PCSK9 Inhibitor Induces a Transient Decrease in Neutrophil-Lymphocyte Ratio and Monocyte-Lymphocyte Ratio in Homozygous/Compound Heterozygous Familial Hypercholesterolemia Patients


 Background: Homozygous/compound heterozygous familial hypercholesterolemia (HoFH/cHeFH) is characterized by extremely elevated low-density lipoprotein-cholesterol (LDL-C) levels that have been reported to contribute to a long-term chronic systemic inflammation. The aims of this study are to describe the inflammatory profile of HoFH/cHeFH patients and explore the effect of PCSK9 inhibitor (PCSK9i) on a series of inflammatory biomarkers, neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), monocyte-HDL ratio (MHR), monocyte-lymphocyte ratio (MLR) and mean platelet volume-lymphocyte ratio (MPVLR). Methods: In this prospective cohort study, 21 definitive HoFH/cHeFH on high-intensity statins plus ezetimibe were placed on subcutaneous injections of PCSK9i 450mg every 4 weeks (Q4W). The biochemical parameters and inflammatory profile were analyzed at the day before PCSK9i therapy, 3 months and 6 months after PCSK9i therapy.Results: We found that HoFH/cHeFH on maximum tolerated statin dose plus ezetimibe displayed an elevated lipid and disturbed blood biomarker profile. After 3 months of add-on PCSK9i therapy, a significant reduction of LDL-C was observed. Meanwhile, percentage and count of neutrophils, monocyte counts, MPV, as well as two inflammatory biomarkers, NLR and MLR were reduced. However, at 6-month PCSK9i treatment, NLR and MLR returned to pre-PCSK9i treatment levels.Conclusions: PCSK9i induces a transient decrease in NLR and MLR in HoFH/cHeFH patients. Our results add evidence in evaluating the effects of PCSK9i on systemic inflammation.


Background
Familial hypercholesterolemia (FH) is an autosomal dominant hereditary disease that is characterized by elevated levels of total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels 1 , premature atherosclerosis (AS) and cardiovascular diseases (CVD). The most frequent reported gene mutated in FH is LDL-receptor (LDLR), accounting for 85-90% of reported cases. In addition, mutation in apolipoprotein B (APOB), proprotein convertase subtilisin/kexin-9 (PCSK9), low-density lipoprotein receptor adaptor protein 1(LDLRAP1) have also been described in causing FH 2 . In the general population, the prevalence of heterozygous FH (HeFH) patients who carry a mutation in one of the alleles is about 1 in 300 3,4 , while homozygous/compound heterozygous FH (HoFH/cHeFH) patients in which both of their alleles harbor mutations may affect 1 in 160,000-300,000 individuals 5 , and usually have higher LDL-C levels and poorer clinical prognosis than HeFH 5 .
Of note, in addition to causing abnormally high blood lipid levels, hypercholesterolemia induces chronic systemic and vascular in ammation 6,7,8,9 . A number of immunocytes and blood components such as monocytes 10 , macrophages 11 , dendritic cells 12 , lymphocytes 13 , neutrophils 14 , platelets 15 and complement system 16 , have been reported to contribute to the pro-in ammatory environment in AS, promoting the development of atherogenesis, plaque destabilization and plaque erosion. A series of blood cellular component-related parameters, such as neutrophil-lymphocyte ratio (NLR) 17,18 , plateletlymphocyte ratio (PLR) 19,20 , monocyte-HDL ratio (MHR) 21,22 , monocyte-lymphocyte ratio (MLR) 23,24 and MPV-lymphocyte ratio (MPVLR) 25 have been used to illustrate systemic in ammation status and evaluate the risk of future CVD events. The treatment of hypercholesterolemia patients should focus not only on lipid lowering but also on reducing chronic in ammation 26,27 .
As the rst-line pharmacological treatment for dyslipidemia, statins can signi cantly reduce the level of LDL-C and the risk of cardiovascular events 28 . However, the majority of FH patients 29 , especially HoFH patients 30 , could not achieve optimal LDL-C reduction even with the maximum tolerated doses of statins, and remain a high CVD risk. Recently, several large randomized clinical trials have shown that the addition of PCSK9 inhibitors (PCSK9i) to statins lead to a further reduction in LDL-C and cardiovascular risk 31,32 , even in HoFH/cHeFH patients that were characterized by high LDL-C levels and CVD risk 33,34 . In addition, PCSK9i showed anti-in ammatory and immunomodulatory effects in FH patients. Roberto et al. 35 found that in HeFH patients, six-month add-on PCSK9i signi cantly reduced LDL-C levels, neutrophils count and in ammatory marker MHR, while NLR was not altered. However, HoFH/cHeFH and HeFH usually respond differently to PCSK9i, and no data exist regarding the effects of PCSK9i on systemic in ammation in HoFH/cHeFH patients exclusively.
In the present study, we rstly described the systemic in ammation pro le of HoFH/cHeFH patients, and then evaluated the effects of PCSK9i on these systemic in ammatory biomarkers at 3 months and 6 months after PCSK9i treatment.

Study design and participants
This study protocol was reviewed and approved by the ethics committees of Beijing Anzhen Hospital, Capital Medical University. All subjects voluntarily participated in the study and signed informed consent, and cooperated with the medical staff to complete the follow-up.
Eligible participants were HoFH/cHeFH patients diagnosed by genetic testing (two alleles both carry mutation in the region of LDLR, APOB, PCSK9 or LDLRAP1). The pathogenic genes were detected by the second-generation sequencing technique. Eligibility criteria were age between 12-75 years old; bodyweight ≥ 40kg; fasting triglyceride (TC) ≤ 4.5 mmol/L; and fasting LDL-C ≥ 3.4 mmol/L after at least 4 weeks of a stable high intensity statins plus ezetimibe therapies. Exclusion criteria included uncontrolled cardiac arrhythmias, myocardial infarction, unstable angina, percutaneous coronary intervention, coronary artery bypass grafting, stroke, deep vein thrombosis, pulmonary embolism (< 3 months prior to study start), systolic blood pressure > 180mmHg and/or diastolic blood pressure > 110 mmHg, a con rmed or potential ineffectiveness of PCSK9i and had received other PCSK9i and cholesteryl ester transfer protein inhibitors (> 6 months prior to study start).
From May 2019 to August 2021, 62 probable HoFH/cHeFH patients were enrolled from Beijing Anzhen Hospital, Capital Medical University. All participants had received stable maximum statin therapy with ezetimibe at least 4 weeks (that is, atorvastatin 40mg/d or rosuvastatin 20mg/d, ezetimibe 10mg/d) before they started to add on PCSK9i 450mg administered subcutaneously every 4 weeks (Q4w). Biochemical analyses were performed at the day before PCSK9i administration (T0), 1 month (T1), 2 months (T2), 3 months (T3) and 6 months (T6) after the start of PCSK9i administration. At T3, the lipidlowering effects of PCSK9i were evaluated. If they did not attain LDL-target, the follow-up would be terminated at T3. LDL-target was de ned by the reduction of mean level of LDL-C at T1, T2 and T3 > 5% compared to T0. Others continued to receive follow-up visits.

Statistical analysis
The continuous data are expressed as mean ± standard deviation (SD), and categorical data as frequency (percentage). SPSS software version 25.0 (SPSS, Inc., Chicago, IL) are used for statistical analyses. Normality distribution was determined by Kolmogorov-Smirnov test. For continuous variables that satisfy normal distribution, independent two-sample t-tests or paired t-test were used; otherwise, the Mann-Whitney U test was used. Categorical data were compared by Chi-square test. For all analyses, Pvalues < 0.05 were considered statistically signi cant.

Results
In our study, we evaluated 62 probable HoFH/cHeFH patients. Of these, 21 de nitive patients that received stable maximum statins plus ezetimibe therapy at least one month started to add on PCSK9i 450mg Q4W and were followed up once a month until T6. Of these, two patients withdrew at T1; two patients withdrew at T2; four patients did not meet LDL-target and the follow-up was terminated at T3; and three patients withdrew at T4 (Figure 1). Meanwhile, 47 healthy donors (HD) were recruited as controls.

Baseline Characteristics Of The Participants Before Pcsk9i Therapy
The baseline characteristics of the participants before PCSK9i therapy were summarized in Table 1. Among 21 patients enrolled in our study, ve patients had homozygous mutations (four in LDLR gene and one in LDLRAP1 gene), and 16 patients had compound heterozygous mutations (15 patients harbored two LDLR mutation sites; one patient had two mutation sites in LDLR gene, one mutation site in APOB gene). Compared to HD, HoFH/cHeFH patients had dramatically higher levels of TC and LDL-C. Of note, HoFH/cHeFH patients displayed a disturbed blood biomarker pro le. Among eight platelets and white blood cell (WBC) parameters, seven were signi cantly different between HoFH/cHeFH patients and HD, including increased MPV, neutrophil counts and percentages, decreased lymphocytes counts and percentages, monocyte percentages, as well as platelets counts in HoFH/cHeFH patients. The monocyte counts were comparable between HoFH/cHeFH patients and HD. The systemic in ammatory biomarkers NLR, MHR, MLR and MPVLR that derived from above parameters was signi cantly higher in HoFH/cHeH patients (76.22%, 117.78%, 27.27%, 30.22% higher than in HD, respectively).
Effects of PCSK9i therapy on in ammatory biomarkers in HoFH/cHeFH patients After 3 months of add-on PCSK9i therapy, the levels of TC and LDL-C were signi cantly reduced by 14.15% and 21.14% (from 11.17±3.32 mmol/L to 9.59±3.56 mmol/L and from 9.65±3.24 mmol/L to 7.61±3.25 mmol/L, respectively). Meanwhile, four of seven above disturbed platelets and WBC parameters statistically recovered, including MPV, percentage and count of neutrophils, percentage of lymphocytes. Monocyte count signi cantly decreased after 3-month PCSK9i therapy. Furthermore, PCSK9i therapy reduced two in ammatory biomarkers NLR and MLR (-25.43% and -23.33%, respectively) ( Table 2).
We further analyzed NLR and MLR of ten patients who completed 6 months therapy. We found that the LDL levels at T6 were lower than the levels before PCSK9i treatment. However, NLR and MLR at T6 returned to the levels of T0. Meanwhile, cell counts of monocytes and lymphocytes at T6 were statistically comparable to the levels before PCSK9i treatment (Figure 2). Discussion Being exposed to high LDL levels since birth, FH patients have high-risk of premature AS and CVD. Since AS and CVD are associated with hypercholesterolemia-induced in ammation, anti-in ammatory effects of lipid lowering drugs should receive great attention 36 . In general, the lipid-lowering effect of statins differs between HeFH and HoFH/cHeFH 37 . Around 20% HeFH can meet LDL-target after the use of statins 38 , while LDL levels in most HoFH patients merely attained around 10 mmol/L even with highest doses of most e cacious statins 5 . In the present study, we enrolled 21 HoFH/cHeFH patients with maximum tolerated statin dose plus ezetimibe. These patients had higher LDL-C levels than HD (9.70±3.62 mmol/L vs 1.36±0.30 mmol/L), and displayed a hyper-in ammatory state, as indicated by an abnormal blood biomarker pro le that related to neutrophils, monocytes, lymphocytes and platelets.
Previous studies have shown that combination therapy of PCSK9i and statins can further reduce LDL-C by 45.7% 39 and 23.1% 33 for HeFH and HoFH/cHeFH patients, respectively. Consistently, we found a signi cant reduction in LDL-C (from 9.65±3.24 mmol/L to 7.61±3.25 mmol/L) after adding-on threemonth PCSK9i. Although the levels of LDL-C at this time-point (T3) did not achieve LDL target recommended by ECS guidelines 28 , the blood biomarker pro le was partially recovered. This nding is consistent with previous studies that revealed the relation between PCSK9 and chronic in ammation 40,41 .
To the best of our knowledge, our study rst reported the effects of PCSK9i on systemic in ammatory status in HoFH/cHeFH patients exclusively.
It should be emphasized that NLR and MLR were signi cantly reduced after three-month add-on PCSK9i therapy. Since lymphocyte counts were comparable during this period, the reduction of NLR and MLR should be attributed to a signi cant decrease in neutrophil counts (from 7.27±3.94 to 4.54±1.32 ×10 9 /L) and monocyte counts (from 0.76±0.50 to 0.47±0.14 ×10 9 /L ). Although further investigations are still needed, several studies have provided clues that PCSK9i alters systemic in ammation levels through regulating neutrophils and monocytes. Clinical trials found that the concentration of PCSK9 in serum was positively correlated with neutrophils and lymphocytes numbers in CAD patients 42 . Bernelot et al. 43 reported that monocytes pro-in ammatory phenotypes of FH were reversed after 24-week PCSK9i treatment, as shown by the decreased monocytes migration capacity and in ammatory responses. The role of monocytes and macrophages has been extensively studied in AS. There are growing evidence suggesting that neutrophils also contribute to cardiovascular in ammation and development of atherosclerotic plaques 44,45 . For example, neutrophils stimulate the activation and dysregulation of the endothelial cell through secreting reactive oxygen species (ROS) 46 and myeloperoxidase (MPO) 47 . MPO also mediate oxidation of LDL, promoting the formation of foam cells.
Unfortunately, we noticed that at 6-month after PCSK9i treatment, NLR and MLR returned to pre-PCSK9i treatment levels. It would be of great interest to investigate why PCSK9i induces a transient decrease in NLR and MLR and whether HoFH/cHeFH patients with reduction of NLR or MLR might have more potential to gain bene t from PCSK9i therapy. Our present study was limited by the case number. In-depth investigation regarding changes in neutrophils function should be conducted in the further studies.

Conclusions
In HoFH/cHeFH patients, PCSK9i induces a transient decrease in systemic in ammatory biomarker NLR and MLR. We notice that NLR and MLR are reduced after 3-month PCSK9i therapy, while returned to the baseline levels after 6-month PCSK9i therapy. These transient changes are mostly attributed to the changes of neutrophil and monocytes counts. Our results add evidence in evaluating the effects of PCSK9i on systemic in ammation. This study protocol was reviewed and approved by the ethics committees of Beijing Anzhen Hospital, Capital Medical University. The consent was signed by all the participants prior to their enrollment in the study.

Consent for publication
Not applicable.

Availability of data and materials
All data generated and analyzed in this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.  Study population owchart