Characterization and Quantification of Innate Lymphoid Cell Subsets in Human Lung.

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.The aim of this study was to characterize and enumerate the different ILC subsets in human lung by multi-color flow cytometry.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.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

ILC subsets in control subjects versus patients with COPD.

<p><b>A,</b> The frequency of ILC1 (CD45⁺, Lin^-, CD127⁺, CD56^-, IL12Rβ2⁺), ILC2 (CD45⁺, Lin^-, CD127⁺, CRTH2⁺), NCR⁺ ILC3 (CD45⁺, Lin^-, CD127⁺, CD117⁺, NKp44⁺) and NCR^- ILC3 (CD45⁺, Lin^-, CD127⁺, CD117⁺, NKp44^-) in digested human lung from control (n = 5) and COPD patients (n = 11) was determined by flow cytometry. ILC numbers were expressed as percentages (%) of the CD45⁺ population (mean ± SEM). <b>B,</b> Pie chart of the relative abundance of ILC1, ILC2, NCR⁺ ILC3 and NCR^- ILC3 subsets in control subjects and patients with COPD.</p

Intracellular cytokine production in the pulmonary ILC population.

<p>Several signature cytokines in pulmonary ILC (gated as CD45⁺, Lin^- CD127⁺ cells) were determined on single cell suspensions of digested human lung (n = 8). Since NK cells could contaminate the non-toxic ILC1 subset, CD56⁺ cells were excluded to investigate the IFN-γ production. For the production of these cytokines, lung cells were first stimulated for 15 hours with PMA/ionomycin (+ transport inhibitors). <b>A,</b> IFN-γ production in ILC. <b>B,</b> Production of IL-5 in the pulmonary ILC population. <b>C,</b> IL-17 production in ILC. <b>D,</b> ILC production of IL-22. <b>E,</b> Production of GM-CSF in the ILC population. The bottom panels represent the isotype controls of the specific cytokine staining. <b>F,</b> Frequency of IFN-γ, IL-5, IL-17A, IL-22 and GM-CSF positive cells within the ILC (CD45⁺, Lin^- CD127⁺) population (n = 8, mean ± SEM).</p

Intracellular staining of transcription factors in pulmonary ILC subsets.

<p>The developmental transcription factors were determined in the specific ILC subsets on single cell suspensions of digested human lung. <b>A,</b> Expression of T-bet (black line) versus isotype control (grey line) in the ILC1 (CD45⁺, Lin^- CD127⁺, CD56^-, CRTH2^-, CD117^-), ILC2 (CD45⁺, Lin^-, CD127⁺, CRTH2⁺), ILC3 (CD45⁺, Lin^-, CD127⁺, CD117⁺) population. <b>B,</b> GATA-3 expression (black line) versus isotype control (grey line) in the different ILC subsets. <b>C,</b> Expression of RORγT (black line) versus isotype control (grey line) in ILC1, ILC2 and ILC3 population.</p

Overview of innate lymphoid cell subsets in human lung tissue.

<p>The presence of CD45⁺, Lin^- (i.e. CD3, CD19, CD11c, CD11b) and CD127⁺ ILC in pulmonary tissue was demonstrated. These ILC were further subdivided in a CD56^- IL12Rβ2⁺ ILC1 subset, CRTH2⁺ ILC2 subset, CD117⁺ NKp44⁺ (NCR⁺) ILC3 subset and CD117⁺ NKp44^- (NCR^-) ILC3 subset. Further, expression of signature transcription factors (i.e. T-bet, GATA-3 and RORγT) within the specific ILC subset and cytokine production (i.e. IFN-γ, IL-5, IL-17A, IL-22 and GM-CSF) within the pulmonary ILC population was demonstrated.</p

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 α (RORα)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

Quantification and role of innate lymphoid cell subsets in Chronic Obstructive Pulmonary Disease

Objectives: Innate lymphoid cells (ILCs) secrete cytokines, such as IFN-γ, IL-13 and IL-17, which are linked to chronic obstructive pulmonary disease (COPD). Here, we investigated the role of pulmonary ILCs in COPD pathogenesis. Methods: Lung ILC subsets in COPD and control subjects were quantified using flow cytometry and associated with clinical parameters. Tissue localisation of ILC and T-cell subsets was determined by immunohistochemistry. Mice were exposed to air or cigarette smoke (CS) for 1, 4 or 24 weeks to investigate whether pulmonary ILC numbers and activation are altered and whether they contribute to CS-induced innate inflammatory responses. Results: Quantification of lung ILC subsets demonstrated that ILC1 frequency in the total ILC population was elevated in COPD and was associated with smoking and severity of respiratory symptoms (COPD Assessment Test [CAT] score). All three ILC subsets localised near lymphoid aggregates in COPD. In the COPD mouse model, CS exposure in C57BL/6J mice increased ILC numbers at all time points, with relative increases in ILC1 in bronchoalveolar lavage (BAL) fluid. Importantly, CS exposure induced increases in neutrophils, monocytes and dendritic cells that remained elevated in Rag2/Il2rg-deficient mice that lack adaptive immune cells and ILCs. However, CS-induced CXCL1, IL-6, TNF-α and IFN-γ levels were reduced by ILC deficiency. Conclusion: The ILC1 subset is increased in COPD patients and correlates with smoking and severity of respiratory symptoms. ILCs also increase upon CS exposure in C57BL/6J mice. In the absence of adaptive immunity, ILCs contribute to CS-induced pro-inflammatory mediator release, but are redundant in CS-induced innate inflammation

Preclinical evidence for the role of stem/stromal cells in COPD

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death worldwide and there are currently limited treatment options for these patients. The disease is characterized by a reduction in airflow due to chronic bronchitis, as well as airspace enlargement in the distal lung, resulting in a loss of surface area available for gas exchange. At end-stage disease, oxygen therapy and lung transplantation remain the only potential options. The disease is heterogeneous and both inflammatory cells as well as structural cells are thought to play a role in disease onset and progression. Pharmaceutical approaches are ineffective at reversing disease pathology and currently aim only to provide symptomatic relief. A recent area of investigation focuses on exogenous cell therapy, including stem cell administration, and its potential for directing lung regeneration. Cell therapies from a variety of sources, as well as cell-derived products such as extracellular vesicles, have recently shown efficacy in animal models of COPD, but early clinical trials have not yet shown efficacy. In this chapter, we discuss the different animal models of COPD as well as the studies which have been conducted to date with cell therapies. We conclude the chapter with a discussion regarding the limitations of current animal models and discuss potential areas for future study