Discussion
In this single-centre retrospective study, a strong association was observed between neutrophil levels lymphopaenia and NLR, but not monocyte levels with FVC decline. Instead, monocytes (and neutrophils) were associated with mortality. This finding extends that from Scott and Kreuter,4 9 who found that monocyte levels were associated with mortality and a composite outcome measure respectively in IPF patients. There were key differences between our study and these previous publications. First, we examined all the three main parameters of the blood leucocyte profile—neutrophils, lymphocytes and monocytes while Scott and Kreuter focused on monocyte levels only. We did this in part, because of our previous findings of correlation between neutrophils and progression of iUIP to definite/probable UIP on CT scans,10 but also because there is ample evidence that neutrophils are involved in the immune-pathogenesis of IPF (described further below).11 Although Scott also examined the transcriptome of blood immune cells, these were on RNA from stored peripheral blood mononuclear cells, where the neutrophil component would not have survived storage.
The second major difference were the outcome measures. In our study, we specifically separated disease progression from all-cause mortality. We defined disease progression by relative decline in absolute FVC and/or progression in CT abnormalities to provide a more IPF-specific measure of deterioration in disease state. This complemented all-cause mortality, which in an elderly IPF cohort is likely to have other contributory factors to mortality. By contrast, in the larger studies Scott et al4 only report all-cause mortality and Kreuter et al9 define disease progression as a composite outcome of mortality, lung function decline and decline in 6 min walk test. Our study also had a longer follow-up period (median of 31 months for the cohort with a 5-year recruiting period, as opposed to 1 year for the other studies). As such we were able to calculate annualised FVC decline over a larger time period and identify FVC decline >10%/yr more accurately. Scott’s cases were also identified by International Classification of Disease-10 coding of clinical records which may pose limitation to the findings. These differences may account for the differences in findings between these studies.
Thus, while there is corroboration for the association between peripheral monocyte count and all-cause mortality observed by Scott, and with the composite outcome in Kreuter et al9 that included mortality, our study suggests that when divided into outcomes that relate more specifically to disease progression (FVC decline), neutrophil and lymphopaenia appear more sensitive compared with monocytes as a correlate.
Mechanistically, our findings could be explained by experimental evidence implicating neutrophils, monocytes and lymphocytes in immunopathogenesis of IPF. Strong evidence supports monocyte and monocyte-derived macrophages in the aetiopathogenesis lung fibrosis from animal and human studies.17 18 The contribution of neutrophils and lymphocytes to the pathogenesis of IPF is becoming increasingly understood. Neutrophils secrete proteases, cytokines and chemokines which can propagate inflammatory responses.19 Neutrophilic bronchoalveolar lavage (BAL) specimens taken from IPF patients are associated with earlier mortality.20 Neutrophil elastases (NE) are elevated in IPF BAL samples,21 and experimental data using murine models implicate NE in activation of the transforming growth factor-ß (TGF-ß) pathway and fibroblast proliferation.22 Indeed, lymphocytic aggregates are a recognised pathologic feature of IPF lesions.18 BAL specimens from IPF patients are also enriched for several T lymphocytes populations,11 and Th17 lymphocytes isolated from peripheral blood of patients with IPF have been identified as a source of TGF-β.23
The differences in statistical significance observed here between neutrophils, lymphocytes and monocytes in our multivariate analysis is probably a question of sensitivity. The HRs generated by our modelling should be interpreted as association between these immune cells and progression, which is statistically significant. Therefore, as a single point biomarker for progression of fibrosis, in established IPF (as opposed to very early disease), neutrophils and lymphocytes may be more sensitive than monocytes. Even more sensitive is NLR, which may well prove to be the biomarker for disease progression. NLR is a recognised surrogate marker of immune response, and a recognised correlate of disease severity, hospitalisation and mortality.24 25 A retrospective cross-sectional study demonstrated NLR, MLR and SIRI measurements to be greater in patients with IPF versus healthy controls,26 providing further support for our findings in progression of this disease.
Several limitations should be considered when interpreting the results of this analysis. The most obvious is the relatively small number of patients included, although compensated by more detailed characterisation and follow-up in the patients. The single-centre and retrospective nature of this study should also be taken forward by prospective, intervention and validation studies in a different cohort. Mortality and FVC decline reported in this study are higher than reported in other studies, most likely because of the long duration of follow-up. Also, study recruitment started before antifibrotic prescribing became available in the UK and, not unexpectedly in this cohort, baseline FVC% was lower in patients that died. However, strong points include statistical analyses including proportional hazards modelling of time-dependent covariates by multivariate analyses and longer median length of follow (31.0 months) will have adjusted for this. The effect of comorbidity burden, which is not insignificant in this and other IPF cohorts, on observed association between blood leucocytes and study outcomes is unknown. We also acknowledge that antifibrotic treatment and corticosteroid therapy may have affected blood leucocyte measurement, however, when adjusting for these covariates using multivariate regression significance was preserved (online supplemental table 1). In total only 11 patients within our cohort received Prednisolone (all prescribed for suspected acute exacerbation) during the 5-year follow-up. One patient was prescribed corticosteroids at the time of baseline assessment and prior blood draw. We do not believe this alters our interpretation of the data.
In summary, we report that neutrophil levels and lymphopaenia, and NLR measured from full blood count analysis were significantly associated with, and predictive of, FVC decline in this IPF cohort. Peripheral blood leucocyte measurements taken from full blood count analysis could be utilised to stratify IPF patients to those more likely to progress, and perhaps prioritised for anti-fibrotic treatment. This readily accessible measurement can also help inform prognosis, and identify patients for therapeutic trials.