Inflammatory triggers associated with exacerbations of COPD orchestrate plasticity of group 2 innate lymphoid cells in the lungs.

Innate lymphoid cells (ILCs) are critical mediators of mucosal immunity, and group 1 ILCs (ILC1 cells) and group 3 ILCs (ILC3 cells) have been shown to be functionally plastic. Here we found that group 2 ILCs (ILC2 cells) also exhibited phenotypic plasticity in response to infectious or noxious agents, characterized by substantially lower expression of the transcription factor GATA-3 and a concomitant switch to being ILC1 cells that produced interferon-γ (IFN-γ). Interleukin 12 (IL-12) and IL-18 regulated this conversion, and during viral infection, ILC2 cells clustered within inflamed areas and acquired an ILC1-like phenotype. Mechanistically, these ILC1 cells augmented virus-induced inflammation in a manner dependent on the transcription factor T-bet. Notably, IL-12 converted human ILC2 cells into ILC1 cells, and the frequency of ILC1 cells in patients with chronic obstructive pulmonary disease (COPD) correlated with disease severity and susceptibility to exacerbations. Thus, functional plasticity of ILC2 cells exacerbates anti-viral immunity, which may have adverse consequences in respiratory diseases such as COPD.

IL-1ß, IL-4 and IL-12 control the fate of group 2 innate lymphoid cells in human airway inflammation in the lungs.

Group 2 innate lymphoid cells (ILC2s) secrete type 2 cytokines, which protect against parasites but can also contribute to a variety of inflammatory airway diseases. We report here that interleukin 1ß (IL-1ß) directly activated human ILC2s and that IL-12 induced the conversion of these activated ILC2s into interferon-γ (IFN-γ)-producing ILC1s, which was reversed by IL-4. The plasticity of ILCs was manifested in diseased tissues of patients with severe chronic obstructive pulmonary disease (COPD) or chronic rhinosinusitis with nasal polyps (CRSwNP), which displayed IL-12 or IL-4 signatures and the accumulation of ILC1s or ILC2s, respectively. Eosinophils were a major cellular source of IL-4, which revealed cross-talk between IL-5-producing ILC2s and IL-4-producing eosinophils. We propose that IL-12 and IL-4 govern ILC2 functional identity and that their imbalance results in the perpetuation of type 1 or type 2 inflammation.

Airway hyperresponsiveness correlates with airway TSLP in asthma independent of eosinophilic inflammation.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is released from the airway epithelium in response to various environmental triggers, inducing a type-2 inflammatory response, and is associated with airway inflammation, airway hyperresponsiveness (AHR), and exacerbations. TSLP may also induce AHR via a direct effect on airway smooth muscle and mast cells, independently of type-2 inflammation, although association between airway TSLP and AHR across asthma phenotypes has been described sparsely. OBJECTIVES: This study sought to investigate the association between AHR and levels of TSLP in serum, sputum, and bronchoalveolar lavage in patients with asthma with and without type-2 inflammation. METHODS: A novel ultrasensitive assay was used to measure levels of TSLP in patients with asthma (serum, n = 182; sputum, n = 81; bronchoalveolar lavage, n = 85) and healthy controls (serum, n = 47). The distribution and association among airway and systemic TSLP, measures of AHR, type-2 inflammation, and severity of disease were assessed. RESULTS: TSLP in sputum was associated with AHR independently of levels of eosinophils and fractional exhaled nitric oxide (ρ = 0.49, P = .005). Serum TSLP was higher in both eosinophil-high and eosinophil-low asthma compared to healthy controls: geometric mean: 1600 fg/mL (95% CI: 1468-1744 fg/mL) and 1294 fg/mL (95% CI: 1167-1435 fg/mL) versus 846 fg/mL (95% CI: 661-1082 fg/mL), but did not correlate with the level of AHR. Increasing age, male sex, and eosinophils in blood were associated with higher levels of TSLP in serum, whereas lung function, inhaled corticosteroid dose, and symptom score were not. CONCLUSIONS: The association between TSLP in sputum and AHR to mannitol irrespective of markers of type-2 inflammation further supports a role of TSLP in AHR that is partially independent of eosinophilic inflammation.

Cigarette smoke silences innate lymphoid cell function and facilitates an exacerbated type I interleukin-33-dependent response to infection.

Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease and is presumed to be central to the altered responsiveness to recurrent infection in these patients. We examined the effects of smoke priming underlying the exacerbated response to viral infection in mice. Lack of interleukin-33 (IL-33) signaling conferred complete protection during exacerbation and prevented enhanced inflammation and exaggerated weight loss. Mechanistically, smoke was required to upregulate epithelial-derived IL-33 and simultaneously alter the distribution of the IL-33 receptor ST2. Specifically, smoke decreased ST2 expression on group 2 innate lymphoid cells (ILC2s) while elevating ST2 expression on macrophages and natural killer (NK) cells, thus altering IL-33 responsiveness within the lung. Consequently, upon infection and release, increased local IL-33 significantly amplified type I proinflammatory responses via synergistic modulation of macrophage and NK cell function. Therefore, in COPD, smoke alters the lung microenvironment to facilitate an alternative IL-33-dependent exaggerated proinflammatory response to infection, exacerbating disease.

Airway hyperresponsiveness reflects corticosteroid-sensitive mast cell involvement across asthma phenotypes.

BACKGROUND: Airway hyperresponsiveness is a hallmark of asthma across asthma phenotypes. Airway hyperresponsiveness to mannitol specifically relates to mast cell infiltration of the airways, suggesting inhaled corticosteroids to be effective in reducing the response to mannitol, despite low levels of type 2 inflammation. OBJECTIVE: We sought to investigate the relationship between airway hyperresponsiveness and infiltrating mast cells, and the response to inhaled corticosteroid treatment. METHODS: In 50 corticosteroid-free patients with airway hyperresponsiveness to mannitol, mucosal cryobiopsies were obtained before and after 6 weeks of daily treatment with 1600 µg of budesonide. Patients were stratified according to baseline fractional exhaled nitric oxide (Feno) with a cutoff of 25 parts per billion. RESULTS: Airway hyperresponsiveness was comparable at baseline and improved equally with treatment in both patients with Feno-high and Feno-low asthma: doubling dose, 3.98 (95% CI, 2.49-6.38; P < .001) and 3.85 (95% CI, 2.51-5.91; P < .001), respectively. However, phenotypes and distribution of mast cells differed between the 2 groups. In patients with Feno-high asthma, airway hyperresponsiveness correlated with the density of chymase-high mast cells infiltrating the epithelial layer (ρ, -0.42; P = .04), and in those with Feno-low asthma, it correlated with the density in the airway smooth muscle (ρ, -0.51; P = .02). The improvement in airway hyperresponsiveness after inhaled corticosteroid treatment correlated with a reduction in mast cells, as well as in airway thymic stromal lymphopoietin and IL-33. CONCLUSIONS: Airway hyperresponsiveness to mannitol is related to mast cell infiltration across asthma phenotypes, correlating with epithelial mast cells in patients with Feno-high asthma and with airway smooth muscle mast cells in patients with Feno-low asthma. Treatment with inhaled corticosteroids was effective in reducing airway hyperresponsiveness in both groups.

Oxidised IL-33 drives COPD epithelial pathogenesis via ST2-independent RAGE/EGFR signalling complex.

BACKGROUND: Epithelial damage, repair and remodelling are critical features of chronic airway diseases including chronic obstructive pulmonary disease (COPD). Interleukin (IL)-33 released from damaged airway epithelia causes inflammation via its receptor, serum stimulation-2 (ST2). Oxidation of IL-33 to a non-ST2-binding form (IL-33ox) is thought to limit its activity. We investigated whether IL-33ox has functional activities that are independent of ST2 in the airway epithelium. METHODS: In vitro epithelial damage assays and three-dimensional, air-liquid interface (ALI) cell culture models of healthy and COPD epithelia were used to elucidate the functional role of IL-33ox. Transcriptomic changes occurring in healthy ALI cultures treated with IL-33ox and COPD ALI cultures treated with an IL-33-neutralising antibody were assessed with bulk and single-cell RNA sequencing analysis. RESULTS: We demonstrate that IL-33ox forms a complex with receptor for advanced glycation end products (RAGE) and epidermal growth factor receptor (EGFR) expressed on airway epithelium. Activation of this alternative, ST2-independent pathway impaired epithelial wound closure and induced airway epithelial remodelling in vitro. IL-33ox increased the proportion of mucus-producing cells and reduced epithelial defence functions, mimicking pathogenic traits of COPD. Neutralisation of the IL-33ox pathway reversed these deleterious traits in COPD epithelia. Gene signatures defining the pathogenic effects of IL-33ox were enriched in airway epithelia from patients with severe COPD. CONCLUSIONS: Our study reveals for the first time that IL-33, RAGE and EGFR act together in an ST2-independent pathway in the airway epithelium and govern abnormal epithelial remodelling and muco-obstructive features in COPD.

Neutrophil phenotypes in bronchial airways differentiate single from dual responding allergic asthmatics.

INTRODUCTION: Allergic asthmatics with both an early (EAR) and a late allergic reaction (LAR) following allergen exposure are termed 'dual responders' (DR), while 'single responders' (SR) only have an EAR. Mechanisms that differentiate DR from SR are largely unknown, particularly regarding the role and phenotypes of neutrophils. Therefore, we aimed to study neutrophils in DR and SR asthmatics. METHODS: Thirty-four allergic asthmatics underwent an inhaled allergen challenge, samples were collected before and up to 24 h post-challenge. Cell differentials were counted from bronchial lavage, alveolar lavage and blood; and tissue neutrophils were quantified in immune-stained bronchial biopsies. Lavage neutrophil nuclei lobe segmentation was used to classify active (1-4 lobes) from suppressive neutrophils (≥5 lobes). Levels of transmigration markers: soluble (s)CD62L and interleukin-1Ra, and activity markers: neutrophil elastase (NE), DNA-histone complex and dsDNA were measured in lavage fluid and plasma. RESULTS: Compared with SR at baseline, DR had more neutrophils in their bronchial airways at baseline, both in the lavage (p = .0031) and biopsies (p = .026) and elevated bronchial neutrophils correlated with less antitransmigratory IL-1Ra levels (r = -0.64). DR airways had less suppressive neutrophils and more 3-lobed (active) neutrophils (p = .029) that correlated with more bronchial lavage histone (p = .020) and more plasma NE (p = .0016). Post-challenge, DR released neutrophil extracellular trap factors in the blood earlier and had less pro-transmigratory sCD62L during the late phase (p = .0076) than in SR. CONCLUSION: DR have a more active airway neutrophil phenotype at baseline and a distinct neutrophil response to allergen challenge that may contribute to the development of an LAR. Therefore, neutrophil activity should be considered during targeted diagnosis and bio-therapeutic development for DR.

Heparin fragments induce cervical inflammation by recruiting immune cells through Toll-like receptor 4 in nonpregnant mice.

Inflammation is a hallmark in the human cervix remodelling. A possible candidate inducing the inflammatory driven ripening of the cervix is the matrix component heparan sulphate, which has been shown to be elevated in late pregnancy in the cervix and uterus. Heparin and a glycol-split low molecular weight heparin (gsHep) with low anticoagulant potency has been shown to enhance myometrial contraction and interleukin (IL)-8 production by cervical fibroblasts. The aim of this study was to investigate the mechanism by which heparin promotes cervical inflammation. Wild-type, Toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88) and Interferon regulatory factor 3 (IRF3)-deficient mice were treated by deposition of gsHep into the vaginas of nonpregnant mice. To identify which cells that responded to the heparin fragments, a rhodamine fluorescent construct of gsHep was used, which initially did bind to the epithelial cells and were at later time points located in the sub-mucosa. The heparin fragments induced a strong local inflammatory response in wild-type mice shown by a rapid infiltration of neutrophils and to a lesser extent macrophages into the epithelium and the underlying extracellular matrix of the cervix. Further, a marked migration into the cervical and vaginal lumen was seen by both neutrophils and macrophages. The induced mucosal inflammation was strongly reduced in TLR4- and IRF3-deficient mice. In conclusion, our findings suggest that a TLR4/IRF3-mediated innate immune response in the cervical mucosa is induced by gsHep. This low anticoagulant heparin version, a novel TLR4 agonist, could contribute to human cervical ripening during the initiation of labour.

RORγt inhibitors block both IL-17 and IL-22 conferring a potential advantage over anti-IL-17 alone to treat severe asthma.

BACKGROUND: RORγt is a transcription factor that enables elaboration of Th17-associated cytokines (including IL-17 and IL-22) and is proposed as a pharmacological target for severe asthma. METHODS: IL-17 immunohistochemistry was performed in severe asthma bronchial biopsies (specificity confirmed with in situ hybridization). Primary human small airway epithelial cells in air liquid interface and primary bronchial smooth muscle cells were stimulated with recombinant human IL-17 and/or IL-22 and pro-inflammatory cytokines measured. Balb/c mice were challenged intratracheally with IL-17 and/or IL-22 and airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. Balb/c mice were sensitized intraperitoneally and challenged intratracheally with house dust mite extract and the effect of either a RORγt inhibitor (BIX119) or an anti-IL-11 antibody assessed on airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. RESULTS: We confirmed in severe asthma bronchial biopsies both the presence of IL-17-positive lymphocytes and that an IL-17 transcriptome profile in a severe asthma patient sub-population. Both IL-17 and IL-22 stimulated the release of pro-inflammatory cytokine and chemokine release from primary human lung cells and in mice. Furthermore, IL-22 in combination with IL-17, but neither alone, elicits airway hyperresponsiveness (AHR) in naïve mice. A RORγt inhibitor specifically blocked both IL-17 and IL-22, AHR and neutrophilia in a mouse house dust mite model unlike other registered or advanced pipeline modes of action. Full efficacy versus these parameters was associated with 90% inhibition of IL-17 and 50% inhibition of IL-22. In contrast, anti-IL-17 also blocked IL-17, but not IL-22, AHR or neutrophilia. Moreover, the deregulated genes in the lungs from these mice correlated well with deregulated genes from severe asthma biopsies suggesting that this model recapitulates significant severe asthma-relevant biology. Furthermore, these genes were reversed upon RORγt inhibition in the HDM model. Cell deconvolution suggested that the responsible cells were corticosteroid insensitive γδ-T-cells. CONCLUSION: These data strongly suggest that both IL-17 and IL-22 are required for Th2-low endotype associated biology and that a RORγt inhibitor may provide improved clinical benefit in a severe asthma sub-population of patients by blocking both IL-17 and IL-22 biology compared with blocking IL-17 alone.

Basophil sensitivity reflects long-term clinical outcome of subcutaneous immunotherapy in grass pollen-allergic patients.

BACKGROUND: Allergic rhinoconjunctivitis is a public health problem. Allergen Immunotherapy is an effective and safe treatment, that modifies the natural course of allergic disease and induces long-term tolerance. OBJECTIVE: To correlate basophil and antibody biomarkers of subcutaneous immunotherapy to clinical outcomes and cellular changes in target tissue. METHODS: Adults suffering from allergic rhinoconjunctivitis due to grass pollen allergy were randomized to receive subcutaneous immunotherapy (n = 18) or to an open control group (n = 6). Patients reported daily symptom and medication scores and weekly rhinitis related quality of life scores during four pollen seasons. Biomarkers were measured every 3 months for three years treatment and every 6 months in the follow-up year. Nasal and cutaneous allergen challenge tests were performed annually. Leukocyte subsets were assessed in nasal mucosa biopsies at baseline and after treatment. RESULTS: Subcutaneous immunotherapy led to a 447-fold decrease in basophil sensitivity during the first treatment year. This remained 100-fold lower than baseline during the 3 year-treatment period and 10-fold lower during the follow-up year (n = 18, P = .03). Decrease in basophil sensitivity after three weeks of treatment predicted long-term improvement in seasonal combined symptom and medication scores (á¿¥=-0.69, P = .0027) during three years of treatment. AUC of IgE-blocking factor correlated to nasal allergen challenge (á¿¥ = 0.63, P = .0012) and SPT (á¿¥ = 0.45, P = .03). Plasma cell numbers in the nasal mucosa increased during treatment (P = .02). CONCLUSION: Decrease in basophil sensitivity after three weeks of subcutaneous allergen immunotherapy predicted the clinical outcome of this treatment.

Eosinophils, basophils and type 2 immune microenvironments in COPD-affected lung tissue.

Although elevated blood or sputum eosinophils are present in many patients with COPD, uncertainties remain regarding the anatomical distribution pattern of lung-infiltrating eosinophils. Basophils have remained virtually unexplored in COPD. This study mapped tissue-infiltrating eosinophils, basophils and eosinophil-promoting immune mechanisms in COPD-affected lungs.Surgical lung tissue and biopsies from major anatomical compartments were obtained from COPD patients with severity grades Global Initiative for Chronic Obstructive Lung Disease stages I-IV; never-smokers/smokers served as controls. Automated immunohistochemistry and in situ hybridisation identified immune cells, the type 2 immunity marker GATA3 and eotaxins (CCL11, CCL24).Eosinophils and basophils were present in all anatomical compartments of COPD-affected lungs and increased significantly in very severe COPD. The eosinophilia was strikingly patchy, and focal eosinophil-rich microenvironments were spatially linked with GATA3+ cells, including type 2 helper T-cell lymphocytes and type 2 innate lymphoid cells. A similarly localised and interleukin-33/ST2-dependent eosinophilia was demonstrated in influenza-infected mice. Both mice and patients displayed spatially confined eotaxin signatures with CCL11+ fibroblasts and CCL24+ macrophages.In addition to identifying tissue basophilia as a novel feature of advanced COPD, the identification of spatially confined eosinophil-rich type 2 microenvironments represents a novel type of heterogeneity in the immunopathology of COPD that is likely to have implications for personalised treatment.

Comparison of Normal and Metaplastic Epithelium in Patients with Stable versus Persistently Symptomatic Severe Asthma Using Laser-Capture Microdissection and Data-Independent Acquisition-Mass Spectrometry.

A proportion of patients with severe asthma (SA) show poor responses to traditional asthma medications; however, it remains unknown why some patients remain persistently symptomatic. Our objective was to explore the use of laser-capture microdissection of specific epithelial structures combined with quantitative data-independent acquisition mass spectrometry to elucidate differences in protein composition in patients with SA with varying symptom control. Unbiased label-free quantitative proteome analyses were performed on laser-capture-microdissected areas of specific epithelial structures from patients with SA with varying degrees of symptom control. A total of 1993 stable SA and 1652 symptomatic SA proteins in normal epithelium and 1458 stable SA and 1647 symptomatic SA proteins in metaplastic epithelium were quantified. When comparing proteome profiles based on symptom control, 33 proteins in patients with stable SA (≥twofold change; P ≤ 0.05) and 13 proteins in patients with persistently symptomatic SA (≥twofold change; P ≤ 0.05) were enriched significantly. When comparing proteome profiles based on epithelial status, 21 proteins in normal epithelium (≥twofold change; P ≤ 0.05) and 6 proteins in metaplastic epithelium (≥twofold change; P ≤ 0.05) were enriched significantly. New treatment strategies are needed for patients with severe asthma and exploratory studies of unbiased nature such as this may help when searching for new mechanisms and potential targets involved in the disease pathology.

IL-33 drives influenza-induced asthma exacerbations by halting innate and adaptive antiviral immunity.

BACKGROUND: Influenza virus triggers severe asthma exacerbations for which no adequate treatment is available. It is known that IL-33 levels correlate with exacerbation severity, but its role in the immunopathogenesis of exacerbations has remained elusive. OBJECTIVE: We hypothesized that IL-33 is necessary to drive asthma exacerbations. We intervened with the IL-33 cascade and sought to dissect its role, also in synergy with thymic stromal lymphopoietin (TSLP), in airway inflammation, antiviral activity, and lung function. We aimed to unveil the major source of IL-33 in the airways and IL-33-dependent mechanisms that underlie severe asthma exacerbations. METHODS: Patients with mild asthma were experimentally infected with rhinovirus. Mice were chronically exposed to house dust mite extract and then infected with influenza to resemble key features of exacerbations in human subjects. Interventions included the anti-IL-33 receptor ST2, anti-TSLP, or both. RESULTS: We identified bronchial ciliated cells and type II alveolar cells as a major local source of IL-33 during virus-driven exacerbation in human subjects and mice, respectively. By blocking ST2, we demonstrated that IL-33 and not TSLP was necessary to drive exacerbations. IL-33 enhanced airway hyperresponsiveness and airway inflammation by suppressing innate and adaptive antiviral responses and by instructing epithelial cells and dendritic cells of house dust mite-sensitized mice to dampen IFN-ß expression and prevent the TH1-promoting dendritic cell phenotype. IL-33 also boosted luminal NETosis and halted cytolytic antiviral activities but did not affect the TH2 response. CONCLUSION: Interventions targeting the IL-33/ST2 axis could prove an effective acute short-term therapy for virus-induced asthma exacerbations.

IL-33 blockade affects mediators of persistence and exacerbation in a model of chronic airway inflammation.

BACKGROUND: Severe inflammatory airway diseases are associated with inflammation that does not resolve, leading to structural changes and an overall environment primed for exacerbations. OBJECTIVE: We sought to identify and inhibit pathways that perpetuate this heightened inflammatory state because this could lead to therapies that allow for a more quiescent lung that is less predisposed to symptoms and exacerbations. METHODS: Using prolonged exposure to house dust mite in mice, we developed a mouse model of persistent and exacerbating airway disease characterized by a mixed inflammatory phenotype. RESULTS: We show that lung IL-33 drives inflammation and remodeling beyond the type 2 response classically associated with IL-33 signaling. IL-33 blockade with an IL-33 neutralizing antibody normalized established inflammation and improved remodeling of both the lung epithelium and lung parenchyma. Specifically, IL-33 blockade normalized persisting and exacerbating inflammatory end points, including eosinophilic, neutrophilic, and ST2+CD4+ T-cell infiltration. Importantly, we identified a key role for IL-33 in driving lung remodeling because anti-IL-33 also re-established the presence of ciliated cells over mucus-producing cells and decreased myofibroblast numbers, even in the context of continuous allergen exposure, resulting in improved lung function. CONCLUSION: Overall, this study shows that increased IL-33 levels drive a self-perpetuating amplification loop that maintains the lung in a state of lasting inflammation and remodeled tissue primed for exacerbations. Thus IL-33 blockade might ameliorate symptoms and prevent exacerbations by quelling persistent inflammation and airway remodeling.

Bronchoscopic mucosal cryobiopsies as a method for studying airway disease.

BACKGROUND: Investigating disease mechanisms and treatment responses in obstructive airway diseases with invasive sampling are hampered by the small size and mechanical artefacts that conventional forceps biopsies suffer from. Endoscopic cryobiopsies are larger and more intact and are being increasingly used. However, the technique has not yet been explored for obtaining mucosa biopsies. OBJECTIVE: To investigate differences in size and quality of endobronchial mucosal biopsies obtained with cryotechnique and forceps. Further, to check for eligibility of cryobiopsies to be evaluated with immunohistochemistry and in situ hybridization and to investigate tolerability and safety of the technique. METHODS: Endobronchial mucosal biopsies were obtained with cryotechnique and forceps from patients with haemoptysis undergoing bronchoscopy and evaluated by quantitative morphometry, automated immunohistochemistry and in situ hybridization. RESULTS: A total of 40 biopsies were obtained from 10 patients. Cross-sectional areas were threefold larger in cryobiopsies (median: 3.08 mm2 (IQR: 1.79) vs 1.03 mm2 (IQR: 1.10), P < 0.001). Stretches of intact epithelium were 8-fold longer (median: 4.61 mm (IQR: 4.50) vs 0.55 mm (IQR: 1.23), P = 0.001). Content of glands (median: 0.095 mm2 (IQR: 0.30) vs 0.00 mm2 (IQR: 0.01), P = 0.002) and airway smooth muscle (median: 0.25 mm2 (IQR: 0.30) vs 0.060 mm2 (IQR: 0.11), P = 0.02) was higher in the cryobiopsies compared with forceps biopsies. Further, the cryobiopsies had well-preserved protein antigens and mRNA. Mild to moderate bleeding was the only complication observed. CONCLUSION AND CLINICAL RELEVANCE: By yielding significantly larger and more intact biopsies, the cryotechnique represents a valuable new research tool to explore the bronchi in airway disease. Ultimately with the potential to create better understanding of underlying disease mechanisms and improvement of treatments.

Unravelling the complexity of tissue inflammation in uncontrolled and severe asthma.

PURPOSE OF REVIEW: The clinical and inflammatory heterogeneity in asthma constitutes a major challenge for improved treatment. This review describes the nature of the inflammatory complexity and how it can be decoded to yield improved disease understanding and personalized treatment. The focus is on the difficult task of revealing the immunological complexity as it occurs inside diseased patient tissues. RECENT FINDINGS: The inflammatory heterogeneity in asthma stretches beyond the classical division into allergic Th2 eosinophilic versus Th1 and/or Th17 neutrophilic (or paucigranulocytic) phenotypes. Rather than having one distinct type of inflammation, many patients display a patchwork of overlapping immune signatures. The patient diversity is further increased by differences in regard of distal lung involvement. Faced with this staggering complexity, calls have been made for a pragmatic biomarker-guided identification of treatable traits. In parallel, novel high-dimensional analyses and multiplex imaging aid the long-term goal of decoding the underlying molecular endotypes. SUMMARY: Asthma is vastly heterogeneous with multiple and superimposed inflammatory and anatomical phenotypes. Despite the intensive research and introduction of highly immune-selective dugs, basic questions remain; especially as still too many of today's uncontrolled patients remain poorly understood. Here, pragmatic biomarker strategies, combined with novel methodological approaches that ultimately reveal the complete immunological complexity, will pave the way for improved differential diagnosis and personalized medication.

Composition and functionality of the intrahepatic innate lymphoid cell-compartment in human nonfibrotic and fibrotic livers.

Human innate lymphoid cells have been described to exist in different organs, with functional deregulation of these cells contributing to several disease states. Here, we performed the first detailed characterization of the phenotype, tissue-residency properties, and functionality of ILC1s, ILC2s, and ILC3s in the human adult and fetal liver. In addition, we investigated changes in the ILC compartment in liver fibrosis. A unique composition of tissue-resident ILCs was observed in nonfibrotic livers as compared with that in mucosal tissues, with NKp44- ILC3s accounting for the majority of total intrahepatic ILCs. The frequency of ILC2s, representing a small fraction of ILCs in nonfibrotic livers, increased in liver fibrosis and correlated directly with the severity of the disease. Notably, intrahepatic ILC2s secreted the profibrotic cytokine IL-13 when exposed to IL-33 and thymic stromal lymphopoetin (TSLP); these cytokines were produced by hepatocytes, hepatic stellate cells (HSCs), and Kupffer cells in response to TLR-3 stimulation. In summary, the present results provide the first detailed characterization of intrahepatic ILCs in human adult and fetal liver. The results indicate a role for ILC2s in human liver fibrosis, implying that targeting ILC2s might be a novel therapeutic strategy for its treatment.

Intratumoural leukocyte infiltration is a prognostic indicator among pancreatic cancer patients with type 2 diabetes.

BACKGROUND: The life expectancy of pancreatic cancer patients remains minimal. The disease progression may be influenced by type 2 diabetes (T2D) and inflammatory status, although important gaps persist around their joint effects on disease outcome. The aim of this study was to investigate the clinical significance of the tumour immune microenvironment on pancreatic cancer prognosis in relation to T2D status. METHOD: Tumour-infiltrating macrophages, neutrophils and eosinophils were studied in primary pancreatic tumours and paired lymph node metastases in relation to patient and tumour characteristics, T2D status and overall survival in a retrospective cohort of patients with resectable pancreatic cancer in Sweden. RESULTS: Of the 80 included pancreatic cancer patients, 22 (27.2%) had T2D. The diabetic pancreatic cancer patients had significantly higher systemic high white blood cell count than those without diabetes (P = 0.028). Macrophage infiltration levels were higher in lymph node metastases compared with primary tumours (P = 0.040) among pancreatic cancer patients with diabetes. Type 2 diabetes or intra-tumoural leukocyte (macrophage, neutrophil or eosinophil) infiltration alone did not significantly influence pancreatic cancer prognosis. However, among cancer patients with T2D high macrophage or neutrophil tumour-infiltration was associated with a significant reduction in overall survival (adjusted hazard ratio [HR] 7.2; 95% CI 1.5-35.0 and HR 5.4; 95% CI 1.1-26.3, respectively). CONCLUSION: These results demonstrate associations between T2D and enhanced inflammatory processes with significant implications on survival among pancreatic cancer patients with T2D. Validation in larger independent patient cohorts may identify additional prognostic tools and improved treatment strategies for specific patient subsets.

Eosinophilic airway inflammation in asthmatic patients is associated with an altered airway microbiome.

BACKGROUND: Asthmatic patients have higher microbiome diversity and an altered composition, with more Proteobacteria and less Bacteroidetes compared with healthy control subjects. Studies comparing airway inflammation and the airway microbiome are sparse, especially in subjects not receiving anti-inflammatory treatment. OBJECTIVE: We sought to describe the relationship between the airway microbiome and patterns of airway inflammation in steroid-free patients with asthma and healthy control subjects. METHODS: Bronchoalveolar lavage fluid was collected from 23 steroid-free nonsmoking patients with asthma and 10 healthy control subjects. Bacterial DNA was extracted from and subjected to Illumina MiSeq sequencing of the 16S rDNA V4 region. Eosinophils and neutrophils in the submucosa were quantified by means of immunohistochemical identification and computerized image analysis. Induced sputum was obtained, and airway hyperresponsiveness to mannitol and fraction of exhaled nitric oxide values were measured. Relationships between airway microbial diversity and composition and inflammatory profiles were analyzed. RESULTS: In asthmatic patients airway microbial composition was associated with airway eosinophilia and AHR to mannitol but not airway neutrophilia. The overall composition of the airway microbiome of asthmatic patients with the lowest levels of eosinophils but not asthmatic patients with the highest levels of eosinophils deviated significantly from that of healthy subjects. Asthmatic patients with the lowest levels of eosinophils had an altered bacterial abundance profile, with more Neisseria, Bacteroides, and Rothia species and less Sphingomonas, Halomonas, and Aeribacillus species compared with asthmatic patients with more eosinophils and healthy control subjects. CONCLUSION: The level of eosinophilic airway inflammation correlates with variations in the microbiome across asthmatic patients, whereas neutrophilic airway inflammation does not. This warrants further investigation on molecular pathways involved in both patients with eosinophilic and those with noneosinophilic asthma.