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. Author manuscript; available in PMC: 2025 Apr 1.
Published in final edited form as: Cytokine. 2024 Feb 6;176:156536. doi: 10.1016/j.cyto.2024.156536

Delayed CCL23 response is associated with poor outcomes after cardiac arrest

Joanne T deKay 1, Elena Chepurko 1, Vadim Chepurko 1, Lacey Knudsen 1, Christine Lord 2, Meghan Searight 2, Sergey Tsibulnikov 1, Michael P Robich 4, Douglas B Sawyer 1, David J Gagnon 1,3,4, Teresa May 1,2, Richard Riker 1,2, David B Seder 1,2, Sergey Ryzhov 1
PMCID: PMC10915974  NIHMSID: NIHMS1966700  PMID: 38325139

Abstract

Chemokines, a family of chemotactic cytokines, mediate leukocyte migration to and entrance into inflamed tissue, contributing to the intensity of local inflammation. We performed an analysis of chemokine and immune cell responses to cardiac arrest (CA). Forty-two patients resuscitated from cardiac arrest were analyzed, and twenty-two patients who underwent coronary artery bypass grafting (CABG) surgery were enrolled. Quantitative antibody array, chemokines, and endotoxin quantification were performed using the patients blood. Analysis of CCL23 production in neutrophils obtained from CA patients and injected into immunodeficient mice after CA and cardiopulmonary resuscitation (CPR) were done using flow cytometry. The levels of CCL2, CCL4, and CCL23 are increased in CA patients. Temporal dynamics were different for each chemokine, with early increases in CCL2 and CCL4, followed by a delayed elevation in CCL23 at forty-eight hours after CA. A high level of CCL23 was associated with an increased number of neutrophils, neuron-specific enolase (NSE), worse cerebral performance category (CPC) score, and higher mortality. To investigate the role of neutrophil activation locally in injured brain tissue, we used a mouse model of CA/CPR. CCL23 production was increased in human neutrophils that infiltrated mouse brains compared to those in the peripheral circulation. It is known that an early intense inflammatory response (within hours) is associated with poor outcomes after CA. Our data indicate that late activation of neutrophils in brain tissue may also promote ongoing injury via the production of CCL23 and impair recovery after cardiac arrest.

Keywords: Cardiac arrest, inflammation, neutrophils, CCL23, mouse model of cardiac arrest and resuscitation

Graphical Abstract

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Late chemokine response is associated with poor outcomes in patients after cardiac arrest

1. INTRODUCTION

Global ischemia and reperfusion injury after cardiac arrest and cardiopulmonary resuscitation is followed by an inflammatory response with increased production of proinflammatory factors1, 2. Pro-inflammatory cytokines in the circulation are increased for a short period early after the resuscitation event3, 4, leading some to suggest that inflammation plays a role only within the first hours after injury5, 6. Recent studies, however, demonstrated the presence of an inflammatory cell response characterized by changes in the number and activity of circulating immune cells that continues for several days after the arrest event7–9. Increased immature neutrophils and intermediate monocytes in the systemic circulation may indicate the activation of emergency myelopoiesis and the egress of myeloid cells from bone marrow as a part of the progression of post-cardiac arrest syndrome (PCAS). An increased number of immature neutrophils and intermediate monocytes in peripheral circulation two to three days after the return of spontaneous circulation (ROSC) is associated with worse outcomes10, 11, suggesting that ongoing inflammatory injury may continue even after cytokine levels in the circulation have normalized. The identification of specific injurious immune mechanisms after cardiac arrest may help to develop a targeted approach to reducing immune cell-mediated tissue damage.

Chemokines are major contributors to immune cell mobilization from bone marrow and trafficking through blood and injured tissues12. Clinical studies have documented increased chemokines, including CCL2, CCL3, CCL4, CCL5, and CCL11, in circulation after cardiac arrest13, 14. CCL2 directly regulates the mobilization of neutrophils and monocytes from bone marrow15, 16, increasing the number of myeloid cells in circulation and their consequent infiltration into the injured tissue, including the heart and brain17–19. While CCL3, CCL4, CCL5, and CCL11 are not involved in the regulation of myeloid cell egress from bone marrow, these chemokines promote the migration of inflammatory cells into tissue, thus contributing to inflammation-driven secondary tissue damage20–24.

In addition, many chemokines, including those increased after cardiac arrest, are produced by neutrophils25 and monocytes26 and may further contribute to the mobilization and recruitment of inflammatory cells. The high number of neutrophils and monocytes is associated with an increased risk of cardiovascular injury27, 28, and therefore, the chemokines produced only by myeloid cells, such as CCL2329, may further promote the myeloid cell-driven inflammatory response. It has also been shown that CCL23 is involved in brain tissue damage30, 31.

The goal of the current study was to characterize temporal cellular and chemokine responses to cardiac arrest. We measured the associations of chemokines at different time points with circulating white blood cells and outcomes in human subjects after resuscitation from cardiac arrest and then employed a mouse model to understand the migration of cardiac arrest neutrophils into brain tissue and their production of CCL23.

2. METHODS

All data that support the findings of this study are available from the corresponding author upon reasonable request.

2.1. Enrollment.

Human research was performed in accordance with study protocols approved by the Maine Medical Center Institutional Review Board, which is accredited by the Association for the Accreditation of Human Research Protection Programs. Study enrollment was conducted between February 2016 and August 2019. Patients aged ≥ 18 years, admitted to the intensive care unit after resuscitation from a cardiopulmonary arrest with encephalopathy (unable to follow verbal commands), and treated with targeted temperature management (TTM) were enrolled following informed consent of the legally authorized representative. Because the dynamics of systemic inflammation over time were fundamental to our study, patients not anticipated to survive ≥48 hours were excluded. Study subjects underwent phlebotomy at 6, 24, 48, and 72 hours after resuscitation (ROSC).

Control subjects were patients with coronary artery disease immediately prior to elective cardiac surgery, as these individuals share many comorbidities and risk factors with study patients and may represent a close match for baseline immune system activity. Control subjects underwent phlebotomy before coronary artery bypass grafting (CABG) surgery at Maine Medical Center (MMC) in Portland, Maine. All subjects were over 18 years of age. Exclusion criteria included known active myocarditis, hypertrophic cardiomyopathy, constrictive pericarditis, significant pericardial disease, severe pulmonary hypertension, severe ventricular arrhythmias, significant hypotension (systolic blood pressure <90mm Hg), pregnancy, known malignancy other than non-melanoma skin cancers, and expected survival less than one year. Relevant clinical data were collected from the electronic medical records for all study participants.

2.2. Clinical management after cardiac arrest.

Research subjects were managed according to American Heart Association Guidelines for Post-Resuscitation Care32. This included normalization of hemodynamic, biochemical, and metabolic parameters33, targeted temperature management at either 33°C or 36°C for 24 hours beginning as soon as possible after ROSC, then gradual rewarming over 12 to 18 hours and controlled normothermia through 72 hours, monitoring with bispectral index and continuous electroencephalography, cardiac catheterization or mechanical circulatory support as indicated, and delayed multimodal neuroprognostication to allow for drug metabolism and delayed awakening; our standardized post-resuscitation care protocol has been described elsewhere34–36.

2.3. Blood sample collection.

Whole blood (8.5mL) was collected from CA and control CABG subjects using BD Vacutainer ACD tubes. Blood aliquots (50μL) were taken for flow cytometric analysis and determination of the white blood cell (WBC) count.

Platelet-free plasma (PFP) was prepared via consecutive centrifugations, each at 2000 × g for 20 minutes at room temperature. PFP samples were stored at −80°C until further analysis. No more than one freeze/thaw cycle was permitted for PFP samples to avoid protein degradation.

2.4. Quantitative antibody array.

Expression of cytokines and soluble factors in human plasma was analyzed in control (CABG pre-op) and cardiac arrest (24 hours post-ROSC) subjects. A Quantibody® Human Antibody Array (#QAH-CAA-640) was conducted by RayBiotech Quantitative Proteomics Services (Norcross, GA).

2.5. Chemokine and cytokine quantification.

Circulating CCL2, CCL4, CCL23, and TNFα protein levels were determined using Human CCL2, CCL4, CCL23, and TNFα DuoSet ELISA (Bio-Techne/ R&D Systems) kits.

2.6. Endotoxin quantification.

Circulating endotoxin levels were measured using the Pierce™ Chromogenic Quantification Kit and its pamphlet protocol. Plasma samples were diluted 1:10 in Endotoxin-Free Water (EFW) and pretreated with incubation at 70°C for 15 minutes.

2.7. Human neutrophil purification.

The erythrocytes and platelets were lyzed with ammonium chloride lysing solution (150 mmol/L NH4Cl, 10 mmol/L NaHCO3, and 1 mmol/L EDTA, pH 7.4), and WBC was incubated for 10 min at 4°C with a combination of APC-conjugated antibodies including CD3 (clone UCHT1), CD19 (clone HIB19), CD42b (clone HIP1), CD94 (clone DX22), CD235a (HI264), and HLA-DR (clone Tu36) (all are purchased from BioLegend) to target T cells, B cells, platelets, NK cells, erythrocytes, and monocytes respectively. After washing out the unbound antibodies, the cells were incubated with anti-APC microbeads (#130-090-855, Miltenyi Biotec Inc.) and passed through the LS columns (#130-090-855, Miltenyi Biotec Inc.). The purity of flowthrough neutrophils was analyzed using flow cytometry. The purified human neutrophils (>95%) were used in experiments.

2.8. Animals.

All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health. Animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of MaineHealth Institute for Research (MHIR). CA/CPR and i.v. injection of human neutrophils was performed in immunodeficient NSG mice (#005557, The Jackson Laboratory).

2.9. CA/CPR induction in mice.

Cardiac arrest and CPR were performed as described37, 38. In brief, mice were intubated, mechanically ventilated using a MidiVent ventilator (Model 845, Harvard Apparatus, Holliston, MA), and instrumented under anesthesia with isoflurane (EZ-108SA, E-Z Systems, Bethlehem, PA). The fluid-filled microcatheter (PE-10, BD Intramedic™, USA) was inserted into the left femoral artery to monitor blood pressure. PC running data acquisition system (LabChart 8.0, PowerLab ADInstruments, Colorado Springs, CO, USA) was used to record blood pressure and needle-probe electrocardiogram. CA was induced by the administration of 80 μg KCl per gram body weight via the left femoral vein and confirmed by loss of arterial pressure and asystolic rhythm. After eight minutes of cardiac arrest, chest compressions were delivered at a rate of 300–350 per minute. ROSC was defined as the return of sinus rhythm with a mean arterial pressure higher than 60 mmHg lasting at least 10 seconds. Throughout the surgical procedure, body temperature was maintained between 36°C-37°C.

2.10. Injection of human neutrophils into the blood of immunodeficient NSG mice after CA/CPR.

Human neutrophils were isolated from the blood of patients following resuscitation from CA at 48 hours after ROSC (n=3, from patients subsequently enrolled in the study described in 2.1; no selection of patients was made) and purified as described above in 2.7. Freshly isolated human neutrophils were resuspended in phosphate-buffered solution (PBS) at the cell concentration of 5 × 107 cells/ml. Then, 5 × 106 cells in a volume of 100 μl were injected intravenously into the mice that underwent CA/CPR (experimental group). The control group of mice received the same volume of PBS (100μl). Injections were performed 10 minutes after ROSC.

Three independent experiments (n=3) were performed on different days depending on the enrollment of patients with CA. Each experiment was carried out using twelve NSG mice (n=12; 6:6, male: female), including six mice intravenously injected with human neutrophils (n=6; 3:3, male: female) and six mice injected with PBS (n=6; 3:3, male: female). Twenty-four hours later, peripheral blood was collected from mice, the mice were euthanized and whole-body perfused with PBS, and the brains were isolated for preparation of cell suspension for flow cytometric analysis.

2.11. Flow cytometric analysis.

Human studies. Blood aliquots (50 μl) for flow cytometric analysis underwent erythrocyte lysis with ammonium chloride lysing solution (150 mmol/L NH4Cl, 10 mmol/L NaHCO3, and 1 mmol/L EDTA, pH 7.4). White blood cells (WBC, 106 cells/ml) were treated with whole molecule mouse and human IgGs to prevent nonspecific binding, followed by incubation with relevant antibodies for 25 min at 4°C. Cells were washed once with 10 volumes of cold PBS/0.5% BSA/2 mM EDTA before data acquisition. The following antibodies were used: CD3 (clone UCHT1), CD14 (clone HCD14), CD16 (clone 3G8), CD19 (clone HIB19), CD56 (clone HCD56), and HLA-DR (clone L243) (all obtained from BioLegend). CD3 and CD19 positive/SSC-Alow cells were identified as T and B cells, respectively. Monocytes were defined as CD14bright/HLA-DR positive/SSC-Aintermediate cells, and SSC-Ahigh/CD16bright/HLA-DRnegative cells were neutrophils. The number of cells in subpopulations was determined using the total cell number and percentage of cells in each subpopulation.

Mouse experiments. Mouse blood was obtained from the submandibular vein and lyzed using an ammonium chloride lysing solution. WBC were resuspended in PBS/0.5% BSA/2 mM EDTA and used for flow cytometric analysis.

To obtain cells from the mouse brain, whole-body perfusion was done with PBS. Mouse brain was isolated, chopped into small pieces, and incubated with an enzymatic digestion solution of 10 mg/ml collagenase II, 2.5 U/ml dispase II, 1 μg/ml DNase I, and 2.5 mM CaCl2 for 25 min at 37°C. Myelin debris was removed using Myelin Removal Beads II (#130-096-733, Miltenyi Biotec Inc.). Cells from mouse brains were resuspended in PBS/0.5% BSA/2 mM EDTA and used for flow cytometric analysis.

The intracellular staining for CCL23 in human neutrophils injected into CA/CPR mouse was performed with biotin-conjugated anti-CCL23 antibody (BAF371, Bio-Techne/RD System) in combination with streptavidin-FITC (BioLegend) in fixed and permeabilized cells using BD Cytofix/Cytoperm™ (BD Biosciences). Human and mouse immune cells were distinguished using PE-conjugated human CD45 (clone HI30) and PeCy7-conjugated mouse CD45 (clone 30-F11).

Data acquisition was performed on a MacsQuant Analyzer 10 (Miltenyi Biotec, Inc). Data were analyzed with WinList 5.0 software. Viable and nonviable cell/cell debris were distinguished using DAPI (to detect dead nucleated cells) and LIVE/DEAD® Fixable Violet Stain kit for the detection of non-nucleated cell debris (Life Technologies, Carlsbad, CA).

2.12. Statistical analysis.

The Kolmogorov-Smirnov test was used as a test for normality. Normally distributed variables are expressed as mean±SEM and skewed data were presented as median and interquartile range. Comparisons between the two groups were performed using two-tailed unpaired t tests (normal distribution) or Mann-Whitney test (skewed). Comparisons between ≥3 groups were performed using ordinary one-way ANOVA with Tukey’s multiple-comparisons post-test or Kruskal Wallis test with Dunn’s multiple comparison test, for normal and skewed data, respectively. Analyses of repeated measures were performed using two-way ANOVA with Sidak’s multiple comparisons test. Correlation analyses were performed using the Spearman correlation coefficient. A p-value less than 0.05 was considered statistically significant.

3. RESULTS

3.1. Study Subjects.

Convenience samples from 42 patients resuscitated from cardiac arrest were analyzed. A total of 22 patients who underwent CABG surgery (control subjects) were enrolled in this study. There were no differences in age or sex between CA and control groups. Demographics and clinical characteristics of CA and control subjects are provided in Table 1.

Table 1.

Characteristics of Control Subjects and Subjects with CA

Characteristic Control Subjects (n=26)* Subjects with CA (n=42)
Age, median (IQR), y 65 (56–71) 64 (53–69)
Male, n (%) 17 (65) 31 (74)
Witnessed, n (%) … 32 (76)
Bystander CPR, n (%) … 36 (86)
Time to ROSC, median (IQR), min … 16.5 (8–27)
Out-of-hospital CA, n (%) … 32 (76)
Shockable rhythm, n (%) … 27 (64)
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CA indicates cardiac arrest; CPR, cardiopulmonary resuscitation; IQR, interquartile range; ROSC, return of spontaneous circulation.

*

Plasma samples from control (patients undergoing coronary artery bypass grafting) were taken immediately before surgery (after heparin administration).

3.2. Changes in the levels of CCL2, CCL4, and CCL23 are characterized by different temporal dynamics after cardiac arrest and resuscitation.

To determine if cardiac arrest is associated with changes in the levels of chemokines, we first performed an analysis of twenty-three chemokines using the Quantibody® Multiplex ELISA service (RayBiotech Life, Inc) in three plasma samples from the cohort of patients with cardiac arrest (24 hr post-ROSC), and three plasma samples obtained from control subjects. Since chemokines mediate immune cell trafficking and accumulation, we used flow cytometric analysis to determine the number of WBC in the peripheral circulation. The samples with the number of leukocytes closest to the median values of WBC in the group of patients with CA (9.1 (IQR: 7.4, 28.3) × 103/μl) and control subjects (6.8 (IQR: 4.8, 17.0) × 103/μl) were selected.

As shown in Figure 1A, most chemokines demonstrated significant inter-individual variability. Although no statistical significance was found, CCL2, CCL4, and CCL23 were consistently increased in all samples obtained from resuscitated patients 24 hours after ROSC compared to pre-operative samples from patients who underwent open heart surgery. These three soluble factors were selected for further, more detailed characterization using sandwich ELISA.

Figure 1. The levels of CCL2, CCL4, and CCL23 chemokines in the peripheral blood are increased after cardiac arrest.

Figure 1.

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A. Chemokines in blood plasma samples from patients with CA (24 hr after ROSC, n=3) and patients undergoing CABG surgery (pre-operative, n=3) were determined using a Quantitative Antibody Array as described in the Methods section. Chemokine levels after CA (floating bars; minimum, median, and maximum values are shown) were normalized to levels of chemokines in CABG patients (control, blue line) and represented as fold-over control. Red floating bars denote chemokines in CA with the levels above the control in all three samples tested. P-values were calculated using the Mann-Whitney test. B-D. The levels of CCL2 (B), CCL4 (C), and CCL23 (D) in patients with CA (n=42) and control subjects (CABG, pre-operative, n=26) were determined using ELISA. Statistical significance between control and different time points after ROSC was calculated using the Kruskal Wallis test with Dunn’s multiple comparison test. Friedman’s test with Dunn’s multiple comparison test was used for comparisons within the group of patients with CA. Asterisk, dagger, and double dagger symbols denote statistical significance vs. control subjects, 6 hours and 24 hours after ROSC, respectively. One, two, or three symbols indicate p<0.05, p<0.01, or p<0.001, respectively.

As demonstrated in Figures 1B and C, the levels of CCL2 and CCL4 were significantly increased in patients with cardiac arrest at 6 hours and 24 hours after ROSC compared to the control group. No differences were found in the level of these chemokines between CA and control groups later at 48 hours and 72 hours after ROSC.

The changes in the levels of CCL23 demonstrated different temporal dynamics (Figure 1D). No early increase in CCL23 was found after CA. However, the level of CCL23 was significantly higher at 24 hr and 48 hr in the circulation of post-CA patients, compared to control subjects or the early timepoint 6 hours after ROSC.

3.3. Early and late associations of CCL2, CCL4, and CCL23 with poor outcome after CA.

We conducted a correlation analysis between the levels of CCL2, CCL4, and CCL23, patient demographics, and clinical indicators of the severity of ischemia-reperfusion injury (Figure 2A). Associations between the levels of CCL2 and CCL4 were found at all four time points after CA, which agrees with their highly similar temporal changes in circulation. Correlations between CCL2 and CCL23 were found only early at 6 hours and late at 72 hours post-ROSC. No associations between CCL4 and CCL23 were found.

Figure 2. The early high levels of CCL2 and late increase in CCL23 are associated with poor outcomes after cardiac arrest.

Figure 2.

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A. Heatmap correlation matrix of chemokine levels, subject demographics, and outcomes after CA. Color represents the Spearman correlation coefficient. The value inside each box represents the P-value. Bystander, bystander CPR; CPC, cerebral performance category score; HxCAD, history of coronary artery disease; TTROSC, time to ROSC; SR6, electroencephalographic suppression ratio at 6 hours after ROSC. B-D. The levels of CCL2 (B), CCL4 (C), and CCL23 (D) in patients who survived (surv, n=23) and non-survivors (non-surv, n=19) after CA/CPR to discharge from the hospital. Repeated measure two-way ANOVA with Sidak’s multiple comparison test (P-values indicated on the graphs).

Our analysis also revealed a presence of a positive association between the level of CCL2 but not CCL4 or CCL23 after CA and the age of patients. While no significant correlations were found between the levels of chemokines and the time to ROSC, a positive trend was identified for CCL2 and CCL4 but not CCL23 and TTROSC, indicating that the initial ischemia may contribute to the induction of CCL2 and CCL4.

There were positive correlations between the level of CCL2 and poor outcomes, including higher CPC score and increased mortality, at 6 hours after ROSC. Conversely, the level of CCL23 was correlated with poor outcomes at 24 and 48 hours after CA. No associations between CCL4 and outcomes were identified. Two-way ANOVA analysis confirmed the early association between CCL2 and death (Figures 2B and C), and the association of a late increase in CCL23 with death (Figure 2D).

3.4. CCL23 but not CCL2 or CCL4 correlates with systemic cellular response.

Since chemokines are involved in inflammatory cell trafficking, we analyzed the associations between CCL2, CCL4, and CCL23 chemokines and circulating leukocytes. As shown in Figure 3A, the number of WBC is increased after cardiac arrest. Correlation analysis revealed a trend toward association at 24 hours and a significant correlation at 48 hours after ROSC between the levels of CCL23 and the total number of WBC (Figure 3B). No significant associations were found between CCL2, CCL4, and total WBC.

Figure 3. CCL23 but not CCL2 or CCL4 is positively correlated with WBC after CA.

Figure 3.

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A. The number of WBC in patients with CA (n=42) and control subjects (CABG, pre-operative, n=26). CA vs. Control: Kruskal Wallis test with Dunn’s multiple comparison test (asterisks). Different time points within the cohort of CA patients: Friedman’s test with Dunn’s multiple comparison test (daggers). One, two, or three symbols indicate p<0.05, p<0.01, or p<0.001, respectively. B. Heatmap correlation matrix of chemokines and WBC after CA. Color - Spearman correlation coefficient. The value inside each box represents the P-value.

3.5. CCL23 correlates with the number of neutrophils and with neuron-specific enolase.

The number of cells in the major subpopulations of WBC was determined using flow cytometry. Neutrophils but not monocytes or lymphocytes correlated with levels of CCL23 (Figure 4A), indicating the potential contribution of neutrophils to the level of circulating CCL23. We also found an association between CCL23 and NSE at 48 hours, the time point associated with the highest levels of CCL23 after CA, suggesting a potential link between neutrophils and neuronal injury.

Figure 4. A high level of CCL23 is associated with increased numbers of neutrophils, and elevated levels of NSE forty-eight hours after CA.

Figure 4.

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A. Heatmap correlation matrix of CCL23 levels, subpopulations of white blood cells, and NSE after CA. Color represents the Spearman correlation coefficient. P-values are indicated inside each box. Ly, lymphocytes; Mon, monocytes; Neu, neutrophils; NLR, neutrophil to lymphocyte ratio; NSE, neuron-specific enolase. B. The number of neutrophils in patients with CA (n=42) and control subjects (CABG, pre-operative, n=26). CA vs. Control: Kruskal Wallis test with Dunn’s multiple comparison test (asterisks). Different time points within the cohort of CA patients: Friedman’s test with Dunn’s multiple comparison test (daggers). One, two, or three symbols indicate p<0.05, p<0.01, or p<0.001, respectively. C. Number of neutrophils in patients who survived (surv, n=23) and non-survivors (non-surv, n=19) after CA/CPR to discharge from the hospital. Repeated measure two-way ANOVA with Sidak’s multiple comparison test.

The neutrophils contribute largely to the elevation of WBC, and their number is high for several days after the CA compared to control subjects (Figure 4B). However, no association was found between the total number of neutrophils and survival (Figure 4C). This may suggest that the activation of circulating neutrophils and not their absolute number contributes to neuronal injury and poor outcomes after CA.

3.6. Lack of association after cardiac arrest between CCL23 and circulating endotoxin and TNFα, two key factors in neutrophil activation.

To examine the role of endotoxin and TNFα in the activation of neutrophils and CCL23 production, we determined their levels in circulation and performed correlation analysis. As shown in Figures 5A and B, no associations were found between the endotoxin levels at 24 hours and 48 hours after ROSC and levels of CCL23 or patient survival after CA.

Figure 5. No associations were found between the levels of CCL23 and Endotoxin or TNFα in peripheral circulation.

Figure 5.

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A. Associations between the levels of CCL23 and endotoxin at 24 hours (left) and 48 hours (right) after ROSC. n=42, Spearman correlation. B. The levels of endotoxin in survivors (surv, n=23) and non-survivors (non-surv, n=19) were measured at 24 hr (left) and 48 hr (right) after CA/CPR at the discharge from the hospital. Mann-Whitney test. C. Associations between circulating levels of CCL23 and TNFα at 24 hours (left) and 48 hours (right) after ROSC. n=42, Spearman correlation. D. The levels of TNFα in survivors (surv, n=23) and non-survivors (non-surv, n=19) after CA/CPR to discharge from the hospital. Mann-Whitney test.

As shown in Figure 5C, the median TNFα was low at 24 hours and 48 hours post-ROSC. Similar to endotoxin, no correlations between TNFα and CCL23 or patient survival (Figure 5D) were noted.

3.7. Neutrophils that accumulate in the brain tissue after cardiac arrest produce a high level of CCL23.

To determine the role of the local tissue microenvironment in the activation of neutrophils and the production of CCL23, we injected human neutrophils into the blood of mice who underwent experimental CA/CPR and determined levels of CCL23 in circulating and brain-infiltrating neutrophils. Because the properties of steady-state neutrophils and those mobilized from bone marrow in response to inflammation might be different, the neutrophils for the experiments were isolated from the blood of patients with cardiac arrest. The neutrophils were purified using an immunomagnetic approach, as shown in Figure 6A. These neutrophils were injected intravenously into immunodeficient NSG mice resuscitated after potassium chloride–induced cardiac arrest, as shown in Figures 6B and C. The next day, the cell suspension obtained from mouse blood and brain was analyzed using flow cytometry using the strategy shown in Figure 6D. Human neutrophils were identified using antibodies against the human pan-immune cell marker, CD45. As shown in Figures 6E and F, the neutrophils that infiltrated the brain tissue produced a higher level of CCL23 compared to neutrophils in the blood.

Figure 6. Human neutrophils infiltrate the mouse brain after cardiac arrest and produce a higher level of CCL23 compared to circulating neutrophils.

Figure 6.

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A. Human neutrophils were purified (immunomagnetic enrichment) from peripheral blood obtained 48 hr after ROSC from patients (n=3) following CA. B-C. Representative arterial blood pressure (B) and ECG measurements (C) in mice with CA/CPR induced by potassium chloride as described in the Methods section. D. Flow cytometric plots showing the strategy to determine the production of CCL23 by human CD45 positive neutrophils in peripheral blood (upper panel) and brain tissue cell suspension (lower panel) obtained from CA/CPR mice 24 hr after ROSC. F. Graphical representation of flow cytometric data showing the level of CCL23 production in human neutrophils in the blood and brain tissue. The level of CCL23 was determined in fixed and permeabilized cells after subtraction of the mean fluorescence intensity (MFI) of isotype-matched controls from the MFI of specific antibodies and represented as a delta mean fluorescence intensity (ΔMFI); n=3, Unpaired t test.

4. DISCUSSION

This study demonstrated increased systemic levels of CCL2, CCL4, and CC23 chemokines after the global ischemia-reperfusion injury of cardiopulmonary arrest and an association of delayed CCL23 response with the number of neutrophils and with poor outcomes. We also showed that human neutrophils infiltrate brain tissue on a delayed basis after experimental cardiac arrest, which extends well beyond the brief cytokine response that has been previously described and is characterized by increased production of the neurotoxic chemokine CCL23.

We and others have demonstrated the shift in circulating immune cells toward higher neutrophils and lower lymphocyte counts after cardiac arrest7, 9. Neutrophils are essential players in antimicrobial defense and clearance of cellular debris after tissue injury39. However, the sustained neutrophilic response may also contribute to secondary tissue injury40, even when pro-inflammation cytokines are not increased in systemic circulation. Our data provide new insight into the potential mechanism for neutrophil activation after global ischemia-reperfusion. We found that the levels of CCL2, CCL4, and CCL23 chemokines are increased in the peripheral circulation of patients with cardiac arrest. All three receptors, including CCR2 (CCL2), CCR5 (CCL4), and CCR1 (CCL23), are expressed in human neutrophils and involved in neutrophil recruitment41, 42. CCL4/CCR5 and CCL23/CCR1 signaling also promote the recruitment of lymphocytes43–45. However, the regulation of lymphocyte trafficking and recruitment after the injury is characterized by more sophisticated mechanisms and also involves sympathetic activation-induced lymphocyte arrest in lymphoid tissue46.

One important finding from our study is related to different temporal changes in chemokines response. Our data showed that CCL2 and CCL4 increased early after the injury. The levels of both chemokines are correlated. Both CCL2 and CCL4 are produced by various cells, including endothelial cells, fibroblasts, and immune cells, in response to pro-inflammatory factors47, 48. Therefore, their early increase after the ischemia-reperfusion injury probably reflects the initial tissue stress and damage. This is confirmed by the correlation between the level of CCL2 early at 6 hours after cardiac arrest and poor survival and neurological outcome. In addition, a trend toward a correlation between CCL2 and CCL4 and a time to ROSC indicate the association of these chemokines with the severity of the initial injury.

In contrast to CCL2 and CCL4, the level of CCL23 was elevated later at 24 and 48 hours after the cardiac arrest. In agreement with previous observation showing that CCL23 is produced by neutrophils29, our data demonstrated a positive correlation between the CCL23 but not CCL2 or CCL4 and the number of neutrophils. This may indicate that CCL23 is associated with the late activation of neutrophils. Positive associations were found between CCL23, increased level of NSE, a marker of brain injury, and poor survival and neurological outcome, indicating the potential contribution of late neutrophils activation to the progression of postcardiac arrest syndrome.

Cardiac arrest is associated with the systemic increase in the levels of endotoxin, at least in some patients, and TNFα49, 50. These factors are known activators of neutrophils and major inducers of CCL23 synthesis and secretion29. Our data, however, demonstrated no associations between endotoxin, TNFα and CCL23 in the peripheral circulation. This observation raised a question about the potential activation of neutrophils locally in the injured and inflamed tissue areas, where concentrations of TLR ligands contributing to CCL23 production are expected to be higher51, 52. Previous studies indicated the detrimental role of CCL23 in brain injury31 and the association of CCL23 level with blood neutrophil count and poor outcomes53.

Blood-brain-barrier disruption after cardiac arrest is patchy in nature54, and tends to peak around 48 hours after resuscitation55. It is caused in part by disruption of the glycocalyx56, is exacerbated by epinephrine57, and ameliorated by hypothermia58. Blood-brain barrier dysfunction after cardiac arrest is linked to cerebral edema. It may be a cause of immune activation in the brain tissue by facilitating the migration of systemic neutrophils into the central nervous system.

We tested the hypothesis of local brain tissue activation of neutrophils using a mouse model of cardiac arrest. CCL23 is not expressed in mice59, providing an excellent opportunity to analyze CCL23-expressing human cells. Our data demonstrated that neutrophil transplantation from patients with cardiac arrest into resuscitated mice had been associated with neutrophil infiltration into the mouse brain. Analysis of human neutrophils revealed significantly increased levels of CCL23 in neutrophils that infiltrated the brain compared to neutrophils in the peripheral circulation.

This study has several limitations. The CCL23, and its receptors, CCR1, are also involved in monocyte activation and recruitment60, 61. The number of monocytes is increased after cardiac arrest, however, for a shorter, early post-injury period compared to neutrophils7. Although we did not find an association between CCL23 and monocytes, we can not exclude the contribution of monocytes, specifically an intermediate subset62, to the total level of CCL23 in blood.

Endotoxemia after cardiac arrest is the result of prolonged intestinal ischemia with translocation of gut flora into the systemic circulation. This is related to the duration (total ischemic time) and severity of the ischemic insult (relative periods of no-flow – without CPR and low flow – with CPR)63. The lack of association between endotoxin levels and CCL23 may also be explained by the presence of these and other confounders, including the administration of antibiotics, the presence or absence of diabetes and hyperglycemia, and other comorbid conditions.

The other limitation is related to our approach to identify chemokines after cardiac arrest. We have used a small sample size for the systemic analysis of twenty-three chemokines in the blood of patients with cardiac arrest and control subjects. Considering a large inter-individual variability, further studies are warranted to investigate the role of CCL23 and other chemokines in CA. Nonetheless, this study is among the first to demonstrate the delayed CCL23 response and its association with neutrophils and poor outcomes after cardiac arrest. Identification of the correlation between the level of CCL23 and poor outcomes in a small cohort with variability makes the strength of our findings even more relevant and clinically important. Clinical trials targeting immunoinflammatory responses64–66 will help to understand better the role of immune cells after global ischemia-reperfusion injury.

In summary, our data indicate that a delayed inflammatory chemokine response may contribute to poor outcomes after cardiac arrest. We have, for the first time, demonstrated local activation of neutrophils infiltrating the injured brain after experimental cardiac arrest and that those neutrophils generate increased production of CCL23. Increased secretion of CCL23 by brain neutrophils may contribute to the recruitment of circulating neutrophils into the brain, creating a vicious cycle that further promotes local brain tissue inflammation. It has been shown previously that the CCL23 receptor, CCR1, mediates the extravasation of neutrophils into postischemic tissue67, promotes neuroinflammation after an experimental intracerebral hemorrhage68, and is involved in the breakdown of blood-brain barrier integrity69. These data indicate that a therapeutic approach targeting CCL23/CCR1 signaling may provide benefits by limiting sustained tissue inflammation and ongoing brain injury after cardiac arrest.

Highlights.

  • Circulating chemokines demonstrate different temporal dynamics after cardiac arrest

  • CCL23 and neutrophil count are related to clinical outcomes after cardiac arrest

  • Late neutrophil activation may promote ongoing tissue injury after cardiac arrest

  • Cardiac arrest neutrophil activation and migration to brain tissue in mouse model

Funding

This work was supported by the Maine Medical Center Cardiovascular Research Institute 2015 Pilot Project Program, the National Heart, Lung, and Blood Institute of the National Institutes of Health under grants U01 HL100398, R01 HL136560, R01 HL139887 the American Heart Association under grant 17POST33410474. We utilized Maine Medical Center’s Progenitor Cell Analysis Core Facility, which is supported by NIH/NIGMS grants P30GM106391, COBRE in Stem and Progenitor Cell Biology and Regenerative Medicine, and U54GM115516, Northern New England Clinical and Translational Research Network (Translational Technologies Core), and P20GM139745, COBRE in Acute Care Research and Rural Disparities. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

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CRediT author statement

Joanne T deKay: Methodology, Samples processing, Data collection, Investigation, Data analysis, Writing-original draft preparation. Elena Chepurko: Methodology, Samples collection and processing. Vadim Chepurko: Methodology, Samples collection and processing, Data analysis. Lacey Knudsen: Methodology, Samples processing, Writing –Reviewing and Editing. Christine Lord: Enrollment, Samples collection, Data collection. Meghan Searight: Enrollment, Samples collection, Data collection. Sergey Tsibulnikov: Methodology, Samples collection and processing. Michael P. Robich: Enrollment, Data collection. Douglas B. Sawyer: Conceptualization. David J Gagnon: Enrollment, Writing- Reviewing and Editing. Teresa May: Conceptualization, Enrollment, Writing- Reviewing and Editing. Richard Riker: Conceptualization, Enrollment, Writing- Reviewing and Editing. David B. Seder: Conceptualization, Enrollment, Methodology, Samples processing, Data collection, Investigation, Data analysis, Writing-original draft preparation. Sergey Ryzhov: Conceptualization, Methodology, Samples processing, Data collection, Investigation, Data analysis, Writing-original draft preparation.

Declaration of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

REFERENCES

  • 1.Jou C, Shah R, Figueroa A and Patel JK. The Role of Inflammatory Cytokines in Cardiac Arrest. J Intensive Care Med. 2020;35:219–224. [DOI] [PubMed] [Google Scholar]
  • 2.Niemann JT, Rosborough JP, Youngquist S, Shah AP, Lewis RJ, Phan QT and Filler SG. Cardiac function and the proinflammatory cytokine response after recovery from cardiac arrest in swine. J Interferon Cytokine Res. 2009;29:749–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bro-Jeppesen J, Kjaergaard J, Wanscher M, Nielsen N, Friberg H, Bjerre M and Hassager C. Systemic Inflammatory Response and Potential Prognostic Implications After Out-of-Hospital Cardiac Arrest: A Substudy of the Target Temperature Management Trial. Crit Care Med. 2015;43:1223–32. [DOI] [PubMed] [Google Scholar]
  • 4.Obling LER, Beske RP, Wiberg S, Folke F, Moeller JE, Kjaergaard J and Hassager C. Steroid treatment as anti-inflammatory and neuroprotective agent following out-of-hospital cardiac arrest: a randomized clinical trial. Trials. 2022;23:952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Bro-Jeppesen J, Kjaergaard J, Stammet P, Nielsen N and Hassager C. Reply to Letter: ‘Corticosteroids and inflammation after cardiac arrest’. Resuscitation. 2016;99:e9. [DOI] [PubMed] [Google Scholar]
  • 6.Pantazopoulos IN, Zakynthinos SG and Mentzelopoulos SD. Corticosteroids and inflammation after cardiac arrest. Resuscitation. 2016;99:e7–8. [DOI] [PubMed] [Google Scholar]
  • 7.Ryzhov S, May T, Dziodzio J, Emery IF, Lucas FL, Leclerc A, McCrum B, Lord C, Eldridge A, Robich MP, Ichinose F, Sawyer DB, Riker R and Seder DB. Number of Circulating CD 73-Expressing Lymphocytes Correlates With Survival After Cardiac Arrest. J Am Heart Assoc. 2019;8:e010874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Huang L, Peng J, Wang X and Li F. High platelet-lymphocyte ratio is a risk factor for 30-day mortality in in-hospital cardiac arrest patients: a case-control study. Expert Rev Clin Immunol. 2021;17:1231–1239. [DOI] [PubMed] [Google Scholar]
  • 9.Villois P, Grimaldi D, Spadaro S, Shinotsuka CR, Fontana V, Scolletta S, Franchi F, Vincent JL, Creteur J and Taccone FS. Lymphopaenia in cardiac arrest patients. Annals of intensive care. 2017;7:85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Başer K, Baş HD, Attaluri P, Rodrigues T, Nichols J and Nugen K. Changes in neutrophil-to-lymphocyte ratios in postcardiac arrest patients treated with targeted temperature management. Anatol J Cardiol. 2017;18:215–222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Krychtiuk KA, Lenz M, Richter B, Hohensinner PJ, Kastl SP, Mangold A, Huber K, Hengstenberg C, Wojta J, Heinz G and Speidl WS. Monocyte subsets predict mortality after cardiac arrest. J Leukoc Biol. 2021;109:1139–1146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Sokol CL and Luster AD. The chemokine system in innate immunity. Cold Spring Harb Perspect Biol. 2015;7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Podolec J, Trąbka-Zawicki A, Badacz R, Siedliński M, Tomala M, Bartuś K, Legutko J, Przewłocki T, Żmudka K and Kabłak-Ziembicka A. Chemokine RANTES and IL-1β in mild therapeutic hypothermia-treated patients after out-of-hospital sudden cardiac arrest. Postepy w kardiologii interwencyjnej = Advances in interventional cardiology. 2019;15:98–106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Bisschops LL, van der Hoeven JG, Mollnes TE and Hoedemaekers CW. Seventy-two hours of mild hypothermia after cardiac arrest is associated with a lowered inflammatory response during rewarming in a prospective observational study. Crit Care. 2014;18:546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Tsou CL, Peters W, Si Y, Slaymaker S, Aslanian AM, Weisberg SP, Mack M and Charo IF. Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites. J Clin Invest. 2007;117:902–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Deshmane SL, Kremlev S, Amini S and Sawaya BE. Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res. 2009;29:313–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Chen Y, Hallenbeck JM, Ruetzler C, Bol D, Thomas K, Berman NE and Vogel SN. Overexpression of monocyte chemoattractant protein 1 in the brain exacerbates ischemic brain injury and is associated with recruitment of inflammatory cells. J Cereb Blood Flow Metab. 2003;23:748–55. [DOI] [PubMed] [Google Scholar]
  • 18.Losy J and Zaremba J. Monocyte Chemoattractant Protein-1 Is Increased in the Cerebrospinal Fluid of Patients With Ischemic Stroke. 2001;32:2695–2696. [DOI] [PubMed] [Google Scholar]
  • 19.Morimoto H and Takahashi M. Role of monocyte chemoattractant protein-1 in myocardial infarction. Int J Biomed Sci. 2007;3:159–67. [PMC free article] [PubMed] [Google Scholar]
  • 20.Pelisch N, Rosas Almanza J, Stehlik KE, Aperi BV and Kroner A. CCL3 contributes to secondary damage after spinal cord injury. J Neuroinflammation. 2020;17:362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Estevao C, Bowers CE, Luo D, Sarker M, Hoeh AE, Frudd K, Turowski P and Greenwood J. CCL4 induces inflammatory signalling and barrier disruption in the neurovascular endothelium. Brain, Behavior, & Immunity - Health. 2021;18:100370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Lin J, Xu Y, Guo P, Chen Y-J, Zhou J, Xia M, Tan B, Liu X, Feng H and Chen Y. CCL5/CCR5-mediated peripheral inflammation exacerbates blood‒brain barrier disruption after intracerebral hemorrhage in mice. J Transl Med. 2023;21:196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Montecucco F, Braunersreuther V, Lenglet S, Delattre BM, Pelli G, Buatois V, Guilhot F, Galan K, Vuilleumier N, Ferlin W, Fischer N, Vallée JP, Kosco-Vilbois M and Mach F. CC chemokine CCL5 plays a central role impacting infarct size and post-infarction heart failure in mice. Eur Heart J. 2012;33:1964–74. [DOI] [PubMed] [Google Scholar]
  • 24.Li Y, Zhao Y, Qiu C, Yang Y, Liao G, Wu X, Zhang X, Zhang Q, Zhang R and Wang ZJA. Role of eotaxin-1/CCL11 in sepsis-induced myocardial injury in elderly patients. 2020;12:4463–4473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Tecchio C, Micheletti A and Cassatella MA. Neutrophil-derived cytokines: facts beyond expression. Front Immunol. 2014;5:508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Ruytinx P, Proost P, Van Damme J and Struyf S. Chemokine-Induced Macrophage Polarization in Inflammatory Conditions. Front Immunol. 2018;9:1930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Gaul DS, Stein S and Matter CM. Neutrophils in cardiovascular disease. Eur Heart J. 2017;38:1702–1704. [DOI] [PubMed] [Google Scholar]
  • 28.Shahid F, Lip GYH and Shantsila E. Role of Monocytes in Heart Failure and Atrial Fibrillation. 2018;7:e007849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Arruda-Silva F, Bianchetto-Aguilera F, Gasperini S, Polletti S, Cosentino E, Tamassia N and Cassatella MA. Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα. Frontiers in cellular and infection microbiology. 2017;7:176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Bonaventura A and Montecucco F. CCL23 is a promising biomarker of injury in patients with ischaemic stroke. 2018;283:476–478. [DOI] [PubMed] [Google Scholar]
  • 31.Simats A, García-Berrocoso T, Penalba A, Giralt D, Llovera G, Jiang Y, Ramiro L, Bustamante A, Martinez-Saez E, Canals F, Wang X, Liesz A, Rosell A and Montaner J. CCL23: a new CC chemokine involved in human brain damage. 2018;283:461–475. [DOI] [PubMed] [Google Scholar]
  • 32.Panchal AR, Bartos JA, Cabañas JG, Donnino MW, Drennan IR, Hirsch KG, Kudenchuk PJ, Kurz MC, Lavonas EJ, Morley PT, O’Neil BJ, Peberdy MA, Rittenberger JC, Rodriguez AJ, Sawyer KN, Berg KM, Arafeh J, Benoit JL, Chase M, Fernandez A, Paiva EFd, Fischberg BL, Flores GE, Fromm P, Gazmuri R, Gibson BC, Hoadley T, Hsu CH, Issa M, Kessler A, Link MS, Magid DJ, Marrill K, Nicholson T, Ornato JP, Pacheco G, Parr M, Pawar R, Jaxton J, Perman SM, Pribble J, Robinett D, Rolston D, Sasson C, Satyapriya SV, Sharkey T, Soar J, Torman D, Schweinitz BV, Uzendu A, Zelop CM and Magid DJ. Part 3: Adult Basic and Advanced Life Support: 2020 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. 2020;142:S366–S468. [DOI] [PubMed] [Google Scholar]
  • 33.Pitcher JH, Dziodzio J, Keller J, May T, Riker RR and Seder DB. Hemodynamic, Biochemical, and Ventilatory Parameters are Independently Associated with Outcome after Cardiac Arrest. Neurocrit Care. 2018;29:69–76. [DOI] [PubMed] [Google Scholar]
  • 34.Seder DB, Lord C and Gagnon DJ. The Evolving Paradigm of Individualized Postresuscitation Care After Cardiac Arrest. Am J Crit Care. 2016;25:556–564. [DOI] [PubMed] [Google Scholar]
  • 35.Seder DB. Management of Comatose Survivors of Cardiac Arrest. Continuum (Minneapolis, Minn). 2018;24:1732–1752. [DOI] [PubMed] [Google Scholar]
  • 36.Seder DB and Van der Kloot TE. Methods of cooling: Practical aspects of therapeutic temperature management. 2009;37:S211–S222. [DOI] [PubMed] [Google Scholar]
  • 37.Abella BS, Zhao D, Alvarado J, Hamann K, Vanden Hoek TL and Becker LB. Intra-arrest cooling improves outcomes in a murine cardiac arrest model. Circulation. 2004;109:2786–91. [DOI] [PubMed] [Google Scholar]
  • 38.Ikeda K, Liu X, Kida K, Marutani E, Hirai S, Sakaguchi M, Andersen LW, Bagchi A, Cocchi MN, Berg KM, Ichinose F and Donnino MW. Thiamine as a neuroprotective agent after cardiac arrest. Resuscitation. 2016;105:138–44. [DOI] [PubMed] [Google Scholar]
  • 39.Rosales C Neutrophil: A Cell with Many Roles in Inflammation or Several Cell Types? 2018;9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Castanheira FVS and Kubes P. Neutrophils and NETs in modulating acute and chronic inflammation. Blood. 2019;133:2178–2185. [DOI] [PubMed] [Google Scholar]
  • 41.Reichel CA, Rehberg M, Lerchenberger M, Berberich N, Bihari P, Khandoga AG, Zahler S and Krombach F. Ccl2 and Ccl3 mediate neutrophil recruitment via induction of protein synthesis and generation of lipid mediators. Arterioscler Thromb Vasc Biol. 2009;29:1787–93. [DOI] [PubMed] [Google Scholar]
  • 42.Berahovich RD, Miao Z, Wang Y, Premack B, Howard MC and Schall TJ. Proteolytic activation of alternative CCR1 ligands in inflammation. J Immunol. 2005;174:7341–51. [DOI] [PubMed] [Google Scholar]
  • 43.Estevao C, Bowers CE, Luo D, Sarker M, Hoeh AE, Frudd K, Turowski P and Greenwood J. CCL4 induces inflammatory signalling and barrier disruption in the neurovascular endothelium. Brain Behav Immun Health. 2021;18:100370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Pham K, Luo D, Liu C and Harrison JK. CCL5, CCR1 and CCR5 in murine glioblastoma: immune cell infiltration and survival rates are not dependent on individual expression of either CCR1 or CCR5. J Neuroimmunol. 2012;246:10–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Chen C, Peng H, Zeng Y and Dong G. CD14, CD163, and CCR1 are involved in heart and blood communication in ischemic cardiac diseases. J Int Med Res. 2020;48:300060520951649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Nakai A, Hayano Y, Furuta F, Noda M and Suzuki K. Control of lymphocyte egress from lymph nodes through β2-adrenergic receptors. J Exp Med. 2014;211:2583–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Zhang J, Patel L and Pienta KJ. Targeting chemokine (C-C motif) ligand 2 (CCL2) as an example of translation of cancer molecular biology to the clinic. Prog Mol Biol Transl Sci. 2010;95:31–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Maurer M and von Stebut E. Macrophage inflammatory protein-1. Int J Biochem Cell Biol. 2004;36:1882–6. [DOI] [PubMed] [Google Scholar]
  • 49.Adrie C, Laurent I, Monchi M, Cariou A, Dhainaou JF and Spaulding C. Postresuscitation disease after cardiac arrest: a sepsis-like syndrome? Curr Opin Crit Care. 2004;10:208–12. [DOI] [PubMed] [Google Scholar]
  • 50.Niemann JT, Youngquist ST, Shah AP, Thomas JL and Rosborough JP. TNF-α blockade improves early post-resuscitation survival and hemodynamics in a swine model of ischemic ventricular fibrillation. Resuscitation. 2013;84:103–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Gesuete R, Kohama SG and Stenzel-Poore MP. Toll-like receptors and ischemic brain injury. J Neuropathol Exp Neurol. 2014;73:378–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Feng Y and Chao W. Toll-like receptors and myocardial inflammation. International journal of inflammation. 2011;2011:170352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Lin H, Shen J, Zhu Y, Zhou L, Wu F, Liu Z, Zhang S and Zhan R. Elevated Serum CCL23 Levels at Admission Predict Delayed Cerebral Ischemia and Functional Outcome after Aneurysmal Subarachnoid Hemorrhage. Journal of clinical medicine. 2022;11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Kim HI, Lee IH, Park JS, Kim DM, You Y, Min JH, Cho YC, Jeong WJ, Ahn HJ, Kang C and Lee BK. The Usefulness of Quantitative Analysis of Blood-Brain Barrier Disruption Measured Using Contrast-Enhanced Magnetic Resonance Imaging to Predict Neurological Prognosis in Out-of-Hospital Cardiac Arrest Survivors: A Preliminary Study. Journal of clinical medicine. 2020;9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Park JS, You Y, Min JH, Yoo I, Jeong W, Cho Y, Ryu S, Lee J, Kim SW, Cho SU, Oh SK, Ahn HJ, Lee J and Lee IH. Study on the timing of severe blood-brain barrier disruption using cerebrospinal fluid-serum albumin quotient in post cardiac arrest patients treated with targeted temperature management. Resuscitation. 2019;135:118–123. [DOI] [PubMed] [Google Scholar]
  • 56.Zhu J, Li X, Yin J, Hu Y, Gu Y and Pan S. Glycocalyx degradation leads to blood-brain barrier dysfunction and brain edema after asphyxia cardiac arrest in rats. J Cereb Blood Flow Metab. 2018;38:1979–1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Semenas E, Sharma HS and Wiklund L. Adrenaline increases blood-brain-barrier permeability after haemorrhagic cardiac arrest in immature pigs. Acta Anaesthesiol Scand. 2014;58:620–9. [DOI] [PubMed] [Google Scholar]
  • 58.Li J, Li C, Yuan W, Wu J, Li J, Li Z and Zhao Y. Mild hypothermia alleviates brain oedema and blood-brain barrier disruption by attenuating tight junction and adherens junction breakdown in a swine model of cardiopulmonary resuscitation. PLoS One. 2017;12:e0174596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Viola A and Luster AD. Chemokines and Their Receptors: Drug Targets in Immunity and Inflammation. 2008;48:171–197. [DOI] [PubMed] [Google Scholar]
  • 60.Kaufmann A, Salentin R, Gemsa D and Sprenger H. Increase of CCR1 and CCR5 expression and enhanced functional response to MIP-1 alpha during differentiation of human monocytes to macrophages. J Leukoc Biol. 2001;69:248–52. [PubMed] [Google Scholar]
  • 61.Nardelli B, Tiffany HL, Bong GW, Yourey PA, Morahan DK, Li Y, Murphy PM and Alderson RF. Characterization of the signal transduction pathway activated in human monocytes and dendritic cells by MPIF-1, a specific ligand for CC chemokine receptor 1. J Immunol. 1999;162:435–44. [PubMed] [Google Scholar]
  • 62.Kapellos TS, Bonaguro L, Gemünd I, Reusch N, Saglam A, Hinkley ER and Schultze JL. Human Monocyte Subsets and Phenotypes in Major Chronic Inflammatory Diseases. 2019;10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Grimaldi D, Sauneuf B, Guivarch E, Ricome S, Geri G, Charpentier J, Zuber B, Dumas F, Spaulding C, Mira JP and Cariou A. High Level of Endotoxemia Following Out-of-Hospital Cardiac Arrest Is Associated With Severity and Duration of Postcardiac Arrest Shock. Crit Care Med. 2015;43:2597–604. [DOI] [PubMed] [Google Scholar]
  • 64.Meyer MAS, Wiberg S, Grand J, Meyer ASP, Obling LER, Frydland M, Thomsen JH, Josiassen J, Møller JE, Kjaergaard J and Hassager C. Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial). 2021;143:1841–1851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Gagnon DJ, Ryzhov SV, May MA, Riker RR, Geller B, May TL, Bockian S, deKay JT, Eldridge A, Van der Kloot T, Lerwick P, Lord C, Lucas FL, Mailloux P, McCrum B, Searight M, Wirth J, Zuckerman J, Sawyer D and Seder DB. Ceftriaxone to PRevent pneumOnia and inflammaTion aftEr Cardiac arresT (PROTECT): study protocol for a randomized, placebo-controlled trial. Trials. 2022;23:197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.François B, Cariou A, Clere-Jehl R, Dequin P-F, Renon-Carron F, Daix T, Guitton C, Deye N, Legriel S, Plantefève G, Quenot J-P, Desachy A, Kamel T, Bedon-Carte S, Diehl J-L, Chudeau N, Karam E, Durand-Zaleski I, Giraudeau B, Vignon P and Le Gouge A. Prevention of Early Ventilator-Associated Pneumonia after Cardiac Arrest. 2019;381:1831–1842. [DOI] [PubMed] [Google Scholar]
  • 67.Metzemaekers M, Gouwy M and Proost P. Neutrophil chemoattractant receptors in health and disease: double-edged swords. Cell Mol Immunol. 2020;17:433–450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Yan J, Zuo G, Sherchan P, Huang L, Ocak U, Xu W, Travis ZD, Wang W, Zhang JH and Tang J. CCR1 Activation Promotes Neuroinflammation Through CCR1/TPR1/ERK1/2 Signaling Pathway After Intracerebral Hemorrhage in Mice. Neurotherapeutics. 2020;17:1170–1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Yan J, Xu W, Lenahan C, Huang L, Ocak U, Wen J, Li G, He W, Le C, Zhang JH, Mo L and Tang J. Met-RANTES preserves the blood-brain barrier through inhibiting CCR1/SRC/Rac1 pathway after intracerebral hemorrhage in mice. Fluids and barriers of the CNS. 2022;19:7. [DOI] [PMC free article] [PubMed] [Google Scholar]

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