruginosa chronic infection, RvD1 regulated the expression of Toll-like receptors, downstream genes, and microRNA (miR)-21 and 155, resulting in reduced inflammatory signaling. In vitro, RvD1 enhanced phagocytosis of P. aeruginosa by neutrophils and macrophages, recapitulating its in vivo actions. These results unveil protective functions and mechanisms of action of RvD1 in acute and chronic P. aeruginosa pneumonia, providing evidence for its potent pro-resolution and tissue protective properties on airway mucosal tissue during infection.

Introduction

Pseudomonas aeruginosa is a major opportunistic pathogen that can cause a wide range of nosocomial infections with fatal outcome in immunocompromised or hospitalized patients. It is also the etiologic agent of chronic airway infections in patients with cystic fibrosis, bronchiectasis, and chronic bronchitis. Antibiotics are rarely successful, due to the intrinsic and acquirable resistance mechanisms exploited by P. aeruginosa.¹ In the United States, >6,000 healthcare-associated infections due to multi-drug resistant P. aeruginosa strains are reported every year.² In Europe, up to 50% of clinical isolates in 2014 were resistant to at least one antibiotic class (including β lactams, aminoglycosides, and fluoroquinolones) and ∼14% of strains were found resistant to >3 antimicrobials.³ In addition, P. aeruginosa exploits adaptation mechanisms to escape killing by the immune system and establish chronic infections that do not resolve.^{4, 5, 6} Hence, both US and European health surveillance agencies classify P. aeruginosa as a “serious threat”, emphasizing the urgent need for new strategies to combat its infections, limit the consequent exacerbated inflammation without immune suppression, and promote resolution.^{2, 3}

Pioneer work from Serhan and colleagues has demonstrated that resolution, the ideal outcome of acute inflammation,⁷ is an active process controlled by endogenous specialized proresolving lipid mediators (SPM), i.e., Lipoxins (LX),⁸ resolvins (Rv),^{9, 10} protectins (PD)¹¹ and maresins (Mar)¹² that are locally produced from polyunsaturated fatty acids in select phases of inflammation and resolution.¹³ Established SPM bioactions encompass the ability to limit excessive leukocyte infiltration and responses to pathogens without immune-suppression, stimulate the removal of microbes and apoptotic cells via monocyte/macrophages (MΦs) phagocytosis, and protect the host from collateral tissue damage.¹⁴ Accruing evidence signifies that SPM have potent host-protective and proresolutive bioactions in experimental inflammatory diseases, including asthma,¹⁵ arthritis¹⁶ and infections^{17, 18, 19, 20, 21, 22, 23} (see ref. 24 for a recent review), unveiling new strategies for the pharmacotherapy of human pathologies in light of the safety and effectiveness demonstrated by SPM in preclinical and initial clinical studies.^{14, 25, 26}

Resolvin D1 (7S, 8R, 17S-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z –docosahexaenoic acid (DHA)) is an endogenous SPM enzymatically biosynthesized from DHA via lipoxygenases (LO).^{27, 28} It binds 2 specific GPCRs, ALX/FPR2 and GPR32/DRV1 to trigger cell-specific responses aimed at limiting excessive inflammation and stimulating return to homeostasis.²³ RvD1 stops polymorphonuclear neutrophil (PMN)-endothelial cell interactions and recruitment at sites of inflammation,²⁹ regulates specific MΦ microRNAs (miR) that control the release of inflammatory cytokines/chemokines and lipid mediators,^{30, 31} and stimulates MΦ phagocytosis,^{18, 32, 33} polarizing these cells towards a pro-resolutive phenotype.³⁴ In vivo, RvD1 promotes resolution of allergen³⁵ or LPS³⁶ -induced airway inflammation and enhances bacterial killing during acute peritonitis, skin infection, and sepsis.^{18, 19} Thus, its role in P. aeruginosa lung infections is of wide interest.

Here, we demonstrate host-directed anti-microbial, anti-inflammatory, and pro-resolutive actions of RvD1 in acute and chronic lung infection triggered by a multi-drug resistant clinical strain of P. aeruginosa.

Results

Lung inflammation associated with P. aeruginosa infection is markedly reduced by RvD1

To determine RvD1 actions on inflammation and bacterial clearance in preclinical models of P. aeruginosa lung infection reminiscent of human pathology, it was important to establish the temporal evolution of self-limited versus long-term lung infection with the clinical strain of P. aeruginosa RP73. Following intratracheal (i.t.) instillation of a planktonic suspension of RP73 cells (∼1 × 10⁶ CFU), the bacterial titer raised up to ∼6.0 × 10⁶ CFU/lung 1 day post infection (DPI), decreased to ∼4.0 × 10⁵ CFU/lung by 3 DPI, and further dropped to <10¹ CFU/lung 7 DPI (Figure 1a) demonstrating bacterial clearance. Total leukocytes and PMN in BAL also rapidly increased 1 DP1 returning to normal by 7 days (Figures 1b and c), whereas monocytes and MΦs gradually increased and equaled PMN at 5 DPI, indicating resolution of inflammation. To better define resolution of acute P. aeruginosa pneumonia, we calculated mathematical resolution indices as defined by Bannenberg et al:¹³ (a) Ψ_max=maximum PMN number (∼3.4 × 10⁶ cells/lung); (b) Ψ₅₀=50% reduction in PMN (∼ 1.7 × 10⁶ cells/lung); (c) T_max=time when Ψ_max was reached (∼1 DPI); (d) T₅₀=time of 50% in PMN (∼ 2 DPI); (e) Resolution interval (R_i) =T₅₀−T_max, namely the time required for resolution; (f) T_PMN=Mono/MΦ=point of intersection of PMN and monocyte+MΦ numbers (Figure 1c).

Figure 1

Distinct outcomes of self-limited versus non resolving P. aeruginosa lung infection. (a) Number of living bacteria in BAL and lungs from mice infected with ∼3.5 × 10⁶ CFU/mouse of planktonic or agar-embedded RP73. Total airway bacterial load was determined as the sum of the CFU found in BAL and lung suspensions upon serial dilution, spreading onto TSA, and o.n. growth. (b, c) Total and differential leukocyte counts in BAL from infected mice. c shows mathematical resolution indices: Ψ_max, maximum PMN number; T_max, time of Ψ_max; Ψ₅₀, 50% decrease in PMN number; T₅₀, time for Ψ₅₀; R_i, resolution interval (T₅₀–T_max); T_PMN=_Mono/MΦ, time of equivalence of PMN and monocytes/MΦ. (d) Weight changes in mice receiving i.t. inoculum of RP73-laden agar beads (∼3.5 × 10⁶ CFU/mouse) and relative total leukocytes (e), PMN, and monocyte/MΦ (f) number in BAL at the indicated time point following challenge with agar-enmeshed RP73. (g) Histopathology of lung microsections during chronic RP73 infection. Photomicrographs are representative from 4–10 mice/data point. (h) Levels of LTB₄ and the biogenic precursor of RvD1 17-HDoHE in lung homogenates at early and late phase of chronic RP73 infection. Amount of each lipid mediator was determined by taking into account the total homogenate volume. Results are mean±s.e.m. from 3 independent experiments with 15 mice. The lower panel shows a representative multiple reaction monitoring MS/MS spectrum of 17-HDoHE identified by the LC-MS/MS diagnostic ions indicated in Table 2. (i) Levels of select cytokines and chemokines in lung homogenates during RP73 chronic infection. *P<0.05; **P=0.005, ***P<0.001 vs. uninfected mice. Results are mean±s.e.m. from 2 (panels A-C) or 3 (d–h) independent experiments with 4 (0, 3, 5 DPI) or 10 mice (1, 7, 14, 21 DPI)/data point. Results in panel I are mean±s.e.m. from three independent experiments with three (0 DPI) and five (5 and 21 DPI) mice/group.

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In sharp contrast, the inoculum of RP73 cells embedded in agar beads, which allows bacteria to survive in the host airways for a long time by growing in a microaerobic/anaerobic environment, as in the mucus of chronically infected patients,^{4, 37} caused a high-titer infection (up to ∼1–2 × 10⁷ CFU/lung at 5 DPI) and produced a stable colonization (∼1 × 10⁶ CFU/lung) up to 21 days (Figure 1a), in keeping with recent results.³⁸ This was accompanied by a steep decline in body weight in the first 3 DPI that was followed by a gradual recover during the late infection phase (Figure 1d). A persistent significant accumulation of total leukocyte, PMN, and monocytes/MΦs in BAL was also evident up to 21 DPI (Figures 1e and f). Histological analysis revealed massive infiltration of PMN and lymphocytes in bronchial, peribronchial, alveolar and parenchymal areas in infected mice, whereas only sparse MΦs were present in lungs of uninfected animals. Metaplasia of mucous secretory cells (MuSC) was also detected, as indicated by the presence of PAS⁺ cells in lung sections at 21 DPI, but not in uninfected controls (Figure 1g).

To characterize the soluble components of inflammation during chronic P. aeruginosa infection, lipidometabolomics and proteomics analyses were carried out. As shown (Figure 1h and Table 1), leukotriene (LT) B₄, and LTD₄ were markedly elevated at early phases (i.e. 5 DPI) of infection, while prostaglandin (PG) E₂ and other prostanoids involved in chronic inflammation were higher at 21 DPI. Of interest, several SPM (including RvD1), were below the lowest detection limit for LC-MS/MS, whereas their precursors accumulated in late phases of RP73 infection: i.e., 15-HETE for LXA₄, 17-HDoHE for RvD1 and 14-HDoHE for Mar-1. These findings indicate a timely-regulated activation of distinct biosynthetic pathways in select phases of P. aeruginosa infection and may also suggest a defect in SPM production in chronic lung infection.

Table 1 Chronic P. aeruginosa infection-triggered lipidometabolomics

Levels of the pro-inflammatory proteins interleukin-1β (IL-1β), CXCL1/KC (the mouse IL-8), and CCL5/RANTES were also significantly higher at 21 DPI compared to non-infected mice (Figure 1i), providing a further biochemical evidence for a non resolving inflammatory state associated with a long term P. aeruginosa infection.

RvD1 treatment regulates leukocyte infiltration and lowers bacteria burden during P. aeruginosa lung infection

In this setting, we tested actions of RvD1 on P. aeruginosa clearance and inflammation. In mice challenged with planktonic RP73, a single oral administration of RvD1 (100 ng/mouse) by intragastric gavage (chosen as a delivery mode given the lower invasiveness compared to intravenous or intratracheal injection) significantly reduced lung bacterial titer by ∼50% (6.03±1.9 × 10⁶ (RvD1) vs. 12.90±1.3 × 10⁶ CFU (Veh)) and BAL accumulation of total leukocytes (2.71±0.47 × 10⁶ (RvD1) vs. 4.73±0.73 × 10⁶ cells (Veh)), and PMN (1.70±0.39 × 10⁶ (RvD1) vs. 3.34±0.66 × 10⁶ PMN (Veh)) at 1 DPI (Figures 2a and b). In contrast, oral gavage of RvE1 (100 ng/mouse), which also carries potent bioactions in airway tissues,¹⁵ did not reduce infection (11.25±1.9 × 10⁶ CFU) and inflammatory cell accumulation in BAL (total leukocytes: 4.43±0.62 × 10⁶; PMN: 3.52±0.70 × 10⁶) indicating selective RvD1 bioactions against P. aeruginosa infection. When RvD1 was given to mice infected with agar-embedded RP73 starting from 3 h post bacterial challenge for 4 consecutive days (100 ng/mouse/day), it significantly reduced mortality (Figure 2c) and cachexia (Figure 2d) in the first days following infection. This was associated to a significant reduction in IL-1β and total protein levels in BAL as well as in Evan’s blue leakage in lungs (Figure 2e) in RvD1-treated mice, which suggest that early protection conferred by RvD1 can be secondary to diminishing vascular and epithelial permeability following bacterial challenge.

Figure 2

RvD1 reduces RP73 load and leukocyte infiltration in acute and chronic lung infection. (a, b) Total numbers of live bacteria and inflammatory cells in mice receiving gavage (200 μL of saline) plus vehicle (0.5%_vol/vol EtOH/mouse) or RvD1 (100 ng/mouse) 3 h post intratracheal inoculum with planktonic RP73. BAL and lungs were collected at 1 DPI. *P=0.021; **P=0.041; ***P=0.027. Results are mean±s.e.m. from 2 independent experiments with 11 (vehicle-treated group) and 12 (RvD1-treated group) mice. (c–e) Differences in survival, weight loss, and indexes of lung permeability. Mice receiving intratracheal inoculation of RP73-charged agar beads were treated with vehicle (0.05%_vol/vol EtOH/mouse, per os) or RvD1 (100 ng/mouse/day per os) via gavage 24 h following inoculum. BAL levels of IL-1β, total proteins, and Evan’s Blue amounts were determined at 5 DPI. *P=0.018; **P=0.002; ***P<0.001. Results are pooled from 2 independent experiments with 12 mice/group. V, vehicle-treated mice; R, RvD1-treated mice. (f, g) Total CFU and leukocytes in vehicle- or RvD1-treated mice infected with agar-embedded RP73 determined 5 days post agar bead inoculum. *P=0.023; ***P<0.001. Results are mean±s.e.m. from 2 experiments with 12 mice/group.

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P. aeruginosa burden (Figure 2f), total leukocytes, and PMN (Figure 2g) were also significantly lowered by RvD1 at 5 DPI, whereas monocyte/MΦ and lymphocyte numbers were unchanged, indicating enhancement of bacterial containment and counter-regulatory actions on leukocyte trafficking. Furthermore, while vehicle-treated animals were profoundly lethargic and poorly responsive to stimuli, mice receiving RvD1 showed a normal behavior with no apparent signs of pain (e.g., self-isolation, reduced motility, and tremor) (Supplementary Movie S1 online).

Thus, RvD1 limits the severity of P. aeruginosa pneumonia when administered early after infection.

RvD1 promotes the resolution of inflammation in infected airways and protects from tissues damage

We next asked whether RvD1 was also effective when given at the peak of infection. To this end, mice were infected with agar-embedded RP73 and left untreated for 5 days, when they were randomized to receive RvD1 (100 ng/mouse) or vehicle every 48 h up to 21 DPI. Treatment with RvD1 led to a significant reduction in lung RP73 titer both at 14 (1.09±0.11 × 10⁶ (RvD1) vs. 1.81±0.23 × 10⁶ CFU (Veh)) and 21 DPI (2.99±2.76 × 10⁵ CFU (RvD1) vs. 2.03±0.13 × 10⁶ CFU (Veh)) (Figure 3a). Remarkably, PMN and lymphocyte numbers in BAL decreased from 5 to 14 or 21 DPI in veh-treated mice, indicating the existence of endogenous resolution programs in response to Pseudomonas infection. RvD1 treatment further activated this program, reducing both PMN and lymphocyte numbers in BAL at 14 and 21 DPI (Figures 3b and c), giving in a marked change in resolution indices. In particular, RvD1 reduced PMN T₅₀ from ∼12 to ∼10 DPI and lymphocyte T₅₀ from ∼13 to ∼11 DPI, giving a left-shift in the R_i of ∼2 days (Table 2).

Figure 3

RvD1 enhances bacterial clearance and resolution in chronic P. aeruginosa infection synergizing with ciprofloxacin. (a-c) Bacterial load and inflammatory cell infiltrates in mice infected with RP73 agar beads (∼ 3.5 × 10⁶ CFU/mouse) that received oral gavage with vehicle or RvD1 (100 ng/mouse) every 48 h starting from day 5 post inoculum. Numbers of live RP73 CFU, PMN, and lymphocytes were determined at 14 and 21 DPI. Quantitative resolution indices for PMN and lymphocyte removal are indicated in panels B and C. Results are mean±s.e.m. from 2 separate experiments with 10 mice/group. ^#P<0.05 vs. 5 DPI; *P<0.05; **P<0.01; vs. veh-treated mice. (d) Effect of ciprofloxacin on chronic P. aeruginosa lung infection. Mice infected via i.t. inoculum of agar-embedded RP73 treated with vehicle or ciprofloxacin (200, 400 μg/day, i.p.). RP73 titer in BAL and lung homogenates was determined 5 days post infection. Results are mean±s.e.m. from 7-10 mice/group. (e, f) Effect of combined RvD1 and ciprofloxacin treatment on RP73 infection, inflammation, and bacterial clearance. Bacterial load in BAL and lung homogenates and BAL cell counts were determined 5 days following inoculum of RP73 in agar beads in mice treated daily with vehicle (0.5% EtOH) or RvD1 (100 ng, os) alone or in combination with ciprofloxacin (200 μg, i.p.). Results are mean±s.e.m. from 2 separate experiments with 10–15 mice/group. *P<0.05; **P<0.01; ***P<0.001 vs. veh-treated mice.

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Table 2 Resolution indices for chronic P. aeruginosa infection

These and previous findings showing adjunctive effects of SPM and antibiotics^{18, 21, 23} prompted us to test RvD1 in combination with ciprofloxacin and levofloxacin, leading drugs for P. aeruginosa infections. To this end, we first determined susceptibility of RP73 to both antimicrobials. As shown (Table 3) ciprofloxacin was more potent than levofloxacin in inhibiting in vitro growth of RP73, although MIC values of both antimicrobials on RP73 were higher than those required for inhibiting the PA01 reference strain, confirming that clinical isolates of P. aeruginosa are prone to acquire resistance.³⁹ Consistent with this, ciprofloxacin at 200 and 400 μg/day gave a dose-dependent reduction in RP73 titer in vivo without leading to complete eradication (Figure 3d). Remarkably, administration of RvD1 (as low as 100 ng/day) was as potent as ciprofloxacin (given at 200 μg/day daily dose) in reducing P. aeruginosa burden (Figure 3e) and inflammatory cell numbers in BAL (Figure 3f). Of interest, the combination of the two compounds further diminished bacterial load in the airways, demonstrating that RvD1 provides additional benefits to the sole antibiotic therapy (Figure 3e), while such additive effect was not evident on BAL leukocyte numbers.

Table 3 Susceptibility of P. aeruginosa strains to ciprofloxacin and levofloxacin

To determine mechanisms underlying RvD1 actions, we measured phagocytosis of P. aeruginosa in vitro and in vivo, which is a defining bioaction of SPM pivotal for microbial killing.^{7, 14} In vitro treatment of murine RAW 264.7 MΦs with RvD1 (0.01–100 nM) prior infection with fluorescent RP73 cells resulted in a dose-dependent enhancement of phagocytosis (Figure 4a). To measure in vivo phagocytosis, BAL leukocytes collected 5 days following infection with agar-embedded RP73 were permeabilized and incubated with an anti-P. aeruginosa antibody in order to detect ingested bacteria with flow cytometry. As shown, RvD1 gave a significant enhancement in phagocytosis of P. aeruginosa by BAL PMN and MΦs 5 days following infection, which was further potentiated by ciprofloxacin co-administration as indicated by (Figure 4b). Also, since Abdulnour and colleagues recently showed that 17R-RvD1 increases the antimicrobial protein lipocalin 2 during E. coli acute pneumonia,²¹ we measured this endogenous peptide in RvD1 treated mice. As shown, RvD1 significantly increased BAL levels of lipocalin 2 at 5 DPI (Figure 4c), demonstrating the ability to boost innate host defenses.

Figure 4

Resolvin D1 enhances phagocytosis of P. aeruginosa and leukocytes by MΦs. (a) Concentration-dependent increase in phagocytosis of RP73 cells induced by RvD1 in murine RAW 264.7 MΦs. FITC-labeled RP73 (1 × 10⁶, 37 °C, 90 min) was added to cells (2 × 10⁵) treated with RvD1 or vehicle (15 min, 37 °C). Phagocytosis was assessed by measuring intracellular FITC Mean Fluoresce Intensity (MFI). Results are mean±s.e.m. from three experiments with quadruplicates. **P<0.01; ***P<0.001 vs. vehicle-treated cells. (b) In vivo phagocytosis in mice infected with RP73-laden agar beads was determined with flow cytometry upon permeabilization and staining of BAL neutrophils (CD11b⁺/Ly6G⁺ leukocytes) and MΦ (CD11b⁺/F4/80⁺ leukocytes) with an anti-P. aeruginosa antibody (1 μg/5 × 10⁵ cells) followed by an Alexa 647-tagged secondary antibody (10 μg/ml). BAL were collected 5 DPI. Right panel shows a representative flow cytometry dot plots. Results are mean±s.e.m. from 8–15 mice/group. *P<0.05; **P<0.01; ***P<0.001 vs. vehicle-treated mice. (c) Lipocalin 2 levels in BAL collected 5 DPI from vehicle- or RvD1-treated mice. Results are mean±s.e.m. from 8 to 9 mice/group. *P<0.05; **P<0.01; ***P<0.001 vs. vehicle-treated mice. (d) RvD1 stimulates phagocytosis of PMN and lymphocytes by lung MΦs in vivo. Lung MΦs from infected mice treated with vehicle or RvD1 (100 ng/mouse) every 48 h from 5 DPI were collected at the indicated time points, labeled (15 min, 4 °C) with PE-anti-F4/80 (0.2 μg/5 × 10⁵ cells), permeabilized, and counter-stained (30 min, 4 °C) with BV421-anti-Ly6G (for PMN) or APC-Cy7-anti-CD3ɛ (for T lymphocytes) antibodies. Intracellular fluorescence of F4/80⁺-MΦ associated with ingested PMN or lymphocytes was determined by flow cytometry. *P<0.05; **P<0.01. Results are mean±s.e.m. from 5 to 8 mice/data point. Representative flow cytometry histograms of intracellular staining of MΦs from RvD1- (filled histograms) vs. vehicle- (open histograms) treated mice are shown on right. Gray histograms are negative controls from F4/80 and Ly6G-or CD3ɛ-stained MΦs without permeabilization.

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Since RvD1 treatment gave a significant reduction in PMN and lymphocyte accumulation in BAL of chronically infected mice, we examined if RvD1 stimulated their active removal in vivo. To this end, BAL MΦs collected 14 and 21 DPI from veh- or RvD1-treated mice were permeabilized and counterstained with anti-Ly6G and CD3ɛ antibodies to detect ingested neutrophils and lymphocytes with flow cytometry (see Supplementary Method). As shown, RvD1 significantly increased phagocytosis of PMN and lymphocytes at 14 and 21 DPI, as indicated by the enhanced Ly6G- or CD3ɛ-intracellular staining of MΦs (Figure 4d). Hence, these results highlight the existence of endogenous resolution mechanisms that are potentiated by RvD1.

To better define pro-resolution activity of RvD1, we assessed histological signs of lung inflammation and damage. Histopathological analyses revealed that RvD1 significantly decreased intraluminal granulocyte and peribronchial lymphocyte infiltrates, as well as parenchymal involvement and MuSC metaplasia (Figure 5a). Moreover, it significantly diminished bronchiolar cell hyperplasia, as indicated by a reduction in epithelial thickness (Figure 5b), an established histological marker of lung pathology and respiratory impairment.

Figure 5

Resolvin D1 is organ protective in chronic P. aeruginosa infection and enhances non phlogistic clearance of P. aeruginosa and inflammatory cells by macrophages. Histopathology and semiquantitative scores of (a) inflammation and mucous metaplasia and (b) bronchiolar epithelial hyperplasia of lung sections collected at 21 DPI from infected mice receiving vehicle or RvD1 (100 ng/mouse) every 48 h from day 5 following inoculum. Photomicrographs are representative from 5 mice/group. Semiquantitative scores are mean±s.e.m. from two experiments with five mice/group. *P<0.05; **P<0.01; ***P<0.001.

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Thus, RvD1 lowered P. aeruginosa-triggered lung inflammation, enhanced bacterial and leukocyte clearance and protected lung tissue from non resolving P.aeruginosa infection.

RvD1 regulates select genes and miRNAs in lung MΦs and carries direct actions on human phagocytes

In order to define molecular mechanisms of RvD1 bioactions, on the basis of early data showing that it regulates select genes and miRNAs in MΦs,^{30, 31, 33} we investigated RvD1-induced changes in expression levels of genes involved in innate and adaptive immune responses in lung MΦs in vivo. To this end, lung MΦs expressing the CD45⁺/CD11b⁺/CD11c⁺/F4/80⁺ phenotype (Figure 6a) were sorted from mice bearing chronic infection and treated with RvD1 or vehicle. Differential analyses carried out on a panel of 84 genes, using quantitative PCR and bioinformatic analyses, showed that RvD1 significantly downregulated several genes linked to pathogen recognition and downstream pro-inflammatory signaling pathways. In particular, RvD1 reduced transcripts of TLR2, 4 and 6, Myeloid differentiation primary response gene 88 (MyD88), and TIR Domain-Containing Adapter Protein Inducing IFN-Beta (TRIF) by >1.5 fold (Figures 6b and c). Genes activated by MyD88 and TRIF, such as Myxovirus resistance 1 (Mx1), C reactive protein (CRP), myeloperoxidase (MPO), CCR4, CCL5/RANTES, and CXCL10/IP10 (Figures 6c and 7a) were also significantly reduced by RvD1. Of note, although tumor necrosis factor-α (TNF-α), TNF receptor-associated factor 6 (TRAF6), IL-1β, and IL-17 mRNAs showed a reduction in MΦs from RvD1-treated mice, this did not attain significance (TNF-α=−1.67 fold change, P=0.057; TRAF6=−1.68, P=0.058; IL-1β=−2.49, P=0.24; IL-17=−1.28, P=0.82).

Figure 6

Select genes regulated by RvD1 in lung MΦs during chronic P. aeruginosa infection. (a) Representative FACS dot plots showing gating strategies for the isolation of CD11b⁺/CD11c⁺/F4/80⁺ lung MΦs. (b) The Volcano plot displays significant changes in gene expression determined 5 days post infection with agar-embedded RP73 in lung MΦs sorted from RvD1- or vehicle-treated mice. Real time PCR analyses were carried out from 4 mice/group using the 2^−ΔCt method with GAPDH (NM_008084), GUSB (NM_010368), actin B (NM_007393), beta-2 microglobulin (NM_009735) and HSP90AB1 (NM_008302) as housekeeping reference genes. Fold changes were calculated as ratios of 2^−ΔCt values between MΦs from RvD1-and vehicle-treated mice. The dashed line represents the P=0.05 value used as a cut-off for selecting genes significantly regulated by RvD1. (c) Relative expression of genes with a significant (P<0.05) fold change induced by RvD1. Results are mean±s.e.m. from four mice/group.

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Figure 7

RvD1-regulated molecular pathway controlling inflammation and resolution signaling in MΦs during chronic P. aeruginosa infection. (a) The IPA map displays connections between RvD1-regulated genes and miRNAs and their functional roles in the regulation of MΦ responses to pathogens. The figure shows direct (solid lines) or indirect (dashed lines) connections between genes and miRNAs that lead to activation/causation or inhibition of indicated functions. Genes found significantly regulated by RvD1 using real time PCR analyses are indicated by filled labels. Genes encoding for proteins whose levels were determined are within boxes. (b) miRNAs regulated by RvD1 in sorted lung MΦs during RP73 infection, determined using real-time PCR. Results are mean±s.e.m. from 4 mice/group. *P=0.029; ***P<0.001 vs. MΦs from vehicle-treated infected mice. (c) Proteins encoded by RvD1-regulated genes, determined in lung homogenates 21 after infection with agar-embedded RP73 in mice treated with RvD1 or vehicle starting from day 5. Results are mean±s.e.m. from 6 to 9 mice/group. *P<0.05; **P<0.01.

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Since RvD1 regulates miRNAs^{30, 31} that are pivots in dampening TLR signaling pathways in MΦs during inflammation,⁴⁰ we searched the Ingenuity Pathway Analysis (IPA) database for miR with binding sequences to the 3′ untranslated region of RvD1-downregulated genes and assessed their level in MΦs sorted from murine lungs. IPA indicated that miR-10, -146b, and -155 can decrease MyD88 expression by binding its 3′ untranslated region mRNA, whereas the TLR2 mRNA is a direct target of miR-21 and miR-21 and -155 regulate CXCL10, TNF-α, and IL-17 (Figure 7a). As shown in Figure 7b, RvD1 significantly increased miR-21 and miR-155 levels, but not miR-10 and -146b, suggesting that selected microRNAs are part of the molecular mechanisms of RvD1 actions on MΦs during chronic P. aeruginosa infection.

To further validate these results, we measured cytokines, chemokines, and growth factors involved in lung inflammation in lung fluids from RvD1- or vehicle-treated infected mice. Consistent with mRNA and miRNA analyses, RvD1 significantly reduced protein levels of CCL5, CXCL10/IP10, CXCL1/KC, IL-1β, IL-17, and vascular endothelial growth factor (VEGF), all downstream effectors of the TLR-MyD88/TRIF pathway (Figure 7c).

Together, these results provide molecular mechanisms of RvD1 action that lower inflammatory signaling in lung MΦs in response to P. aeruginosa.

For translational purposes, we assessed bioactions of RvD1 in human systems. As shown, RvD1 significantly enhanced phagocytosis of fluorescent RP73 by peripheral blood human PMN (Figure 8a) and monocyte-derived MΦs (Figure 8b), recapitulating in vivo actions. In contrast, RvD1 diminished TNF-α-induced activation of pulmonary artery endothelial cells (indicated by CD62E/E-selectin and CD54/ICAM1 surface expression) and endothelial-PMN interactions (assessed by monitoring changes in electric impedance to quantify neutrophil diapedesis across endothelial cells). In addition, RvD1 blunted the decrease in endothelia cell layer impedance induced by IL-1β, a measure of loss in barrier function and increase of vascular permeability in response to inflammatory stimuli (Supplementary Figure S1).

Figure 8

RvD1 enhances phagocytosis of P. aeruginosa by human leukocytes. Phagocytosis of FITC-labeled RP73 by human PMN in whole blood (a) or MΦs obtained from differentiated blood monocytes (b) treated (15 min, 37 °C) with the indicated concentrations of RvD1. Phagocytosis of RP73 was assessed by measuring fluorescence associated with PMN or MΦs using a flow cytometer or a plate reader, respectively. *P<0.05; vs. vehicle. Results are mean±s.e.m. from three experiments with determinations carried out as a quadruplicate. MFI, mean fluorescence intensity.

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Collectively, these results indicate that RvD1 carries multipronged actions on human cells that boost host-defensive phagocytic activity against P. aeruginosa infection while limiting leukocyte recruitment and vascular responses.

Discussion

P. aeruginosa is a global health threat since it sustains infections often difficult to eradicate, triggering non resolving inflammatory processes that lead to disability and death.¹ Therefore, there is an unmet need for new strategies to combat P. aeruginosa infections and inflammation. Here, we provide preclinical evidence that RvD1 is protective in acute and chronic lung infection caused by a clinical strain of P. aeruginosa. When given therapeutically to P. aeruginosa-infected mice, RvD1 markedly reduced bacterial load, dampened leukocyte accumulation, enhanced bacterial clearance potentiating the efficacy of ciprofloxacin, and activated removal of infiltrated leukocytes by MΦs. RvD1 protected lung tissue, reducing parenchymal inflammation, mucus metaplasia, and bronchiolar hyperplasia. Mechanistically, we demonstrated that RvD1 regulated select genes and miRNAs involved in pathogen recognition and inflammatory signaling in MΦs in vivo, blunted PMN-EC interactions, and enhanced P. aeruginosa phagocytosis by human leukocytes.

The RP73 strain of P. aeruginosa used in this study is a clinical isolate derived from the airways of a cystic fibrosis patient at a late stage of infection. It is endowed with virulence factors, phenotypic traits, and adaptation capacity that confer advantages for establishing long-term infections and resistance to antibiotics.^{5, 41} In order to create a stable colonization of mice lungs, P. aeruginosa must be immobilized in polysaccharide-rich matrices to mimic the growth in the mucus of infected patients³⁷ and reproduce histological hallmarks of chronic human infections.³⁸ Congruently, challenge with agar-embedded RP73 triggered a long lasting bacterial infection in mice and a sustained inflammatory reaction characterized by leukocyte recruitment, airway tissue damage, and enhanced release of pro-inflammatory cytokines (Figure 1). Moreover, we observed changes in the lipid mediator profile of lung tissues chronically infected with RP73. For instance, 5-LO-derived LTB₄, a primary leukocyte-derived chemoattractant, and LTD₄, which mediates vascular permeability and bronchoconstriction,⁴² as well as 5-HETE, increased rapidly after infection. This is consistent with the initial recruitment in the infected lung of PMN, which express high levels of metabolically active 5-LO. On the contrary, PGE₂, PGF_2α, 6-keto-PGF_1α (a PGI₂ further breakdown product) and thromboxane (TX) B₂ (a TXA₂ biomarker), involved in oedema formation, vascular, and regulation of endothelial-leukocyte interactions⁴² reached a maximum 21 DPI, likely as a reflection of changes in cellularity and activation of parenchymal cells. 14-HDoHE and 17-HDoHE, precursors of D-series resolvins and maresins, also increased during infection. Of note, although RvD1 has been identified in several mouse tissues^{13, 18, 27} and, more recently, in human plasma from patients with pulmonary tuberculosis,⁴³ and arthritic synovial fluids,¹⁶ it was below the LC-MS/MS detection limit in infected mice, likely reflecting the lack of proper transcellular exchanges required for RvD1 biosynthesis.²⁸ Lack of biosynthesis of SPM investigated here could also represent a distinct defect of chronic P. aeruginosa infection. Along this line, recent studies demonstrate that a secreted P. aeruginosa protein abundantly found in sputum of patients with cystic fibrosis disrupts the biosynthesis of 15-epi-LXA4.⁴⁴ Overall, our data are consistent with the switch, observed in humans⁴⁵ as well as in preclinical models,^{13, 45, 46} in the biosynthesis of AA-derived PG and LT, which are predominant in the early phase of an inflammatory response, to SPM, which accumulate at a later time.

SPM limit the severity of infection caused by several bacterial species and promote timely resolution. For instance, RvD2 reduces mortality and bacterial dissemination after cecal ligation puncture-induced sepsis,¹⁷ 17R-RvD1 stimulates resolution of E. coli acute pneumonia,²¹ and RvD1 and D5 both enhance clearance of E. coli peritonitis and S. aureus skin infection.¹⁸ In these early studies, RvD1 and 17R-RvD1 proved to potentiate in vivo efficacy of antibiotics in eradicating infections and lowering leukocyte numbers in exudates.^{18, 21} Here, effects of RvD1 plus ciprofloxacin were additive on RP73 titer, but not on BAL leukocyte and PMN counts. This may depend on the greater complexity of chronic lung infection used in this study compared to the self-resolving systems used in previous reports, or by the fact that the RP73 strain used here is a multi-drug resistant isolate evolutionarily adapted to cause persistent, non resolving lung inflammation that are resistant to therapies.

Like other SPM, does not inhibit bacterial growth per se.^{17, 18} Thus, enhancement of phagocytosis represents a main cellular mechanism by which they enhance resolution of infection. Consistent with this, RvD1 significantly lowered RP73 burden in self-limited and chronic infection and stimulated its phagocytosis by mouse and human phagocytes. In addition, RvD1 increased levels of lipocalin, an iron chelating protein involved in microbial killing that is also regulated by 17R-RvD1, indicating that SPM can boost both cell-mediated and humoral defense mechanisms. Together with the evidence that RvD1 can be detected in human tissues^{16, 43} these results suggest that this SPM can be involved in the host response to bacterial colonization and that it has a therapeutic potential in human pharmacology, specially in clinical settings in which infections caused by multiple multi-drug resistant bacteria can occur, like in patients with cystic fibrosis.

MΦ from RvD1-treated mice showed enhanced capability to phagocytose PMN and lymphocytes (Figure 4), which is in keeping with the proresolution action of RvD1, since clearance of infiltrated leukocytes by MΦs is the hallmark of the resolution phase of inflammation.⁷ Histological analysis of the lung parenchyma confirmed that RvD1 potently reduced RP73-triggered inflammation by reducing leukocyte and lymphocyte tissue infiltration, mucous secretion and bronchiolar epithelium thickness (Figure 5). These results also emphasize the concept that SPM are crucial in preventing exacerbated leukocyte infiltration following infections, stimulating their removal, and ensuring tissue protection.

In vivo and in isolated cell systems RvD1 also stopped recruitment and infiltration of PMN, which have essential roles in innate defenses and clearance of P. aeruginosa, as demonstrated by preclinical and clinical studies. In experimental lung infections, neutrophil depletion in mice results in hypersusceptibility, with P. aeruginosa spreading, uncontrolled cytokine storm, and rapid death^{47, 48} and neutropenic patients have a higher risk of mortality due to P. aeruginosa bacteremia and septic shock.⁴⁹ In addition, recent clinical trial evaluating the safety of BIIL 284, a LTB4 receptor antagonist that blocks PMN recruitment in the lung, was prematurely terminated because of the increased incidence of pulmonary exacerbations in patients treated with the investigational drug, likely as a consequence of the impairment in neutrophil-mediated bacterial killing,⁵⁰ highlighting the narrow risk:benefit ratio of anti-inflammatories in the context of bacterial infections. Hence, dual actions of RvD1 on neutrophils (i.e., stop of their further infiltration and enhancement of phagocytic activity) reported here and in previous studies are remarkable.

We also investigated molecular mechanisms underlying RvD1 actions in chronic P. aeruginosa lung infection and demonstrated that RvD1 regulates specific genes and proteins in lung MΦs involved in leukocyte chemotaxis and infiltration (e.g., CCL5, CXCL10, and CXCL1), Th1 immune response and chronicity of airway inflammation (e.g., MyD88 and IL17), and cell activation (MyD88, TRIF, IL17) (Figures 6 and 7). RvD1 also reduced VEGF and IL17, which stimulate cell proliferation, airway remodeling, and metaplasia^{51, 52} (Figures 6 and 7). These results are in agreement with our findings that RvD1 limited excessive leukocyte infiltration, lung histopathology, and hyperplasia in vivo.

Of interest, many of the genes diminished by RvD1 in MΦ in vivo are controlled by miR-155 and miR-21, which were also upregulated by RvD1 during chronic P. aeruginosa infection. miR-155 and miR-21 have important roles in limiting the extent and the duration of inflammation. Indeed, by targeting TLRs and NF-κB, they prevent excessive or persistent stimulation of MΦs that can lead to unrestrained cell activation, overshooting cytokine and ROS release, and tissue destruction, which contribute to the pathogenesis of many diseases, including chronic pneumonia and cystic fibrosis.^{40, 53, 54} RvD1 carries remarkable regulatory actions on MΦs via miRNAs and their target genes without suppressing their responsiveness to bacteria or other dangerous stimuli (e.g., microbial particles). During acute peritonitis induced by zymosan (a yeast cell wall component and a TLR2 ligand), and also in human MΦs, RvD1 regulates miR-21, miR-146b, miR-208a, and miR-219 and their target genes as an underlying mechanism to limit overzealous MΦ activation, reduce inflammation, and promote resolution.^{30, 31} For instance, the RvD1-regulated miR-146b targets IL-8, miR-208a controls IL-10, and miR-219 reduces 5-LO protein and biosynthesis LTB₄, which stimulates PMN recruitment.³⁰ In E. coli-infected MΦ, RvD1 dampens the expression of pro-inflammatory genes regulated via TLRs and NF-κB such as COX-2 together with stimulating bacterial uptake.¹⁸ Along these lines, RvD2 also reduces TLR4 expression and signaling in human monocytes via miR-146a⁵⁵ and 17R-RvD1 dampens endotoxin- and E. coli-induced activation of human macrophages,⁵⁶ indicating that regulation of MΦ responses through miRNAs and TLRs is a common mechanism of action of SPM. Therefore, current findings on regulation of TLRs are congruent with the established concept that RvD1 (like other SPM) finely tunes MΦ responses to avoid perpetuation of inflammation without blunting their host defensive properties, a concept further corroborated by the increase in P. aeruginosa clearance and phagocytosis observed in RvD1-treated mice and cells shown in the current study.

In conclusion, here we demonstrate that RvD1 is protective against persistent, non resolving inflammation caused by chronic lung infections with a clinical, multi resistant strain of P. aeruginosa. Our results unveil key cellular functions and molecular actions of RvD1 in host defense of airways mucosal tissues to pathogens, pointing to this SPM as to a prototype of new drugs to combat P. aeruginosa infections.

Methods

Bacterial growth and mice infection. For acute infection with planktonic bacteria, the clinical strain of P. aeruginosa RP73⁴¹ was grown in tryptic soy broth (TSB) to mid-log phase (OD_{600 nm}=0.45±0.05; ∼2 × 10⁸ CFU/mL). Bacteria were washed, suspended in Dulbecco’s phosphate buffered saline (DPBS) at ∼2 × 10⁷ CFU/mL, and injected i.t. into mice. For chronic infection, 16 OD (∼50 ml) of RP73 were included into 100-200 μm diameter tryptic soy agar (TSA) beads and used within 24 h (see Supplementary Materials and Methods). Male and female C57Bl6/N mice (8-12 week-old, 20-22 g) from Charles River (Calco, Italy) were husbanded in semi-barrier cages and fed Altromin (Rieper, Bolzano, Italy) chow pellet diet containing 7.5 g/kg ω-6 linoleic acid and 1.2 g/kg (as a mixture of 18:3, 18:4, 20:5, and 22:6) fatty acids. Upon anesthesia with 2,2,2,-tribromoethanol (500 mg/kg, i.p.), 30–50 μl of planktonic or agar embedded RP73 (∼3.5 × 10⁶ CFU/mouse) were injected i.t. into lungs with 22-g 0.9 × 25 mm i.v. catheters. RvD1 (100 ng/mouse) or equal amount of vehicle (0.5%_vol/vol EtOH) were administered via intragastric gavage of 0.2 ml of saline. Mice were monitored daily for clinical signs of disease and those that lost ≥ 20% body weight or showed evidence of severe clinical disease were euthanized before the termination of the experiment. In vitro minimal inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin on the RP73 and the PA01 reference strain (LMG 27638) were determined with ɛ-test strips (Liofilchem, Roseto degli Abruzzi, Italy).

BAL collection, lung homogenization, and analyses. BAL was collected after injection of 1 ml (× 3) sterile DPBS i.t. Total leukocytes were counted using Turk’s solution and stained (15 min, 4 °C) with fluorochrome-tagged antibodies (0.2 μg/5 × 10⁵ cells) (Supplementary Methods and Methods) before acquisition with a FACS Canto II flow cytometer (BD). Viable RP73 cells in BAL and aseptically dissociated lungs were determined upon serial dilutions (10⁻¹ down to 10⁻⁶), plating on TSA, and o.n. growth at 37 °C. Cytokines/chemokines/growth factors were measured with Luminex (Millipore, Vimodrone, Italy) multiplex arrays except for CXCL10, that was determined with a sandwich ELISA (Peptotech, London, UK). For LC-MS/MS-based lipidomics, lungs non subjected to BAL (which could markedly modify the lipid mediator profiles) were rapidly dissociated in ice, mixed with 2 × ice-cold methanol, and snap frozen (−80 °C) to prevent further degradation of lipid mediators (see Supplementary Methods and Methods for further details). To assess changes in vascular permeability, Evan’s blue (100 μl/mouse of a 1% solution in saline) was injected i.p. 24 h prior lung collection. Lungs were fixed with 10% neutral buffered formalin, placed in dimethylformamide (1 ml/g of tissue) and the amount of extravasated dye was quantified on a spectrophotometer using the formula: as in (ref. 57). BAL total proteins were quantified with the Bradford method.

Lung examination and histology. Lungs excised en bloc were inflated with 1 ml of DPBS to permit even organ expansion (critical for quantitative morphometry), fixed, and cut transversally to the trachea into 5, 2 mm thick, parallel slabs/lung starting from the top 2 mm of the lung to ensure uniform random sampling. Slabs were embedded and cut surface-down into 2 μm sections that were stained with H&E or PAS (BioOptica, Milan, Italy) to detect inflammatory cell infiltrates or MuSC. Semi-quantitative scores of lung pathology were assigned on a 0–3 scale, based on criteria described in Table 1, minimally modified from (ref. 57). Epithelium thickness (μm) was determined by measuring 15 random fields/lung with the QWin Leica software. Tissue blocks received a numerical code at time of embedding and slides were scored by an investigator blinded to specimen group.

Cell sorting and real time PCR analysis of lung MΦs. Cells obtained upon dissociation of lungs using the GentleMACS dissociator (Miltenyi, Calderara di Reno, Italy) were stained (15 min, 4 °C) with fluorescent anti CD45, EpCAM (CD326), CD11b, F4/80, and CD11c antibodies and sorted using a BD FACS Aria II system. Total RNA and miRNA were isolated with Norgen isolation kits following digestion (18 h, 55 °C) with SDS (∼1%) and proteinase K (∼1 mg) and quantified with a Nanodrop 2000 spectrophotometer and a Quibit 3.0 fluorimeter (Thermo Fisher Scientific, Waltham, MA). RNA was reverse transcribed using the RT² PreAMP cDNA Synthesis Kit (SA Biosciences) and loaded into a real time PCR array (SA Biosciences Cat. # PAMM-052Z). For miRNA expression analysis, 1 ng of miRNAs from sorted MΦs was converted into cDNA with the miScript II system (Qiagen, Milan, Italy) and 50 pg of cDNA were used in real time PCR reactions with miScript primers for: mmu-miR-146b-5p (Cat # MS00001645), mmu-miR-155-5p (Cat # MS00001701), mmu-miR-10b-5p (Cat # MS00032249), mmu-miR-21a-5p (Cat # MS00011487) and SNORD61 (Cat # MS00033705) used as a control of equal cDNA input. Reactions were carried out using an ABI 7900 HT thermal cycler and results were analyzed using the 2^−ΔCt method as in (refs 30, 33).

Statistics. Results are reported as arithmetic mean±s.e.m., unless otherwise indicated. Statistical analysis of in vivo and in vitro experiments was carried out using one-way ANOVA followed by Holm–Sidak or Dunn’s post hoc test, as appropriate, for the distribution of variance among groups. HPAEC experiments and real time PCR data were analyzed with paired t-test. Survival curves and weight loss grade among vehicle- or RvD1-treated infected mice were compared using LogRank Mantel-Cox and χ²–test, respectively. P-values<0.05 were considered statistically significant.

Study approval. Experimental procedures involving the use of mice were done in accordance with protocols approved by the Ethics Committee of the G. D’Annunzio University of Chieti-Pescara (Protocol #65/2013/CEISA/COM/PROG55).