Krebs von den Lungen‐6 (KL‐6) is a pathophysiological biomarker of early‐stage acute hypersensitivity pneumonitis among pigeon fanciers

Identifying early stages of hypersensitivity pneumonitis (HP) is hampered by variable presentation, heterogeneous or undetected causal antigens and lack of gold‐standard biomarkers. Krebs von den Lungen (KL)‐6 is pathophysiological biomarker of alveolar epithelial damage. Pigeon fanciers, susceptible to HP, provide a model to investigate early HP.


| INTRODUC TI ON
Early detection of hypersensitivity pneumonitis (HP) is crucial for prompt intervention. 1,2 This can be compromised by heterogeneous clinical presentation and delay in identifying often cryptic causal antigens 3,4 ; meanwhile, HP can become insidious and progress to treatment-refractory chronic cHP. 5 Objective tests to assess the pathophysiological significance of early mild symptoms would provide timely biomarkers for subjects at-risk of progression. This knowledge gap in the pathogenesis of early HP may be addressed by investigating HP among pigeon fanciers. This population is at known risk for HP, where the causal exposure to antigenic dust from pigeons and the immune hypersensitivity responses can be measured, along with clinical responses. These include interstitial inflammation and restrictive lung dysfunction causing shortness of breath, 6 and acute symptoms described as influenza-like with pyrexia and myalgia suggest systemic inflammation. 7 The determinants of these outcomes are unresolved, for example persistently raised serum IgG antibody against pigeon antigens can occur in asymptomatic fanciers suggesting that antibody may be necessary but not sufficient for progression. 4 Additional biomarkers indicative of a pathophysiological response may help explain early progression.
Krebs von den Lungen-6 (KL-6) is a mucinous extracellular high-molecular-weight (~200kDa) sialylated glycoprotein fragment of the transmembrane mucin MUC-1 (CD227), classified as cluster 9 of lung tumour and differentiation antigens. 8,9 It is expressed on the apical surface of type 2 alveolar epithelial cells (AEC2). 10 KL-6 is cleaved at the cysteine bond near the epithelial membrane surface, released into lung lining fluid and is measurable in BAL fluid. 11   diffuses into the circulation 11,12 providing evidence of increased lung epithelial/endothelial permeability. 12,13,14 AEC2 replicate homeostatically to replace alveolar epithelial cells and proliferate to repair damaged alveolar epithelium, for example in pulmonary alveolar proteinosis 10 and following oxidative stress and epithelial apoptosis in ILD including IPF and cHP. 15 The density of AEC2 reflects the severity of recent alveolar damage 16 ; therefore, the associated increased KL-6 production measured in plasma may be a sensitive indicator of alveolitis. In ILD, plasma KL-6 is a particularly useful molecular biomarker, with concentrations reflecting disease activity, 2 severity 8 and the effectiveness of corticosteroid therapy 8,[17][18][19] conferring value as a diagnostic and prognostic biomarker of progression or survival. 20 In a study of ILD, HP had the highest BALF KL-6 and the most prominent alveolitis compared with IPF and sarcoidosis, and the serum KL-6 correlated with BALF KL-6, albumin and lymphocyte counts, particularly cytotoxic CD8 T cells. 21,22 High concentrations of serum KL-6 have been reported in diagnosed HP associated with domestic 23 and occupational 24,25 environments. KL-6 is higher in acute than chronic HP 26 suggesting that alveolitis may already be prominent early in the disease trajectory of HP. We hypothesized that KL-6 may be an early biomarker of HP and tested this among undiagnosed pigeon fanciers with intermittent acute symptoms associated with antigen exposure. We identified an endotype of increased KL-6 associated with IgG antibody and lymphocyte profile, linking lung pathophysiology with immunity that was indicative of early HP in this at-risk population.

| Participants
Participants were recruited at a community-based cross-sectional study of early-stage acute HP at a national convention of pigeon fanciers where our clinical research team provided advice about pigeon fancier's lung, colloquial for hypersensitivity pneumonitis. Those interested to participate were fully informed of the detail and purpose of the study and provided signed informed consent. All information collected was anonymized and linked by a unique study number for each person. with the culture of pigeon fanciers. They recorded their recollection of any of the following symptoms: dyspnoea, influenza-like symptoms, chest tightness, cough, polymyalgia, fever, fatigue, diaphoresis and wheeze, occurring 4-12 hours after exposure to undue amounts of pigeon dust (eg cleaning-out pigeon loft and/or during the moult when feathers are shed copiously), that resolved usually by the next day. A history of at least one respiratory symptom simultaneously with at least one systemic symptom on at least three occasions in the last year was recorded. Although this was insufficient for a diagnosis of HP, 3,6 for the purposes of this study this symptom profile was used to categorize a group with "early-stage acute" HP 27,28 who reported recurrent episodes of acute symptoms after contact with pigeons, that resolved quickly, but who had not (yet) had an established diagnosis of pathogenesis by linking lung pathophysiological changes with an endotype of immune hypersensitivity.

K E Y W O R D S
biomarkers, hypersensitivity pneumonitis, ILD, KL-6 HP by a hospital specialist or general practitioner. We excluded those who did not currently keep pigeons, anyone with symptoms suggestive of a recent respiratory tract infection or any current symptoms, those with a history of any respiratory disease or co-morbid conditions (such as heart disease), those who were taking anti-inflammatory medications, those with a history of a dusty occupation and current and recent ex-smokers (within the last 10 years). We also excluded those with an established diagnosis of HP after medical investigations as we wished to focus on those with early stage or subclinical disease. Of 155 interviewed, 47 pigeon fanciers fulfilled study criteria and were enrolled; 20 had a history of acute symptoms indicative of early-stage acute HP, and 27 subjects had a history of no symptoms. These subjects performed pulmonary function spirometry and donated a 7 mL heparin blood sample. A control group of 10 volunteers from the research study team who fulfilled the same exclusion criteria but without any significant avian exposure donated a 7 mL blood sample.

| Measurement of pigeon antigen-specific IgG antibody
The predominant species-specific antigen in the respirable dust in pigeon lofts is pigeon serum gamma-globulin, 29 and the subjects' antibody activity against this antigen was measured by indirect enzyme immunoassay (EIA). 30 Briefly, 96-well polystyrene microtitre EIA plates (Dynatech Ltd, UK) were coated with purified antigen (donated by Dr P. Lynch); 5 μg/mL in bicarbonate buffer (0.02 mol/L, pH 9.6), 100 μL/well 24 hour at 4ᵒC. Plate wells were washed three times in detergent buffer (phosphate-buffered saline, 0.02 mol/L, pH7.4, containing 0.05% Tween-20, PBS-T). Plasma samples were diluted optimally at 1:200 with PBS-T and incubated in duplicate at 100 μL/well for 1 hour at room temperature, after which the plates were washed as above. Bound antibody was quantified using alkaline phosphatase conjugated anti-human IgG at 1:10 000 dilution in PBS-T (Sigma, UK).
After washing as before, the plate wells were incubated with the colourless substrate p-nitrophenyl phosphate (Sigma, UK) at 1 mg/mL in 10% diethanolamine, pH10. This is converted by enzyme activity to a yellow product with an absorbance at 405 nm. After approximately 30 minutes, the optical density was measured by spectrophotometer (Dynatech Ltd UK). If all other reagents are in excess, the E405 nm was proportional to the antibody activity and this was quantified by interpolation using an optical density standard curve from serial dilutions of a standard serum with known antibody concentration previously titrated by quantitative precipitation.

| Blood leucocyte cytology and lymphocyte culture
Total and differential white blood cell counts were determined by Coulter counter (CBC5, Coulter Electronics) and by flow cytometry (FACScan, BD Biosciences, UK) using FITC-anti-CD45 and PE-anti-CD14 (Sigma, UK). The major T lymphocyte subsets were counted by FACS using FITC-anti-CD3 with either PE-anti-CD4 or PE-anti-CD8 (Sigma). The cell counts and proportions are listed in Supplementary Table S1.
Antigen-specific lymphocyte responses were measured by whole blood assay. Briefly, each blood bottle was resuspended by inversion, and 20 μL was added to 80 μL DMEM containing penicillin and streptomycin (Gibco, UK) per well in 96-well cell culture plates (Fisher Scientific, UK). Each blood was tested in quadruplicate, with and without 5 μg/mL of pigeon serum gamma-globulin antigen.
Plates were incubated at 37ᵒC in a humidified 5% CO 2 atmosphere.

| Plasma KL-6
Plasma KL-6 was measured by sandwich enzyme immunoassay (Eitest KL-6 kit, Sekisui Medical Co. Ltd. Japan) according to the manufacturer's instructions. Reference levels are listed in Table S2.

| Statistical analysis
The primary aim was to test the working hypothesis that the plasma KL-6 concentration was increased in pigeon fanciers with a symptom history indicative of risk of progression to hypersensitivity pneumonitis.
Secondary aims included investigating linear relationships between KL-6 with antigen exposure, lung function and immune reactivity. Variables were summarized as median and interquartile range; between-group comparisons were tested by Mann-Whitney U test. The linear relationships were tested by Spearman's rho. Analysis was performed using Minitab software (Minitab Inc, State College, PA). Secondary aims were considered exploratory, and there was no correction for multiple comparisons. P < .05 was considered statistically significant.

| Categorization of pigeon fanciers according to symptom history
Of 155 interviewed, 20 pigeon fanciers described a history of intermittent acute symptoms following exposure to pigeon dust (Methods), and 27 had a history of no symptoms (Table 1). There was no difference between the "early-stage acute" and "asymptomatic" categories for age, pigeon antigen exposure or spirometric lung function; therefore, these groups were considered suitable for a comparative investigation for biomarkers of risk of progression to HP.

| Blood leucocytes
Compared with control subjects (Table S1), the pigeon fanciers had lower total blood leucocyte counts (P = .041), and lower relative proportions (P = .003) and absolute counts (P = .01) of CD8 lymphocytes. Within the categories of pigeon fanciers (Table 1), those with early-stage acute HP had a lower proportion of CD4+ lymphocytes (P = .045) and a higher proportion of CD8+ lymphocytes (P = .02) than their asymptomatic counterparts.

| Immune reactivity against pigeon antigens and symptom category
Pigeon fanciers had high IgG antibody ( Figure 1A) and modest in vitro lymphocyte proliferative responses ( Figure 1B) against pigeon antigens, that were negligible among control subjects with no significant avian exposure. There was no correlation between the IgG antibody concentration or the lymphocyte proliferative response with age, antigen exposure, lung function or blood cytology in all pigeon fanciers and in the "early-stage acute" and "asymptomatic" categories (Table S3). There was a significant correlation between the humoral (IgG antibody titre) and the cellular (lymphocyte proliferation) responses, r = .429, P = .003 compared with asymptomatic fanciers. However, there were many asymptomatic subjects with evidence of immune reactivity, and several symptomatic subjects with relatively low immune reactivity and this overlap suggested that the immune response profile was insufficient to cleanly differentiate asymptomatic from early-stage acute HP; therefore, we investigated KL-6 as a pathophysiological biomarker between these categories.

| Plasma KL-6 as a pathophysiological biomarker of symptoms indicative of early-stage acute HP
There was no linear relationship between plasma KL-6 concentration and cumulative exposure to pigeon antigens, spirometric lung function or blood cytology (Table S3)   category of "early-stage acute" HP (r = .146, P = .539) or "asymptomatic" fanciers (r = .152, P = .451).

| Comparison between objective categories of normal and high plasma KL-6 concentrations
A 95th centile of normality for KL-6 was calculated from control subjects. This generated an upper cut-off normal limit at 452 µ/mL which was consistent with published levels (Table S2). We used this to as an objective discriminator of "normal" and "high" categories of KL-6 and used this, rather than subjective symptom-recall, to compare lung function, antigen exposure and immune response among the pigeon fanciers (

| Evaluation of serum KL-6 and IgG antibody in factory workers exposed a contaminated humidification system
For comparison, we conducted a serological study of KL-6 and IgG antibody activity among undiagnosed factory workers, some with acute HP symptoms associated with exposure to recognized but uncharacterized antigens aerosolized from the water sump of a

| Main findings
To

| Comparison with other studies
The published studies of KL-6 in HP generally report diagnosed cases. Our findings extend and complement these by investigating KL-6 in "pre-clinical" or undiagnosed early-stage acute HP.

| What can KL-6 reveal about the pathogenesis of early-stage acute HP
The pathogenic determinants of onset and progression of HP are unresolved. Antigen-specific antibody and cellular immune responses appear necessary but not sufficient for the development of symptoms.
Our findings highlight KL-6 as a useful candidate biomarker for early progression. KL-6 produced by regenerating type-2 alveolar epithelial cells (AEC2) is increased with the proliferation required to repair apoptotic epithelial cells associated with alveolar damage in HP. 23 Although KL-6 is associated with changes in lung pathophysiology, it might directly cause pathogenic remodelling. KL-6 is chemotactic for human fibroblasts in vitro and may contribute to the intra-alveolar fibrosis in HP. 46 Speculating on this observation, resolution or increase of KL-6 might usefully discriminate stable or progressive cHP phenotypes. 31 HP likely has clinical phenotypes beyond the inadequate classification of acute, subacute and chronic. 47 These phenotypes could be determined by combinations of immunological, for example antigen-specific antibody and lymphocyte activities, and remodelling mediators, for example KL-6. A combination of these might reflect an endotype of lower-lung pathology that could guide appropriate anti-inflammatory or anti-fibrotic therapies. Treatment and monitoring of HP that is chronic with fibrosis is difficult 2 ; therefore, therapeutic targeting KL-6, for example with corticosteroids early in the disease trajectory, would have optimum impact to forestall fibrosis before disease becomes refractory and plasma KL-6 levels might identify and stratify HP patients for relevant therapies or trials.

| Strengths and limitations
Strength Classification of HP based on symptoms into acute, subacute or chronic categories is inadequate, with subacute HP particularly difficult to define. 47,48 The excellent engagement with pigeon fanciers as a homogeneous study group at known risk of HP, and their familiarity with symptom patterns of "pigeon lung" helped provide well-categorized groups based on post-exposure symptoms which underpinned this study. as a prognostic marker. SNP genotype analysis will be included in a follow-up study to address these limitations.

| CON CLUS ION
This study demonstrated that early-stage acute HP can be identified by raised plasma KL-6, and commensurate increased antibody is evidence of an underlying immunological diathesis. These findings should stimulate further research to facilitate and hasten the identification of HP and improve the clinical management and monitoring of individuals at-risk of HP, for example screening agricultural workers, and in antigen-occult HP, for example humidifier fever.
Immunological and pathophysiological biomarkers add endotype evidence for dynamic clinical phenotypes. Among these, early-stage acute HP implicates AEC2 as an axis of homeostatic or aberrant remodelling and therefore a potential target for early therapeutic intervention in hypersensitivity pneumonitis.

ACK N OWLED G EM ENTS
This study is testament to the support of pigeon fanciers who vol-

CO N FLI C T O F I NTE R E S T
Nobuoki Kohno holds a patent for KL-6. The remaining authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.