Increased B Cell ADAM10 in Allergic Patients and Th2 Prone Mice

Abstract

ADAM10, as the sheddase of the low affinity IgE receptor (CD23), promotes IgE production and thus is a unique target for attenuating allergic disease. Herein, we describe that B cell levels of ADAM10, specifically, are increased in allergic patients and Th2 prone WT mouse strains (Balb/c and A/J). While T cell help augments ADAM10 expression, Balb WT B cells exhibit increased ADAM10 in the naïve state and even more dramatically increased ADAM10 after anti-CD40/IL4 stimulation compared C57 (Th1 prone) WT B cells. Furthermore, ADAM17 and TNF are reduced in allergic patients and Th2 prone mouse strains (Balb/c and A/J) compared to Th1 prone controls. To further understand this regulation, ADAM17 and TNF were studied in C57Bl/6 and Balb/c mice deficient in ADAM10. C57-ADAM10B-/- were more adept at increasing ADAM17 levels and thus TNF cleavage resulting in excess follicular TNF levels and abnormal secondary lymphoid tissue architecture not noted in Balb-ADAM10B-/-. Moreover, the level of B cell ADAM10 as well as Th context is critical for determining IgE production potential. Using a murine house dust mite airway hypersensitivity model, we describe that high B cell ADAM10 level in a Th2 context (Balb/c WT) is optimal for disease induction including bronchoconstriction, goblet cell metaplasia, mucus, inflammatory cellular infiltration, and IgE production. Balb/c mice deficient in B cell ADAM10 have attenuated lung and airway symptoms compared to Balb WT and are actually most similar to C57 WT (Th1 prone). C57-ADAM10B-/- have even further reduced symptomology. Taken together, it is critical to consider both innate B cell levels of ADAM10 and ADAM17 as well as Th context when determining host susceptibility to allergic disease. High B cell ADAM10 and low ADAM17 levels would help diagnostically in predicting Th2 disease susceptibility; and, we provide support for the use ADAM10 inhibitors in treating Th2 disease.

Fig 1. Allergic patient B cells exhibit increased ADAM10 and sCD23 but decreased ADAM17 and TNF.

(A) Total ADAM10 in naïve CD19⁺ B cells from control (thin line, open dot) and allergic (bold line, black dot) patients; dot plot (right) shows percent of total B cells staining high in ADAM10 (grey gate). Isotype control is shaded grey in histogram. (B,C) Total ADAM10 on naïve T cells (B) and monocytes (C). (D) sCD23 from control (open dot) or allergic (black dot) supernatants. (E) Naïve B cells from 4 allergic and 4 control patients analyzed for ADAM10, ADAM17, and TNF message normalized to GAPDH. Significance (*) indicates ≥ 2 fold change between allergic and control B cells for respective gene. (F) HDM specific IgE levels in sera of control (open dot) or allergic (black dot) patients determined by ImmunoCAP. <0.35kuA/l considered a negative result. *p<0.05, **p<0.005, ***p<0.0005.

Fig 2. Increased B-ADAM10 and decreased ADAM17 and TNF in Th2 biased strains.

Naïve B220⁺ B cells (A, B) stained for total ADAM10 (A) or ADAM17 (B) expression by flow cytometry. (C) TNF coexpression on 3 day stimulated (anti-CD40+IL4) B220⁺ B cells by TSA. (A,B,C) Bar graph represents percent total B cells staining high in ADAM10 (A), ADAM17 (B), or TNF (C). (A,B,C) Balb/c (──)A/J (——), C57Bl/6 (▬▬), SJL (▪▪▪▪▪), respective isotype control (shaded grey) in representative overlay (left); grey gate demonstrates high stained population. n = 8 per group, 3 independent studies. *p<0.05, **p<0.005, ***p<0.0005. (D) TNF message from 3 day stimulated (anti-CD40 + IL4) B220⁺ B cells. Presented as fold change from C57Bl/6 WT. * signifies ≥2 fold change between C57Bl/6 WT and the listed WT strain.

Fig 3. B cell TNF and ADAM17 regulation in C57Bl/6 and Balb/c ADAM10^B-/- (A10K0) and WT.

(A) sTNF or (B) mTNF from 3 day stimulated (LPS + IL4) B cell cultures; (B) Balb/c WT (black), Balb/c A10KO (red), C57Bl/6 WT (green), and C57Bl/6 A10KO (blue) B cells. (C) Fold change (A10KO over WT) in relative TNF message from naïve (white) or 3 day stimulated (anti-CD40+IL4) (black) B cells normalized to 18s. (D) Fold change (A10KO over WT) in relative ADAM17 expression normalized to 18s for naïve (white) or 3 day stimulated (black) B cells. (C,D) * signifies ≥2 fold change between groups. (E) ADAM17 (~93kDa) and actin (~42kDa) from 5 day stimulated (anti-CD40 + IL4) B cells (left) and band densitometry (right). KO = A10KO. n = 9 per group, 3 independent studies. *p<0.05, **p<0.005.

Fig 4. Balb-ADAM10^B-/- LN exhibit WT architecture unlike C57-ADAM10^B-/- nodes.

Naïve LN sections from C57Bl/6 and Balb/c WT and ADAM10^B-/-; (A) B cell (blue, B220), T cell (red, Thy1.2), and HEV (green, pNAD); (B) FDC reticula (red,CR1/2), collagen (green), and cortico-medullary junction (dotted line in inset box); (C) TNF staining (green), B cell (blue, B220) follicle. (D) Average TNF staining intensity representing 12 follicle sections per group. Scale bar, 50μm. **p<0.005.

Fig 5. B-ADAM10 deletion attenuates bronchoconstriction and HDM specific IgE.

(A) Airway resistance (cmH20.s/mL) with increasing doses of methacholine (left); Balb-WT (──,○), Balb-ADAM10^B-/-(——,●), C57-WT (──,□), C57-ADAM10^B-/- (——,○), Saline (──,●) presented as fold increase from saline control. Bar graph (right) represents 25 mg/mL methacholine dose. (B) Percent macrophages (left) and eosinophils (right) from total BALF determined by flow cytometry; Saline control (white), Balb WT (checkered), Balb-ADAM10^B-/- (dotted), C57 WT (slash), and C57-ADAM10^B-/- (black). (C) HDM specific IgE production. n = 7–9 per group, 3 independent experiments. All mice immunized with saline demonstrated comparable results. For simplicity, saline represents Balb WT mice given saline. *p<0.05, **p<0.005, ***p<0.005.

Fig 6. B-ADAM10 deletion reduces cellular infiltration, goblet cell metaplasia, and mucus production in a strain dependent manner.

(A) H&E stain for lung tissue demonstrates peribronchiolar and perivascular inflammatory cellular infiltration (red box); 10x magnification, scale bar 200 μm. (B) Goblet cell metaplasia determined by PAS stain; note dark pink mucin producing cells (black arrow) and alveolar epithelium thickness; 20x magnification, scale bar 100 μm. (C) Lung pathology score representing quantitation of peribronchiolar and perivascular inflammatory cellular infiltration on H&E stained lung sections; 2 sections per mouse and at least 4 mice per group were assessed. (D) MUC5AC protein. n = 7–9 per group, 3 independent experiments. All mice immunized with saline demonstrated comparable results. For simplicity, saline represents Balb WT mice given saline. *p<0.05, **p<0.005.