Katrien De Grove

Characterization and Quantification of Innate Lymphoid Cell Subsets in Human Lung

Background Innate lymphoid cells (ILC) are a new family of innate immune cells that have emerged as important regulators of tissue homeostasis and inflammation. However, limited data are available concerning the relative abundance and characteristics of ILC in the human lung. Methods The aim of this study was to characterize and enumerate the different ILC subsets in human lung by multi-color flow cytometry. Results Within the CD45+ Lin- CD127+ pulmonary ILC population, we identified group 1 (ILC1), group 2 (ILC2) and group 3 (ILC3) innate lymphoid cells using specific surface markers (i.e. IL12Rβ2, CRTH2 and CD117 respectively) and key transcription factors (i.e. T-bet, GATA-3 and RORγT respectively). Based on the presence of NKp44, ILC3 were further subdivided in natural cytotoxicity receptor (NCR)+ and NCR- ILC3. In addition, we demonstrated the production of signature cytokines IFN-γ, IL-5, IL-17A, IL-22 and GM-CSF in the pulmonary ILC population. Interestingly, we observed a tendency to a higher frequency of NCR- ILC3 in lungs of patients with chronic obstructive pulmonary disease (COPD) compared with controls. Conclusions We show that the three main ILC subsets are present in human lung. Importantly, the relative abundance of ILC subsets tended to change in COPD patients in comparison to control individuals.

Dysregulation of type 2 innate lymphoid cells and TH2 cells impairs pollutant-induced allergic airway responses.

Background: Although the prominent role of TH2 cells in type 2 immune responses is well established, the newly identified type 2 innate lymphoid cells (ILC2s) can also contribute to orchestration of allergic responses. Several experimental and epidemiologic studies have provided evidence that allergen‐induced airway responses can be further enhanced on exposure to environmental pollutants, such as diesel exhaust particles (DEPs). However, the components and pathways responsible remain incompletely known. Objective: We sought to investigate the relative contribution of ILC2 and adaptive TH2 cell responses in a murine model of DEP‐enhanced allergic airway inflammation. Methods: Wild‐type, Gata‐3+/nlslacZ (Gata‐3–haploinsufficient), RAR‐related orphan receptor &agr; (ROR&agr;)fl/flIL7RCre (ILC2‐deficient), and recombination‐activating gene (Rag) 2−/− mice were challenged with saline, DEPs, or house dust mite (HDM) or DEP+HDM. Airway hyperresponsiveness, as well as inflammation, and intracellular cytokine expression in ILC2s and TH2 cells in the bronchoalveolar lavage fluid and lung tissue were assessed. Results: Concomitant DEP+HDM exposure significantly enhanced allergic airway inflammation, as characterized by increased airway eosinophilia, goblet cell metaplasia, accumulation of ILC2s and TH2 cells, type 2 cytokine production, and airway hyperresponsiveness compared with sole DEPs or HDM. Reduced Gata‐3 expression decreased the number of functional ILC2s and TH2 cells in DEP+HDM‐exposed mice, resulting in an impaired DEP‐enhanced allergic airway inflammation. Interestingly, although the DEP‐enhanced allergic inflammation was marginally reduced in ILC2‐deficient mice that received combined DEP+HDM, it was abolished in DEP+HDM‐exposed Rag2−/− mice. Conclusion: These data indicate that dysregulation of ILC2s and TH2 cells attenuates DEP‐enhanced allergic airway inflammation. In addition, a crucial role for the adaptive immune system was shown on concomitant DEP+HDM exposure. GRAPHICAL ABSTRACT Figure. No caption available.

The IL-33/ST2 axis is crucial in type 2 airway responses induced by Staphylococcus aureus–derived serine protease–like protein D

Background: Chronic airway inflammatory diseases, such as chronic rhinosinusitis with nasal polyps and asthma, show increased nasal Staphylococcus aureus colonization. Staphylococcus aureus–derived serine protease–like protein (Spl) D and other closely related proteases secreted by S aureus have recently been identified as inducers of allergic asthma in human subjects and mice, but their mechanism of action is largely unknown. Objective: We investigated the role of recombinant SplD in driving TH2‐biased responses and IgE formation in a murine model of allergic asthma. Methods: Allergic asthma was induced in C57BL/6 J wild‐type mice, Toll‐like receptor (TLR) 4 knockout (Tlr4−/−) mice, and recombination‐activating gene (Rag2) knockout (Rag2−/−) mice by means of repeated intratracheal applications of SplD. Inflammatory parameters in the airways were assessed by means of flow cytometry, ELISA, Luminex, and immunohistochemistry. Serum SplD‐specific IgE levels were analyzed by using ELISA. Results: We observed that repeated intratracheal exposure to SplD led to IL‐33 and eotaxin production, eosinophilia, bronchial hyperreactivity, and goblet cell hyperplasia in the airways. Blocking IL‐33 activity with a soluble ST2 receptor significantly decreased the numbers of eosinophils, IL‐13+ type 2 innate lymphoid cells and IL‐13+CD4+ T cells and IL‐5 and IL‐13 production by lymph node cells but had no effect on IgE production. SplD‐induced airway inflammation and IgE production were largely dependent on the presence of the functional adaptive immune system and independent of TLR4 signaling. Conclusion: The S aureus–derived protein SplD is a potent allergen of S aureus and induces a TH2‐biased inflammatory response in the airways in an IL‐33–dependent but TRL4‐independent manner. The soluble ST2 receptor could be an efficient strategy to interfere with SplD‐induced TH2 inflammation but does not prevent the allergic sensitization. Graphical abstract Figure. No caption available.

Pro- and Anti-Inflammatory Role of ChemR23 Signaling in Pollutant-Induced Inflammatory Lung Responses

Inhalation of traffic-related particulate matter (e.g., diesel exhaust particles [DEPs]) is associated with acute inflammatory responses in the lung, and it promotes the development and aggravation of allergic airway diseases. We previously demonstrated that exposure to DEP was associated with increased recruitment and maturation of monocytes and conventional dendritic cells (DCs), resulting in TH2 polarization. Monocytes and immature DCs express the G-protein coupled receptor chemR23, which binds the chemoattractant chemerin. Using chemR23 knockout (KO) and corresponding wild-type (WT) mice, we determined the role of chemR23 signaling in response to acute exposure to DEPs and in response to DEP-enhanced house dust mite (HDM)-induced allergic airway inflammation. Exposure to DEP alone, as well as combined exposure to DEP plus HDM, elevated the levels of chemerin in the bronchoalveolar lavage fluid of WT mice. In response to acute exposure to DEPs, monocytes and monocyte-derived DCs accumulated in the lungs of WT mice, but this response was significantly attenuated in chemR23 KO mice. Concomitant exposure to DEP plus HDM resulted in allergic airway inflammation with increased eosinophilia, goblet cell metaplasia, and TH2 cytokine production in WT mice, which was further enhanced in chemR23 KO mice. In conclusion, we demonstrated an opposing role for chemR23 signaling depending on the context of DEP-induced inflammation. The chemR23 axis showed proinflammatory properties in a model of DEP-induced acute lung inflammation, in contrast to anti-inflammatory effects in a model of DEP-enhanced allergic airway inflammation.

Aggravation of Allergic Airway Inflammation by Cigarette Smoke in Mice Is CD44-Dependent

Background Although epidemiological studies reveal that cigarette smoke (CS) facilitates the development and exacerbation of allergic asthma, these studies offer limited information on the mechanisms involved. The transmembrane glycoprotein CD44 is involved in cell adhesion and acts as a receptor for hyaluronic acid and osteopontin. We aimed to investigate the role of CD44 in a murine model of CS-facilitated allergic airway inflammation. Methods Wild type (WT) and CD44 knock-out (KO) mice were exposed simultaneously to house dust mite (HDM) extract and CS. Inflammatory cells, hyaluronic acid (HA) and osteopontin (OPN) levels were measured in bronchoalveolar lavage fluid (BALF). Proinflammatory mediators, goblet cell metaplasia and peribronchial eosinophilia were assessed in lung tissue. T-helper (Th) 1, Th2 and Th17 cytokine production was evaluated in mediastinal lymph node cultures. Results In WT mice, combined HDM/CS exposure increased the number of inflammatory cells and the levels of HA and OPN in BALF and Th2 cytokine production in mediastinal lymph nodes compared to control groups exposed to phosphate buffered saline (PBS)/CS, HDM/Air or PBS/Air. Furthermore, HDM/CS exposure significantly increased goblet cell metaplasia, peribronchial eosinophilia and inflammatory mediators in the lung. CD44 KO mice exposed to HDM/CS had significantly fewer inflammatory cells in BALF, an attenuated Th2 cytokine production, as well as decreased goblet cells and peribronchial eosinophils compared to WT mice. In contrast, the levels of inflammatory mediators were similar or higher than in WT mice. Conclusion We demonstrate for the first time that the aggravation of pulmonary inflammation upon combined exposure to allergen and an environmental pollutant is CD44-dependent. Data from this murine model of concomitant exposure to CS and HDM might be of importance for smoking allergic asthmatics.