Adaptive Immunity to Nontypeable Haemophilus influenzae Paul T. King, Paul E. Hutchinson, Paul D. Johnson, Peter W. Holmes, Nicholas J. Freezer, and Stephen R. Holdsworth Departments of Respiratory Medicine and Medicine, Monash Medical Centre, Monash University, Melbourne; and Department of Infectious Disease, Austin and Repatriation Medical Centre, Melbourne, Australia

Nontypeable Haemophilus influenzae (NTHi) colonizes the upper respiratory tract of most healthy people and is also a major cause of infection in chronic obstructive lung disease. The immune response to this bacterium has not been well characterized. We tested the hypothesis that recurrent airway infection with NTHi may be associated with nonclearing adaptive immunity. Study subjects were healthy control subjects and patients with idiopathic bronchiectasis who had severe chronic infection with H. influenzae. We established that all subjects in both groups had detectable antibody to NTHi, suggesting that most normal people have developed an adaptive immune response. To characterize the nature of the immune response, we measured antigen-specific production of T helper cell cytokines and CD40 ligand by flow cytometry and immunoglobulin subclass levels in peripheral blood. We found that normal control subjects made Th1 response to NTHi with distinct CD40 ligand production. In contrast, subjects with bronchiectasis had predominant production of Th2 cytokines, decreased expression of CD40 ligand, and different immunoglobulin G subclass production. Therefore, chronic infection with NTHi in bronchiectasis is associated with a change in adaptive immunity that may be important in the pathogenesis of bronchial infection. Keywords: Haemophilus influenzae; bronchiectasis; lymphocyte; immunoglobulin

Nontypeable Haemophilus influenzae (NTHi) lacks a polysaccharide capsule (distinguishing it from encapsulated forms like type b) and colonizes the upper respiratory tract of up to 75% of normal adults (1). Because of the high rate of colonization and the fact that it was often previously misidentified as type b (1), the role of NTHi as a pathogen has been unclear. However, it has now been recognized that this bacterium is also a major cause of respiratory infection, which tends to be chronic and recurrent and includes sinusitis, otitis media, tonsillitis, pneumonia, and chronic bronchitis (1–4). NTHi may cause systemic infection (1, 5). In the context of infection, it has the capacity to live intracellularly (6–13), especially in macrophages (6, 11–13). In chronic bronchitis, studies have shown that a large proportion of patients has persistent infection with NTHi (8, 9, 14, 15) and may show extensive invasion of the lung (8). H. influenzae is the most frequently isolated bacterium in subjects with bronchiectasis (16). Despite the fact that NTHi is perhaps the most common

(Received in original form July 22, 2002; accepted in final form October 22, 2002) Supported by a grant from the National Health and Medical Research Council of Australia (P.K.). Correspondence and requests for reprints should be addressed to Paul T. King, M.D., Department of Respiratory Medicine, Monash Medical Centre, 246 Clayton Road, Clayton, Melbourne, Victoria, 3168, Australia. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents online at www.atsjournals.org Am J Respir Crit Care Med Vol 167. pp 587–592, 2003 Originally Published in Press as DOI: 10.1164/rccm.200207-728OC on November 14, 2002 Internet address: www.atsjournals.org

cause of chronic respiratory infection, the nature of the adaptive immune response to it has not been well defined. The adaptive immune response is mediated by T helper (Th/ CD4⫹) cells, which become activated after presentation of antigen by an antigen-presenting cell. The activated Th cell then directs the immune response by the production of cytokines and signaling back to the antigen-presenting cell. The concept of Th1/Th2 cytokine differentiation in coordinating adaptive immunity has been well established. Th1 (cell-mediated) responses are directed against intracellular pathogens, whereas Th2 (humoral) responses are directed against extracellular pathogens. Initiation of an inappropriate response can lead to unhindered spread of infection resulting in severe host pathology (17). CD40 ligand (CD40L/CD154) is produced by Th cells to signal back to the antigen-presenting cell and is important in B cell proliferation and isotype switching and also for activating macrophages (18). Adaptive immune responses also result in the production of immunoglobulin G (IgG), which has four subclasses, each with distinct functional properties (19). We tested the hypothesis that recurrent airway infection with NTHi may be associated with nonclearing adaptive immunity. To define the immune response, we studied subjects with idiopathic bronchiectasis who were compared with healthy control subjects. We performed a comprehensive assessment of the antigen-specific adaptive immune response to NTHi by measuring (1) the seroprevalence rate, (2) cytokine production, (3) CD40L production, and (4) IgG subclass production. METHODS Patients A cohort of 15 patients who had had bronchiectasis diagnosed by computed tomography scanning was studied at Monash Medical Centre. Subjects had severe and long-standing (more than 10 years) symptoms (Table 1) and significant destruction of lung tissue (Figure 1A). The subjects had all had multiple isolates of H. influenzae from their sputum in the past 5 years. In all subjects, H. influenzae was the predominant species isolated from their sputum. The subjects with bronchiectasis also had a detailed clinical review to screen them for any predisposing conditions for their bronchiectasis and on clinical grounds were classified as having idiopathic bronchiectasis. Subjects were nonsmokers and did not have any other major illnesses. They were compared with 24 healthy control subjects (aged 46 ⫾ 3 years; range, 27–78 years). Ethical approval for this project was obtained from the Southern Health Ethics Committee, Monash Medical Centre.

Screening for Immune Function in Bronchiectasis Troup All of the patients with bronchiectasis had a comprehensive screen of their immune function where peripheral blood was taken and analyzed for a variety of parameters, including full blood examinations, immunoglobulins and complement levels, lymphocyte subsets/proliferation, and cystic fibrosis mutations.

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TABLE 1. BRONCHIECTASIS PATIENT DEMOGRAPHICS/ SYMPTOMS/SPIROMETRY n Age, yr Age range, yr Patients with fatigue Patients with dyspnea Exacerbations per year Duration of chronic cough, yr FEV1, % predicted FVC, % predicted

15 54 ⫾ 3 33–68 13 12 6⫾2 37 ⫾ 7 62 ⫾ 4 77 ⫾ 5

NTHi Antigen NTHi is a heterogeneous species, with multiple outer membrane protein subtypes (20). Therefore, nine sputum isolates of NTHi were heat inactivated, sonicated, and combined to make a pooled antigen. The nine samples were also analyzed for their outer membrane protein by a previously published method (21) and shown to have distinct subtypes (Figure 1B).

Measurement of Total Ig/IgG Subclasses by Enzyme-linked Immunosorbent Assay A standard sandwich enzyme-linked immunosorbent assay was used to measure immunoglobulin (total Ig and IgG subclasses) responses to the NTHi antigen in 13 control subjects and 13 bronchiectasis subjects.

Measurement of Antigen-specific Th Cell Responses by Flow Cytometry A previously published flow cytometry technique (22) was used to establish the nature of the Th cell cytokine and CD40L production to NTHi. Four milliliters of blood was obtained from each subject; 2 ml was used as a control sample and 2 ml as the antigen sample. Costimulatory antibodies were added to both samples and 100 ␮L of NTHi antigen to the antigen sample. Blood was incubated for 6 hours. Cells were then permeablized with saponin and incubated with fluorescent-labeled antibodies, and four-color flow cytometry was performed. The proportion of antigen-responding CD4⫹ cells was determined by gating on the CD4⫹CD69⫹ cells and measuring their cytokine staining. For each cytokine, we screened an average of 100,000 CD4⫹-stimulated cells. The response to tuberculosis antigen was tested by adding 100 ␮L of purified protein derivative instead of NTHi to subjects’ blood. In addition the interferon-␥ (IFN-␥) production to NTHi antigen was measured by the enzyme-linked immunospot assay.

Statistical Methods Differences between the two groups were analyzed by Mann Whitney U-test or the Student’s unpaired t-test as appropriate using Prism 2.0 (Graphpad Software, San Diego, CA). Differences with a p value of less than 0.05 were considered statistically significant. Results are expressed as means ⫾ SEM (see the online supplement for further details about the methods).

RESULTS Immune Function of Bronchiectasis Group

The subjects with bronchiectasis had severe clinical disease (Table 1), which was idiopathic and characterized by recurrent infection with H. influenzae. These patients had no major abnormalities on comprehensive testing of their immune responses. None of them had cystic fibrosis mutations. One subject had a lymphocyte proliferation that was low (720 CPM/1,000 lymphocytes, a normal range of more than 1,000). Thus, on both clinical and laboratory testing, subjects were considered to be immunocompetent and not to have any underlying immune deficiency that might make them susceptible to recurrent infection.

Figure 1. Responses to NTHi. (A ) Chest computed tomography scan of one of the subjects with bronchiectasis and chronic NTHi infection with an acute exacerbation. It shows widespread destruction of lung tissue and associated consolidation. (B ) Nine samples of NTHi were obtained from sputum to make up a pooled antigen. The nine different samples of NTHi were analyzed for their outer membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane protein preparation stained with Commassie blue showing distinct subtypes of the specimens.

Control Subjects and Subjects with Bronchiectasis All Had Detectable Antibody to NTHi

The seroprevalence rate to NTHi of the general population is not known. Using enzyme-linked immunosorbent assay, we measured the total Ig to NTHi antigen in patients with bronchiectasis and chronic infection with NTHi (n ⫽ 13) and healthy control subjects (n ⫽ 13). Both patients and control subjects all produced antibody to NTHi antigen, which was of similar total and end-point titer (Figure E1 in the online supplement). This result suggests that most normal people have developed an adaptive immune response to NTHi, and a significant proportion may have detectable production of cytokines and other immune mediators. Control and Bronchiectasis Groups Had Differing Th Cytokine Production

The production of the cytokines IFN-␥, interleukin (IL)-2, IL-4, and IL-10 by activated Th (CD4⫹CD69⫹) cells in response to stimulation by pooled NTHi antigen was measured in control subjects (n ⫽ 24) and bronchiectasis (n ⫽ 15) subjects. Results were expressed as the number of CD4⫹ cells per 100,000 screened. The profile of cytokine production was distinctly different between control and bronchiectasis groups. The predominant cytokines produced by the control group were the Th1 cytokines IFN-␥ and IL-2. In contrast, the predominant cytokines produced by the bronchiectasis group were the Th2 cytokines IL-4 and IL-10 (Figure 2). The number of activated Th cells producing IFN-␥ was significantly higher in the control subjects (70 ⫾ 30) than in subjects with bronchiectasis (2 ⫾ 0.5) (p ⬍ 0.0001). The number of acti-

King, Hutchinson, Johnson, et al.: Immunity to Haemophilus influenzae

Figure 2. Th cytokine production in control and bronchiectasis groups. Antigen-specific responses to NTHi by activated Th (CD4⫹CD69⫹) cells were measured by flow cytometry using intracellular cytokine staining. (A ) IFN-␥ production was significantly higher in the control group (p ⬍ 0.0001). (B ) IL-4 production was significantly higher in the bronchiectasis group (p ⬍ 0.05). (C ) IL-2 production was significantly higher in the control group (p ⬍ 0.005). (D ) IL-10 production was higher in the bronchiectasis group, but this did not achieve statistical significance.

vated Th cells producing IL-2 was significantly higher in the control subjects (21 ⫾ 6) than in subjects with bronchiectasis (3 ⫾ 1) (p ⬍ 0.005). In contrast, the number of activated Th cells producing IL-4 was significantly higher in the bronchiectasis group (10 ⫾ 2) when compared with the control group (2 ⫾ 0.5) (p ⬍ 0.05). IL10 production was also higher in the bronchiectasis group, but this did not achieve statistical significance (p ⫽ 0.10) (examples of Th1 and Th2 responses in control and bronchiectasis subjects are shown in Figure E2 in the online supplement). The IFN-␥ response to NTHi antigen was also measured by enzyme-linked immunospot assay in six control subjects and five bronchiectasis subjects. This confirmed that control subjects had a significantly higher production of IFN-␥ than bronchiectasis subjects (see Figure E3 in the online supplement for further details about enzyme-linked immunospot results).

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Figure 4. IgG subclass end point titers to NTHi in control and bronchiectasis groups. (A ) IgG1 levels were significantly higher in the bronchiectasis group (p ⬍ 0.05). (B ) IgG2 titers were similar between the two groups. (C ) IgG3 levels were significantly higher in the bronchiectasis group (p ⬍ 0.01). (D ) IgG4 levels were higher in the bronchiectasis group due to very high levels of three subjects, but this did not achieve statistical significance.

The number of activated Th cells producing CD40L was significantly higher in the control subjects (121 ⫾ 28) than in subjects with bronchiectasis (25 ⫾ 6) (p ⬍ 0.001). The number of activated Th cells producing both CD40L and IFN-␥ was significantly higher in the control subjects (40 ⫾ 11) than in subjects with bronchiectasis (2 ⫾ 0.5) (p ⬍ 0.0001). Subjects with Bronchiectasis Produced High Titers of IgG1 and IgG3 to NTHi

Although the IgG subclass levels were similar to control values, the antigen-specific IgG subclass production to NTHi antigen was quite different in the control and the bronchiectasis groups. We measured the IgG subclass production to NTHi antigen by enzyme-linked immunosorbent assay and found that the levels of IgG1 (p ⬍ 0.05) and IgG3 (p ⬍ 0.01) made by the subjects with bronchiectasis were significantly elevated compared with control subjects (Figure 4). The IgG4 levels were higher in the bronchiectasis group chiefly because of the very high levels of three subjects, but this did not achieve statistical significance.

Subjects with Bronchiectasis Had Decreased Production of CD40 Ligand

Control Subjects and Subjects with Bronchiectasis Made Similar Th1 Responses to Tuberculosis Antigen

The production of the CD40L by activated Th (CD4⫹CD69⫹) cells was also quite different between the two groups (Figure 3).

We tested the hypothesis that patients with bronchiectasis had a generalized defect in their antigen-specific responses. We studied

Figure 3. CD40L and CD40L/IFN-␥ expression in control and bronchiectasis groups. (A ) CD40L expression was significantly higher in the control group (p ⬍ 0.001). (B ) CD40L/IFN-␥ expression was significantly higher in the control group (p ⬍ 0.0001).

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the response to purified protein derivative of tuberculosis antigen, which is expected to be a Th1 response. We assessed the response to purified protein derivative in control subjects and subjects with bronchiectasis who had previously received BCG vaccination. Blood was taken from five control subjects and five subjects with bronchiectasis and incubated with purified protein derivative. Antigen-specific responses were measured by flow cytometry. Both the control subjects and the bronchiectatic subjects produced a Th1 predominant response with similar expression of CD40L (Figure E4 in the online supplement).

DISCUSSION NTHi is a major cause of adult respiratory infection (in contrast to type b, which usually does not infect children older than 6 years old) (1). The nature of the adaptive immune response to NTHi has not been clearly defined, although several studies have looked at Th responses with varying conclusions (23–25). We found that all of the normal control subjects that we tested had clearly detectable antibody, which was of similar titer to the bronchiectasis subjects. The data from our small sample of 13 control subjects suggest that NTHi may be a common bacterial infection in healthy adults. There are no absolute criteria for designation of a Th1 and Th2 response. However, in control subjects, the predominant cytokines produced were IFN-␥ and IL-2, consistent with a Th1 response. In the case of clinical infection, NTHi is often found intracellularly and particularly in macrophages (6–15). In this circumstance, the appropriate clearing response would be predicted to be Th1, as occurs with other intracellular bacteria such as Mycobacterium tuberculosis. Normal control subjects also had production of CD40L a critical requirement in the activation of macrophages (in association with IFN-␥). Thus, the responses made by healthy control subjects were characterized by the predominance of Th1 cytokines and CD40L. In contrast, subjects with bronchiectasis made a completely different immune response. These subjects who had recurrent infection with NTHi had a Th2 predominant response with the production of IL-4 and IL-10. Such a response would not be protective against an invasive intracellular pathogen. The number of cells producing Th2 cytokines was significantly lower than the number of cells producing Th1 cytokines, but it is known that Th2 cytokines are very potent in their biologic action and are found in significantly smaller quantities than in Th1 responses (26, 27). We also found that the expression of CD40L, particularly in association with IFN-␥, was significantly lower in the bronchiectasis group, and this would be associated with decreased activation of macrophages. The expression of CD40L is also important in the production of IL-12 (18) by the macrophage, a key event driving Th1 cytokine production. Subjects with bronchiectasis had higher levels of antigenspecific IgG1 and IgG3. IgG1 and IgG3 are the strongly opsonizing subclasses that are also potent activators of complement (19). In humans, these subclasses are thought to associate more with Th1 responses, but in contrast to mouse data (Th1 subclasses; IgG2a and IgG3) (19), this is still controversial. The different response made by the subjects with bronchiectasis may reflect an inherent susceptibility to NTHi and may be a key factor in the development of their disease. This could occur at multiple sites, including defects in antigen processing/ presentation, cell signaling, and cytokine production (see the online supplement for further discussion). There are a number of immune disorders that are associated with bronchiectasis both innate and adaptive, which include major histocompatibility complex (MHC) class I deficiency (28), human immunodeficiency virus

infection (29), neutrophil (30), and ciliary disorders. In this study, which primarily looked at adaptive immunity to NTHi, we did not screen for all the conditions that have been associated with bronchiectasis. Alternatively the responses that we measured may have developed in the context of chronic infection and be a form of immune tolerance. There is some evidence to suggest that tolerance occurs in infections such as hepatitis B (31) and trypanosomiasis (32). Recent work has highlighted the role of the dendritic cell in chronic infection and some chronic infectious agents such as herpes simplex (33), cytomegalovirus (34), and plasmodium falciparum (35) may inhibit DC maturation and decrease the efficacy of host immunity. It has been suggested that human immunodeficiency virus may induce tolerance by inducing regulatory T cells or depleting reactive T cells (36). Other chronic infections may also induce regulatory T cells (37). Chronic illness may also be associated with a change in adaptive immunity, and this could contribute to the different immune responses measured in these subjects with bronchiectasis. However, the patients despite their symptoms were all living independently and had normal proliferative responses to mitogenic stimulus and to purified protein derivative. Intracellular pathogens associated with a spectrum of clinical disease and immune responses include Leishmania major and Mycobacterium leprae. In both of these infections, host protective responses have been shown to be Th1 predominant, whereas Th2 responses (visceral leishmaniasis and lepromatous leprosy) (38, 39) are associated with progressive infection. It has also been realized that host-protective responses in these infections are associated with production of CD40L, whereas progressive infection is associated with significantly less expression (40, 41). In addition, patients with visceral leishmaniasis and lepromatous leprosy produce higher levels of antigen-specific IgG1 and IgG3 (42–44). The reason for the differing adaptive immune responses in leishmaniasis and leprosy is not well understood. In leprosy and leishmaniasis in which subjects do not make a clearing immune response, cytokine therapy can be helpful. Trials have shown that cytokines, particularly in combination with other agents such as antibiotics, may cause clearing of the infectious agent (38, 45–48). Inhaled IFN-␥, which in normal subjects is easy to administer and produces potent activation of pulmonary macrophages with no systemic side effects, is a potential option (49). In subjects with multidrug-resistant tuberculosis, aerosolized IFN-␥ was well-tolerated, stabilized body weight, decreased size of cavitary lesions, and reduced time to negative cultures (50). We hypothesize that the addition of cytokine therapy could be effective in our patients with bronchiectasis who have intractable symptoms despite full medical treatment. We have looked at the response to NTHi in the context of bronchiectasis. The group of patients that we examined had no clear cause for their illness, and we propose that the chronic infection with NTHi and its associated immune response has a major role in the pathogenesis of their condition. We also believe that our findings may be applicable to the most common cause of chronic lung disease, chronic obstructive pulmonary disease (COPD). COPD is a major and increasing problem worldwide that affects more than 50-million people and caused 2.74-million deaths in 2000 (51). Smoking is the major factor associated with COPD; however, only 10 to 15% of smokers will develop emphysema/bronchitis (52, 53), and it has also been shown that airway inflammation persists in COPD after the cessation of smoking (54). Thus, there are other factors that may be important, such as bronchial infection (55, 56). The exact role of infection in the pathogenesis of COPD is still contentious (55, 57, 58), but many subjects who have chronic bronchitis in this context have recur-

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rent infection with NTHi (8, 9, 14, 15). In addition, acute exacerbations of COPD are commonly due to bacterial infection (57), especially with NTHi (7, 9, 59), and chronic colonization with H. influenzae is associated with increased severity of exacerbations (60). In summary, NTHi is an important respiratory pathogen, which in the context of infection is often found intracellularly. Our data suggest that NTHi is a common bacterial infection, and most normal people have developed protective immunity to this bacterium with the production of Th1 cytokines and CD40L. In contrast, patients with bronchiectasis and persistent infection with H. influenzae had an immune response characterized by Th2 predominance, decreased CD40L production, and higher levels of IgG1 and IgG3. In these subjects with bronchiectasis who were otherwise immunocompetent, we propose their response to NTHi is a key factor in the pathogenesis of their lung disease. Cytokine therapy may be useful for these patients and also may have a role in other subjects who have chronic nonclearing infection with NTHi.

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Acknowledgment : The authors thank K. Edgerton for help with the enzyme-linked immunosorbent assays, K. Forshaw and D. du Sart for cystic fibrosis mutation analysis, P. Midolo and J. Hamblin for help with the NTHi specimens, F. Oppesdisano for the outer membrane protein preparation, and V. Kovacs and G. Barker for graphics and photography.

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