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.
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•There is spontaneous accumulation of type 2 immune cells in the developing lung•Perinatal type 2 immunity depends on IL-33 and not on T or B cells•Postnatal lung DCs are very scarce yet very efficient at inducing Th2 cell adaptive immunity•The IL-33 axis is more important for asthma development at young age
It is not known why allergy often begins in early life. de Kleer et al. show that shortly after birth, type 2 innate immune cells accumulate in an IL-33-dependent manner in the developing lung. Interleukin-33 furthermore boosts the function of neonatal dendritic cells to promote long-lasting Th2-cell-mediated immunity.
Background
Short‐chain fatty acids (SCFAs) are fermented dietary components that regulate immune responses, promote colonic health, and suppress mast cell–mediated diseases. However, the effects of ...SCFAs on human mast cell function, including the underlying mechanisms, remain unclear. Here, we investigated the effects of the SCFAs (acetate, propionate, and butyrate) on mast cell–mediated pathology and human mast cell activation, including the molecular mechanisms involved.
Method
Precision‐cut lung slices (PCLS) of allergen‐exposed guinea pigs were used to assess the effects of butyrate on allergic airway contraction. Human and mouse mast cells were co‐cultured with SCFAs and assessed for degranulation after IgE‐ or non–IgE‐mediated stimulation. The underlying mechanisms involved were investigated using knockout mice, small molecule inhibitors/agonists, and genomics assays.
Results
Butyrate treatment inhibited allergen‐induced histamine release and airway contraction in guinea pig PCLS. Propionate and butyrate, but not acetate, inhibited IgE‐ and non–IgE‐mediated human or mouse mast cell degranulation in a concentration‐dependent manner. Notably, these effects were independent of the stimulation of SCFA receptors GPR41, GPR43, or PPAR, but instead were associated with inhibition of histone deacetylases. Transcriptome analyses revealed butyrate‐induced downregulation of the tyrosine kinases BTK, SYK, and LAT, critical transducers of FcεRI‐mediated signals that are essential for mast cell activation. Epigenome analyses indicated that butyrate redistributed global histone acetylation in human mast cells, including significantly decreased acetylation at the BTK, SYK, and LAT promoter regions.
Conclusion
Known health benefits of SCFAs in allergic disease can, at least in part, be explained by epigenetic suppression of human mast cell activation.
Short‐chain fatty acids suppress primary human mast cell activation via HDAC inhibition and not via PPAR or GPR signaling. Butyrate induces transcriptional changes of genes important for signaling and activation of human mast cells. Butyrate triggers a redistribution of global H3K27 acetylation levels, leading to decreased acetylation at the transcription start site of genes encoding key FcεRI‐mediated signaling molecules.
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.
Induction of group 2 innate lymphoid cells (ILC2s) in house dust mite (HDM)‐mediated allergic airway inflammation requires signals from activated T cells. Therefore, in this asthma model, ILC2s are not an early source of Th2 cytokines. Nevertheless, optimal cytokine production by ILC2s also depends on the presence of IL‐33.
Group 2 innate lymphoid cells (ILC2s; also called nuocytes, innate helper cells, or natural helper cells) provide protective immunity during helminth infection and play an important role in ...influenza-induced and allergic airway hyperreactivity. Whereas the transcription factor GATA binding protein 3 (Gata3) is important for the production of IL-5 and -13 by ILC2s in response to IL-33 or -25 stimulation, it is not known whether Gata3 is required for ILC2 development from hematopoietic stem cells. Here, we show that chimeric mice generated with Gata3 -deficient fetal liver hematopoietic stem cells fail to develop systemically dispersed ILC2s. In these chimeric mice, in vivo administration of IL-33 or -25 fails to expand ILC2 numbers or to induce characteristic ILC2-dependent IL-5 or -13 production. Moreover, cell-intrinsic Gata3 expression is required for ILC2 development in vitro and in vivo. Using mutant and transgenic mice in which Gata3 gene copy number is altered, we show that ILC2 generation from common lymphoid progenitors, as well as ILC2 homeostasis and cytokine production, is regulated by Gata3 expression levels in a dose-dependent fashion. Collectively, these results identify Gata3 as a critical early regulator of ILC2 development, thereby extending the paradigm of Gata3 -dependent control of type 2 immunity to include both innate and adaptive lymphocytes.
Bruton's tyrosine kinase (Btk) is a signaling molecule involved in development and activation of B cells through B-cell receptor (BCR) and Toll-like receptor (TLR) signaling. We have previously shown ...that transgenic mice that overexpress human Btk under the control of the CD19 promoter (CD19-hBtk) display spontaneous germinal center formation, increased cytokine production, anti-nuclear autoantibodies (ANAs), and systemic autoimsmune disease upon aging. As TLR and BCR signaling are both implicated in autoimmunity, we studied their impact on splenic B cells. Using phosphoflow cytometry, we observed that phosphorylation of ribosomal protein S6, a downstream Akt target, was increased in CD19-hBtk B cells following BCR stimulation or combined BCR/TLR stimulation, when compared with wild-type (WT) B cells. The CD19-hBtk transgene enhanced BCR-induced B cell survival and proliferation, but had an opposite effect following TLR9 or combined BCR/TLR9 stimulation. Although the expression of TLR9 was reduced in CD19-hBtk B cells compared to WT B cells, a synergistic effect of TLR9 and BCR stimulation on the induction of CD25 and CD80 was observed in CD19-hBtk B cells. In splenic follicular (Fol) and marginal zone (MZ) B cells from aging CD19-hBtk mice BCR signaling stimulated
IL-10 production in synergy with TLR4 and particularly TLR9 stimulation, but not with TLR3 and TLR7. The enhanced capacity of CD19-hBtk Fol B cells to produce the pro-inflammatory cytokines IFNγ and IL-6 compared with WT B cells was however not further increased following
BCR or TLR9 stimulation. Finally, we used crosses with mice deficient for the TLR-associated molecule myeloid differentiation primary response 88 (MyD88) to show that TLR signaling was crucial for spontaneous formation of germinal centers, increased IFNγ, and IL-6 production by B cells and anti-nuclear autoantibody induction in CD19-hBtk mice. Taken together, we conclude that high Btk expression does not only increase B cell survival following BCR stimulation, but also renders B cells more sensitive to TLR stimulation, resulting in increased expression of CD80, and IL-10 in activated B cells. Although BCR-TLR interplay is complex, our findings show that both signaling pathways are crucial for the development of pathology in a Btk-dependent model for systemic autoimmune disease.
Abstract
CD4
+
T helper 2 (Th2) cells and group 2 innate lymphoid cells are considered the main producers of type-2 cytokines that fuel chronic airway inflammation in allergic asthma. However, CD8
+
...cytotoxic T (Tc) cells - critical for anti-viral defense - can also produce type-2 cytokines (referred to as ‘Tc2’ cells). The role of Tc cells in asthma and virus-induced disease exacerbations remains poorly understood, including which micro-environmental signals and cell types promote Tc2 cell formation. Here we show increased circulating Tc2 cell abundance in severe asthma patients, reaching peak levels during exacerbations and likely emerging from canonical IFNγ
+
Tc cells through plasticity. Tc2 cell abundance is associated with increased disease burden, higher exacerbations rates and steroid insensitivity. Mouse models of asthma recapitulate the human disease by showing extensive type-2 skewing of lung Tc cells, which is controlled by conventional type-1 dendritic cells and IFNγ. Importantly, we demonstrate that the alarmin interleukin-33 (IL-33) critically promotes type-2 cytokine production by lung Tc cells in experimental allergic airway inflammation. Our data identify Tc cells as major producers of type-2 cytokines in severe asthma and during exacerbations that are remarkably sensitive to alterations in their inflammatory tissue micro-environment, with IL-33 emerging as an important regulator of Tc2 formation.
On antigen binding by the B-cell receptor (BCR), B cells up-regulate protein expression of the key downstream signaling molecule Bruton tyrosine kinase (Btk), but the effects of Btk up-regulation on ...B-cell function are unknown. Here, we show that transgenic mice overexpressing Btk specifically in B cells spontaneously formed germinal centers and manifested increased plasma cell numbers, leading to antinuclear autoantibody production and systemic lupus erythematosus (SLE)–like autoimmune pathology affecting kidneys, lungs, and salivary glands. Autoimmunity was fully dependent on Btk kinase activity, because Btk inhibitor treatment (PCI-32765) could normalize B-cell activation and differentiation, and because autoantibodies were absent in Btk transgenic mice overexpressing a kinase inactive Btk mutant. B cells overexpressing wild-type Btk were selectively hyperresponsive to BCR stimulation and showed enhanced Ca2+ influx, nuclear factor (NF)–κB activation, resistance to Fas-mediated apoptosis, and defective elimination of selfreactive B cells in vivo. These findings unravel a crucial role for Btk in setting the threshold for B-cell activation and counterselection of autoreactive B cells, making Btk an attractive therapeutic target in systemic autoimmune disease such as SLE. The finding of in vivo pathology associated with Btk overexpression may have important implications for the development of gene therapy strategies for X-linked agammaglobulinemia, the immunodeficiency associated with mutations in BTK.
Recently, human ILCs that express CD117 and CD127 but lack CRTH2 and NKp44 have been shown to contain precursors of ILC1, ILC2, and ILC3. However, these ILCs have not been extensively characterized. ...We performed an unbiased hierarchical stochastic neighbor embedding (HSNE) analysis of the phenotype of peripheral blood CD117
ILCs, which revealed the presence of three major subsets: the first expressed NKp46, the second expressed both NKp46 and CD56, and the third expressed KLRG1, but not NKp46 or CD56. Analysis of their cytokine production profiles and transcriptome revealed that NKp46
ILCs predominantly develop into ILC3s; some of them can differentiate into ILC1/NK-like cells, but they are unable to develop into ILC2s. In contrast, KLRG1
ILCs predominantly differentiate into ILC2s. Single-cell cultures demonstrate that KLRG1
ILCs can also differentiate into other ILC subsets depending on the signals they receive. Epigenetic profiling of KLRG1
ILCs is consistent with the broad differentiation potential of these cells.
BCR signaling, involving phosphorylation of various downstream molecules, including kinases, lipases, and linkers, is crucial for B cell selection, survival, proliferation, and differentiation. ...Phosphoflow cytometry (phosphoflow) is a single-cell-based technique to measure phosphorylated intracellular proteins, providing a more quantitative read-out than Western blotting. Recent advances in phosphoflow basically allow simultaneous analysis of protein phosphorylation in B cell (sub)populations, without prior cell sorting. However, fixation and permeabilization procedures required for phosphoflow often affect cell surface epitopes or mAb conjugates, precluding the evaluation of the phosphorylation status of signaling proteins across different B cell subpopulations present in a single sample. In this study, we report a versatile phosphoflow protocol allowing extensive staining of B cell subpopulations in human peripheral blood or various anatomical compartments in the mouse, starting from freshly isolated or frozen cell suspensions. Both human and mouse B cell subpopulations showed different basal and BCR stimulation-induced phosphorylation levels of downstream signaling proteins. For example, peritoneal B-1 cells and splenic marginal zone B cells exhibited significantly increased basal (ex vivo) signaling and increased responsiveness to in vitro BCR stimulation compared with peritoneal B-2 cells and splenic follicular B cells, respectively. In addition, whereas stimulation with anti-IgM or anti-Igκ L chain Abs resulted in strong pCD79a and pPLCγ2 signals, IgD stimulation only induced CD79a but not pPLCγ2 phosphorylation. In summary, the protocol is user friendly and quantifies BCR-mediated phosphorylation with high sensitivity at the single-cell level, in combination with extensive staining to identify individual B cell development and differentiation stages.
Background
Asthma exacerbations are frequently induced by respiratory tract infections (RTIs). Bacterial lysates have been described to possess immune‐modulatory effects and reduce RTIs as well as ...asthma symptoms in children. However, whether bacterial lysates have similar effects in adult asthma patients is unknown.
Aims
To reduce asthma exacerbations by add‐on bacterial lysate therapy in adults with severe asthma and to characterize the clinical and immune‐modulatory effects of this treatment.
Methods
Asthma patients (GINA 4) with ≥2 annual exacerbations in the previous year were included. The intervention regimen consisted of OM‐85/placebo for 10 consecutive days per month for 6 months during two winter seasons. Primary end‐point was the number of severe asthma exacerbations within 18 months. The study was approved by the national and local ethical review board and registered in the Dutch Trial Registry (NL5752). All participants provided written informed consent.
Results
Seventy‐five participants were included (38 OM‐85; 37 placebo). Exacerbation frequencies were not different between the groups after 18 months (incidence rate ratio 1.07, 95%CI 0.68–1.69, p = 0.77). With the use of OM‐85, FEV1% increased by 3.81% (p = 0.04) compared with placebo. Nasopharyngeal swabs taken during RTIs detected a virus less frequently in patients using OM‐85 compared to placebo (30.5% vs. 48.0%, p = 0.02).
In subjects with type 2 inflammation adherent to the protocol (22 OM‐85; 20 placebo), a non‐statistically significant decrease in exacerbations in the OM‐85 group was observed (IRR = 0.71, 95%CI 0.39–1.26, p = 0.25). Immune‐modulatory effects included an increase in several plasma cytokines in the OM‐85 group, especially IL‐10 and interferons. Peripheral blood T‐ and B cell subtyping, including regulatory T cells, did not show differences between the groups.
Conclusion
Although OM‐85 may have immune‐modulatory effects, it did not reduce asthma exacerbations in this heterogeneous severe adult asthma group. Post hoc analysis showed a potential clinical benefit in patients with type 2 inflammation.
Seventy‐five severe asthma patients were included (38 OM‐85; 37 placebo). While intention to treat (ITT) analysis revealed no difference in exacerbation frequencies between the groups after 18 months, per protocol analysis in patients with type 2 inflammation (PPT2) showed a non‐significant decrease. Nasopharyngeal swabs detected a virus less frequently in the OM‐85 group during respiratory tract infections. Immune‐modulatory effects were seen in several plasma cytokines. To conclude, OM‐85 might be an effective add‐on therapy in severe asthma patients with type 2 inflammation.
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