Acute exposure to air pollution particulate matter aggravates experimental myocardial infarction in mice by potentiating cytokine secretion from lung macrophages

Clinical, but not experimental evidence has suggested that air pollution particulate matter (PM) aggravates myocardial infarction (MI). Here, we aimed to describe mechanisms and consequences of PM exposure in an experimental model of MI. C57BL/6J mice were challenged with a PM surrogate (Residual Oil Fly Ash, ROFA) by intranasal installation before MI was induced by permanent ligation of the left anterior descending coronary artery. Histological analysis of the myocardium 7 days after MI demonstrated an increase in infarct area and enhanced inflammatory cell recruitment in ROFA-exposed mice. Mechanistically, ROFA exposure increased the levels of the circulating pro-inflammatory cytokines TNF-α, IL-6, and MCP-1, activated myeloid and endothelial cells, and enhanced leukocyte recruitment to the peritoneal cavity and the vascular endothelium. Notably, these effects on endothelial cells and circulating leukocytes could be reversed by neutralizing anti-TNF-α treatment. We identified alveolar macrophages as the primary source of elevated cytokine production after PM exposure. Accordingly, in vivo depletion of alveolar macrophages by intranasal clodronate attenuated inflammation and cell recruitment to infarcted tissue of ROFA-exposed mice. Taken together, our data demonstrate that exposure to environmental PM induces the release of inflammatory cytokines from alveolar macrophages which directly worsens the course of MI in mice. These findings uncover a novel link between air pollution PM exposure and inflammatory pathways, highlighting the importance of environmental factors in cardiovascular disease. Electronic supplementary material The online version of this article (doi:10.1007/s00395-016-0562-5) contains supplementary material, which is available to authorized users.

Freiburg, Germany, and every procedure was carried out in accordance with institutional guidelines.
Murine model of myocardial infarction (MI). MI was induced to C57BL/6J mice by permanent ligation of the left anterior descending coronary artery (LAD) as described earlier [3]. Mice were anesthetized with isoflurane (2%/2 litters O 2 ), immobilized on a heating pad, intubated, and mechanically ventilated with a rodent respirator. Left thoracotomy was performed in the fourth intercostal space after shaving the chest wall. The left ventricle was visualized and the LAD was ligated with monofilament 8-0 suture (Ethicon, Somerville, NJ, US). Pallor of the perfusion bed ensured myocardial ischemia. The chest and skin were closed with a 7-0 nylon suture (Ethicon) after removal of air from the thorax via a pleural catheter. Mice were exposed to sterile saline solution or ROFA particles on a daily basis, starting one day before the surgery and up to one week. Electrocardiography (ECG). Electrodes were implanted subcutaneously in a Lead II configuration (right arm, left leg, and right leg) in mice under isoflurane anesthesia. Data were acquired after the ROFA exposure on days 0, 1, 3, and 7 after LAD ligation in a 701 3 EGM module (Harvard Apparatus, March-Hugstetten, Germany) equipped with a PowerLab 8/35 data acquisition system (ADInstruments, Oxford, United Kingdom). ECG data was analyzed with LabChart Pro (ADInstruments).
Mice were kept on a heating pad to maintain body temperature and intravital microscopy was performed as previously described [11]. A loop of ileum was exteriorized by a longitudinal abdominal skin incision and subsequent dissection of facial and peritoneal layers. A small mesenteric vein with a diameter of approximately 100 µm was chosen for analysis. Videos were taken with an intravital microscope (AxioScope Vario, Carl Zeiss, Germany) fitted with a saline immersion objective (WPlan-APOCHROMAT 20x/1,0DIC IR, Carl Zeiss, Germany) and a high sensitivity camera system (AxioCam MRm, Carl Zeiss, Germany) for 30 seconds.
Rolling leukocyte flux was defined as the number of leukocytes moving at a lower velocity than erythrocytes. Rolling velocity was calculated from the average time needed for one single leukocyte to roll over the endothelium for a distance of 100 µm. Adherent leukocytes were defined as cells that remained stationary for at least 30 seconds. Measurements were performed 3 hours after the acute ROFA exposure. As positive control, non-exposed mice received an i.p. injection of 200 ng murine TNF-α (R&D Systems, Minneapolis, MN, US) 4 hours prior to imaging. Rolling flux, adhering leukocytes, and rolling velocity were quantified by blinded investigators.

Primary mouse endothelial cells (mEC)
. mEC were isolated from non-exposed C57BL/6J mice as previously described [11]. Briefly, animals were euthanized with CO 2 and lungs, heart, brain, spleen, and liver were harvested in a sterile environment. Tissue was minced were then separated and washed using a magnetic particle concentrator (Dynal Biotech), and seeded into gelatin-coated T25 plates. After reaching confluence, a second magnetic sorting was performed with an anti-mouse CD102/ICAM-2 antibody (clone 3C4, BD Biosciences). After being separated and washed, isolated endothelial cells were seeded into gelatin-coated T75 plates. Cells were grown in DMEM high glucose supplemented with 20% fetal bovine serum, 1% sodium pyruvate, 1% heparin, 1% bovine endothelial growth factor, 0.6% non-essential amino acids, and 1% penicillin/streptomycin.
After 24 hours at 37 °C, mEC culture supernatants were frozen and kept at -20 °C until were isolated by negative selection from a splenocyte cell suspension, obtained from the spleen by nicking the capsule and gently rotating two microscope slides. The obtained cell suspension was filtered through a 100 µm nylon mesh, pelleted, and used for cell isolation.
Cell purity was >98% in every leukocyte subtype preparation as assessed by flow cytometry.
In vitro cell activation assays. As indicated, isolated leukocytes were seeded into 96-weel plates at 1x10 5 cells/ml RPMI media, and incubated with plasma (1% v/v) from saline-or ROFA-exposed mice, or ROFA particles (1 μg/ml). To evaluate the contribution of plasma TNF-α, IL-6, and MCP-1 on leukocyte activation, plasma samples were pre-incubated on ice for 10 min with a blocking anti-TNF-α, anti-IL-6, or anti-MCP-1 antibody (BD Biosciences) at 10 μg/ml. After 24 hours at 37 °C, cell culture supernatants were frozen and kept at -20 °C until analyzed for cytokine levels by the CBA assay. Leukocytes were washed with 0.1% BSA/PBS, detached using Accutase (Life Technologies), and analyzed by flow cytometry.

Bronchoalveolar lavage (BAL).
Mice were euthanized with CO 2 3 hours after the ROFA exposure and a BAL was performed as previously described. Briefly, the trachea was cannulated and lungs were flushed 3 times with 1 ml 0.1% BSA/PBS each. BAL fluid total cells were counted in a hemocytometer. Samples were centrifuged at 600 g for 5 min at 4 °C and BAL fluid supernatant was stored at -20 °C until assayed for cytokine levels by the CBA assay. The obtained cell pellet was assayed for leukocyte subsets, TLR4 expression, and intracellular cytokines by flow cytometry. Alveolar macrophages were identified as CD45 + , CD11b low , Siglec-F + , CD11c + , CD64 + , F4/80 int cells (Supplemental Fig. 2).
Macrophage depletion. Alveolar macrophages were depleted from C57BL/6J mice before ROFA exposure by an i.n. instillation of 50 μl dichloromethylene bisphosphonate (clodronate) liposomes (5 mg/mL) (VU University Medical Center, Amsterdam, The Netherlands) as 8 previously described [12]. Alternatively, 200 μl clodronate liposomes were injected i.v. for systemic depletion of macrophages before ROFA exposure. PBS-loaded liposomes were used as control. Clodronate i.n. resulted in a selective depletion of alveolar macrophages by ~90% after 24 hours (Supplemental Fig. 5a) while other leukocyte populations, such as myeloid cells in blood and spleen, were not significantly affected (Supplemental Fig. 5b, c).
Alveolar macrophage depletion in ROFA-exposed mice lasts for up to 5 days after a single i.n. clodronate dose (Supplemental Fig. 6). As alveolar macrophages start to significantly repopulating the lung within days 3 and 4 after i.n. clodronate delivery, mice received i.n.
clodronate every third day in the combined model of alveolar macrophage depletion, ROFA exposure, and MI.
Statistics. Data are presented as mean ± SEM. Unpaired Student's t-test was used to analyze differences between two groups. ANOVA followed by the Student-Newman-Keuls post-hoc test was performed to evaluate differences between more than two groups.
Statistical significance was considered at p<0.05.  indicates clodronate-containing liposomes. P-values > 0.05 are not indicated.