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Modulation of inflammation by treatment with tocilizumab after out-of-hospital cardiac arrest and associations with clinical status, myocardial- and brain injury

Open AccessPublished:December 23, 2022DOI:https://doi.org/10.1016/j.resuscitation.2022.109676

      Abstract

      Aim

      To investigate how the inflammatory response after out-of-hospital cardiac arrest (OHCA) is modulated by blocking IL-6-mediated signalling with tocilizumab, and to relate induced changes to clinical status, myocardial- and brain injury.

      Methods

      This is a preplanned substudy of the IMICA trial (ClinicalTrials.gov, NCT03863015). Upon admission 80 comatose OHCA patients were randomized to infusion of tocilizumab or placebo. Inflammation was characterized by a cytokine assay, CRP, and leukocyte differential count; myocardial injury by TnT and NT-proBNP; brain injury by neuron-specific enolase (NSE) and Neurofilament Light chain (NFL), while sequential organ assessment (SOFA) score and Vasoactive-Inotropic Score (VIS) represented overall clinical status.

      Results

      Responses for IL-5, IL-6, IL-17, neutrophil as well as monocyte counts, and VIS were affected by tocilizumab treatment (all p < 0.05), while there was no effect on levels of NFL. IL-5 and IL-6 were substantially increased by tocilizumab, while IL-17 was lowered. Neutrophils and monocytes were lower at 24 and 48 hours, and VIS was lower at 24 hours, for the tocilizumab group compared to placebo. Multiple correlations were identified for markers of organ injury and clinical status versus inflammatory markers; this included correlations of neutrophils and monocytes with TnT, NSE, NFL, SOFA- and VIS score for the tocilizumab but not the placebo group. NT-proBNP, NFL and SOFA score correlated with CRP in both groups.

      Conclusions

      Treatment with tocilizumab after OHCA modulated the inflammatory response with notable increases for IL-5, IL-6, and decreases for neutrophils and monocytes, as well as reduced vasopressor and inotropy requirements.

      Introduction

      Comatose patients resuscitated from out-of-hospital cardiac arrest (OHCA) are susceptible to a high degree of morbidity and mortality owing to the post cardiac arrest syndrome (PCAS). As part of PCAS a profound degree of systemic inflammation develops after resuscitation, the severity of which is associated with severity of PCAS and mortality.
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      The inflammatory response after out-of-hospital cardiac arrest is not modified by targeted temperature management at 33°C or 36°C.
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      Systemic Inflammatory Response and Potential Prognostic Implications after Out-of-Hospital Cardiac Arrest: A Substudy of the Target Temperature Management Trial.
      • Bro-Jeppesen J.
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      • et al.
      Predictive value of interleukin-6 in post-cardiac arrest patients treated with targeted temperature management at 33°C or 36°C.
      • Peberdy M.A.
      • Andersen L.W.
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      Inflammatory markers following resuscitation from out-of-hospital cardiac arrest-A prospective multicenter observational study.
      This association includes a range of inflammatory markers such as multiple cytokines and C-reactive protein (CRP). In OHCA patients, Interleukin (IL) 6 was identified as a potential target for intervention as elevated IL-6 levels are predictive of morbidity and mortality,
      • Bro-Jeppesen J.
      • Kjaergaard J.
      • Wanscher M.
      • et al.
      Systemic Inflammatory Response and Potential Prognostic Implications after Out-of-Hospital Cardiac Arrest: A Substudy of the Target Temperature Management Trial.
      • Bro-Jeppesen J.
      • Kjaergaard J.
      • Stammet P.
      • et al.
      Predictive value of interleukin-6 in post-cardiac arrest patients treated with targeted temperature management at 33°C or 36°C.
      • Peberdy M.A.
      • Andersen L.W.
      • Abbate A.
      • et al.
      Inflammatory markers following resuscitation from out-of-hospital cardiac arrest-A prospective multicenter observational study.
      and a specific antibody targeting the IL-6 receptor exists.
      • Tanaka T.
      • Narazaki M.
      • Kishimoto T.
      Interleukin (IL-6) immunotherapy.
      IL-6 is a pleiotropic inflammatory cytokine central to initiation of inflammation and acute phase response,
      • Tanaka T.
      • Narazaki M.
      • Kishimoto T.
      Il-6 in inflammation, Immunity, And disease.
      and may have deleterious effects on the cardiovascular system.
      • Tanaka T.
      • Narazaki M.
      • Kishimoto T.
      Interleukin (IL-6) immunotherapy.
      • Krüttgen A.
      • Rose-John S.
      Interleukin-6 in sepsis and capillary leakage syndrome.
      Leukocytes also increase in the circulation after cardiac arrest,
      • Bernard S.A.
      • Gray T.W.
      • Buist M.D.
      • et al.
      Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia.
      • Meyer A.S.P.
      • Johansson P.I.
      • Kjaergaard J.
      • et al.
      Endothelial Dysfunction in Resuscitated Cardiac Arrest (ENDO-RCA): Safety and efficacy of low-dose Iloprost, a prostacyclin analogue, in addition to standard therapy, as compared to standard therapy alone, in post-cardiac-arrest-syndrome patients.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      and although leukocyte subtyping or their association with outcomes has not been investigated to the same extent as for cytokines, levels of different leukocyte subtypes are predictive for outcomes after cardiac arrest.
      • Weiser C.
      • Schwameis M.
      • Sterz F.
      • et al.
      Mortality in patients resuscitated from out-of-hospital cardiac arrest based on automated blood cell count and neutrophil lymphocyte ratio at admission.
      • Miyatake H.
      • Fujino K.
      • Tanaka S.
      • Tsujita Y.
      • Horie M.
      • Eguchi Y.
      Association between lymphocyte count and neurological outcomes in post-cardiac arrest patients treated with mild therapeutic hypothermia.
      To address the possible link between systemic inflammation,
      • Jou C.
      • Shah R.
      • Figueroa A.
      • Patel J.K.
      The Role of Inflammatory Cytokines in Cardiac Arrest.
      IL-6 and outcomes,
      • Bro-Jeppesen J.
      • Kjaergaard J.
      • Stammet P.
      • et al.
      Predictive value of interleukin-6 in post-cardiac arrest patients treated with targeted temperature management at 33°C or 36°C.
      we conducted the “Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response after Out-of-Hospital Cardiac Arrest” (IMICA) trial,
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • Kjaergaard J.
      • Hassager C.
      Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response after Out-of-Hospital Cardiac Arrest (IMICA): study protocol for a double-blinded, placebo-controlled, single-center, randomized clinical trial.
      which was designed to limit systemic inflammation after OHCA with a presumed cardiac cause, and thereby possibly improve patient outcomes by treatment with the IL-6 receptor anti-body, tocilizumab.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • Kjaergaard J.
      • Hassager C.
      Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response after Out-of-Hospital Cardiac Arrest (IMICA): study protocol for a double-blinded, placebo-controlled, single-center, randomized clinical trial.
      The main results showed a marked reduction in CRP-production following IL-6 inhibition compared to placebo, a faster normalization of leukocytes, and an apparent cardio protective effect as illustrated by lower levels of Troponin T (TnT) and N-terminal pro-BNP (NT-proBNP) in patients treated with tocilizumab.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      The primary objective of the present substudy is to assesses if IL-6 receptor inhibition induces changes in the inflammatory response after cardiac arrest, and if changes are induced, to relate these to clinical status, myocardial- and brain injury in exploratory analyses for hypothesis generation.

      Methods

      Trial design, approvals, patients, and randomization

      This is a pre-planned substudy of the IMICA trial. For a comprehensive description of trial rationale and design, see published protocol.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • Kjaergaard J.
      • Hassager C.
      Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response after Out-of-Hospital Cardiac Arrest (IMICA): study protocol for a double-blinded, placebo-controlled, single-center, randomized clinical trial.
      The main results have been published.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      The trial was investigator initiated without support from the pharmaceutical industry, and was a randomized, placebo-controlled, double-blinded, single centre, clinical phase II trial. Patients were randomized to infusion of a single dose of either tocilizumab at 8 mg/kg (maximum 800 mg) or placebo at the earliest possible time after hospital admittance. The trial was conducted in compliance with the Helsinki declaration and was approved by relevant authorities prior to initiation (ethical approval from The Capital Region of Denmark: H-18037286; approval from the Danish Medicines Agency: 2018-002686-19; approval of data handling agreement by the legal department of Rigshospitalet: VD-2019-26), and the trial was registered at clinicaltrials.gov prior to enrolment of the first subject (NCT03863015). The trial was externally monitored for Good Clinical Practice (GCP). For screening, randomization, consent, and intervention procedures as well as a description of concomitant care, please see supplementary material.

      Trial endpoints

      The primary objective is to asses if tocilizumab induces changes in the inflammatory response; these endpoints are pre-defined secondary endpoints of the IMICA trial; an exploratory analyses will assess affected biomarkers correlates with markers of clinical status, myocardial- and brain injury.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • Kjaergaard J.
      • Hassager C.
      Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response after Out-of-Hospital Cardiac Arrest (IMICA): study protocol for a double-blinded, placebo-controlled, single-center, randomized clinical trial.
      Blood samples were obtained at 0, 24, 48 and 72 hours following admission. Leukocyte differential count was performed using a Sysmex XN; CRP, TnT, N-terminal pro B-type natriuretic peptide (NT-proBNP), and neuron-specific enolase (NSE) measurements were performed using a COBAS 8000; analyses were performed in a DS/EN ISO 15189 standardized laboratory as part of routine biochemistry. Samples were prepared for biobank storage by spinning pre-treated blood at 2000 g for 10 minutes to yield EDTA-plasma and serum that was stored at −80 °C. Cytokines were measured in EDTA biobank samples not previously thawed. A 17-plex human cytokine assay (Bio-Rad Laboratories, Hercules, California) was used, and individual cytokine measurements below the lower level of detection (LLD) were assigned the value of the LLD (see supplementary materials for specific cytokines and LLDs). Neurofilament Light chain (NFL) was measured in serum biobank samples by ELISA using a R-PLEX Human Neurofilament L Antibody assay on a MESO QuickPlex SQ120 (MSD, Rockville, Maryland). Overall clinical status was assessed by the Sequential Organ Assessment (SOFA) Score.
      • Vincent J.L.
      • Moreno R.
      • Takala J.
      • et al.
      The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine.
      Hemodynamic status was characterised by heart rate, mean arterial pressure (MAP), and Vasoactive-Inotropic Score (VIS).
      • Belletti A.
      • Lerose C.C.
      • Zangrillo A.
      • Landoni G.
      Vasoactive-Inotropic Score: Evolution, Clinical Utility, and Pitfalls.

      Statistics

      Results reported in the study are based on the modified intention-to-treat population of the IMICA trial in accordance with the published protocol and as used for the reporting of the main results.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • Kjaergaard J.
      • Hassager C.
      Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response after Out-of-Hospital Cardiac Arrest (IMICA): study protocol for a double-blinded, placebo-controlled, single-center, randomized clinical trial.
      For statistical analyses SAS Enterprise Guide 7.1 (SAS-Institute Inc., Cary, NC) was used. Continuous endpoints were transformed by use of log2 for cytokines and NFL, and square root function for leukocyte subtypes (which included “0″ values not suitable for log-transformation) and analysed by baseline corrected repeated measurement mixed models (SAS, PROC MIXED), and results where back-transformed utilizing square root function or square-function as appropriate. Missing values were estimated by maximum likelihood inference as by default by the SAS PROC MIXED function when performing mixed model analyses. Results from mixed models are reported as geometric means with 95% confidence intervals for the estimate, and p values are reported for both treatment-by-time interactions and group differences at corresponding time points. Variables pertaining to hemodynamic status are reported as median (interquartile range) and comparisons between treatment groups were assessed by non-parametric methods. Possible relationships between markers of organ injury, clinical status and inflammatory markers modulated in their response by tocilizumab, were investigated by Spearman’s correlation analyses of summarized values by use of area-under-the-curve (AUC) per hour for bloodborne biomarkers and mean over time for clinical status-variables. Categorical endpoints were investigated by Fishers exact test. The two-sided significance level was p < 0.05 for all analyses.

      Results

      The trial included 80 patients, whereof 39 were randomized to tocilizumab, and 41 to placebo. The median age was 65 years (IQR 53–73) and 60 years (57–70), time-to-ROSC was 20 (14–27) minutes and 17 (11–28) minutes, and 30-day mortality was 35.9% (14 out of 39) and 34.1% (14 out of 41) in the tocilizumab group and placebo group respectively. There was no difference in levels of brain injury assessed by NFL between the tocilizumab and placebo group (Fig. 1A and Fig. S1 in the supplementary materials), and there was no difference in the occurrence of seizures, as 9 (11.3%) and 8 (10%) patients experienced an adverse event of seizures in the tocilizumab and placebo groups respectively, p = 0.79 (no serious adverse events of seizures were reported).
      Figure thumbnail gr1
      Fig. 1Biomarker of brain injury, and inflammatory markers affected by tocilizumab. A: Neurofilament Light Chain. B: IL-5. C: IL-6. D: IL-17. E: Neutrophils. F: Monocytes. Data presented as geometric means with 95% confidence intervals for the estimate based on baseline corrected linear mixed-model analysis; for individual values and BOX-plot with no baseline correction see in the supplementary materials. * indicates p < 0.01 for the tocilizumab group versus placebo at corresponding timepoints. Abbreviations: NFL, Neurofilament Light Chain; IL, Interleukin.

      Cytokines and leukocyte subtypes

      Most cytokines underwent time related changes during the time course from admission till 72 h for both treatment groups (Table 1 and Fig. S2 in the supplementary materials). However, group differences between tocilizumab and placebo were only identified for IL-5, IL-6, and IL-17 (Fig. 1B-D). IL-5 levels in the tocilizumab group were elevated compared to placebo at 24 and 48 hours (both p < 0.01), and IL-6 was markedly elevated at 24, 48, and 72 hours compared to placebo (all p < 0.001), while IL-17 only differed at 72 hours, with IL-17 levels being lesser in the tocilizumab group compared to placebo (p = 0.014).
      Table 1Cytokines and leukocyte differential count.
      GroupT0T24T48T72p for timep for treatment-by-time
      Cytokines (pg/mL)
      IL-1βTocilizumab0.45 (0.35–0.59)0.32 (0.25–0.42)0.28 (0.22–0.36)0.0120.063
      Placebo0.30 (0.25–0.36)0.35 (0.27–0.46)0.29 (0.22–0.38)0.37 (0.29–0.48)
      IL-2Tocilizumab3.14 (2.39–4.13)2.27 (1.74–2.95)2.17 (1.71–2.77)0.0050.664
      Placebo2.22 (1.83–2.70)2.84 (2.18–3.70)2.39 (1.85 – 3.08)2.40 (1.89–3.05)
      IL-4Tocilizumab0.51 (0.37–0.69)0.37 (0.27–0.51)0.31 (0.23–0.41)<0.0010.236
      Placebo0.21 (0.17–0.26)0.38 (0.28–0.51)0.31 (0.23–0.42)0.37 (0.28–0.48)
      IL-5Tocilizumab9.07 (5.89–13.96)*5.09 (3.36–7.72)*3.38 (2.30–4.97)<0.0010.006
      Placebo3.05 (2.25–4.15)3.42 (2.25–5.18)2.12 (1.41–3.18)2.27 (1.55–3.31)
      IL-6Tocilizumab708.9 (46.3–1070.7)*511.3 (342.5–763.4)*246.6 (170.0–357.8)*<0.001<0.001
      Placebo46.3 (34.7–61.8)106.4 (71.5–158.3)33.8 (22.9–49.9)10.81 (7.52–15.55)
      IL-7Tocilizumab3.18 (2.38–4.25)2.52 (1.89–3.36)2.74 (2.07–3.63)0.0080.330
      Placebo2.19 (1.8–2.67)3.3 (2.52–4.34)3.51 (2.65–4.64)3.47 (2.64–4.55)
      IL-8Tocilizumab16.39 (12.08–22.22)10.1 (7.55–13.51)6.94 (5.29–9.1)<0.0010.870
      Placebo19.17 (15.45–23.77)14.71 (11.03–19.61)8.47 (6.38–11.26)6.5 (4.98–8.48)
      IL-10Tocilizumab1.15 (0.72–1.83)0.91 (0.58–1.44)0.82 (0.53–1.27)<0.0010.835
      Placebo15.67 (11.43–21.48)1.16 (0.75–1.8)0.86 (0.55–1.35)0.63 (0.41–0.97)
      IL-12Tocilizumab2.07 (1.43–3.01)1.65 (1.16–2.36)1.79 (1.29–2.49)0.4090.674
      Placebo1.68 (1.29–2.2)1.63 (1.15–2.32)1.67 (1.18–2.36)1.94 (1.4–2.68)
      IL-13Tocilizumab0.57 (0.4–0.8)0.53 (0.38–0.74)0.52 (0.38–0.71)0.0100.481
      Placebo0.43 (0.34–0.54)0.6 (0.43–0.83)0.56 (0.41–0.77)0.68 (0.5–0.92)
      IL-17Tocilizumab2.41 (1.82–3.19)1.94 (1.47–2.55)1.8 (1.39–2.34)*0.0010.026
      Placebo1.57 (1.29–1.9)1.96 (1.5–2.55)2.13 (1.63–2.78)2.74 (2.13–3.53)
      IFN-γTocilizumab7.75 (5.32–11.29)3.98 (2.77–5.7)2.8 (2–3.91)<0.0010.174
      Placebo7.15 (5.49–9.32)6.74 (4.73–9.62)4.62 (3.25–6.56)4.04 (2.91–5.62)
      TNF-αTocilizumab32.4 (26.77–39.2)22.08 (18.4–26.49)17.5 (14.77–20.73)<0.0010.728
      Placebo28.35 (24.78–32.44)27.89 (23.3–33.38)20.05 (16.79–23.95)17.82 (15.09–21.05)
      MCP-1Tocilizumab56.1 (40.8–77.0)22.4 (16.5–30.4)11.4 (8.6–15.2)<0.0010.940
      Placebo89.6 (71.9–111.6)48.2 (35.7–64.9)18.8 (14.0–25.3)10.9 (8.2–14.4)
      MIP-1bTocilizumab10.47 (7.63–14.37)4.56 (3.36–6.2)2.72 (2.04–3.63)<0.0010.178
      Placebo20.27 (16.29–25.24)7.43 (5.52–10.01)2.85 (2.11–3.84)1.96 (1.48–2.6)
      G-CSFTocilizumab8.91 (5.26–15.1)4.51 (2.72–7.5)3.4 (2.12–5.47)<0.0010.911
      Placebo5.88 (4.08–8.5)7.9 (4.81–12.98)3.55 (2.17–5.83)2.79 (1.75–4.45)
      GM-CSFTocilizumab0.31 (0.2–0.47)0.28 (0.19–0.42)0.26 (0.17–0.38)0.0910.589
      Placebo0.35 (0.26–0.47)0.26 (0.18–0.39)0.2 (0.13–0.3)0.26 (0.18–0.39)
      Leukocyte differential count (10^9/L)
      NeutrophilsTocilizumab6.4 (5.22–7.71)*7.52 (6.26–8.89)*7.43 (6.26–8.69)<0.0010.005
      Placebo12.13 (10.97–13.36)9.93 (8.47–11.51)9.41 (8.04–10.89)8.1 (6.91–9.38)
      LymphocytesTocilizumab1.66 (1.25–2.13)1.14 (0.82–1.51)1.27 (0.96–1.63)<0.0010.330
      Placebo2.14 (1.8–2.51)1.43 (1.06–1.86)0.96 (0.68–1.3)0.96 (0.7–1.27)
      MonocytesTocilizumab0.56 (0.45–0.69)*0.56 (0.45–0.68)*0.67 (0.56–0.8)0.0990.002
      Placebo0.58 (0.5–0.67)0.85 (0.71–0.99)0.8 (0.67–0.93)0.67 (0.56–0.79)
      BasophilsTocilizumab0.03 (0.02–0.05)0.01 (0–0.02)0 (0–0.01)<0.0010.860
      Placebo0.03 (0.02–0.05)0.02 (0.01–0.04)0.01 (0–0.02)0 (0–0.01)
      EosinophilsTocilizumab0.13 (0.09–0.19)0.05 (0.02–0.08)0.06 (0.03–0.1)<0.0010.566
      Placebo0.09 (0.06–0.12)0.11 (0.07–0.16)0.03 (0.01–0.06)0.03 (0.01–0.06)
      Data presented as geometric means with 95% confidence intervals for the estimate based on baseline corrected linear mixed-model analysis; baseline values are shown jointly for both groups due to the baseline correction; for individual values and BOX-plot with no baseline correction see Fig. S2 in supplemental materials. * indicates p < 0.05 for the tocilizumab group versus placebo at corresponding timepoints. Abbreviations: IL, Interleukin; IFN- γ, interferon gamma; TNF-α, tumor necrosis factor alpha; MCP-1, monocyte chemoattractant protein-1; MIP-1β, macrophage inflammatory protein-1 beta; G-CSF, granulocyte-colony stimulation factor; GM-CSF, granulocyte–macrophage colony-stimulating factor.
      For the leukocyte subtypes, similarly, there were temporal changes for all except for monocytes (these only trended temporal changes, p = 0.099), and there were also group differences for neutrophils and monocytes (Fig. 1E-F). Neutrophils and monocytes were both lower in the tocilizumab group compared to placebo at 24 hours (p < 0.001 and p = 0.002) and 48 hours (p = 0.043 and p = 0.006).

      Hemodynamic status

      The tocilizumab group had lower vasopressor/inotropy usage at 24 hours, defined by VIS, compared to placebo (Table 2). There were no differences in heart rate or MAP between the tocilizumab and placebo groups.
      Table 2Hemodynamic status.
      GroupT0T24T48T72
      Heart rateTocilizumab71 (36–87)64 (51–83)85 (69–96)88 (74–103)
      Placebo75 (66–85)73 (57.5–87)88 (76–99)88 (77–95)
      MAPTocilizumab73 (65–79)71 (67–78)79 (68–84)86 (76–100)
      Placebo76 (67–84)71 (65–7676 (68–86)88 (76–99)
      VISTocilizumab10 (0.0–20.0)16.0 (9.9–34.0) *16.0 (6.0–32.0)8.0 (4.0–14.3)
      Placebo8.0 (0.0–18.0)32.3 (13.8–54.5)18.8 (10.0–35.5)17.3 (5.0–26.8)
      Data presented as median (interquartile range). * indicates p < 0.05 for the tocilizumab group versus placebo at corresponding timepoints. Abbreviations: MAP, mean arterial pressure; VIS, Vasoactive-Inotropic Score.

      Correlations of organ injury, clinical status and inflammatory markers modulated by tocilizumab

      In the exploratory analyses of correlations, multiple associations were identified for summarized values of markers for organ injury and clinical status versus those inflammatory markers altered by tocilizumab (Table 3 and Fig. 2). TnT was found for the tocilizumab group to correlate positively with neutrophils, monocytes as well as CRP (all p < 0.05), while no correlations were found for the placebo group. NSE had no correlations in common for the two groups, as in the tocilizumab group it correlated with neutrophils, monocytes, as well as IL-5 and IL-6, while for the placebo group, NSE only correlated with CRP (all p < 0.05). The mean SOFA score on day 1–3 correlated for the tocilizumab group with neutrophils, monocytes, CRP, IL-5, and IL-6; for the placebo groups correlations were found for CRP, IL-5, IL-6, and IL-17. NT-proBNP correlated with CRP in both groups, and for the tocilizumab group it also corelated with IL-5 and IL-6.
      Table 3Correlations of organ injury, clinical status and inflammatory markers modulated by tocilizumab..
      GroupTime to ROSCNeutrophilsMonocytesCRPIL-5IL-6IL-17
      Time to ROSCTocilizumabrho-0.530.400.420.280.52−0.15
      p<0.0010.0130.0090.0890.0010.387
      Placeborho-0.240.090.220.120.220.33
      p0.1480.5960.1740.4570.1750.039
      TnTTocilizumabrho0.680.340.330.340.090.29−0.15
      p<0.0010.0340.0440.0320.6050.0850.370
      Placeborho0.280.270.160.030.070.000.06
      p0.0820.0880.3350.8560.6740.9950.714
      NT-proBNPTocilizumabrho0.280.250.260.370.390.400.23
      p0.1130.1560.1300.0320.0210.0180.184
      Placeborho−0.070.170.120.330.03−0.020.01
      p0.6870.3050.4700.0430.8590.9190.974
      NSETocilizumabrho0.710.430.450.260.440.440.12
      p<0.0010.0090.0050.1130.0060.0060.473
      Placeborho0.460.26−0.040.490.230.250.28
      p0.0040.1080.8240.0020.1720.1320.091
      NFLTocilizumabrho0.440.470.480.390.500.570.15
      p0.0060.0030.0030.0180.002<0.0010.365
      Placeborho0.230.260.120.460.300.190.39
      p0.1540.0990.4630.0030.0610.2500.014
      SOFA scoreTocilizumabrho0.390.500.470.490.510.770.14
      p0.0160.0010.0030.0020.001<0.0010.396
      Placeborho0.300.240.100.600.310.510.39
      p0.0630.1370.545<0.0010.0490.0010.013
      VIS ScoreTocilizumabrho0.410.600.370.380.320.470.029
      p0.009<0.0010.0210.0170.0510.0030.866
      Placeborho0.07−0.210.110.060.130.170.10
      p0.6700.1860.4880.7280.4140.2830.521
      Data for correlations between markers of cardiac and brain injury and SOFA score versus markers of inflammation presented as Spearmans rho (correlation coefficient), p-values for the correlation. Correlation analyses were based on area under the curve (AUC) per hour for inflammatory markers versus TnT, NT-proBNP, NFL and NSE until 72 h, and mean SOFA score on days 1 to 3, as well as the mean of VIS scores at 0, 24, 48 and 72 h (for n patients with available data for correlation analysis and all leukocyte subtypes see supplementary Table S1 in supplementary materials). Abbreviations: ROSC, return of spontaneous circulation; TnT, Troponin T; NT-proBNP, N-terminal pro B-type natriuretic peptide; NSE, neuron-specific enolase; NFL, Neurofilament Light chain; SOFA, sequential organ failure assessment; VIS, Vasoactive-Inotropic Score.
      Figure thumbnail gr2
      Fig. 2Correlations of markers of organ injury and inflammation. A: TnT versus neutrophils. B: TnT versus CRP. C: AUC of NSE versus IL-6. Results of Spearman’s correlation shown in figure. Abbreviations: AUC/h, area under the curve per hour; TnT, Troponin T; CRP, C-reactive protein; IL, Interleukin; NSE, Neuron-specific enolase.
      Time to ROSC correlated in the tocilizumab group with neutrophils, monocytes, CRP, IL-6, TnT, NSE, NFL, SOFA and VIS; in the placebo group it correlated with NSE.
      Additionally, for both the treatment groups NSE was found to correlate with TnT (tocilizumab: r = 0.56, p < 0.001; placebo: r = 0.35, p = 0.033) and mean SOFA score (tocilizumab: r = 0.43, p = 0.008; placebo: r = 0.36, p = 0.025).

      Discussion

      In the current study, nearly all cytokines and leukocyte subtypes underwent time related changes after OHCA. Treatment with tocilizumab modulated the responses compared to the placebo group with notable increases for IL-5 and IL-6 and decreases for neutrophils and monocytes, as well as decreased vasopressor/inotropy usage at 24 h, while there were no differences in levels of brain injury assessed by NFL, or NSE as previously reported.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      Many of the inflammatory markers affected by treatment with tocilizumab, were found to correlate with markers of myocardial and brain injury, especially in the tocilizumab group, while only to a lesser extent in the placebo group. Further, correlations were observed for the clinical status of the patients and these inflammatory markers.
      IL-6 levels were markedly elevated in patients treated with tocilizumab compared to placebo at all time points post infusion, and IL-6 correlated with SOFA score for both groups, and with ROSC, NSE, NFL, NT-proBNP as well as VIS in the tocilizumab group. The highly increased levels of IL-6 in the tocilizumab group compared to the placebo likely reflect that IL-6 receptors, both circulating and membrane bound, were blocked by tocilizumab and IL-6 therefore remained in circulation rather than binding to receptors,
      • Tanaka T.
      • Narazaki M.
      • Kishimoto T.
      Interleukin (IL-6) immunotherapy.
      thereby escaping normal routes of internalization and degradation.
      • Nesbitt J.E.
      • Fuller G.M.
      Dynamics of interleukin-6 internalization and degradation in rat hepatocytes.
      • Nishimoto N.
      • Terao K.
      • Mima T.
      • Nakahara H.
      • Takagi N.
      • Kakehi T.
      Mechanisms and pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease.
      Increases in IL-6 levels as a consequence of treatment with tocilizumab have also been demonstrated in patients with non-ST-elevation myocardial infarction,
      • Kleveland O.
      • Kunszt G.
      • Bratlie M.
      • et al.
      Effect of a single dose of the interleukin-6 receptor antagonist tocilizumab on inflammation and troponin T release in patients with non-ST-elevation myocardial infarction: A double-blind, randomized, placebo-controlled phase 2 trial.
      as well as other non-cardiac related diseases such as rheumatoid arthritis.
      • Nishimoto N.
      • Terao K.
      • Mima T.
      • Nakahara H.
      • Takagi N.
      • Kakehi T.
      Mechanisms and pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease.
      As we have previously published, CRP production was markedly reduced by tocilizumab in the IMICA trial,
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      and since CRP is normally produced in hepatocytes in response to IL-6 stimulation,
      • Tanaka T.
      • Narazaki M.
      • Kishimoto T.
      Il-6 in inflammation, Immunity, And disease.
      the biological function of IL-6 was plausibly blunted to a high extent despite the high IL-6 levels. Similarly, there was a trend towards a lesser occurrence of fever in patients treated with tocilizumab compared to placebo (15% vs 34%, p = 0.07),
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      even though IL-6 is known to mediate fever,
      • Conti B.
      • Tabarean I.
      • Andrei C.
      • Bartfai T.
      Cytokines and fever.
      • Eskilsson A.
      • Mirrasekhian E.
      • Dufour S.
      • Schwaninger M.
      • Engblom D.
      • Blomqvist A.
      Immune-induced fever is mediated by IL-6 receptors on brain endothelial cells coupled to STAT3-dependent induction of brain endothelial prostaglandin synthesis.
      and despite that tocilizumab has been described to have limited penetration to the central nervous system; therefore it’s possible that IL-6 may still have some biologic effect in this setting, not reflected in our findings.
      • Kotch C.
      • Barrett D.
      • Teachey D.T.
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      • Nellan A.
      • McCully C.M.L.
      • Cruz Garcia R.
      • et al.
      Improved CNS exposure to tocilizumab after cerebrospinal fluid compared to intravenous administration in rhesus macaques.
      • Gust J.
      • Hay K.A.
      • Hanafi L.-A.
      • et al.
      Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells.
      • Lee D.W.
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      • Porter D.L.
      • et al.
      Current concepts in the diagnosis and management of cytokine release syndrome.
      IL-6 has also been described to affect the vasculature and the lower vasopressor/inotropy usage in the tocilizumab group could represent a more stable overall cardiovascular status due to the lesser IL-6 signalling either directly affecting the vasculature or due to a lesser overall activity of other inflammatory mediators.
      • Tanaka T.
      • Narazaki M.
      • Kishimoto T.
      Interleukin (IL-6) immunotherapy.
      • Bro-Jeppesen J.
      • Johansson P.I.
      • Kjaergaard J.
      • et al.
      Level of systemic inflammation and endothelial injury is associated with cardiovascular dysfunction and vasopressor support in post-cardiac arrest patients.
      • Langeland H.
      • Damås J.K.
      • Mollnes T.E.
      • et al.
      The inflammatory response is related to circulatory failure after out-of-hospital cardiac arrest: A prospective cohort study.
      • Fajgenbaum D.C.
      • June C.H.
      Cytokine Storm.
      The correlation between markers of inflammation including IL-6, and SOFA score after OHCA has previously been demonstrated,
      • Bro-Jeppesen J.
      • Kjaergaard J.
      • Wanscher M.
      • et al.
      The inflammatory response after out-of-hospital cardiac arrest is not modified by targeted temperature management at 33°C or 36°C.
      and in the present trial a broad range of the markers affected by tocilizumab treatment was also observed to correlate with SOFA score. Interestingly, although levels of IL-6 differed substantially between the tocilizumab and placebo group because of the treatment, these levels correlated with SOFA-score in both groups. There were no group differences found in SOFA scores on days 1–3 in the IMICA trial, and the correlation between IL-6 and SOFA score seems to confirm IL-6 as a marker of illness severity with no indication of increased biologic function of IL-6 in the tocilizumab group despite highly elevated levels.
      • Bro-Jeppesen J.
      • Kjaergaard J.
      • Wanscher M.
      • et al.
      The inflammatory response after out-of-hospital cardiac arrest is not modified by targeted temperature management at 33°C or 36°C.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      Besides IL-6, the only other measured cytokine assessed to have potential clinically relevant magnitudes of changes was IL-5. Like IL-6, IL-5 levels were elevated in the tocilizumab group compared to placebo, and IL-5 correlated with SOFA-score for both treatment groups, and correlated with NT-proBNP, NSE and NFL in the tocilizumab group. This cytokine is predominantly excreted by T-helper 2 cells,
      • Akdis M.
      • Burgler S.
      • Crameri R.
      • et al.
      Interleukins, from 1 to 37, and interferon-γ: receptors, functions, and roles in diseases.
      • Walker J.A.
      • McKenzie A.N.J.
      T(H)2 cell development and function.
      and the biologic function of IL-5 has primarily been related to the development and function of eosinophils.
      • Akdis M.
      • Burgler S.
      • Crameri R.
      • et al.
      Interleukins, from 1 to 37, and interferon-γ: receptors, functions, and roles in diseases.
      • Simon H.-U.
      • Yousefi S.
      • Germic N.
      • et al.
      The Cellular Functions of Eosinophils: Collegium Internationale Allergologicum (CIA) Update 2020.
      Eosinophils are involved in a variety of immunologic reactions ranging from protective functions against various infections, to pathological actions in a number of diseases including asthma, but they are also active during tissue repair, and have many regulatory functions.
      • Wechsler M.E.
      • Munitz A.
      • Ackerman S.J.
      • et al.
      Eosinophils in Health and Disease: A State-of-the-Art Review.
      • Jackson D.J.
      • Akuthota P.
      • Roufosse F.
      Eosinophils and eosinophilic immune dysfunction in health and disease.
      In an experimental setting, IL-5 has been demonstrated to limit infarct size in response to ischemia and improve cardiac recovery, and this is hypothesized to be facilitated by eosinophils.
      • Xu J.-Y.
      • Xiong Y.-Y.
      • Tang R.-J.
      • et al.
      Interleukin-5-induced eosinophil population improves cardiac function after myocardial infarction.
      While we found no difference in eosinophil levels, we currently have no measure of eosinophilic activity to substantiate if such infarct limiting mechanisms were present.
      CRP levels were substantially lower in the tocilizumab group compared to placebo as previously reported,
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      and in the present study we found that CRP levels correlated with NT-proBNP, NFL, as well as SOFA score in both treatment groups; correlations were also identified for time to ROSC, TnT and VIS in the tocilizumab group, and for NSE in the placebo group. As CRP is produced mainly in response to IL-6 stimulation, the correlation of CRP in the tocilizumab group could possibly reflect baseline activity prior to treatment, or response to treatment.
      Patients treated with tocilizumab also had lower counts of neutrophils and monocytes at 24 and 48 h compared to placebo. The AUC of both correlated with time to ROSC, TnT, NSE, NFL, SOFA and VIS score in the tocilizumab group, while no correlations were found for the placebo group. We have previously reported that treatment with tocilizumab in these patients led to a faster normalization of the overall leukocyte count compared to placebo,
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      and the present investigation indicates that the decrease in neutrophil count is the main factor for this. Neutrophils generally increase in the circulation in response to various inflammatory stimuli and this is at least partially mediated by IL-6.
      • Suwa T.
      • Hogg J.C.
      • English D.
      • Van Eeden S.F.
      Interleukin-6 induces demargination of intravascular neutrophils and shortens their transit in marrow.
      • Hashizume M.
      • Higuchi Y.
      • Uchiyama Y.
      • Mihara M.
      IL-6 plays an essential role in neutrophilia under inflammation.
      Neutrophils are central to host defences against infections,
      • Burn G.L.
      • Foti A.
      • Marsman G.
      • Patel D.F.
      • Zychlinsky A.
      The Neutrophil.
      however, they’ve also been linked to pathological functions including involvement in reperfusion injury after myocardial ischemia.
      • Jordan J.E.
      • Zhao Z.Q.
      • Vinten-Johansen J.
      The role of neutrophils in myocardial ischemia-reperfusion injury.
      While, the exact mechanism for lowering of neutrophil counts in response to blockage with tocilizumab is unknown,
      • Moots R.J.
      • Sebba A.
      • Rigby W.
      • et al.
      Effect of tocilizumab on neutrophils in adult patients with rheumatoid arthritis: pooled analysis of data from phase 3 and 4 clinical trials.
      similar findings have been made in STEMI,
      • Broch K.
      • Anstensrud A.K.
      • Woxholt S.
      • et al.
      Randomized Trial of Interleukin-6 Receptor Inhibition in Patients With Acute ST-Segment Elevation Myocardial Infarction.
      rheumatoid arthritis patients,
      • Moots R.J.
      • Sebba A.
      • Rigby W.
      • et al.
      Effect of tocilizumab on neutrophils in adult patients with rheumatoid arthritis: pooled analysis of data from phase 3 and 4 clinical trials.
      and in healthy volunteers.
      • Lok L.S.C.
      • Farahi N.
      • Juss J.K.
      • et al.
      Effects of tocilizumab on neutrophil function and kinetics.
      Furthermore, in relation to our finding in the IMICA trial of lower levels of TnT in the tocilizumab group as compared to placebo, the ASSAIL-MI trial found improved myocardial salvage after STEMI in tocilizumab-treated patients compared to placebo,
      • Broch K.
      • Anstensrud A.K.
      • Woxholt S.
      • et al.
      Randomized Trial of Interleukin-6 Receptor Inhibition in Patients With Acute ST-Segment Elevation Myocardial Infarction.
      and a mechanistic study found inverse relationships between myocardial injury and neutrophil levels and indirect measures of activity.
      • Huse C.
      • Anstensrud A.K.
      • Michelsen A.E.
      • et al.
      Interleukin-6 inhibition in ST-elevation myocardial infarction: Immune cell profile in the randomised ASSAIL-MI trial.
      Of note, a greater range of investigated parameters of organ injury and clinical status were correlated with markers of inflammation in the tocilizumab group than in the placebo group, and a similar tendency was identified for the relationship of these parameters and time to ROSC. It has previously been hypothesized that in patients with inflammatory diseases in which IL-6 has been found to be a major contributor to disease burden, the measured plasma levels of IL-6 better reflected disease severity after treatment with tocilizumab than prior to initiation of treatment.
      • Nishimoto N.
      • Terao K.
      • Mima T.
      • Nakahara H.
      • Takagi N.
      • Kakehi T.
      Mechanisms and pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease.
      Possibly, the increased tendency for correlations in the tocilizumab group between markers of organ injury, clinical status and inflammatory markers could similarly be caused by the degree of inflammation in these patients primarily being related to the initial insult of the cardiac arrest – as reflected in the correlation with time to ROSC, and only to a lesser extent secondary insults occurring in the post resuscitation phase. We thus hypothesise that by limiting further activation of the inflammatory response, the initial insult of the cardiac arrest remains the main driver for changes in inflammatory markers downstream from this, while for the placebo group, ongoing or secondary insults – e.g., prolonged periods of hypoperfusion, makes the associations between the initial insults and downstream measurements less distinct.
      While most cytokines and leukocyte subtypes had time related changes in their concentrations, only a subset of the responses were affected by tocilizumab. In the trial we found indications of a cardioprotective effect, but no evidence of reductions in brain injury.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      We cannot determine based on these findings if other anti-inflammatory pharmaceuticals with broader anti-inflammatory effects or better CNS-penetration could have more potent organ protecting effects, with possible reductions in brain injury as well.
      • Kotch C.
      • Barrett D.
      • Teachey D.T.
      Tocilizumab for the treatment of chimeric antigen receptor T cell-induced cytokine release syndrome.
      • Nellan A.
      • McCully C.M.L.
      • Cruz Garcia R.
      • et al.
      Improved CNS exposure to tocilizumab after cerebrospinal fluid compared to intravenous administration in rhesus macaques.
      • Gust J.
      • Hay K.A.
      • Hanafi L.-A.
      • et al.
      Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells.
      • Lee D.W.
      • Gardner R.
      • Porter D.L.
      • et al.
      Current concepts in the diagnosis and management of cytokine release syndrome.
      In summary, the IMICA trial demonstrated a clinically relevant reduction in the otherwise substantial inflammation after OHCA, and further found an apparent cardioprotective effect by treatment with tocilizumab, as these patients had lower levels of biomarkers of myocardial injury and stress represented by TnT, CKMB, as well as NT-proBNP.
      • Meyer M.A.S.
      • Wiberg S.
      • Grand J.
      • et al.
      Treatment Effects of Interleukin-6 Receptor Antibodies for Modulating the Systemic Inflammatory Response After Out-of-Hospital Cardiac Arrest (The IMICA Trial): A Double-Blinded, Placebo-Controlled, Single-Center, Randomized, Clinical Trial.
      An association between the cardioprotective effect of tocilizumab and the changes in cytokine-responses and circulating counts of leukocyte subtypes is possible although such determination cannot be made based on the present study. Also, we investigated a sub-set of 17 cytokines and since blood was only sampled at 24-hour intervals it is possible that a cardioprotective effect was induced by yet un-investigated mechanisms, or changes occurring at unobserved time points. Finally, whether the finding of lesser levels of TnT, NT-proBNP and vasopressor/inotropy usage after treatment with tocilizumab compared to placebo is due to a “direct effect” on the myocardium by less local inflammation and thereby lesser injury and better function,
      • Murphy A.J.
      • Febbraio M.A.
      Immune-based therapies in cardiovascular and metabolic diseases: past, present and future.
      • Frangogiannis N.G.
      The inflammatory response in myocardial injury, repair, and remodelling.
      or due to an “indirect effect” due to less systemic inflammation and influences on the vasculature in general cannot be determined by the present study.
      • Langeland H.
      • Damås J.K.
      • Mollnes T.E.
      • et al.
      The inflammatory response is related to circulatory failure after out-of-hospital cardiac arrest: A prospective cohort study.

      Conclusions

      Treatment with tocilizumab after OHCA modulated the inflammatory responses with notable increases for IL-5, IL-6, and decreases for neutrophils and monocytes, together with reduced vasopressor and inotropy requirements, while there was no influence on markers of brain injury. The highly elevated levels of IL-6 after treatment with tocilizumab indicates that the IL-6 receptors were blocked and circulating IL-6 increased because of this.

      CRediT authorship contribution statement

      Martin Abild Stengaard Meyer: Conceptualization, Methodology, Funding acquisition, Investigation, Resources, Data curation, Formal analysis, Visualization, Project administration, Writing – original draft. Mette Bjerre: Conceptualization, Methodology, Investigation, Data curation, Resources, Project administration, Writing – review & editing, Supervision. Sebastian Wiberg: Conceptualization, Methodology, Investigation, Writing – review & editing. Johannes Grand: Methodology, Investigation, Data curation, Writing – review & editing. Laust Emil Roelsgaard Obling: Methodology, Investigation, Data curation, Writing – review & editing. Anna Sina Pettersson Meyer: Methodology, Investigation, Writing – review & editing. Jakob Josiassen: Methodology, Investigation, Writing – review & editing. Martin Frydland: Methodology, Investigation, Writing – review & editing. Jakob Hartvig Thomsen: Methodology, Investigation, Writing – review & editing. Ruth Frikke-Schmidt: Methodology, Investigation, Data curation, Resources, Project administration, Writing – review & editing. Jesper Kjaergaard: Conceptualization, Methodology, Investigation, Resources, Project administration, Writing – review & editing, Supervision. Christian Hassager: Conceptualization, Methodology, Investigation, Resources, Project administration, Funding acquisition, Writing – review & editing, Supervision, Validation.

      Conflicts of interest

      MASM, MB, SW, JG, LERO, JJ, MF, JHT, RFS, JK, CH declares no conflicts of interest. ASPM has after the completion of the trial been employed by Novo Nordisk, Denmark.

      Acknowledgements

      The study was supported by research grants from “Hjerteforeningen” (The Danish Heart Foundation, Denmark) award no 19-R135-A9302-22125, and “Hjertecenterets Forsningsudvalg” (The Heart Center Research Council, Rigshospitalet, Denmark). The funding sources had no involvement in any aspects of the study including design, conduct, data analysis or reporting.

      Appendix A. Supplementary material

      The following are the Supplementary data to this article:

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