Anti-bacterial antibody and T cell responses in bronchiectasis are differentially associated with lung colonization and disease

Anti-bacterial antibody and T cell responses in bronchiectasis are differentially associated with... Background: As a way to determine markers of infection or disease informing disease management, and to reveal disease-associated immune mechanisms, this study sought to measure antibody and T cell responses against key lung pathogens and to relate these to patients’ microbial colonization status, exacerbation history and lung function, in Bronchiectasis (BR) and Chronic Obstructive Pulmonary Disease (COPD). Methods: One hundred nineteen patients with stable BR, 58 with COPD and 28 healthy volunteers were recruited and spirometry was performed. Bacterial lysates were used to measure specific antibody responses by ELISA and T cells by ELIspot. Cytokine secretion by lysate-stimulated T cells was measured by multiplex cytokine assay whilst activation phenotype was measured by flow cytometry. Results: Typical colonization profiles were observed in BR and COPD, dominated by P.aeruginosa, H.influenzae, S. pneumoniae and M.catarrhalis. Colonization frequency was greater in BR, showing association with increased antibody responses against P.aeruginosa compared to COPD and HV, and with sensitivity of 73% and specificity of 95%. Interferon-gamma T cell responses against P.aeruginosa and S.pneumoniae were reduced in BR and COPD, whilst reactive T cells in BR had similar markers of homing and senescence compared to healthy volunteers. Exacerbation frequency in BR was associated with increased antibodies against P. aeruginosa, M.catarrhalis and S. maltophilia. T cell responses against H.influenzae showed positive correlation with FEV %(r = 0.201, p = 0.033) and negative correlation with Bronchiectasis Severity Index (r = − 0.287, p = 0.0035). Conclusion: Our findings suggest a difference in antibody and T cell immunity in BR, with antibody being a marker of exposure and disease in BR for P.aeruginosa, M.catarrhalis and H.influenzae, and T cells a marker of reduced disease for H.influenzae. Keywords: Bronchiectasis, Antibodies, T cells, Lung function, Exacerbation, COPD * Correspondence: stephen.todryk@northumbria.ac.uk Anthony De Soyza and Stephen M. Todryk contributed equally to this work. Faculty of Health & Life Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, UK Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, UK Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jaat et al. Respiratory Research (2018) 19:106 Page 2 of 12 Background appear in individuals with defined immunodeficiencies The chronic lung diseases of bronchiectasis (BR) and [21], underlining the role of antibodies and phagocytes in chronic obstructive pulmonary disease (COPD) are both protection. Whilst healthy individuals are exposed to the associated with recurrent airway infections. COPD is a same pathogenic organisms as diseased individuals, major cause of death globally, with numbers of deaths healthy lungs typically have low levels of bacterial species, rising [1], and BR is underestimated with incidence ris- reflecting the naso-pharynx [22]. Immune responses ing in the UK by around 6% annually [2]. Whilst they against pathogenic microbes do not cause overt immuno- differ in disease causation, established disease in both is pathology in healthy individuals, but may contribute to mainly characterised by repeated or persistent heavy disease in colonized patients due to continuous immune bacterial colonization of the damaged lower respiratory stimulation by the localised high antigen doses, particu- tract. Such infection is associated with inflammation, larly through excessive Th17 responses that promote mucus production, and reduced ciliary action, which neutrophil infiltration [23]. Together with inflammatory promotes further infection, inflammation and tissue cytokines, neutrophils are abundant in the sputum of BR damage, in a vicious cycle [3]. Studies have suggested patients, and decline after antibiotic treatment [24]. It is that infection causes disease exacerbation and dimin- possible that dysfunction of both innate and adaptive im- ished lung function, which are often proportional to the munity contribute directly or indirectly to disease in both bacterial load and to reduced diversity [4, 5]. More re- BR and COPD. The aim of this study was to characterise cent findings propose more species-rich lung ecologies antibody and T cell responses against key lung microbes where alterations in specific bacterial populations, dys- in disease-stable patients with BR and COPD, charac- biosis, is at the heart of clinical disease [6, 7]. Pathogenic terised by the Bronchiectasis Severity Index (BSI) and bacteria, as determined clinically by microbiological cul- GOLD guidelines, respectively, in comparison to controls ture of expectorated sputum, are dominated by organ- (healthy volunteers), and to relate the immune responses isms specific to these diseases including Pseudomonas to culture-based bacterial colonization, lung function and aeruginosa, Haemophilus influenzae, Streptococcus pneu- frequency of exacerbation. moniae and Moraxella catarrhalis [8]. Recent studies using DNA-sequencing technology reveal more detailed Methods bacterial ecosystems in the lungs of diseased patients, Study participants and samples but with culture approaches mainly corroborated [9, 10]. Ethical approval for the project was granted by the local P.aeruginosa is considered the major cause of morbidity NHS Research Ethics Committee, the NRES Committee (increased exacerbations and reduced lung function) and North East – County Durham & Tees Valley (ref 12/NE/ mortality in BR [11], particularly during chronic infection 0248). Adult patients with (non-CF) BR, COPD and and mucoid characteristics of the bacterium [12], which healthy volunteer (HV) controls, were recruited at the may allow evasion of host immunity. Non-typeable strains Freeman Hospital, Newcastle upon Tyne. Female to of Haemophilus influenzae (NTHi) are frequently found male ration was about 1.5:1. BR is routinely confirmed in BR [13] and are not targeted by current vaccines. Both by high-resolution computed tomography (HCRT), and pathogens are also common in COPD albeit with a re- COPD according to prevailing GOLD guidelines (BTS duced frequency of Pseudomonas infections as compared and NICE 2010, [25]). Diverse aetiologies of BR were in- to BR [14]. Furthermore, less frequent suppurative infec- cluded in the study, with the exception of known tion and sputum production in COPD results in lower de- immunodeficiency. tection of pathogenic microbes, implying fewer infections Patients were clinically stable at the time of assess- than BR. Failure to produce sputum for microbiology, ment. They underwent spirometry to determine forced particularly in younger BR patients and in many COPD expiratory volume in 1 s (FEV ), and Forced Vital patients, as well as intermittent negative cultures, means Capacity (FVC), from which FEV1% predicted, FEV1/ that immune biomarkers of disease may provide a useful FVC ratio and FVC % predicted were obtained. The adjunct for directing clinical management. bronchiectasis severity index (BSI) score, as previously Knowledge of immunity in BR is limited, but studies sug- validated [26], was assessed. Patients were divided into 2 gest immune system genes that are involved in presentation groups: either those with one severe exacerbation requir- of antigens to CD4 T cells, such as HLA-DR1 and DQ5, ing hospitalisation or those with 3 or more exacerbations playarole[15, 16]. Notably, a role for adaptive immune re- per year, compared to those not requiring hospitalisation sponses (specific antibodies and T cells) in protection and having less than 3 exacerbations per year. The exac- against P.aeruginosa and H.influenzae, has been demon- erbations were determined for the preceding 12 months. strated in human vaccine trials in cystic fibrosis-related Colonization history of patients was also available going bronchiectasis [17, 18] and in mouse vaccination models back at least 4 years. Patients were categorised by patho- [19, 20]. Furthermore, the above-mentioned lung pathogens gen status based on positive sputum cultures. ‘Chronic Jaat et al. Respiratory Research (2018) 19:106 Page 3 of 12 colonization’ was defined here as 2 positive sputum cul- data t-test and Pearson correlation was used whilst for not tures at least 3 months apart in 12 months. ‘Chronic normally distributed data the Mann-Whitney U test and currently’ was defined as a positive sputum culture at Spearman’s correlation was used. A priori calculations time of blood sampling (for immune responses), and based on our previous data suggested that sufficient num- more than 2 positive sputum cultures in 12 months. bers were included to detect a modest effect with 0.9 ‘Previously chronic’ was defined as more than 2 positive power to a significance level of 0.05. SPSS v.15 and Graph sputum cultures in 12 months > 2 years ago. ‘Occasional’ Pad Prism were used for analysis. The cut-off value for infection was ≥ 1 positive sputum culture per year. ‘No statistical significance was p <0.05. colonization’ was sputum culture negative over at last 3 years (Table 3). Results Clinical data Sample processing and bacterial culture This study examined antigen-specific immune responses Heparinized venous blood samples from patients and in 119 BR and 58 COPD patients, and in 28 HV (Table 1), healthy controls were processed to give plasma for against lung pathogens that are commonly isolated from ELISA and peripheral blood mononuclear cells (PBMC) these patient groups. The patient groups showed typical for T cell assays (detailed in Additional file 1). Sputum clinical features, similar to previously-published reports samples were cultured in the Microbiology Department, e.g. average FEV % predicted, which was 68 for BR, 49 for Freeman Hospital, according to national standards. COPD and 113 for HV. FVC % predicted and FEV1/FVC ratio were also reduced in the patient groups compared to Enzyme-linked immunosorbent assay (ELISA) for serum HV. The BR group was predominantly of a post-infection antibody measurement aetiology, whilst COPD was smoking-related, and all pa- An indirect enzyme-linked immunosorbent assay (ELISA) tients with BR and COPD were in a clinically stable state method was used: to determine optimal dilutions for the with no current exacerbation. At the time of taking the coating of microbe-derived antigens, for initial serum blood samples, the BR group had a higher proportion of screening, and to undertake titration for total IgG (all patients producing sputum (93%) that could be microbio- subclasses combined) to give an end-point titre (e.g. 1 in logically tested than COPD (66%), and BR patients showed 1000), and for the measurement of individual Ig subclasses greater overall populations infected with the main bacter- (given as absorbance for 1 in 25 dliution), as described ial species (Table 2), many with multiple species. H.influ- previously [27](see Additional file 1). enzae was the most commonly identified species in both BR and COPD, followed by P.aeruginosa, S.pneumoniae T cell responses to microbial antigens and M.catarrhalis in BR, and M.catarrhalis, S.pneumoniae ELIspot as previously optimised and described [28] (see and P.aeruginosa in COPD. Additional file 1) was used to measure T cell responses against a range of stimuli including bacterial lysates plus Antibody responses selected peptide epitopes where available. For cell activa- The first experiments involved ELISA to determine anti- tion and surface staining of cells, PBMC were thawed body levels (total specific IgG), by end-point titre, and activated with stimuli for 20 h as detailed in the against the main bacterial species in BR, COPD and HV. supplement. Intracellular staining was performed to de- BR showed significantly higher levels of antibody against termine the number and phenotype of IFNγ-producing or activated CD69 T cells following stimulation. Table 1 Demographics of the subjects included in this study Characteristics BR COPD HV n = 119 n =58 n =28 Measurement of cytokines in culture supernatants using multiplex ELISA Sex (no.) 45/74 Not av. Not av. Male/female) Cytokines in supernatants from stimulated PBMCs were Age (y) 65 ± 1.08 69 ± 1.23 54 ± 3.01 measured using the Mesoscale Scale Discovery (MSD) multiplex cytokine Kit (Meso Scale Diagnostic, LLC, Exacerbations (per year) 4 ± 0.29 3 ± 0.38 Not app. Gaithersburg, USA). Multiplex kit – pro-inflammatory Smoking history (pack years) 8 ± 1.33 47 ± 4.08 Not av. panel 1 (for IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, FEV (% predicted) 68 ± 2.68 49 ± 2.70 113 ± 2.83 IL-12p70, TNFα) and cytokine panel 2 (IL-17A, IL-5) FVC (% predicted) 82 ± 2.48 77 ± 2.40 118 ± 2.7 were used. See Additional file 1. FEV /FVC ratio 66 ± 1.52 50 ± 2.17 83 ± 1.75 Values are presented as means ± SEM, exacerbations represents the number Statistics per year. FEV represent forced expiratory volume in the first second and FVC, The immunological data was tested for normal distribu- forced vital capacity. Not av., indicates the data are not available, whereas Not tion using the Shapiro-Wilk test. For normally distributed app., means that the category was not applicable Jaat et al. Respiratory Research (2018) 19:106 Page 4 of 12 Table 2 Microorganisms isolated from the sputum of patients associate with an occasional number of exposures, as with BR and COPD opposed to none or chronic, this was not significant. Microorganism Identified Bronchiectasis Chronic obstructive patients (%) pulmonary disease Exacerbation and lung function patients (%) The contribution of bacterial colonisation, as per Table 3, NT. H.influenzae 63.8 31 to lung function as measured by FEV % predicted, was ex- P. aeruginosa 56.3 12.0 amined in the BR cohort. Overall there was a downward S. pneumoniae 44.5 17.2 trend in lung function as bacterial colonisation became more frequent and with current positive cultures (Fig. 2a), M. catarrhalis 37.8 24.1 with P.aeruginosa, M.catarrhalis and S.maltophilia all A. fumigatus 22.6 3.4 showing a significant reduction. The numbers with M.- S. maltophilia 15.9 3.4 catarrhalis and S.maltophilia were low (n =10 and 9, Candida. sp 14.4 6.9 respectively) with 52 and 75% of these, respectively, being S aureus 30.3 1.7 co-colonised with P.aeruginosa. E.coli 20.1 5.2 Since exacerbation of disease is a key event in need of urgent clinical management, and makes up part of the No pathogen isolated 2.5 17.2 validated BSI scoring, exacerbation history of the BR No sputum produced 6.8 43.7 patients was used to determine whether there were any BR patients n = 119; COPD patients n = 58 informative associations with immunological responses. NT = non-typeable The patients were compared based on them having less P.aeruginosa, H.influenzae and S.maltophilia compared than 3 exacerbations, greater than or equal to 3, and to HV (Fig. 1a). COPD failed to show significant in- those that were hospitalised within the last year. This crease from HV for any of the bacteria. HV only showed breakdown of the patients was first validated by examin- significantly higher antibody responses than BR and ing the FEV % predicted within the groups compared to COPD against S.pneumoniae. HV (Fig. 2b). A significant decrease in lung function was The next aim was to relate specific IgG antibody levels seen moving from HV, through < 3 exacerbations, ≥ 3 against bacteria, to bacterial colonization status and his- exacerbations, to hospitalised. Since the hospitalised tory. Because of the relatively low infection rates in COPD, group comprise a variable causality (and not necessarily the numbers were insufficient to determine significance, greater disease) and was relatively small in number, this and so we focussed on BR. BR patient pathogen status was was omitted from further analysis. Small but statistically analysed and condensed into a categorization shown in significant increases in IgG titres against P.aeruginosa, Table 3,indicating ‘current chronic’, ‘previous chronic’, ‘oc- H.influenzae and M.catarrhalis were seen between < 3 casional’,or ‘no colonization’. Whilst there was an overall and ≥ 3 exacerbations (Fig. 3a-c). For T cell responses no trend for antibody titre to increase based on colonization significant differences were seen between the 2 groups (Fig. 1b), this was only significant for P.aeruginosa and for any bacterium (Fig. 3d-f). S.maltophilia. We further studied immunoglobulins by class and isotype across the disease groups and found in T cell phenotypes general higher levels of IgG1 and IgA in BR than COPD Since T cell responses of a single Th type (Th1/IFNγ) (Fig. 1e-g). may not be the only response against the lung bacteria, antigen-specific T cell cytokine responses were also mea- T cell responses sured in the supernatants of antigen-stimulated PBMC T cell responses, in the form of IFNγ spot-forming cells by multiplex cytokine analysis (Fig. 4a-h). A sub-group per 10 PBMC, were measured against the bacterial anti- of BR patients who were good responders to the anti- gens as for the antibodies. In contrast to the antibody gens, determined by both IFNγ ELIspot and by CD69 responses, there was an overall trend towards the BR expression following antigen-stimulation, were selected and COPD groups having lower T cell responses than for stimulation, as well as similarly good HV responders. the HV group (Fig. 1c). BR and COPD both had signifi- For IFNγ, IL-2 and IL-17 responses the BR patients cantly lower responses than HV against P.aeruginosa showed a trend, though not significant, towards reduced and S.pneumoniae. As with the antibody responses, the levels for both P.aeruginosa and H.influenzae compared relatively low infection rates in COPD meant that there to HV. For IL-4, BR patients showed marginally reduced were insufficient numbers of infected patients to deter- responses for P.aeruginosa but significantly increased re- mine significance and so BR was focussed upon for relat- sponses to H.influenzae compared to HV, although the ing to colonization (Fig. 1d). Whilst there was an overall responses showed considerable variability. Finally, IL-10 trend for T cell responses against all antigens to responses showed a trend for increased response in BR Jaat et al. Respiratory Research (2018) 19:106 Page 5 of 12 a c b d e f Fig. 1 Anti-bacterial antibody and T cells responses. Antibody levels against bacterial antigens were measured by ELISA: (a) IgG titres (1/value) against bacteria in BR, COPD and HV groups; (b) Antibody responses against bacteria were compared based on bacterial colonization. T cell responses against bacterial antigens were measured by IFNγ ELIspot: (c) IFNγ spot-forming cells per 10 PBMC against bacteria in BR, COPD and HV groups; (d) IFNγ spot-forming cells per 10 PBMC against bacteria were compared based on bacterial colonization. Anti-pseudomonas Ig subclasses and IgG isotypes in BR (e), COPD (f) and HV (g) subgroups. Mean value + SEM are given. *p < 0.05, ***p < 0.001. Mann-Whitney tests were performed Table 3 Classification of patients based on sputum microbiological results 0 No pathogen isolated (NPI) 1 Occasional ≥ 1 isolation in a year Chronic colonization is defined by the isolation of bacteria in sputum culture on 2 or more occasions, 3 months apart, within 1 year 2 Chronic previously Chronic in preceding 5 years but not last 2 years 3 Chronic currently Current, and chronic in last 2 years prior to recruitment Jaat et al. Respiratory Research (2018) 19:106 Page 6 of 12 Fig. 2 Lung function and exacerbation in BR. FEV1% predicted is a measure of lung function. (a) FEV1% predicted in BR patients grouped based on their bacterial colonization. (b) FEV1% predicted in BR patients based on exacerbation in the last year, and in HV – healthy subjects. Mean values + SEM are given. Mann-Whitney tests were performed over HV for both antigens, but again with much variabil- examined, a significant positive correlation (Fig. 6a), ity. All other cytokine responses tested were equivocal. albeit weak (r = 0.201; p = 0.033), was found only with T Flow cytometry analysis was used to further character- cell response against H.influenzae. With removal of the ise antigen-responding T cells in the same subgroups of outlier, significance was still retained (r =0.186, p =0.049). BR and HV. Similar proportions of activated CD69 Furthermore, a stronger (negative) correlation was ob- CD4 T cells were seen in BR and HV following stimula- served between BSI and anti-H.influenzae T cell re- tion with P.aeruginosa and H.influenzae, all being sponses (Fig. 6c)(r = − 0.287; p = 0.003), and again significantly higher than the unstimulated (medium) significance remained upon removal of an outlier control (Fig. 5a). Co-staining of the activated CD4 T (r = − 0.265, p = 0.0035). Conversely, a moderate cells with CD69 and for IFNγ intracellularly showed a negative correlation was seen (Fig. 6b) with antibodies concordance of the two forms of activation but showed against H.influenzae (r = − 0.224; p = 0.018), but none + + that there were more CD69 cells than IFNγ cells with BSI (Fig. 6d). This suggests that T cell responses (Additional file 1). Staining of the CD69 stimulated are associated with improved lung function and less cells for other potentially important markers (see severe disease, whereas higher levels of H. influenzae Additional file 1: Figure S1), showed significant levels of antibodies are associated with poorer lung function. + + staining (above isotype controls) on CD4 CD69 T cells Overall, no relationships were evident between the (Fig. 5b-e), but no significant difference between BR magnitudes of antibody and T cell responses against patient and HV cells. any of the bacteria examined. Furthermore, T cell and When the relationship between T cell responses (IFNγ antibody responses against bacteria showed no rela- ELIspot) and lung function (FEV % predicted) was tionships with one another (data not shown). 1 Jaat et al. Respiratory Research (2018) 19:106 Page 7 of 12 a d Fig. 3 Antibody and T cell responses in BR groups of different recent exacerbation history. Antibody levels against bacterial antigens were measured by ELISA to give IgG titres (1/value) in BR groups with < 3 compared to > 3 exacerbations in the last 12 months against bacteria. T cell responses were measured by IFNγ ELIspot and expressed as spot-forming cells per 10 PBMC. (a, d) P.aeruginosa (b, e) M. catarrhalis (c, f) H.influenzae. Mean values + SEM are given. Mann-Whitney tests were performed Discussion may suggest that COPD has reduced isotype switching, This study began by comparing immune responses which is usually controlled by cognate T cell responses, against common lung pathogens in BR, COPD and HV. through CD40:CD40L interaction and through cyto- The clinical categorisation of the patients followed kines. Reduced or altered antibody responses as we have standard processes and was in keeping with other stud- seen here could be due to increased regulatory T cells, ies in the field, as were the microbiology results ob- as have been demonstrated in COPD, which may depress tained. One expectation was that the degree of exposure protective immunity [29]. to the microbes will be proportional to the magnitude of Having found specific antibody responses to be in- immune response measured. This was broadly the case creased in BR, the question was whether these responses for antibody responses, which were higher in BR than showed a direct dynamic relationship with colonization COPD and HV, particularly against P.aeruginosa, levels. Sufficient numbers for this analysis were only H.influenzae and S.maltophilia, reflecting rates of posi- available in the BR group. Whilst there was a trend for tive sputum cultures in BR and COPD. Measurement of increasing antibodies with colonisation for each individual isotype components of the antibody responses against pathogen, only P.aeurginosa and S.maltophilia showed P.aeruginosa showed a high IgG1 component in BR and significance. We categorised patients based on their HV, compared to COPD which had a higher IgM. This exacerbation frequency (< 3, ≥ 3) which were validated by Jaat et al. Respiratory Research (2018) 19:106 Page 8 of 12 a e b f c g d h Fig. 4 Cytokine responses in a subset of BR patients and HV. PBMC from BR and HV were stimulated with bacterial antigens P.aeruginosa (PSA) and H.influenzae (Hi), and supernatants were tested for the following cytokines: (a) IL-2, (b) IL-4, (c) IL-12, (d) IL-17, (e) IL-2, (f) IL-5, (g) IL-13, (h) IL-10. Mean values + SEM are given in pg/ml. Mann-Whitney tests were performed showing reducing lung function. Although significant, FEV % predicted, suggesting it to be a marker of disease only modest increases in antibody against P.aeruginosa, and exposure. M.catarrhalis and H.influenzae were found in BR with ≥ 3 The measurement of T cell responses against lung exacerbations compared to < 3. Antibody response only pathogens may be useful for the diagnosis of latent against H.influenzae showed a negative correlation with infection, as is the case of the Quantiferon test for Jaat et al. Respiratory Research (2018) 19:106 Page 9 of 12 a b d c e Fig. 5 Flow cytometry responses on a subset of BR patients and HV. Flow cytometry was performed on PBMC from BR and HV stimulated with bacterial antigens. (a) CD69 activation marker following stimulation with medium only, P.aeruginosa or H.influenzae. The % of CD4+ CD69+ activated T cells with phenotypic markers following stimulation with (b) P.aeruginosa,(c) anti-CD3 monoclonal antibody, (d) H.influenzae and (e) medium only. Mean % values + SEM are given. Mann-Whitney tests were performed Mycobacterium tuberculosis (Mtb). In this study T cell Within the BR group T cell responses showed a trend for responses showed an overall tendency for reduction in BR being highest in the group that had occasional infections, and COPD compared to HV, associated with colonisation for all pathogens tested. The highest T cell responses were status, with responses to P.aeruginosa and S.pneumoniae found for H.influenzae and M.catarrhalis which coincides being significantly reduced. This suggests that increased with them having intracellular phases that require T cells infection and exposure may exhaust the T cell response. for efficient immune protection or eradication. T cell a b Fig. 6 Lung function and BSI related to immune responses against H.influenzae in BR. (a) T cell responses against H.influenzae measured by IFNγ ELIspot and expressed as spot-forming cells per 10 PBMC plotted against FEV1% predicted. (b) T cells responses against H.influenzae plotted against BSI. (c) Antibody levels against H.influenzae were measured by ELISA to give IgG titres (1/value) in BR plotted against FEV1% predicted. (d) Antibody responses against H.influenzae plotted against BSI. Spearman correlation was performed Jaat et al. Respiratory Research (2018) 19:106 Page 10 of 12 responses did not show any associations with exacerbation particularly be a useful way to identify a frequent exacer- level. However, increased IFNγ ELIspot T cell responses bator phenotype. With regard to an antibody marker of against H.influenzae showed significant positive associ- current colonization with P.aeruginosa, this data showed ation, albeit weak, with lung function (FEV %) and nega- 92% specificity (ability to show true negatives) and 73% tive association with BSI, which may suggest that T cells sensitivity (ability to show true positives) based on the HV are protective against disease, in contrast to antibody re- mean + 2 sd. This is similar to previous findings [31]. sponses which showed a negative correlation with FEV %, The strengths of the study were the extensive nature and may simply be associated with more infection. The of the immunological investigations carried out on pa- next aim was to investigate further the nature of the T tients, particularly those with BR, who were well charac- cells reactive against the two major pathogens, P.aerugi- terised clinically and microbiologically. One weakness is nosa and H.influenzae, in a sub-group of BR patients who that numbers of COPD patients producing sputum, and were good responders to the antigens and in comparison thus with positive cultures, was too low to allow a suffi- to good-responding HV. There was a tendency for IFNγ, ciently powered analysis to be undertaken for COPD IL-2 and IL-17 to be reduced in BR patients compared to and so the study focussed on BR after the initial obser- HV, suggesting greater antigen exposure, where memory vations (Fig.1). Furthermore, it would have been useful T cells producing IL-2 convert to T cells secreting effector to have longitudinal data of immune responses and cytokines. Conversely, there was a tendency for IL-10 to microbiology, and this is the subject of a future study. be increased in BR for both antigens suggesting their con- Another weakness is that microbiological culture is not version to a regulatory (Tr1) phenotype due to high and able to determine the complete microbial makeup in a sustained antigen exposure at the mucosal surface. IL-4 sample if it contains fastidious unculturable bacteria. We responses showed a significant increase in BR against are currently addressing this by carrying out genomic H.influenzae, similar to published work on COPD [30], analysis of patient sputum samples as well as microbio- but a tendency for the opposite for P.aeruginosa. This sug- logical culture. Finally, the cytokine secretion data would gests a discrepancy in immune responses between BR and have benefited from larger numbers, particularly for HV, HV, and against the two pathogens, reflecting the fact that and again this is the subject of ongoing work. Tcell response against H.influenzae was protective against disease. When pathogen-reactive T cells, based on CD69 Conclusion and CD4 staining, were examined for further key pheno- In conclusion, exposure to these lung pathogens generates typic markers no differences were found between BR and antibody responses of magnitudes that are broadly propor- HV. All reactive cells had high levels of CD49d, a lung tional to the level of exposure and thus disease (exacerba- homing receptor, but low levels of inflammatory homing tion, reduced lung function), and may be useful markers of receptors and the marker of senescence PD-1. disease. T cell responses appear to be reduced in patients The measurement of antibodies and T cells specific for with increased infection rates, and are proportional to lung P.aeruginosa and H.influenzae in patients with BR [31, function and BSI for H.influenzae, suggesting that they 32] and COPD [30, 33] has previously revealed increased maybeprotectiveagainst such apathogenthatispartially antibody responses associated with repeated infection, intracellular. The T cell responses in patients differ little in but decreased T cell responses, despite CD4 T cell pres- phenotype from HV, apart from possible subtle cytokine ence and oligoclonal TcR T cell expansion in the lungs differences that are currently being examined further. The [34, 35], suggesting immune dysregulation such as T cell interaction between T cells and antibody-producing B cells, exhaustion. Thus, while immune responses may be and how the two arms of the adaptive immune response protective, or a marker of infection by microbes, their interact and influence innate immunity, and ultimately im- dysregulation may be detrimental to the patient due to pact on bacterial infection and disease, is likely to be com- reduced protection from infection or through immuno- plex and multifactorial. The data in this study suggests the pathology as suggested in cystic fibrosis [36]. Studying use of antibodies for Pseudomonas-inducing disease diag- responses in disease states is important as this may reveal nosis, whilst T cells may indicate protective immunity mechanisms of disease that are direct (via immunopathol- against Haemophilus, suggesting a possible benefit of T ogy) or indirect (via anti-microbial effects) that may pro- cell-inducing vaccines. vide therapeutic targets. Furthermore, studies of such blood-based immunodiagnostics may be useful for diagno- sis and stratification of patients, and their responses to Additional file treatment [31], when microbiology or genomic analysis is not possible or reliable (young BR patients, no sputum, Additional file 1: Supplement: The significance of anti-bacterial immune responses in Bronchiectasis and Chronic Obstructive Pulmonary Disease. difficult to culture microbes, false negative). Baseline (DOCX 235 kb) immunity related to contemporaneous microbiota may Jaat et al. Respiratory Research (2018) 19:106 Page 11 of 12 Abbreviations 8. Rogers GB, Zain NM, Bruce KD, Burr LD, Chen AC, Rivett DW, McGuckin MA, BR: Bronchiectasis; COPD: Chronic obstructive pulmonary disease; Serisier DJ. A novel microbiota stratification system predicts future ELISA: Enzyme linked immunosorbent assay; ELIspot: Enzyme linked exacerbations in bronchiectasis. Ann Am Thorac Soc. 2014;11:496–503. immunospot; FEV: Forced expiratory volume; Hi: Haemophilus influenzae; 9. Rogers GB, van der Gast CJ, Cuthbertson L, Thomson SK, Bruce KD, Martin HV: Healthy volunteers; PSA: Pseudomonas aeruginosa ML, Serisier DJ. Clinical measures of disease in adult non-CF bronchiectasis correlate with airway microbiota composition. Thorax. 2013;68:731–7. Acknowledgements 10. Wang Z, Singh R, Miller BE, Tal-Singer R, Van Horn S, Tomsho L, Mackay A, We thank John Davison for clinical assistance in this study, and Jem Palmer Allinson JP, Webb AJ, Brookes AJ, George LM, Barker B, Kolsum U, Donnelly for help with LPS assays. LE, Belchamber K, Barnes PJ, Singh D, Brightling CE, Donaldson GC, Wedzicha JA, Brown JR, Sputum COPDMAP. Microbiome temporal variability and dysbiosis in chronic obstructive pulmonary disease exacerbations: an Availability of data and materials analysis of the COPDMAP study. Thorax. 2017;73:331–38. Reasonable request for raw data and materials relating to this work can be 11. McDonnell MJ, Jary HR, Perry A, MacFarlane JG, Hester KL, Small T, requested from the corresponding author. Molyneux C, Perry JD, Walton KE, De Soyza A. Non cystic fibrosis bronchiectasis: a longitudinal retrospective observational cohort study of Authors’ contributions Pseudomonas persistence and resistance. Respir Med. 2015;109:716–26. SMT, ADS and FGJ contributed to conception and design of the study. FGJ, 12. Jones CJ, Wozniak DJ. Psl produced by mucoid Pseudomonas aeruginosa SFH, AP, SC, JDP, SM, ADS, SMT collected and processed samples, acquired contributes to the establishment of biofilms and immune evasion. MBio. the data, analysed and interpreted the data. SMT and ADS wrote the 2017;8(3) manuscript. All authors read and critically revised the manuscript, and gave 13. Angrill J, Agustí C, De Celis R, Filella X, Rañó A, Elena M, De La Bellacasa JP, final approval for the submitted manuscript. Xaubet A, Torres A. Bronchial inflammation and colonization in patients with clinically stable bronchiectasis. Am J Respir Crit Care Med. Ethics approval and consent to participate 2001;164:1628–32. Ethical approval for the project was granted by the local NHS Research 14. Erb-Downward JR, Thompson DL, Han MK, Freeman CM, McCloskey L, Ethics Committee, the NRES Committee North East – County Durham & Schmidt LA, Young VB, Toews GB, Curtis JL, Sundaram B, Martinez FJ, Tees Valley (ref 12/NE/0248). All participants were adults who gave their Huffnagle GB. Analysis of the lung microbiome in the "healthy" smoker and informed consent. in COPD. PLoS One. 2011;6:e16384. 15. Boyton RJ, Smith J, Jones M, Reynolds C, Ozerovitch L, Chaudhry A, Competing interests Wilson R, Rose M, Altmann DM. Human leucocyte antigen class II ADS has received medical education grant support for a UK bronchiectasis association in idiopathic bronchiectasis, a disease of chronic lung network from GSK, Gilead, Chiesi and Forest labs. ADS’s employing institution infection, implicates a role for adaptive immunity. Clin Exp Immunol. receives fees for his work as Coordinating investigator in a phase III trial in 2008 Apr;152(1):95–101. Bronchiectasis sponsored by Bayer. All other authors have no competing 16. Boyton RJ, Altmann DM. Bronchiectasis: current concepts in pathogenesis, interests. immunology, and microbiology. Annu Rev Pathol. 2016;11:523–54. 17. Döring G, Meisner C, Stern M. Flagella vaccine trial study group. A double- blind randomized placebo-controlled phase III study of a Pseudomonas Publisher’sNote aeruginosa flagella vaccine in cystic fibrosis patients. Proc Natl Acad Springer Nature remains neutral with regard to jurisdictional claims in Sci U S A. 2007;104:11020–5. published maps and institutional affiliations. 18. Bumann D, Behre C, Behre K, Herz S, Gewecke B, Gessner JE, von Specht BU, Baumann U. Systemic, nasal and oral live vaccines against Pseudomonas Author details aeruginosa: a clinical trial of immunogenicity in lower airways of human Faculty of Health & Life Sciences, Northumbria University, Newcastle upon volunteers. Vaccine. 2010;28:707–13. Tyne NE1 8ST, UK. Department of Microbiology, Freeman Hospital, 19. Wu W, Huang J, Duan B, Traficante DC, Hong H, Risech M, Lory S, Priebe GP. Newcastle upon Tyne NE7 7DN, UK. Adult Bronchiectasis Service, Freeman Th17-stimulating protein vaccines confer protection against Pseudomonas Hospital, Newcastle upon Tyne NE7 7DN, UK. Institute of Cellular Medicine, aeruginosa pneumonia. Am J Respir Crit Care Med. 2012;186:420–7. Newcastle University, Newcastle upon Tyne NE2 4HH, UK. Zawia University, 20. Murphy TF. Vaccines for Nontypeable Haemophilus influenzae: the future is Zawia, Libya. College of Pharmacy, University of Kerbala, Kerbala, Iraq. now. Clin Vaccine Immunol. 2015;22:459–66. 21. Aguilar C, Malphettes M, Donadieu J, et al. Prevention of infections during Received: 16 February 2018 Accepted: 14 May 2018 primary immunodeficiency. Clin Infect Dis. 2014;59:1462–70. 22. Marsland BJ, Gollwitzer ES. Host-microorganism interactions in lung diseases. Nat Rev Immunol. 2014;14:827–35. References 23. McAleer JP, Kolls JK. Directing traffic: IL-17 and IL-22 coordinate pulmonary 1. World Health Organisation. Global burden of diseases. 2016. immune defense. Immunol Rev. 2014;260:129–44. 2. McDonnell MJ, Aliberti S, Goeminne PC, Restrepo MI, Finch S, Pesci A, 24. Chalmers JD, Smith MP, McHugh BJ, Doherty C, Govan JR, Hill AT. 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Host response to the lung microbiome in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2015;192:438–45. 35. Sullivan AK, Simonian PL, Falta MT, Mitchell JD, Cosgrove GP, Brown KK, Kotzin BL, Voelkel NF, Fontenot AP. Oligoclonal CD4+ T cells in the lungs of patients with severe emphysema. Am J Respir Crit Care Med. 2005;172:590–6. 36. Robinson KM, Alcorn JF. T-cell immunotherapy in cystic fibrosis: weighing the risk/reward. Am J Respir Crit Care Med. 2013;187(6):564. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Respiratory Research Springer Journals

Anti-bacterial antibody and T cell responses in bronchiectasis are differentially associated with lung colonization and disease

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Abstract

Background: As a way to determine markers of infection or disease informing disease management, and to reveal disease-associated immune mechanisms, this study sought to measure antibody and T cell responses against key lung pathogens and to relate these to patients’ microbial colonization status, exacerbation history and lung function, in Bronchiectasis (BR) and Chronic Obstructive Pulmonary Disease (COPD). Methods: One hundred nineteen patients with stable BR, 58 with COPD and 28 healthy volunteers were recruited and spirometry was performed. Bacterial lysates were used to measure specific antibody responses by ELISA and T cells by ELIspot. Cytokine secretion by lysate-stimulated T cells was measured by multiplex cytokine assay whilst activation phenotype was measured by flow cytometry. Results: Typical colonization profiles were observed in BR and COPD, dominated by P.aeruginosa, H.influenzae, S. pneumoniae and M.catarrhalis. Colonization frequency was greater in BR, showing association with increased antibody responses against P.aeruginosa compared to COPD and HV, and with sensitivity of 73% and specificity of 95%. Interferon-gamma T cell responses against P.aeruginosa and S.pneumoniae were reduced in BR and COPD, whilst reactive T cells in BR had similar markers of homing and senescence compared to healthy volunteers. Exacerbation frequency in BR was associated with increased antibodies against P. aeruginosa, M.catarrhalis and S. maltophilia. T cell responses against H.influenzae showed positive correlation with FEV %(r = 0.201, p = 0.033) and negative correlation with Bronchiectasis Severity Index (r = − 0.287, p = 0.0035). Conclusion: Our findings suggest a difference in antibody and T cell immunity in BR, with antibody being a marker of exposure and disease in BR for P.aeruginosa, M.catarrhalis and H.influenzae, and T cells a marker of reduced disease for H.influenzae. Keywords: Bronchiectasis, Antibodies, T cells, Lung function, Exacerbation, COPD * Correspondence: stephen.todryk@northumbria.ac.uk Anthony De Soyza and Stephen M. Todryk contributed equally to this work. Faculty of Health & Life Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, UK Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, UK Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jaat et al. Respiratory Research (2018) 19:106 Page 2 of 12 Background appear in individuals with defined immunodeficiencies The chronic lung diseases of bronchiectasis (BR) and [21], underlining the role of antibodies and phagocytes in chronic obstructive pulmonary disease (COPD) are both protection. Whilst healthy individuals are exposed to the associated with recurrent airway infections. COPD is a same pathogenic organisms as diseased individuals, major cause of death globally, with numbers of deaths healthy lungs typically have low levels of bacterial species, rising [1], and BR is underestimated with incidence ris- reflecting the naso-pharynx [22]. Immune responses ing in the UK by around 6% annually [2]. Whilst they against pathogenic microbes do not cause overt immuno- differ in disease causation, established disease in both is pathology in healthy individuals, but may contribute to mainly characterised by repeated or persistent heavy disease in colonized patients due to continuous immune bacterial colonization of the damaged lower respiratory stimulation by the localised high antigen doses, particu- tract. Such infection is associated with inflammation, larly through excessive Th17 responses that promote mucus production, and reduced ciliary action, which neutrophil infiltration [23]. Together with inflammatory promotes further infection, inflammation and tissue cytokines, neutrophils are abundant in the sputum of BR damage, in a vicious cycle [3]. Studies have suggested patients, and decline after antibiotic treatment [24]. It is that infection causes disease exacerbation and dimin- possible that dysfunction of both innate and adaptive im- ished lung function, which are often proportional to the munity contribute directly or indirectly to disease in both bacterial load and to reduced diversity [4, 5]. More re- BR and COPD. The aim of this study was to characterise cent findings propose more species-rich lung ecologies antibody and T cell responses against key lung microbes where alterations in specific bacterial populations, dys- in disease-stable patients with BR and COPD, charac- biosis, is at the heart of clinical disease [6, 7]. Pathogenic terised by the Bronchiectasis Severity Index (BSI) and bacteria, as determined clinically by microbiological cul- GOLD guidelines, respectively, in comparison to controls ture of expectorated sputum, are dominated by organ- (healthy volunteers), and to relate the immune responses isms specific to these diseases including Pseudomonas to culture-based bacterial colonization, lung function and aeruginosa, Haemophilus influenzae, Streptococcus pneu- frequency of exacerbation. moniae and Moraxella catarrhalis [8]. Recent studies using DNA-sequencing technology reveal more detailed Methods bacterial ecosystems in the lungs of diseased patients, Study participants and samples but with culture approaches mainly corroborated [9, 10]. Ethical approval for the project was granted by the local P.aeruginosa is considered the major cause of morbidity NHS Research Ethics Committee, the NRES Committee (increased exacerbations and reduced lung function) and North East – County Durham & Tees Valley (ref 12/NE/ mortality in BR [11], particularly during chronic infection 0248). Adult patients with (non-CF) BR, COPD and and mucoid characteristics of the bacterium [12], which healthy volunteer (HV) controls, were recruited at the may allow evasion of host immunity. Non-typeable strains Freeman Hospital, Newcastle upon Tyne. Female to of Haemophilus influenzae (NTHi) are frequently found male ration was about 1.5:1. BR is routinely confirmed in BR [13] and are not targeted by current vaccines. Both by high-resolution computed tomography (HCRT), and pathogens are also common in COPD albeit with a re- COPD according to prevailing GOLD guidelines (BTS duced frequency of Pseudomonas infections as compared and NICE 2010, [25]). Diverse aetiologies of BR were in- to BR [14]. Furthermore, less frequent suppurative infec- cluded in the study, with the exception of known tion and sputum production in COPD results in lower de- immunodeficiency. tection of pathogenic microbes, implying fewer infections Patients were clinically stable at the time of assess- than BR. Failure to produce sputum for microbiology, ment. They underwent spirometry to determine forced particularly in younger BR patients and in many COPD expiratory volume in 1 s (FEV ), and Forced Vital patients, as well as intermittent negative cultures, means Capacity (FVC), from which FEV1% predicted, FEV1/ that immune biomarkers of disease may provide a useful FVC ratio and FVC % predicted were obtained. The adjunct for directing clinical management. bronchiectasis severity index (BSI) score, as previously Knowledge of immunity in BR is limited, but studies sug- validated [26], was assessed. Patients were divided into 2 gest immune system genes that are involved in presentation groups: either those with one severe exacerbation requir- of antigens to CD4 T cells, such as HLA-DR1 and DQ5, ing hospitalisation or those with 3 or more exacerbations playarole[15, 16]. Notably, a role for adaptive immune re- per year, compared to those not requiring hospitalisation sponses (specific antibodies and T cells) in protection and having less than 3 exacerbations per year. The exac- against P.aeruginosa and H.influenzae, has been demon- erbations were determined for the preceding 12 months. strated in human vaccine trials in cystic fibrosis-related Colonization history of patients was also available going bronchiectasis [17, 18] and in mouse vaccination models back at least 4 years. Patients were categorised by patho- [19, 20]. Furthermore, the above-mentioned lung pathogens gen status based on positive sputum cultures. ‘Chronic Jaat et al. Respiratory Research (2018) 19:106 Page 3 of 12 colonization’ was defined here as 2 positive sputum cul- data t-test and Pearson correlation was used whilst for not tures at least 3 months apart in 12 months. ‘Chronic normally distributed data the Mann-Whitney U test and currently’ was defined as a positive sputum culture at Spearman’s correlation was used. A priori calculations time of blood sampling (for immune responses), and based on our previous data suggested that sufficient num- more than 2 positive sputum cultures in 12 months. bers were included to detect a modest effect with 0.9 ‘Previously chronic’ was defined as more than 2 positive power to a significance level of 0.05. SPSS v.15 and Graph sputum cultures in 12 months > 2 years ago. ‘Occasional’ Pad Prism were used for analysis. The cut-off value for infection was ≥ 1 positive sputum culture per year. ‘No statistical significance was p <0.05. colonization’ was sputum culture negative over at last 3 years (Table 3). Results Clinical data Sample processing and bacterial culture This study examined antigen-specific immune responses Heparinized venous blood samples from patients and in 119 BR and 58 COPD patients, and in 28 HV (Table 1), healthy controls were processed to give plasma for against lung pathogens that are commonly isolated from ELISA and peripheral blood mononuclear cells (PBMC) these patient groups. The patient groups showed typical for T cell assays (detailed in Additional file 1). Sputum clinical features, similar to previously-published reports samples were cultured in the Microbiology Department, e.g. average FEV % predicted, which was 68 for BR, 49 for Freeman Hospital, according to national standards. COPD and 113 for HV. FVC % predicted and FEV1/FVC ratio were also reduced in the patient groups compared to Enzyme-linked immunosorbent assay (ELISA) for serum HV. The BR group was predominantly of a post-infection antibody measurement aetiology, whilst COPD was smoking-related, and all pa- An indirect enzyme-linked immunosorbent assay (ELISA) tients with BR and COPD were in a clinically stable state method was used: to determine optimal dilutions for the with no current exacerbation. At the time of taking the coating of microbe-derived antigens, for initial serum blood samples, the BR group had a higher proportion of screening, and to undertake titration for total IgG (all patients producing sputum (93%) that could be microbio- subclasses combined) to give an end-point titre (e.g. 1 in logically tested than COPD (66%), and BR patients showed 1000), and for the measurement of individual Ig subclasses greater overall populations infected with the main bacter- (given as absorbance for 1 in 25 dliution), as described ial species (Table 2), many with multiple species. H.influ- previously [27](see Additional file 1). enzae was the most commonly identified species in both BR and COPD, followed by P.aeruginosa, S.pneumoniae T cell responses to microbial antigens and M.catarrhalis in BR, and M.catarrhalis, S.pneumoniae ELIspot as previously optimised and described [28] (see and P.aeruginosa in COPD. Additional file 1) was used to measure T cell responses against a range of stimuli including bacterial lysates plus Antibody responses selected peptide epitopes where available. For cell activa- The first experiments involved ELISA to determine anti- tion and surface staining of cells, PBMC were thawed body levels (total specific IgG), by end-point titre, and activated with stimuli for 20 h as detailed in the against the main bacterial species in BR, COPD and HV. supplement. Intracellular staining was performed to de- BR showed significantly higher levels of antibody against termine the number and phenotype of IFNγ-producing or activated CD69 T cells following stimulation. Table 1 Demographics of the subjects included in this study Characteristics BR COPD HV n = 119 n =58 n =28 Measurement of cytokines in culture supernatants using multiplex ELISA Sex (no.) 45/74 Not av. Not av. Male/female) Cytokines in supernatants from stimulated PBMCs were Age (y) 65 ± 1.08 69 ± 1.23 54 ± 3.01 measured using the Mesoscale Scale Discovery (MSD) multiplex cytokine Kit (Meso Scale Diagnostic, LLC, Exacerbations (per year) 4 ± 0.29 3 ± 0.38 Not app. Gaithersburg, USA). Multiplex kit – pro-inflammatory Smoking history (pack years) 8 ± 1.33 47 ± 4.08 Not av. panel 1 (for IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, FEV (% predicted) 68 ± 2.68 49 ± 2.70 113 ± 2.83 IL-12p70, TNFα) and cytokine panel 2 (IL-17A, IL-5) FVC (% predicted) 82 ± 2.48 77 ± 2.40 118 ± 2.7 were used. See Additional file 1. FEV /FVC ratio 66 ± 1.52 50 ± 2.17 83 ± 1.75 Values are presented as means ± SEM, exacerbations represents the number Statistics per year. FEV represent forced expiratory volume in the first second and FVC, The immunological data was tested for normal distribu- forced vital capacity. Not av., indicates the data are not available, whereas Not tion using the Shapiro-Wilk test. For normally distributed app., means that the category was not applicable Jaat et al. Respiratory Research (2018) 19:106 Page 4 of 12 Table 2 Microorganisms isolated from the sputum of patients associate with an occasional number of exposures, as with BR and COPD opposed to none or chronic, this was not significant. Microorganism Identified Bronchiectasis Chronic obstructive patients (%) pulmonary disease Exacerbation and lung function patients (%) The contribution of bacterial colonisation, as per Table 3, NT. H.influenzae 63.8 31 to lung function as measured by FEV % predicted, was ex- P. aeruginosa 56.3 12.0 amined in the BR cohort. Overall there was a downward S. pneumoniae 44.5 17.2 trend in lung function as bacterial colonisation became more frequent and with current positive cultures (Fig. 2a), M. catarrhalis 37.8 24.1 with P.aeruginosa, M.catarrhalis and S.maltophilia all A. fumigatus 22.6 3.4 showing a significant reduction. The numbers with M.- S. maltophilia 15.9 3.4 catarrhalis and S.maltophilia were low (n =10 and 9, Candida. sp 14.4 6.9 respectively) with 52 and 75% of these, respectively, being S aureus 30.3 1.7 co-colonised with P.aeruginosa. E.coli 20.1 5.2 Since exacerbation of disease is a key event in need of urgent clinical management, and makes up part of the No pathogen isolated 2.5 17.2 validated BSI scoring, exacerbation history of the BR No sputum produced 6.8 43.7 patients was used to determine whether there were any BR patients n = 119; COPD patients n = 58 informative associations with immunological responses. NT = non-typeable The patients were compared based on them having less P.aeruginosa, H.influenzae and S.maltophilia compared than 3 exacerbations, greater than or equal to 3, and to HV (Fig. 1a). COPD failed to show significant in- those that were hospitalised within the last year. This crease from HV for any of the bacteria. HV only showed breakdown of the patients was first validated by examin- significantly higher antibody responses than BR and ing the FEV % predicted within the groups compared to COPD against S.pneumoniae. HV (Fig. 2b). A significant decrease in lung function was The next aim was to relate specific IgG antibody levels seen moving from HV, through < 3 exacerbations, ≥ 3 against bacteria, to bacterial colonization status and his- exacerbations, to hospitalised. Since the hospitalised tory. Because of the relatively low infection rates in COPD, group comprise a variable causality (and not necessarily the numbers were insufficient to determine significance, greater disease) and was relatively small in number, this and so we focussed on BR. BR patient pathogen status was was omitted from further analysis. Small but statistically analysed and condensed into a categorization shown in significant increases in IgG titres against P.aeruginosa, Table 3,indicating ‘current chronic’, ‘previous chronic’, ‘oc- H.influenzae and M.catarrhalis were seen between < 3 casional’,or ‘no colonization’. Whilst there was an overall and ≥ 3 exacerbations (Fig. 3a-c). For T cell responses no trend for antibody titre to increase based on colonization significant differences were seen between the 2 groups (Fig. 1b), this was only significant for P.aeruginosa and for any bacterium (Fig. 3d-f). S.maltophilia. We further studied immunoglobulins by class and isotype across the disease groups and found in T cell phenotypes general higher levels of IgG1 and IgA in BR than COPD Since T cell responses of a single Th type (Th1/IFNγ) (Fig. 1e-g). may not be the only response against the lung bacteria, antigen-specific T cell cytokine responses were also mea- T cell responses sured in the supernatants of antigen-stimulated PBMC T cell responses, in the form of IFNγ spot-forming cells by multiplex cytokine analysis (Fig. 4a-h). A sub-group per 10 PBMC, were measured against the bacterial anti- of BR patients who were good responders to the anti- gens as for the antibodies. In contrast to the antibody gens, determined by both IFNγ ELIspot and by CD69 responses, there was an overall trend towards the BR expression following antigen-stimulation, were selected and COPD groups having lower T cell responses than for stimulation, as well as similarly good HV responders. the HV group (Fig. 1c). BR and COPD both had signifi- For IFNγ, IL-2 and IL-17 responses the BR patients cantly lower responses than HV against P.aeruginosa showed a trend, though not significant, towards reduced and S.pneumoniae. As with the antibody responses, the levels for both P.aeruginosa and H.influenzae compared relatively low infection rates in COPD meant that there to HV. For IL-4, BR patients showed marginally reduced were insufficient numbers of infected patients to deter- responses for P.aeruginosa but significantly increased re- mine significance and so BR was focussed upon for relat- sponses to H.influenzae compared to HV, although the ing to colonization (Fig. 1d). Whilst there was an overall responses showed considerable variability. Finally, IL-10 trend for T cell responses against all antigens to responses showed a trend for increased response in BR Jaat et al. Respiratory Research (2018) 19:106 Page 5 of 12 a c b d e f Fig. 1 Anti-bacterial antibody and T cells responses. Antibody levels against bacterial antigens were measured by ELISA: (a) IgG titres (1/value) against bacteria in BR, COPD and HV groups; (b) Antibody responses against bacteria were compared based on bacterial colonization. T cell responses against bacterial antigens were measured by IFNγ ELIspot: (c) IFNγ spot-forming cells per 10 PBMC against bacteria in BR, COPD and HV groups; (d) IFNγ spot-forming cells per 10 PBMC against bacteria were compared based on bacterial colonization. Anti-pseudomonas Ig subclasses and IgG isotypes in BR (e), COPD (f) and HV (g) subgroups. Mean value + SEM are given. *p < 0.05, ***p < 0.001. Mann-Whitney tests were performed Table 3 Classification of patients based on sputum microbiological results 0 No pathogen isolated (NPI) 1 Occasional ≥ 1 isolation in a year Chronic colonization is defined by the isolation of bacteria in sputum culture on 2 or more occasions, 3 months apart, within 1 year 2 Chronic previously Chronic in preceding 5 years but not last 2 years 3 Chronic currently Current, and chronic in last 2 years prior to recruitment Jaat et al. Respiratory Research (2018) 19:106 Page 6 of 12 Fig. 2 Lung function and exacerbation in BR. FEV1% predicted is a measure of lung function. (a) FEV1% predicted in BR patients grouped based on their bacterial colonization. (b) FEV1% predicted in BR patients based on exacerbation in the last year, and in HV – healthy subjects. Mean values + SEM are given. Mann-Whitney tests were performed over HV for both antigens, but again with much variabil- examined, a significant positive correlation (Fig. 6a), ity. All other cytokine responses tested were equivocal. albeit weak (r = 0.201; p = 0.033), was found only with T Flow cytometry analysis was used to further character- cell response against H.influenzae. With removal of the ise antigen-responding T cells in the same subgroups of outlier, significance was still retained (r =0.186, p =0.049). BR and HV. Similar proportions of activated CD69 Furthermore, a stronger (negative) correlation was ob- CD4 T cells were seen in BR and HV following stimula- served between BSI and anti-H.influenzae T cell re- tion with P.aeruginosa and H.influenzae, all being sponses (Fig. 6c)(r = − 0.287; p = 0.003), and again significantly higher than the unstimulated (medium) significance remained upon removal of an outlier control (Fig. 5a). Co-staining of the activated CD4 T (r = − 0.265, p = 0.0035). Conversely, a moderate cells with CD69 and for IFNγ intracellularly showed a negative correlation was seen (Fig. 6b) with antibodies concordance of the two forms of activation but showed against H.influenzae (r = − 0.224; p = 0.018), but none + + that there were more CD69 cells than IFNγ cells with BSI (Fig. 6d). This suggests that T cell responses (Additional file 1). Staining of the CD69 stimulated are associated with improved lung function and less cells for other potentially important markers (see severe disease, whereas higher levels of H. influenzae Additional file 1: Figure S1), showed significant levels of antibodies are associated with poorer lung function. + + staining (above isotype controls) on CD4 CD69 T cells Overall, no relationships were evident between the (Fig. 5b-e), but no significant difference between BR magnitudes of antibody and T cell responses against patient and HV cells. any of the bacteria examined. Furthermore, T cell and When the relationship between T cell responses (IFNγ antibody responses against bacteria showed no rela- ELIspot) and lung function (FEV % predicted) was tionships with one another (data not shown). 1 Jaat et al. Respiratory Research (2018) 19:106 Page 7 of 12 a d Fig. 3 Antibody and T cell responses in BR groups of different recent exacerbation history. Antibody levels against bacterial antigens were measured by ELISA to give IgG titres (1/value) in BR groups with < 3 compared to > 3 exacerbations in the last 12 months against bacteria. T cell responses were measured by IFNγ ELIspot and expressed as spot-forming cells per 10 PBMC. (a, d) P.aeruginosa (b, e) M. catarrhalis (c, f) H.influenzae. Mean values + SEM are given. Mann-Whitney tests were performed Discussion may suggest that COPD has reduced isotype switching, This study began by comparing immune responses which is usually controlled by cognate T cell responses, against common lung pathogens in BR, COPD and HV. through CD40:CD40L interaction and through cyto- The clinical categorisation of the patients followed kines. Reduced or altered antibody responses as we have standard processes and was in keeping with other stud- seen here could be due to increased regulatory T cells, ies in the field, as were the microbiology results ob- as have been demonstrated in COPD, which may depress tained. One expectation was that the degree of exposure protective immunity [29]. to the microbes will be proportional to the magnitude of Having found specific antibody responses to be in- immune response measured. This was broadly the case creased in BR, the question was whether these responses for antibody responses, which were higher in BR than showed a direct dynamic relationship with colonization COPD and HV, particularly against P.aeruginosa, levels. Sufficient numbers for this analysis were only H.influenzae and S.maltophilia, reflecting rates of posi- available in the BR group. Whilst there was a trend for tive sputum cultures in BR and COPD. Measurement of increasing antibodies with colonisation for each individual isotype components of the antibody responses against pathogen, only P.aeurginosa and S.maltophilia showed P.aeruginosa showed a high IgG1 component in BR and significance. We categorised patients based on their HV, compared to COPD which had a higher IgM. This exacerbation frequency (< 3, ≥ 3) which were validated by Jaat et al. Respiratory Research (2018) 19:106 Page 8 of 12 a e b f c g d h Fig. 4 Cytokine responses in a subset of BR patients and HV. PBMC from BR and HV were stimulated with bacterial antigens P.aeruginosa (PSA) and H.influenzae (Hi), and supernatants were tested for the following cytokines: (a) IL-2, (b) IL-4, (c) IL-12, (d) IL-17, (e) IL-2, (f) IL-5, (g) IL-13, (h) IL-10. Mean values + SEM are given in pg/ml. Mann-Whitney tests were performed showing reducing lung function. Although significant, FEV % predicted, suggesting it to be a marker of disease only modest increases in antibody against P.aeruginosa, and exposure. M.catarrhalis and H.influenzae were found in BR with ≥ 3 The measurement of T cell responses against lung exacerbations compared to < 3. Antibody response only pathogens may be useful for the diagnosis of latent against H.influenzae showed a negative correlation with infection, as is the case of the Quantiferon test for Jaat et al. Respiratory Research (2018) 19:106 Page 9 of 12 a b d c e Fig. 5 Flow cytometry responses on a subset of BR patients and HV. Flow cytometry was performed on PBMC from BR and HV stimulated with bacterial antigens. (a) CD69 activation marker following stimulation with medium only, P.aeruginosa or H.influenzae. The % of CD4+ CD69+ activated T cells with phenotypic markers following stimulation with (b) P.aeruginosa,(c) anti-CD3 monoclonal antibody, (d) H.influenzae and (e) medium only. Mean % values + SEM are given. Mann-Whitney tests were performed Mycobacterium tuberculosis (Mtb). In this study T cell Within the BR group T cell responses showed a trend for responses showed an overall tendency for reduction in BR being highest in the group that had occasional infections, and COPD compared to HV, associated with colonisation for all pathogens tested. The highest T cell responses were status, with responses to P.aeruginosa and S.pneumoniae found for H.influenzae and M.catarrhalis which coincides being significantly reduced. This suggests that increased with them having intracellular phases that require T cells infection and exposure may exhaust the T cell response. for efficient immune protection or eradication. T cell a b Fig. 6 Lung function and BSI related to immune responses against H.influenzae in BR. (a) T cell responses against H.influenzae measured by IFNγ ELIspot and expressed as spot-forming cells per 10 PBMC plotted against FEV1% predicted. (b) T cells responses against H.influenzae plotted against BSI. (c) Antibody levels against H.influenzae were measured by ELISA to give IgG titres (1/value) in BR plotted against FEV1% predicted. (d) Antibody responses against H.influenzae plotted against BSI. Spearman correlation was performed Jaat et al. Respiratory Research (2018) 19:106 Page 10 of 12 responses did not show any associations with exacerbation particularly be a useful way to identify a frequent exacer- level. However, increased IFNγ ELIspot T cell responses bator phenotype. With regard to an antibody marker of against H.influenzae showed significant positive associ- current colonization with P.aeruginosa, this data showed ation, albeit weak, with lung function (FEV %) and nega- 92% specificity (ability to show true negatives) and 73% tive association with BSI, which may suggest that T cells sensitivity (ability to show true positives) based on the HV are protective against disease, in contrast to antibody re- mean + 2 sd. This is similar to previous findings [31]. sponses which showed a negative correlation with FEV %, The strengths of the study were the extensive nature and may simply be associated with more infection. The of the immunological investigations carried out on pa- next aim was to investigate further the nature of the T tients, particularly those with BR, who were well charac- cells reactive against the two major pathogens, P.aerugi- terised clinically and microbiologically. One weakness is nosa and H.influenzae, in a sub-group of BR patients who that numbers of COPD patients producing sputum, and were good responders to the antigens and in comparison thus with positive cultures, was too low to allow a suffi- to good-responding HV. There was a tendency for IFNγ, ciently powered analysis to be undertaken for COPD IL-2 and IL-17 to be reduced in BR patients compared to and so the study focussed on BR after the initial obser- HV, suggesting greater antigen exposure, where memory vations (Fig.1). Furthermore, it would have been useful T cells producing IL-2 convert to T cells secreting effector to have longitudinal data of immune responses and cytokines. Conversely, there was a tendency for IL-10 to microbiology, and this is the subject of a future study. be increased in BR for both antigens suggesting their con- Another weakness is that microbiological culture is not version to a regulatory (Tr1) phenotype due to high and able to determine the complete microbial makeup in a sustained antigen exposure at the mucosal surface. IL-4 sample if it contains fastidious unculturable bacteria. We responses showed a significant increase in BR against are currently addressing this by carrying out genomic H.influenzae, similar to published work on COPD [30], analysis of patient sputum samples as well as microbio- but a tendency for the opposite for P.aeruginosa. This sug- logical culture. Finally, the cytokine secretion data would gests a discrepancy in immune responses between BR and have benefited from larger numbers, particularly for HV, HV, and against the two pathogens, reflecting the fact that and again this is the subject of ongoing work. Tcell response against H.influenzae was protective against disease. When pathogen-reactive T cells, based on CD69 Conclusion and CD4 staining, were examined for further key pheno- In conclusion, exposure to these lung pathogens generates typic markers no differences were found between BR and antibody responses of magnitudes that are broadly propor- HV. All reactive cells had high levels of CD49d, a lung tional to the level of exposure and thus disease (exacerba- homing receptor, but low levels of inflammatory homing tion, reduced lung function), and may be useful markers of receptors and the marker of senescence PD-1. disease. T cell responses appear to be reduced in patients The measurement of antibodies and T cells specific for with increased infection rates, and are proportional to lung P.aeruginosa and H.influenzae in patients with BR [31, function and BSI for H.influenzae, suggesting that they 32] and COPD [30, 33] has previously revealed increased maybeprotectiveagainst such apathogenthatispartially antibody responses associated with repeated infection, intracellular. The T cell responses in patients differ little in but decreased T cell responses, despite CD4 T cell pres- phenotype from HV, apart from possible subtle cytokine ence and oligoclonal TcR T cell expansion in the lungs differences that are currently being examined further. The [34, 35], suggesting immune dysregulation such as T cell interaction between T cells and antibody-producing B cells, exhaustion. Thus, while immune responses may be and how the two arms of the adaptive immune response protective, or a marker of infection by microbes, their interact and influence innate immunity, and ultimately im- dysregulation may be detrimental to the patient due to pact on bacterial infection and disease, is likely to be com- reduced protection from infection or through immuno- plex and multifactorial. The data in this study suggests the pathology as suggested in cystic fibrosis [36]. Studying use of antibodies for Pseudomonas-inducing disease diag- responses in disease states is important as this may reveal nosis, whilst T cells may indicate protective immunity mechanisms of disease that are direct (via immunopathol- against Haemophilus, suggesting a possible benefit of T ogy) or indirect (via anti-microbial effects) that may pro- cell-inducing vaccines. vide therapeutic targets. Furthermore, studies of such blood-based immunodiagnostics may be useful for diagno- sis and stratification of patients, and their responses to Additional file treatment [31], when microbiology or genomic analysis is not possible or reliable (young BR patients, no sputum, Additional file 1: Supplement: The significance of anti-bacterial immune responses in Bronchiectasis and Chronic Obstructive Pulmonary Disease. difficult to culture microbes, false negative). Baseline (DOCX 235 kb) immunity related to contemporaneous microbiota may Jaat et al. Respiratory Research (2018) 19:106 Page 11 of 12 Abbreviations 8. Rogers GB, Zain NM, Bruce KD, Burr LD, Chen AC, Rivett DW, McGuckin MA, BR: Bronchiectasis; COPD: Chronic obstructive pulmonary disease; Serisier DJ. A novel microbiota stratification system predicts future ELISA: Enzyme linked immunosorbent assay; ELIspot: Enzyme linked exacerbations in bronchiectasis. Ann Am Thorac Soc. 2014;11:496–503. immunospot; FEV: Forced expiratory volume; Hi: Haemophilus influenzae; 9. Rogers GB, van der Gast CJ, Cuthbertson L, Thomson SK, Bruce KD, Martin HV: Healthy volunteers; PSA: Pseudomonas aeruginosa ML, Serisier DJ. Clinical measures of disease in adult non-CF bronchiectasis correlate with airway microbiota composition. Thorax. 2013;68:731–7. Acknowledgements 10. Wang Z, Singh R, Miller BE, Tal-Singer R, Van Horn S, Tomsho L, Mackay A, We thank John Davison for clinical assistance in this study, and Jem Palmer Allinson JP, Webb AJ, Brookes AJ, George LM, Barker B, Kolsum U, Donnelly for help with LPS assays. LE, Belchamber K, Barnes PJ, Singh D, Brightling CE, Donaldson GC, Wedzicha JA, Brown JR, Sputum COPDMAP. Microbiome temporal variability and dysbiosis in chronic obstructive pulmonary disease exacerbations: an Availability of data and materials analysis of the COPDMAP study. Thorax. 2017;73:331–38. Reasonable request for raw data and materials relating to this work can be 11. McDonnell MJ, Jary HR, Perry A, MacFarlane JG, Hester KL, Small T, requested from the corresponding author. Molyneux C, Perry JD, Walton KE, De Soyza A. Non cystic fibrosis bronchiectasis: a longitudinal retrospective observational cohort study of Authors’ contributions Pseudomonas persistence and resistance. Respir Med. 2015;109:716–26. SMT, ADS and FGJ contributed to conception and design of the study. FGJ, 12. Jones CJ, Wozniak DJ. Psl produced by mucoid Pseudomonas aeruginosa SFH, AP, SC, JDP, SM, ADS, SMT collected and processed samples, acquired contributes to the establishment of biofilms and immune evasion. MBio. the data, analysed and interpreted the data. SMT and ADS wrote the 2017;8(3) manuscript. All authors read and critically revised the manuscript, and gave 13. Angrill J, Agustí C, De Celis R, Filella X, Rañó A, Elena M, De La Bellacasa JP, final approval for the submitted manuscript. 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Respiratory ResearchSpringer Journals

Published: May 30, 2018

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