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[Preprint]. 2024 Sep 11:2024.09.06.611551.
doi: 10.1101/2024.09.06.611551.

The Cardioprotective Role of Neutrophil-Specific STING in Myocardial Ischemia/Reperfusion Injury

Maegan L Brockman  1 , Triniti A Scruggs  1 , Lanfang Wang  1 , Gabriella Kabboul  1   2 , John W Calvert  3   4 , Rebecca D Levit  1
Affiliations

The Cardioprotective Role of Neutrophil-Specific STING in Myocardial Ischemia/Reperfusion Injury

Maegan L Brockman et al. bioRxiv. .

Abstract

Background: Neutrophils are the most rapid and abundant immune cells to infiltrate the myocardium following myocardial ischemia/reperfusion injury (MI/R). Neutrophil heterogeneity has not been well characterized in MI/R, and studies have shown conflicting results regarding the impact of neutrophil depletion on cardiac injury. We thus aim to study the impact of neutrophils with enriched type I interferon signature and the role of STING (stimulator of interferon genes) signaling in neutrophils on cardiac reperfusion injury.

Methods: We utilized single-cell RNA sequencing to study neutrophil heterogeneity in response to MI/R. We generated a neutrophil-specific STING knockout mouse to assess the role of neutrophil STING in a model of MI/R. We examined cardiac function following injury via echocardiography and assessed the immune cell trajectory following injury utilizing flow cytometry.

Results: We identified a population of neutrophils with enriched type I interferon signaling and response to type I interferon following MI/R. We found that genetic deletion of neutrophil-specific STING led to worsened cardiac function following MI/R. Further investigation of the immune response by flow cytometry revealed decreased neutrophil infiltration into the myocardium and a shift in macrophage polarization.

Conclusions: Our findings suggest that neutrophil-specific STING is cardioprotective in MI/R, partly due to its effects on downstream immune cells. These results demonstrate that early alterations or therapeutic interventions can influence key events in the resolution of inflammation following MI/R.

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Figures

Figure 1.
Figure 1.. Single-cell RNA Sequencing of Neutrophils Post-MI/R Reveals Type I Interferon Responding Neutrophil Population:
Schematic of experimental design (A). t-SNE plots of sham and MI/R integrated (left) and segregated (right) neutrophils colored by clusters (B). Heat map plotting the top marker genes for each cluster (C). Dot plot of the expression of specific marker genes used to identify neutrophil subtypes (D). Gene set enrichment analysis of MI/R neutrophil clusters (E). Violin plots showing the expression of representative interferon stimulated genes in each neutrophil cluster (F). Violin plot of aggregate ISG score in sham and MI/R neutrophils (G). ***p<0.001, Mann-Whitney test.
Figure 2.
Figure 2.. Type I IFN Signaling Early Post-MI/R:
Flow cytometry analysis of neutrophils isolated from cardiac tissue of sham and IR mice 1 day after reperfusion (A). Values are mean ± SD. Unpaired one-tailed t test. N=4–5. Gene expression levels of Ifna1 and Ifnb1 in cardiac tissue collected from sham and IR mice 1 day after reperfusion (B). Values are mean ± SD. Unpaired one-tailed t test. N=6. Gene expression levels of interferon stimulated genes in cardiac tissue collected from sham and IR mice 1 day after reperfusion (C). Values are mean ± SD. Unpaired one-tailed t test. N=5–6. Flow cytometry analysis of type I IFN signaling in neutrophils isolated from cardiac tissue of sham and IR mice 1 day after reperfusion (D). Values are mean ± SD. Unpaired one-tailed t test. N=4–5. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Figure 3.
Figure 3.. Knockout of STING in Neutrophils Worsens Cardiac Injury:
Representative immunoblot (A) and quantification of STING knockout efficiency in neutrophils and non-neutrophils (B). Values are mean ± SD. Unpaired two-tailed t test. N=3. Representative echocardiography images of parasternal long-axis left ventricular M-mode (C). Left ventricular end-systolic diameter, left ventricular end-diastolic diameter, (D) left ventricular ejection fraction, and fractional shortening (E) from wild-type and neutrophil-specific STING KO mice at baseline and two weeks after ischemia/reperfusion. Values are mean ± SD. Two-way RM ANOVA with Bonferroni. $p<0.0.05; $$p<0.01; $$$p<0.001; $$$$p<0.0001. vs WT baseline. #p<0.0.05; ##p<0.01; ###p<0.001; ####p<0.0001. vs KO baseline. N=5. Ratios of ventricular weight to tibia length were used as a measure of cardiac hypertrophy (F) and Masson’s Trichrome was used to stain for fibrosis and quantified two weeks after ischemia/reperfusion (G,H). Scale bare equals 2.5 mm. Values are mean ± SD. Unpaired one-tailed t test. N=5. LV = left ventricle; LVESD = left ventricular end systolic diameter; LVEDD = left ventricular end diastolic diameter; EF = ejection fraction; FS = fractional shortening; VW/TL = ventricular weight/ tibia length. *p<0.0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Figure 4.
Figure 4.. Knockout of STING in Neutrophils alters Immune Response in MI/R:
Flow cytometry analysis of total leukocytes, myeloid cells, and lymphocytes isolated from cardiac tissue of sham and IR mice 1- and 3- days after reperfusion in wild-type and neutrophil-specific STING knockout mice (A). Flow cytometry analysis of neutrophils, CD64+ cells, and CCR2+ monocytes isolated from cardiac tissue of sham and IR mice 1- and 3- days after reperfusion in wild-type and neutrophil-specific STING knockout mice (B). Flow cytometry analysis of pro- and anti-inflammatory macrophages isolated from cardiac tissue of sham and IR mice 3- days after reperfusion in wild-type and neutrophil-specific STING knockout mice (C). Percentage of pro- and anti-inflammatory macrophages isolated from cardiac tissue of sham and IR mice 3- days after reperfusion in wildtype and neutrophil-specific STING knockout mice (D). Representative flow cytometry plots of CD206 expression on macrophages isolated from cardiac tissue of wild-type and neutrophil-specific STING KO mice 3-days after reperfusion. (E). Flow cytometry analysis of peripheral blood leukocytes, neutrophils, and monocytes taken from sham and IR mice 1- and 3- days after reperfusion in wild-type and neutrophil-specific STING knockout mice (F). Values are mean ± SD. N=4–6. *p<0.0.05; **p<0.01; ***p<0.001; ****p<0.0001. $p<0.0.05; $$p<0.01; $$$p<0.001; $$$$p<0.0001. vs WT sham. #p<0.0.05; ##p<0.01; ###p<0.001; ####p<0.0001. vs KO sham.
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