Melanie Lukkes

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.

Enhanced Bruton's Tyrosine Kinase Activity in Peripheral Blood B Lymphocytes From Patients With Autoimmune Disease

Brutons tyrosine kinase (BTK) transmits crucial survival signals from the B cell receptor (BCR) in B cells. Pharmacologic BTK inhibition effectively diminishes disease symptoms in mouse models of autoimmunity; conversely, transgenic BTK overexpression induces systemic autoimmunity in mice. We undertook this study to investigate BTK expression and activity in human B cells in the context of autoimmune disease.

Enhanced Bruton's tyrosine kinase activity in peripheral blood B lymphocytes of autoimmune disease patients

Brutons tyrosine kinase (BTK) transmits crucial survival signals from the B cell receptor (BCR) in B cells. Pharmacologic BTK inhibition effectively diminishes disease symptoms in mouse models of autoimmunity; conversely, transgenic BTK overexpression induces systemic autoimmunity in mice. We undertook this study to investigate BTK expression and activity in human B cells in the context of autoimmune disease.

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 J&kgr; region (RBPJ&kgr;), 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, RBPJ&kgr;‐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. Graphical abstract Figure. No Caption available.

Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation

Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL) fluid, lung, draining lymph nodes, and spleen. Whereas in vivo IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25+CD127+T1/ST2+ICOS+KLRG1+ phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25lowCD127+T1/ST2low ICOSlowKLRG1low, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25low ILC2s in BAL fluid and lung rapidly reverted to CD25high ILC2s upon in vivo stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than previously thought, whereby their surface marker and gene expression profile are highly dynamic.

Increased surface expression of NOTCH on memory T cells in peripheral blood from asthma patients

Using flow cytometry we found that circulating T cell subsets from asthma patients contained higher proportions of NOTCH receptor positive cells compared with healthy controls, suggesting enhanced NOTCH signaling in T cells from asthma patients.

A pathophysiological role of PDE3 in allergic airway inflammation

Phosphodiesterase 3 (PDE3) and PDE4 regulate levels of cyclic AMP, which are critical in various cell types involved in allergic airway inflammation. Although PDE4 inhibition attenuates allergic airway inflammation, reported side effects preclude its application as an antiasthma drug in humans. Case reports showed that enoximone, which is a smooth muscle relaxant that inhibits PDE3, is beneficial and lifesaving in status asthmaticus and is well tolerated. However, clinical observations also showed antiinflammatory effects of PDE3 inhibition. In this study, we investigated the role of PDE3 in a house dust mite-driven (HDM-driven) allergic airway inflammation (AAI) model that is characterized by T helper 2 cell activation, eosinophilia, and reduced mucosal barrier function. Compared with wild-type (WT) littermates, mice with a targeted deletion of the PDE3A or PDE3B gene showed significantly reduced HDM-driven AAI. Therapeutic intervention in WT mice showed that all hallmarks of HDM-driven AAI were abrogated by the PDE3 inhibitors enoximone and milrinone. Importantly, we found that enoximone also reduced the upregulation of the CD11b integrin on mouse and human eosinophils in vitro, which is crucial for their recruitment during allergic inflammation. This study provides evidence for a hitherto unknown antiinflammatory role of PDE3 inhibition in allergic airway inflammation and offers a potentially novel treatment approach.

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/ST2highICOShighKLRG1highCD25high 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 &agr;‐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‐&ggr; 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‐&ggr; 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 &ggr;t 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.The Notch signaling pathway has been implicated in the pathogenesis of allergic airway inflammation. Targeting the active Notch transactivation complex by the cell-permeable, hydrocarbon-stapled synthetic peptide SAHM1 was results in genome-wide suppression of Notch-activated genes in leukemic cells and other models. However, efficacy of SAHM1 in allergic asthma models has remained unexplored. We aimed to investigate therapeutic efficacy of SAHM1 in a house dust mite (HDM)-driven asthma model. Topical therapeutic intervention with SAHM1 or a control peptide was performed during sensitization and/or challenge with HDM in mice. Airway inflammation was assessed by multi-color flow cytometry and bronchial hyperreactivity (BHR) was studied. Additionally, SAHM1 therapy was investigated in established asthma and in a model in which we neutralized IFNγ during HDM challenge to support the Th2 response and exacerbate asthma. SAHM1 treatment during the challenge phase led to a marked reduction of eosinophils and T-cells in bronchoalveolar lavage (BAL), compared with diluent-treated or control peptide-treated mice. Likewise, T-cell cytokine content and BHR were reduced. SAHM1 treatment dampened Th2-inflammation during ongoing HDM challenge and enhanced recovery following established asthma. Additionally, in the presence of anti-IFNγ antibodies, SAHM1 downregulated expression of the key Th2 transcription factor (TF) GATA3 and intracellular IL-4 in BAL T-cells, but expression of the Th17 TF RORγt or intracellular IL-17 was not affected. SAHM1 therapy also reduced serum IgE levels. Therapeutic intervention of Notch signaling by SAHM1 inhibits allergic airway inflammation in mice and is therefore an interesting new topical treatment opportunity in asthma