What Role Do Immunological and Inflammatory Biomarkers Play on Mortality and Exacerbations in COPD Patients?

Background: COPD phenotypes have been identified according to patient clinical characteristics and, more recently, to their immunological markers. However, the role of these biomarkers in relation to exacerbations in different COPD phenotypes is not yet clear. Thus it is important to continue investigating this topic to plan the most appropriate treatment, to contrast the decline of respiratory function and to reduce subsequent exacerbations in COPD patients.

Therefore the aim of this study was to assess whether biomarkers are only predictors of exacerbations and mortality in COPD patients or instead play a causal role in these adverse COPD developments.

Methods: COPD phenotypes were identified according to patient clinical characteristics or immunological markers. Immunological and inflammatory biomarkers were identified as predictors of mortality and exacerbations.

COPD patients were identified from the hospital discharge register of Pisa (Italy) in 2000-2006, on the basis of diagnoses and a FEV1/FVC < 0.70, and enrolled in 2003-2006 according to a longitudinal approach. They were characterized at enrollment by age, sex, comorbidities, immunological and inflammatory biomarkers. The phenotype was assigned at enrollment as eosinophilic (eosinophils>2%) or neutrophilic (leukocytes>11.0x109/L and/or neutrophils>65%). Mortality and exacerbations registered in the period 2003-2012 were assessed by phenotype and other possible predictors in regression models.

Results: Mortality rates reached 43.3% in COPD patients: 69.1% and 36.5% in neutrophilic and eosinophilic, respectively. The exacerbation rate was higher in neutrophilic (54.4%) than in eosinophilic patients (48.4%). Uni-variable regression confirmed that mortality was related to age, Congestive Heart Failure (CHF), Ischemic Heart Disease (IHD), elevated neutrophil count and Neutrophil to Lymphocyte Ratio (NLR). At multivariable analysis only age was associated with mortality, whereas inflammatory biomarkers induced exacerbations.

Conclusion: Mortality was influenced by phenotype and age, exacerbations by COPD severity and NLR to suggest that an appropriate treatment and the biomarker’s monitoring might help prevent exacerbations.

Chronic Obstructive Pulmonary Disease (COPD) is “a common, preventable and treatable disease that is characterized by persistent respiratory symptoms and airflow limitations usually caused by significant exposure to noxious particles or gases” [1].

Exacerbations defined as “acute worsening of respiratory symptoms beyond day-to-day variation that leads to a change in medication”, and comorbidities defined as “concomitant chronic diseases of COPD”, both have a significant impact on the disease prognosis, increasing COPD severity, hospitalizations and mortality [1-3].

Different phenotypes of COPD have been identified in recent years on the basis of their clinical characteristics and progression. The inflammatory characteristics of phenotypes have been related to the etiology and prognosis of fixed airflow obstruction, given that patients with a history of asthma showed significantly more eosinophils in blood, sputum and airway mucosa, and greater reversibility with bronchodilators and steroids [4-6] than COPD patients showing, in contrast, neutrophil exacerbations rather than eosinophilic inflammation [7], were usually corticosteroid resistant [8] and showed a significantly worse survival rate than asthmatics [9,10].

An interesting recent paper [11] reminds us that not all COPD exacerbations are alike, and even questions the possibility of identifying etiology or planning the treatment of COPD patients without the support of immunological and inflammatory biomarkers. Moreover, Bafadhel [12,13] had already identified four phenotypes of COPD (bacterial, viral, eosinophilic and pauci-inflammatory) referring to their etiology taking also into account the immune systemic response which aids in choosing an effective treatment for these patients. The role of immunological and inflammatory biomarkers in COPD needs further study, since inflammation and exacerbation do not always overlap in COPD, especially in patients with a neutrophilic phenotype, for whom bacterial clearance of macrophages is defective [14].

The role of inflammatory biomarkers is not very clear as a predictor of exacerbation: only C Reactive Protein (CRP), together with at least one major COPD symptom, has proved helpful in diagnosing exacerbation, but changes are unrelated to both clinical severity and recovery according to other authors [15]. In addition, exacerbations are not associated with high levels of CRP in eosinophilic phenotypes [13]. More recently, increasing erythrocyte sedimentation rate and Neutrophil to Lymphocyte Ratio (NLR) were reported as biomarkers of COPD exacerbations [16,17] but these results are not yet well established.

On the other hand, immunological and inflammatory biomarkers have been identified as predictors of mortality and frequent hospital readmissions in COPD patients. For example, the persistence of a high serum CRP concentration predicts recurrent exacerbation episodes [18]. A high NLR predicts both frequent hospitalizations and an increased mortality rate [19]. Finally, a recent systematic review and meta-analysis [20] reported a higher risk of mortality for COPD patients with increasing fibrinogen, CRP and white cell counts as well as a higher risk of exacerbations with elevated fibrinogen and CRP levels.

In the light of these observations, it seems reasonable to investigate the role of inflammatory biomarkers on COPD exacerbations and mortality, and whether phenotype identification by means of immunological and clinical criteria, helps indicate the most appropriate treatment to retard the decline of respiratory function and reducing subsequent exacerbations. We aimed to assess whether biomarkers are only predictors of COPD exacerbations and mortality or play a causal role respect these COPD adverse developments.

In this study, we identified a group of COPD patients in terms of their clinical, immunological and inflammatory characteristics and studied subsequent exacerbations and mortality by phenotype, taking into account other well-known predictors of outcomes such as age, comorbidities [1] and COPD severity [21].

The study population

We assumed that immunological and inflammatory biomarkers CRP and NLR, predict outcomes (both mortality and exacerbations) in COPD patients, though their causal role is not clear.

A COPD case was defined as a 40-plus-year-old subject discharged from hospital with COPD [ICD-9: codes 490-492, 494, 496] as principal or secondary diagnosis, being heart failure or pneumonia, or respiratory failure the first diagnosis.

A COPD case was also a 40-plus-year-old subject with an outpatient diagnosis of COPD confirmed by a ratio of One-Second Forced Expiratory Volume (FEV1) to Forced Vital Capacity (FVC) <0.70, at spirometry [1].

COPD cases were obtained from Pisa (88,627 inhabitants), in central Italy. All the data were from the city’s Hospital Discharge Register (HDR). Hospital cases were enrolled in the period 2003-2006 among all COPD hospitalized patients and according to a 4-year longitudinal approach as described in a previous study [22]. Outpatient cases were sought between 2000 and 2006 and enrolled between 2003 and 2006 as were hospital cases. COPD patients were characterized at enrollment by demographic and clinical characteristics: age, sex, and comorbidities reported at hospital diagnoses in the 3 years preceding enrollment; immunological markers (total leukcocytes, neutrophils, eosinophils, lymphocytes); inflammatory biomarkers (CRP and NLR); and severity of airway obstruction, in accordance with the progressive reduction of FEV1. Laboratory and spirometry data were from the Institute of Clinical Physiology (ICP) of the National Research Council (NRC). Subjects did not participate in person in the study, administrative and medical databases being used in accordance with the privacy laws in effect in Italy; clinical charts were consulted by researchers from the ICP–NRC and patient records were anonymized and de-identified prior to statistical analysis.

Phenotype assessment

Phenotypes were assigned to patients at enrollment on the basis of one or more (up to 4 consecutive days in a week) blood counts carried out during the three months preceding enrollment, or in the course of the three months following it (for a total of 96 out of 289 patients) [18]. A further 131 out of 289 patients were phenotipically classified on the basis of blood tests available 1-year-before and 5-years-after enrollment. This second group was included after comparing their demographic and clinical characteristics with those of the "three months preceding enrollment" group and if their phenotype was confirmed by at least one other blood test in the range longer - to support the underlying hypothesis of a stable phenotype.

The phenotype was defined as eosinophilic when the proportion of eosinophils to leukocytes was >2%. It was defined as neutrophilic when the leukocyte count was >11.0x109/L and/or the neutrophil percentage >65% [23]. The phenotype was not assigned when only one blood test was performed (without a retest) or when both neutrophils and eosinophils were in the reference ranges.

The outcomes

Mortality and exacerbations occurred from 2003 to 2012 were the outcomes, with a period of follow-up ranging from 6 to 10 years after enrollment. Mortality data were from the city mortality register, and exacerbations were identified from the city hospital register, which show both of these data for all residents. We assumed that hospitalizations were the best approximation of severe exacerbations [9,13] which rely only on clinical symptoms (dyspnea, cough, and/or sputum production) in COPD patients [1]. To be analyzed as exacerbations, the hospitalizations should be urgent and show COPD as the principal diagnosis - or the secondary diagnosis when the principal one is a serious comorbidity. In addition, patients’ hospitalizations were spaced 3 months apart to be included as independent events.

The analyses

Our main research questions were whether mortality and exacerbations are influenced by the COPD phenotype and whether other predictors modifiy or confound the principal associations between phenotypes and outcomes.

We therefore first assessed mortality and exacerbation rates in COPD patients with eosinophil and neutrophil phenotypes, unassigned phenotypes, and total COPD population (Tables 1 &2); the second comparison also aims to exclude a possible selection of cases, due to the higher probability of tracing blood tests for patients hospitalized at the ICP than elsewhere. Secondly, we assessed the distribution of other possible predictors of COPD outcomes, such as age, comorbidities, airway obstruction severity, immunological and inflammatory biomarkers by phenotype (Table A1). We also assessed the possible interaction of each predictor (one at a time) with the main associations between phenotype and each outcome.

Multiple regression models were then used to assess the confounding of the main associations between phenotype and outcomes, due to the other factors. Data of five under 50 years old subjects and those of 62 subjects without an assigned phenotype were excluded from both regression analysis.

Our secondary questions were whether changes in biomarkers are related to death or to hospitalizations in COPD patients and whether these associations differ between phenotypes. This second analysis also allows us to assess whether biomarkers found in exacerbations relate to the phenotype assigned at enrollment. The cut-offs for immunological markers during exacerbations matched those at enrollment, while the cut-offs of the two inflammatory markers were 3.29 for NLR and 1.17 mg/dl for CRP [16].

Overall, 2337 subjects in Pisa received a diagnosis of COPD during the studied period. Of these, 2048 (87.6%) did not respond to our definition of “case” or had no available respiratory function tests (n.1) or blood count tests (n.31). Of the 289 patients included in the analysis, 159 (55.0%) were classified as eosinophilic phenotype and 68 (23.5%) as neutrophilic phenotype; 24 (8.3%) showed both eosinophils and neutrophils in the normal range, and 38 (13.1%) had only one blood count test, without confirmation. No phenotype was assigned to patients in the last two groups. Thus the phenotype was assigned to 227 patients. An additional table shows the different phenotypic COPD according to different standards in more detail (see Additional file 1, table A1). We have an initial comment about the higher proportion of eosinophilic as compared to neutrophil phenotypes in our population This is unusual among COPD patients, in that the burden of eosinophil phenotype is estimated as nearly 30% [12, 23] and about 40% in the USA population [24]. Only a large population cohort in the USA (NHANES) [25], reports a higher proportion of COPD patients with eosinophil than with neutrophil phenotype. The eosinophil phenotype patients in this study are of relatively advanced age and males and suffered from severe asthma or hay fever. In our data, asthma is reported in only 2 out of 159 eosinophilic COPD patients. Then we cannot exclude the shadow of a selection of less serious cases among our hospitalized COPD patients.

Mortality rates (Table 1) reached 43.3% in all the COPD patients we studied, with notable differences between phenotypes: the rate was 69.1% (95%CI:58.1%–80.1%), 36.5% (95%CI:30.0%–44.0%) and 32.3% (95%CI:20.6%–43.9%) in patients with neutrophil, eosinophil and unassigned phenotype, respectively. Rates are clearly higher among neutrophil phenotype patients than in those with an unassigned phenotype, and also higher than in patients with an eosinophilic phenotype. An additional table shows the mortality in more detail (see Additional file 1, table A2).

Neutrophil phenotype patients also showed a less clear trend of mortality across the age groups than either eosinophilic and unassigned phenotype patients. A high level of neutrophils in peripheral blood was related to the progression of the COPD independently of the different mechanisms of their increase [26]. This could explain what we observed, though it is not sufficient to prove that clinical worsening is more responsible for inducing death than age in neutrophil phenotype patients (mean age 73.7 yrs) as compared with those with a normal white blood cell count (mean age 66.4 yrs).

Among the other possible predictors of mortality, in neutrophil phenotype patients more than in those with an eosinophilic phenotype were more frequent cardiovascular diseases, more severe airway obstruction and higher levels of both inflammatory biomarkers (CRP and NLR) (Table A1).

Of the 289 COPD patients enrolled, 237 (82%) had at least one hospitalization subsequent to enrollment, in 138 (58.2%) of them hospitalization was due to COPD exacerbations, and in the other 99 (41.8%) hospitalization was due to health problems unrelated to COPD. Thus, exacerbations reached a barely higher rate than that of mortality, with once again a considerably higher result for neutrophil (54.4%; 95%CI:42.6%-66.2%, 37) than for eosinophil phenotype (48.4%; 95%CI:40.7%–56.2%, 77). Mortality following exacerbation also showed a higher rate (78.4%; 95%CI:65.1%–91.6%) in neutrophil than in eosinophil phenotype (46.8%; 95%CI:35.6%–57.9%). Finally, patients with unassigned phenotype had a lower rate of exacerbations (38.7%; 95%CI:26.6%–50.1%), than either eosinophil or neutrophil phenotype patients.

Among COPD patients with at least one hospital exacerbation, 50% of both neutrophil and eosinophil phenotype patients had only one re-hospitalization, while the other 50% of both groups had repeated exacerbations. These proportions differ in patients with unassigned phenotype, of whom only 29% had repeated exacerbations, and 71% had only one re-hospitalization (Table 2). These data, together with the lowest rate of hospital exacerbations, would suggest that COPD patients with unassigned phenotype have a less serious form of the disease in our population. Again, among neutrophil phenotype patients, there was no appreciable exacerbation trend across the age groups, unlike eosinophilic patients, suggesting that an increased neutrophil count is related to subsequent exacerbations, as it is for mortality.

Patients without hospital COPD exacerbations were 151 (52.2% of total COPD population), 52 of whom (34%; 95%CI:21.5%–47.4%) were not hospitalized thereafter, and 99 (66%; 95%CI:56.2%–74.9%) were hospitalized with diseases unrelated to COPD. Among patients without exacerbations, mortality reached 31.4% (95%CI:18.6%–44.1%) in eosinophil and 60% (95%CI:38.5%–81.5%, 12) in neutrophil phenotypes, respectively. These frequencies are very similar to those observed in patients with repeated COPD exacerbations, suggesting that COPD patients have a high mortality risk from diseases other than exacerbations, probably from comorbidities. The single variable regression models (Table 3) confirm that the neutrophil phenotype is related, in our data, to a higher mortality than that of the eosinophil phenotype (OR = 3.70; 95%CI:2.01-6.81). Mortality is also related to increasing age (OR = 1.09; 95%CI:1.05-1.13, p<0.001) but not to sex. It is strongly influenced by heart diseases but with a wide variability: particularly by congestive heart failure (CHF) (OR = 5.39; 95%CI:1.14-25.50, p = 0.020), and Ischemic Heart Disease (IHD) (OR = 4.63; 95%CI:1.79–11.97,p = 0.001), but not by airway obstruction severity. Finally, mortality is related to a higher count of neutrophils (OR = 1.57; 95%CI:1.33-1.86, p < 0.001) at enrollment - independently of the phenotype assessed just the same at enrollment - as well as to a higher NLR (OR = 1.52; 95%CI:1.27-1.81, p < 0.001) but unrelated to CRP levels. In multivariable analysis (Table 4), only advanced age at enrollment was confirmed to be associated with mortality (OR = 1.07; 95%CI:1.00–1.13, p = 0.04), whereas the role of inflammatory biomarkers emerged in inducing exacerbations. In particular, only NLR was identified as independently related to exacerbations (OR = 1.87; 95%CI:1.10-3.16, p = 0.02), unlike CRP, for which correlation to exacerbations did not reach statistical significance. When we included only the other risk factors in the analysis (Table 5), both a neutrophil phenotype and CHF emerged as predictors of mortality in COPD patients, whereas COPD severity was related to a higher probability of exacerbation.

We found differences between patients with neutrophil and those with eosinophil phenotype, in outcomes, severity of obstruction and cardiovascular comorbidities. Mortality and hospital exacerbations were more frequent in neutrophil than in eosinophil phenotype patients and even more so in the total COPD population.

Given that a prospective validation regarding clinical outcomes is an essential point in defining phenotypes [27], we might conclude that including immunological biomarkers to assess phenotypes could help in choosing a specific treatment for bacterial infections, in preventing the decline of respiratory function, and in managing cardiovascular comorbidities.

However, the so-called clinical phenotypes [28] include diagnoses frankly oriented towards specific treatments, such as alfa-1 antitrypsin phenotype or emphysema/hyperinflation, which improves with the surgical reduction of lung volume. Other clinical phenotypes named “frequent exacerbators” or “persistent inflamed” show elevated levels of inflammatory or immunological markers. Finally, the “chronic bronchitis phenotype” is characterized by frequent exacerbations, greater airway obstruction and more frequent cardiovascular comorbidities, which are all characteristics that closely resemble our neutrophil phenotype. It is worth remembering that a neutrophil phenotype for COPD was proposed by Sin who hypothesized neutrophil mediated lung damage [26].

In our population, other factors predict, independently, either mortality or exacerbations in both phenotypes, i.e., age, comorbidities, declined respiratory function and systemic inflammation. We observed that neutrophil phenotype patients are older and have more frequent cardiovascular comorbidities and a greater severity of airway obstruction, as well as higher levels of both inflammatory and immunological markers.

Regarding age, a recent analysis reinforced the evidence of the COPD mortality trend due to demographic changes, by excluding the mortality proportion attributable to gross national income per capita and cumulative smoking exposure [29].

Since the '90s it is generally known that cardiovascular comorbidities and mortality are both higher in COPD cohorts than in the general population [30]. More recently, it has been recognized that the prognosis for combined COPD and CHF is poorer than for either disease alone, and present many therapeutic challenges [31]. Long-term mortality is also higher for COPD patients after a myocardial infarction [32]. In addition, patients with combined COPD and IHD have a worse outcome than those suffering from COPD or IHD only and also show an increasing risk of hospital readmissions for recurrent myocardial infarction, heart failure, coronary revascularization, and acute exacerbation of COPD [33]. The increased risk of death in COPD patients with both CHF and IHD, is no longer appreciable in multivariable models. Paucity of patients with complete data about immunological and inflammatory biomarkers (Table 5) or possible selection of patients at hospitalization might explain this result in our data.

We measured COPD severity by FEV1, although a faster progression of functional impairment occurs in GOLD stage II [34]. Lung function decline is more clearly defined by severity of airflow obstruction as proposed by the GOLD in 2018 [35], but the clinical data was not then available. The sensitivity analysis (based on the LLN threshold) showed a somewhat lower COPD prevalence [22]. In the literature, COPD severity also refers to exacerbation frequency [36] in critically ill patients requiring Intensive Care Unit (ICU), who show higher mortality than exacerbated patients who do not need ICU [37]. We did not find that the number of previous hospital exacerbations is a predictor of mortality. The results on this topic are inconsistent in the literature: some authors conclude that previous hospitalization is the main risk factor for exacerbations [38], whereas other authors observe no relationship between mortality and number of previous exacerbations [36].

Among inflammatory biomarkers, CRP has been reported in the literature since 2007 as a predictor of all-cause mortality in COPD patients [39]. COPD patients with high levels of CRP, fibrinogen and white blood cell counts have increased mortality and exacerbation frequency [40,41].

In addition, the NLR plays an important role in predicting both mortality [42] and exacerbations [19]. These results, however, do not definitively clarify whether these outcomes are independently predicted by NLR or are related in sequence in the same chain of events.

We conclude that mortality in our COPD population was influenced by immunological phenotype and age, as well as by CHF, whereas exacerbations were influenced by the severity of air-way obstruction and were predicted by inflammatory biomarkers, such as the NLR. All of which suggests that an appropriate treatment of chronic COPD and the monitoring of biomarkers could help prevent hospital exacerbations. Moreover immunological phenotype, age, CHF, inflammatory biomarkers such as NLR could be considered in an integrated approach in order to aid doctors in identifying high-risk individuals. This could facilitate implementation of tailored interventions to prevent exacerbations.

A brief comment deserves to be made on the nature of the explicative factors used in this study. Risk factors and predictors are frequently used interchangeably in both medical and epidemiological literature. This is acceptable when no hypothesis is made about the possible causal role of analyzed factors and is supported also by the same statistical methods, like regression. However, the predictive (or prognostic) factors allow to anticipate the disease progression but not to assess its causes. Nevertheless, a temporal relationship exists between predictors and patient worsening, given that clinicians use predictors of disease progression to adjust the patients’ therapy. In contrast, when it is hypothesized that the factors represent a condition, a characteristic or an exposure that increases the probability of suffering an event, they should better defined as risk factors and the statistical models should take into account their hypothesized role.

Not applicable.

Authors’ contribution

Conceived and designed the experiments: AMR RP AP. Analyzed the data: AMR MR.

Wrote the paper: AMR.

All authors contributed toward data analysis critically and interpretation revising the paper, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Availability of data and materials

The datasets used and/or generated and/or used during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declared no potential conflict of interest whit respect to the research, authorship, and/or publication of this article.

Supplementary tables (A1, A2, A3).

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