The role of ADAM10, ADAM17, and Spag6 in humoral immunity and secondary lymphoid tissue architecture

ADAM10, ADAM17, and SPAG6 contribute significantly to humoral immunity and secondary lymphoid tissue architecture. ADAM10 and ADAM17 are two closely related zinc-metalloproteinases. Through cleavage of their ligands CD23 and TNF, respectively, they greatly influence IgE production and secondary lymphoid tissue architecture maintenance. Th1 prone WT strains initially exhibit increased ADAM17 and TNF yet reduced ADAM10 relative to Th2 prone WT strains. In the absence of B cell ADAM10, a compensatory increase in ADAM17 and TNF cleavage is noted only in Th1 prone C57Bl/6, not Th2 prone Balb/c. B cell TNF homeostasis is important for maintaining secondary lymphoid tissue architecture. We show for the first time that excessive B cell TNF production in C57-ADAM10B-/- lymph nodes contributes to loss of B/T segregation, increased HEV number and size, fibrosis, loss of FDC networks, and impaired germinal center formation. Furthermore, B cell ADAM10, which enhances IgE production through CD23 cleavage, is shown to be a marker of Th2 susceptibility. B cell ADAM10 is elevated in Th2 prone mouse strains and allergic patients compared to Th1 prone controls and as B cell ADAM10 level increases, so does IgE production. Lastly, the B cell profile of allergic patients is determined to be B cell ADAM10highADAM17lowTNFlow. Furthermore, the mechanism underlying reduced class-switched antibody production in C57-ADAM10B-/- mice is explored. C57-ADAM10B-/- B cells exhibit a B10, or IL-10 producing, phenotype, which is linked to reduced antibody production. Furthermore, increased Tregs noted in C57-ADAM10B-/- mice contributed to reduced class switched IgE production and disease parameters following a house dust mite airway inflammation challenge. SPAG6, a component of the central apparatus of the “9+2” axoneme, plays a central role in flagellar stability and motility. Immune cells lack cilia, but the immunological synapse is a surrogate cilium as it utilizes the same machinery as ciliogenesis including the nucleation of microtubules at the centrosome. We demonstrate that Spag6 localizes in the centrosome and is critical for centrosome polarization at and actin clearance away from the synapse between CTL and target cells. Furthermore, improper synapse formation and function likely explains reduced CTL function and class-switched antibody production in Spag6KO mice

Increased B Cell ADAM10 in Allergic Patients and Th2 Prone Mice

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

Revisiting One-Stage Urethroplasties for Distal Urethral Strictures

Background: Reconstructive approaches for distal urethral strictures range from simple meatotomy to utilizing grafts or flaps depending on the etiology, length and location. We describe a contemporary cohort of distal urethral strictures and report a surgical technique termed distal one-stage urethroplasty developed to address the majority of distal urethral strictures encountered. Methods: Thirty-four patients were included. The mean age was 56.7 years (range 15.7–84.9 years), the mean stricture length was 1.1 cm (0.5–1.5) and the mean follow-up was 42.5 months (28–61.3). Results: The vast majority of distal strictures (27/34 (79.4%)) were treated with our hybrid one-stage approach combining a distal urethral reconstruction with excision of the scar tissue without the need to use grafts or flaps. The average stricture length was 0.68 cm and average operative time was 24.43 min. Post-operative spraying was reported in a minority of patients (4/27 (14.8%)). The length of stricture and surgery were significantly longer in those 7/34 (20.6%) patients in whom grafts or flaps were used (2.88 cm and 154.8 min, respectively, p < 0.001 for both when compared to the hybrid one-stage approach). We noted 6/34 (17.6%) recurrences of distal urethral strictures, all of which were treated successfully with graft and flap repairs. Conclusions: The vast majority of distal urethral strictures are amenable to a distal one-stage urethroplasty, avoiding the use of grafts and/or flaps while achieving reasonable outcomes. This limited approach, at least initially, is associated with shorter operative time and time of catheter placement and avoids morbidity associated with graft or flap harvesting. Spraying of urine is seldomly encountered and comparable to other approaches addressing distal urethral strictures

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

<p>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.</p

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

<p>(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.</p

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

<p>(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.</p

B-ADAM10 deletion attenuates bronchoconstriction and HDM specific IgE.

<p>(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.</p