Abstract
Background:
Sufficient levels of club cell secretory protein (CC16) are essential to protect against lung function impairments. Experimental studies have demonstrated that CC16 modulates inflammatory responses and protects against airway hyperresponsiveness following Mycoplasma pneumoniae (Mp) infection. Individuals with asthma have low CC16 levels and increased susceptibility to Mp infection. Here we determine whether low CC16 and Mp seropositivity have combined effects on lung function deficits predisposing to airflow limitation, particularly in asthma.
Methods:
Serum levels of CC16 and IgG antibodies against Mp (MpIgG) were measured in adult participants from cohorts BAMSE, MAAS, LSC, and TESAOD. Participants were then stratified into four groups: normal CC16/MpIgG−, normal CC16/MpIgG+, low CC16/MpIgG−, low CC16/MpIgG+. Associations between these groups and lung function (FEV1 and FEV1/FVC) were assessed by linear regression, adjusting for covariates. Meta-analyzed estimates were calculated.
Results:
Low CC16 was associated with decreased lung function in the total population, but no combined effects of CC16 and MpIgG were observed. Among asthmatic participants, the low CC16/MpIgG+ group had remarkably lower FEV1/FVC z-scores (−0.84, CI: −1.29, −0.38) compared to the reference group, and Mp seropositivity was associated with significant deficits in FEV1/FVC z-scores among those with low CC16 (−0.60, CI: −1.08, −0.12), but not among those with normal CC16 (−0.10, CI: −0.56, 0.36).
Conclusion:
This suggests that individuals with asthma with low levels of CC16 combined with a history of Mp infection may be more susceptible to deficits in FEV1/FVC, the hallmark of airflow limitation, emphasizing the need for prospective studies designed to test this hypothesis.
Keywords: Asthma, CC16, lung function, Mycoplasma pneumoniae
Introduction
Club cell secretory protein (CC16) is a pneumoprotein whose circulating levels are readily measured in serum. Low serum CC16 levels have been implicated in asthma and chronic obstructive pulmonary disease (COPD), and their associated lung function deficits[1-5]. Indeed, prior studies have robustly established the association between low circulating levels of CC16 with negative lung health outcomes in multiple cohorts: ie. accelerated decline in FEV1[6], prevalence of chronic bronchitis[7], and incident COPD[8]. Moreover, in the Severe Asthma Research Program, low expression levels of CC16 from human bronchial epithelial cells not only correlated with asthma and asthma severity, but also with increased corticosteroid use and exacerbation frequency[9].
Experimental models provide supporting evidence for a role for CC16 in attenuating severity of respiratory infections[10], modulating inflammation[11] and protecting against airway remodeling[12]. In this context, multiple experimental CC16 studies have focused on infection by Mycoplasma pneumoniae (Mp), an “atypical” bacterial pathogen that is frequently implicated in community-acquired pneumonia and linked to progression and clinical severity of asthma[13-16]. Infection with Mp of well-differentiated airway epithelial cells, grown at an air-liquid interface in vitro, upregulates secretion of apical proteins related to airway remodeling in mice lacking CC16, which is not evident in their wild-type counterparts[17]. Additionally, in in vivo mouse models of pulmonary infection, a deficiency in CC16 leads to increased airway hyperresponsiveness after Mp infection[18].
Taken together, evidence suggests that low CC16 levels may have combined effects with Mp respiratory infections on lung function, particularly among individuals with asthma. In this study, we used data from participants in four population-based cohorts to examine whether a positive antibody status against Mp influences the documented relation of CC16 and lung function in both the general population and in individuals with asthma.
Methods
Cross-sectional data from the population-based cohorts of the Swedish Child (Barn) Allergy, Milieu, Stockholm Epidemiological Study (BAMSE, age 21 to 25 years)[19], the Manchester Asthma and Allergy Study (MAAS, age 18 to 22 years)[20], the Lovelace Smokers Cohort (LSC, age 40 to 76 years)[21], and the Tucson Epidemiological Study of Airway Obstructive Disease (TESAOD, age 28 to 70 years)[22] were analyzed for this study. Lung function was measured according to ATS/ERS spirometry criteria in respective cohorts[8, 21, 23, 24]. Cryopreserved serum samples from the four cohorts were analyzed in a central laboratory (Airway Genetics and Immunology Laboratory, University of Arizona, Tucson AZ) for CC16 using a commercially available enzyme-linked immunosorbent assay (ELISA) (BioVendor, Asheville NC). Consistent with previous work[12, 25, 26], in main analyses, CC16 measurements were stratified into tertiles and the lowest tertile of CC16 levels (low CC16) was then contrasted against the two higher tertiles combined (normal CC16). CC16 was also used as a continuous variable in sensitivity analyses (see below). Antibodies against Mp (MpIgG) were measured in the same samples for BAMSE (N=496), MAAS (N=309), and LSC (N=437) by the same central laboratory, using target-specific ELISA (Serion Diagnostics, Wurzburg, Germany) that provides cut-off values to determine positivity (MpIgG+) or negativity (MpIgG−). In TESAOD (N=803), Mp IgG levels were measured using a bead-based multiplex assay (Myriad-RBM, Austin TX), which provides ratios of median fluorescence intensities (MFIs) from target-specific microspheres relative to MFIs from negative control microspheres[27]. Multiplex assays do not provide cut-off values to determine positivity. Because the mean seroprevalence of Mp IgG in BAMSE (28.6%), MAAS (27.5%) and LSC (16.7%) was 24.3%, in TESAOD, the highest quartile of Mp IgG levels (MpIgG+) was contrasted against the three lower quartiles of Mp IgG levels (MpIgG−). In all cohorts, individuals were classified into one of the following CC16/MpIgG groups: 1) normal CC16/MpIgG− (the reference group throughout this study), 2) normal CC16/MpIgG+, 3) low CC16/MpIgG−, or 4) low CC16/MpIgG+.
The association of the CC16/MpIgG status with race-neutral Global Lung Initiative (GLI)-derived z-scores of FEV1 and FEV1/FVC was evaluated for each cohort using multivariable linear regressions adjusted for age, sex, categorical body mass index (BMI), and smoking. BMI categories were as follows: underweight (less than 18.5), healthy weight (18.5 to less than 25), overweight (25 to less than 30), and obese (30 or greater). In addition, number of pack-years was included in the regression models for older cohorts (ie. LSC and TESAOD). Sensitivity analyses were performed: 1) using raw values of FEV1 and FEV1/FVC, with additional adjustment for height, 2) using race-neutral GLI-derived percent-predicted (%-predicted) values of FEV1 and FEV1/FVC, 3) including additional adjustment for available environmental and occupational exposures to main models, and 4) testing CC16 as a continuous variable (instead of a categorical low vs non-low CC16 variable). Crude CC16 values, which had a skewed distribution, were log-transformed prior to analysis. All statistical analyses were performed using Stata 18.5 (College Station, TX).
Results
Results confirmed previous findings[1, 12] – including from these same cohorts[7, 8] – that low levels of CC16, regardless of Mp IgG status, are associated with significant decreases in FEV1 and FEV1/FVC, as shown by significant and comparable deficits of the meta-analyzed estimates in the two groups with low CC16 compared to the normal CC16/MpIgG− reference group. This is regardless of whether z-scores, raw values, or %-predicted values were used for FEV1 and FEV1/FVC (Fig 1A-B, SuppFig 1A-B, SuppFig 2A-B, respectively). In contrast, no effects were seen in the normal CC16/MpIgG+ group compared to the reference group (Fig 1A-B, SuppFig 1A-B, SuppFig 2A-B). These results were confirmed when models were adjusted for additional environmental and occupational exposures (SuppFig 3). In addition, the main effects of CC16 were also observed when CC16 levels were tested as a continuous variable (Supp Table 1). Next, as shown in Table 1, the effect of Mp IgG positivity among all individuals was evaluated in the normal and the low CC16 groups. After adjustment for covariates, Mp IgG positivity was not associated with FEV1 or FEV1/FVC among participants with normal or with low CC16. Thus, in the total population, we confirmed decreases in lung function with low levels of CC16 but found no evidence of combined effects between low CC16 and presence of Mp IgG on lung function.
Figure 1. Meta-analysis of associations of CC16 / Mp IgG Ab groups with FEV1 and FEV1/FVC z-scores in the BAMSE, MAAS, LSC and TESAOD cohorts.a.
Forest plots for meta-analyzed estimates (CI) for FEV1 and FEV1/FVC z-scores for A-B) all participants and C-D) asthmatic participants. All tests for heterogeneity, p > 0.05.
Sample sizes for meta-analysis among all participants:
BAMSE = 496, MAAS = 309, LSC = 437, TESAOD = 803; Combined groups of normal CC16/MpIgG− = 1023, normal CC16/MpIgG+ = 353, low CC16/MpIgG− = 523, low CC16/MpIgG+ = 146
Sample sizes for meta-analysis among asthmatic participants:
BAMSE = 65, MAAS = 50, LSC = 134, TESAOD = 96; Combined groups of normal CC16/MpIgG− = 157, normal CC16/MpIgG+ = 48, low CC16/MpIgG− = 113, low CC16/MpIgG+ = 27
aLinear regression covariates: age, sex, smoking status, pack-years (for LSC and TESAOD), BMI
Table 1.
Effect of Mp IgG positivity on lung function in all participants with normal and low CC16a
| Among participants with normal CC16 | Among participants with low CC16 | |||
|---|---|---|---|---|
| FEV1 z-scores | N | Estimate (CI) | N | Estimate (CI) |
| BAMSE (N = 496) | 339 | −0.07 (−0.26, 0.12) | 157 | −0.03 (−0.31, 0.26) |
| MAAS (N = 309) | 207 | −0.01 (−0.29, 0.27) | 102 | 0.13 (−0.28, 0.54) |
| LSC (N = 437) | 294 | −0.10 (−0.43, 0.24) | 143 | 0.09 (−0.44, 0.62) |
| TESAOD (N = 803) | 536 | −0.11 (−0.34, 0.11) | 267 | 0.18 (−0.17, 0.52) |
| Meta-analysis | 1376 | −0.07 (−0.19, 0.05) | 669 | 0.07 (−0.11, 0.26) |
| FEV1/FVC z-scores | ||||
| BAMSE (N = 496) | 339 | −0.10 (−0.31, 0.11) | 157 | −0.09 (−0.41, 0.22) |
| MAAS (N = 309) | 207 | 0.03 (−0.30, 0.36) | 102 | −0.34 (−0.82, 0.15) |
| LSC (N = 437) | 294 | −0.24 (0.53, 0.06) | 143 | −0.05 (−0.53, 0.42) |
| TESAOD (N = 803) | 536 | 0 (−0.25, 0.25) | 267 | 0.01 (−0.37, 0.39) |
| Meta-analysis | 1376 | −0.08 (−0.21, 0.05) | 669 | −0.10 (−0.30, 0.10) |
Linear regression covariates: age, sex, smoking status, pack-years (for LSC and TESAOD), BMI
The meta-analysis was then restricted to participants with asthma in each of the cohorts to test the interrelationships among low CC16, Mp IgG positivity, and lung function. Again, the groups with low CC16, as compared to the reference group, had large deficits for both FEV1 and FEV1/FVC, regardless of whether z-scores, raw values, or %-predicted values were used (Fig 1C-D, SuppFig 1C-D, SuppFig 2C-D, respectively). However, for FEV1/FVC (though not FEV1), as compared to the reference group, meta-analyzed deficits were most pronounced in the low CC16/MpIgG+ group at a z-score of −0.84 (CI: −1.29, −0.38) (Fig 1D), which equates to a %-predicted deficit of −7.6% (CI: −11.7, −3.6) (SuppFig 2D). In line with these results, Mp IgG positivity had a significant effect on FEV1/FVC z-scores among asthmatic participants with low CC16 (−0.60, CI: −1.08, −0.12) but not among asthmatic participants with normal CC16 (−0.10, CI: −0.56, 0.36) (Table 2).
Table 2.
Effect of Mp IgG positivity on lung function in asthmatic participants with normal and low CC16a
| Among participants with normal CC16 | Among participants with low CC16 | |||
|---|---|---|---|---|
| FEV1 z-scores | N | Estimate (CI) | N | Estimate (CI) |
| BAMSE (N = 65) | 37 | −0.02 (−0.81, 0.77) | 28 | −0.04 (−0.78, 0.71) |
| MAAS (N = 50) | 32 | −0.09 (−0.93, 0.75) | 18 | 0.39 (−0.77, 1.55) |
| LSC (N = 134) | 78 | −0.14 (−0.87, 0.58) | 56 | −0.29 (−1.17, 0.58) |
| TESAOD (N = 96) | 58 | −0.28 (−1.06, 0.50) | 38 | −0.27 (−1.55, 1.01) |
| Meta-analysis | 205 | −0.14 (−0.53, 0.25) | 140 | −0.07 (−0.55, 0.40) |
| FEV1/FVC z-scores | ||||
| BAMSE (N = 65) | 37 | 0.60 (−0.25, 1.44) | 28 | −0.87 (−1.67, −0.07) |
| MAAS (N = 50) | 32 | −0.45 (−1.26, 0.36) | 18 | −0.51 (−1.62, 0.61) |
| LSC (N = 134) | 78 | −0.41 (−1.05, 0.23) | 56 | −0.12 (−0.89, 0.64) |
| TESAOD (N = 96) | 58 | 0.02 (−0.76, 0.81) | 38 | −1.33 (−2.62, −0.05) |
| Meta-analysis | 205 | −0.10 (−0.56, 0.36) | 140 | −0.60 (−1.08, −0.12) |
Linear regression covariates: age, sex, smoking status, pack-years (for LSC and TESAOD), BMI
Discussion
These studies with population-based cohorts confirm the association of low CC16 with deficits of FEV1 and FEV1/FVC both in the general population and among asthmatic participants, and suggest that individuals with asthma who have low levels of CC16, accompanied by a history of Mp infection, are more susceptible to having decreased FEV1/FVC, the hallmark of airflow limitation.
While this combined effect is consistent with previous studies showing the protective contributions of CC16 against inflammation and airway remodeling resulting from Mp infections[10, 17, 18], these results should be considered in the context of some limitations. The combined effect of low CC16 and MpIgG+ among participants with asthma was present for levels of FEV1/FVC ratio and not for FEV1, although the reduced sample size of analyses restricted to asthmatic individuals may have limited statistical power. There were cohort differences in multiple factors (including age ranges and prevalence and intensity of smoking) that are known to influence CC16 levels[25, 28] as well as susceptibility to infections[29, 30]. For instance, participants in LSC and TESAOD included older adults, while BAMSE and MAAS only included young adult participants. Similarly, while individuals in LSC were all smokers with ≥10 pack-years, less than 20% of BAMSE and MAAS participants were active smokers. Methods to determine Mp IgG positivity varied between TESAOD and the other cohorts, and blood samples came from different temporal windows. Although we recognize this inter-cohort heterogeneity, and variability in available data across cohorts limited our ability to systematically test the effects of these differences, it is notable that the main finding of lower FEV1/FVC z-scores among asthmatic participants with low CC16 and positive Mp IgG was consistent across the four cohorts (Fig 1D) and meta-analyses that were conducted did not return a significant heterogeneity test.
Serological testing at a single time point cannot comprehensively capture temporal trends of respiratory infections and immune responses against Mycoplasma pneumoniae. Thus, although our study indicates possible effects of Mp infections in asthmatic participants with low CC16, these results warrant future prospective studies specifically designed to determine whether low CC16 levels in individuals with asthma are linked to increased susceptibility to respiratory infections by Mp (and other pathogens) and, in turn, to the resulting lung function impairments related to airflow limitation in both children and adults[31].
Supplementary Material
Highlights.
• Low circulating CC16 levels are associated with significant deficits in lung function in the total population.
• Asthmatic individuals with a combination of low circulating CC16 levels and Mp seropositivity have lower FEV1/FVC levels.
• Mp seropositivity is linked to FEV1/FVC deficits in asthmatic individuals with low CC16, but not in those with normal CC16.
Acknowledgements
The authors sincerely thank Debra Stern, MS, MEd for her critical review of the methods of analysis of this work and Gang Wang, PhD for his efforts in coordinating data sharing.
Declaration of funding sources
This work was funded by the following grants: NIH T32HL007249 (ABCD); NIH HL142769 (JGL); NIH AI135108 (SG); NIH P30CA118100 (Y. Sanchez for SAB); NIHR203308 (AG and CM)
Footnotes
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Declaration of interests
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Clare Murray reports a relationship with AstraZeneca that includes: speaking and lecture fees. Clare Murray reports a relationship with Sanofi that includes: speaking and lecture fees. Clare Murray reports a relationship with GlaxoSmithKline that includes: speaking and lecture fees. Stefano Guerra and Julie Ledford are co-founders of a start-up, Aspiro Therapeutics, with the goal of developing novel CC16-based therapeutics. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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