Proprotein convertase subtilisin/kexin type 9 regulates the production of acute‐phase reactants from the liver

Proprotein convertase subtilisin/kexin type 9 (PCSK9) controls blood cholesterol levels by fostering the LDL receptor (LDLR) degradation in hepatocytes. Additionally, PCSK9 has been suggested to participate in immunoregulation by modulating cytokine production. We studied the immunological role of PCSK9 in Streptococcus pneumoniae bacteraemia in vivo and in a human hepatocyte cell line.


| INTRODUC TI ON
Proprotein convertase subtilisin/kexins (PCSKs) are serine endoproteases that enzymatically process and convert biologically inactive proproteins into functional end-products. PCSKs are important not only in maintaining homeostasis in the body, but also in a number of pathological conditions, such as malignancies and inflammatory disorders. 1,2 In contrast to the other PCSK enzymes, PCSK9 has a fundamental nonenzymatic role in cholesterol homeostasis by targeting the LDL receptor (LDLR) for lysosomal degradation. 3,4 Consequently, loss-of-function mutations of PCSK9 are associated with decreased plasma LDLcholesterol levels and protection against coronary heart disease (CHD), 5 whereas gain-of-function PCSK9 variants have been reported to cause hypercholesterolemia. 6 Collectively, mechanistic and genetic insights into the PCSK9-mediated regulation of hepatic clearance of blood LDLcholesterol have paved the way for the existing anti-PCSK9 antibodies and small interfering RNAs (siRNAs) as therapeutics to prevent CHD. [7][8][9] In addition to its undisputable significance in lipid metabolism, PCSK9 has recently been suggested to affect the pathogenesis of dengue virus infection 10 as well as to regulate the production of inflammatory cytokines. In fact, although tomographic analysis in patients receiving PCSK9 inhibitors has suggested that the arterial inflammation is not affected by the PCSK9 levels, 11 experimental evidence from mice argues that PCSK9 can increase the magnitude of the pro-inflammatory response in macrophages, and subsequently exacerbate foam cell formation and atherosclerosis. 12,13 Moreover, the PCSK9-controlled production of inflammatory cytokines, such as tumour necrosis factor (TNF) and interleukin 6 (IL6), leads to a dysregulated systemic inflammatory response in septic infections. 14,15 Importantly, since lipoprotein particles, particularly VLDL, LDL, and HDL, are able to bind and neutralize pathogen-associated lipids such as lipopolysaccharide (LPS) and lipoteichoic acid from the circulation, 14,16 the inhibition of PCSK9 has potentially a dual beneficial effect in treating septic patients. Accordingly, improved survival has been reported in septic patients with loss-offunction mutations in PCSK9 as well as in situations where the levels of PCSK9 in the plasma are lowered. 14,15,17 In contrast, recent clinical data have suggested that neither loss-nor gain-of-function variants of PCSK9 are associated with morbidity, 18 and that low PCSK9 levels in bacteraemia patients are associated with increased mortality. [19][20][21] All in all, the contradictory clinical data necessitates additional research into the role of PCSK9 in systemic bacterial infections.
We have previously shown that a Streptococcus pneumoniae infection can model the pathogenesis of streptococcal bacteraemia in both zebrafish (Danio rerio) larvae and in adult fish. 22,23 Although the larvae can be specifically used to study the innate immunity against bacteria, the adult zebrafish model enables systemic and organ-specific studies of the immune response as a whole. 24,25 Furthermore, the zebrafish CRISPR/Cas9 mutagenesis system enables targeted modification of the fish genome to create gene knockout (KO) animals. 26 In this study, we used CRISPR/Cas9 to create two pcsk9 KO zebrafish lines (pcsk9 tpu-13 and pcsk9 tpu-2,+15 ) and determined whether pcsk9 is critical for zebrafish survival in a systemic S pneumoniae infection. Additionally, we used RNA profiling in the Conclusions: We demonstrate that PCSK9 is not critical for zebrafish survival in a systemic pneumococcal infection. However, PCSK9 deficiency was associated with the lower expression of APR genes in zebrafish and altered the expression of innate immunity genes in a human hepatocyte cell line. Overall, our data suggest an evolutionarily conserved function for PCSK9 in APR in the liver.

K E Y W O R D S
innate immunity, Pneumococcus, sepsis, zebrafish

Lay summary
We deleted the proprotein convertase pcsk9 gene in zebrafish and demonstrated that the lack of pcsk9 does not cause developmental defects or impact the survival from pneumococcal infection. However, gene expression studies indicated that deleting PCSK9 is associated with lower production of the acute-phase response genes from the liver. Our findings unravel a novel immunoregulatory function for the proprotein convertase PCSK9 in the liver.

Key points
• Deleting pcsk9 in zebrafish using CRISPR/Cas9 mutagenesis does not cause developmental defects or morphological abnormalities.
• Pcsk9 is dispensable for zebrafish survival in a systemic S. pneumoniae infection.
• Pcsk9 KO is associated with reduced expression of acute-phase reaction genes in pneumococcus infected zebrafish.
• PCSK9 siRNA alters the expression of innate immunity genes in a human hepatocellular carcinoma cell line.
in vivo zebrafish pneumococcal model and in human HepG2 cells to evaluate the immunoregulatory function of PCSK9 at the molecular level in the liver.

| Zebrafish use and ethics statement
The zebrafish maintenance and the experiments followed the Gemma Micro 500 (Skretting, Stavanger, Norway). S pneumoniae infected adults were kept in a separate flow through system (Aqua Schwarz GMbH, Göttingen, Germany) or conductivity-(800 µs) and pH-(7.6) adjusted water was manually changed twice a day and the tanks kept at 28.5℃ for the duration of the experiment. An identical light/dark cycle compared with the unchallenged fish was used and the fish were fed once a day with Gemma Micro 500 (Skretting).
Adult zebrafish infected with pneumococcus were monitored at least two times a day to follow the humane endpoint criteria as defined in the permit for animal experiments.

| RNA isolation and quantitative PCR
Adult zebrafish tissues or HepG2 cells were homogenized and the re- The target gene expression was normalized to either the eukaryotic translation elongation factor 1 alpha 1, like 1 (eef1a1l1 or ef1a, zebrafish data) expression 32 or to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, HepG2 data) using the 2^(-ΔCt) method. PCR contamination as well as the specificity of the qPCR products were monitored using non-template (H 2 O) as well as no-reverse transcriptase controls and performing a melt curve analysis and 1.5% agarose TAE gel electrophoresis. qPCR primer sequences are depicted in Table S1.

| RNA sequencing
The RNA quality was controlled with the Fragment Analyzer system

| Cell culture and transfections
The human HCC cell line HepG2 was cultured in RPMI 1640 (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum, 2 mM l-glutamine and 6 mM of both penicillin and streptomycin at 37℃ and 5% CO 2 . ON-TARGETplus Non-targeting Control Pool (Dharmacon, Colorado, USA) or ON-TARGETplus Human PCSK9 small interfering RNA (siRNA)-SMART pool (GE Dharmacon) was transfected at a final concentration of 6 nM using Transfection Reagent 4 (GE Dharmacon).

| Statistical analysis
Sample sizes are based on our previous calculations and experimental observations. 23,33 Statistical analyses were performed with the Prism program (v. 5.02, GraphPad Software, Inc, California, USA). In zebrafish survival experiments a log-rank (Mantel-Cox) test was used to compare differences between the experimental groups, whereas a nonparametric two-tailed Mann-Whitney was used to analyse the zebrafish qPCR data. A 2-tailed t test was used in the analysis of HepG2 qPCR results. Statistics of the RNA sequencing have been described in the Supplementary Methods. P-values (or the adjusted P-value in RNA sequencing data) less than .05 was considered statistically significant.

| RE SULTS
3.1 | Nonsense pcsk9 tpu-13/tpu-13 and pcsk9 tpu-2,+15/ tpu-2,+15 mutant zebrafish have diminished pcsk9 expression and are morphologically comparable with WT fish The engineered type II CRISPR/Cas system known as CRISPR/Cas9 is an efficient tool for reverse genetics, 27 Our previous work has demonstrated that CRISPR/Cas9mediated indel mutations not only affect the protein translation, causing inability to produce intact protein, but can also lead to changes in the transcription of the target gene. 26 In line with this, qPCR quantification of pcsk9 expression in adult steady-state zebrafish revealed significantly lower pcsk9 mRNA levels in homozygous pcsk9 tpu-13/tpu-13 and pcsk9 tpu-2,+15/tpu-2,+15 mutants (residual expression medians of 15.9% and 8.0%; P = .008 in both comparisons) compared with their WT siblings ( Figure 1D). Importantly, although earlier knock-down studies using morpholinos in zebrafish have indicated a crucial role for pcsk9 in neural development, 35 pcsk9 KO zebrafish did not have any apparent developmental abnormalities during the first 7 dpf ( Figure S1). Furthermore, as adults, pcsk9 tpu-13/ tpu-13 and pcsk9 tpu-2,+15/tpu-2,+15 mutants were morphologically similar to the WT fish ( Figure 1E).
The host defense against pneumococcus is critically dependent on the innate immune response. 22 Consequently, we next used zebrafish larvae to specifically address if PCSK9 is important for the protective innate immunity in a pneumococcal infection. To this end, we microinjected S pneumoniae (240 CFU; SD 76 CFU) into the blood circulation valley of the F2-generation progeny of heterozygous pcsk9 tpu-13/+ and pcsk9 tpu-2,+15/+ zebrafish at 2 dpf and followed their survival for 5 days. At the end of the experiment, an average of 25.7% (pcsk9 tpu-13 background) and 77.7% (pcsk9 tpu-2,+15 background) survival was observed ( Figure 2C and 2D). More specifically, 20.8% of the pcsk9 tpu-13/tpu-13 , 39.6% of the pcsk9 tpu-13/+ and 16.7% of the WT (pcsk9 tpu-13 background) fish ( Figure 2C) had survived, whereas survival fractions of 58.8% in the pcsk9 tpu-2,+15/ tpu-2,+15 , 87.5% in the pcsk9 tpu-2,+15/+ and 86.7% in the WT (pcsk9 tpu-2,+15 ) fish were seen ( Figure 2D). Similarly to our experiments in adult zebrafish, no statistically significant differences in the survival rates of the pcsk9 KO and WT larvae were observed (P = .57 and P = .078 in pcsk9 tpu-13 and pcsk9 tpu-2,+15 lines respectively), although the difference between pcsk9 KO and heterozygous pcsk9 tpu-2,+15/+ larvae was significant (P = .038). Overall, we conclude that pcsk9 is F I G U R E 1 Homozygous pcsk9 tpu-13/tpu-13 and pcsk9 tpu-2,+15/tpu-2,+15 mutant zebrafish show reduced pcsk9 expression but normal morphology. A, A schematic representation of zebrafish pcsk9 (ENSDARG00000074185), and the gRNA target site for CRISPR/Cas9 mutagenesis in the third exon. B, The in vivo mutagenesis efficiency was estimated in the gRNA and Cas9 protein injected F0-generation embryos and in the uninjected controls using a T7 endonuclease I (T7EI) assay and 2.5% agarose TAE gel electrophoresis. The uninjected controls: a 169-bp WT PCR product (lanes 8 and 9); the gRNA and Cas9-injected mutant embryos: three bands of 169 bp (WT), ~100 bp and ~70 bp (lanes 1-7). The mutagenesis efficiency was calculated as described previously. 27 C, A schematic representation of the indel mutations (−13 bp deletion, pcsk9 tpu-13 and −2 bp deletion, +15 bp insertion, pcsk9 tpu-2,+15 ), leading to truncated protein products of 173 and 159 amino acids (aa) respectively. Red boxes indicate altered amino acids caused by the frameshift. D, The expression of pcsk9 was determined in the brain of pcsk9 tpu-13/tpu-13 (n = 5; 2 females, 3 males) and pcsk9 tpu-2,+15/tpu-2,+15 zebrafish (n = 5; 2 females, 3 males) and in the WT siblings (n = 5 in both control groups; 2 females, 3 males) with qPCR. Gene expression levels were normalized to eef1a1l1 and a 2-tailed Mann-Whitney test was used for statistics. E, Anesthetized WT and homozygous pcsk9 mutant zebrafish were imaged using a Canon EOS 7D Mark II camera with an exposure time of 17 ms. Fish were kept submerged in water during image acquisition. Images in (B) and (E) were cropped to exclude empty background from the figure not critical for the adult or larvae zebrafish survival on a S pneumoniae infection.

| Expression of pcsk9 is upregulated on a S pneumoniae infection, and it is required for a normal acute-phase response
Previously, it has been shown that hepatic PCSK9 expression is increased in LPS-induced inflammation, 37 and that plasma PCSK9 levels are upregulated in a liver-dependent manner in bacteraemia patients. 21 To directly test whether hepatic PCSK9 is upregulated in a pneumococcal infection, we injected adult WT zebrafish (AB line) with PBS or S pneumoniae (635 000 CFU; SD 276 000 CFU) into the abdominal cavity and quantified the expression of pcsk9 in the liver at 7 dpi. Our qPCR analysis revealed a 4.5-fold increase (P = .008) in the relative levels of pcsk9 mRNA in the pneumococcus infected zebrafish ( Figure 3A), suggesting that the inflammation-mediated inducibility of hepatic PCSK9 is evolutionarily conserved.
To gain a genome-wide perspective on the PCSK9-dependent liver-specific transcriptional response in a S pneumoniae infection, we performed RNA sequencing on the livers of unchallenged, PBS injected and S pneumoniae infected (1 dpi, 3 370 000 CFU; SD 840 000 CFU) adult pcsk9 tpu-13/tpu-13 zebrafish and their WT siblings.
To decipher the immunoregulatory functions of PCSK9 at a molecular level in vivo on infection, we next focused on the 112 upand 77 down-regulated protein coding genes in the S pneumoniae challenged pcsk9 tpu-13/tpu-13 mutants compared with the WT controls. To this end, gene ontology (GO) enrichment analysis of the induced transcripts in the pcsk9 KO zebrafish, revealed a total of 15 enriched processes that were related to hormonal responses, such F I G U R E 3 pcsk9 is up-regulated on a pneumococcal infection and associated with the expression of acute-phase reactants. A, The relative expression of pcsk9 was determined in the liver of PBS injected (n = 5; all males) and S pneumoniae infected (635 000 CFU; SD 276 000 CFU, n = 5; all males) WT AB zebrafish at 7 dpi using qPCR. Gene expression levels were normalized to eef1a1l1 expression and target genes were run once as technical duplicates. A 2-tailed Mann-Whitney test was used for statistics. B-C, RNA was isolated from the liver of unchallenged (n = 2 in both groups; 2 females/group), PBS injected (n = 3 in both groups; 3 females/ group) and S pneumoniae infected (3 370 000 CFU; SD 840 000 CFU, n = 3 in both groups; 3 females/group) adult pcsk9 tpu-13/tpu-13 and WT (pcsk9 tpu-13 ) zebrafish at 1 dpi and the transcriptome was analysed using RNA sequencing. B, Venn diagram for the differentially expressed genes within treatment groups. C, The top 25 up-and down-regulated genes in S pneumoniae infected pcsk9 tpu-13/ tpu-13 zebrafish in comparison with the WT controls are depicted using a heat-map. Statistics were done using DESeq2 and adjusted using the Benjamini-Hochberg (BH) method. D, The relative expression of selected genes up-or down-regulated in the RNA sequencing data (ldlrap1a, hamp and socs3a) was quantified using qPCR. Gene expression levels were normalized to eef1a1l1 expression and target genes were run once as technical duplicates as the cellular response to an estrogen stimulus (GO:0071391), but also to a lipid metabolism, e.g., the response to lipids (GO:0033993) and lipid transport (GO:0006869) ( Table 1). Among the up-regulated genes in pcsk9 KO fish, we found stearoyl-CoA desaturase (scd, log 2fold change 5.1, P < .001), estrogen receptor 1 (esr1, log 2-fold change 3.5, P < .001) as well as several vitellogenin (eg vtg3, log 2-fold change 2.3, P = .010) genes ( Figure 3C, Table S5). It is additionally important to note that the PCSK9 promoter region contains estrogen response elements (EREs), 38 which can in principle explain the effects on esr1 expression in the pcsk9 tpu-13/tpu-13 mutants. Interestingly, among the genes induced in pcsk9 tpu-13/tpu-13 zebrafish we also observed a 2.5fold induction (log 2 change, P = .049) in the low-density lipoprotein receptor adaptor protein 1a (ldlrap1a) gene, encoding a homologue of the human mediator of LDLR endocytosis. 39 Importantly, the GO-analysis of the down-regulated genes showed that from a total of six processes two were directly related to the immune system; the immune system process (GO:0002376) and the humoral immune response (GO:0006959) ( Table 2). In fact, among the 78 genes whose expression levels were reduced in the

| PCSK9 regulates the expression of innate immunity genes in human HepG2 cells
Suggesting a direct regulatory function for PCSK9 in the immune response in the absence of a microbial insult, in vitro administration of recombinant PCSK9 into a mouse and human macrophage culture was shown to induce the expression of genes coding for proinflammatory cytokines such as TNF, IL6 and IL1B. 13 To address whether PCSK9 controls the expression of the above identified innate immunity genes also in human hepatocytes, we knocked- In mammals, PCSK9 is expressed not only in the liver but also in, e.g., the pancreas and brain. 40,41 Our previous analysis of genes of the pcsk family in adult zebrafish tissues revealed high relative pcsk9 expression in the fish brain, 42 which is in accordance with an earlier report, where pcsk9 was inhibited with morpholinos and a critical role for PCSK9 in neural tissues and early fish development were described. 35 By creating targeted nonsense mutations, we have demonstrated that the CRISPR/Cas9 mutagenesis method can be efficiently used to knock out genes of interest in zebrafish. 26,29 Accordingly, we created 2 pcsk9 KO zebrafish lines; pcsk9 tpu-13 and pcsk9 tpu-2,+15 , with disrupted open reading frames (residual expression medians of the mutated mRNAs in the brain; 15.9% and 8.0%, respectively). However, in contrast with previously published data demonstrating the absence of tectum and midbrain-hindbrain boundary in the pcsk9 morphants at 24 hours post fertilization, 35 homozygous pcsk9 tpu-13/tpu-13 and pcsk9 tpu-2,+15/tpu-2,+15 mutants did Sepsis is a clinical syndrome caused by the interplay of microorganisms and an exacerbated immune reaction leading to multiple organ failure. 46 Although mammalian sepsis models remain highly important for pre-clinical drug development, they are laborious, expensive and raise ethical concerns. Zebrafish is a non-mammalian alternative for studying host-pathogen interactions in vivo. As it takes several weeks for lymphocytes and the adaptive immune system to develop in zebrafish, the fish larvae can be used to specifically study the innate immunity. 24 Importantly, adult zebrafish have a highly similar immune system compared with humans with both innate immune cells, lymphocytes 24 as well as humoral components like complement components. 47 To assess how the KO of pcsk9 affects zebrafish mortality on a pneumococcal challenge, we infected both zebrafish larvae and adult fish with S pneumoniae and followed their survival until 5 or 7 dpi respectively. Similarly to studies using LPSinduced endotoxemia in Pcsk9 KO mice, 36 the mortality of pneumococcus infected homozygous pcsk9 tpu-13/tpu-13 and pcsk9 tpu-2,+15/ tpu-2,+15 mutants was comparable with the WT controls in both the larvae and adult zebrafish. Overall, we conclude that PCSK9 is not essential for zebrafish survival in a systemic S pneumoniae infection.
More than 70% of the human genes have zebrafish orthologues, 48 and consequently gene KO zebrafish accompanied with genomewide transcriptome analysis can be used to model both systemic and tissue-specific functions of human genes. 25,26 Here, our genome-wide transcriptomic analysis of pneumococcus infected pcsk9 tpu-13/tpu-13 mutants and WT fish revealed a substantial proportion ( 54 It is important to note that conventional PCSK enzymes FURIN, PCSK5, PCSK6 and PCSK7 have been demonstrated to directly process pro-hepcidin, 55 whereas FURIN has been reported to process PCSK9. 56 It remains to be confirmed whether there is a direct causal relationship between lowered HAMP levels and PCSK9 or if this is also affected by the other PCSK family members. Collectively, our F I G U R E 4 Silencing PCSK9 influences the expression of genes of the innate immune response in HepG2 cells. The relative expression levels of PCSK9, HAMP, C7, SOCS3, TNF, TNFAIP3, C6 and LDLRAP1 were determined in control (n = 3) and PCSK9 siRNA (n = 3) transfected HepG2 cells using qPCR. Gene expression levels were normalized to GAPDH expression and target genes were run once as technical duplicates. A 2-tailed t test was used for statistics gene expression analyses unravel that the lack of pcsk9/PCSK9 transcriptionally impacts APR, arguing for an immunostimulatory role for PCSK9 on a bacterial challenge and independent of infection.
The dual role of PCSK9 in regulating hepatocytic lipoprotein uptake 3,4 and the inflammatory response 13,14 has made this proprotein convertase an attractive candidate for studying sepsis.
Although some data indicate that normal PCSK9 levels are associated with a better prognosis, 20,21 and that this effect could be age dependent, 19 other studies support the idea that blocking PCSK9 activity could improve the outcome of treatment in septic patients. 14,15,17 In fact, the PCSK9 inhibiting antibodies alirocumab (Praluent, Sanofi-Regeneron) and evolocumab (Repatha, Amgen) are being tested in clinical trials to treat septic patients (NCT03634293 and NCT03869073 respectively). Although we did not see any difference in the survival of S pneumoniae infected pcsk9 KO and WT zebrafish, our transcriptomic data show that PCSK9 regulates the production of acute-phase reactants such as hepcidin and complement components. Although the direct role of LDLR-signalling remains to be investigated, inhibition of PCSK9 may cause immunodeficiency by dampening APR. Further studies are clearly warranted to decipher the role of PCSK9 in S pneumoniae host responses in tissue-specific infections such as pneumonia as well as in the context of infections in which an optimal liver immune response is necessary.

ACK N OWLED G EM ENTS
The authors thank the Tampere Zebrafish Core

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
RNA sequencing data has been submitted to Gene Expression Omnibus (GEO) repository (identifier code: GSE16 5508). Other generated and analyzed data are available on reasonable request from the corresponding author.