The secretome of irradiated peripheral blood mononuclear cells attenuates activation of mast cells and basophils

Abstract

Background: IgE-mediated hypersensitivity is becoming increasingly prevalent and activation of mast cells and basophils represent key events in the pathophysiology of allergy. We have previously reported that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCsec) exerts beneficial anti-inflammatory effects. Yet, its ability to alleviate allergic symptoms has not been investigated so far.

Methods: Several experimental in vitro and in vivo models have been used in this basic research study. A murine ear swelling model was used to study the effects of PBMCsec on 48/80-induced mast cell degranulation in vivo. The transcriptional profile of murine mast cells was analysed by single cell RNA sequencing (scRNAseq). Mast cell activation was studied in vitro using primary skin mast cells. Basophils from individuals allergic to birch pollens were used to investigate basophile activation by allergens. Transcriptomic and lipidomic analyses were used to identify mRNA expression and lipid species present in PBMCsec, respectively.

Findings: Topical application of PBMCsec on mouse ears (C57BL/6) significantly reduced tissue swelling following intradermal injection of compound 48/80, an inducer of mast cell degranulation. Single cell RNA sequencing of PBMCsec-treated murine dermal mast cells (Balb/c) revealed a downregulation of genes involved in immune cell degranulation and Fc-receptor signalling. In addition, treatment of primary human dermal mast cells with PBMCsec strongly inhibited compound 48/80- and α-IgE-induced mediator release in vitro. Furthermore, PBMCsec remarkably attenuated allergen driven activation of basophils from allergic individuals. Transcriptomic analysis of these basophils showed that PBMCsec downregulated a distinct gene battery involved in immune cell degranulation and Fc-receptor signalling, corroborating results obtained from dermal mast cells. Finally, we identified the lipid fraction of PBMCsec as the major active ingredient involved in effector cell inhibition.

Interpretation: Collectively, our data demonstrate that PBMCsec is able to reduce activation of mast cells and basophils, encouraging further studies on the potential use of PBMCsec for treating allergy.

Funding: Austrian Research Promotion Agency (852748 and 862068, 2015-2019), Vienna Business Agency (2343727, 2018-2020), Aposcience AG, Austrian Federal Ministry of Education, Science and Research (SPA06/055), Danube Allergy Research Cluster, Austrian Science Fund (I4437 and P32953).

Keywords: Anti-allergic therapeutic secretome; Basophil activation; Birch pollen allergy; Mast cell degranulation.

Figure 1

PBMCsec prevents mast cells activation in vivo. (a) Experimental protocol of topical PBMCsec administration and induction of in vivo mast cell degranulation. (b) Ear thickness after intradermal injection of compound 48/80 with topical PBMCsec or medium application. Data show arithmetic means and standard errors of the mean of n=9 mice per treatment condition and n=6 mice for untreated controls. [Treatment groups were compared by two-way, repeated measurements ANOVA and Sidak's multiple comparisons test]. Numbers indicate p values of PBMCsec versus medium.

Figure 2

PBMCsec downregulated Fc receptor signalling and mast cell degranulation in dermal mast cells. (a) Experimental approach of subcutaneous PBMCsec application in vivo and scRNAseq. (b) Uniform manifold approximation and projection (UMAP) plot of PBMCsec- and medium-treated skin. Each dot represents one cell. Colour code indicates identified cell clusters. Dashed circle indicates mast cell population. EC endothelial cells, FB fibroblasts, KC1 keratinocyte cluster 1, KC2 keratinocyte cluster 2, KC3 keratinocyte cluster 3, LC Langerhans cells, MC mast cells, mel melanocytes, mono monocytes, MP macrophages, PC pericytes, TC T cells. (c) Feature plots of biomarker genes used to identify epidermal and dermal cell types. Colour intensities indicate average gene expression levels. Pan keratin feature plot shows blended expressions of keratin 5 (Krt5) and Krt10. Cd3d Cluster of Differentiation 3d, Cd207 Cluster of Differentiation 207, Col1a1 Collagen Type I Alpha 1 Chain, Lyz2 lysozyme, Pecam1 Platelet And Endothelial Cell Adhesion Molecule 1, Pmel Premelanosome Protein, Rgs5 Regulator Of G Protein Signaling 5, Tpsab1 Tryptase alpha/beta 1. (d) Expression of mast cell-specific genes across all cell clusters. Tpsab1 Tryptase alpha/beta 1, Tpsb2 Tryptase beta 2, Cma1 Chymase 1, Fcer1a Fc receptor IgE high affinity I alpha polypeptide, Kit KIT proto-oncogene receptor tyrosine kinase. (e) Bar diagram showing up- (red) and downregulated genes (green) by PBMCsec compared to medium in dermal mast cells. DEGs were calculated for the mast cell cluster comparing PBMCsec-treated skin versus medium. Genes with average logarithmic fold change of >1.5 or <0.66 were included. (f) GO terms associated with PBMCsec-downregulated genes in mast cells. Each circle represents one GO term. Colour codes indicate biological processes with high amounts of shared genes. (g) Expression of mast cell-specific genes. Colours indicate average gene expressions in medium- and PBMCsec-treated mast cells, respectively. Il13 interleukin 13, Il4ra interleukin 4 receptor alpha, Il4 interleukin 4, Stxbp2 syntaxin binding protein 2, Gab2 growth factor receptor bound protein 2-associated protein 2, Fcer1g Fc receptor IgE high affinity I gamma polypeptide, Ms4a2 membrane-spanning 4-domains subfamily A member 2, Fcer1a Fc receptor, IgE, high affinity I, alpha polypeptide, Syk spleen tyrosine kinase, Lyn LYN proto-oncogene Src family tyrosine kinase.

Figure 3

PBMCsec prevents compound 48/80- and IgE/α-IgE-induced mediator release by human dermal mast cells. (a) Human dermal mast cells, indicated by tryptase-positive staining, were isolated from trunk skin and (b) enriched by CD117 cell sorting. Scale bar in (a) 100 µm. Scale bars in (b) 50 µm. Mast cell degranulation was assessed by β-hexaminidase release upon (c) compound 48/80 and (d) IgE/α-IgE stimulation of primary human dermal mast cells. Controls refer to medium routinely used to culture mast cells (DMEM alone), while medium represents the medium used for PBMC culture and secretome generation. Dashed horizontal lines in (c) and (d) indicate levels of mediator release of non-stimulated mast cells. Data show averages of n=4 donors from independent experiments. [P values were computed using one-way ANOVA and Dunnett's multiple comparisons tests].

Figure 4

PBMCsec prevents allergen-mediated basophil activation. Representative histograms of (a) non-stimulated, (c) Bet v 1-, and (e) α-IgE-induced basophil activation after gating on lymphocytes and monocytes in FSC/SSC and CD123⁺, CCR3⁺ cells. Statistical analyses of (b) non-stimulated, (d) Bet v 1-, and (f) α-IgE-induced activation of PBMCsec- or medium-treated human basophils. Controls refer to cells treated with 0.9 % NaCl. Each symbol identifies one individual donor. Horizontal lines indicate arithmetic means and standard deviations, respectively. [Data were compared by ordinary one-way ANOVA and Dunnett's multiple comparisons post hoc analysis].

Figure 5

PBMCsec downregulates mast cell degranulation-associated genes in individuals allergic to birch pollens. (a) Volcano plot of down- (green) and up- (red) regulated genes by PBMCsec compared to medium. Each dot represents one gene. Cut-offs of average <0.5 and >2-fold change were set. Bar diagram indicates absolute numbers of differentially expressed genes by PBMCsec compared to medium. Gene ontologies associated with (b) up- and (c) down-regulated genes by PBMCsec compared to medium. Each circle represents one gene ontology. Colours indicate p-values. (d) Downregulated genes associated with ontologies. Colour code indicates gene expression levels of medium- and PBMCsec-treated human basophils.

Figure 6

Lipids prevent mast cell degranulation and basophil activation. Mast cells were pre-treated with medium, PBMCsec, or PBMCsec-derived lipids and mediator release was assessed following (a) compound 48/80 and (b) IgE/α-IgE stimulation. Bars show mean ± s.e.m. of n=4 donors for medium and PBMCsec and n=2 for lipids. Data were compared by ANOVA and Sidak's multiple comparisons test. Basophil activation test after pre-incubation with PBMCsec-derived lipids upon (c) α-IgE and (d) Bet v 1 (1 ng/mL) stimulation. Each dot represents one donor. [Data were compared by ANOVA and Dunnett's multiple comparisons test]. (e) PS and (f) LPC lipid species in PBMCsec and medium quantified by lipidomics. LPC lysophosphatidylcholine, PS phosphatidylserine. (g) Gene expression of non-receptor protein tyrosine and lipid phosphatases in basophils obtained from individuals allergic to birch pollen. Data of n=3 individual donors were compared by empirical Bayes method and are presented as mean±s.e.m. INPPL1 inositol polyphosphate phosphatase-like 1, INPP5D inositol polyphosphate-5-phosphatase D, PTPN protein tyrosine phosphatase non-receptor type, PTP4A1 protein tyrosine phosphatase type IVA member 1, PTP4A2 protein tyrosine phosphatase type IVA member 2, PTPRA protein tyrosine phosphatase receptor type A.