Severe Asthma, Inflammation, and Lung Repair

Employing models of microbial colonization, pathogen infection, chronic inflammation and tissue repair, research in the Artis lab is examining how mammalian host genetics and signals derived from the environment and the microbiota influence innate and adaptive immune cell responses at the body’s barrier surfaces. We are employing gnotobiotic mice to examine the influence of defined beneficial microbial communities on intestinal and peripheral immune cell development, function and influence on tissue homeostasis. Our recent findings indicate that beneficial microbes have a significant regulatory influence on lymphocyte, innate lymphoid cell and granulocyte function associated with susceptibility to cancer and multiple infectious, inflammatory and metabolic disease processes. We have also developed a number of translational immunology projects, including analysis of tissue samples from patients with diseases of the barrier surfaces including atopic dermatitis, food allergy, obesity and IBD. It is hoped that the results of these basic and translational studies will advance understanding of the pathophysiology of multiple diseases associated with chronic inflammation, including asthma, allergy, inflammatory bowel disease, obesity and cancer and provide a framework to test new therapeutic pathways to prevent and treat these diseases.

Most cases of asthma are associated with the expression of T2 cytokines.  T2-high asthma is associated airway hyperresponsiveness (AHR), an exaggerated response to inhaled methacholine and other bronchoconstrictive agonists.  The excessive airway narrowing is mediated by contraction of airway smooth muscle (ASM).  ASM ex vivo does not show an increase in contractile properties, therefore modulation of ASM properties by inflammatory mediators is the likely culprit for AHR.  T cells infiltrate ASM bundles and are often juxtaposed to ASM cells.  Although the effector subset that these cells comprise is as yet uncharacterized, it seems plausible to consider that many will be Th2 cells.  Contact between T cells and ASM cells may have several outcomes, of which one is promoting proliferation of the ASM.  Contact-dependent prolongation of T cell survival also occurs and may contribute to chronicity of disease.   Proliferation of ASM is expected to reduce contractility of ASM but this may be transient whereas the acquisition of increased ASM mass will in itself promote bronchoconstriction.

Among the T2 cytokines interleukin-13 (IL-13) has been most explored for its effects on bronchoconstriction.  Exogenous administration of IL-13 to mice induces AHR whereas neutralization of IL-13 abrogates AHR caused by a number of stimuli including allergen and isocyanate.  AHR caused by IL-13 administration has been shown to be epithelial-dependent but deficiency of the IL-4 receptor alpha chain on ASM has also been shown to be necessary for IL-4 and IL-13 induced AHR.   However, deficiency of the IL-4Ralpha chain in ASM does not prevent allergen induced AHR in mice suggesting that the effects of IL-13 following allergen challenge are not mediated by direct effects on ASM.   No such experiments appear to have been done, so far, on larger mammals to test the relevance of the murine models for human asthma.

Several cytokines have been shown to augment ASM responses ex vivo.  Treatment of ASM in culture with IL-13 causes an enhanced calcium transient after stimulation with a contractile agonist and an enhanced force generation.  There is also an up-regulation of RhoA, a small GTPase associated with sensitization of the contractile apparatus, by IL-13. This is mediated by down-regulation of miR-133a, a microRNA that is inhibitory of RhoA synthesis.  In addition to effects on contraction, IL-13 attenuates β-agonist induced ASM relaxation.  Autocrine actions of ASM synthesized IL-5 and IL-13, triggered by contact with activated T cells, have been implicated in augmented responses of isolated ASM.

Clinical application of anti-IL-13 in severe asthma has been somewhat disappointing although combined anti-IL-13/anti-IL4 may have more promise.  The predominant effect of these treatments has been to reduce exacerbation rates, although some biologics also improve baseline lung function.  Whether any of these therapeutic effects are mediated by direct actions on ASM is unclear.

Immunoglobulin E (IgE) plays an important role in allergic diseases. Nevertheless, the source of IgE serological memory remains controversial. We re-examined the mechanism of serological memory in allergy using a dual-reporter system to track IgE plasma cells (PCs) in mice. Short-term allergen exposure resulted in the generation of IgE plasma cells that resided mainly in secondary-lymphoid organs and produced IgE that was unable to degranulate mast cells. In contrast, chronic allergen exposure led to the generation of long-lived IgE plasma cells that were primarily derived from sequential class switching of IgG1, accumulated in the bone marrow (BM) and produced IgE capable of inducing anaphylaxis. Most importantly, IgE plasma cells were found in the BM of human allergic, but not non-allergic donors, and allergen-specific IgE produced by these cells was able to induce mast cell degranulation when transferred to mice. These data demonstrate that long-lived IgE BMPCs arise during chronic allergen exposure and establish serological memory in both mice and humans.

Sensing of the gut microbiota, including fungi, regulates mucosal immunity. Whether fungal sensing in the gut can influence immunity at other body sites is unknown. Here we show that fluconazole-induced gut fungal dysbiosis has persistent effects on allergic airway disease in a house dust mite challenge model. Mice with a defined community of bacteria, but lacking intestinal fungi were not susceptible to fluconazole-induced dysbiosis, while colonization with a fungal mixture recapitulated the detrimental effects. Gut-resident mononuclear phagocytes (MNPs) expressing the fractalkine receptor CX3CR1 were essential for the effect of gut fungal dysbiosis on peripheral immunity. However, how mycobiota influence immunity in gut distal sites is not well understood. We developed protocols for gut-targeted depletion of phagocytes to investigate the influence of fungi on gut-lung crosstalk. Depletion of CX3CR1+ MNPs or selective inhibition of Syk signaling downstream of fungal sensing in these cells ameliorated lung allergy. These results indicate that disruption of intestinal fungal communities can mediate gut-lung-directed immune crosstalk and aggravate disease severity through fungal sensing by gut resident CX3CR1+ MNPs to prime fungal-specific Th2 cells. Specific targeting CX3CR1+ MNPs or Syk signaling within these cells might be useful clinically to alleviate allergic airway diseases related to fungal dysbiosis.

According to the World Health Organization, an estimated 300 million people worldwide suffer from asthma, with 250,000 annual deaths attributed to the disease. By 2025, it is expected that an additional 100 million people will have asthma. Although the prevalence of asthma in different countries varies widely, the World Allergy Organization reports that rates are rising in low and middle income countries and plateauing in high income countries.

This dramatically rising trend has been impacted by industry-derived emissions, motor vehicles, tobacco smoke, and global climate change. Increased concentrations of greenhouse gases, and especially carbon dioxide (CO2), in the atmosphere have already warmed the planet substantially, causing more severe and prolonged heat waves, variability in temperature, intensified pollen seasons, increased air pollution, forest fires, droughts, and floods — all of which can put the respiratory health of the public at risk.

While there is much work to be done to combat these environmental factors, research strives to better understand the Atopic March and the role of environmental triggers in Asthma exacerbations in order to mitigate the effects with targeted preventions and therapeutics. Use of high-dimensional approaches, such as mass cytometry, illuminate the effects of primary and secondary impacts of these exposures on immune cells, including T Cells, that may lead to diseases such as asthma and allergy. And through animal models and clinical trials, new therapies and interventions are emerging for severe asthma, including targeted biologics.

Pulmonary neuroendocrine cells (PNECs) are rare airway epithelial cells that are preferentially localized to airway branch points. They are innervated by nerves, and can produce potent neuropeptides, neurotransmitters and amines. Our data suggest that these cells are key sensors of allergen. In the absence of these cells, lung response to allergen is drastically dampened. We are currently testing the hypothesis that these cells serve as key nodes that mediate lung, environment, immune and nervous system interactions.

Asthma is a common, complex disease where both genetic factors and environmental exposures control susceptibility and disease progression. With the publication of initial efforts in sequencing the human genome the opportunity to genotype markers directly in genes of interest was greatly expanded.  Relying upon one of the simplest of these polymorphisms, single nucleotide polymorphisms (SNPs), this advancement allowed researchers to expand genetic studies beyond linkage toward the genetic association study design, and ultimately, genome-wide association studies (GWAS).  To date, 629 associations from >70 GWAS have been identified for asthma and its related traits.  However, most of the significant GWAS associations involve intergenic and non-coding variants tagging regulatory elements controlling biological processes, and most studies have been performed in Europeans. This is problematic because, in the U.S., there remains an epidemic of asthma that disproportionately affects underrepresented minorities and creates a major public health burden. Individuals of African ancestry have greater asthma morbidity and mortality both within and outside the U.S., supporting a role for genetics.  Although GWAS alone can provide little mechanistic insight into the loci associated with risk of asthma, when combined with an integrative, multi-omics approach, our ability to understand the molecular architecture underlying asthma is enhanced. Studies addressing these unmet needs are underway.

Charcot-Leyden protein crystals (CLC) have been described in the airways of asthmatics already in 1853. They consist of galectin-10 (Gal10), a protein that is abundantly produced by eosinophils. CLCs are merely seen as markers of eosinophilia, but we hypothesized they ight contribute to disease. Release of Gal10 was associated with EEtosis of eosinophils in human primary eosinophils. We found that recombinant crystalline Gal10 was completely biosimilar to in vivo obtained CLCs and induced innate airway inflammation, whereas a soluble Gal10 engineered to resist crystallization was inert in the airways. When co-administered with harmless antigens, only crystalline Gal10 stimulated adaptive immunity, Th2 sensitization, goblet cell metaplasia and airway eosinophilia. Antibodies directed against key epitopes of the crystallization interface dissolved pre-existing CLC in patient-derived mucus within hours, and reversed crystal driven inflammation, goblet cell metaplasia, IgE synthesis and bronchial hyperreactivity in a humanized asthma model. Thus, CLC promote key features of asthma and can be targeted by crystal dissolving antibodies.

Coauthors: E.K. Persson[1], K. Verstraete[2], I. Heyndrickx[1], H. Aegerter[1], E. Gevaert[5], J-M Percier[3], K. Deswarte[1], K. Verschueren[2], A. Dansercoer[2], D. Gras[4], P. Chanez[4], C. Bachert[5].

1. Immunoregulation Unit
2. Unit for Structural Biology, VIB Center for Inflammation Research, Ghent University
3. arGEN-X, Ghent, Belgium
4. Department of Respiratory Diseases, Aix-Marseille University, U1067, Marseille
5. Upper Airways Research Laboratory, ENT Department, Ghent University Hospital

My research focuses on deciphering mechanisms involved in lung repair and regeneration, with the aim to identify novel therapeutic targets relevant for chronic lung diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). In particular, my translational research program has focused on the comprehensive characterization of primary lung epithelial cells from experimental models and human tissue samples from IPF and COPD patients. Out of these studies, we identified the developmental WNT signaling pathway as a potent contributor to impaired lung repair and epithelial cell reprogramming,which is amenable to therapy and have further characterized features of epithelial cell reprogramming, such as cellular senescence. We apply primary cell isolates to study lung epithelial-mesenchymal interactions in decelluarized scaffolds and organoid cultures. We further pioneered and apply patient-derived 3D Lung Tissue Cultures that allow for high resolution cell-ECM imaging and assessment and further to test potential novel drugs in an individualized fashion, thus are suitable to enhance translation into clinical practice. I have been continuously funded externally over the past years and have the expertise, leadership, and motivation required to successfully carry out the proposed research project and will apply my extensive lung cell biology and injury/fibrosis expertise to this proposal.