Indocyanine-enhanced mouse model of bleomycin-induced lung fibrosis with hallmarks of progressive emphysema.

The development of new drugs for idiopathic pulmonary fibrosis strongly relies on preclinical experimentation, which requires the continuous improvement of animal models and integration with in vivo imaging data. Here, we investigated the lung distribution of bleomycin (BLM) associated with the indocyanine green (ICG) dye by fluorescence imaging. A long-lasting lung retention (up to 21 days) was observed upon oropharyngeal aspiration (OA) of either ICG or BLM + ICG, with significantly more severe pulmonary fibrosis, accompanied by the progressive appearance of emphysema-like features, uniquely associated with the latter combination. More severe and persistent lung fibrosis, together with a progressive air space enlargement uniquely associated with the BLM + ICG group, was confirmed by longitudinal micro-computed tomography (CT) and histological analyses. Multiple inflammation and fibrosis biomarkers were found to be increased in the bronchoalveolar lavage fluid of BLM- and BLM + ICG-treated animals, but with a clear trend toward a much stronger increase in the latter group. Similarly, in vitro assays performed on macrophage and epithelial cell lines revealed a significantly more marked cytotoxicity in the case of BLM + ICG-treated mice. Also unique to this group was the synergistic upregulation of apoptotic markers both in lung sections and cell lines. Although the exact mechanism underlying the more intense lung fibrosis phenotype with emphysema-like features induced by BLM + ICG remains to be elucidated, we believe that this combination treatment, whose overall effects more closely resemble the human disease, represents a valuable alternative model for studying fibrosis development and for the identification of new antifibrotic compounds.

Impaired pulmonary vasomotor control in exercising swine with multiple comorbidities.

Pulmonary hypertension is common in heart failure with preserved ejection fraction (HFpEF). Here, we tested the hypothesis that comorbidities [diabetes mellitus (DM, streptozotocin), hypercholesterolemia (HC, high-fat diet) and chronic kidney disease (CKD, renal microembolization)] directly impair pulmonary vasomotor control in a DM + HC + CKD swine model. 6 months after induction of DM + HC + CKD, pulmonary arterial pressure was similar in chronically instrumented female DM + HC + CKD (n = 19) and Healthy swine (n = 18). However, cardiac output was lower both at rest and during exercise, implying an elevated pulmonary vascular resistance (PVR) in DM + HC + CKD swine (153 ± 10 vs. 122 ± 9 mmHg∙L-1∙min∙kg). Phosphodiesterase 5 inhibition and endothelin receptor antagonism decreased PVR in DM + HC + CKD (- 12 ± 12 and - 22 ± 7 mmHg∙L-1∙min∙kg) but not in Healthy swine (- 1 ± 12 and 2 ± 14 mmHg∙L-1∙min∙kg), indicating increased vasoconstrictor influences of phosphodiesterase 5 and endothelin. Inhibition of nitric oxide synthase produced pulmonary vasoconstriction that was similar in Healthy and DM + HC + CKD swine, but unmasked a pulmonary vasodilator effect of endothelin receptor antagonism in Healthy (- 56 ± 26 mmHg∙L-1∙min∙kg), whereas it failed to significantly decrease PVR in DM + HC + CKD, indicating loss of nitric oxide mediated inhibition of endothelin in DM + HC + CKD. Scavenging of reactive oxygen species (ROS) had no effect on PVR in either Healthy or DM + HC + CKD swine. Cardiovascular magnetic resonance imaging, under anesthesia, showed no right ventricular changes. Finally, despite an increased contribution of endogenous nitric oxide to vasomotor tone regulation in the systemic vasculature, systemic vascular resistance at rest was higher in DM + HC + CKD compared to Healthy swine (824 ± 41 vs. 698 ± 35 mmHg∙L-1∙min∙kg). ROS scavenging induced systemic vasodilation in DM + HC + CKD, but not Healthy swine. In conclusion, common comorbidities directly alter pulmonary vascular control, by enhanced PDE5 and endothelin-mediated vasoconstrictor influences, well before overt left ventricular backward failure or pulmonary hypertension develop.

PDE3-inhibitor enoximone prevented mechanical ventilation in patients with SARS-CoV-2 pneumonia.

BACKGROUND: Standard care in severe SARS-CoV-2 pneumonia complicated by severe dyspnea and respiratory failure, consists of symptom reduction, ultimately supported by mechanical ventilation. Patients with severe SARS-CoV-2, a prominent feature of COVID-19, show several similar symptoms to Critical Asthma Syndrome (CAS) patients, such as pulmonary edema, mucus plugging of distal airways, decreased tissue oxygenation, (emergent) exhaustion due to severe dyspnea and respiratory failure. Prior application of elective phosphodiesterase (PDE)3-inhibitors milrinone and enoximone in patients with CAS yielded rapid symptomatic relief and reverted the need for mechanical ventilation, due to their bronchodilator and anti-inflammatory properties. Based on these observations, we hypothesized that enoximone may be beneficial in the treatment of patients with severe SARS-CoV-2 pneumonia and prominent CAS-features. METHODS: In this case report enoximone was administered to four consecutive patients (1 M; 3 F; 46-70 y) with emergent respiratory failure due to SARS-CoV-2 pneumonia. Clinical outcome was compared with three controls who received standard care only. RESULTS: After an intravenous bolus of enoximone 20 mg followed by 10 mg/h via perfusor, a rapid symptomatic relief was observed: two out of four patients recovered within a few hours, the other two (with comorbid COPD GOLD II/III) responded within 24-36 h. Compared to the controls, in the enoximone-treated patients respiratory failure and further COVID-19-related deterioration was reverted and mechanical ventilation was prevented, leading to reduced hospital/ICU time. DISCUSSION: Our preliminary observations suggest that early intervention with the selective PDE3-inhibitor enoximone may help to revert respiratory failure as well as avert mechanical ventilation, and reduces ICU/hospital time in patients with severe SARS-CoV-2 pneumonia. Our findings warrant further research on the therapeutic potential of PDE3-inhibition, alone or in combination with other anti-COVID-19 strategies.

Involvement of Dendritic Cells and Th17 Cells in Induced Tertiary Lymphoid Structures in a Chronic Beryllium Disease Mouse Model.

OBJECTIVE: To study airway pathophysiology and the role of dendritic cells (DCs) and IL-17 receptor (IL-17R) signals in a mouse model for CBD. METHODS: Here, we present a CBD mouse model in which mice were exposed to beryllium during three weeks. We also exposed IL-17R-deficient mice and mice in which DCs were depleted. RESULTS: Eight weeks after the initial beryllium exposure, an inflammatory response was detected in the lungs. Mice displayed inflammation of the lower airways that included focal dense infiltrates, granuloma-like foci, and tertiary lymphoid structure (TLS) containing T cells, B cells, and germinal centers. Alveolar cell analysis showed significantly increased numbers of CD4+ T cells expressing IFNγ, IL-17, or both cytokines. The pathogenic role of IL-17R signals was demonstrated in IL-17R-deficient mice, which had strongly reduced lung inflammation and TLS development following beryllium exposure. In CBD mice, pulmonary DC subsets including CD103+ conventional DCs (cDCs), CD11b+ cDCs, and monocyte-derived DCs (moDCs) were also prominently increased. We used diphtheria toxin receptor-mediated targeted cell ablation to conditionally deplete DCs and found that DCs are essential for the maintenance of TLS in CBD. Furthermore, the presence of antinuclear autoantibodies in the serum of CBD mice showed that CBD had characteristics of autoimmune disease. CONCLUSIONS: We generated a translational model of sarcoidosis driven by beryllium and show that DCs and IL-17R signals play a pathophysiological role in CBD development as well as in established CBD in vivo.

T cells and ILC2s are major effector cells in influenza-induced exacerbation of allergic airway inflammation in mice.

Influenza virus infection is an important cause of severe asthma exacerbations, but it remains unclear how a Th1-mediated antiviral response triggers a prototypical Th2 disease. We investigated CD4+ T cells and group 2 innate lymphoid cells (ILC2s) in influenza virus-infected mice. We found that ILC2s accumulated in the lung rapidly after influenza virus infection, but the induction of IL-5 and IL-13 secretion was delayed and concomitant with T cell activation. In an influenza-induced exacerbation of allergic airway inflammation model we noticed an initial reduction of ILC2 numbers and cytokine production in broncho-alveolar lavage compared to chronic house dust mite (HDM)-mediated airway inflammation alone. ILC2s phenotype was characterized by low T1/ST2, ICOS, KLRG1, and CD25 expression, resembling naïve ILC2s. The contribution of ILC2s to type 2 cytokine production in the early stage of the influenza-induced exacerbation was limited. In contrast, T cells showed increased IL-4 and IL-5 production when exposed to both HDM and influenza virus. Upon virus clearance, ILC2s regained an activated T1/ST2high ICOShigh KLRG1high CD25high phenotype paired with cytokine production and were major contributors to the type 2 cytokine milieu. Collectively, our data indicate that both T cells and ILC2s contribute to influenza-induced exacerbation of allergic airway inflammation, but with different kinetics.

The Notch pathway inhibitor stapled α-helical peptide derived from mastermind-like 1 (SAHM1) abrogates the hallmarks of allergic asthma.

BACKGROUND: The Notch signaling pathway has been implicated in the pathogenesis of allergic airway inflammation. Targeting the active Notch transactivation complex by using the cell-permeable, hydrocarbon-stapled synthetic peptide stapled α-helical peptide derived from mastermind-like 1 (SAHM1) resulted in genome-wide suppression of Notch-activated genes in leukemic cells and other models. However, the efficacy of SAHM1 in allergic asthma models has remained unexplored. OBJECTIVE: We aimed to investigate the therapeutic efficacy of SAHM1 in a house dust mite (HDM)-driven asthma model. METHODS: Topical therapeutic intervention with SAHM1 or a control peptide was performed during sensitization, challenge, or both with HDM in mice. Airway inflammation was assessed by using multicolor flow cytometry, and bronchial hyperreactivity was studied. Additionally, SAHM1 therapy was investigated in mice with established allergic airway inflammation and in a model in which we neutralized IFN-γ during HDM challenge to support the TH2 response and exacerbate asthma. RESULTS: SAHM1 treatment during the challenge phase led to a marked reduction of eosinophil and T cell numbers in bronchoalveolar lavage fluid compared with those in diluent-treated or control peptide-treated mice. Likewise, T-cell cytokine content and bronchial hyperreactivity were reduced. SAHM1 treatment dampened TH2 inflammation during ongoing HDM challenge and enhanced recovery after established asthma. Additionally, in the presence of anti-IFN-γ antibodies, SAHM1 downregulated expression of the key TH2 transcription factor GATA3 and intracellular IL-4 in bronchoalveolar lavage fluid T cells, but expression of the TH17 transcription factor retinoic acid-related orphan receptor γt or intracellular IL-17 was not affected. SAHM1 therapy also reduced serum IgE levels. CONCLUSIONS: Therapeutic intervention of Notch signaling by SAHM1 inhibits allergic airway inflammation in mice and is therefore an interesting new topical treatment opportunity in asthmatic patients.

DPP4, the Middle East Respiratory Syndrome Coronavirus Receptor, is Upregulated in Lungs of Smokers and Chronic Obstructive Pulmonary Disease Patients.

Background: Middle East respiratory syndrome coronavirus (MERS-CoV) causes pneumonia with a relatively high case fatality rate in humans. Smokers and chronic obstructive pulmonary disease (COPD) patients have been reported to be more susceptible to MERS-CoV infection. Here, we determined the expression of MERS-CoV receptor, dipeptidyl peptidase IV (DPP4), in lung tissues of smokers without airflow limitation and COPD patients in comparison to nonsmoking individuals (never-smokers). Methods: DPP4 expression was measured in lung tissue of lung resection specimens of never-smokers, smokers without airflow limitation, COPD GOLD stage II patients and in lung explants of end-stage COPD patients. Both control subjects and COPD patients were well phenotyped and age-matched. The mRNA expression was determined using qRT-PCR and protein expression was quantified using immunohistochemistry. Results: In smokers and subjects with COPD, both DPP4 mRNA and protein expression were significantly higher compared to never-smokers. Additionally, we found that both DPP4 mRNA and protein expression were inversely correlated with lung function and diffusing capacity parameters. Conclusions: We provide evidence that DPP4 is upregulated in the lungs of smokers and COPD patients, which could partially explain why these individuals are more susceptible to MERS-CoV infection. These data also highlight a possible role of DPP4 in COPD pathogenesis.

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.

Notch signaling in T cells is essential for allergic airway inflammation, but expression of the Notch ligands Jagged 1 and Jagged 2 on dendritic cells is dispensable.

BACKGROUND: Allergic asthma is characterized by a TH2 response induced by dendritic cells (DCs) that present inhaled allergen. Although the mechanisms by which they instruct TH2 differentiation are still poorly understood, expression of the Notch ligand Jagged on DCs has been implicated in this process. OBJECTIVE: We sought to establish whether Notch signaling induced by DCs is critical for house dust mite (HDM)-driven allergic airway inflammation (AAI) in vivo. METHODS: The induction of Notch ligand expression on DC subsets by HDM was quantified by using quantitative real-time PCR. We used an HDM-driven asthma mouse model to compare the capacity of Jagged 1 and Jagged 2 single- and double-deficient DCs to induce AAI. In addition, we studied AAI in mice with a T cell-specific deletion of recombination signal-binding protein for immunoglobulin Jκ region (RBPJκ), a downstream effector of Notch signaling. RESULTS: HDM exposure promoted expression of Jagged 1, but not Jagged 2, on DCs. In agreement with published findings, in vitro-differentiated and HDM-pulsed Jagged 1 and Jagged 2 double-deficient DCs lacked the capacity to induce AAI. However, after in vivo intranasal sensitization and challenge with HDM, DC-specific Jagged 1 or Jagged 2 single- or double-deficient mice had eosinophilic airway inflammation and a TH2 cell activation phenotype that was not different from that in control littermates. In contrast, RBPJκ-deficient mice did not experience AAI and airway hyperreactivity. CONCLUSION: Our results show that the Notch signaling pathway in T cells is crucial for the induction of TH2-mediated AAI in an HDM-driven asthma model but that expression of Jagged 1 or Jagged 2 on DCs is not required.

T cells are necessary for ILC2 activation in house dust mite-induced allergic airway inflammation in mice.

Allergic asthma is a chronic inflammation of the airways mediated by an adaptive type 2 immune response. Upon allergen exposure, group 2 innate lymphoid cells (ILC2s) can be rapidly activated and represent an early innate source of IL-5 and IL-13. Here, we used a house dust mite (HDM)-driven asthma mouse model to study the induction of ILC2s in allergic airway inflammation. In BALF, lungs, and lymph nodes, ILC2 activation is critically dependent on prior sensitization with HDM. Importantly, T cells are required for ILC2 induction, whereby T-cell activation precedes ILC2 induction. During HDM-driven allergic airway inflammation the accumulation of ILC2s in BALF is IL-33 independent, although infiltrating ILC2s produce less cytokines in Il33(-/-) mice. Transfer of in vitro polarized OVA-specific OT-II Th2 cells alone or in combination with Th17 cells followed by OVA and HDM challenge is not sufficient to induce ILC2, despite significant eosinophilic inflammation and T-cell activation. In this asthma model, ILC2s are therefore not an early source of Th2 cytokines, but rather contribute to type 2 inflammation in which Th2 cells play a key role. Taken together, ILC2 induction in HDM-mediated allergic airway inflammation in mice critically depends on activation of T cells.

Airway inflammation in asthma: key players beyond the Th2 pathway.

PURPOSE OF REVIEW: The Th2 pathway starts with the binding of IL-4 to the IL-4 receptor followed by the phosphorylation of signal transducer and activator of transcription 6 and the activation of GATA3. The most important question relates to the sources of IL-4 and IL-4 related inflammation. Which cells other than Th2 cells are responsible for airway inflammation in asthma? RECENT FINDINGS: Accumulating data indicate that basophils contribute to endothelium-related IL-4-dependent inflammation. There is also a dendritic cell-related alternative for the induction of Th2 cells via Notch signalling. GATA3 deoxyribozyme improves asthma that is not clearly related to T-cells. The innate immune response in allergy is linked to mast cells, basophils, and the innate lymphoid cell type 2 (ILC2). ILC2s respond to IL-25, IL-33, thymic stromal lymphopoietin, and leukotrienes by producing IL-4, IL-5, and IL-13. In addition to all this inflammatory-cell-driven asthma, increasing evidence has emerged relating to smooth muscle cell activation, the endothelial and epithelial barrier functions, and improvements in the barrier function. The elevation of intracellular cyclic adenosine monophosphate because of the use of phosphodiesterase inhibitors adds to the prevention of epithelial-endothelial leakage, supports airway smooth muscle relaxation, and is immunosuppressive. CONCLUSION AND SUMMARY: IL-4 is the predominant Th2 cell cytokine. Many more cells, including eosinophils, basophils, mast cells, and ILC2, contribute to the production of IL-4 in the airways. Epithelial cells and endothelial cells lose barrier function in the context of allergic airway inflammation, and this could be at least partially remedied by increasing the intracellular cyclic adenosine monophosphate levels through phosphodiesterase inhibition.

Enforced expression of Gata3 in T cells and group 2 innate lymphoid cells increases susceptibility to allergic airway inflammation in mice.

Airway inflammation in allergic asthma reflects a threshold response of the innate immune system, including group 2 innate lymphoid cells (ILC2), followed by an adaptive Th2 cell-mediated response. Transcription factor Gata3 is essential for differentiation of both Th2 cells and ILC2. We investigated the effects of enforced Gata3 expression in T cells and ILC2 on the susceptibility of mice to allergic airway inflammation (AAI). We used CD2-Gata3 transgenic (Tg) mice with enforced Gata3 expression driven by the CD2 promoter, which is active both in T cells and during ILC2 development. CD2-Gata3 Tg mice and wild-type (WT) littermates were analyzed in mild models of AAI without adjuvants. Whereas OVA allergen exposure did not induce inflammation in WT controls, CD2-Gata3 Tg mice showed clear AAI and enhanced levels of IL-5 and IL-13 in bronchoalveolar lavage. Likewise, in house dust mite-driven asthma, CD2-Gata3 Tg mice were significantly more susceptible to AAI than WT littermates, whereby both ILC2 and Th2 cells were important cellular sources of IL-5 and IL-13 in bronchoalveolar lavage and lung tissue. Compared with WT littermates, CD2-Gata3 Tg mice contained increased numbers of ILC2, which expressed high levels of IL-33R and contributed significantly to early production of IL-4, IL-5, and IL-13. CD2-Gata3 Tg mice also had a unique population of IL-33-responsive non-B/non-T lymphoid cells expressing IFN-γ. Enforced Gata3 expression is therefore sufficient to enhance Th2 and ILC2 activity, and leads to increased susceptibility to AAI after mild exposure to inhaled harmless Ags that otherwise induce Ag tolerance.

Impaired survival of regulatory T cells in pulmonary sarcoidosis.

BACKGROUND: Impaired regulatory T cell (Treg) function is thought to contribute to ongoing inflammatory responses in sarcoidosis, but underlying mechanisms remain unclear. Moreover, it is not known if increased apoptotic susceptibility of Tregs may contribute to an impaired immunosuppressive function in sarcoidosis. Therefore, the aim of this study is to analyze proportions, phenotype, survival, and apoptotic susceptibility of Tregs in sarcoidosis. METHODS: Patients with pulmonary sarcoidosis (n = 58) were included at time of diagnosis. Tregs were analyzed in broncho-alveolar lavage fluid and peripheral blood of patients and healthy controls (HC). RESULTS: In sarcoidosis patients no evidence was found for a relative deficit of Tregs, neither locally nor systemically. Rather, increased proportions of circulating Tregs were observed, most prominently in patients developing chronic disease. Sarcoidosis circulating Tregs displayed adequate expression of FoxP3, CD25 and CTLA4. Remarkably, in sarcoidosis enhanced CD95 expression on circulating activated CD45RO(+) Tregs was observed compared with HC, and proportions of these cells were significantly increased. Specifically sarcoidosis Tregs--but not Th cells--showed impaired survival compared with HC. Finally, CD95L-mediated apoptosis was enhanced in sarcoidosis Tregs. CONCLUSION: In untreated patients with active pulmonary sarcoidosis, Tregs show impaired survival and enhanced apoptotic susceptibility towards CD95L. Increased apoptosis likely contributes to the insufficient immunosuppressive function of sarcoidosis Tregs. Further research into this field will help determine whether improvement of Treg survival holds a promising new therapeutic approach for chronic sarcoidosis patients.

Pulmonary innate lymphoid cells are major producers of IL-5 and IL-13 in murine models of allergic asthma.

Allergic asthma is characterized by chronic airway inflammation and hyperreactivity and is thought to be mediated by an adaptive T helper-2 (Th2) cell-type immune response. Here, we demonstrate that type 2 pulmonary innate lymphoid cells (ILC2s) significantly contribute to production of the key cytokines IL-5 and IL-13 in experimental asthma. In naive mice, lineage-marker negative ILC2s expressing IL-7Rα, CD25, Sca-1, and T1/ST2(IL-33R) were present in lungs and mediastinal lymph nodes (MedLNs), but not in broncho-alveolar lavage (BAL) fluid. Upon intranasal administration of IL-25 or IL-33, an asthma phenotype was induced, whereby ILC2s accumulated in lungs, MedLNs, and BAL fluid. After IL-25 and IL-33 administration, ILC2s constituted ∼50 and ∼80% of IL-5(+) /IL-13(+) cells in lung and BAL, respectively. Also in house dust mite-induced or ovalbumin-induced allergic asthma, the ILC2 population in lung and BAL fluid increased significantly in size and ILC2s were a major source of IL-5 or IL-13. Particularly in OVA-induced asthma, the contribution of ILC2s to the total population of intracellular IL-5(+) and IL-13(+) cells in the lung was in the same range as found for Th2 cells. We conclude that both ILC2s and Th2 cells produce large amounts of IL-5 and IL-13 that contribute to allergic airway inflammation.

Increased IL-17A expression in granulomas and in circulating memory T cells in sarcoidosis.

OBJECTIVE: Sarcoidosis is a systemic inflammatory disorder characterized by granulomas. Although the aetiology is unknown, sarcoidosis is thought to be mediated by Th1 lymphocytes. Recently, IL-17A has been implicated in granuloma formation in various diseases, including tuberculosis. Therefore, we hypothesized that Th17 cells play a role in sarcoidosis, paralleling recent findings in autoimmune diseases such as RA. The aim of our study was to investigate the role of Th17 cells in sarcoidosis. METHODS: T cells were investigated by intracellular flow cytometry and immunohistochemistry, in blood, bronchoalveolar lavages (BALs) and bronchial mucosal biopsies from a cohort of newly diagnosed sarcoidosis patients and healthy controls. RESULTS: Circulating memory CD4(+) T-cell populations of sarcoidosis patients contained significantly increased proportions of IL-17A(+) cells when compared with healthy controls. Interestingly, proportions of IL-17A/IFN-γ and IL-17A/IL-4 double-producing cells were significantly increased in blood of sarcoidosis patients and were present in substantial numbers in BAL. In granuloma-containing, but not in non-granulomatous sarcoidosis biopsies, we found significantly increased numbers of IL-17A(+) T cells, located in and around granulomas throughout the lamina propria. IL-22(+) T cells were increased in the subepithelial layer. CONCLUSIONS: Enhanced IL-17A expression in granulomas and the presence of IL-17A(+), IL-17A(+)IFN-γ(+) and IL-17A(+)IL-4(+)memory Th cells in the circulation and BAL indicate Th17 cell involvement in granuloma induction or maintenance in sarcoidosis. Therefore, neutralization of IL-17A activity may be a novel strategy to treat sarcoidosis.

Evidence for local dendritic cell activation in pulmonary sarcoidosis.

BACKGROUND: Sarcoidosis is a granulomatous disease characterized by a seemingly exaggerated immune response against a difficult to discern antigen. Dendritic cells (DCs) are pivotal antigen presenting cells thought to play an important role in the pathogenesis. Paradoxically, decreased DC immune reactivity was reported in blood samples from pulmonary sarcoidosis patients. However, functional data on lung DCs in sarcoidosis are lacking. We hypothesized that at the site of disease DCs are mature, immunocompetent and involved in granuloma formation. METHODS: We analyzed myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in broncho-alveolar lavage (BAL) and blood from newly diagnosed, untreated pulmonary sarcoidosis patients and healthy controls using 9-color flowcytometry. DCs, isolated from BAL using flowcytometric sorting (mDCs) or cultured from monocytes (mo-DCs), were functionally assessed in a mixed leukocyte reaction with naïve allogeneic CD4+ T cells. Using Immunohistochemistry, location and activation status of CD11c+DCs was assessed in mucosal airway biopsies. RESULTS: mDCs in BAL, but not in blood, from sarcoidosis patients were increased in number when compared with mDCs from healthy controls. mDCs purified from BAL of sarcoidosis patients induced T cell proliferation and differentiation and did not show diminished immune reactivity. Mo-DCs from patients induced increased TNFα release in co-cultures with naïve allogeneic CD4+ T cells. Finally, immunohistochemical analyses revealed increased numbers of mature CD86+ DCs in granuloma-containing airway mucosal biopsies from sarcoidosis patients. CONCLUSION: Taken together, these finding implicate increased local DC activation in granuloma formation or maintenance in pulmonary sarcoidosis.

Oral enoximone allows the reduction and discontinuation of inhaled steroids and beta2 agonists in asthmatic children.

In the last two decades, improvement on asthma treatment has been merely marginal for both adults and children; inhaled corticosteroids (ICS) combined with ß-2-mimetics remain the main therapy [3,4]. "New" therapies are just variations on ICS or, for children, on various other drugs that were allowed for adult asthma patients (clinicaltrials.gov). Although currently monoclonal antibodies have been introduced to the field, there is still a large therapeutic burden, given the mortality rate and widespread prevalence of uncontrolled asthma [2]. A simple and adequate way to reduce distress and costs would have great merit. PDE3 inhibitor enoximone was used earlier in successful treatment of life-threatening bronchial asthma (status asthmaticus) as well as in preoperative settings to prevent patients with severe asthma from suffering major surgery-related exacerbations; also, translational mice models showed the anti-inflammatory effects when PDE3 was targeted. Both outcomes suggested a beneficial effect of enoximone in severe chronic asthma. We hypothesized that enoximone might also be helpful in patients with severe chronic asthma; hence, we treated (and followed) > 70 patients (age 0-77, all volunteers) with personalized low doses of enoximone (orally), among them 11 minors, who are described here. Both children and adults reported improvement and/or alleviation of their asthma symptoms. All patients reported a better quality of life and greater drug compliance. The drug was well tolerated and showed no/negligible side effects. Notable bonus: asthma-related comorbidities (allergies, eczema, and rhinitis) were reported also to be less severe or even to disappear. The evaluation shows that PDE3 inhibitor enoximone is an adequate alternative for or addition to current asthma therapeutics, as add-on as well as stand-alone, considerably reducing the use of ß-2-mimetics/ICS, with no or negligible side effects. Additional studies are advisable.