Myocardial ischemia-reperfusion injury (MIRI) is defined as tissue damage that occurs when early and fast coronary flow returns to the heart after ischemia, which often exacerbates the damage caused by previous myocardial ischemia (1, 2). A growing number of studies have demonstrated that the reasons behind the condition mainly lie in three aspects: dysfunction of energy metabolism due to intracellular calcium overload, cardiomyocyte death due to increased oxygen free radicals, and cardiomyocyte death in response to inflammation (3). According to the time of reperfusion after ischemia, reperfusion can be divided into early (≤ 6 h) or late stages (4–7 days post-reperfusion) (4). During early reperfusion, a coordinated upregulation of inflammatory processes was observed in the ischemic area, whereas interstitial proteins, angiogenesis, and cardio-renal signaling processes were found to be increased at later reperfusion (days 4 and 7) (5). However, the mechanism of myocardial IRI, especially the role of the immune microenvironment in myocardial IRI, still needs to be further explored.

Nuclear Factor Erythroid 2 Like 2 (Nuclear factor erythroid 2-related factor 2; Nrf2; NFE2L2) is an important member of the cap´n´collar (CNC) basic leucine zipper family of transcription factors, characterized structurally by the presence of Nrf2-ECH (embedded contact homology) homology domains (6). Moreover, as the master regulator of the cellular oxidative stress response, Nrf2 regulates many cytoprotective genes and plays a central role in defense mechanisms against oxidative and electrophilic insults (7). Peroxisome proliferator-activated receptor-gamma co-activator 1α (PGC-1α), an important antioxidant molecule, interacts with Nrf2 to inhibit mitochondrial oxidative stress, promotes the clearance of damaged mitochondria, enhances mitochondrial biogenesis, and reduces the burden of IRI (8). Under oxidative stress, Kelch-like ECH-associated protein 1 (Keap1) is inactivated; Nrf2 escapes degradation based on the ubiquitin-proteasome pathway. Stabilized Nrf2 proteins form heterodimers with small Maf proteins in the nucleus and induce target gene expression for antioxidant reactions and detoxification by binding to antioxidant/electrophile response elements (9). Therefore, it plays an important role in the damage caused by oxidative stress. Please note, henceforth for the sake of uniformity, whether it’s human NRF2 or mouse Nrf2, it’s written Nrf2.

Programmed cell death 4 (Pdcd4), is a novel tumor suppressor to show multi-functions inhibiting cell growth, tumor invasion, metastasis, and inducing apoptosis (10). As a downstream target of miR-499a-5p, Pdcd4 reduced the infarct damage and cortical neuron apoptosis caused by I/R injury, which might represent a novel target that regulates brain injury by inhibiting Pdcd4-mediating apoptosis (11). In a rat model of IRI, overexpression of miR-21 in the heart could reduce cardiomyocyte apoptosis and myocardial infarct size, as well as protect the myocardium from ischemia/reperfusion by inhibiting Pdcd4 transcription (12). Additionally, a circular RNA, circPVT1, protects the myocardium from myocardial infarction (MI) and hypoxia/reoxygenation (H/R) injury by miR-125b/miR-200 sponges and then regulating Keap1/Nrf2- and Pdcd4-mediated apoptotic signaling (13). Pdcd4 contributes to c-Jun repression and p21 induction, and leads to the ubiquitination of Keap1-dependent Nrf2, which is the key factor of the cellular oxidative stress response (14). Hence, PDCD4 cooperates with Nrf2 to regulate apoptosis-related signaling pathways.

Myocardial ischemia-reperfusion (MI/R) promotes the polarization of proinflammatory M1 macrophages and aggravates heart injury (15). Macrophages promote endothelial-to-mesenchymal transition via MT1-MMP/TGFbeta1 after myocardial infarction (16). Macrophage AXL receptor tyrosine kinase worsens cardiac repair after reperfused myocardial infarction (17). Under the coating of platelet-like fusogenic liposomes (PLPs), mesoporous silica nanospheres with a payload of miR-21, an anti-inflammatory agent, can be specifically delivered to inflammatory monocytes in the blood circulation of MI/R-induced mice; they directly enter the cytoplasm of monocytes through membrane fusion, thereby realizing the reparative reprogramming of the inflamed macrophages derived from it. Ultimately, miRNA-21 delivered into macrophages via guidance and protection preserves the cardiac function of mice undergoing MI/R after being guided and protected (18). Hence, macrophages not only often play a pro-inflammatory role in myocardial IRI, but can also be reprogrammed to evolve as a therapeutic vector.

In this study, on the basis of confirming that early MI/R (MI/R for 6 h, IR6h) promoted myocardial apoptosis and inflammatory response, we hypothesize that Nrf2 promotes the transcription initiation of chemokines in the myocardial tissues of mice treated with IR6h, and then the latter contributes to the infiltration of pro-inflammatory M1 macrophages and the release of inflammatory factors. Therefore, we first investigated the inflammatory cells significantly associated with Nrf2 and Pdcd4 in the early stage of MI/R. Then, we explored the mechanisms by which Nrf2 promotes inflammatory cell infiltration. Finally, we investigated the effect of inflammatory cell infiltration on cardiomyocytes. We present the following article in accordance with the MDAR reporting checklist. ¹

In this study, on the basis of confirming that early MI/R (MI/R for 6 h, IR6h) promoted myocardial apoptosis and inflammatory response, we found that Nrf2, cooperating with Pdcd4, promoted the transcription initiation of Ccl3 in the myocardial tissues of mice treated with IR6h. Moreover, Ccl3 contributed to a high signature score of Ccr1-positive macrophages. The high signature score of the Ccr1-positive macrophages led to the release of pro-inflammatory factors Il-1β and Il-6 ( Figure 5I ).

In the early stages of MI/R, neutrophil-derived cathelicidin aggravates MI/R injury by over-activating TLR4 signaling and the P2X7R/NLRP3 inflammasome in heart-infiltrating neutrophils, which leads to the excessive secretion of IL-1β and subsequent inflammatory injury (27). N,N-dimethylsphingosine (DMS) has been documented to have protective effects against myocardial ischemia-reperfusion injury (IRI) by recruiting CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) via the PI3K/Akt pathway (28). The binding of CD137 on natural killer cells to CD137L on tubular epithelial cells triggers the production of the chemokine (C-X-C motif) receptor 2 ligands CXCL1 and CXCL2, and the subsequent induction of neutrophil recruitment, resulting in a cascade of proinflammatory events during kidney IRI (29). In this study, it’s Pdcd4 rather than Nrf2 that regulates neutrophil infiltration, but the specific regulatory mechanism needs to be further explored in the future. Of course, it cannot be ruled out that the lack of statistical difference in the correlation between Nrf2 and neutrophil infiltration may be due to the small number of samples, which need to be further explored in future studies ( Figures S2 , S3 ). Additionally, the following were observed in myocardial tissues treated with ischemia and reperfusion for 6 h: the expression of some macrophage-related genes was significantly upregulated ( Figure 1A ); the infiltration rate of macrophages was significantly increased ( Figure 2A ); the chemokines and phagocytosis-related signaling pathways were significantly activated ( Figures 1B, E ); and myocardial apoptosis-, inflammation-, and macrophage-related modules were abnormally activated ( Figures 1C, D ). Subsequently, our results suggested that, macrophages were the only inflammatory immune cells associated with both Nrf2 and PDCD4 ( Figures 2A, B ). Moreover, the infiltration rate of M1 macrophages, rather than that of M2 macrophages, was significantly upregulated in the IR6h group, as compared with the control group ( Figures 2C–G ). Eventually, we considered M1 macrophages as inflammatory immune cells associated with both Nrf2 and PDCD4, which was consistent with some previous studies. Fibrinogen-like protein 2 (FGL2) deficiency decreases macrophage infiltration and shifts the macrophage phenotype from a pro-inflammatory (M1) to an anti-inflammatory (M2) pattern in the early stage of coronary microvascular obstruction (29). Conversely, postponed activation of the M2a macrophage subpopulation resulted in a prolonged inflammatory response and adverse myocardial remodeling in CB2-deficient hearts (30).

Ccl2 secretion by epidermal keratinocytes is directly orchestrated by Nrf2, a prominent transcriptional regulator of tissue regeneration that is activated early after cutaneous injury. Epidermal keratinocyte-derived Ccl2 not only drives chemotaxis of macrophages into the wound, but also triggers macrophage expression of EGF, which in turn activates basal epidermal keratinocyte proliferation (31). Nrf2 knockout results in a decrease in Tnfα, Ccl3, and Cxcl2 gene transcription in zymosan-stimulated polymorphonuclear neutrophils, suggesting a role for Nrf2 in the regulation of proinflammatory cytokine production (32). Although our analysis indicated that Ccl2, Ccl3, and Ccl7 may all be potential genes directly transcribed by Nrf2 according to the authoritative JASPAR database ( Figure 3 ), the analysis of available chromatin immunoprecipitation sequencing (ChIP-Seq) dependent on anti-Nrf2 antibody suggests that Ccl3, rather than Ccl2 or Ccl7, was found to directly initiate transcription by Nrf2 ( Figure 4A ).

Interestingly, Nrf2 knockout abolished the neuroprotective effects of SIRT6 overexpression (33). Nrf2 knockout suppressed the amelioration of cardiac function and histological alterations induced by myocardial ischemia-reperfusion injury after hyperbaric oxygen preconditioning, and increased oxidative products and proinflammatory cytokines via the PI3K/Akt pathway (34). Deficiency of Nrf2 suppressed cell proliferation, improved cell apoptosis with an increase in ROS and HO-1, and significantly increased the levels of chemokine 2 (Ccl2), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), angiotensin II receptor type 1 (AT1R), and reactive oxygen species (ROS) in the embryonic tissues, providing theoretical guidance for the application of Nrf2 in the treatment of preeclampsia (35). These findings are consistent with our analysis that Nrf2 silencing significantly promoted functional enrichment of T cell differentiation involved in immune response, apoptotic cell clearance, negative regulation of chemokine production, and regulation of macrophage cytokine production ( Figures 4B–D ).

A growing body of evidence suggests that Nrf2, as the upstream regulator of cytokine production, opposes transcriptional upregulation of proinflammatory cytokine genes and establishes a molecular basis for an Nrf2-mediated anti-inflammatory approach (36). Additionally, its protective role is associated with increased expression of antioxidant genes (ho-1, gclc, and nqo1) and decreased chemokine production (Ccl2, Ccl4, and Ccl11) (37). However, high Nrf2 was found to result in the upregulation of Ccl2, Ccl3, Ccl7 expression in the cardiac muscle tissues treated with IR6h ( Figures 3G–I ). These findings are completely contrary to those reports elaborating on its protective effects. The root of the contradiction with the existing understanding lies in that the perspective of the study may be different. Previous studies have focused on the cardiomyocyte level, while our current study mainly explores the relationship between multi-cells through deconvolution. Besides, a thought-provoking phenomenon was that the expression of Nrf2 was fluctuant, which was up-regulated during ischemia or early reperfusion and down-regulated during mid and late reperfusion.

Recruitment of monocytes into sites of inflammation is essential for immune response; Ccl2 is a major chemokine in the recruitment of monocyte-derived macrophages. Monocytes recruited via Ccl2/Ccr2, rather than Ccl5/Ccr5, propagate inflammation and tissue damage in osteoarthritis, thereby representing a promising therapeutic approach (38). However, Ccl3 also plays an important role in the recruitment of monocyte-derived macrophages. Ccl3 contributes to the formation of CD8⁺ T-cells and macrophage-enriched cardiac inflammation in chronic Trypanosoma cruzi infection; it creates a scenario with abundant IFNγ and TNF associated with cardiomyocyte injury, heart dysfunction, and QTc prolongation, which are biomarkers of Chagas heart disease (39). Pretreatment of wild-type mice with the specific Ccr1 antagonist BX471 also suppressed neutrophil and macrophage infiltration, suggesting a leukocyte source for these inflammatory chemokines and the existence of a Ccr1-dependent positive feedback loop for leukocyte infiltration in the model of renal ischemia-reperfusion injury (40). As expected, given that Nrf2 promoted Ccl3 expression rather than inhibiting it in the cardiac muscle tissues treated with IR6h, it is not surprising that Nrf2 subsequently promoted, rather than inhibited, macrophage infiltration ( Figures 5A, B ), especially the infiltration of Ccr1-positive macrophages ( Figures 5C, D ). Notably, Ccr5 is a receptor for Ccl3. However, since there was no significant difference in the expression of Ccr5 between the IR6h and control groups, we did not consider the infiltration of Ccr5⁺ macrophages, but directly considered the infiltration of Ccr1⁺ macrophages. In the tissue, immune cells trigger inflammation that, when uncontrolled, leads to fibrosis. Among the immune cells, macrophages play a special role in fibrosis formation, as macrophage depletion reflects less collagen deposition (41). Hence, a high score of Ccr1⁺ M1 macrophages resulted in a high expression of Il1b and Il6 ( Figures 5E–H ). In other words, Ccl3-recruited macrophages resulted in the release of inflammatory factors, which is consistent with our previous findings.

Admittedly, there are still some limitations to this study. Above all, we cannot completely deny the possibility that Nrf2 directly orchestrates the transcription of Ccl2 and Ccl7; the reasons are as follows: the samples for CHIP-seq analysis were not samples of mice treated with myocardial ischemia-reperfusion for 6h; CHIP-seq analysis dependent on anti-Nrf2 antibody does not necessarily reflect true and accurate results in the samples of mice treated with IR6h. Secondly, the conclusions of our study need to be further analyzed by flow cytometry, although the logic of our data analysis is quite rigorous. Thirdly, no article related to the data (No. GSE160516) has been published. The presented data did not provide the ischemia-reperfusion protocol, but according to conventional understanding, we can speculate that normal mice were treated with sham surgery, while mice in the IR6h group were subjected to ischemia for 30 mins with ligation of the left anterior descending coronary artery, subsequently subjected to reperfusion for 6 hours. As for the extent of myocardial injury, infarct size, and impaired contractile function, we may have to wait until the relevant data (No. GSE160516) is published to find a detailed explanation in their paper. However, although we only know 6 hours of reperfusion after ischemia and do not know more details, it does not affect the reliability of our results and conclusions, as well as the methods of data analysis. Fourthly, although caspase 8 was activated, caspase 3 and caspase 9 were not activated, the root cause of which may be that the pro-inflammatory macrophages recruited by Nrf2 did not promote myocardial apoptosis through mitochondria or death receptor pathways dependent on caspas3/9, but promoted apoptosis through apoptosis-inducing factor pathways. Fifthly, the root of the contradiction about the proinflammatory role of Nrf2 with the existing understanding lies in the fact that the expression of Nrf2 was fluctuant, which is up-regulated during ischemia or early reperfusion and down-regulated during mid and late reperfusion.

In conclusion, our study found that Nrf2, cooperating with PDCD4, promoted the infiltration of M1 macrophages and release of proinflammatory cytokines Il1b and Il6 by initiating the transcription of Ccl3 in the myocardial tissues of mice treated with IR6h. This study is the first to elucidate the damaging effect of Nrf2 via remodeling of the immune microenvironment during early myocardial ischemia-reperfusion. Our findings may provide new perspectives and treatment strategies for myocardial ischemia-reperfusion. Our results may also be a powerful supplement to those reports previously elaborating the protective effect of Nrf2.

The datasets used during the current study and related scripts of bioinformatics analysis are available from the corresponding author on reasonable request. All data of OEP002796 can be viewed in NODE (http://www.biosino.org/node) by pasting the accession OEP002796 into the text search box or through the URL: http://www.biosino.org/node/project/detail/OEP002796.

This study was reviewed and approved by the Animal Experiment Ethics Review Committee of Nantong University (S20210227-005).

Study concept and design: HZ and JS. Technical and material support: XQC, WW, XY, XHC, and YW. Analysis and interpretation of data, drafting of the manuscript: HZ and YL. Obtained funding and study supervision: HZ and JS. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (81770266); China Postdoctoral Science Foundation (2019M661907); Jiangsu Postdoctoral Science Foundation (2019K159, 2019Z153); General project of Jiangsu Provincial Health Committee (H2019101), Clinical Medical Research Center of Cardiothoracic Diseases in Nantong (HS2019001), Innovation Team of Cardiothoracic Disease in Affiliated Hospital of Nantong University (TECT-A04), and Nantong Key Laboratory of Translational Medicine of Cardiothoracic.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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