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In the world today, asthma affects more than 235 million people. The widespread prescription of inhaled corticosteroids—the current gold standard of asthma control medication—allows many asthmatics to live symptom-free and has significantly reduced the number of deaths due to asthma. However, when the disease is poorly controlled, for example due to ubiquitous exposure to airborne fungal conidia, this chronic inflammatory disease often results in lung dysfunction caused by airway architectural changes. The role of B lymphocytes in allergic asthma has been relegated to the production of IgE with relatively little being known about the trafficking of these cells in the tissues or their role(s) in the affected tissue. As a first step in ascertaining their function, the initial aim of this project was to characterize the recruitment and localization of B cells in the murine lung in response to Aspergillus fumigatus inhalation. We found that CD19+CD23+ B2 lymphocytes were recruited to the lungs after fungal inhalation and that IgA-, IgE-, IgG-producing cells localized around the large airways. The second aim of the project was to begin defining the impact that these B lymphocytes have on the allergic lung. By using mice that were deficient of conventional B cells, we were able to demonstrate that the allergic phenotype was retained, although the impact of tissue B1 B cells cannot yet be ruled out. We then investigated the ability of hyaluronic acid (HA), a major component of the extracellular matrix (ECM) generated at sites of chronic inflammation, to recruit and modulate B lymphocyte functions in allergic fungal disease. We found that B lymphocytes undergo chemotaxis in response to LMM HA, while HMM HA had little to no effect on B cell chemotaxis. Furthermore, HA-mediated B lymphocyte chemotaxis was significantly inhibited by blocking the CD44 HA receptor. We also demonstrated that LMM HA fragments elicit the production of the pro-fibrotic cytokines IL-10 and TGF-β1 by B lymphocytes. These observations suggest a previously unrecognized role for B lymphocytes and HA in the context of allergy and represent novel pathways by which B lymphocytes may contribute to airway inflammation and airway remodeling.

The lung was recently identified to consist of a complex microenvironment made up of microorganisms that interact with one another and the host cells via direct and indirect interactions. As a result, understanding the dynamic of the microbiome in chronic respiratory diseases has become the focus of pulmonary researches. In cystic fibrosis (CF), chronic infections are a comorbidity associated with the genetic disorder. Recently, it was noted that the interactions of the fungus, Aspergillus fumigatus, and the bacterium, Pseudomonas aeruginosa together contribute to more severe disease outcomes in CF patients. In vitro co-cultures show that P. aeruginosa and A. fumigatus can affect one another’s growth and pathogenicity, but very few studies have attempted to model interactions of these microorganisms in vivo. Based on clinical and basic research, we developed a co-exposure model in which we could compare non-allergic and allergic animals co-exposed to Pseudomonas aeruginosa and Aspergillus fumigatus. While both groups had significant neutrophilia and production of acute phase response cytokines and chemokines, the allergic co-exposed group had a greater mortality with 34.8% of the animals expiring by 24h in comparison to 12.5% for the non-allergic co-exposed animals and 100% survival in the controls. A contributing factor to the more severe disease outcomes in the allergic co-exposed group is the increase in eosinophilic inflammation and IL-17A production, which only occurs when both microorganisms are viable. In addition, it was found that viable P. aeruginosa but not A. fumigatus causes interstitial inflammation, significant neutrophilia, and even death during co-exposures. The decline in health of animals co-exposed to the fungus and bacteria could be attributed not only to the host’s inflammatory response, but also to the spatial and temporal co-localization in the lung. To address this, we performed in vitro studies finding an aggregation of the microorganisms that could also be identified in vivo. This current research emphasizes the need for in vivo studies on polymicrobial interactions. / ND Agricultural Experiment Station; National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R155AI137886

Pulmonary tuberculosis (TB) remains one of the most infectious causes of death worldwide. Mycobacterium tuberculosis (M.tb), the causative agent of TB is transmitted via infectious aerosols, and in the majority of cases the bacteria is effectively controlled, by the host, resulting in a chronic latent infection. Currently, the only available vaccine is the Bacillus Calmette-Guérin (BCG), which despite being successful in preventing childhood disseminated forms of TB, has failed to control the adult pulmonary TB epidemic. One of the major contributing factors in the failure of the BCG is that although antigen-specific T cells are present at the time of M.tb infection, the recruitment of such T cells into the site of infection is significantly delayed. This delay, while reduced compared to non-vaccinated hosts, allows the bacteria to replicate unchecked within the lung and establish a “foothold” prior to the arrival of protective T cells and subsequent immune control. Thus, novel initiatives seek to close this “immunological gap” through increasing the level of protective T cell responses within the airway mucosa immediately following M.tb infection. We therefore investigated the impact of deliberate modulation of T cell geography following BCG vaccination on the outcome of pulmonary M.tb infection. In addition, a number of environmental factors are also thought to affect the site of M.tb infection: the respiratory mucosa. However, little is currently known about the effects of environmental exposure to allergens and other substances such as cigarette smoke on the outcome of pulmonary TB. Throughout this thesis we have investigated the mechanisms of immune protection and failure of protection against pulmonary M.tb infection within the respiratory mucosa. / Thesis / Doctor of Philosophy (Medical Science)

Regulatory T cells (Treg) are essential for the induction and maintenance of immunological tolerance to self and foreign antigens. The development of allergic asthma is mediated by T helper cell type-2 (Th2) inflammatory mechanisms and may also involve, based on murine and human studies of allergic asthma, compromised Treg immune regulation. Our overall objective was to more thoroughly elucidate the biology of Treg in allergic asthma, and to better understand their potential as a treatment for the disease. Initially, we characterized three different Treg phenotypes based on frequency and functional capacity. We showed both quantitative and functional heterogeneity in circulating Treg. Quantitative variability was also observed in circulating, but not airway, Treg when comparisons were made between healthy controls and asthmatic subjects. These findings emphasize the need for clear definitions of Treg phenotypes, and that interpretation of their frequency and function in health and disease needs to be phenotype-specific. Next, we assessed the Treg response in mild allergic asthmatic isolated early responders and dual responders after allergen inhalation challenge. We observed a reduced frequency of airway Treg after allergen challenge in DR, but not IER, associated with a smaller ratio of Treg to CD4+ cells. These data suggest that Treg to T effector cell (Teff) balance is important in the regulation of late asthmatic responses. Lastly, we evaluated the effects of two novel monoclonal anti-asthma therapies on circulating Treg after allergen inhalation challenge. We demonstrated that neither anti-OX40L nor anti-TSLP therapy altered circulating Treg frequency, while anti-TSLP, but not anti-OX40L, was effective in attenuating allergen-induced airway responses. These observations demonstrate the need for further investigation into the effects of anti-asthma therapies on Treg as well as the development of novel therapies aimed at manipulating Treg in order to better control immune responses. The findings of this thesis enhance our understanding of Treg in allergic asthma. Treg, utilized as stand-alone or adjunct therapy, may provide a novel therapy in the treatment of allergic asthma. / Thesis / Doctor of Philosophy (PhD)

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<p><strong>Background:</strong> Early infancy is a critical period during which the interplay between host and environmental factors influences susceptibility to allergic sensitization, a process that can also be construed as a failure to induce tolerance. Indeed, allergic asthma emerges, in most instances, in early childhood although the specific intervals of protection or susceptibility remain to be elucidated. We found that exposure to a concentration of allergen, house dust mite (HDM), that normally induces robust airway inflammation in adult mice elicits negligible immune-inflammatory responses in infant mice.</p> <p><strong>Methods:</strong> We investigated immune-inflammatory responses in mice exposed to 25 μg of HDM intranasally for 10 consecutive days at different points in development (3, 4, 5 and 7 weeks of age). We delineated the immune cell profile in the lungs of naïve mice from birth to adulthood, focusing on markers of immune maturation and immunosuppression. Moreover, we studied the impact of T-regulatory cell (Treg) depletion with the use of α-CD25 antibodies administered intraperitoneally one day prior to the start of HDM exposures, and then again on day 6 of the above protocol.</p> <p><strong>Results:</strong> Our data show that there is a progressive acquisition of immune-inflammatory responsiveness to HDM in BALB/c mice as exposures are initiated later in development, evidenced by total cell number and eosinophilia in the BAL and serum HDM-specific IgG₁ levels. Additionally, there is an immunological shift that occurs in the infant lung during development in that the early immunosuppressive environment, defined by T-regulatory cells and immunosuppressive alveolar macrophages, subsides as the capacity to respond to ensuing immune challenges, defined by natural killer (NK) cell, dendritic cell (DC) and alveolar macrophage (AM) maturation, increases. Specifically, in regards to the immunosuppressive lung environment during infancy, we identified higher baseline levels of CD25⁺Foxp3⁺CD101⁺ and CD25⁺Foxp3^highTregs, i.e. those with more potent suppressive ability. These populations also expand following HDM exposure in both adult and infant mice. Interestingly, 2 week-old infant mice depleted of Tregs or exposed to a very high dose of HDM (125 μg) overcome the natural immunosuppressive environment resulting in the acquisition of HDM responsiveness, as manifested by robust Th2 immune-inflammatory responses, comparable to that observed in 8 week-old adult mice.</p> <p><strong>Conclusion/Implications: </strong>Together, our data suggest that the hyporesponsiveness to HDM very early in life may be explained by two connected events: a) the inherent immunosuppressive environment in the lung, and b) the immaturity of the machinery for effective immune responses. Furthermore, we demonstrate that a disruption in the homeostatic immune balance in infancy, Treg depletion in this case, may lead to the imprinting of aberrant immune-inflammatory responses, like allergen sensitization. Thus, the inherent immunosuppressive environment in infancy may have long-term implications on allergen responsiveness.</p> / Master of Science in Medical Sciences (MSMS)

Yes / Background:To inform the development of the European Academy of Allergy and Clinical Immunology’s (EAACI) Guidelines on Allergen Immunotherapy (AIT) for allergic asthma, we assessed the evidence on the effectiveness, cost-effectiveness and safety of AIT. Methods:We performed a systematic review, which involved searching nine data-bases. Studies were screened against predefined eligibility criteria and critically appraised using established instruments. Data were synthesized using random-effects meta-analyses.Results:98 studies satisfied the inclusion criteria. Short-term symptom scores were reduced with a standardized mean difference (SMD) of 1.11 (95% CI 1.66, 0.56). This was robust to a prespecified sensitivity analyses, but there was evidence suggestive of publication bias. Short-term medication scores were reduced SMD 1.21 (95% CI 1.87, 0.54), again with evidence of potential publication bias. There was no reduction in short-term combined medication and symptom scores SMD 0.17 (95% CI 0.23, 0.58), but one study showed a beneficial long-term effect. For secondary outcomes, subcutaneous immunotherapy (SCIT) improved quality of life and decreased allergen-specific airway hyperreactivity (AHR), but this was not the case for sublingual immunotherapy (SLIT). There were no consistent effects on asthma control, exacerbations, lung function, and nonspecific AHR. AIT resulted in a modest increased risk of adverse events (AEs). Although relatively uncommon, systemic AEs were more frequent with SCIT; however no fatalities were reported. The limited evidence on cost-effectiveness was mainly available for sublingual immunotherapy (SLIT) and this suggested that SLIT is likely to be cost-effective. Conclusions: AIT can achieve substantial reductions in short-term symptom and medication scores in allergic asthma. It was however associated with a modest increased risk of systemic and local AEs. More data are needed in relation to secondary outcomes, longer-term effectiveness and cost-effectiveness. / EAACI; BM4SIT. Grant Number: 601763; European Union's Seventh Framework Programme FP7

A asma alérgica é uma doença inflamatória crônica das vias aéreas que envolve ativação de fibroblastos pulmonares. Esta ativação é induzida por TGF-<font face=\"Symbol\">b e este processo é regulado por moléculas anti-fibróticas. Nosso objetivo foi elucidar mecanismos envolvidos na fibrose das vias aéreas em um modelo de asma. Na primeira parte, investigamos o eixo síntese/resposta da PGE2. A PGE2 e seu análogo forskolina inibiram síntese de colágeno I e proliferação de fibroblastos. Estas células apresentaram perda tempo-dependente na capacidade de sintetizar PGE2 sob estímulo com IL-1<font face=\"Symbol\">b, e menor expressão de COX-2 e mPGEs-1. Na segunda parte, estudamos a relação TGF-<font face=\"Symbol\">b1/BMP-7 na fibrose das vias aéreas. Há predomínio da molécula pró-fibrótica TGF-<font face=\"Symbol\">b1 sobre a molécula anti-fibrótica BMP-7 nos pulmões de animais asmáticos. Em fibroblastos, a BMP-7 inibe a síntese de colágeno tipo I induzida pelo TGF-<font face=\"Symbol\">b1 e as vias de SMAD-2, SMAD-3 e p38. O tratamento dos animais com BMP-7 causou diminuição significativa da fibrose. Os resultados implicam estes mecanismos na fibrose das vias aéreas na asma. / Allergic asthma is a chronic inflammatory disease of the airways that involves activation of lung fibroblasts. This activation is induced by TGF-<font face=\"Symbol\">b and this process is regulated by anti-fibrotic molecules. Our goal was to elucidate mechanisms involved in airway fibrosis in an animal model of asthma. In the first part, we investigated the PGE2 synthesis/response axis. PGE2 and its analog forskolin inhibited collagen I synthesis and proliferation of fibroblasts. These cells showed a time-dependent loss in the ability to synthesize PGE2 under IL-1<font face=\"Symbol\">b stimulation, and downregulated COX-2 and mPGEs-1. In the second part, we studied the ratio TGF-<font face=\"Symbol\">b1/BMP-7 in airway fibrosis. There is predominance of the pro-fibrotic TGF-<font face=\"Symbol\">b1 over the anti-fibrotic BMP-7 in the lungs of asthmatic animals. In fibroblasts, BMP-7 inhibits TGF-<font face=\"Symbol\">b1-induced type I collagen synthesis and the SMAD-2, SMAD-3 and p38 pathways. Treatment of the animals with BMP-7 caused significant decrease in fibrosis. The results implicate these mechanisms in airway fibrosis in asthma.

Background: It is unclear how far the superior efficacy of omalizumab, established in randomized controlled clinical trials of patients with severe allergic asthma (SAA), translates into routine practice and when compared to matched controls. Methods: New-onset omalizumab-treated (OT) patients with SAA (n = 53) were compared to a matched control group of usual-care (UC) patients (n = 53). Treatment and procedures were naturalistic. Subsequent to a baseline assessment, patients were followed up over at least 6 months with at least two follow-up assessments. Primary clinical outcomes were the number of asthma attacks, persistence of asthma symptoms and degree of control [asthma control test (ACT), Global Initiative for Asthma]. Secondary outcome criteria were quality of life (Euro-Qol 5D) and number of medications. For each outcome we compared within-group effects from baseline to 6-month follow-up as well as between-group effects. Results: OT patients showed significant improvements in number [effect size (ES) = 0.03] and frequency (ES = 0.04) of asthma attacks as well as asthma control (ES = 0.09), whereas controls revealed no significant improvements in these measures. Further improvements in the OT group were found for ‘perceived control always’ (ACT, p = 0.006), no impairment (ACT, p = 0.02), reduction of sickness days (p = 0.002) and number of medications needed (p = 0.001). Conclusions: Substantial beneficial effects of omalizumab, similar to those observed in controlled trials and after marketing studies, were confirmed, particularly with regard to the reduction of asthma attacks, persistence of symptoms, asthma control and reduction of concomitant asthma medications. This study provides a tougher test and generalizable evidence for the effectiveness of omalizumab in routine care.