lgM-deficiency leads to reduced airway hyperresponsiveness and class switching to IgE in HDM-induced asthma.

a Schematic diagram showing sensitisation and challenge protocol where mice (IgM-/-) and wild type littermate control (WT) were sensitised with HDM 1 μg intra-tracheally on days 0 and challenged with HDM 3 μg on days 8-12. Analysis was done on day 15.

b Airway resistance and elastance were measured with increasing doses of acetyl methacholine (0 -40 mg/mL).

c Total lung eosinophil numbers (live+Siglec-F+CD11c-) and B cells (live+B220+CD19+MHCII+) were stained and analysed by Flow cytometry and enumerated from % of live cells.

d Muc5a gene expression in whole lung tissue.

e Total IgE and HDM-specific IgE in serum.

f IgG1 and IgM surface expression in mediastinal lymph node B cells of WT, IgM-/- and μMT-/- mice.

g Marginal Zone (live+B220+CD19+MHCII+CD21/CD35+CD23-), follicular (live+B220+CD19+MHCII+CD23+CD21/CD35+) and Germinal Centre (live+B220+CD19+MHCII+GL7+FAS+) B cells in the mediastinal lymph node of WT and IgM-/- mice challenged with HDM.

Shown is mean ± SEM from two pooled experiments (n=7 - 10). Significant differences between groups were performed by Student t-test (Mann-Whitney) (c, d, e) or by Two-Way ANOVA with Benforroni post-test (b) and are described as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

lgM-deficiency does not lead to reduced Th2 allergic airway inflammation and serum transfer restores IgE, but not AHR.

Fig. a-c, mice treated as in Fig. 1a.

a Total mediastinal lymph node CD4 T cell numbers (live+CD3+CD4+) and % of Follicular Helper T cells (live+CD3+CD4+PD-1+CXCR5+) of CD4 T cells were stained and analysed by Flow cytometry and enumerated from % of live cells.

b Mediastinal lymph nodes were stimulated with anti-CD3 (10μg/mL) for 5 days and supernatants were used to measure levels of IL-4 and IL-13. Cytokines were not detected in unstimulated or HDM (30μg) stimulated mLN.

c Representative FACS plots and frequencies of lung CD4 T cells (live+CD3+CD4+) producing IL-4 and IL-13 after 5 hr stimulation with PMA/ionomycin in the presence of monensin.

d Schematic diagram showing serum transfer from WT to IgM-/- which were then sensitised as shown in Fig 1,a.

e Airway resistance was measured with increasing doses of acetyl methacholine (0 - 40 mg/mL).

f Total IgE in serum of mice either transferred with WT serum, IgM-/- serum or no serum.

Shown is the mean ± SEM from two pooled experiments (n=5 - 8). Significant differences between groups were performed by Student t-test (Mann-Whitney) (C, D, E) or by Two-Way ANOVA with Benforroni post-test (B) and are described as: *p<0.05, **p<0.01, ***p<0.001, ****p< 0.0001.

Genes associated with muscle contraction are downregulated in IgM-deficient mice.

WT and IgM-/- mice were treated as in Figure 1A and RNA was collected from the whole lung for RNA sequencing.

a Principal-component (PC) analysis showing variation in the global gene expression profiles across the different groups. PC1 (60%) and PC2 (18%), which capture the greatest variation in gene expression, are shown. Orange colour represents WT HDM, green colour represents WT PBS, Blue colour represents IgM-/- HDM and purple crosses represent IgM-/- PBS. Each dot represents an individual mouse.

b Number of differentially expressed genes between groups.

c Heatmaps depicting the differently expressed genes between WT and IgM-/- samples from HDM-treated and PBS mice ranked based on hierarchical clustering.

d-e Volcano plots: numbers and colour relate to genes that have an adjusted p value <0.05. Blue, significantly downregulated; red, significantly up regulated; grey, non-differentially expressed. P values were adjusted for multiple testing using the Benjamini-Hochberg method. (D) represent changes between WT and IgM-/- treated with HDM and (E) represents changes between WT and IgM-/- treated with saline.

f Gene set enrichment analysis (GSEA) of hallmark gene sets from the Molecular Signatures Database of the Broad Institute, showing the normalized enrichment scores (NES) for lung RNA-Seq data from WT mice.

BAIAP2L1 is expressed in close contact to smooth muscle.

a Mouse lungs were homogenised in RIPA buffer and blotted on nitrocellulose. Rabbit anti-human BAIAP2L1 and mouse anti-GAPDH was used as primary antibody. Lines 1-3 is WT mice, lines 4-6 is IgM-/- mice sensitised and challenged with HDM.

b Lung sections from WT and IgM-/- mice sensitised and challenged with HDM were immunostained for nuclei stained DAPI (Blue), anti-BAIAP2L1 (red), α smooth muscle actin (green) and merged images (Magenta). Insert below shows zoomed in image of merged BAIAP2L1 and α smooth muscle actin.

c Representative flow cytometry plots showing BAIAP2L1 expression (Live+Singlets+CD45-α SMA+BAIAP2L1+) in WT and IgM-/- treated with HDM or PBS. Quantification of % BAIAP2L1 in α smooth muscle actin is shown.

Shown are representative images from 2 independent experiments (n= 3 mice per group).

CRISPR-based deletion of BAIAP2L1 leads to reduced smooth muscle contraction at a single-cell level.

a, Bronchial smooth muscle cells (1.6×105 cells/well) were transfected with CRISPR-Cas9 single guide RNAs (scramble, BAIAP2L1 and ERDR1), stimulated with recombinant human IL-13 (100 ng/mL) and TNF-α (10 ng/mL) for 48 h. Cells were then transferred to elastomeric micropatterns, stimulated again with rIL-13 and rTNF-α and fixed before imaging on a StellarVision microscope.

b, Representative images of single BSMCs on micropatterns from scramble, BAIAP2L1 and ERDR1 stimulated with 10 ng/mL TNF-α. DNA was stained with DAPI, actin fibers with Phaloidin-565 and elastomeric micropatterns are coated in Fibronectin-488. Merged images are shown on the right.

c, Violin plots showing contraction of 50-100 cells/condition stimulated with 10 ng/mL TNF-α, individual dots represent a single cell contraction, where blue is scramble sgRNA, red is BAIAP2L1 sgRNA and green is ERDR1 is sgRNA.

d, Violin plots showing contraction of 50-100 cells/condition stimulated with 100 ng/mL IL-13, individual dots represent a single cell contraction, where blue is scramble sgRNA, red is BAIAP2L1 sgRNA and green is ERDR1 is sgRNA.

Shown is mean ±SEMs from two pooled experiment (n=50 - 100). Significant differences between groups were performed by student t-test (Mann-Whitney) and p value is shown.

CRISPR-based deletion of BAIAP2L1 reduces smooth muscle contraction upon stimulation with acetylcholine.

a Bronchial smooth muscle cells (1.6×105 cells/well) were transfected with CRISPR-Cas9 single guide RNAs (scramble, BAIAP2L1 and ERDR1), stimulated with Acetylcholine (10µM) for 48 h. Cells were then transferred to elastomeric micropatterns, stimulated again with ACh (10µM) and fixed before imaging on a StellarVision microscope.

b, Representative images of single BSMCs on a single micropattern from unstimulated, scramble, BAIAP2L1 and ERDR1 stimulated with ACh (10µM). Shown are DAPI, actin (red), a green fluorescent micropattern and merged images.

c, Violin plots showing contraction of 50-100 cells/condition stimulated with ACh (10µM) individual dots represent a single cell contraction, where blue is scramble sgRNA, red is BAIAP2L1 sgRNA and green is ERDR1 is sgRNA.

Shown is mean ±SD from 1 representative experiment of 2 independent experiments (n=50 - 100). Significant differences between groups were performed by student t-test (Mann-Whitney) and p value is shown.

Working model showing how Baiap2l1 could influence muscle contraction through influencing myosin and actin filament interaction. Image adapted from Biorender.

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