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PCSK9 monoclonal antibodies reverse the pro-inflammatory profile of monocytes in familial hypercholesterolaemia
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Download the PDF here
Download the PDF here
Eur Heart J May 1 2017
Sophie J. Bernelot Moens1 , Annette E. Neele2 , Jeffrey Kroon1, Fleur M. van der Valk1, Jan Van den Bossche2, Marten A. Hoeksema2,
Renate M. Hoogeveen1, Johan G. Schnitzler3, Marie T. Baccara-Dinet4, Garen Manvelian5, Menno P.J. de Winther2,6* , and Erik S.G. Stroes1*
1Vascular Medicine, Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands; 2Experimental Vascular Biology, Medical Biochemistry, Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands; 3Experimental Vascular Medicine, Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands; 4Sanofi, Clinical Development, R&D, 371 Rue du Professeur Blayac, 34080, Montpellier, France; 5Regeneron Pharmaceuticals, 777 Old Saw Mill River Rd, Tarrytown, NY 10591, USA; and 6Institute for Cardiovascular Prevention (IPEK), Pettenkoferstraβe 8a & 9, 80336 Munich, Germany
Aims
Migration of monocytes into the arterial wall contributes to arterial inflammation and atherosclerosis progression. Since elevated low-density lipoprotein cholesterol (LDL-C) levels have been associated with activation of plasma monocytes, intensive LDL-C lowering may reverse these pro-inflammatory changes. Using proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibodies (mAbs) which selectively reduce LDL-C, we studied the impact of LDL-C lowering on monocyte phenotype and function in patients with familial hypercholesterolaemia (FH) not using statins due to statin-associated muscle symptoms.
Methods and results
We assessed monocyte phenotype and function using flow cytometry and a trans-endothelial migration assay in FH patients (n = 22: LDL 6.8 ± 1.9 mmol/L) and healthy controls (n = 18, LDL 2.9 ± 0.8 mmol/L). Monocyte chemokine receptor (CCR) 2 expression was approximaterly three-fold higher in FH patients compared with controls. C-C chemokine receptor type 2 (CCR2) expression correlated significantly with plasma LDL-C levels (r = 0.709) and was positively associated with intracellular lipid accumulation. Monocytes from FH patients also displayed enhanced migratory capacity ex vivo. After 24 weeks of PCSK9 mAb treatment (n = 17), plasma LDL-C was reduced by 49%, which coincided with reduced intracellular lipid accumulation and reduced CCR2 expression. Functional relevance was substantiated by the reversal of enhanced migratory capacity of monocytes following PCSK9 mAb therapy.
Conclusions
Monocytes of FH patients have a pro-inflammatory phenotype, which is dampened by LDL-C lowering by PCSK9 mAb therapy. LDL-C lowering was paralleled by reduced intracellular lipid accumulation, suggesting that LDL-C lowering itself is associated with anti-inflammatory effects on circulating monocytes.
Whether the reduction in immune cell activity following PCSK9 mAbs will translate into decreased inflammatory activity in atherosclerotic lesions is currently being addressed in the ANITSCHKOW study (NCT02729025).
it should be taken into account that besides potentially beneficial effects, lowering of inflammatory responses may also increase vulnerability for infectious diseases. Since CV disease is widely recognized as a pro-inflammatory state, the use of anti-inflammatory strategies is, however, considered to be pre-dominantly beneficial, underscored by ongoing large outcome studies using anti-inflammatory agents (Cantos, NCT01327846, CIRT, NCT01594333).
In conclusion, we show that lipid lowering with PCSK9 mAbs reduces the pro-inflammatory phenotype of monocytes without affecting CRP, implying that potent lowering of LDL-C has an anti-inflammatory impact in hypercholesterolemic patients. These data highlight that inflammation is not solely captured by CRP measurement and warrant future investigations to assess the role of attenuating cellular inflammation in patients at high CV-risk.
PCSK9 monoclonal antibodies reduce TNF and enhance IL-10 production
Upon entry into the arterial wall, production of (pro-inflammatory) cytokines contributes to plaque destabilization. To assess whether PCSK9 mAbs affected the inflammatory responsiveness of monocytes we measured cytokine production following LPS stimulation in PCSK9 mAb treated subjects (see Supplementary material online, Table S6). Production of TNF was reduced (baseline: 896 ± 593 pg/mL, post-PCSK9 mAbs: 471 ± 272 pg/mL, P < 0.01), whereas secretion of the anti-inflammatory IL-10 was enhanced (baseline: 950 ± 624 pg/mL, post-PCSK9 mAbs: 2097 ± 772 pg/mL, P < 0.001). Cytokine levels of monocytes from PCSK9 mAb treated patients were comparable to those observed in stable statin users (Figure 3I and J).
Introduction
The cardiovascular (CV) benefit conveyed by statins is attributed to low-density lipoprotein cholesterol (LDL-C) lowering1,2 as well as to anti-inflammatory effects,3,4 evidenced by an independent linear relation between CV-benefit and both LDL-C as well as C-reactive protein (CRP) lowering in statin trials.4 Mendelian randomization studies, however, have not substantiated a causal role of CRP in atherogenesis.5 Subsequently, focus shifted towards immune cells which are considered causal players in atherogenesis and plaque progression.6,7 Monocytes infiltrate atherosclerotic lesions,8 where monocyte-derived macrophages contribute to a local pro-inflammatory milieu.9 Distinct monocyte subsets, based on CD14 and CD16 expression, are distinguished, displaying differential surface expression of membrane receptors and concomitant variable migratory behavior.7 The receptor for monocyte chemo-attractant protein 1 (MCP-1), C-C chemokine receptor type 2 (CCR2), is of particular importance in the recruitment of monocytes to the arterial wall10 and its expression is increased in patients with hypercholesterolaemia.11,12
Since statins decrease both LDL-C and CRP,13 it is difficult to disentangle the impact of lipid lowering from inflammatory changes. Several proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibodies (mAbs) are now available, which increase hepatic LDL-receptor (LDLR) expression following antibody-mediated scavenging of the free PCSK9 protein.14 PCSK9 mAbs injected subcutaneously once every 2-4 weeks provide a consistent and potent LDL-C reduction of 60%15-17 with only minimal changes in other lipoprotein fractions, such as high-density lipoprotein (HDL)-cholesterol and triglycerides (TAGs). In contrast to statin therapy, the marked reduction in LDL-C upon PCSK9 mAb administration is not accompanied by a CRP reduction,18 implying the absence of an anti-inflammatory effect by this class of therapeutic agents. However, the impact of PCSK9 mAbs on other key inflammatory mediators, including monocytes, has not been reported.
Here, we assessed the impact of elevated LDL-C levels on monocyte phenotype and function in patients with familial hypercholesterolaemia. (FH) not using statins due to statin-associated muscle symptoms (SAMS) versus normolipidemic control subjects. Subsequently, we assessed the impact of PCSK9 mAbs in FH patients on monocytes, compared with a reference group of FH patients on stable statin dose for at least 6 months. Overall, these studies demonstrate that selective lowering of LDL-C with PCSK9 mAbs reduces the pro-inflammatory profile of circulating monocytes.
Patient selection
This single-center study comprised 22 patients with definite or probable FH19 not receiving statin therapy due to SAMS and 18 age- and gender-matched healthy controls. In 17 patients with FH who started PCSK9 mAb treatment, we assessed the effect of LDL-C lowering following 24 weeks of PCSK9 mAb administration. Patients received either Alirocumab (n = 10; 150 mg every 4 weeks or 75 mg every 2; see Supplementary material online, methods) or Evolocumab (n = 7; 140 mg every 2 weeks). For comparison of treatment effects, we also included age and gender matched FH patients (n = 14) with stable statin treatment (>24 weeks). Exclusion criteria for both patients and controls included recent CV-events in the past 12 months, infection or diabetes. Subjects visited the hospital after an overnight fast for physical examination, medical history recording and blood withdrawal.
The study protocol was approved by the institutional review board of the Academic Medical Center in Amsterdam, the Netherlands, and written informed consent was obtained from each participant.
Discussion
We show that monocytes from patients with FH not using statins due to SAMS, display pro-inflammatory and migratory changes, which coincide with an increase in cytoplasmic lipid droplets. The latter implies a direct relation between intracellular lipid accumulation and inflammatory changes in monocytes (Figure4). Following 24 weeks of treatment with a PCSK9 mAb monocyte migratory capacity, lipid content, as well as inflammatory responsiveness decreased towards levels observed in FH patients on stable statin use.
These data imply an LDL-C mediated pro-inflammatory effect on circulating monocytes in patients with FH, which reverses upon LDL-C lowering by PCSK9 mAbs. A potential explanation for these observations is the marked accumulation of lipids in circulating monocytes of FH patients. In contrast to previous findings in macrophages showing that PCSK9 modulates macrophage LDL-C uptake by altering LDL-C receptor expression in macrophages,21,22 LDLR expression was virtually absent in circulating monocytes in the present study. This data implies that the anti-inflammatory effect observed in monocytes following PCSK9 mAbs is predominantly an indirect effect mediated via lowering of plasma LDL-C. In addition, our findings indicate that the LDL receptor pathway does not play an important role in monocyte lipid accumulation. This is corroborated by a previous report by Mosig et al.23 showing accumulation of lipids in monocytes of patients with homozygous FH, characterized by absence of functional LDL receptors. Potential other pathways contributing to monocyte lipid uptake comprise scavenger receptors. In support, the increased lipid content in FH patients coincided with higher surface expression of CD36 on classical monocytes, and SR-A on intermediate monocytes. As expected, lipid accumulation also resulted in increased expression of efflux mediators (ABCA1 and ABCG1).24 Nonetheless, the increase in intracellular lipid content in FH subjects supports that this accumulation is dominated by the increased levels of LDL-C in the plasma.
The increased lipid content in monocytes coincided with elevated CCR2 expression in FH patients. Previous in vitro data support the uptake of native LDL in monocytes eliciting increased CCR2 expression and monocyte chemotaxis.11 The strong correlation between intracellular lipid accumulation and CCR2 expression observed in monocytes of FH patients implies a causal relation between cytoplasmatic lipid increase and pro-inflammatory changes. The functional relevance of these changes is substantiated by the marked increase in the ex vivo endothelial migration rate of monocytes obtained from FH patients. PCSK9 mAbs reduced plasma LDL-C levels by 49% and also reduced the intracellular lipid content in circulating monocytes. In conjunction, monocyte CCR2 expression was reduced to levels comparable to those observed in FH patients using statins, whereas statin therapy has been previously shown to prospectively lower monocyte CCR2 expression.25 In parallel, migratory capacity of monocytes as well as the response of circulating monocytes to an inflammatory challenge was also attenuated following PCSK9 mAbs, illustrated by decreased TNF release with increased secretion of the anti-inflammatory cytokine IL-10. These data imply that decreased intracellular lipid accumulation leads to an attenuated inflammatory activity of the monocytes, which is independent of the mechanism by which LDL-C reduction is established.
Interestingly, the anti-inflammatory changes in monocyte phenotype and responsiveness following PCSK9 mAbs were not accompanied by a decrease in plasma CRP levels. The reduction in CRP following statin therapy4,26 reflects a direct effect of statins in hepatocytes,27 which is not present following PCSK9 mAb administration.18 Whereas the clinical relevance of the absence of a CRP change remains to be established, recent studies have revealed that CRP is not a mediator but merely a marker for CVD risk.5,28 In parallel, the reduction in arterial inflammation following statin therapy is not correlated to statin-induced changes in CRP.29,30 Whether the reduction in immune cell activity following PCSK9 mAbs will translate into decreased inflammatory activity in atherosclerotic lesions is currently being addressed in the ANITSCHKOW study (NCT02729025).
Limitations
Several limitations merit attention. First, this was a proof-of-concept study using mAbs against PCSK9 to lower LDL-C without a placebo arm. Since the wide array of laboratory tests in the present study consistently show an anti-inflammatory effect of PCSK9 mAbs on monocytes in conjunction with lowering of both plasma LDL-C and intracellular lipid content in monocytes, this study does support an anti-inflammatory effect of potent LDL-C lowering in patients with elevated LDL-C levels not receiving statins. Future randomized, placebo-controlled trials are recommended to further elaborate on these findings.
Secondly, the interpretation of the findings is limited by the lack of a statin intervention comparison group, which is first-line therapy in FH. To show the independent effect of PCSK9 mAbs, we included FH patients with intolerable SAMS to at least three different statins,31 which precludes adding an additional statin intervention group. Therefore, we reverted to the inclusion of FH patients on stable statin use. These subjects had a more favourable CVD risk profile compared with PCSK9 mAb treated subjects, with a lower BMI. Nonetheless, the similarity of monocyte inflammatory status as well as LDL-C levels in PCSK9 mAb and statin users implies that LDL-C lowering per se is the pre-dominant actor in the pro-inflammatory effects, independent from the pathway by which LDL-C is reduced.
Third, although murine data is consistent, the role of CCR2 expression on monocytes in predicting CVD risk has not been established prospectively in patients.10 Moreover, final evaluations on the role of PCSK9 lowering on plaque regression (GLAGOV, NCT01813422) and definitive end point studies (ODYSSEY Outcomes NCT01663403, FOURIER NCT01764633), awaited in 2017 and 2018), are paramount to assess functional relevance of the present mechanistical insights.
Finally, it should be taken into account that besides potentially beneficial effects, lowering of inflammatory responses may also increase vulnerability for infectious diseases. Since CV disease is widely recognized as a pro-inflammatory state, the use of anti-inflammatory strategies is, however, considered to be pre-dominantly beneficial, underscored by ongoing large outcome studies using anti-inflammatory agents (Cantos, NCT01327846, CIRT, NCT01594333).
In conclusion, we show that lipid lowering with PCSK9 mAbs reduces the pro-inflammatory phenotype of monocytes without affecting CRP, implying that potent lowering of LDL-C has an anti-inflammatory impact in hypercholesterolemic patients. These data highlight that inflammation is not solely captured by CRP measurement and warrant future investigations to assess the role of attenuating cellular inflammation in patients at high CV-risk.
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