Selective Interleukin-6 Trans-Signaling Blockade Is More Effective Than Panantagonism in Reperfused Myocardial Infarction

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SUMMARY
Interleukin (IL)-6 is an emerging therapeutic target in myocardial infarction (MI). IL-6 has 2 distinct signaling pathways: trans-signaling, which mediates inflammation, and classic signaling, which also has antiinflammatory effects. The novel recombinant fusion protein sgp130Fc achieves exclusive trans-signaling blockade, whereas anti-IL-6 antibodies (Abs) result in panantagonism. In a rat model of reperfused MI, sgp130Fc, but not anti-IL-6-Ab, attenuated neutrophil and macrophage infiltration into the myocardium, reduced infarct size, and preserved cardiac function 28 days after MI. These data demonstrate the efficacy of exclusive IL-6 trans-signaling blockade and support further investigation of sgp130Fc as a potential novel response (3)(4)(5) and contributes directly to neutrophilmediated myocyte necrosis (6). Third, its signaling can lead to excessive fibrosis and adverse remodeling, culminating in reduced cardiac function (7,8).
These and other data have prompted 2 clinical trials of IL-6 antagonism in MI. In the first, a single dose of the human anti-IL-6 receptor (IL-6R) monoclonal antibody (Ab) tocilizumab (TCZ) was administered to 117 patients presenting with non-ST-segment-elevation MI (NSTEMI), resulting in reduced C-reactive protein and troponin-T (Trop-T) (9). Crucially, these reductions and antiinflammatory effects of TCZ on the proteome (10) were seen only in the patients that underwent percutaneous coronary intervention.
Therefore, targeting IL-6 is likely to be more effective in the context of reperfusion.
The follow-up ASSAIL-MI study (11) was performed in 199 patients with STEMI undergoing percutaneous coronary intervention (12). Preliminary data from the study have recently been presented showing that TCZ improved myocardial salvage index (12).
However, despite the promising results from these studies, it is unclear if targeting IL-6R is the best approach to modulating IL-6 in MI, given the complexities of its signaling and pleiotropic nature. IL-6 has 2 distinct signaling pathways termed classic and trans. Classic signaling is achieved by IL-6 binding to its membrane-bound a-receptor, which associates with a dimer of the receptor b-subunit glycoprotein 130 (gp130) to initiate intracellular signaling (13) (Supplemental Figure 1A). Classic signaling stimulates only cells that express membrane-bound IL-6R, including hepatocytes and leukocytes (13). Tissues may also respond to IL-6 via trans-signaling (Supplemental Figure 1B). This is achieved by IL-6 binding a circulating soluble form of the receptor (sIL-6R), and then the IL-6/sIL-6R complex subsequently binds to a dimer of membrane-bound gp130 (13). Because gp130 is ubiquitously expressed, this pathway enables IL-6 to stimulate all tissues regardless of their expression of membrane-bound IL-6R.
Because it has no affinity for free IL-6, it is an exclusive trans-signaling inhibitor (16) (Supplemental Figure 1D) and therefore leaves the anti-inflammatory effects of IL-6 classic signaling intact. sgp130Fc has been shown to reduce atherosclerosis in mice (17), but it has never been investigated in a model of MI.
In this paper, we present data demonstrating that sgp130Fc has greater anti-inflammatory effects than an anti-IL-6 antibody and is more effective in   Minnesota) was administered at a dose of 0.5 mg/g as previously described (17). Rabbit polyclonal anti-IL-6-Ab (AF506; R&D Systems) was administered at 0.1 mg/g. Because the intention was to provide a single bolus dose at reperfusion, this dose is 5Â the previously reported daily intraperitoneal (IP) dose (20).
However, because this antibody had not previously been administered in this manner, plasma measurements were taken to guide dosing (Supplemental Methods, Supplemental Figure 3). Based on these, a second dose of 0.1 mg/g was administered IP in 0.5 mL PBS 3 days after MI to ensure pan-IL-6 antagonism for at least 7 days after MI (total dose per rat 0.2 mg/g).
PBS, rather than isotype controls, were used in the control group, after the relevant isotype controls were shown to be inactive in vitro (Supplemental Figure 4) and to avoid including additional groups of rats.

INFARCT SIZE MEASUREMENT. Infarct size and area at risk (AAR) in excised hearts was measured with
Evans Blue and 2,3,5-triphenyl-tetrazolium-chloride dyes as previously described (21). Staining was per- production ( Figure 1B). The addition of sIL-6R did not significantly affect the expression of either ( Figure 1B). The cells shed sIL-6R, which was not influenced by IL-6 stimulation ( Figure 1C).
Of a series of antibodies tested (Supplemental Table 3), all but 1 failed to antagonize IL-6 (Supplemental Figure 5). This was the goat polyclonal anti-IL-6 antibody AF506, which antagonized IL-6 in a concentration-dependent manner ( Figure 1D).
Therefore, AF506 (subsequently referred to as anti-IL-6-Ab) was used as an effective pan-IL-6 inhibitor.
George et al. There was a significant increase in numbers of 2 macrophage populations, major histocompatibility complex (MHC) II pos and MHC-II neg , 3 days after MI, corresponding with the second peak in IL-6. This was most dramatic in the MHC-II neg subset, which increased w20-fold from baseline ( Figure 2D). This subset also had the highest surface CD11b expression ( Figure 2E) and intracellular IL-6 production (Supplemental Figure 7).
These data informed the design of the subsequent experiments. First, they highlight 4 h, 1 day, and 3 days after MI as key time points in the inflammatory response that may be modulated by targeting IL-6.
Second, for therapeutic experiments, they provided a rationale for targeting IL-6 at reperfusion and for at least 7 days thereafter. The design of these experiments is outlined in Supplemental Table 4.
Relative to vehicle control, total mononuclear phagocyte ( Figure 3E) and neutrophil ( Figure 3G) counts were both reduced in the sgp130Fc group, and lymphocytes ( Figure 3F) were reduced in both drugs groups 1 day after MI ( Figure 3F). Crucially, at this time point the number of classic monocytes was significantly higher in the anti-IL-6-Ab group compared with the sgp130Fc group ( Figure 3H).
Neither drug had an effect on nonclassical monocytes ( Figure 3I). Similarly, 3 days after MI, the number of highly inflammatory MHC-II neg macrophages was significantly reduced only in the sgp130Fc group ( Figure 3J). In blood, neither drug altered leukocyte numbers (Supplemental Figure 8G).
Interestingly, only anti-IL-6-Ab had a significant effect on leukocyte CD11b expression, suppressing it on neutrophils (1 day) ( Figure 3G) and both macrophage subtypes (3 days) ( Figures 3J and 3K), suggesting that this effect is classic signaling dependent. LGE  rise has been described in humans (23). Circulating levels returned to baseline 1 day after MI, whereas in humans the levels may be raised for several days (24,25). The second peak at 3 days, which was restricted to the myocardium, appears to be largely due to the massive influx of mononuclear phagocytes, which stained strongly for IL-6. This was preceded by a rapid rise in sIL-6R concentration 1 day after MI, coinciding with peak neutrophil numbers which shed it to drive trans-signaling (13).

DIFFERENTIAL EFFECTS OF anti-IL-6-AB AND
sgp130Fc ON IL-6 SIGNALING. We used anti-IL-6-Ab to achieve panantagonism and sgp130Fc for exclusive trans-signaling blockade. The in vitro RCAEC data demonstrated that anti-IL-6-Ab is able to bind and neutralize IL-6, which prevents both classic and trans-signaling, as has been extensively described (13,15). In contrast, sgp130Fc had no effect on free IL-6 in vitro, only blocking its action in the presence of sIL-6R and is therefore an exclusive trans-signaling antagonist. In vivo, only anti-IL-6-Ab, not sgp130Fc, reduced IL-6 concentration, providing direct evidence of their different effects on classic signaling.
Although it is not possible to provide similar direct in vivo evidence for equipotent effects on transsignaling, both reduced the trans-signaling-dependent chemokine CCL2 (4) to a similar degree, which is strongly suggestive that this was indeed the case.
Thus, the two drugs achieved the intended differential effects on IL-6 signaling.

THE ROLE OF IL-6 CLASSIC AND TRANS-SIGNALING
AFTER REPERFUSED MI. There were reductions in total mononuclear phagocytes and neutrophils 1 day after MI in those that received sgp130Fc, suggesting that this was a trans-signaling-dependent effect. There was no corresponding reduction in a specific chemokine to account for this observation. However, blockade of IL-6 transsignaling reduces expression of endothelial adhesion molecules such as ICAM-1, which has been shown to attenuate cell trafficking (3,5). In addition, the reduced inflammatory cell numbers may also be due to increased apoptosis, given the increase in c-caspase-3 expression in nonmyocytes 4 h after MI. This is in keeping with data showing that IL-6 signaling has antiapoptotic effects on leukocytes (26).
Interestingly, 4 h after MI, anti-IL-6-Ab, but not sgp130Fc, increased the plasma concentration of CXCL1, suggesting that IL-6 classic signaling negatively regulates this chemokine, as previously described (27). CXCL1 drives monocyte trafficking (28)  In summary, we demonstrated that proinflammatory effects of IL-6 after MI are trans-signaling dependent, whereas classic signaling has antiinflammatory effects. Therefore, specific targeting of IL-6 trans-signaling had more pronounced antiinflammatory effects than pan-blockade (the proposed mechanism is shown in Supplemental Figure 9).

FIGURE 3 Continued
Rats were subjected to surgical myocardial infarction with 50 min of ischemia before reperfusion. One minute before reperfusion, vehicle (comparing sham with vehicle controls; gray asterisks and daggers). Where ANOVA was significant but differences between vehicle and the drug groups were not, a post hoc unpaired Student's t-test was performed between the two drug groups (red asterisk). Multiplicity-adjusted (asterisks) and ANOVA (daggers) p < 0.05 was considered to be significant. * †p<0.05; ** † †p<0.01; *** † † †p<0.001. Ab ¼ antibody; MHC ¼ major histocompatibility complex; Mo ¼ monocytes; other abbreviations as in Figures 1 and 2.  Furthermore, results of ASSAIL-MI suggest improved myocardial salvage index with the use of TCZ after STEMI (12). Therefore, there may be interspecies variability in the effect of pan-IL-6 blockade in reperfused MI. One such difference may be the effect on neutrophils. In the NSTEMI study, a significant reduction in circulating neutrophil count was observed in the TCZ-treated group, which was proposed to mediate the beneficial effects of the drug (25). However, in the present study we did not observe a reduction in circulating neutrophil count in either drug group.
Although myocardial salvage index was reportedly improved by TCZ in the ASSAIL-MI trial, infarct size, Trop-T, N-terminal pro-B-type natriuretic peptide, and LV end-diastolic volume at 6 months were not (12). Therefore, while TCZ shows some promise in In the characterization studies, we did not observe a rise in myocardial lymphocytes after MI, which are increasingly recognized to play an important role in myocardial injury and remodeling (33). However, our flow cytometry panel was not designed to identify individual lymphocyte subsets and therefore we can not exclude the possibility that there was an increase in specific cell types, such as CD4 T cells.
Finally, while we have demonstrated that sgp130Fc reduces infarct size and suggest that this is due to its anti-inflammatory effects, further work is required to discern the precise mechanisms which underpin this observation.

CONCLUSIONS
We demonstrated for the first time that exclusive IL-6 trans-signaling antagonism with sgp130Fc is more effective than pan-blockade in reperfused MI. This approach attenuated inflammation, reduced infarct size, and preserved cardiac function. Given the pressing need to reduce the incidence of heart failure after MI, sgp130Fc warrants further investigation as a potential novel therapeutic in this setting.