Dissecting causal relationships between immune cells, plasma metabolites, and COPD: a mediating Mendelian randomization study

Objective This study employed Mendelian Randomization (MR) to investigate the causal relationships among immune cells, COPD, and potential metabolic mediators. Methods Utilizing summary data from genome-wide association studies, we analyzed 731 immune cell phenotypes, 1,400 plasma metabolites, and COPD. Bidirectional MR analysis was conducted to explore the causal links between immune cells and COPD, complemented by two-step mediation analysis and multivariable MR to identify potential mediating metabolites. Results Causal relationships were identified between 41 immune cell phenotypes and COPD, with 6 exhibiting reverse causality. Additionally, 21 metabolites were causally related to COPD. Through two-step MR and multivariable MR analyses, 8 cell phenotypes were found to have causal relationships with COPD mediated by 8 plasma metabolites (including one unidentified), with 1-methylnicotinamide levels showing the highest mediation proportion at 26.4%. Conclusion We have identified causal relationships between 8 immune cell phenotypes and COPD, mediated by 8 metabolites. These findings contribute to the screening of individuals at high risk for COPD and offer insights into early prevention and the precocious diagnosis of Pre-COPD.


Introduction
Chronic Obstructive Pulmonary Disease (COPD) is a heterogeneous disorder primarily characterized by airway pathologies (bronchitis, bronchiolitis) and/or alveolar abnormalities (emphysema), leading to chronic respiratory symptoms (dyspnea, cough, sputum production) and progressively worsening airflow limitation (1).Globally, COPD accounts for more than half of all chronic respiratory disease cases (2), gradually becoming the third leading cause of death worldwide (3).With the increasing prevalence of an aging population, both the incidence and mortality rates of COPD are on the rise annually (4), imposing a significant economic burden on society (5).
Immune cells possess multifaceted functions in maintaining homeostasis and facilitating repair after injury (6).The lungs may serve as a battleground for the interaction between various microbes and the host's innate and adaptive immune defenses (7).Consequently, the immune system could be a pivotal driving force in the pathogenesis of COPD, with immune responses being significantly associated with acute exacerbations of COPD (8).However, the detailed physiological mechanisms remain insufficiently explored (9).Most existing evidence, primarily from observational studies, indicates that compared to healthy controls, individuals with COPD have an increased presence of immune cells in lung tissue (10,11) and an upregulated immune cell response (12).Cells such as CD68+ myeloid antigen-presenting cells, CD4+ T cells, and CD8 T cells are found to proliferate in the lungs of patients with COPD, potentially leading to persistent inflammation (13,14).Certain immune cells exhibit a negative correlation with the frequency of COPD exacerbations (15), such as CD4 T cells and resting natural killer cells (16).Consequently, the causal relationship and underlying mechanisms between immune cells and COPD remain unclear.Metabolites, as intermediates of metabolic reactions, can influence disease progression (17) and serve as targets for therapeutic intervention (18).They have the potential to improve the diagnosis and treatment of COPD (19) and may play a synergistic role in its pathogenesis (20), possibly mediating important immunoregulatory functions (21).Compared to healthy controls, COPD patients exhibit reduced levels of the metabolites 1-methylnicotinamide creatinine, and lactate (17).Glutamylphenylalanine may serve as a biomarker for acute exacerbations of COPD (22), while sphingolipids are associated with pulmonary function (23).Therefore, we hypothesize a causal relationship between immune cells, metabolites, and COPD.Elucidating these associations and understanding the true causal relationships among immune cells, metabolites, and COPD could aid in the early identification, prevention, and management of COPD.
Mendelian Randomization (MR) represents a potential method for causal inference, designed to estimate the causal effects of exposure factors on outcomes, with the capability to control for potential confounding factors and circumvent reverse causation biases (24).Utilizing the methodology of MR, we are poised to conduct a bidirectional MR study concerning immune cells and COPD, concurrently undertaking two mediating analyses to dissect the causal relationships among immune cells, metabolites, and COPD.

Study design
Grounded in two-sample Mendelian Randomization, our study initially assessed the causal relationship between 731 immune cell phenotypes across 7 panels (B cell, cDC, TBNK, Treg, Myeloid cell, Maturation stages of T cell and Monocyte) and COPD.Proceeding with COPD as the exposure factor and employing the Inverse Variance Weighted (IVW) method to select immune cells as the outcome factor, we conducted reverse Mendelian Randomization to ascertain the presence of a reverse causal relationship.Utilizing both the two-step MR (TSMR) and multivariable MR approaches, with 1400 plasma metabolites serving as mediating factors, we aimed to elucidate the significant mediatory role that plasma metabolites may play in the causal pathway between immune cells and COPD (Figure 1).

Data sources
The genetic information pertinent to COPD was sourced from the GWAS database (https://gwas.mrcieu.ac.uk/), with the selected dataset bearing the identifier ebi-a-GCST90018807, encompassing 468,475 samples and 24,180,654 SNPs, all of which pertain to the European population.The genetic data related to 731 immune cell phenotypes were derived from a 2020 study (25), all pertaining to the European demographic, with the catalog identifiers ranging from ebi-a-GCST90001391 to ebi-a-GCST90002121.The GWAS data for 1,400 plasma metabolites, hailing from a 2023 study (18), are accessible from the GWAS database, with identifiers spanning from GCST90199621 to GCST90201020, all associated with the European population.

Instrumental variable selection
The selection of instrumental variables necessitates adherence to several assumptions (26), to fulfill their relevance (27), we conducted an association analysis on 731 immune cell phenotypes and 1,400 plasma metabolites, uniformly applying a threshold of P<1×10 −5 (28,29).Subsequently, SNPs exhibiting linkage disequilibrium were filtered out using criteria of R^2<0.001 and Kb=10,000 (30), followed by the calculation of the F-statistic for the selected SNPs to eliminate weak instrumental variables.An Fstatistic greater than 10 is considered indicative of the absence of weak instrumental variables (31, 32).

Statistical analysis
We employed five methodologies to assess causality: Inverse Variance Weighted (IVW), MR-Egger, Weighted Median, Simple Mode, and Weighted Mode methods, with IVW serving as the primary approach (33, 34).P<0.05 was indicative of a causal relationship (35), while the other four methods served as supplementary analyses (36).To evaluate the robustness of our results, we conducted sensitivity analysis using the "leave-one-out" approach, further examining pleiotropy and heterogeneity, P>0.05 suggesting the absence of both (37,38).Utilizing the TSMR approach, we first calculated the total effect from immune cells to COPD, the effect of immune cells on metabolites (b1), and the effect of metabolites on COPD (b2), followed by the calculation of the mediating effect (b1*b2), with the direct effect being the total effect minus the mediating effect (39).All analyses were conducted using the R language (version 4.3.2),with the TwoSampleMR package at version 0.6.0.

Genetic causality between immune cells and COPD
Through the selection of quantitative tools, we conducted an associative analysis, eliminating linkage disequilibrium and weak instrumental variables, thereby identifying 13,318 SNPs associated with immune cells, with the smallest F-value being 19.53.Preliminary investigations via the Inverse Variance Weighted (IVW) method revealed 41 immune cell phenotypes correlated with COPD, including but not limited to IgD+ CD38br %B cell, CD19 on IgD-CD38br, CD19 on PB/PC, and CD24 on memory B cell within the B cell category; TCRgd %T cell, HLA DR+ T cell%T cell, NKT %lymphocyte, and HLA DR+ NK AC within the TBNK category; CCR2 on plasmacytoid DC, CCR2 on CD62L+ plasmacytoid DC, CD80 on granulocyte, and CD62L on monocyte within the cDC category; and CD28+ CD45RA-CD8br %T cell, CD45RA+ CD28-CD8br %CD8br, CD25hi %T cell, and CD25++ CD8br %CD8br within the Treg category.In our study, we conducted a reverse Mendelian randomization analysis with COPD as the exposure factor and 41 immune cell phenotypes as the outcome factors.Our findings revealed that COPD does not exhibit a reverse causal relationship with 35 of the immune cell phenotypes (r-Pvalue > 0.05).However, a reverse causality was observed in six immune cell phenotypes (r-Pvalue < 0.05), specifically CD14+ CD16+ monocyte AC, CD4+ CD8dim % lymphocyte, CD4+ CD8dim %leukocyte, CD3-lymphocyte AC, CD3 on EM CD8br, and CD45 on Im MDSC.Furthermore, 21 immune cell phenotypes demonstrated a negative correlation with COPD, while 20 showed a positive correlation.Concurrently, tests for pleiotropy and heterogeneity yielded results (P> 0.05), with the direction of OR values being consistent, and leave-one-out sensitivity analysis confirmed the robustness of the MR findings (Table 1, Figure 2).

Mediated Mendelian randomization analysis
Building upon the previously identified immune cells and plasma metabolites, we employed the TSMR approach to further compute mediation through Mendelian randomization.Utilizing the 35 selected immune cell phenotypes as exposure factors and the 21 plasma metabolites as outcome measures, we conducted a MR analysis from immune cell phenotypes to plasma metabolites.This analysis revealed causal relationships between 20 immune cell phenotypes and 14 plasma metabolites, yielding the effect size b1 from immune cell phenotypes to metabolites.Our research has uncovered that there is a negative correlation between CD62Lmyeloid DC AC and 1-palmitoyl-GPG (16:0), a positive correlation between TCRgd %T cell and 1-methylnicotinamide, and a positive correlation between HLA DR+ T cell%T cell and Alpha-tocopherol levels, among other findings.Further analysis has revealed that a single immune cell phenotype can have causal relationships with When considering the 14 plasma metabolites as exposure factors and COPD as the outcome, an MR analysis was conducted along with an MR-PRESSO test (p > 0.05), indicating no pleiotropy and unbiased SNPs.This led to the determination of the effect size b2 from metabolites to COPD, and subsequently, the overall effect from immune cells to COPD was calculated (Figure 4, Table 3).

Mediation analysis
In our final analysis, we conducted a mediation analysis to elucidate the causal relationship between immune cell phenotypes and COPD, mediated by plasma metabolites.We discovered that 8 plasma metabolites mediated the relationship between 8 immune cell phenotypes and COPD (P < 0.05), among which 7 are known plasma metabolites and one remains unidentified.Notably, the CD24 on memory B cells was mediated by two distinct plasma metabolites.The mediation proportion of 1-methylnicotinamide was found to be the highest at 26.4% (P=0.013),followed by the unidentified metabolite X-19438 with a mediation proportion of 21.8% (P=0.004),Taurochenodeoxycholate at 18.8% (P=0.008), and Alpha-hydroxyisovalerate at 14.2% (P=0.036),among others (Figure 5, Table 4).

Discussion
In our MR study, the findings indicated a causal relationship between 41 immune cell phenotypes and COPD.However, a reverse MR analysis revealed that 35 immune cell phenotypes bore no causal relationship with COPD, while 6 did exhibit a causal connection.Further employing TSMR and MVMR for mediation analysis, we identified that 8 cell phenotypes could be causally linked to COPD through 8 plasma metabolites (including one unidentified), among which the mediation proportion of 1methylnicotinamide levels was the highest at 26.4%.Our research has corroborated the existence of a causal relationship between 41 immune cell phenotypes and COPD, aligning with previous studies that posit chronic inflammation leading to compromised immunity and immunosuppression as pivotal in the pathogenesis of COPD (40), a condition persistently present in the disease (41).It has been observed that, compared to healthy individuals, patients with COPD exhibit an increase in B cells and their products in the blood and lungs (42), alongside an upsurge in the expression of genes related to inflammation, B cell activation, and proliferation.This activation of B cells is associated with an autoimmune-mediated mechanism of COPD pathogenesis (43,44).However, the association between B cells and COPD does not imply causality (45).Our study, however, confirms a causal relationship between specific B cell phenotypes and COPD, with an increase in memory B-cells being linked to impaired lung function and small airway dysfunction (46), consistent with our findings that CD24 on memory B cells increases the risk of COPD.Furthermore, the subgroups of peripheral blood TBNK lymphocytes in COPD patients show a certain correlation with COPD (47) and its severity (48), aligning with our MR results and suggesting a causal relationship.Regulatory T (Treg) cells play a crucial role in the immune system by suppressing excessive immune responses and maintaining immune balance.The relationship between Treg cells and lung function (49), the imbalance of Treg cells during COPD progression (50), and the potential of modulating Treg cells to improve COPD (51) and lung inflammation underscore their significance (52).Myeloid cells, capable of phagocytosing pathogens, initiating inflammatory responses, and presenting antigens to other immune cells, contribute to tissue repair and remodeling.Our analysis identified six immune cell phenotypes with a causal relationship to COPD in both directions, namely CD14+ CD16+ monocyte AC, CD4+ CD8dim %lymphocyte, CD4+ CD8dim %leukocyte, CD3-lymphocyte AC, CD3 on EM CD8br, and CD45 on Im MDS, all associated with a reduced risk of COPD.The CD14+ CD16+ monocyte AC, a monocyte subgroup expressing CD14 and CD16, plays a role in modulating inflammatory responses and promoting tissue repair, with monocytes being etiologically related to COPD (53) and influencing its pathogenesis and diagnosis (54,55), serving as key drivers of lung inflammation and tissue remodeling (56).The CD4+ CD8dim %lymphocyte, a unique lymphocyte, plays a role in regulating immune responses and maintaining immune balance, with CD4-regulated T cells controlling autoimmunity and thus managing lung inflammation in COPD (57, 58), while CD4 and CD8 are related to bronchiolar wall remodeling in COPD ( 59) and the reduction of terminal bronchioles (60).
Metabolites play a crucial role in the early identification of individuals at high risk and in the prevention of diseases (61).Clinically, they enable us to differentiate the disease characteristics of COPD (62), identify diagnostic biomarkers (63,64), and evaluate the efficacy indicators of COPD treatments (65).Our study has discovered a causal relationship between CD24 on memory B cells     (72).Vitamin D may be a risk factor for COPD (73), though specific studies on COPD are lacking.Lipid metabolism plays a key role in the adaptive immune response to chronic inflammation (74) and is associated with lung inflammation in mice (75), with COPD patients exhibiting higher lipid expression (76).Our MR analysis concludes a causal relationship between CD24 on memory B cells and COPD through 1-palmitoyl-GPG (16:0).Taurochenodeoxycholate could potentially serve as an intermediary in the causal relationship between CD27 on unsw mem and COPD, demonstrating a negative correlation with both CD27 on unsw mem and COPD.Taurochenodeoxycholate, a bile acid functionally related to chenodeoxycholic acid, is involved in inflammatory responses (77), immune cell regulation (78), endoplasmic reticulum stress inhibition (79), and is associated with pulmonary fibrosis (80).However, research specifically targeting its role in COPD is scarce.MR analysis suggests that CD27 on unsw mem may have a causal relationship with COPD through the mediation of taurochenodeoxycholate.Alphatocopherol, the most active form of Vitamin E, has been observed in studies to reduce the risk of COPD in women (81), exhibiting anti-inflammatory and antioxidant properties that improve bronchial epithelial thickening, alveolar destruction, and lung function (82).Consistent with our MR analysis, alpha-tocopherol is negatively correlated with the risk of COPD, mediating a causal relationship between COPD and the percentage of HLA DR+ T cells among T cells.Alpha-hydroxyisovalerate, initially identified in studies related to human aging and early development (83), is associated with the severity of bronchiolitis (84) and, consistent with our MR analysis, positively correlated with the risk of COPD.It mediates a causal relationship between COPD and the phenotype of CD28+ CD45RA-CD8 bright %T cells.Cinnamoylglycine, with limited research related to COPD, has been found through MR analysis to be positively correlated with COPD.CD25 on Forest plots of immune cells, plasma metabolites, and COPD (A) Plasma metabolites mediate the causal relationship between immune cells and COPD (red is a risk factor, green is a protective factor) (B).

Conclusion
This study represents a comprehensive assessment of the causal relationships between immune cell phenotypes, plasma metabolites, and COPD.We have identified 8 immune cell phenotypes that exhibit a causal relationship with COPD, mediated by 8 metabolites.These findings illuminate the significance of the underlying mechanisms between immune cells, metabolites, and COPD.They contribute to the screening of individuals at high risk for COPD and offer insights into early prevention and the preemptive diagnosis of Pre-COPD conditions.

4
FIGURE 4 Forest plot of immune cells and metabolites (A) Partial scatter plot of immune cells and metabolites (B) Test of MR-PRESSO of metabolites to COPD (C) Partial leave-one-out method sensitivity analysis of metabolites to COPD (D).

3
FIGURE 3 Circle plot of five Mendelian randomization methods (p <0.05) (A) Forest plot of causality of 21 metabolites and COPD (B) Scatter plot of 7 metabolites with increased COPD risk (C) Scatter plot of the 9 metabolites reducing the risk of COPD (D).
and COPD, mediated by two intermediaries: 1-methylnicotinamide and 1-palmitoyl-GPG (16:0).There is a positive correlation between CD24 on memory B cells and COPD, where an increase in CD24 on memory B cells elevates the risk of COPD.Furthermore, CD24 on memory B cells is positively correlated with 1-methylnicotinamide, which, in turn, is positively associated with COPD.1methylnicotinamide, a primary metabolite found in all living organisms and involved in growth, development, or reproduction, possesses various immunomodulatory properties.It is linked to inflammatory responses in lung epithelial cells(66)and the activation of the NLRP3 inflammasome (67), a significant mediator in COPD inflammation (68).Through NLRP3, lung inflammation can be regulated (69, 70), indicating a certain correlation between 1-methylnicotinamide and COPD.Our research confi rms a caus al relationship between 1methylnicotinamide and COPD, with CD24 on memory B cells influencing COPD risk through the mediation of 1methylnicotinamide.Conversely, CD24 on memory B cells is negatively correlated with 1-palmitoyl-GPG (16:0), which is positively associated with COPD.Research on 1-palmitoyl-GPG (16:0) is limited, but it is generally considered part of lipid metabolism, related to vitamin D deficiency(71) and pulmonary hypertension

TABLE 1
MR analysis of immune cells and COPD.

TABLE 1 Continued
Circle plot of five Mendelian randomized methods (P<0.05) (A) Forest plot of causality between 35 immune cells and COPD (r-Pvalue is the result of reverse MR) (B) Forest plot of causality between six immune cells and COPD (C) Scatter plot of six immune cells reducing COPD risk (D).

TABLE 2
MR analysis of metabolites and COPD.

TABLE 3
MR analysis of immune cells and metabolites.

TABLE 3 Continued
ExposureOutcome Method Nsnp Beta Se P-val Pleiotropy Heterogeneity Cao et al. 10.3389/fimmu.2024.1406234Frontiers in Immunology frontiersin.orgtransitional cells has a causal relationship with COPD mediated by cinnamoylglycine.N-oleoylserine, a secondary metabolite functionally related to oleic acid and with scant research in the context of lung inflammation (85), has been found through MR analysis to be negatively correlated with COPD, suggesting a protective factor.

TABLE 4
Mendelian randomization analyses of the causal effects between immune cells, plasma metabolites and COPD.