Associations of Monocyte Count and Other Immune Cell Types with Interstitial Lung Abnormalities

Abstract

Rationale

Higher blood monocyte counts are associated with worse survival in adults with clinically diagnosed pulmonary fibrosis. Their association with the development and progression of interstitial lung abnormalities (ILA) in humans is unknown.

Objectives

We evaluated the associations of blood monocyte count, and other immune cell types, with ILA, high-attenuation areas, and FVC in four independent cohorts.

Methods

We included participants with measured monocyte counts and computed tomographic (CT) imaging enrolled in MESA (Multi-Ethnic Study of Atherosclerosis, n = 484), AGES-Reykjavik (Age/Gene Environment Susceptibility Study, n = 3,547), COPDGene (Genetic Epidemiology of COPD, n = 2,719), and the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points, n = 646).

Measurements and Main Results

After adjustment for covariates, a 1-SD increment in blood monocyte count was associated with ILA in MESA (odds ratio [OR], 1.3; 95% confidence interval [CI], 1.0–1.8), AGES-Reykjavik (OR, 1.2; 95% CI, 1.1–1.3), COPDGene (OR, 1.3; 95% CI, 1.2–1.4), and ECLIPSE (OR, 1.2; 95% CI, 1.0–1.4). A higher monocyte count was associated with ILA progression over 5 years in AGES-Reykjavik (OR, 1.2; 95% CI, 1.0–1.3). Compared with participants without ILA, there was a higher percentage of activated monocytes among those with ILA in MESA. Higher monocyte count was associated with greater high-attenuation areas in MESA and lower FVC in MESA and COPDGene. Associations of other immune cell types were less consistent.

Conclusions

Higher blood monocyte counts were associated with the presence and progression of interstitial lung abnormalities and lower FVC.

At a Glance Commentary

Scientific Knowledge on the Subject

Prior studies have demonstrated the critical role the innate immune system has in the pathogenesis of pulmonary fibrosis and in particular the mononuclear phagocyte system. Absolute blood monocyte count may be a useful prognostic biomarker in adults with idiopathic pulmonary fibrosis. A higher monocyte count in the blood is strongly associated with disease progression and worse survival.

What This Study Adds to the Field

In this analysis across four independent population and smoker-based cohorts (MESA [Multi-Ethnic Study of Atherosclerosis], AGES-Reykjavik [Age, Gene/Environment Susceptibility-Reykjavik], COPDGene [Genetic Epidemiology of Chronic Obstructive Pulmonary Disease (COPD)], and ECLIPSE [Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points]), a higher absolute blood monocyte count was associated with a higher odds ratio for interstitial lung abnormalities (ILA) and their progression on computed tomography imaging. A higher monocyte count was also associated with more high-attenuation areas on computed tomography imaging and a lower FVC. Adults with ILA had a higher percentage of monocytes expressing tissue factor than adults without ILA. These findings suggest monocytes may have a critical role in the early development of interstitial lung disease.

Interstitial lung disease (ILD) is a group of chronic respiratory illnesses characterized by varying degrees of inflammation and fibrosis of the lung parenchyma (1). Fibrotic ILDs are believed to be driven by recurrent lung injury and abnormal wound healing, but the underlying pathways and potential therapeutic targets have not been fully defined (2). Recent studies have focused on the role of innate immunity in initiating and propagating the fibrotic cascade in the lungs (3). Monocyte-derived macrophages have emerged as important drivers of lung fibrogenesis (4, 5). Higher blood monocyte counts may be prognostic and are associated with an accelerated decline in lung function and worse survival in adults with pulmonary fibrosis (6–10). However, whether monocyte counts and monocyte activation (i.e., tissue factor [TF] expression) (11) are associated with the development and progression of interstitial lung abnormalities is unknown.

Studies that leverage computed tomography (CT)-based assessments of the lungs to identify ILD-related radiologic patterns (i.e., traction bronchiectasis, reticular opacities) in high-risk and general population-based cohorts have enabled research into novel risk factors and mechanisms of early pulmonary fibrosis in humans (12). Visually identifiable interstitial lung abnormalities (ILAs) are found on chest CT imaging in 7–10% of community-dwelling adults and are associated with worse lung function, higher overall mortality, and respiratory-related illness (13–15). A recent Fleischner Society position paper attempted to standardize the definitions and noted that ILA can be a clinically meaningful finding in individuals not otherwise suspected of having clinical ILD (12). Smoking, air pollution, and genetic polymorphisms are related to ILA, but there remains a critical need for a better understanding of the mechanisms involved in the development and progression of ILA (16–18). Automated, quantitative measurement of ILD-related changes on CT, including measures of increased lung density such as high-attenuation areas (HAA), can further aid in identifying novel risk factors for pulmonary fibrosis (19, 20).

We hypothesized that a higher monocyte count would be associated with ILA on CT imaging among adults in four independent cohorts. We also investigated whether participants with ILA have a higher percentage of activated monocytes, as defined by TF expression (11). We secondarily examined associations of blood monocyte count and other immune cell types with HAA and FVC. Some of the results have been previously reported in form of an abstract (21).

Methods

Study Participants

MESA (the Multi-Ethnic Study of Atherosclerosis) is an NHLBI-sponsored prospective cohort study that originally enrolled 6,814 adults between the ages of 45 and 84 years without clinical cardiovascular disease between 2000 and 2002 from six U.S. communities and followed over 18 years (22). The AGES-Reykjavik (Age, Gene/Environment Susceptibility-Reykjavik) study is a longitudinal birth cohort of 5,764 men and women from the Reykjavik study of Icelanders who were 66–96 years old at time of study enrollment in the years 2002–2006 (23). The COPDGene (Genetic Epidemiology of COPD) study enrolled 10,198 non-Hispanic white and African American participants, aged 45–80 years, with ⩾10 pack-years of smoking history (24). COPDGene started as a cross-sectional COPD case-control study and was extended into a longitudinal study with 5- and 10-year follow-up visits. The ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points) study was a 3-year longitudinal study of 3,291 participants aged 40–75 years with ⩾10 pack-years of smoking. The goal of ECLIPSE was to identify surrogate endpoints and predictors associated with COPD exacerbations and progression (25).

Immune Cell Phenotyping

In MESA, whole blood ethylenediaminetetraacetic acid samples were collected and shipped overnight at room temperature to the University of Vermont from 930 randomly selected participants who returned for Exam 4 (2005–2007). Full description of the methods and protocol used in MESA has been previously published (11, 26). Total white blood cell count and leukocyte subsets (i.e., white blood cell count with differential) were analyzed at the University of Vermont Medical Center. Peripheral lymphocyte subsets were measured by flow cytometry, and percentage B cells and CD8+ cells were estimated from other cell counts. White blood cell count and its differential were measured in fasting whole blood samples from 5,572 AGES-Reykjavik participants using an automated cell counter, Coulter HmX AL Hematology Analyzer (Beckman Coulter). In COPDGene, whole blood samples were collected from 4,895 individuals who returned for their 5-year follow-up visit, and detailed methods have been previously reported (27). Total white cell counts and leukocyte subsets were assessed by complete blood count with differential analysis. In ECLIPSE, whole blood samples were collected at the time of study enrollment, and sample handling has been previously described (28). Total leukocyte counts and subsets were assessed by complete blood count and differential analysis.

Chest CT Analysis

ILA was defined as nondependent abnormalities involving >5% of a lung zone, which include ground-glass and/or reticular abnormalities, nonemphysematous cysts, honeycombing, and traction bronchiectasis, in accordance with the 2020 Fleischner Society position paper (12). ILA was assessed in MESA at Exam 5 (2010–2012) on full-lung scans using the MESA/SPIROMICS protocol, approximately 5 years after Exam 4 when immune cell phenotyping was performed (29). ILA was determined from thoracic CT images obtained on study enrollment in AGES-Reykjavik, COPDGene, and ECLIPSE (14). One of five trained radiologists interpreted CT scans for ILA in MESA and with three readers (one pulmonologist and two radiologists) by consensus in the AGES-Reykjavik, COPDGene, and ECLIPSE cohorts (14, 19). A subset of participants in AGES-Reykjavik had a repeat CT scan approximately 5 years later with ILA assessment. Those with ILA on either scan had both images evaluated for ILA progression by simultaneous comparison, with the final assessment dependent on the consensus of multiple readers as previously described (30). Participants with findings indeterminate for ILA were excluded from the study.

Pulmonary Analysis Software Suite was used to semiautomatically segment MESA Exam 5 full-lung CT scans for quantitative assessment of lung attenuation, with correction by a trained technician at the University of Iowa’s Advanced Pulmonary Physiomic Imaging Laboratory (Iowa City, IA) (31). HAA was defined as the percentage of lung volume with a range of Hounsfield units (HU) of −600 to −250 (19).

Monocyte Activation

Monocyte activation, which can lead to macrophage polarization that is critical in the immunomodulatory response to stimuli and has been implicated in lung fibrogenesis, was measured at MESA Exam 4 (32). Whole blood heparin samples maintained for 24 hours at room temperature were stimulated with Escherichia coli LPS for 4 hours at 37°C. Side-scatter and CD14+ fluorescence were used to determine monocytes. TF expression, a marker of monocyte activation on stimulation from agonists including LPS, was measured while a CD14+ tube was used to enhance gate setting for TF+ cells as previously described (11, 33). Monocytes expressing TF (CD14+, TF+) were reported as a percentage of CD14+ cells. This was also performed in an unstimulated assay using a buffer in place of LPS.

Spirometry

FVC was measured by spirometry in MESA at Exam 5 in accordance with guidelines (34, 35). Spirometry measures were obtained in the COPDGene and ECLIPSE studies at each visit, and American Thoracic Society/European Respiratory Society standards of repeatability and acceptability were applied along with manual review by pulmonologists (24, 25, 34, 35). In COPDGene, FVC measures at the 5-year follow-up visit were used to coincide with white blood cell count measurements. In ECLIPSE, baseline FVC measures were used to coincide with white blood cell count measurements. Timing of blood sample collection and outcome assessments are summarized in Figure E1 in the online supplement.

Statistical Analysis

We used logistic regression models to examine associations of absolute monocyte count with baseline ILA in all cohorts. Linear regression models were used to examine associations of monocyte count with HAA in MESA and FVC in all of the cohorts except for AGES-Reykjavik. Model 1 was unadjusted. Model 2 adjusted for potential confounders, which included age, sex, smoking status, cigarette pack-years, and body mass index. Self-reported race/ethnicity was additionally adjusted for in MESA and COPDGene models. We used random-effects meta-analysis to assess the combined cohort effect estimate of overall monocyte-ILA association using the “meta” package from R version 4.0.4 (R Foundation for Statistical Computing). I² was used to assess heterogeneity. Study site was adjusted for in HAA models. Logistic regression was used to investigate the associations of monocyte count with ILA progression in AGES-Reykjavik using the same approach for the ILA cross-sectional analyses. As in previous analyses of ILA progression in AGES-Reykjavik, participants with new or progressing ILA were compared with those without ILA on either CT (30). Participants who had indeterminate findings at baseline or at the repeat scan and those with stable or regressing ILA were excluded from the progression analysis. Generalized additive models were used to assess the linearity of associations for ILA and HAA. For regression purposes, we modeled HAA as (−1/[HAA]²), which appeared to show better homoscedasticity based on visualization of residual plots than absolute HAA or log-transformed HAA (36).

We performed a sensitivity analysis in which we adjusted for percentage emphysema (defined as percentage of low-attenuation areas <−950 HU on CT scan), which was available in MESA, COPDGene, and ECLIPSE (37). We examined effect modification by smoking status and sex using log-likelihood ratio tests. For the smoking-stratified analyses, “nonsmoker” was defined as “never” in MESA and AGES-Reykjavik and “former” in COPDGene and ECLIPSE. “Smoker” was defined as “former/current” in MESA and AGES-Reykjavik and “current” in COPDGene and ECLIPSE. To test whether the lack of association of monocyte counts with FVC in ECLIPSE could be attributable to study enrollment selection criteria, we used propensity score–matched COPDGene and ECLIPSE participants based on age, sex, pack-years of smoking, current smoking status, and annual exacerbation frequency (MatchIt R package) (38). We then repeated the association analyses between monocyte counts and FVC in this matched COPDGene sample and in a subset of these participants reporting one or more exacerbations per year. We additionally examined whether age modified the association between monocyte count and ILA by performing stratified analyses using 65 years as a cutoff in MESA, COPDGene, and ECLIPSE. We used a cutoff of 75 years for AGES-Reykjavik because it comprised an older population at enrollment. For the final analysis, we included participants with complete covariate data. Results are presented as 1-SD increment of absolute monocyte count. The 1-SD values for each cohort were the following: 0.1389616 (MESA), 0.1847431 (AGES-Reykjavik), 0.2017182 (COPDGene), and 0.2345582 (ECLIPSE). We used a similar approach with other immune cell types as predictor variables. Analyses were performed with SAS version 9.4 (SAS Foundation) and R version 4.0.4 (R Foundation for Statistical Computing).

Results

Baseline characteristics of participants with absolute monocyte counts and ILA assessments from each cohort are summarized in Table 1. Among those with ILA assessments and monocyte count, fewer than 2% had missing covariate data in each cohort. Adults with ILA in each cohort were older than those without ILA. With the exception of MESA, there was a higher prevalence of men among those with ILA than those without ILA.

Table 1.

Baseline Characteristics of Participants with Monocyte Count and ILA Assessments

Characteristic	MESA			AGES-Reykjavik			COPDGene			ECLIPSE
	Overall	No ILA	ILA	Overall	No ILA	ILA	Overall	No ILA	ILA	Overall	No ILA	ILA
No. participants	484	421	63	3,547	3,211	336	2,719	2,256	463	646	509	137
Age, yr, mean (SD)	64 (9)	63 (9)	68 (10)	76 (5)	76 (5)	78 (6)	65 (9)	64 (8)	69 (9)	63 (7)	62 (7)	65 (7)
Female, %	54	53	62	58	59	45	49	49	48	33	35	25
Race/ethnicity, %
Non-Hispanic White	47	45	54	100	100	100	73	72	78	100	100	100
Asian	11	12	5	0	0	0	0	0	0	0	0	0
African American	19	20	19	0	0	0	27	28	22	0	0	0
Hispanic	23	23	22	0	0	0	0	0	0	0	0	0
Smoking status, %
Never	49	50	38	44	46	29	0	0	0	0	0	0
Former	42	41	48	44	43	55	64	64	65	61	61	59
Current	9	9	14	12	12	16	36	36	35	39	39	41
Height, cm, mean (SD)	166 (10)	166 (10)	165 (11)	167 (9)	167 (9)	168 (10)	170 (10)	170 (9)	169 (10)	170 (9)	169 (9)	170 (9)
Weight, kg, mean (SD)	79 (18)	79 (18)	78 (18)	76 (15)	76 (15)	77 (16)	84 (20)	84 (20)	85 (20)	78 (19)	78 (19)	77 (18)
Body mass index, kg/m2, mean (SD)	29 (6)	29 (6)	29 (6)	27 (4)	27 (4)	27 (5)	29 (6)	29 (6)	30 (6)	27 (6)	27 (6)	27 (5)
Absolute white blood cell count, ×103 μl, median (IQR)	5.5 (4.6–6.5)	5.4 (4.5–6.4)	5.6 (4.8–7.0)	4.7 (4.8–6.8)	5.7 (4.8–6.7)	6.1 (5.1–7.2)	6.8 (5.7–8.3)	6.8 (5.6–8.2)	7.2 (6.1–8.6)	7.4 (6.3–8.9)	7.3 (6.2–8.6)	8.3 (6.9–9.6)
Absolute monocyte count, ×103 μl, median (IQR)	0.3 (0.2–0.4)	0.3 (0.2–0.4)	0.4 (0.3–0.5)	0.5 (0.4–0.6)	0.5 (0.4–0.6)	0.6 (0.5–0.7)	0.5 (0.4–0.7)	0.5 (0.4–0.7)	0.6 (0.5–0.7)	0.5 (0.3–0.6)	0.5 (0.3–0.6)	0.5 (0.4–0.6)

Definition of abbreviations: AGES-Reykjavik = Age, Gene/Environment Susceptibility-Reykjavik; COPD = chronic obstructive pulmonary disease; COPDGene = Genetic Epidemiology of COPD; ECLIPSE = Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points; ILA = interstitial lung abnormalities; IQR = interquartile range; MESA = Multi-Ethnic Study of Atherosclerosis.

Absolute Monocyte Count

A higher absolute monocyte count was associated with the presence of ILA in MESA with an adjusted odds ratio (OR) of 1.3 (95% confidence interval [CI], 1.0–1.8) per 1-SD increment in monocyte count (Figure 1). This finding was replicated in the other three independent cohorts (Figure 1). For every 1-SD increment in monocyte count, there was an adjusted OR of 1.2 (95% CI, 1.1–1.3), 1.3 (95% CI, 1.2–1.4), and 1.2 (95% CI, 1.0–1.4) in AGES-Reykjavik, COPDGene, and ECLIPSE, respectively. A higher monocyte count was associated with an OR of 1.3 (95% CI, 1.2–1.4) with minimal heterogeneity (I² = 0) when all four cohorts were meta-analyzed. The association between monocyte count and probability of ILA was mostly linear in MESA and ECLIPSE, with flattening at much higher monocyte count levels (P values for linearity = 0.04; Figure 2). The associations were largely linear in AGES-Reykjavik and ECLIPSE (Figure 2). The association with ILA was monotonic in all four cohorts when we examined monocyte count by tertiles (P values for trend ⩽ 0.03) (Table E1). There was no effect modification by smoking status, sex, or age in any of the cohorts (Figure E2). Associations persisted after adjustment for percentage emphysema in MESA, COPDGene, and ECLIPSE (Table E2).

Figure 1.

Forest plot of associations between absolute monocyte count and interstitial lung abnormalities. Model 1 is unadjusted. Model 2 is adjusted for age, sex, smoking status, cigarette pack-years, and body mass index. Self-reported race/ethnicity also adjusted for in MESA (Multi-Ethnic Study of Atherosclerosis) and COPDGene (Genetic Epidemiology of COPD). The I² was <1% for pooled effect estimates for both model 1 and model 2. Boxes represent the effect estimates, and horizontal lines represent their 95% confidence intervals (95% CI). AGES-Reykjavik = Age, Gene/Environment Susceptibility-Reykjavik; COPD = chronic obstructive pulmonary disease; ECLIPSE = Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points; ILA = interstitial lung abnormalities.

Figure 2.

Continuous associations of absolute monocyte count with interstitial lung abnormalities in (A) MESA (Multi-Ethnic Study of Atherosclerosis), (B) AGES-Reykjavik (Age, Gene/Environment Susceptibility-Reykjavik), (C) COPDGene (Genetic Epidemiology of COPD), and (D) ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points). P values for linearity are (A) 0.04, (B) 0.002, (C) <0.001, and (D) 0.04. Models adjusted for age, sex, smoking status, cigarette pack-years, and body mass index. Self-reported race/ethnicity is also adjusted for in MESA and COPDGene. The solid line represents the overall effect estimate; thin dashed lines indicate the 95% confidence interval bands. In A, C, and D, each vertical hashmark on the x-axis represents an individual participant. In B, the proportion of participants is denoted by the height of the hashmarks. COPD = chronic obstructive pulmonary disease; ILA = interstitial lung abnormalities.

Among AGES-Reykjavik participants with a baseline CT scan, 1,659 had a repeat CT scan approximately 5 years later with ILA assessment (Figure E1). A 1-SD increment in baseline monocyte count was associated with ILA progression over time, with an adjusted OR of 1.2 (95% CI, 1.0–1.3) (Table 2). There was no effect modification by smoking status or sex on the association between monocyte count and ILA progression in either cohort.

Table 2.

Absolute Monocyte Count and Interstitial Lung Abnormalities Progression

Model	AGES-Reykjavik
	No. Participants	OR for ILA Progression (95% CI)	P Value
Model 1	1,659	1.3 (1.1–1.5)	<0.001
Model 2	1,659	1.2 (1.0–1.3)	0.04
Smoking status
Nonsmoker	751	1.1 (0.8–1.4)	0.67
Smoker	908	1.2 (1.0–1.4)	0.04
Sex
Female	966	1.3 (1.0–1.5)	0.03
Male	693	1.1 (0.9–1.3)	0.38

Definition of abbreviations: AGES = Age, Gene/Environment Susceptibility Study; CI = confidence interval; ILA = interstitial lung abnormalities; OR = odds ratio.

Model 1: Unadjusted. Model 2: Adjusted for age, sex, smoking status, cigarette pack-years, and body mass index. Stratified models include interaction term “effect modifier × monocyte count.” P value for smoking interaction = 0.42. P value for sex interaction = 0.28. Results are reported per 1-SD increment of absolute monocyte count.

In MESA, greater absolute monocyte count was associated with greater HAA overall and among ever-smokers in a linear fashion (Figure 3). A 1-SD increment of absolute monocyte count was associated with a 21.4-unit increment in HAA (95% CI, 3.6–39.3). Associations were stronger among ever-smokers than never-smokers (P value for smoking interaction = 0.04). Sex did not modify the association, and adjustment for percentage emphysema did not significantly attenuate the overall association (Table E3). After adjustment for covariates, a 1-SD increment in monocyte count was associated with an FVC difference of −70 ml (95% CI, −122 to −18 ml) in MESA, −100 ml (95% CI, −130 to −72 ml) in COPDGene, and 3 ml (95% CI, −50 to 57 ml) in ECLIPSE (Table 3). To determine whether the association between monocyte count and FVC in ECLIPSE was attributable to study selection criteria, we repeated these analyses in a subset of COPDGene participants (n = 645) matched to ECLIPSE on the basis of demographic and exacerbation metrics (Table E4). Among matched COPDGene participants, we observed an association of monocyte counts with FVC (−77 ml per 1-SD increment in monocyte count; 95% CI, −140 to −14) (Table E5). Among COPDGene individuals reporting at least one exacerbation per year (n = 139), this association was attenuated (−45 ml; 95% CI, −160 to 69 ml). Associations were not significantly different in stratified analyses.

Figure 3.

Continuous associations of absolute monocyte count with high attenuation areas in MESA (Multi-Ethnic Study of Atherosclerosis) overall and stratified by smoking status. Overall model is adjusted for age, sex, race/ethnicity, smoking status, cigarette pack-years, body mass index, and study site. P values for linearity are 0.03 (overall, n = 603), 0.002 (ever-smokers, n = 328), and 0.96 (never-smokers, n = 275). P value for smoking interaction = 0.04. Each vertical hashmark on the x-axis represents an individual participant.

Table 3.

Cross-Sectional Associations of Absolute Monocyte Count with FVC

Model	MESA			COPDGene			ECLIPSE
	No. Participants	Difference in FVC (95% CI) (ml)	P Value	No. Participants	Difference in FVC (95% CI) (ml)	P Value	No. Participants	Difference in FVC (95% CI) (ml)	P Value
Model 1	594	65 (−19 to 148)	0.13	2,680	8 (−31 to 46)	0.70	645	79 (17 to 140)	0.01
Model 2	594	−70 (−122 to −18)	0.008	2,680	−100 (−130 to −72)	<0.001	645	3 (−50 to 57)	0.90
Smoking status
Nonsmoker	277	−63 (−153 to 27)	0.17	1,717	−96 (−130 to −60)	<0.001	394	−25 (−100 to 55)	0.54
Smoker	317	−79 (−144 to −14)	0.02	963	−110 (−160 to −60)	<0.001	251	25 (−48 to 98)	0.50
Sex
Female	315	−34 (−106 to 39)	0.36	1,310	−89 (−120 to −54)	<0.001	210	3 (−95 to 100)	0.96
Male	279	−106 (−184 to −28)	0.01	1,370	−110 (−160 to −71)	<0.001	435	4 (−61 to 69)	0.89

Definition of abbreviations: CI = confidence interval; COPD = chronic obstructive pulmonary disease; COPDGene = Genetic Epidemiology of COPD; ECLIPSE = Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points; MESA = Multi-Ethnic Study of Atherosclerosis.

Model 1: Unadjusted. Model 2: Adjusted for age, sex, race/ethnicity, smoking status, cigarette pack-years, and body mass index. Stratified models include interaction term “effect modifier × monocyte count.” P value for smoking interaction = 0.77 (MESA), 0.99 (COPDGene), and 0.53 (ECLIPSE). P value for sex interaction = 0.17 (MESA), 0.29 (COPDGene), and 0.97 (ECLIPSE). Results are reported per 1-SD increment of absolute monocyte count.

Monocyte Activation in MESA

There were 441 and 440 MESA participants who had TF expression measured from unstimulated and LPS-stimulated assays, respectively. In the LPS-unstimulated assay, participants with ILA had a mean (SD) of 3.4% (0.8%) TF+ monocytes, compared with 4.0% (0.7%) for those without ILA (adjusted mean difference, −0.55; P value = 0.75; Figure 4). After LPS stimulation, participants with ILA had a higher percentage of TF+ monocytes (mean, 21.3%; SD, 1.6%) than those without ILA (mean, 18.8%; SD, 1.6%; adjusted mean difference, 2.5; P value = 0.04; Figure 4). Smoking status did not modify these findings (Table E6 and Figure E3), and there was no significant difference in percentage of monocytes expressing TF+ by smoking status overall (Table E7).

Figure 4.

Boxplots of percentage of monocytes expressing tissue factor in (A) unstimulated, and (B) LPS-stimulated assays by participants with and without interstitial lung abnormalities. Dots represent individual predicted values adjusted for age, sex, race/ethnicity, smoking status, cigarette pack-years, body mass index, and study site. P value = 0.75 (unstimulated assay) and P value = 0.04 (LPS-stimulated assay). Solid horizontal lines represent the median percentage of monocytes expressing tissue factor. Boxes are bound by the upper and lower quartiles, and whiskers extend to 1.5 times the interquartile range. ILA = interstitial lung abnormalities.

Other Immune Cell Subsets

Associations of other leukocyte subsets with ILA are summarized in Table E8. A higher absolute count of neutrophils was associated with a higher OR for ILA in AGES-Reykjavik, COPDGene, and ECLIPSE. Smoking status did not modify this association (Table E9). Neutrophil count was associated with a lower FVC in MESA and COPDGene, but not in ECLIPSE (Table E10). Associations of other immune cell subsets with ILA, HAA, and FVC in MESA are summarized in Tables E11–E13 and were largely inconsistent compared with monocytes.

Discussion

Higher blood monocyte counts were associated with the presence of ILA and its progression, more HAA, and a lower FVC among adults. These associations persisted after adjustment for potential ILD-related confounders, including age and smoking. The percentage of monocytes expressing TF after stimulation by LPS was higher among those with ILA than those without ILA.

There is a growing body of evidence that points to the critical role of innate immunity in the pathogenesis of pulmonary fibrosis with a particular focus on the mononuclear phagocyte system. At sites of injury, including the lung, monocytes differentiate into macrophages and dendritic cells, where they facilitate clearance of pathogens and inflammatory responses and regulate wound healing through the upregulation of TGF-β (transforming growth factor-β), IL-10, and arginase-1, all of which are important in the composition and production of collagen (5, 39). Depletion of alveolar macrophages attenuates bleomycin-induced lung fibrosis in mouse models, and profibrotic genes are upregulated in macrophages from human fibrotic lungs compared with normal (4). Peripheral monocytes also differentiate into fibrocytes, mesenchymal cells that promote wound healing through secretion of TGF-β1, matrix metalloproteinases (MMPs), and fibroblast growth factor, and have been implicated in lung fibrosis (40).

Among immune cell types, aberrant populations of peripheral monocytes are strongly associated with worse transplant-free survival in patients with idiopathic pulmonary fibrosis (IPF) (6). In the same study, higher absolute monocyte count was associated with worse survival across multiple cohorts of patients with IPF and has been replicated (7–9). Although these studies have focused on adults with clinically diagnosed pulmonary fibrosis, it is unclear if elevated monocyte counts and their activation are related to markers of lung injury and scarring in more general-based population and smoker cohorts.

We leveraged data from four independent cohorts, both population and smoker-based, for which extensive existing radiologic and clinical phenotype data enabled us to account for potential confounders. We also demonstrated that the proportion of activated monocytes (i.e., TF-expressing monocytes after LPS stimulation) was higher among those with ILA on CT scan than those without ILA and suggests a “hyperactive” monocyte state may contribute to lung injury and aberrant healing.

In a COPDGene sample matched to ECLIPSE participants based on demographic measures, we observed a significant yet reduced magnitude of effect of monocyte counts on reduced FVC. This effect was attenuated among those reporting exacerbations; however, we do note that the direction of effect was still negative, and there were only 645 participants in this analysis, so this result could reflect diminished power. Nevertheless, our results suggest that the selection criteria for ECLIPSE, and in particular, selection for those with recent exacerbations, may have at least partially attenuated the association between monocyte counts and FVC. Further investigation into which factors mediate the monocyte and FVC association is needed.

The association of monocyte count with HAA was stronger among ever-smokers in MESA. Smoking is a risk factor for pulmonary fibrosis, and our study suggests one of the mechanisms may be related to their effect on the mononuclear phagocyte system (41). Reduced expression of the transferrin receptor CD71 in monocytes and macrophages, which can be triggered by cigarette smoke exposure, leads to activation of fibrosis-related genes (42, 43). Human monocytes and monocyte-derived macrophages exposed to cigarette smoke generate procoagulant microvesicles, which have been linked to lung injury and fibrosis (4, 44). Specific smoking-related interstitial lung patterns on CT imaging (e.g., respiratory-bronchiolitis-ILD) were not ascertained in the cohorts and may be interesting to investigate in future studies. The adjustment for percentage emphysema in sensitivity analyses did not significantly attenuate the associations we observed. One potential theory that merits further investigation is that the mononuclear phagocyte system plays a role in differentiating the different disease trajectories (obstructive versus restrictive) triggered by smoking. Associations of monocyte count with ILA and FVC were also stronger among smokers, but they did not reach statistical significance for effect modification. The percentage of activated monocytes by ILA status was also not influenced by smoking status and suggests the relationship between monocytes and markers of lung injury and remodeling cannot be entirely explained by smoking. Further research is needed to identify the potential synergistic effects of smoking and innate immunity on lung fibrosis.

Our study has several limitations. We did not have repeat subsequent measurements of monocyte counts or any other immune cell subtypes, which limited us from examining longitudinal associations with our outcomes. However, a prior study did not find any association of change in monocyte count with outcomes among patients with IPF (9). Although LPS is considered a broad activator of monocytes, we did not have more detailed phenotyping (i.e., expression of cellular markers related to activation) in MESA and did not have data for replication in the other cohorts. Further investigation of monocyte activation and downstream differentiation into macrophages, particularly from lung samples of adults with ILA, will be informative. The cohorts used in our study comprise older community-dwelling adults, which limits the generalizability of the findings. Although we adjusted for age in our models and it did not appear to modify associations between monocyte count and ILA, we cannot completely discount a confounding effect by age. Whether markers of accelerated biological aging modify the association between monocyte count and ILA in cohorts comprising younger adults is a future area of research. Mortality was one of the main reasons for loss of participants between study phases in our ILA progression analysis in AGES-Reykjavik. It is possible that some participants without ILA or with mild ILA at baseline developed rapidly progressive ILA/ILD and died before the repeat evaluation, especially because individuals with ILAs at highest risk for progression are also at increased risk for death. These participants would have been excluded from the ILA progression analyses. We would expect that this would bias our results to the null, because we were not capturing participants with a more rapid progressive type of ILA, and the association between monocyte count and progression of ILA may actually be stronger.

The associations between monocyte count and ILA in MESA and ECLIPSE were less linear at much higher monocyte levels, which is likely driven by a small number of participants, as the association was uniformly linear in the other two cohorts. Although the criteria to define ILA were the same in all of the cohorts, there were some differences in the approach to reading scans. Unless systematically different based on the exposure (i.e., monocyte levels), this should bias our findings to the null, as the actual associations may be stronger than shown. Although higher neutrophil count appeared to be associated with ILA and lower FVC in some of the cohorts, we caution overinterpretation, given the exploratory nature of the analysis with other immune cell types, and the associations were not consistent in all of the available cohorts. The association of monocytes with lower FVC was found in MESA and COPDGene, but not in ECLIPSE. This difference may be related to ascertainment for enrollment into the ECLIPSE study, which primarily selected for smokers with diminished spirometry measures or recent chronic obstructive pulmonary disease exacerbations. Last, the identification of a clinically relevant cutoff for monocyte count in relation to ILA was not an objective of this study. There are key differences in the type of study population (patients with IPF versus population-based/at-risk cohorts) and outcomes (disease severity and progression vs. markers of lung injury/remodeling) between previous studies that have used monocyte count cutoffs and our approach. Importantly, our findings show largely linear and monotonic associations between monocyte count and our outcomes, suggesting a lack of a threshold effect. Research remains ongoing regarding the clinical implications of early detection of interstitial lung changes and what is the appropriate management (12).

In summary, a higher blood monocyte count was associated with a greater burden of interstitial lung abnormalities and its progression on CT imaging and lower FVC among adults. Future human studies that examine monocyte/macrophage subsets and function in early stages of ILD will be informative.