Role of infections in the developmental origins of asthma and other diseases
Infections in early life have long-term consequences on respiratory and general health. This is because immunity in early life is plastic and can be altered and moulded by factors and events such as exposure to infections and allergens, as well as different diets and obesity.Reference Bush 1 – Reference Jensen, Collins, Gibson and Wood 8 Infections in early life alter immunity by affecting the phenotype of immune responses, especially of T helper cells, macrophages and other immune and structural cells.Reference Culley, Pollott and Openshaw 9 – Reference Horvat, Starkey and Kim 11 Infections may also reduce lung function, which becomes irreversible as the lungs mature during the first 2 years of life. Thus, infections may play a major role in the developmental origins of health and disease. They may either protect against or predispose to the development of many other conditions, for example, asthma.Reference Hansbro, Beagley, Horvat and Gibson 3 , Reference Hansbro, Horvat, Wark and Hansbro 4 , Reference Horvat, Beagley and Wade 10 – Reference Dharmage, Erbas and Jarvis 17
The nature of immune responses to infectious agents and allergens is the result of coordinated responses of immune cells that regulate each other through complex signalling networks. Innate and adaptive immune cell activity and cytokine release may be critically important in driving pathogenic responses to infections and to allergens. In asthma, T helper type 2 (Th2) cells and the cytokines they release, interleukin (IL)-4, IL-5 and IL-13, mediate pathogenesis (reviewed inReference Hansbro, Kaiko and Foster 18 ). IL-4 is necessary for Th2 cell differentiation, promotes immunoglobulin E production, allergic inflammation and the development of mucus hypersecretion and airway hyper-responsiveness (AHR). IL-5 is required for the development and influx of eosinophils and may contribute to AHR. IL-13 is a critical mediator of Th2 responses and hallmark features of allergic airway disease (e.g. inflammation, mucus hypersecretion and AHR). Antigen-presenting cells (APCs), plasmacytoid and myeloid dendritic cells (DCs) and alveolar macrophages, are innate immune cells that are critical mediators of adaptive responses to infection and are important in the mechanisms that lead to alterations in T-cell programming. APCs recognize pathogens and present antigen as part of the major histocompatibility complexes to T cells, which drives the development of effector T cells with particular functions (e.g. protective or destructive).Reference Kaiko, Phipps and Hickey 19 – Reference Yang and Gao 21 Infections may alter the phenotype of APCs by altering surface molecule expression, inducing Th2 and inflammatory responses that promote allergic airway diseases, such as asthma.
Numerous pathogens that infect the lung are associated with the development of asthma. Early-life infections with viruses (respiratory syncytial virus, rhinovirus, influenza) and bacteria (Chlamydia and Mycoplasma) have deleterious effects and may drive the development of wheezing, asthma and stress and potentially have systemic effects.Reference Hansbro, Beagley, Horvat and Gibson 3 , Reference Hansbro, Horvat, Wark and Hansbro 4 , Reference Wynne, Horvat and Kim 22 – Reference Wynne, Horvat and Osei-Kumar 24 Others, such as probiotics, and as we have shown Streptococcus pneumoniae, may be beneficial and suppress pro-asthmatic responses.Reference Thorburn and Hansbro 12 , Reference Singh and Ranjan Das 25 – Reference Thorburn, Foster, Gibson and Hansbro 29 There has also been substantial recent interest in the role of the entire lung microbiome in the development of asthma.Reference Hilty, Burke and Pedro 30 – Reference Huang, Nelson and Brodie 32 There is an increase in the numbers and species (i.e. the bioburden) of the lung microbiome in asthma, and reduced lung function is related to bioburden and diversity. Nevertheless, the mechanisms that underpin the effects of infections remain poorly understood. If we can elucidate the mechanisms, we may be able to develop new treatments for asthma that either target deleterious pathogens or utilize beneficial ones.
The association of chlamydial respiratory tract infections in early life and asthma
Chlamydial respiratory tract infections (RTIs) are an example of how infections in early life may promote the developmental origins of disease and asthma. RTIs with Chlamydia pneumoniae are very common and frequently cause asymptomatic lung infection and acute respiratory disease in children and adults. At any particular time, 5% of children and adults have asymptomatic chlamydial respiratory infection.Reference Gnarpe, Gnarpe and Sundelöf 33 , Reference Schmidt, Muller, Mahner and Wiersbitzky 34 Importantly, 80% of adults have anti-chlamydial antibodies, indicating that most individuals have been infected at some stage of their lives.Reference Kuo, Jackson, Campbell and Grayston 35 Substantial epidemiological, clinical and experimental evidence associates infection with C. pneumoniae with the development and exacerbation of asthma, particularly severe asthma in children and adults.Reference Hansbro, Beagley, Horvat and Gibson 3 , Reference Webley, Tilahun and Lay 13 , Reference Wark, Johnston, Simpson, Hensley and Gibson 36 – Reference von Hertzen 40 Recent studies specifically implicate early-life chlamydial infection in the development of reduced lung function and asthma later in life.Reference Horvat, Beagley and Wade 10 , Reference Horvat, Starkey and Kim 11 , Reference Webley, Tilahun and Lay 13 , Reference Sutherland and Martin 37 – Reference von Hertzen 40 Early C. pneumoniae infection in children is strongly associated with acute wheezing, disease progression and asthma.Reference Webley, Tilahun and Lay 13 , Reference Webley, Salva and Andrzejewski 38 , Reference Normann, Gnarpe and Wettergren 39 , Reference Cunningham, Johnston, Julious, Lampe and Ward 41 – Reference Zaitsu 45 Indeed, 50% and 70% of children with severe asthma have culturable and detectable C. pneumoniae and/or Chlamydia trachomatis in their lungs, respectively. The role of Chlamydia in asthma is almost certainly underappreciated because of the following: the need to assess lower respiratory tract tissues, difficulties in detection, lack of standardized methods, assessment not being routine in clinical laboratories, infections often being asymptomatic and the effects of infection persisting after the clearance of the bacteria.Reference Hansbro, Beagley, Horvat and Gibson 3 , Reference Wang, He and Baines 46 , Reference Webley and Hahn 47 Given that many individuals are infected in the respiratory tract with Chlamydiae, it is likely that ‘two hits’ are required for asthma development and both environmental and genetic factors are likely to contribute.Reference Holt and Sly 48 Such factors include the status of maturation of the immune system and allergic sensitization. Indeed, some individuals may be genetically predisposed to aberrant immune responses (i.e. Th2 responses) to environmental factors (e.g. chlamydial RTIs) that may inhibit the clearance of infectious agents and promote the development of allergy and asthma.Reference Hansbro, Beagley, Horvat and Gibson 3
The mechanisms of the association between early-life chlamydial RTI, reduced lung function and enhanced asthma in later life remain little known and there are currently no effective treatments for infection-associated asthma. It is likely that infection induces permanent immune and structural alterations that play crucial pathogenic roles,Reference Brunetti, Colazzo and Francavilla 49 and we have investigated these possibilities (see subsequent sections). In addition, we, and others, have also shown that Chlamydia can infect and activate airway epithelial cells and induce the release of IL-8, IL-13 and granulocyte macrophage-colony-stimulating factor that could enhance airway inflammation and asthma severity.Reference Hansbro, Kaiko and Foster 18 , Reference Krüll, Bockstaller and Wuppermann 50 – Reference Jahn, Krüll and Wuppermann 52 Chlamydiae can also infect endothelial cells and induce the release of pro-fibrotic factors such as basic fibroblast growth factor and cytokines such as IL-8 and monocyte chemotactic protein-1, as well as soluble factors that may contribute to inflammation and remodelling in asthma.Reference Prochnau, Rödel, Hartmann, Straube and Figulla 53 – Reference Coombes and Mahony 55 These bacteria also have the capacity to infect airway smooth muscle cells,Reference Rodel, Woytas and Groh 56 which may alter muscle tone and induce AHR. Chlamydiae may also affect the innervation of the airways and can infect cells of the nervous system such as microglia and astrocytes and promote the release of pro-inflammatory cytokines such as IL-6, which may induce Th2 cell development and expansion, as well as AHR.Reference Doganci, Sauer, Karwot and Finotto 57 Detailed studies that determine how infections promote the development and exacerbation of asthma are required to facilitate the design of effective interventions.Reference Wills-Karp, Santeliz and Karp 58
Chlamydial RTI in early life causes permanent alterations in immunity and lung structure and function, and increases the severity of asthma
Our group, and others, have investigated the mechanisms of how chlamydial RTIs are associated with the induction and increased severity of allergic airway disease in later life using mouse models.Reference Horvat, Beagley and Wade 10 , Reference Horvat, Starkey and Kim 11 , Reference Jupelli, Murthy and Chaganty 59 We developed a model of neonatal chlamydial RTI and combined it with the classic model of ovalbumin-induced allergic airway disease. We used Chlamydia muridarum, which is a natural mouse respiratory pathogen and is the most appropriate chlamydial species to investigate host–pathogen relationships and the impact of infection on allergic airway disease in mice.Reference Horvat, Beagley and Wade 10 , Reference Horvat, Starkey and Kim 11 We infected neonatal mice on the 1st day of life with 400 inclusion-forming units of C. muridarum. The bacteria replicated in the lungs and infection peaked after 10 days, declined after 15 days and was cleared by 21 days.Reference Horvat, Beagley and Wade 10 Infection was mirrored by histopathological evidence of inflammation in the lung. Infection was allowed to resolve and allergic airway disease was induced on day 45. We then assessed the impact of infection on allergic airway disease.Reference Horvat, Beagley and Wade 10 , Reference Horvat, Starkey and Kim 11 Controls were: (1) infection without allergic airway disease, (2) allergic airway disease without infection and (3) no infection or allergic airway disease.
The induction of allergic airway disease resulted in the development of hallmark features of asthma with increased numbers of mucus-secreting cells around the airways, IL-13 expression in the lungs and increased AHR. Neonatal chlamydial RTI increased the severity of allergic airway disease in later life by further increasing mucus-secreting cell numbers, IL-13 expression and AHR. The mechanisms involved were then investigated. The development of allergic airway disease was also characterized by eosinophilic inflammation of airway tissue, increased Th2 cytokine release (IL-5 and IL-13) from ovalbumin-restimulated T cells in the lung-draining mediastinal lymph nodes (MLN) and the influx of activated mDCs into the lungs. Surprisingly, all of these pro-inflammatory responses that are normally associated with enhancing allergic airway disease were suppressed by the prior neonatal chlamydial RTI, with infection instead inducing a mixed Th2/Th1 response. The effects of infection on systemic DC-induced T-cell responses were then assessed. DCs were isolated from spleens of adult (9-week-old) mice that were infected or sham-exposed as neonates. DCs were pulsed with ovalbumin and cultured with ovalbumin-peptide T-cell receptor-specific transgenic (ovalbumin TCR Tg) T cells. Neonatal infection resulted in the increased release of IL-13 in DC and T-cell co-cultures. IL-13 can induce all the hallmark features of asthma in mice, and this effect of infection may account for some/all of the increases in severity of allergic airway disease. The effect of infection on lung structure was also assessed. Neonatal infection had striking and permanent effects on lung structure. Infection resulted in alveolar wall destruction, which substantially increased alveolar diameter in adulthood, which was reminiscent of emphysema in the parenchyma. These destructive effects were permanent as they were present in adult mice 9 weeks after neonatal infection. Infection had little observable effect on airway structure (thickness of airways-associated basement membrane, epithelial cell or smooth muscle layers), apart from mucus-secreting cell hyperplasia. These effects on parenchymal structure have recently been confirmed by othersReference Jupelli, Murthy and Chaganty 59 and may contribute to the induction of AHR and reduced lung function by neonatal infection. It is not known how these specific changes in lung structure enhance AHR, which was detected by increases in transpulmonary resistance and decreases in dynamic compliance (i.e. the inverse of elastic recoil). It is likely that reductions in alveolar attachments to the airway wall decreased airway support and elastic recoil, leading to alterations in resistance and compliance. Furthermore, the level of resistance is mostly driven by the larger airways, although the smaller airways contribute, whereas the smaller airways are primarily responsible for compliance. The consequences of infection that resulted in parenchymal destruction may have led to a stiffening of the smaller airways, which could enhance their contribution to resistance and reduce compliance. It is also possible that infection of the larger airways and smooth muscle may have altered their reactivity without changing their structure, although this possibility remains untested.
We also examined the effect of chlamydial infection during the infant and adult stages.Reference Horvat, Starkey and Kim 11 Infant mice were infected at 3 weeks of age just after weaning, which equates to a 2-year-old human infant, whereas adult mice were infected at 6 weeks of age. Allergic airway disease was induced 6 weeks after infection (i.e. when mice were 9 or 12 weeks old). Infant infection also increased the severity of allergic airway disease by increasing mucus-secreting cell numbers, IL-13 expression and reducing lung function. However, these effects occurred through very different mechanisms compared with neonatal infection. Infant infection increased Th2-mediated allergic inflammation (increased IL-5 and IL-13 release from MLN T cells stimulated with ovalbumin, and eosinophil influx into the airways) and systemic DC-induced IL-13 release, but had no effects on lung structure. By contrast, adult infection had no effect on lung structure and/or any of the features of allergic airway disease that were altered by neonatal and/or infant infections.
Thus, early-life infection increases the severity of allergic airway disease and potentially asthma, but occurs through different mechanisms in different age groups.
Chlamydial RTI, IL-13 and asthma
The role of IL-13 in chlamydial infections was further investigated.Reference Kaiko, Phipps and Hickey 19 , Reference Asquith, Horvat and Kaiko 51 Chlamydia is known to grow in innate immune cells such as neutrophils and macrophages,Reference Beagley, Huston, Hansbro and Timms 60 and these observations were extended to show that bone marrow DCs could also be infected.Reference Kaiko, Phipps and Hickey 19 Infection induced the production of IL-13 by DCs and increased the proliferation of ovalbumin TCR Tg T cells and promoted a Th2 phenotype. Adoptive transfer of infected and ovalbumin-pulsed DCs along with ovalbumin TCR Tg T cells into naïve adult mice increased IL-13 release into bronchoalveolar lavage fluid and AHR compared with similarly treated but uninfected DCs.Reference Kaiko, Phipps and Hickey 19 These studies suggest that chlamydial RTI may subvert DC function to promote Th2 responses and AHR in asthma. These Th2 responses may be ineffective in removing the bacteria, which requires interferon (IFN)-γ and Th1 responses for clearance.Reference Jupelli, Guentzel and Meier 61 , Reference Jupelli, Selby and Guentzel 62
We have also used adult IL-13-deficient (−/−) mice to demonstrate that infection and disease are markedly reduced in the absence of IL-13.Reference Asquith, Horvat and Kaiko 51 Compared with their wild-type counterparts, IL-13−/− mice did not lose weight and had reduced infection and airway inflammation. Depletion of CD4+ T cells did not affect the clearance of Chlamydia in IL-13−/− mice, suggesting a lack of CD4+ T-cell involvement. We then showed that a lack of IL-13 increased the uptake of Chlamydia by macrophages and decreased the infection of lung epithelial cells (i.e. LA4 murine lower respiratory epithelial cells).Reference Asquith, Horvat and Kaiko 51
Thus, IL-13 promotes chlamydial RTI, and as IL-13 is increased in the airways of asthmatics this may increase their susceptibility to infection and worsen the severity of asthma. We have also shown that an ongoing, rather than cleared, chlamydial RTI in adults increases the severity of asthma by inducing a phenotype resembling neutrophilic asthma that is dependent on infection-induced neutrophils.Reference Horvat, Starkey and Kim 63 We have made similar observations with Haemophilus influenzae that may be driven by infection-induced IL-17/Th17 responses.Reference Essilfie, Simpson and Horvat 64 , Reference Essilfie, Simpson and Dunkley 65
Other infections and the developmental origins of asthma
Infections with respiratory syncytial virus and rhinovirus in early life have also been widely associated with the induction and exacerbation of asthma, which we have extensively reviewed recently,Reference Hansbro, Horvat, Wark and Hansbro 4 and it is not necessary to repeat the details here. This probably occurs through increases in airway inflammation and inappropriate Th2 responses that are induced by these infections, and which are reinforced upon re-infection. A recent study has shown that infection of neonatal mice with pneumonia virus of mice, the murine equivalent of respiratory syncytial virus, also increased the severity of subsequent allergic airway disease.Reference Siegle, Hansbro and Herbert 66 This also occurred through the augmentation of Th2 responses. Again, infection was associated with the development of mucus hypersecretion and AHR, and the effects were dependent on signalling through the IL-4 receptor α.Reference Siegle, Hansbro and Herbert 66
Many studies have investigated the use of including probiotics in early life to inhibit the development of asthma and other allergic diseases. Probiotics can activate common mucosal surfaces, and effects in the gastrointestinal tract can alter responses in the airways.Reference Forsythe 67 They have immune-modulating capacity and can alter DC responses, induce the development of Tregs and attenuate allergic responses. Administration of probiotics in children may have benefits in eczema but trials investigating their capacity to suppress asthma have had variable results and are, as yet, not recommended for management of allergic diseases in children.Reference Forsythe 67 , Reference Yao, Chang, Hsu and Huang 68
We have performed a series of studies that suggest that the Gram-positive bacteria S. pneumoniae may also have beneficial effects in asthma.Reference Thorburn and Hansbro 12 , Reference Preston, Thorburn and Starkey 26 – Reference Thorburn, Foster, Gibson and Hansbro 29 Although this bacterium is a common respiratory pathogen, it is surprising that it has not been widely associated with asthma. The studies that have been performed have shown that infection suppresses peripheral eosinophiliaReference Bass 69 and that S. pneumoniae vaccination reduces asthma exacerbations.Reference Schuller 70 , Reference Ansaldi, Turello and Lai 71 Furthermore, asthmatic children do not necessarily seroconvert upon infection and have low S. pneumoniae antibody levels.Reference Rose, Schubert, Kujumdshiev, Kitz and Zielen 72 , Reference Lee, Kang and Henrichsen 73 Thus, infection or immunization with S. pneumoniae may boost immune responses and moderate allergic responses, thereby reducing the severity of asthma.Reference Thorburn and Hansbro 12 We have shown that S. pneumoniae infection, the killed bacteria or its human vaccines, suppress the asthma phenotype in our models of allergic airway diseases. This occurs through the induction of increased numbers of regulatory T cells that have enhanced suppressive capacity and reduce the proliferation of Th2 cells and their release of cytokines. It is known that S. pneumoniae induces the production of regulatory T cells to a greater extent than other Gram-positive bacteria.Reference Kadioglu, Coward, Colston, Hewitt and Andrew 74 Others have shown similar suppressive effects with daily gavage of mice with Lactobacillus,Reference Karimi, Inman, Bienenstock and Forsythe 15 Helicobacter infectionReference Arnold, Dehzad and Reuter 16 and a range of other bacteria.Reference Thorburn and Hansbro 12
Summary
Infectious challenge in early life can promote the developmental origins of both health and disease, and our hypothesis is that the outcome depends on the nature and age of infection and perhaps also the timing of infection relative to allergen exposure. Respiratory viruses and some bacteria, particularly Chlamydia and Mycoplasma, are associated with the induction or increased severity of asthma in early life. In contrast, exposure to probiotics, S. pneumoniae or other bacteria may be protective. Whether the effects are beneficial or deleterious are likely to result from the phenotype of immune responses to infection, the nature of the interaction of the infectious agent with the immune response and the impact of infection on lung structure and function. Elucidating the relationships between infections, immunity and lung structure in early life is likely to identify new potential avenues for the development of therapeutic approaches in order to prevent the emergence of disease in early life.Reference Szefler 75
Acknowledgements
Our work was supported by grants from the National Health and Medical Research Council of Australia, The Australian Research Council, the Asthma Foundation of NSW and the Hunter Medical Research Institute.
