Severe physical inactivity in chronic obstructive pulmonary disease: changes in systemic inflammation after pulmonary rehabilitation – a pilot study


 Background The purpose was to investigate whether severe physical inactivity (SPI) is associated with altered levels of common inflammatory markers as compared to higher Physical activity levels (PALs), and whether pulmonary rehabilitation would reduce the levels of systemic inflammation in SPI in patients with COPD. Methods This prospective, cohort study was conducted in four rehabilitation centers in Region Zealand, Denmark. We included patients with COPD referred for 7-12 weeks of pulmonary rehabilitation (2-hours of exercise therapy and education twice weekly). We measured b-eosinophils, p-fibrinogen, p-CRP, s-IL-6, s-CD 163, nasal lavage-IL-8, and daily physical activity, using a validated activity monitor, SenseWear®, at baseline and after rehabilitation. SPI was defined as PAL <1.4.Results In total, 31 of 57 patients were SPI at baseline, and seven (23%) of these patients improved to non-SPI after pulmonary rehabilitation. We observed no significant differences in the primary endpoint i.e. change in systemic inflammation between patients who remained SPI at both time pints vs. patients who improved PAL from SPI at baseline to non-SPI at follow-up. At baseline, SPI was associated with significantly higher p-fibrinogen levels (p=0.04) than non-SPI, but no other differences were observed between SPI and non-SPI Conclusion In a cohort of patients with COPD attending pulmonary rehabilitation, we could not demonstrate any differences or changes in systemic inflammation or PAL following rehabilitation between patients with SPI and non-SPI. This might be a true negative finding or due to a high pro-inflammatory drive in all patients regardless of SPI status or study limitations such as suboptimal levels of rehabilitation intensity and SPI cut-off level. The study was registered at ClinicalTrials.gov (NCT01700296), https://register.clinicaltrials.gov. Registered in November 2012.

poor nutrition, low physical activity level (PAL) and obesity are known to increase systemic and/or local in ammation in COPD 10,13,14 . The association between physical inactivity and systemic in ammation is particularly interesting as it is present not only in COPD, but across a range of chronic conditions 15,16 thereby potentially contributing to the link between systemic in ammation, COPD disease severity and comorbidities. As exercise therapy and other types of physical activity is known to be antiin ammatory 17 , physical inactivity constitutes a highly relevant target for treatment to reduce in ammation and disease symptoms. This is further substantiated by the fact that exercise therapy, typically as part of pulmonary rehabilitation, improves health-related quality of life and exercise capacity in the general population of patients with COPD 18,19 . It is unknown, whether SPI is associated with worse systemic in ammation as compared to higher PALs in patients with COPD, and whether pulmonary rehabilitation can reduce systemic in ammation in patients with SPI. This is important to know to be able to individualize treatment in patients with COPD and SPI. Therefore, we aimed at investigating whether SPI is associated with altered levels of common in ammatory markers as compared to higher PALs, and whether pulmonary rehabilitation (exercise therapy and education) would reduce the levels of systemic in ammation in SPI. We hypothesized, that patients with SPI would have increased systemic in ammation and that measurable improvements in PAL after pulmonary rehabilitation -especially changing from SPI to non-SPI -would lead to measurable reduction in systemic in ammation.

Design
We have recently published the clinical data and study design 5 . Brie y, this was a prospective, observational cohort study conducted in 2013-2014 at four pulmonary rehabilitation units in Region Zealand, Denmark. The reporting of this study adheres to the STROBE guidelines 20 The study was approved by the Danish National Ethical Committee, and complied with the Declaration of Helsinki. Written and oral informed consent was obtained from all participants.

Participants
Patients referred for pulmonary rehabilitation were included if they ful lled the following eligibility criteria: physician-diagnosed COPD; no exacerbations in the past four weeks prior to inclusion, ability to give informed consent, no life-threatening comorbidity (eg. malignancy or severe heart failure), and no history of asthma.

Rehabilitation
The reporting of the rehabilitation adheres to the TIDieR guidelines 21 . Pulmonary rehabilitation was conducted according to Danish national guidelines as a multidisciplinary, non-pharmacological intervention using supervised exercise therapy and patient education 22 . Patients attended either one of three municipal rehabilitation centers or a hospital-based rehabilitation program. As this was an observational study, the duration of the program varied between seven and twelve weeks due to differences between the individual rehabilitation programs. Other than that, the content of the rehabilitation program was similar according to the national treatment guidelines. The program consisted of two-hour sessions twice weekly: one hour of exercise therapy, equally distributed between endurance and strength training under supervision of a physical therapist, and one hour of patient education by an assigned nurse or other relevant professionals e.g. oxygen specialist. Initially, 30 min of strength training was performed in weight training machines for mm. pectoralis major, deltoid, biceps brachii, triceps and quadriceps. Patients performed 3 x 12 repetitions at 50-80% of one repetition maximum (1-RM). The 1-RM test was repeated halfway to re-establish the workload. Subsequently, 30 min. of endurance training was performed at moderate intensity adjusted individually to level 14-15 on the Borg scale of perceived exertion. The endurance training included either cycling on an ergometer or walking on a treadmill. Education included disease-, nutritional-and pharmacological education in addition to smoking cessation assistance to optimize the performance and autonomy of patients with chronic respiratory impairment.

Outcomes
The participants were examined twice: at rst day of the rehabilitation course (before the rst rehabilitation session (baseline), and in the week after completing the rehabilitation (follow-up).
Primary endpoint: Between-groups differences in change of in ammatory markers levels between patients with SPI at baseline who at follow-up still have SPI vs. those improving to non-SPI.

Secondary endpoints:
Baseline differences in levels of in ammatory markers between patients with SPI vs. non-SPI.

Protocol deviations
The current study deviates from our original protocol (clinicaltrials.gov number NCT01700296) in two ways. Firstly, the Ethics Committee disapproved of including a control group not attending the physicianrequested pulmonary rehabilitation. Secondly, we chose to examine the short-term outcome as long-term systemic in ammation is affected by many non-COPD related factors, such as rhinitis, cardiovascular diseases, chronic liver disease etc., which we could not control or adjust for.

Basic measurements
Our publication on clinical characterization of SPI in COPD included details on measurements of e.g. forced expiratory volume in rst second (FEV 1 ), 6-minute walk test, and patient-reported outcomes. For further details on the results of these measurements please refer to reference 5 .
Objective measurement of physical activity including Severe Physical Inactivity PAL was measured with an activity monitor (SenseWear © Armband, Bodymedia, Pittsburgh, PA) continuously worn for seven days after each study visit. SenseWear © has previously been validated for use in COPD 1 . The activity monitor is worn on the back of the upper right arm at the level of the triceps. It assesses accelerations in two planes using a bi-axial accelerometer, and measures and stores skin temperature, near body temperature, heat ux, and galvanic skin resistance. Output from the SenseWear © includes total energy expenditure, daily time in sedentary-, light-, moderate-and hard activity as well as number of steps.
In the current study, PAL was calculated as total daily energy expenditure divided by resting energy expenditure in kcal measured by the activity monitor. Based on a previous study cut-offs for PAL were de ned as PAL ≥1.70: active person, 1.40-1.69: sedentary person; and <1.40: SPI 23 .
Data from patients with ≥two days of ≥90% wearing time were included.
Serum and plasma samples: 5 mL samples of both serum and plasma were collected in sterile tubes and centrifuged, and the supernatant was kept for later analysis of the in ammatory markers IL-6, IL-8 and CD 163 at -80 0 C. Levels of biomarkers were measured at The Respiratory Research Unit, Bispebjerg University Hospital, using commercial ELISA kits (Human IL-6, IL-8 and sCD163 Quantikine high sensitivity ELISA; R&D Systems, Minneapolis, MN, USA).

Nasal lavage
Nasal lavage was performed using a technique adapted from Hilding and Hurst 10,26 . A 12F Foley catheter, modi ed by removal of the tip distal to the balloon, was inserted into the nostril and in ated with su cient volume of 0.9% saline solution to form a comfortable seal, typically 7 to 10mL. With the head of the patient exed forward, a volume of 7 mL 0.9% saline solution was instilled through the catheter, and was washed in and out of the nasal cavity three times using a 100 mL syringe. The nasal wash was collected in sterile tubes and stored at -80 0 C, for later analysis of IL-8.

Sample size calculation
The sample size calculation was based on the clinical study 5 . However, assuming a signi cant level (α) of 0.05, power (1-β) of 0.80, minimal clinically important difference (MCID) of 20%, and a standard deviation of 2 times MCID, a total number of 34 patient was needed to detect a 20% difference in change in IL-8 levels between patients with SPI at baseline who at follow-up still have SPI vs. those improving to non-SPI.

Statistics
As data were not normally distributed, we used non-parametric statistics. Continuous data were presented as median (range), and categorical data as n (%). Differences were assessed using Wilcoxon signed-rank test (continuous data) resp. Chi2-test (categorical data). Kruskal-Wallis test for trend was performed for coherence between PAL tertiles and in ammation, and Spearman's rho was used for correlation analyses.

Patients
In total, 31 patients out of the 57 patients were classi ed as SPI at baseline. After rehabilitation, seven of these patients (23%) became non-SPI, as they increased their daily level of objectively measured physical activity (PAL) to ≥1.4. None of the study participants were treated with oral corticosteroids or antibiotics within four weeks prior to baseline or follow-up visits. Complete data sets of SenseWear © and in ammatory markers were available in 66-76% of patients: nl-IL-8 68% (n=40), IL-6 68% (n=40), CD-163 66% (n=39), and brinogen, eosinophils and CRP 76% (n=45).
In ammatory markers at baseline Table 1 shows that patients with SPI had slightly, yet signi cantly, higher brinogen than patients without SPI, (p=0.04), but otherwise no signi cant differences were observed. Subdividing PAL into tertiles did not result in signi cant differences for any of the in ammatory marker ( Table 2). Figure 1a-c shows the lack of association between baseline PAL and in ammatory markers.
In total, 20 (39%) were current smokers (median pack-years: 41), and 30 (59%) used daily-inhaled corticosteroids (ICS). We observed no association between neither ICS use nor current smoking and levels of in ammatory markers (data not shown). Summarized a large deviation on all in ammatory markers were seen.
Changes in in ammatory markers after pulmonary rehabilitation Table 3 depicts that shifting from SPI at baseline to non-SPI at follow-up was only associated with signi cant changes in brinogen (p=0.04) compared to patients with SPI at both time points. Although a lower CD-163 and nl IL-8 was observed in patients with non-SPI after rehabilitation, the difference was not signi cant, potentially due to a small sample size and large variation. Figure 2a-c show that improvement in PAL was not associated with signi cant changes in in ammatory markers.

Discussion
SPI is associated with morbidity and mortality in COPD 2,3 and reduced lung function is associated with systemic in ammation 6,7 , highlighting that interventions aimed at SPI and in ammation are needed. However, in this pilot study, including patients with COPD attending pulmonary rehabilitation (exercise and education), we observed that neither Severe Physical Inactivity (SPI) nor improvement in physical activity levels were associated with levels of in ammatory markers.
We used validated motion sensors 1 , since recollection of performed physical activity is poor 27 , and we used well-validated methods for measuring relevant biomarkers 13 . However, even though it might be true negative ndings, several reasons might account for the non-signi cant ndings. First, our study sample was small, indicating that the lack of signi cant ndings might be a result of a type II error, yet statistically signi cant changes in systemic in ammation has previously been demonstrated in a rehabilitation study with even lower number of patients 28 . Individual variation in in ammatory markers over time 12 , or too small changes in physical activity to elicit in ammatory changes might also explain our negative ndings 13,19,29 . There is no well-established cut-off for the minimal exercise intensity or duration to lower systemic in ammation. The pulmonary rehabilitation offered in our study followed national guidelines 30 . Indeed, we demonstrated at best a modest mean increase in PAL but in the current study we focused on responders to rehabilitation i.e. those who actually improved their daily level of physical activity. Even though PAL improved which lead some patients to go from SPI to non-SPI, the patients still ful lled the criteria of sedentary lifestyle 23 . This suggests that further improvements in PAL is needed in order to reduce in ammation.
In COPD, local or systemic in ammation is in uenced by phenotype, medication as well as several non-COPD factors, such as comorbidities, smoking status, nutrition, physical activity, age and drugs 10,31,32 . Participants in our study were stable with respect to treatment, weight, comorbidities and smoking habits. The study design excluded patients with acute exacerbation four weeks prior to baseline or follow-up.
Researchers have found no relation between smoking status and in ammatory markers 33 , though other studies show an association of smoking and in ammatory status 34 . It can be speculated that the in ammation in COPD patients with SPI is not primarily driven by the low level of physical activity but by all contributing factors leading to SPI 6,10 . Thus, improving PAL affects only one of several proin ammatory causes, which in our low-volume study resulted in an undetectable alteration in in ammation. Yet, Waschki et al prospectively found in 170 patients with stable COPD that PAL was a superior predictor for 2 year-mortality compared to cardiovascular, nutritional, respiratory, muscular, depression status or biomarkers 25 .
Our assessment of in ammation was primarily based on serology. We included no measurements from the lower airways. Nasal lavage served as a proxy marker for lower airway in ammation, using a previously described protocol for sampling, and we found similar levels of nasal IL-8 25,26 . The nose is not the target organ in COPD, and it is likely that bronchoalveolar sampling by sputum or bronchoscopy would allow detection of higher and more physiologically relevant levels of in ammatory markers 35,36 . Future studies should incorporate sampling from lower airways, e.g. spontaneous or induced sputum which is obtainable from the majority of patients with COPD 14 .

Conclusions
In conclusion, we did not nd any differences in systemic in ammation or changes in systemic in ammation or PAL following the rehabilitation between patients with SPI and non-SPI. We speculate that suboptimal intensity and duration of daily physical activity despite improvement as well as measurement of in ammation in clinically irrelevant compartments might play a role for the negative ndings. SPI remains a serious challenge in COPD as a grave prognosticator of morbidity and mortality. The study was approved by the Danish National Ethical Committee, and complied with the Declaration of Helsinki. Written and oral informed consent was obtained from all participants.

List Of Abbreviations
The study was registered at ClinicalTrials.gov (NCT01700296).

Consent for publication
Not applicable.

Availability of data and materials
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request UBT has as a main supervisor of the PhD project also contributed substantially in all aspects, except data collection.
AL has made substantially contributions in the interpretations of data as well as having substantially revised the manuscript.
SS has made substantially contributions in the interpretations of data as well as having substantially revised the manuscript.
JL has made substantially contributions in acquisition and analysis of the laboratory samples.  Association between PAL an log CD-163 at baseline.