Neutrophil proteome shifts over the myocardial infarction time continuum
In response to myocardial infarction (MI), neutrophils (PMNs) are early responders that initiate the inflammatory reaction. Because macrophages and fibroblasts show polarization states after MI, we hypothesized PMNs also undergo phenotypic changes over the MI time course. The objective of the current study was to map the continuum of polarization phenotypes in cardiac neutrophils over the first week of MI. C57BL/6J male mice (3–6 months old) underwent permanent coronary artery ligation to induce MI, and PMNs were isolated from the infarct region at days 1, 3, 5, and 7 after MI. Day 0 served as a no MI negative control. Aptamer proteomics was performed on biological replicates (n = 10–12) for each time point. Day (D)1 MI neutrophils had a high degranulation profile with increased matrix metalloproteinase (MMP) activity. D3 MI neutrophil profiles showed upregulation of apoptosis and induction of extracellular matrix (ECM) organization. D5 MI neutrophils further increased their ECM reorganization profile. D7 MI neutrophils had a reparative signature that included expression of fibronectin, galectin-3, and fibrinogen to contribute to scar formation by stimulating ECM reorganization. Of note, fibronectin was a key modulator of degranulation, as it amplified MMP-9 release in the presence of an inflammatory stimulus. Our results indicate that neutrophils selectively degranulate over the MI time course, reflective of both their intrinsic protein profiles as well as the ECM environment in which they reside. MMPs, cathepsins, and ECM proteins were prominent neutrophil degranulation indicators.
KeywordsMyocardial infarction Neutrophil Proteomics Aptamer LV remodeling Cell polarization
Myocardial infarction (MI) initiates a cardiac wound-healing cascade that originates with myocyte necrosis to stimulate inflammation and leukocyte influx and culminates with scar formation. By 24 h after MI, the prominent leukocyte is the neutrophil . While excess neutrophil influx is detrimental by promoting excessive tissue breakdown to enhance dilation of the left ventricle (LV), neutrophil depletion also amplifies inflammation and LV dilation to reduce cardiac performance in rodent models and humans [17, 18, 41]. Neutrophils, therefore, are essential for the wound-healing process.
Neutrophils undergo degranulation, releasing proteases to degrade the extracellular matrix (ECM) and facilitate leukocyte infiltration [13, 42]. The Steffens laboratory reported that neutrophils also orchestrate MI healing by polarizing macrophages toward a reparative phenotype, assigning an indirect role for neutrophils in LV remodeling . Our laboratory observed that neutrophils themselves undergo polarization after MI, with > 95% of day 1 neutrophils expressing N1 markers (Ccl3, Ccl5, and Tnfα) and 20% of day 7 neutrophils expressing N2 markers (Tgfβ1, Il10, and Cd206) . N1 neutrophils activated by damage-associated molecular patterns (DAMPs) link to an early increase in MI wall thinning. Neutrophils, therefore, have roles in both promoting and turning off inflammation.
The current study extends past reports by mapping neutrophil transitions in response to MI. We analyzed proteomic changes at MI days 1, 3, 5, and 7 to reflect the early inflammatory, proliferative, and maturation phases compared to day 0 no MI neutrophils. We hypothesized that neutrophils would undergo phenotypic changes over the MI time course that range from pro-inflammatory to reparative polarization. We used an aptamer proteomics approach, because mature neutrophils are post-mitotic and have reduced transcriptional capacity compared to dividing cells [15, 34, 43]. To our knowledge, this is the first study to report in detail the full proteome changes that occur in cardiac neutrophils that mediate post-MI wound healing and remodeling.
Animal use, coronary artery ligation, echocardiography, and necropsy
Detailed methods are provided in the Supplemental Methods [5, 21, 23]. Because neutrophils exhibit time of day variation, with MI surgery performed in the evening resulting in higher rupture rates due to higher neutrophil recruitment, all surgeries were performed between 8 am and noon .
Isolation of LV infarct neutrophils
LV neutrophils were isolated from the combined infarct and border region by immunomagnetic separation (Supplemental Methods) [10, 19, 31]. The neutrophils obtained by this method are highly (> 99%) pure .
Neutrophil cell pellets were lysed in Reagent 4 (Sigma C0356; 50 μL per 1 × 106 cells) with 1× protease inhibitor cocktail (Roche), centrifuged at 14,000×g for 5 min, and the supernatant was collected. Total protein was quantified using the Bradford assay. Sample concentrations were adjusted to 200 μg/mL using phosphate-buffered saline (PBS) and 100 μL of sample was sent to SomaLogic (Boulder, CO) for aptamer proteomic evaluation.
Aptamer proteomics uses short single-stranded DNA sequences that bind to proteins. The SOMAscan® multiplex aptamer-based proteomics platform on a custom Agilent hybridization chip was used to analyze 1305 proteins (assay version 3.2) [14, 20, 30, 33, 44]. Aptamers are chemically modified unique and single-stranded DNA segments that bind to specific proteins. Data normalization and calibration were performed according to the SOMAscan® Data Standardization technical note (SSM-071) .
In situ hybridization and immunohistochemistry multiplex imaging
In situ hybridization and immunoblotting histological evaluations were performed according to the guidelines for authors and reviewers on antibody use in physiology studies (Supplemental Methods) .
Isolation and stimulation of bone marrow-derived neutrophils
Bone marrow-derived neutrophils were isolated from the tibias and femurs of control no MI mice under isoflurane anesthesia as previously described . Ly6G + neutrophils were diluted to 2 × 106 cells/mL in RPMI 1640 media with 1% antibiotic solution. Cells (1 × 106) were unstimulated or stimulated with the positive control phorbol 12-myristate 13-acetate (PMA; 20 nM) or fibronectin (100 ng/mL) for 15 min at 37 °C. The cells were centrifuged at 800×g for 10 min, and the supernatant (200 μL) was analyzed using the Proteome Profiler Mouse XL Cytokine Array (R&D Systems, ARY028).
Statistics and bioinformatic analyses of the proteomics dataset
Statistical analyses were performed according to established guidelines . All experiments were performed and analyzed in a blinded design, and data are presented as mean ± SEM unless otherwise noted. For echocardiography, comparisons were made using one-way ANOVA followed by Newman–Keuls post hoc test. A value of p < 0.05 was considered statistically significant.
Aptamer proteomics results are given as relative fluorescence units (RFUs). Prior to bioinformatics analysis, a quality control assessment was performed (Supplemental Methods) and the data log transformed for normalization. Analyses tools available in the online resource MetaboAnalyst 3.0 (http://www.metaboanalyst.ca/) and GraphPad Prism were used for statistical and bioinformatics analyses [47, 48]. One-way ANOVA with Tukey’s post hoc test was performed to determine differentially expressed proteins using a false discovery rate (FDR) adjusted p value cutoff of 0.05. For individual MI days, differential expression was characterized by a fold-change threshold of > 2.0 or < 0.5 compared to day 0 no MI values and a p value of less than 0.05 by two-tailed unpaired t test. Proteins were ranked first by p value and then by fold change.
Enrichment analysis for differentially expressed proteins was performed using Enrichr (http://amp.pharm.msm.edu/Enrichr/) gene ontology (GO) biological processes and Ingenuity Pathway Analysis (Qiagen) canonical pathways. For GO terms, the combined score (calculated from Z score and p value) was reported. Proteomic data comparisons to galectin-3 and fibronectin immunoblotting were made by one-phase association analysis to obtain goodness of fit R2 values.
Neutrophils differentially polarize from MI days 1–7
MI D1 neutrophil polarization phenotype: pro-inflammation, degranulation, and invasion
In a separate Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the top three enriched pathways in MI D1 neutrophils were complement and coagulation cascades (combined score 20.56), platelet activation (combined score 18.84), and cytokine–cytokine receptor interaction (combined score 14.04; all p < 0.001). This indicates that MI D1 neutrophils are predominantly pro-inflammatory. In acute inflammation, neutrophils and platelets are simultaneously activated to regulate the inflammatory response. Heteromers of proteins contributed from both cells form to promote monocyte recruitment , and our results indicate that this happens beginning at day 1. Neutrophils interact with platelets by both binding to form neutrophil–platelet complexes and by phagocytosing activated platelets . While we cannot totally rule out the possibility of contamination of neutrophils by platelet–neutrophil complexes in the LV infarct, platelets were excluded during the cell isolation procedure.
Increased recruitment into the infarct region was demonstrated by a peak in calgranulin B at MI D1 (Fig. 3c) [9, 38, 39]. Further evidence of degranulation was the increased release of matrix metalloproteinase (MMP)-8 and -9 into the infarct region (Fig. 3d, e). MMP-8 and MMP-9 are released into the ECM from neutrophil gelatinase granules to degrade ECM and propagate inflammatory signaling . Overall, the MI D1 neutrophils responded by turning on leukocyte recruitment and stimulating inflammatory signaling through degranulation. Neutrophils at this time reflected the well-characterized response to injury .
MI D3 neutrophil polarization phenotype: similar to D1 cell, along with increased apoptotic signaling, ECM reorganization, and cathepsin activity
Because fibronectin increased 22-fold in MI D3 neutrophils compared to D0 cardiac neutrophils, we evaluated whether the neutrophil was the source or recipient of fibronectin protein. By in situ hybridization, fibronectin-positive cells contributed 32% of the total cells within the MI D3 left ventricle infarct region (Fig. 4d). As expected, fibroblasts were positive for fibronectin . The total percentage of neutrophils was 5% of the infarct region. Within the neutrophil pool, 2% of the total infarct area (or 40% of neutrophils) was also positive for fibronectin. Overall, the MI D3 neutrophil showed increased cathepsin activity and apoptotic signaling along with the initiation of ECM production.
MI D5 neutrophil polarization phenotype: increased ECM reorganization, reduced neutrophil recruitment, and resolution of inflammation
MI D7 neutrophil polarization phenotype: ECM synthesis and organization
Fibronectin stimulates selective neutrophil degranulation
By immunoblotting examination, PMA stimulation increased the release of fibronectin by neutrophils and reduced fibronectin fragmentation to increase fibronectin availability (Fig. 7d). Thus, MMP-9 was not as effective in breaking down fibronectin when in the presence of a pro-inflammatory stimulus. By Enrichr evaluation of protein–protein interactions, the primary transcription factors induced by PMA were Gata3, Foxp3, Tbx21, and Sirt1. Fibronectin shifted the transcription factor profile to Irf4, Ikbkb, Ets2, and JunD (Fig. 7e). These results indicate that fibronectin selectively degranulates neutrophils and in the presence of a pro-inflammatory stimulus amplified the release of MMP-9 available to degrade fibronectin to its 120 kD fragment (Fig. 7f).
Neutrophils are first responders in the inflammatory process . Early after MI, neutrophils infiltrate infarcted tissue and interact with surrounding endogenous cardiac cells to propagate inflammation by interacting with DAMPs such as high mobility group box 1 protein and heat shock protein 60 produced by injured host tissue [35, 45]. Neutrophils are professional phagocytes as well, engulfing debris and degraded materials. Removal of necrotic tissue is a necessary process for healthy scar formation . Our results indicate that there is a larger pool of D0 resident cardiac neutrophils than previously appreciated. The high engulfment of cardiomyocyte proteins (troponins I and T) indicates that the resident cardiac neutrophils may be performing a surveillance function.
At D3 MI, neutrophils begin to undergo apoptosis. In addition to apoptosis, neutrophils undergo NETosis and can undergo polarization to a phenotype expressing anti-inflammatory factors [26, 36]. The ECM proteins identified may actually help to form the NETosis structure. Interactions between macrophages and apoptotic neutrophils induce an M2 anti-inflammatory phenotype in macrophages to initiate the wound-healing phase of cardiac remodeling . While we know neutrophils and macrophages interact, this study provides novel insight into the neutrophil as an active contributor of ECM production and organization.
The initiation of ECM reorganization occurred concomitantly at D3 MI along with increased production of ECM proteins, fibrinogen and fibronectin. Fibrinogen activates fibroblasts during wound healing and thus may play an important role in remodeling and fibrosis in the heart post-MI . Fibronectin has not been reported to be expressed by neutrophils; our results confirmed that 40% of D3 MI neutrophils expressed fibronectin mRNA. Our study revealed a novel feedback loop between fibronectin and MMP-9, both contributed by the neutrophil that would provide a fine layer of regulation of wound healing within the infarct zone localized to the site of neutrophil entry.
Neutrophils were evolutionarily derived as an immediate means to protect against infection, releasing a large variety of antibacterial agents and destructive enzymes through a multi-granule delivery system . Secondary granules contain neutrophil gelatinase-associated lipocalin, and tertiary (gelatinase) granules contain matrix metalloproteinase (MMP)-8 and MMP-9 . The Borregaard laboratory has recently performed proteome profiling of human neutrophils to catalog granule subsets and identified a new granule type, the ficolin-1 rich granule that contains high levels of cathepsins B, D, H, S, and Z . Our results indicate that this granule is highly released at D3–7 MI. Neutrophil numbers are highest at MI days 1 and 3, with numbers returning toward baseline by day 7 due to macrophage phagocytosis of apoptotic neutrophils to remove them from the LV infarct .
Whether the change in protein expression reflects a change in neutrophil subset composition over time (different infiltrating phenotypes) or the day 1 N1 neutrophils develop into pro-resolving day 7 neutrophils remains to be determined. Sorting and analyzing Ly6G+ CD206+ and Ly6G+ CD206− neutrophils separately will be informative, as would single cell and labeling for half-life determination experiments. While neutrophils in circulation mirror some components of tissue neutrophil phenotypes [4, 9], we have shown that circulating neutrophils at day 0 or MI days 1, 3, and 5 were all CD206− . This indicates that CD206+ N2 neutrophils are locally activated in the LV infarct. Our study adds to our current knowledge by revealing distinct neutrophil profiles across the first week of MI.
We are the first to report that neutrophils contribute ECM components. Contribution of ECM components was more prominent in D5–7 MI, correlating with the temporal polarization of neutrophils from N1 to an N2 phenotype. While there are fewer total neutrophils at later times, ECM production is prominent. How neutrophils coordinate with the fibroblasts remains to be revealed; it may be that neutrophils at later time points serve a fine-tuning role to keep the scar localized to the region of the infarct. Fibronectin and fibrinogen are ECM proteins with both structural and signaling properties. Stimulation of neutrophils with fibronectin induced degranulation, and multiplex imaging confirmed neutrophils contain fibronectin mRNA. These results reveal that neutrophils can act in an autocrine manner to induce their own selective degranulation and release the MMPs that shut off further signaling.
In conclusion, neutrophils showed distinct proteomic profiles over the MI time course. Our findings provide novel insights into mechanisms that both regulate and are regulated by neutrophils during MI. Our work indicates that therapeutic strategies for MI remodeling should include consideration for effects on neutrophil subtypes.
We thank Dr. Rugmani P. Iyer, Dr. Mira Jung, Presley L. Cannon, and Elizabeth R. Flynn for their technical support.
We acknowledge funding from National Institutes of Health under Award Numbers HL075360, HL129823, and HL137319, from American Heart Association Award Number 15SDG22930009, and from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development under Award Numbers 5I01BX000505 and IK2BX003922. The content is solely the responsibility of the authors and does not necessarily represent the official views of any of the funding agencies.
Compliance with ethical standards
Conflict of interest
All authors have reviewed and approved the article. All authors have read the journal authorship agreement and policy on disclosure of potential conflicts of interest and have nothing to disclose.
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