Perinatal Activation of the Interleukin-33 Pathway Promotes Type 2 Immunity in the Developing Lung.

Allergic disease originates in early life and polymorphisms in interleukin-33 gene (IL33) and IL1RL1, coding for IL-33R and decoy receptor sST2, confer allergy risk. Early life T helper 2 (Th2) cell skewing and allergy susceptibility are often seen as remnants of feto-maternal symbiosis. Here we report that shortly after birth, innate lymphoid type 2 cells (ILC2s), eosinophils, basophils, and mast cells spontaneously accumulated in developing lungs in an IL-33-dependent manner. During the phase of postnatal lung alveolarization, house dust mite exposure further increased IL-33, which boosted cytokine production in ILC2s and activated CD11b+ dendritic cells (DCs). IL-33 suppressed IL-12p35 and induced OX40L in neonatal DCs, thus promoting Th2 cell skewing. Decoy sST2 had a strong preventive effect on asthma in the neonatal period, less so in adulthood. Thus, enhanced neonatal Th2 cell skewing to inhaled allergens results from postnatal hyperactivity of the IL-33 axis during a period of maximal lung remodeling.

Circulating T cells in sarcoidosis have an aberrantly activated phenotype that correlates with disease outcome.

RATIONALE: Disease course in sarcoidosis is highly variable. Bronchoalveolar lavage fluid and mediastinal lymph nodes show accumulation of activated T cells with a T-helper (Th)17.1 signature, which correlates with non-resolving sarcoidosis. We hypothesize that the peripheral blood (PB) T cell phenotype may correlate with outcome. OBJECTIVES: To compare frequencies, phenotypes and function of circulating T cell populations in sarcoidosis patients with healthy controls (HCs) and correlate these parameters with outcome. METHODS: We used multi-color flow cytometry to quantify activation marker expression on PB T cell subsets in treatment-naïve patients and HCs. The disease course was determined after 2-year follow-up. Cytokine production was measured after T cell stimulation in vitro. MEASUREMENTS AND MAIN RESULTS: We observed significant differences between patients and HCs in several T cell populations, including CD8+ and CD4+ T cells, Th1/Th17 subsets, CD4+ T memory stem cells, regulatory T cells (Tregs) and γδ T cells. Decreased frequencies of CD4+ T cells and increased frequencies of Tregs and CD8+ γδ T cells correlated with worse outcome. Naïve CD4+ T cells displayed an activated phenotype with increased CD25 expression in patients with active chronic disease at 2-year follow-up. A distinctive Treg phenotype with increased expression of CD25, CTLA4, CD69, PD-1 and CD95 correlated with chronic sarcoidosis. Upon stimulation, both naïve and memory T cells displayed a different cytokine profile in sarcoidosis compared to HCs. CONCLUSIONS: Circulating T cell subpopulations of sarcoidosis patients display phenotypic abnormalities that correlate with disease outcome, supporting a critical role of aberrant T cell activation in sarcoidosis pathogenesis.

Peripheral Blood T Cells of Patients with IPAH Have a Reduced Cytokine-Producing Capacity.

Pulmonary arterial hypertension (PAH) is rare disease that is categorized as idiopathic (IPAH) when no underlying cause can be identified. Lungs of most patients with IPAH contain increased numbers of T cells and dendritic cells (DCs), suggesting involvement of the immune system in its pathophysiology. However, our knowledge on circulating immune cells in IPAH is rather limited. We used flow cytometry to characterize peripheral blood DCs and T cells in treatment-naive IPAH patients, compared with connective-tissue disease-PAH (CTD-PAH) patients and healthy controls (HCs). At diagnosis, T-helper (Th) cells of IPAH patients were less capable of producing TNFα, IFNγ, IL-4 and IL-17 compared to HCs. IPAH patients showed a decreased frequency of Th2 cells and significantly enhanced expression of the CTLA4 checkpoint molecule in naive CD4+ T cells and both naive and memory CD8+ T cells. Frequencies and surface marker expression of circulating DCs and monocytes were essentially comparable between IPAH patients and HCs. Principal component analysis (PCA) separated IPAH patients-but not CTD-PAH patients-from HCs, based on T-cell cytokine profiles. At 1-year follow-up, the frequencies of IL-17+ production by memory CD4+ T cells were increased in IPAH patients and accompanied by increased proportions of Th17 and Tc17 cells, as well as decreased CTLA4 expression. Treatment-naive IPAH patients displayed a unique T-cell phenotype that was different from CTD-PAH patients and was characterized by reduced cytokine-producing capacity. These findings point to involvement of adaptive immune responses in IPAH, which may have an implication for the development of therapeutic interventions.

Loss of immune homeostasis in patients with idiopathic pulmonary arterial hypertension.

INTRODUCTION: Autoreactivity against pulmonary vascular structures is thought to be involved in idiopathic pulmonary arterial hypertension (IPAH), but the underlying mechanisms remain poorly understood. We hypothesised that aberrant B-cell activation contributes to IPAH aetiology. METHODS: Mice with enhanced B-cell activation due to B-cell-specific overexpression of the B-cell receptor (BCR) signalling molecule Bruton's tyrosine kinase (BTK) were subjected to lung injury and examined for several pulmonary hypertension (PH) indices. Peripheral blood lymphocytes from patients with IPAH (n=13), connective tissue disease-associated PAH (CTD-PAH, n=9), congenital heart disease PAH (n=7), interstitial lung disease associated PH (n=17) and healthy controls (n=19) were characterised by 14-colour flow cytometry. RESULTS: Following pulmonary injury, BTK-overexpressing mice showed prolonged activation of B cells and CXCR5+ follicular T-helper (Tfh) cells, as well as features of PH development. Patients with CTD-PAH and CHD-PAH displayed reduced proportions of circulating non-switched-memory B cells (p=0.03, p=0.02, respectively). Interestingly, we observed increased BTK protein expression in naive (p=0.007) and memory B-cell subsets of patients with IPAH and CTD-PAH. BTK was particularly high in patients with IPAH with circulating autoantibodies (p=0.045). IPAH patients had low frequencies of circulating CXCR5+ Tfh cells (p=0.005). Hereby, the increased BTK protein expression in B cells was associated with high proportions of Tfh17 (p=0.018) and Tfh17.1 (p=0.007) cells within the circulating Tfh population. CONCLUSIONS: Our study shows that pulmonary injury in combination with enhanced B-cell activation is sufficient to induce PH symptoms in mice. In parallel, immune homeostasis in patients with IPAH is compromised, as evidenced by increased BCR signalling and cTfh17 polarisation, indicating that adaptive immune activation contributes to IPAH disease induction or progression.

Central Role of Dendritic Cells in Pulmonary Arterial Hypertension in Human and Mice.

The pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) is not fully understood, but evidence is accumulating that immune dysfunction plays a significant role. We previously reported that 31-week-old Tnfaip3DNGR1-KO mice develop pulmonary hypertension (PH) symptoms. These mice harbor a targeted deletion of the TNFα-induced protein-3 (Tnfaip3) gene, encoding the NF-κB regulatory protein A20, specifically in type I conventional dendritic cells (cDC1s). Here, we studied the involvement of dendritic cells (DCs) in PH in more detail. We found various immune cells, including DCs, in the hearts of Tnfaip3DNGR1-KO mice, particularly in the right ventricle (RV). Secondly, in young Tnfaip3DNGR1-KO mice, innate immune activation through airway exposure to toll-like receptor ligands essentially did not result in elevated RV pressures, although we did observe significant RV hypertrophy. Thirdly, PH symptoms in Tnfaip3DNGR1-KO mice were not enhanced by concomitant mutation of bone morphogenetic protein receptor type 2 (Bmpr2), which is the most affected gene in PAH patients. Finally, in human IPAH lung tissue we found co-localization of DCs and CD8+ T cells, representing the main cell type activated by cDC1s. Taken together, these findings support a unique role of cDC1s in PAH pathogenesis, independent of general immune activation or a mutation in the Bmpr2 gene.

DNGR1-Cre-mediated Deletion of Tnfaip3/A20 in Conventional Dendritic Cells Induces Pulmonary Hypertension in Mice.

Chronic perivascular inflammation is a prominent feature in the lungs of idiopathic pulmonary arterial hypertension. Although the proportions of conventional dendritic cells (cDCs) and plasmacytoid DCs are increased in idiopathic pulmonary arterial hypertension lungs, it remains unknown whether activated cDCs play a pathogenic role. The Tnfaip3 gene encodes the ubiquitin-binding protein A20, which is a negative regulator of NF-κB, critically involved in DC activation. Targeting of Tnfaip3/A20 in cDCs was achieved by Clec9a (DNGR1)-Cre-mediated excision of the Tnfaip3 gene in Tnfaip3DNGR1-KO mice. Mice were evaluated for signs of pulmonary hypertension (PH) using right heart catheterization, echocardiography, and measurement of the Fulton index. Inflammation was assessed by immunohistochemistry and flow cytometry. Pulmonary cDCs and monocyte-derived DCs from 31-week-old Tnfaip3DNGR1-KO mice showed modulated expression of cell surface activation markers compared with Tnfaip3DNGR1-WT mice. Tnfaip3DNGR1-KO mice developed elevated right ventricular systolic pressure and right ventricular hypertrophy. The lungs of these mice displayed increased vascular remodeling and perivascular and peribronchial immune cell infiltration resembling tertiary lymphoid organs. Proportions of activated T cells and expression of IL-1ß, IL-6, and IL-10 were enhanced in the lungs of Tnfaip3DNGR1-KO mice. Autoreactive IgA and IgG1 was detected in BAL and autoreactive IgA recognizing pulmonary endothelial antigens was present in the serum of Tnfaip3DNGR1-KO mice. All signs of PH were ameliorated in Tnfaip3DNGR1-KO mice by anti-IL-6 antibody treatment. These results indicate that activation of the NF-κB pathway in DCs, through deletion of A20/Tnfaip3, leads to experimental PH with accompanied pulmonary inflammation in an IL-6-dependent fashion.

Tnfaip3 expression in pulmonary conventional type 1 Langerin-expressing dendritic cells regulates T helper 2-mediated airway inflammation in mice.

BACKGROUND: Conventional type 1 dendritic cells (cDC1s) control anti-viral and anti-tumor immunity by inducing antigen-specific cytotoxic CD8+ T-cell responses. Controversy exists whether cDC1s also control CD4+ T helper 2 (Th2) cell responses, since suppressive and activating roles have been reported. DC activation status, controlled by the transcription factor NF-κB, might determine the precise outcome of Th-cell differentiation upon encounter with cDC1s. To investigate the role of activated cDC1s in Th2-driven immune responses, pulmonary cDC1s were activated by targeted deletion of A20/Tnfaip3, a negative regulator of NF-κB signaling. METHODS: To target pulmonary cDC1s, Cd207 (Langerin)-mediated excision of A20/Tnfaip3 was used, generating Tnfaip3fl/fl xCd207+/cre (Tnfaip3Lg-KO ) mice. Mice were exposed to house dust mite (HDM) to provoke Th2-mediated immune responses. RESULTS: Mice harboring Tnfaip3-deficient cDC1s did not develop Th2-driven eosinophilic airway inflammation upon HDM exposure, but rather showed elevated numbers of IFNγ-expressing CD8+ T cells. In addition, Tnfaip3Lg-KO mice harbored increased numbers of IL-12-expressing cDC1s and elevated PD-L1 expression in all pulmonary DC subsets. Blocking either IL-12 or IFNγ in Tnfaip3Lg-KO mice restored Th2 responses, whereas administration of recombinant IFNγ during HDM sensitization in C57Bl/6 mice blocked Th2 development. CONCLUSIONS: These findings indicate that the activation status of cDC1s, shown by their specific expression of co-inhibitory molecules and cytokines, critically contributes to the development of Th2 cell-mediated disorders, most likely by influencing IFNγ production in CD8+ T cells.

An unexpected role for uric acid as an inducer of T helper 2 cell immunity to inhaled antigens and inflammatory mediator of allergic asthma.

Although deposition of uric acid (UA) crystals is known as the cause of gout, it is unclear whether UA plays a role in other inflammatory diseases. We here have shown that UA is released in the airways of allergen-challenged asthmatic patients and mice, where it was necessary for mounting T helper 2 (Th2) cell immunity, airway eosinophilia, and bronchial hyperreactivity to inhaled harmless proteins and clinically relevant house dust mite allergen. Conversely, administration of UA crystals together with protein antigen was sufficient to promote Th2 cell immunity and features of asthma. The adjuvant effects of UA did not require the inflammasome (Nlrp3, Pycard) or the interleukin-1 (Myd88, IL-1r) axis. UA crystals promoted Th2 cell immunity by activating dendritic cells through spleen tyrosine kinase and PI3-kinase δ signaling. These findings provide further molecular insight into Th2 cell development and identify UA as an essential initiator and amplifier of allergic inflammation.

The ubiquitin-editing protein A20 prevents dendritic cell activation, recognition of apoptotic cells, and systemic autoimmunity.

Dendritic cells (DCs) regulate both immunity and tolerance. Here we have shown that the ubiquitin editing enzyme A20 (Tnfaip3) determines the activation threshold of DCs, via control of canonical NF-κB activation. Tnfaip3(fl/fl)Cd11c-cre(+) mice lacking A20 in DCs demonstrated spontaneous proliferation of conventional and double-negative T cells, their conversion to interferon-γ (IFN-γ)-producing effector cells, and expansion of plasma cells. They developed ds-DNA antibodies, nephritis, the antiphospholipid syndrome, and lymphosplenomegaly-features of systemic lupus erythematosus-and extramedullary hematopoiesis. A20-deficient DCs were resistant to apoptosis, caused by increased sensitivity to CD40L and RANKL prosurvival signals and upregulation of antiapoptotic proteins Bcl-2 and Bcl-x. They captured injected apoptotic cells more efficiently, resisted the inhibitory effects of apoptotic cells, and induced self-reactive effector lymphocytes. Because genetic polymorphisms in TNFAIP3 are associated with human autoimmune disorders, these findings identify A20-mediated control of DC activation as a crucial checkpoint in the development of systemic autoimmunity.

Design of a randomized controlled trial to evaluate effectiveness of methotrexate versus prednisone as first-line treatment for pulmonary sarcoidosis: the PREDMETH study.

BACKGROUND: Treatment of pulmonary sarcoidosis is recommended in case of significant symptoms, impaired or deteriorating lung function. Evidence-based treatment recommendations are limited and largely based on expert opinion. Prednisone is currently the first-choice therapy and leads to short-term improvement of lung function. Unfortunately, prednisone often has side-effects and may be associated with impaired quality of life. Methotrexate is presently considered second-line therapy, and appears to have fewer side-effects. OBJECTIVE: The primary objective of this trial is to investigate the effectiveness and tolerability of methotrexate as first-line therapy in patients with pulmonary sarcoidosis compared with prednisone. The primary endpoint of this study will be the change in hospital-measured Forced Vital Capacity (FVC) between baseline and 24 weeks. Secondary objectives are to gain more insights in response to therapy in individual patients by home spirometry and patient-reported outcomes. Blood biomarkers will be examined to find predictors of response to therapy, disease progression and chronicity, and to improve our understanding of the underlying disease mechanism. METHODS/DESIGN: In this prospective, randomized, non-blinded, multi-center, non-inferiority trial, we plan to randomize 138 treatment-naïve patients with pulmonary sarcoidosis who are about to start treatment. Patients will be randomized in a 1:1 ratio to receive either prednisone or methotrexate in a predefined schedule for 24 weeks, after which they will be followed up in regular care for up to 2 years. Regular hospital visits will include pulmonary function assessment, completion of patient-reported outcomes, and blood withdrawal. Additionally, patients will be asked to perform weekly home spirometry, and record symptoms and side-effects via a home monitoring application for 24 weeks. DISCUSSION: This study will be the first randomized controlled trial comparing first-line treatment of prednisone and methotrexate and provide valuable data on efficacy, safety, quality of life and biomarkers. If this study confirms the hypothesis that methotrexate is as effective as prednisone as first-line treatment for sarcoidosis but with fewer side-effects, this will lead to improvement in care and initiate a change in practice. Furthermore, insights into the immunological mechanisms underlying sarcoidosis pathology might reveal new therapeutic targets. TRIAL REGISTRATION: The study was registered on the 19th of March 2020 in the International Clinical Trial Registry, www.clinicaltrials.gov; ID NCT04314193 .

DNGR1-mediated deletion of A20/Tnfaip3 in dendritic cells alters T and B-cell homeostasis and promotes autoimmune liver pathology.

Dendritic cells (DCs) are central regulators of tolerance versus immunity. The outcome depends amongst others on DC subset and activation status. Whereas CD11b+ type 2 conventional DCs (cDC2s) initiate proinflammatory helper T (Th)-cell responses, CD103+ cDC1s are crucial for regulatory T-cell (Treg) induction and CD8+ T-cell activation. DC activation is controlled by the transcription factor NF-κB. Ablation of A20/Tnfaip3, a critical regulator of NF-κB activation, in DCs leads to constitutive DC activation and development of systemic autoimmunity. We hypothesized that the activation status of cDCs controls the development of autoimmunity. To target cDCs, DNGR1(Clec9a)-cre-mediated excision of A20/Tnfaip3 was used through generation of Tnfaip3fl/flxClec9a+/cre (Tnfaip3DNGR1-KO) mice. Immune cell activation was evaluated at 31-weeks of age. We found that DNGR1-cre-mediated deletion of A20/Tnfaip3 resulted in liver pathology characterized by inflammatory infiltrates adjacent to the portal triads. Both cDC subsets as well as monocyte-derived DCs (moDCs) in Tnfaip3DNGR1-KO livers harbored an activated phenotype. Specifically, the costimulatory molecule CD40 in liver cDCs and moDCs was regulated by A20/Tnfaip3 expression. Livers from Tnfaip3DNGR1-KO mice had augmented proportions of Th1, Th17, Treg, and follicular Th (Tfh)-cells compared to control mice, accompanied by an increase in IgA-producing plasma cells. Serum IgA from Tnfaip3DNGR1-KO mice recognized self-proteins, specifically cytoplasmic proteins in liver periportal regions. These data show that enhanced activation of cDCs and moDCs, due to A20/Tnfaip3 ablation, promotes the development of organ-specific autoimmunity but not systemic autoimmunity. This model could be useful to examine the pathobiological processes contributing to autoimmune liver diseases.

Enhanced Bruton's tyrosine kinase in B-cells and autoreactive IgA in patients with idiopathic pulmonary fibrosis.

RATIONALE: Idiopathic Pulmonary Fibrosis (IPF) is thought to be triggered by repeated alveolar epithelial cell injury. Current evidence suggests that aberrant immune activation may contribute. However, the role of B-cell activation remains unclear. We determined the phenotype and activation status of B-cell subsets and evaluated the contribution of activated B-cells to the development of lung fibrosis both in humans and in mice. METHODS: B-cells in blood, mediastinal lymph node, and lung single-cell suspensions of IPF patients and healthy controls (HC) were characterized using 14-color flow cytometry. Mice were exposed to bleomycin to provoke pulmonary fibrosis. RESULTS: More IgA+ memory B-cells and plasmablasts were found in blood (n = 27) and lungs (n = 11) of IPF patients compared to HC (n = 21) and control lungs (n = 9). IPF patients had higher levels of autoreactive IgA in plasma, which correlated with an enhanced decline of forced vital capacity (p = 0.002, r = - 0.50). Bruton's tyrosine kinase expression was higher in circulating IPF B-cells compared to HC, indicating enhanced B-cell activation. Bleomycin-exposed mice had increased pulmonary IgA+ germinal center and plasma cell proportions compared to control mice. The degree of lung fibrosis correlated with pulmonary germinal center B-cell proportions (p = 0.010, r = 0.88). CONCLUSION: Our study demonstrates that IPF patients have more circulating activated B-cells and autoreactive IgA, which correlate with disease progression. These B-cell alterations were also observed in the widely used mouse model of experimental pulmonary fibrosis. Autoreactive IgA could be useful as a biomarker for disease progression in IPF.

Nebulised hypertonic saline in children with bronchiolitis admitted to the paediatric intensive care unit: A retrospective study.

AIM: Bronchiolitis is one of the most common lower respiratory tract infections in young children, associated with significant morbidity, but limited therapeutic options. Nebulised hypertonic saline (HS) has been a supportive treatment until current guidelines advised against its routine use. Accordingly, the University Hospital of Antwerp recently changed their policies to stop using it, allowing us to evaluate retrospectively if HS influences the duration of respiratory support. Because, to our knowledge, the effect of HS on children with severe bronchiolitis admitted to a paediatric intensive care unit (PICU) has not been studied yet, we aimed to investigate the effect in this specific patient group. METHODS: Retrospective study including children up to the age of 2, admitted to the PICU with bronchiolitis from October 2013 until March 2016. The primary end point is the duration of respiratory support, including high flow nasal cannula, continuous positive airway pressure and invasive ventilation. RESULTS: A total of 104 children admitted to the PICU with bronchiolitis were included, with an average age of 3.4 months. In respiratory syncytial virus (RSV) positive patients, the use of nebulised HS was correlated with a decrease in the duration of respiratory support and the length of stay by factors 0.72 (P = 0.01) and 0.81 (P = 0.04), respectively. CONCLUSIONS: A significant correlation was found between the use of HS and a decreased duration of respiratory support and admission in the PICU in patients with RSV bronchiolitis. This finding may warrant new prospective studies investigating HS specifically in children with severe bronchiolitis.

TNF-α-induced protein 3 levels in lung dendritic cells instruct TH2 or TH17 cell differentiation in eosinophilic or neutrophilic asthma.

BACKGROUND: It is currently unknown why allergen exposure or environmental triggers in patients with mild-to-moderate asthma result in TH2-mediated eosinophilic inflammation, whereas patients with severe asthma often present with TH17-mediated neutrophilic inflammation. The activation state of dendritic cells (DCs) is crucial for both TH2 and TH17 cell differentiation and is mediated through nuclear factor κB activation. Ablation of TNF-α-induced protein 3 (TNFAIP3), one of the crucial negative regulators of nuclear factor κB activation in myeloid cells and DCs, was shown to control DC activation. OBJECTIVE: In this study we investigated the precise role of TNFAIP3 in myeloid cells for the development of TH2- and TH17-cell mediated asthma. METHODS: We exposed mice with conditional deletion of the Tnfaip3 gene in either myeloid cells (by using the lysozyme M [LysM] promotor) or specifically in DCs (by using the Cd11c promotor) to acute and chronic house dust mite (HDM)-driven asthma models. RESULTS: We demonstrated that reduced Tnfaip3 gene expression in DCs in either Tnfaip3CD11c or Tnfaip3LysM mice dose-dependently controlled development of TH17-mediated neutrophilic severe asthma in both acute and chronic HDM-driven models, whereas wild-type mice had a purely TH2-mediated eosinophilic inflammation. TNFAIP3-deficient DCs induced HDM-specific TH17 cell differentiation through increased expression of the TH17-instructing cytokines IL-1ß, IL-6, and IL-23, whereas HDM-specific TH2 cell differentiation was hampered by increased IL-12 and IL-6 production. CONCLUSIONS: These data show that the extent of TNFAIP3 expression in DCs controls TH2/TH17 cell differentiation. This implies that reducing DC activation could be a new pharmacologic intervention to treat patients with severe asthma who present with TH17-mediated neutrophilic inflammation.

Increased T-helper 17.1 cells in sarcoidosis mediastinal lymph nodes.

The lung-draining mediastinal lymph nodes (MLNs) are currently widely used to diagnose sarcoidosis. We previously reported that T-helper (Th) 17.1 cells are responsible for the exaggerated interferon-γ production in sarcoidosis lungs. In this study, we aimed to investigate 1) whether Th17.1 cells are also increased in the MLNs of sarcoidosis patients and 2) whether frequencies of the Th17.1 cells at diagnosis may correlate with disease progression.MLN cells from treatment-naive pulmonary sarcoidosis patients (n=17) and healthy controls (n=22) and peripheral blood mononuclear cells (n=34) and bronchoalveolar lavage fluid (BALF) (n=36) from sarcoidosis patients were examined for CD4+ T-cell subset proportions using flow cytometry.Higher proportions of Th17.1 cells were detected in sarcoidosis MLNs than in control MLNs. Higher Th17.1 cell proportions were found in sarcoidosis BALF compared with MLNs and peripheral blood. Furthermore, BALF Th17.1 cell proportions were significantly higher in patients developing chronic disease than in patients undergoing resolution within 2 years of clinical follow-up.These data suggest that Th17.1 cell proportions in pulmonary sarcoidosis can be evaluated as a diagnostic and/or prognostic marker in clinical practice and could serve as a new therapeutic target.

House dust mite-driven neutrophilic airway inflammation in mice with TNFAIP3-deficient myeloid cells is IL-17-independent.

BACKGROUND: Asthma is a heterogeneous disease of the airways that involves several types of granulocytic inflammation. Recently, we have shown that the activation status of myeloid cells regulated by TNFAIP3/A20 is a crucial determinant of eosinophilic or neutrophilic airway inflammation. However, whether neutrophilic inflammation observed in this model is dependent on IL-17 remains unknown. OBJECTIVE: In this study, we investigated whether IL-17RA-signalling is essential for eosinophilic or neutrophilic inflammation in house dust mite (HDM)-driven airway inflammation. METHODS: Tnfaip3fl/fl xLyz2+/cre (Tnfaip3LysM-KO ) mice were crossed to Il17raKO mice, generating Tnfaip3LysM Il17raKO mice and subjected to an HDM-driven airway inflammation model. RESULTS: Both eosinophilic and neutrophilic inflammation observed in HDM-exposed WT and Tnfaip3LysM-KO mice respectively were unaltered in the absence of IL-17RA. Production of IL-5, IL-13 and IFN-γ by CD4+ T cells was similar between WT, Tnfaip3LysM-KO and Il17raKO mice, whereas mucus-producing cells in Tnfaip3LysM-KO Il17raKO mice were reduced compared to controls. Strikingly, spontaneous accumulation of pulmonary Th1, Th17 and γδ-17 T cells was observed in Tnfaip3LysM-KO Il17raKO mice, but not in the other genotypes. Th17 cell-associated cytokines such as GM-CSF and IL-22 were increased in the lungs of HDM-exposed Tnfaip3LysM-KO Il17raKO mice, compared to IL-17RA-sufficient controls. Moreover, neutrophilic chemo-attractants CXCL1, CXCL2, CXCL12 and Th17-promoting cytokines IL-1ß and IL-6 were unaltered between Tnfaip3LysM-KO and Tnfaip3LysM-KO Il17raKO mice. CONCLUSION AND CLINICAL RELEVANCE: These findings show that neutrophilic airway inflammation induced by activated TNFAIP3/A20-deficient myeloid cells can develop in the absence of IL-17RA-signalling. Neutrophilic inflammation is likely maintained by similar quantities of pro-inflammatory cytokines IL-1ß and IL-6 that can, independently of IL-17-signalling, induce the expression of neutrophil chemo-attractants.

Th17-lineage cells in pulmonary sarcoidosis and Löfgren's syndrome: Friend or foe?

Sarcoidosis, a multisystem granulomatous disorder, has historically been classified as Th1-driven disease. However, increasing data demonstrate a key role of Th17-cell plasticity in granuloma formation and maintenance. In Löfgren's syndrome (LS), an acute and distinct phenotype of sarcoidosis with a favorable outcome, differences in Th17-lineage cell subsets, cytokine expression and T-cell suppressive mechanisms may account for differences in clinical presentation as well as prognosis compared to non-LS sarcoidosis. In contrast with LS, up to 20% of non-LS sarcoidosis patients may progress to irreversible pulmonary fibrosis. In non-LS sarcoidosis patients, IFN-γ-producing Th17.1-cells appear to be more pathogenic and possibly linked to disease progression, while a broader range of cytokines is found in bronchoalveolar lavage fluid (BALF) in LS patients. Differences in Cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression on Th17-cells and regulatory T-cells (Treg) could contribute to Th17-cell pathogenicity and consequently to either disease resolution or ongoing inflammation in sarcoidosis. Furthermore, several genes and SNPs are associated with disease susceptibility and outcome in sarcoidosis, the majority of which are involved in antigen presentation, T-cell activation or regulation of T-cell survival. Novel insights into the role of Th17-cells in the pathogenesis of both LS and non-LS sarcoidosis will unravel pathogenic and benign Th17-lineage cell function and drivers of Th17-cell plasticity. This will also help identify new treatment strategies for LS and non-LS sarcoidosis patients by altering Th17-cell activation, suppress conversion into more pathogenic subtypes, or influence cytokine signaling towards a beneficial signature of Th17-lineage cells. In this review, we summarize new insights into Th17-cell plasticity in the complex pathogenesis of sarcoidosis and connect these cells to the different disease phenotypes, discuss the role of genetic susceptibility and autoimmunity and focus on possible treatment strategies.

GATA1-Deficient Dendritic Cells Display Impaired CCL21-Dependent Migration toward Lymph Nodes Due to Reduced Levels of Polysialic Acid.

Dendritic cells (DCs) play a pivotal role in the regulation of the immune response. DC development and activation is finely orchestrated through transcriptional programs. GATA1 transcription factor is required for murine DC development, and data suggest that it might be involved in the fine-tuning of the life span and function of activated DCs. We generated DC-specific Gata1 knockout mice (Gata1-KODC), which presented a 20% reduction of splenic DCs, partially explained by enhanced apoptosis. RNA sequencing analysis revealed a number of deregulated genes involved in cell survival, migration, and function. DC migration toward peripheral lymph nodes was impaired in Gata1-KODC mice. Migration assays performed in vitro showed that this defect was selective for CCL21, but not CCL19. Interestingly, we show that Gata1-KODC DCs have reduced polysialic acid levels on their surface, which is a known determinant for the proper migration of DCs toward CCL21.

A20-deficient mast cells exacerbate inflammatory responses in vivo.

Mast cells are implicated in the pathogenesis of inflammatory and autoimmune diseases. However, this notion based on studies in mast cell-deficient mice is controversial. We therefore established an in vivo model for hyperactive mast cells by specifically ablating the NF-κB negative feedback regulator A20. While A20 deficiency did not affect mast cell degranulation, it resulted in amplified pro-inflammatory responses downstream of IgE/FcεRI, TLRs, IL-1R, and IL-33R. As a consequence house dust mite- and IL-33-driven lung inflammation, late phase cutaneous anaphylaxis, and collagen-induced arthritis were aggravated, in contrast to experimental autoimmune encephalomyelitis and immediate anaphylaxis. Our results provide in vivo evidence that hyperactive mast cells can exacerbate inflammatory disorders and define diseases that might benefit from therapeutic intervention with mast cell function.

IFN-γ-Producing T-Helper 17.1 Cells Are Increased in Sarcoidosis and Are More Prevalent than T-Helper Type 1 Cells.

RATIONALE: Pulmonary sarcoidosis is classically defined by T-helper (Th) cell type 1 inflammation (e.g., IFN-γ production by CD4(+) effector T cells). Recently, IL-17A-secreting cells have been found in lung lavage, invoking Th17 immunity in sarcoidosis. Studies also identified IL-17A-secreting cells that expressed IFN-γ, but their abundance as a percentage of total CD4(+) cells was either low or undetermined. OBJECTIVES: Based on evidence that Th17 cells can be polarized to Th17.1 cells to produce only IFN-γ, our goal was to determine whether Th17.1 cells are a prominent source of IFN-γ in sarcoidosis. METHODS: We developed a single-cell approach to define and isolate major Th-cell subsets using combinations of chemokine receptors and fluorescence-activated cell sorting. We subsequently confirmed the accuracy of subset enrichment by measuring cytokine production. MEASUREMENTS AND MAIN RESULTS: Discrimination between Th17 and Th17.1 cells revealed very high percentages of Th17.1 cells in lung lavage in sarcoidosis compared with controls in two separate cohorts. No differences in Th17 or Th1 lavage cells were found compared with controls. Lung lavage Th17.1-cell percentages were also higher than Th1-cell percentages, and approximately 60% of Th17.1-enriched cells produced only IFN-γ. CONCLUSIONS: Combined use of surface markers and functional assays to study CD4(+) T cells in sarcoidosis revealed a marked expansion of Th17.1 cells that only produce IFN-γ. These results suggest that Th17.1 cells could be misclassified as Th1 cells and may be the predominant producer of IFN-γ in pulmonary sarcoidosis, challenging the Th1 paradigm of pathogenesis.