Blood MMP-9 measured at 2 years after lung transplantation as a prognostic biomarker of chronic lung allograft dysfunction.

BACKGROUND: Long-term outcomes of lung transplantation (LTx) remain hampered by chronic lung allograft dysfunction (CLAD). Matrix metalloproteinase 9 (MMP-9) is a secretory endopeptidase identified as a key mediator in fibrosis processes associated with CLAD. The objective of this study was to investigate whether plasma MMP9 levels may be prognostic of CLAD development. METHODS: Participants were selected from the Cohort in Lung Transplantation (COLT) for which a biocollection was associated. We considered two time points, year 1 (Y1) and year 2 (Y2) post-transplantation, for plasma MMP-9 measurements. We analysed stable recipients at those time points, comparing those who would develop a CLAD within the 2 years following the measurement to those who would remain stable 2 years after. RESULTS: MMP-9 levels at Y1 were not significantly different between the CLAD and stable groups (230 ng/ml vs. 160 ng/ml, p = 0.4). For the Y2 analysis, 129 recipients were included, of whom 50 developed CLAD within 2 years and 79 remained stable within 2 years. MMP-9 plasma median concentrations were higher in recipients who then developed CLAD than in the stable group (230 ng/ml vs. 118 ng/ml, p = 0.003). In the multivariate analysis, the Y2 MMP-9 level was independently associated with CLAD, with an average increase of 150 ng/ml (95% CI [0-253], p = 0.05) compared to that in the stable group. The Y2 ROC curve revealed a discriminating capacity of blood MMP-9 with an area under the curve of 66%. CONCLUSION: Plasmatic MMP-9 levels measured 2 years after lung transplantation have prognostic value for CLAD.

Differential early response of monocyte/macrophage subsets to intra-operative corticosteroid administration in lung transplantation.

Introduction: Lung transplantation often results in primary and/or chronic dysfunctions that are related to early perioperative innate allo-responses where myeloid subsets play a major role. Corticosteroids are administered upon surgery as a standard-of-care but their action on the different myeloid cell subsets in that context is not known. Methods: To address this issue, we used a cross-circulatory platform perfusing an extracorporeal lung coupled to cell mapping in the pig model, that enabled us to study the recruited cells in the allogeneic lung over 10 hours. Results: Myeloid cells, i.e. granulocytes and monocytic cells including classical CD14pos and non-classical/intermediate CD16pos cells, were the dominantly recruited subsets, with the latter upregulating the membrane expression of MHC class II and CD80/86 molecules. Whereas corticosteroids did not reduce the different cell subset recruitment, they potently dampened the MHC class II and CD80/86 expression on monocytic cells and not on alveolar macrophages. Besides, corticosteroids induced a temporary and partial anti-inflammatory gene profile depending on cytokines and monocyte/macrophage subsets. Discussion: This work documents the baseline effects of the standard-of-care corticosteroid treatment for early innate allo-responses. These insights will enable further optimization and improvement of lung transplantation outcomes.

Cell type- and time-dependent biological responses in ex vivo perfused lung grafts.

In response to the increasing demand for lung transplantation, ex vivo lung perfusion (EVLP) has extended the number of suitable donor lungs by rehabilitating marginal organs. However despite an expanding use in clinical practice, the responses of the different lung cell types to EVLP are not known. In order to advance our mechanistic understanding and establish a refine tool for improvement of EVLP, we conducted a pioneer study involving single cell RNA-seq on human lungs declined for transplantation. Functional enrichment analyses were performed upon integration of data sets generated at 4 h (clinical duration) and 10 h (prolonged duration) from two human lungs processed to EVLP. Pathways related to inflammation were predicted activated in epithelial and blood endothelial cells, in monocyte-derived macrophages and temporally at 4 h in alveolar macrophages. Pathways related to cytoskeleton signaling/organization were predicted reduced in most cell types mainly at 10 h. We identified a division of labor between cell types for the selected expression of cytokine and chemokine genes that varied according to time. Immune cells including CD4+ and CD8+ T cells, NK cells, mast cells and conventional dendritic cells displayed gene expression patterns indicating blunted activation, already at 4 h in several instances and further more at 10 h. Therefore despite inducing inflammatory responses, EVLP appears to dampen the activation of major lung immune cell types, what may be beneficial to the outcome of transplantation. Our results also support that therapeutics approaches aiming at reducing inflammation upon EVLP should target both the alveolar and vascular compartments.

A cross-circulatory platform for monitoring innate allo-responses in lung grafts.

Lung transplantation is the only curative option for end-stage chronic respiratory diseases. However the survival rate is only about 50% at 5 years. Although experimental evidences have shown that innate allo-responses impact on the clinical outcome, the knowledge of the involved mechanisms involved is limited. We established a cross-circulatory platform to monitor the early recruitment and activation of immune cells in an extracorporeal donor lung by coupling blood perfusion to cell mapping with a fluorescent marker in the pig, a commonly-used species for lung transplantation. The perfusing pig cells were easily detectable in lung cell suspensions, in broncho-alveolar lavages and in different areas of lung sections, indicating infiltration of the organ. Myeloid cells (granulocytes and monocytic cells) were the dominant recruited subsets. Between 6 and 10 h of perfusion, recruited monocytic cells presented a strong upregulation of MHC class II and CD80/86 expression, whereas alveolar macrophages and donor monocytic cells showed no significant modulation of expression. This cross-circulation model allowed us to monitor the initial encounter between perfusing cells and the lung graft, in an easy, rapid, and controllable manner, to generate robust information on innate response and test targeted therapies for improvement of lung transplantation outcome.

Allergic Sensitization Driving Immune Phenotyping and Disease Severity in a Mouse Model of Asthma.

PURPOSE: Asthma is a frequent chronic inflammatory bronchial disease affecting more than 300 million patients worldwide, 70% of whom are secondary to allergy. The diversity of asthmatic endotypes contributes to their complexity. The inter-relationship between allergen and other exposure and the airway microbiome adds to the phenotypic diversity and defines the natural course of asthma. Here, we compared the mouse models of house dust mite (HDM)-induced allergic asthma. Allergic sensitization was performed via various routes and associated with outcomes. METHODS: Mice were sensitized with HDM via the oral, nasal or percutaneous routes. Lung function, barrier integrity, immune response and microbiota composition were analyzed. RESULTS: Severe impairment of respiratory function was observed in the mice sensitized by the nasal and cutaneous paths. It was associated with epithelial dysfunction characterized by an increased permeability secondary to junction protein disruption. Such sensitization paths induced a mixed eosinophilic and neutrophilic inflammatory response with high interleukin (IL)-17 airway secretion. In contrast, orally sensitized mice showed a mild impairment of respiratory function. Epithelial dysfunction was mild with increased mucus production, but preserved epithelial junctions. Regarding lung microbiota, sensitization provoked a significant loss of diversity. At the genus level, Cutibacterium, Acinetobacter, Streptococcus and Lactobacillus were found to be modulated according to the sensitization pathway. An increase in theanti-inflammatory microbiota metabolites was observed in the oral-sensitization group. CONCLUSIONS: Our study highlights the strong impact of the sensitization route on the pathophysiology and the critical phenotypic diversity of allergic asthma in a mouse model.

French guidelines for the etiological workup of eosinophilia and the management of hypereosinophilic syndromes.

Eosinophilic-related clinical manifestations are protean and the underlying conditions underpinning eosinophilia are highly diverse. The etiological workup of unexplained eosinophilia/hypereosinophilia can be challenging, and can lead sometimes to extensive, inappropriate, costly and/or invasive investigations. To date, guidelines for the etiological workup and management of eosinophilia are mainly issued by hematologists, and thus mostly cover the scope of clonal hypereosinophilic syndromes (HES). Here, thanks to an extensive literature review, and thanks to the joint work of a large panel of experts involving physicians from both adult and pediatric medicine and from various subspecialties (as well as a representative of a patients' association representative), we provide recommendations for both the step-by step diagnostic workup of eosinophilia (whether unexplained or within specific contexts) as well as the management and follow-up of the full spectrum of eosinophilic disorders (including clonal, reactive, lymphocytic and idiopathic HES, as well as single-organ diseases). Didactic prescription summaries intended to facilitate the prescription of eosinophil-targeted drugs are also provided, as are practical diagnostic and therapeutic algorithms. Lastly, this set of recommendations also includes a summary intended for general practitioners, as well as an overview of the therapeutic patient education program set up by the French reference center for HES. Further updates will be mandatory as new validated information emerges.

Systems prediction of chronic lung allograft dysfunction: Results and perspectives from the Cohort of Lung Transplantation and Systems prediction of Chronic Lung Allograft Dysfunction cohorts.

Background: Chronic lung allograft dysfunction (CLAD) is the leading cause of poor long-term survival after lung transplantation (LT). Systems prediction of Chronic Lung Allograft Dysfunction (SysCLAD) aimed to predict CLAD. Methods: To predict CLAD, we investigated the clinicome of patients with LT; the exposome through assessment of airway microbiota in bronchoalveolar lavage cells and air pollution studies; the immunome with works on activation of dendritic cells, the role of T cells to promote the secretion of matrix metalloproteinase-9, and subpopulations of T and B cells; genome polymorphisms; blood transcriptome; plasma proteome studies and assessment of MSK1 expression. Results: Clinicome: the best multivariate logistic regression analysis model for early-onset CLAD in 422 LT eligible patients generated a ROC curve with an area under the curve of 0.77. Exposome: chronic exposure to air pollutants appears deleterious on lung function levels in LT recipients (LTRs), might be modified by macrolides, and increases mortality. Our findings established a link between the lung microbial ecosystem, human lung function, and clinical stability post-transplant. Immunome: a decreased expression of CLEC1A in human lung transplants is predictive of the development of chronic rejection and associated with a higher level of interleukin 17A; Immune cells support airway remodeling through the production of plasma MMP-9 levels, a potential predictive biomarker of CLAD. Blood CD9-expressing B cells appear to favor the maintenance of long-term stable graft function and are a potential new predictive biomarker of BOS-free survival. An early increase of blood CD4 + CD57 + ILT2+ T cells after LT may be associated with CLAD onset. Genome: Donor Club cell secretory protein G38A polymorphism is associated with a decreased risk of severe primary graft dysfunction after LT. Transcriptome: blood POU class 2 associating factor 1, T-cell leukemia/lymphoma domain, and B cell lymphocytes, were validated as predictive biomarkers of CLAD phenotypes more than 6 months before diagnosis. Proteome: blood A2MG is an independent predictor of CLAD, and MSK1 kinase overexpression is either a marker or a potential therapeutic target in CLAD. Conclusion: Systems prediction of Chronic Lung Allograft Dysfunction generated multiple fingerprints that enabled the development of predictors of CLAD. These results open the way to the integration of these fingerprints into a predictive handprint.

Deciphering Differential Behavior of Immune Responses as the Foundation for Precision Dosing in Allergen Immunotherapy.

Like in many fields of medicine, the concept of precision dosing has re-emerged in routine practice in allergology. Only one retrospective study on French physicians' practice has addressed this topic so far and generated preliminary data supporting dose adaptation, mainly based on experience, patient profile understanding and response to treatment. Both intrinsic and extrinsic factors shape the individual immune system response to allergen immunotherapy (AIT). Herein, we focus on key immune cells (i.e., dendritic cells, innate lymphoid cells, B and T cells, basophils and mast cells) involved in allergic disease and its resolution to further understand the effect of AIT on the phenotype, frequency or polarization of these cells. We strive to discriminate differences in immune responses between responders and non-responders to AIT, and discuss the eligibility of a non/low-responder subset for dose adaptation. A differential behavior in immune cells is clearly observed in responders, highlighting the importance of conducting clinical trials with large cohorts of well-characterized subjects to decipher the immune mechanism of AIT. We conclude that there is a need for designing new clinical and mechanistic studies to support the scientific rationale of dose adaptation in the interest of patients who do not properly respond to AIT.

Bronchial smooth muscle cell in asthma: where does it fit?

Asthma is a frequent respiratory condition whose pathophysiology relies on altered interactions between bronchial epithelium, smooth muscle cells (SMC) and immune responses. Those leads to classical hallmarks of asthma: airway hyper-responsiveness, bronchial remodelling and chronic inflammation. Airway smooth muscle biology and pathophysiological implication in asthma are now better understood. Precise deciphering of intracellular signalling pathways regulating smooth muscle contraction highlighted the critical roles played by small GTPases of Rho superfamily. Beyond contractile considerations, active involvement of airway smooth muscle in bronchial remodelling mechanisms is now established. Not only cytokines and growth factors, such as fibroblats growth factor or transforming growth factor-ß, but also extracellular matrix composition have been demonstrated as potent phenotype modifiers for airway SMC. Although basic science knowledge has grown significantly, little of it has translated into improvement in asthma clinical practice. Evaluation of airway smooth muscle function is still limited to its contractile activity. Moreover, it relies on tools, such as spirometry, that give only an overall assessment and not a specific one. Interesting technics such as forced oscillometry or specific imagery (CT and MRI) give new perspectives to evaluate other aspects of airway muscle such as bronchial remodelling. Finally, except for the refinement of conventional bronchodilators, no new drug therapy directly targeting airway smooth muscle proved its efficacy. Bronchial thermoplasty is an innovative and efficient therapeutic strategy but is only restricted to a small proportion of severe asthmatic patients. New diagnostic and therapeutic strategies specifically oriented toward airway smooth muscle are needed to improve global asthma care.

Incidence and risk factors of anastomotic complications after lung transplantation.

BACKGROUND: Anastomotic complications are common after lung transplantation (1.4-33% of cases) and still associated with a high morbi-mortality. METHODS: The current study is a monocenter retrospective analysis of symptomatic anastomotic complications (SAC) occurring after lung transplantation between 2010 and 2016, using the macroscopic, diameter, and suture (M-D-S) classification from consensus of French experts in bronchoscopy. The objectives were to determine incidence from surgery, risk factors, and impact of survival of SAC. We defined SAC as M-D-S abnormalities (stenosis ⩾ 50% or dehiscence) requiring bronchoscopic or surgical interventions. RESULTS: A total of 121 patients were included. SAC occurred in 26.5% of patients (n = 32), divided in symptomatic stenosis for 23.7% (n = 29), and symptomatic dehiscence in 2.5% (n = 3). In multivariate analysis, donor bacterial lung infection [HR 2.08 (1.04-4.17), p = 0.04] and age above 50 years [HR 3.26 (1.04-10.26), p = 0.04] were associated with SAC occurrence. Cystic fibrosis etiology was associated with better survival on Kaplan-Meier curve (p < 0.001). SAC [HR 2.15 (1.07-4.32), p = 0.03] was independently associated with worst survival. The 29 symptomatic patients because of stenosis required endoscopic procedure, of whom 16 patients needed bronchial stent placement. Four patients underwent surgery: three patients because of dehiscence and one because of severe bilateral stenosis (re-transplantation). DISCUSSION: SAC occurred in 26.5% of patients. Donor lung infection was the only alterable identified factors. The increase rate of SAC in older patients above 50 years of age encourages in regular endoscopic monitoring.

Chronic lung allograft dysfunction is associated with an early increase of circulating cytotoxic CD4+CD57+ILT2+ T cells, selectively inhibited by the immune check-point HLA-G.

BACKGROUND: Survival after lung transplantation (LTx) still remains limited by chronic lung allograft dysfunction (CLAD), thought to represent a form of chronic rejection. We investigated whether the immune checkpoint HLA-G/ILT2 expressed by peripheral T-cell subpopulations could predict CLAD. METHODS: We used data for 150 LTx recipients from COLT (Cohort-For-Lung-Transplantation) cohort with ≥1 available blood sample at 1-, 6-, or 12-months post-Tx. Analysis of T cells by flow cytometry focused on the ILT2 receptor of HLA-G and other markers (CD57, CD25, CD127). T-cell subset analyses compared stable patients and those with CLAD at 3 years post-LTx. RESULTS: With data for 78 stable and 72 CLAD patients, among 21 T-cell subsets expressing ILT2, only CD4+CD57+ILT2+ T cells were associated with outcome. At 1-month post-Tx, low proportion of CD4+CD57+ILT2+ T cells was associated with reduced 3-year incidence of CLAD (CD4+CD57+ILT2+ T cells ≤ first IQR [25%] vs > first IQR, log-rank test, p = 0.028). Furthermore, the incidence of CLAD was higher with >2.6- vs ≤2.6-fold increased proportion of CD4+CD57+ILT2+ T cells over the first year post-LTx (3-year freedom frequencies: 27% [95%CI: 8-50] vs 64% [95%CI: 48-77] (log-rank test, p = 0.014). On multivariable analysis, increased proportion of CD4+CD57+ILT2+ T cells over the first year predicted CLAD (hazard ratio 1.25; 95%CI: 1.09-1.44; p = 0.001). Focusing on CD4+CD57+ILT2+ T cells, we demonstrated ex vivo that they are cytotoxic CD4+ T cells, selectively inhibited by HLA-G. CONCLUSIONS: Our data suggest that an early increase of CD4+CD57+ILT2+ T cells after LTx may be associated with CLAD onset.

Immunoglobulin E response in health and disease beyond allergic disorders.

Immunoglobulin E is the latest discovered of immunoglobulin family and has been long associated with anaphylaxis and worm expulsion. Immunoglobulin E, along with mast cells, basophils, and eosinophils, is also a hallmark of type 2 immunity which is dysregulated in numerous diseases such as asthma, rhinitis, atopic dermatitis, and eosinophilic esophagitis in addition to anaphylaxis as aforementioned. However, recent advances have shed light on IgE regulation and memory explaining the low level of free IgE, the scarcity of IgE plasma cells that are mainly short live and the absence of IgE memory B cells in homeostatic conditions. Furthermore, IgE was implicated in inflammatory conditions beyond allergic disorders where IgE-mediated facilitated antigen presentation can enhance cellular and humoral response against autoantigens in systemic lupus or chronic urticaria leading to more severe disease and even against neoantigen facilitating tumor cell lysis. At last, IgE was unexpectedly associated with allograft rejection or atheromatous cardiovascular diseases where precise mechanisms remain to be deciphered. The purpose of this review is to summarize these recent advances in IgE regulation, biology, and physiopathology beyond allergic diseases opening whole new fields of IgE biology to explore.

Is CCL18 a potential biomarker of type-2 asthma endotypes?

Objective: This exploratory cross-sectional study aimed to evaluate the associations between the chemokine ligand 18 (CCL18) blood level and phenotypic characteristics of asthma.Methods: We evaluated in a sample of 173 asthmatic adult patients from the Cohort of Bronchial obstruction and Asthma (63.4% women; median age 50 ± interquartile range 27.5 years; median level of CCL18 was 44.1 ± interquartile range 27.5 ng/mL) the association between CCL18 blood level and allergic features of asthma using a multivariate analysis.Results: We found an association between the log-transformed value of blood CCL18 and age (+0.7% [0.1; 1.3] per 1-year increase, p = 0.033), gender (-25.1% [-42; -3.2] in women, p = 0.029), and nasal polyposis (+38.1% [11.6; 70.9], p = 0.004). No association was observed between CCL18 level and the other main phenotypic characteristics of asthma.Conclusions: Our exploratory study suggests that CCL18 is not an effective biomarker of allergic asthma endotype but may rather be a biomarker of tissue eosinophilia as supported by its association with nasal polyposis.

Separation of the CaV 1.2-CaV 1.3 calcium channel duo prevents type 2 allergic airway inflammation.

BACKGROUND: Voltage-gated calcium (Cav 1) channels contribute to T-lymphocyte activation. Cav 1.2 and Cav 1.3 channels are expressed in Th2 cells but their respective roles are unknown, which is investigated herein. METHODS: We generated mice deleted for Cav 1.2 in T cells or Cav 1.3 and analyzed TCR-driven signaling. In this line, we developed original fast calcium imaging to measure early elementary calcium events (ECE). We also tested the impact of Cav 1.2 or Cav 1.3 deletion in models of type 2 airway inflammation. Finally, we checked whether the expression of both Cav 1.2 and Cav 1.3 in T cells from asthmatic children correlates with Th2-cytokine expression. RESULTS: We demonstrated non-redundant and synergistic functions of Cav 1.2 and Cav 1.3 in Th2 cells. Indeed, the deficiency of only one channel in Th2 cells triggers TCR-driven hyporesponsiveness with weakened tyrosine phosphorylation profile, a strong decrease in initial ECE and subsequent reduction in the global calcium response. Moreover, Cav 1.3 has a particular role in calcium homeostasis. In accordance with the singular roles of Cav 1.2 and Cav 1.3 in Th2 cells, deficiency in either one of these channels was sufficient to inhibit cardinal features of type 2 airway inflammation. Furthermore, Cav 1.2 and Cav 1.3 must be co-expressed within the same CD4+ T cell to trigger allergic airway inflammation. Accordingly with the concerted roles of Cav 1.2 and Cav 1.3, the expression of both channels by activated CD4+ T cells from asthmatic children was associated with increased Th2-cytokine transcription. CONCLUSIONS: Thus, Cav 1.2 and Cav 1.3 act as a duo, and targeting only one of these channels would be efficient in allergy treatment.

Engineering a safe monoclonal anti-human IL-2 that is effective in a murine model of food allergy and asthma.

BACKGROUND: Regulatory T cells (Tregs) are known to protect against allergies. Moreover, the decrease in the frequency and efficiency of Tregs amplifies allergic symptoms. AIM: This study investigated whether expanding Tregs in vivo with an IL-2/IL-2 antibody complex could be safe, well tolerated and efficient in a therapeutic setting in allergies. METHODS: We produced an anti-IL-2 antibody (1C6) and demonstrated that when it is complexed to human IL-2, it increases IL-2 efficiency to induce Tregs in vivo without any detectable side effects. Furthermore, the IL-2/1C6 complex induces an increase in Helios expression by Tregs, suggesting that it not only elevated Treg numbers but also boosted their functions. Using mouse models of house-dust-mite-induced airway inflammation and wheat-gliadin-induced food allergies, we investigated the therapeutic potential of the IL-2/1C6 complex in allergies. RESULTS: IL-2/1C6 treatment significantly reduced allergic symptoms, specific IgE production, the adaptive immune response and tissue damage. Interestingly, IL-2/1C6 treatment modulated innate lymphoid cells by increasing ILC2s in asthma and decreasing ILC3s in food allergies. CONCLUSION: In conclusion,complexed IL-2/anti-IL-2 may restore Treg numbers and function in respiratory and food allergies, thereby improving allergic markers and symptoms. Our IL-2/anti-IL-2 complex offers new hope for reestablishing immune tolerance in patients with allergies.

Type 2 immunity-driven diseases: Towards a multidisciplinary approach.

Asthma, atopic dermatitis and chronic rhinoconjunctivitis are highly heterogeneous. However, epidemiologic associations exist between phenotypic groups of patients. Atopic march is one such association but is not the only common point. Indeed, beyond such phenotypes, hallmarks of type 2 immunity have been found in these diseases involving immune dysregulation as well as environmental triggers and epithelial dysfunction. From the canonical Th2 cytokines (IL-4, IL-5, IL-13), new cellular and molecular actors arise, from the epithelium's alarmins to new innate immune cells. Their interactions are now better understood across the different environmental barriers, and slight differences appeared. In parallel, the development of type 2-targeting biotherapies not only raised hope to treat those diseases but also raised new questions regarding their true pathophysiological involvement. Here, we review the place of type 2 immunity in the different phenotypes of asthma, chronic rhinitis, chronic rhinosinusitis and atopic dermatitis, highlighting nuances between them. New hypotheses rising from the use of biotherapies will be discussed along with the uncertainties and unmet needs of this field.

Time-dependent blood eosinophilia count increases the risk of kidney allograft rejection.

BACKGROUND: Growing evidence suggest that type 2 immune effectors play a role in solid organ transplantation. The aim of this study was to evaluate the impact of blood count eosinophils (BCEo) on immunological outcomes in kidney transplant recipients with stable graft function after 3 months post-transplant. METHOD: We performed cause-specific Cox model considering BCEo, the use of calcineurin inhibitors and systemic corticoids as time-dependent explicative variables on a prospective cohort of 1013 kidney transplant patients who experienced kidney allograft rejection and/or the appearance of de novo donor specific antibodies after excluding common causes of increased BCEo.. FINDINGS: BCEo ≥ 0.3 G/L was associated with a 3-fold increased risk of rejection independent of immunosuppressive regimen after 3 months post-transplant in patients without pre-transplant DSAs and with CNI-based immunosuppression. No association between BCEo either with donor specific antibodies or graft survival was noticed. INTERPRETATION: These observations in this large cohort support the hypothesis of eosinophils in allo-immunity in human and claim for further mechanistic research. FUNDING: This study was supported by the French National Research Agency, The "Institut de Recherche en Santé Respiratoire des Pays de la Loire" and the University hospital of Nantes.

Essential role of smooth muscle Rac1 in severe asthma-associated airway remodelling.

BACKGROUND: Severe asthma is a chronic lung disease characterised by inflammation, airway hyperresponsiveness (AHR) and airway remodelling. The molecular mechanisms underlying uncontrolled airway smooth muscle cell (aSMC) proliferation involved in pulmonary remodelling are still largely unknown. Small G proteins of the Rho family (RhoA, Rac1 and Cdc42) are key regulators of smooth muscle functions and we recently demonstrated that Rac1 is activated in aSMC from allergic mice. The objective of this study was to assess the role of Rac1 in severe asthma-associated airway remodelling. METHODS AND RESULTS: Immunofluorescence analysis in human bronchial biopsies revealed an increased Rac1 activity in aSMC from patients with severe asthma compared with control subjects. Inhibition of Rac1 by EHT1864 showed that Rac1 signalling controlled human aSMC proliferation induced by mitogenic stimuli through the signal transducer and activator of transcription 3 (STAT3) signalling pathway. In vivo, specific deletion of Rac1 in SMC or pharmacological inhibition of Rac1 by nebulisation of NSC23766 prevented AHR and aSMC hyperplasia in a mouse model of severe asthma. Moreover, the Rac1 inhibitor prevented goblet cell hyperplasia and epithelial cell hypertrophy whereas treatment with corticosteroids had less effect. Nebulisation of NSC23766 also decreased eosinophil accumulation in the bronchoalveolar lavage of asthmatic mice. CONCLUSION: This study demonstrates that Rac1 is overactive in the airways of patients with severe asthma and is essential for aSMC proliferation. It also provides evidence that Rac1 is causally involved in AHR and airway remodelling. Rac1 may represent as an interesting target for treating both AHR and airway remodelling of patients with severe asthma.

Overexpression of the MSK1 Kinase in Patients With Chronic Lung Allograft Dysfunction and Its Confirmed Role in a Murine Model.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) and its obstructive form, the obliterative bronchiolitis (OB), are the main long-term complications related to high mortality rate postlung transplantation. CLAD treatment lacks a significant success in survival. Here, we investigated a new strategy through inhibition of the proinflammatory mitogen- and stress-activated kinase 1 (MSK1) kinase. METHODS: MSK1 expression was assessed in a mouse OB model after heterotopic tracheal allotransplantation. Pharmacological inhibition of MSK1 (H89, fasudil, PHA767491) was evaluated in the murine model and in a translational model using human lung primary fibroblasts in proinflammatory conditions. MSK1 expression was graded over time in biopsies from a cohort of CLAD patients. RESULTS: MSK1 mRNA progressively increased during OB (6.4-fold at D21 posttransplantation). Inhibition of MSK1 allowed to counteract the damage to the epithelium (56% restoration for H89), and abolished the recruitment of MHCII+ (94%) and T cells (100%) at the early inflammatory phase of OB. In addition, it markedly decreased the late fibroproliferative obstruction in allografts (48%). MSK1 inhibitors decreased production of IL-6 (whose transcription is under the control of MSK1) released from human lung fibroblasts (96%). Finally, we confirmed occurrence of a 2.9-fold increased MSK1 mRNA expression in lung biopsies in patients at 6 months before CLAD diagnosis as compared to recipients with stable lung function. CONCLUSIONS: These findings suggest the overall interest of the MSK1 kinase either as a marker or as a potential therapeutic target in lung dysfunction posttransplantation.