Lipids, lipid-lowering drug and sepsis: a Mendelian randomization study

Background: To investigate the causal role of lipid in sepsis and determine the effect of lipid-lowering interventions on the disease. Methods: Two-sample Mendelian randomization analyses were conducted to evaluate the associations of high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, apolipoprotein B and apolipoprotein A-I levels with risks for sepsis, and those of low-density lipoprotein cholesterol (HMGCR, PCSK9, NPC1L1), triglycerides (LPL, ANGPTL3, APOC3) and high-density lipoprotein cholesterol (CETP), apolipoprotein A-I (CETP), apolipoprotein B (HMGCR, PCSK9, NPC1L1, LPL, APOC3) with sepsis. Results: HMGCR-mediated low-density lipoprotein cholesterol and apolipoprotein B were associated with an increased risk of sepsis, with an odds ratio value of 1.4 (95% confidence interval (CI): 1.06–1.84, P = 0.017) and 1.41 (95% CI: 1.01–1.98, P = 0.046). CETP-mediated high-density lipoprotein cholesterol and apolipoprotein A-I were associated with a reduced risk of sepsis, with an odds ratio of 0.87 (95% CI: 0.82–0.92, P < 0.01) respectively and 0.84 (95% CI: 0.78–0.9, P < 0.01). Sensitivity analysis showed that the results were robust. Conclusion : HMG-CoA reductase inhibitors and CETP inhibitors may contribute to the prevention and treatment of sepsis.


Introduction
Sepsis ranks as a prime cause of mortality among critically ill global patients, exhibiting an incidence rate of 1-2% in all hospitalized cases.This condition also imposes the most significant financial burden among all diseases due to its high expense of treatment [1].Despite considerable progression in medical technologies, including anti-infection treatments, the incidence rate of sepsis persistently escalates.Furthermore, sepsis patients face profound challenges encompassing physical, psychological, and cognitive impediments [2,3].In 2012, the Global Sepsis Alliance assigned September 13 as World Sepsis Day to enhance public consciousness about this perilous disease.Hence, it is of pressing necessity to find efficient methods for the prevention, diagnosis, and treatment of sepsis to preserve human health.A marked alteration in lipid levels among sepsis patients is linked to organ failure or mortality [4,5].
During sepsis, levels of total cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) decrease [6].Observational clinical studies have reported that low levels of HDL-C are adverse prognostic factors in sepsis patients [7,8].A study investigating hemostatic changes in septic rats, initiated by lipopolysaccharide and resulting in hepatic microvascular thrombosis, discovered that simvastatin potentially exhibits an antithrombotic impact on hepatic endothelial cells [9].With the advent of a series of new lipid-regulating drugs, it is necessary to evaluate the effects of these drugs on sepsis.
Observational studies may exhibit contradictory results due to confounding variables, reverse causality, and survival bias.Overcoming such limitations can be achieved through the application of Mendelian randomization (MR) analysis [10].An individual's random genetic makeup may influence the presence of risk factor gene mutations at birth.If genetic variation is unconnected to confounding variables, any variation within the findings can be ascribed to differences in risk factors.For an effective MR analysis, the instrumental variable should comply with three conditions: (1) it exhibits a close relationship with exposure; (2) it maintains independence from all known confounding variables, and (3) influencing the outcome solely through risk factors [11].Multivariate MR extends the capabilities of MR by enabling the estimation of each exposure's direct causal effect on outcomes.This approach is commonly applied in lipid MR analyses [12].Moreover, MR can generate invaluable data for pharmaceutical studies, including foretelling efficacy and unveiling target-mediated adverse reactions-an approach known as drug target MR.This methodology can display the effect of altering biomarkers through specific treatment targets on long-term health outcomes [13].This research employs drug-targeted MR techniques to investigate the genetic prediction of lipid-regulating activity concerning common lipid-regulating targets and its relation to sepsis risk.The objective is to pinpoint potential drug targets effective for sepsis prevention.

Study design
The research design necessitates the fulfillment of three key assumptions, as illustrated in Figure 1. Figure 2 demonstrates the schematic design of the study.We executed two MR analyses.Firstly, we carried out an MR analysis regarding the overall influence of diverse lipids or apolipoprotein traits (including apolipoprotein A-I (ApoA-I), apolipoprotein B (ApoB), HDL-C, LDL-C, and triglycerides) on the outcome.Secondly, we evaluated the effect of lipid-lowering medications on the outcome via drug-targeted MR.This study utilized data exclusively drawn from research that had secured pertinent participant consent and ethical approval.As such, the need for additional ethical approval from the Institutional Review Board was obviated.

Data source
The GWAS data for sepsis utilized in this research originates from the UK Biobank, encompassing 10,154 sepsis cases alongside 454,764 controls [14].Sepsis is classified as cases explicitly referenced as sepsis in the Ninth or Tenth Revision of the International Classification of Diseases [15].This research utilizes GWAS data of lipids and apolipoproteins from the UK Biobank.The genetic associations were derived for each trait measured among European participants (n = 393,193-441,016) [16].

Instrumental variables
In the MR analysis, we initially identified single nucleotide polymorphisms (SNPs) demonstrating significant association with each trait on a genome-wide level (P < 5e-08).Further, we ensured these were independent by eliminating linkage disequilibrium (r 2 = 0.001, kb = 10000) [17].In the drug-targeted MR analysis, we opted for SNPs showcasing a close relationship with lipid traits (P < 5e-08).These chosen SNPs needed to be situated within ± 100 kb of a particular drug target gene region (r 2 < 0.3, effect allele frequencies > 0.01).To circumvent the bias of a weak instrumental variable, SNPs possessing an F-value exceeding 10 were selected [18][19][20][21][22].We selected instrumental variables for drug targets from the GWAS summary data of LDL-C (HMGCR, PCSK9, NPC1L1), triglyceride (TG) (LPL, ANGPTL3, APOC3), and HDL-C (CETP).Given that ApoB serves as the primary transport protein for LDL-C/TG and ApoA-I for HDL-C, we employed the same criteria to screen for drug-target SNPs for ApoB and ApoA-I.

Mendelian randomization analysis
We used the inverse-variance weighted (IVW) method as our primary MR method [23], which estimates the causal effect of an exposure predicted genetically to increase by one standard deviation on the outcome.We further conducted MR-Egger, Weighted Median, Simple Mode, and Weighted Mode tests to control for pleiotropy [24].For the sensitivity analysis, we carried out MR-Egger and Mendelian Randomization Pleiotropy RESidual Sum and Outlier (MR-PRESSO) global tests to assess the potential pleiotropic effects of the instrumental variables included [25].Moreover, we conducted a leave-one-out sensitivity analysis to validate the accuracy and robustness of the causal effect estimates, ensuring the MR estimates are not substantially influenced by the effects of SNPs [26].We conducted Cochran's Q statistic [27] and an MR-Egger test [28] to check for heterogeneity.All statistical analyses in our work, including MR analysis and sensitivity analysis, were carried out by applying the R package "DrMR" along with publicly available R software (version 4.3.0).

Effect of lipids on sepsis
We observed significant associations between sepsis and two of the five examined lipids: HDL-C and ApoA-I (P < 0.05, Figure 3).For every standard deviation increase in HDL-C and ApoA-I, the odds ratio (OR) for sepsis was 0.91 (95% confidence interval (CI): 0.85-0.98;P = 0.014) and 0.88 (95% CI: 0.81-0.95;P = 7.53E-4), respectively.However, after implementing Bonferroni correction, the association between HDL-C and sepsis did not maintain statistical significance (P = 0.05/5), indicating potential suggestive causal relationships (Figure 4).Following Bonferroni correction, the findings for ApoA-I and sepsis remained statistically significant (P = 0.05/5), suggesting causal associations (Figure 5).Effect estimates derived from MR-Egger, Weighted Median, Simple Mode, and Weighted Mode methods were consistent with IVW estimates (Figure 3).No evidence of horizontal pleiotropy was found, demonstrated by the lack of a significant difference between the intercept in MR-Egger and zero.Although outlier SNPs were identified in some analyses through the MR-PRESSO global test and Cochran's Q test, there were no significant differences between pre-and post-correction outcomes according to the MR-PRESSO distortion test.

Mendelian randomization of sepsis and lipid drug targets
All instrumental variables had a minimum F-value exceeding 10, indicating an absence of weak instrumental variable bias, as illustrated in Table 1.The IVW-MR analysis revealed associations between LDL-C and ApoB, mediated by HMGCR, and an elevated sepsis risk (Figure 6, Figure 7).The OR values were 1.4 (95% CI: 1.06-1.84,P = 0.017) and 1.41 (95% CI: 1.01-1.98,P = 0.046) respectively, as depicted in Figure 8.This supports the potential protective effect of HMGCR inhibitors against sepsis.The mediation of HDL-C and ApoA-I by CETP was associated with a diminished sepsis risk (Figure 9, Figure 10), shown by OR values of 0.87 (95% CI: 0.82-0.92,P < 0.001) and 0.84 (95% CI: 0.78-0.9,P < 0.001), respectively.These findings further reinforce the plausible protective role of CETP inhibitors against sepsis.TG mediated by LPL exhibited a negative association with sepsis risk (Figure 11), showing an OR of 0.88 (95% CI: 0.8-0.97,P < 0.001).Both the MR-Egger and Weighted Median analyses exhibited causal relationships similar to those observed with the IVW method, providing supplementary evidence outlined in Table 1.To validate these results, sensitivity analyses were conducted, which did not detect any potential horizontal pleiotropy via the MR-Egger method (P > 0.05), as elaborated in Table 2.No heterogeneity was detected in either the MR-PRESSO global test or Cochran's Q test.These observations confirm the robustness of our findings regarding the association between drug target-mediated lipids and sepsis.

Discussion
Sepsis is essentially linked to excessive and dysregulated host responses, including hyperinflammation and immunosuppression [29].Lipids associated with pathogens, including lipopolysaccharides in Gram-negative bacteria, lipoteichoic acid in Gram-positive bacteria, and phospholipomannan in Candida albicans, serve as principal ligands for innate immune receptors, such as Toll-like receptors [30].Signaling activation through Toll-like receptors instigates the nuclear factor-ĸB, initiating antigen presentation to eliminate invasive microbes.The attachment of lipopolysaccharides and other toxic pathogenic lipids to various types of lipoproteins can neutralize and potentially deactivate their proinflammatory effects [31,32].The speculated function of this process is to isolate lipopolysaccharides within lipoproteins, thereby preventing their interaction with Toll-like receptors and subsequently mitigating hyperinflammation.Therefore, the level of circulating lipoproteins in plasma could potentially dictate the progression of infection and sepsis by neutralizing the effects of these pathogen-associated lipids.Submit a manuscript: https://www.tmrjournals.com/mdmHigh-density lipoproteins (HDL) exhibit the highest affinity for lipopolysaccharides, while low-density lipoproteins (LDL) show moderate affinity [33].Comprising ApoA-I, cholesterol, and phospholipids, HDL possesses the capacity to isolate and neutralize pathogen-associated lipids and counteract cytokine-induced endothelial activation.Multivariate logistic regression analysis suggests that ApoA-I levels serve as an independent predictor of 30-day mortality [34].A reduction in plasma HDL cholesterol levels can predict a poor prognosis in adult patients with severe sepsis [35] and pediatric patients with severe pneumococcal sepsis [36].CETP plays a pivotal role in HDL metabolism [37].Lowering CETP activity can mitigate the decrease in HDL induced by sepsis [38].Statins, categorized as lipid-reducing drugs, function by inhibiting the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase.This specific enzyme facilitates the conversion of HMG-CoA into mevalonic acid, a critical step in intracellular cholesterol biosynthesis.By inhibiting HMG-CoA reductase, statins halt the synthesis of LDL cholesterol, subsequently reducing its levels [39].

Sepsis
This research established an association between the genetically hypothesized increase in HDL-C and decrease in LDL-C with a reduced sepsis risk.Furthermore, extensive epidemiological studies have corroborated that a higher risk of infectious diseases is associated with low HDL-C and high LDL-C levels [40].In terms of main structural proteins, HDL-C consists of apoA-I while LDL-C incorporates ApoB [41].The study identified that LDL-C and ApoB, facilitated by HMGCR, exhibited a significant positive correlation with sepsis risk, boasting ORs of 1.4 (95% CI: 1.06-1.84,P = 0.017) and 1.41 (95% CI: 1.01-1.98,P = 0.046) respectively.Conversely, HDL-C and ApoA-I, mediated by CETP, demonstrated a negative correlation with sepsis risk, possessing ORs of 0.87 (95% CI: 0.82-0.92,P < 0.001) and 0.84 (95% CI: 0.78-0.9,P < 0.001) respectively.These findings suggest that inhibitors for HMGCR and CETP could potentially aid in sepsis prevention.Further, the robustness of the results was affirmed by the sensitivity analysis.
Inhibitors of HMG-CoA reductase, such as atorvastatin, exhibit anti-inflammatory, antioxidant, and immunomodulatory properties, which may potentially attenuate the progression of sepsis [42].Administering simvastatin demonstrated potential benefits such as mitigation of organ dysfunction, enhancement in the generation of local nitric oxide, improvement in bacterial clearance, and regulation of inflammation in related sepsis models [43].Statins, by targeting the multiple inflammatory and immunomodulatory cascades implicated in sepsis progression, hold potential as innovative therapeutics for sepsis treatment and prevention, as evidenced in mouse models [44].
A study conducted by Brown and colleagues unveiled that the genetic deficiency of CETP substantially augmented both the count and size of HDL particles [45].The primary role of CETP could potentially be to modulate the elevation of HDL, inhibit systemic endotoxemia through lipopolysaccharide binding and instigate a systemic pro-inflammatory response in macrophages to facilitate bacterial clearance [46].Current therapeutic strategies employing CETP inhibitors for lipid regulation can target the pathways implicated in severe infection and sepsis progression [47].Potential links might exist between the immune response, sepsis, and the inhibitors of HMG-CoA reductase and CETP, although further investigation is required to elucidate the specific mechanisms.Given the advent of various new lipid-lowering targets and non-statin lipid-lowering drugs, conducting extensive clinical trials to assess the effects of lipid-lowering therapies on sepsis becomes imperative.MR studies suggest a protective role of HMG-CoA reductase inhibitors and CETP inhibitors in sepsis prevention, warranting future validation from real-world data.This study's strengths lie in the minimization of bias due to confounding factors and reverse causality, as established by the MR analysis.Nevertheless, there are noted limitations: (1) both sets of GWAS data utilized in our MR analysis are sourced from European Submit a manuscript: https://www.tmrjournals.com/mdmpopulations.Consequently, future MR studies probing the causal relationships between lipid-lowering targets and sepsis should aim to involve samples from varied ethnic groups, thereby enhancing the results' generalizability.(2) We were not in a position to execute subgroup analyses given our reliance on the summary data of sepsis rather than the initial dataset.In conclusion, HMGCR inhibitors and CETP inhibitors play a beneficial role in sepsis treatment and prevention.It becomes imperative to undertake extensive clinical trials to evaluate the effect of lipid-lowering therapy on sepsis, thereby expanding the therapeutic options for sepsis patients.

Figure 1
Figure 1 Model of the Mendelian randomization analysis.IVs, instrumental variable selection; SNPs, single nucleotide polymorphisms.

Figure 5
Figure 5 Scatter plot of 5 MR models for ApoA-I and sepsis.MR, Mendelian randomization; ApoA-I, apolipoprotein A-I; SNP, single nucleotide polymorphism.

Figure 6
Figure 6 Scatterplot of LDL-C mediated by HMGCR and 3 MR models of sepsis.LDL-C, low-density lipoprotein cholesterol; MR, Mendelian randomization; SNP, single nucleotide polymorphism.

Figure 10
Figure 10 Scatterplot of ApoA-I mediated by CETP and 3 MR models of sepsis.ApoA-I, apolipoprotein A-I; MR, Mendelian randomization; SNP, single nucleotide polymorphism.

Figure 11
Figure 11 Scatterplot of TG mediated by LPL and 3 MR models of sepsis.TG, triglyceride; MR, Mendelian randomization; SNP, single nucleotide polymorphism.