Innate Lymphoid Cells Promote Recovery of Ventricular Function After Myocardial Infarction

Background Innate lymphoid cells type 2 (ILC2s) play critical homeostatic functions in peripheral tissues. ILC2s reside in perivascular niches and limit atherosclerosis development. Objectives ILC2s also reside in the pericardium but their role in postischemic injury is unknown. Methods We examined the role of ILC2 in a mouse model of myocardial infarction (MI), and compared mice with or without genetic deletion of ILC2. We determined infarct size using histology and heart function using echocardiography. We assessed cardiac ILC2 using flow cytometry and RNA sequencing. Based on these data, we devised a therapeutic strategy to activate ILC2 in mice with acute MI, using exogenous interleukin (IL)-2. We also assessed the ability of low-dose IL-2 to activate ILC2 in a double-blind randomized clinical trial of patients with acute coronary syndromes (ACS). Results We found that ILC2 levels were increased in pericardial adipose tissue after experimental MI, and genetic ablation of ILC2 impeded the recovery of heart function. RNA sequencing revealed distinct transcript signatures in ILC2, and pointed to IL-2 axis as a major upstream regulator. Treatment of T-cell–deficient mice with IL-2 (to activate ILC2) significantly improved the recovery of heart function post-MI. Administration of low-dose IL-2 to patients with ACS led to activation of circulating ILC2, with significant increase in circulating IL-5, a prototypic ILC2-derived cytokine. Conclusions ILC2s promote cardiac healing and improve the recovery of heart function after MI in mice. Activation of ILC2 using low-dose IL-2 could be a novel therapeutic strategy to promote a reparative response after MI.

T he mechanisms that lead to heart failure post-myocardial infarction (MI) are initiated very early after ischemic injury, triggering waves of immune responses that play critical roles in tissue healing and remodeling (1). None of the current therapies aimed at improving cardiac remodeling directly target the immune system around the time of ischemic injury (2). Therefore, important advances in therapeutic management could be expected from a better understanding and early targeting of the post-ischemic immune response.
During the initial inflammatory phase after ischemic injury, the rapid infiltration of neutrophils to the infarct area begins the process of clearing the necrotic tissue. This is aided by Ly6C hi monocytes recruited through secretion of chemokines such as CCL2 and CCL7, which in turn potentially differentiate into activated macrophages. The macrophages are further conditioned during the subsequent regulatory phase (3), shifting the balance from proinflammatory classical activation toward the alternative activation phenotype, which is more conducive for tissue repair (4)(5)(6)(7). Circulating CD4 þ T cells infiltrate the wounded heart early on and their presence helps to slew the macrophage phenotype toward the alternative activation phenotype (8). During the resolution phase, the activation of regulatory T cells (Tregs) is beneficial, reducing effector T-cell activation, and promoting alternative macrophage activation and tissue repair (9,10). Activation of these cells is primarily through the cytokines IL-25 and IL-33, further supported by lymphocyte-derived IL-2 (11). Although ILC2s are present in secondary lymphoid tissues, they are 10fold more prevalent in barrier tissues (12,13). In our previous work, we found that the adventitia of arteries and fat-associated lymphoid clusters in perivascular adipose tissue contain ILC2s, which expand in response to atherosclerosis (14). Further work (15) showed that adventitial stromal cells provide a supportive niche in these tissues, supplying them with IL-33 and thymic stromal lymphopoietin. Perivascular   (14) and quantified using Fiji (21) as % total area.

FLOW
CYTOMETRY. Single-cell suspension for spleen, mesenteric lymph nodes, and pericardial and peri-gonadal white adipose tissue was generated (14).
Cells were washed in phosphate-buffered saline (PBS) 1% fetal calf serum before labeling with desired antibody cocktails (Supplemental Table 1). Intracellular antigens were detected using the IC Fix/perm kit    Figure 2B).
In the PcAT, the ILC2 population expanded 2-fold on D3 before returning to presurgery levels ( Figure 1A) while heart resident ILC2s were largely unchanged.
During this period, ST2 expression within the PcAT compartment increased stepwise with the expansion of ILC2s, before regressing to its initial expression level by D7 ( Figure 1A). Intracellular staining for proliferation marker Ki67 in PcAT-resident ILC2s showed that there was a 3-fold increase in the proliferating population at the D3 time point ( Figure 1B), suggesting that the increase in cell number at this time point was due in part to local proliferation.
In the context of MI, it is possible that release of IL- Cd127 cre mice was sufficient to increase atherosclerotic burden during high-fat feeding (14). Using these mice in the LAD ligation MI model, we observed a trend toward a difference in mortality between ILC2 replete (ILC2 WT ) and ILC2 knockout (ILC2 KO ) cohorts   Flow cytometric analysis detects the expansion of ILC2 3 days post-MI (A) in heart (top) and pericardial adipose tissue accompanied with elevated ST2 surface expression (bottom), and increased proliferation (Ki67) (B). ILC2 proliferation in the PcAT was inhibited by injections of sST2, less pronounced in the heart (C).

Representative images shown. Mann-Whitney
Continued on the next page Yu et al.    Figure 6A). Complementing this, there    Figures 5B and 5C), a pattern also observed in PcAT and in the heart ( Figure 5C). Proportions of splenic macrophages were unchanged; however, the expression of the Type II marker Arginase 1 (Arg1) was decreased. Conversely, within the PcAT macrophage, Arg1 expression was elevated (coinciding with more CD206 in this case, Supplemental Figure 6B) slewing the population toward a wound-healing phenotype.
Macrophages were also detected within the heart tissue in greater abundance, and this was accompanied by a greater proportion of them expressing Arg1 ( Figures 5B and 5C).
Assessing the impact of these phenotypic changes on long-term MI outcome, Rag2 À/À mice received  Figures 7E and 7F), further supporting an IL-2-driven activation of circulating ILC2s.

DISCUSSION
Here, we have shown that ILC2s have a direct and protective role in recovery from experimental MI.
ILC2s were detected in the heart and pericardial adipose tissue in steady state and expanded during the initial inflammatory phase of MI, trafficking from the blood under some conditions. The expansion within the inflamed tissues is rapid but not sustained and falls away within the first week of recovery. This complements earlier observations that there is a cardiac resident population of ILC2s that are present in the heart during periods of pericarditis and MI (16), and we show that cardiac remodeling and wound stability is severely compromised in their absence. As we have seen in other models of ILC2 response to tissue damage (eg, atherosclerosis), the types of wounds that develop in the absence of ILC2s tend to be larger, have less collagen deposition, and are overall more fragile than ILC2-replete mice (14); as a consequence, the trend toward higher mortality rate seen in ILC2 KO mice during experimental MI was due to cardiac rupture.    Figure 1.    The results observed during the direct targeting of ILC2 with IL-2/Jes6-1 complexes were promising, not only preventing a marked decrease in cardiac function during the initial phase but also improving recovery over the subsequent 4 weeks.
Data from our LILACS human clinical trial provide translational relevance of our preclinical findings. We demonstrate that low-dose IL-2 activates ILC2s in patients presenting with ACS, with increases in IL-5 and eosinophil levels as a consequence of ILC2 activation (34). Interestingly, recent data in mice have shown that eosinophils may play a significant role in MI, and their deficiency promoted adverse cardiac remodeling (35). H u m a n c l i n i c a l r e s u l t s . Our data show increased serum IL-5 and blood eosinophil counts after lowdose IL-2, in association with signs of ILC2 activation; however, the potential protective role of such changes will require further investigation.

CONCLUSIONS
We have presented data that highlight a critical role for ILC2 during the response to MI. Expanding early to condition the immune response, their absence increases the accumulation of inflammatory monocytes and macrophages and severely impacts cardiac function after MI. We have shown that expansion of ILC2 with exogenous IL-2 is beneficial, driving heart In this report, we demonstrate that type 2 innate lymphoid cells (ILC2s) are resident in the heart and pericardium, expanding during experimental myocardial infarction.
In mouse models deficient in ILC2s, cardiac recovery and repair are significantly retarded; the recovering scar contains less collagen, fewer T regulatory cells (Tregs) and more inflammatory monocyte/macrophage populations. Supplementing the mice with interleukin (IL)-2 increases ILC2 activation and promotes the secretion of cardioprotective cytokines (eg, IL-5) and ultimately improves recovery. We observe a similar phenotypic shift in ILC2s in patients with acute coronary syndromes after administration of low-dose IL-2, coupled with increased IL-5 secretion and associated eosinophilia.
Yu et al.