Nonredundant protective properties of FPR2/ALX in polymicrobial murine sepsis

Abstract

Sepsis is characterized by overlapping phases of excessive inflammation temporally aligned with an immunosuppressed state, defining a complex clinical scenario that explains the lack of successful therapeutic options. Here we tested whether the formyl-peptide receptor 2/3 (Fpr2/3)--ortholog to human FPR2/ALX (receptor for lipoxin A4)--exerted regulatory and organ-protective functions in experimental sepsis. Coecal ligature and puncture was performed to obtain nonlethal polymicrobial sepsis, with animals receiving antibiotics and analgesics. Clinical symptoms, temperature, and heart function were monitored up to 24 h. Peritoneal lavage and plasma samples were analyzed for proinflammatory and proresolving markers of inflammation and organ dysfunction. Compared with wild-type mice, Fpr2/3(-/-) animals exhibited exacerbation of disease severity, including hypothermia and cardiac dysfunction. This scenario was paralleled by higher levels of cytokines [CXCL1 (CXC receptor ligand 1), CCL2 (CC receptor ligand 2), and TNFα] as quantified in cell-free biological fluids. Reduced monocyte recruitment in peritoneal lavages of Fpr2/3(-/-) animals was reflected by a higher granulocyte/monocyte ratio. Monitoring Fpr2/3(-/-) gene promoter activity with a GFP proxy marker revealed an over threefold increase in granulocyte and monocyte signals at 24 h post-coecal ligature and puncture, a response mediated by TNFα. Treatment with a receptor peptido-agonist conferred protection against myocardial dysfunction in wild-type, but not Fpr2/3(-/-), animals. Therefore, coordinated physio-pharmacological analyses indicate nonredundant modulatory functions for Fpr2/3 in experimental sepsis, opening new opportunities to manipulate the host response for therapeutic development.

Keywords: ALX; annexin peptide; cardiac dysfunction; resolution of inflammation; therapeutic innovation.

Fig. 1.

Fpr2/3 deficiency aggravates response to microbial sepsis. WT and Fpr2/3^−/− mice were subjected to CLP at time 0. (A) At 24 h post-CLP, mice were scored for the presence or absence of six different macroscopic signs of sepsis (SI Methods). A clinical score >3 is considered as severe sepsis. Data are from 11 mice; *P < 0.05 (Fisher’s exact test). (B) Temporal changes in rectal temperature post-CLP in WT and Fpr2/3^−/− animals. (C) Scattergrams illustrating neutrophil (identified as Ly6G+F4/80–) and monocyte–macrophage (identified as Ly6G–F4/80+) positive events in peritoneal lavages from WT and Fpr2/3^−/− mice at 24 h post-CLP. (D and E) Cumulative data for peritoneal Ly6G+ and F4/80+ cells. (Inset) Ratios of neutrophils/monocytes. Data are mean ± SEM of six mice per genotype. ***P < 0.001 versus respective WT value (two-way ANOVA, post hoc Tukey test).

Fig. 2.

Inadequate bacterial clearance in the absence of Fpr2/3. WT and Fpr2/3^−/− mice were subjected to CLP at time 0. (A) Representative flow cytometry scattergrams illustrating bacteria (SYTO BC bacteria dye) positive events in E. coli suspension (Left) as well as 24 h post-CLP peritoneal exudates from WT and Fpr2/3^−/− mice. The density of bacteria in the experimental samples was determined from the ratio of bacterial to microsphere signals. (B) Bacteria levels in peritoneal lavages from WT and Fpr2/3^−/− mice. Data are mean ± SEM of six mice. ***P < 0.001 versus correspondent WT value (two-way ANOVA, post hoc Tukey test). (C and D) In vitro bacteria phagocytosis by zymosan-elicited neutrophils following incubation with pHrodo Red E. coli BioParticles for 90 min at 37 °C. (C) Bacteria phagocytosis represented as percentage of positive cells. (D) Cell-associated fluorescence measured as median fluorescence intensity (MFI) units. Data are mean ± SEM of six mice. *P < 0.05 versus correspondent WT value (Student t test). (E and F) Effect of AnxA1 (10 nM; 4 h at 37 °C) on neutrophil phagocytosis of pHrodo Red E. coli BioParticles and cellular MFI intensity. Data are mean ± SEM of 3–4 mice per group. *P < 0.05 versus vehicle; ⁺P < 0.05 versus correspondent WT value (two-way ANOVA, post hoc Tukey test). (G) Zymosan-induced peritoneal cells were incubated with opsonized E. coli for 2 h and bactericidal activity determined using a gentamicin survival assay. Data are mean ± SEM of three mice. *P < 0.05 versus correspondent WT value (Student t test).

Fig. 3.

Modulation of Fpr2/3 gene expression in immune cells during polymicrobial sepsis. (A) Fpr2/3^−/− mice, bearing an in-frame GFP reporter construct, were subjected to CLP at time 0 and exudates collected at the 24 h time point. (Left) Representative histograms showing GFP fluorescence as quantified by flow cytometry in monocytes (Mo) and neutrophils (PMN) from sham (blue) or CLP (red) exudates. (Right) Cumulative data showing increment over sham in cell-associated GFP. Mean ± SEM, six mice. (B) Biogel-elicited macrophages from Fpr2/3^−/− mice were incubated with the indicated cytokines (all at 50 ng/mL) for 24 h at 37 °C before assessment of GFP fluorescence by flow cytometry. Data are mean ± SEM of 3–4 distinct cell preparations. ***P < 0.001 versus control (one-way ANOVA, post hoc Dunnet test).

Fig. 4.

Cardiac dysfunction following polymicrobial sepsis is exacerbated by Fpr2/3 deficiency. WT and Fpr2/3^−/− mice were subjected to CLP at time 0 before analysis of cardiac function at the 24 h time point by echocardiography. (A) Representative M-mode echocardiograms 24 h post-CLP or sham from WT or Fpr2/3^−/− mice. (B) Cumulative data for percentage ejection fraction measured as percent of the total amount of blood ejected in the left ventricle with each heartbeat. (C) Cumulative data for fractional shortening measured as percent of left ventricle internal diameters between the diastolic and systolic phases. (D) Cumulative data for fractional area of change measured as percent change in left ventricular cross-sectional area between diastole and systole. (E) Myocardial MPO activity 24 h post-CLP or sham samples from WT or Fpr2/3^−/− mice. (F) Plasma troponin I levels 24 h post-CLP or sham samples from WT or Fpr2/3^−/− mice. (B–F) Mean ± SEM of six mice. ***P < 0.001 versus corresponding sham value; ⁺⁺⁺P < 0.001 between genotypes (two-way ANOVA, post hoc Tukey test). (G) WT myocardial Fpr1 and Fpr2 mRNA quantification by real-time PCR. Data are expressed as fold CLP increase over sham (mean ± SEM of six mice).

Fig. 5.

Fpr2/3 agonism modulates organ injury in polymicrobial sepsis. WT and Fpr2/3^−/− mice were subjected to CLP at time 0 and treated with peptide CR-Ac_2–50 1 h and 9 h postsurgery (90 µg/kg i.p.), or with vehicle (100 µL i.p.), before being sacrificed at 24 h post-CLP. (A–C) Assessment of myocardial dysfunction by echocardiography; mean ± SEM of six mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 versus correspondent vehicle value; ⁺⁺⁺P < 0.001 between genotypes (two-way ANOVA, post hoc Tukey test).

Fig. 6.

Loss of host control on polymicrobial sepsis in the absence of Fpr2/3. The control exerted by endogenous engagement of FPR2/ALX (mimicked here by its orthologs Fpr2/3) is dual: regulation for an optimal local reaction with proper dealing by host immune cells with the bacteria load, and modulation of circulating mediators and distant organ functionality (heart and kidney).