Loss of Zbtb32 in NOD mice does not significantly alter T cell responses. [version 2; referees: 2 approved]

Background: We previously identified the transcriptional regulator Zbtb32 as a factor that can promote T cell tolerance in the Non-Obese Diabetic (NOD) mouse, a model of Type 1 diabetes. Antigen targeted to DCIR2+ dendritic cells (DCs) in vivo inhibited both diabetes and effector T cell expansion in NOD mice. Furthermore, Zbtb32 was preferentially induced in autoreactive CD4 T cells stimulated by these tolerogenic DCIR2+ DCs, and overexpression of Zbtb32 in islet-specific T cells inhibited the diabetes development by limiting T cell proliferation and cytokine production. Methods: To further understand the role of Zbtb32 in T cell tolerance induction, we have now used CRISPR to target the Zbtb32 gene for deletion directly in NOD mice and characterized the mutant mice. We hypothesized that the systemic loss of Zbtb32 in NOD mice would lead to increased T cell activation and increased diabetes pathogenesis. Results: Although NOD.Zbtb32-/- male NOD mice showed a trend towards increased diabetes incidence compared to littermate controls, the difference was not significant. Furthermore, no significant alteration in lymphocyte number or function was observed. Importantly, in vitro stimulation of lymphocytes from NOD.Zbtb32-/- mice did not produce the expected hypersensitive phenotype observed in other genetic strains, potentially due to compensation by homologous genes. Conclusions: The loss of Zbtb32 in the NOD background does not result in the expected T cell activation phenotype

Loss of Zbtb32 in NOD mice does not significantly alter T cell responses. [version 1; referees: 2 approved]

Background: We previously identified the transcriptional regulator Zbtb32 as a factor that can promote T cell tolerance in the Non-Obese Diabetic (NOD) mouse, a model of Type 1 diabetes. Antigen targeted to DCIR2+ dendritic cells (DCs) in vivo inhibited both diabetes and effector T cell expansion in NOD mice. Furthermore, Zbtb32 was preferentially induced in autoreactive CD4 T cells stimulated by these tolerogenic DCIR2+ DCs, and overexpression of Zbtb32 in islet-specific T cells inhibited the diabetes development by limiting T cell proliferation and cytokine production. Methods: To further understand the role of Zbtb32 in T cell tolerance induction, we have now used CRISPR to target the Zbtb32 gene for deletion directly in NOD mice and characterized the mutant mice. We hypothesized that the systemic loss of Zbtb32 in NOD mice would lead to increased T cell activation and increased diabetes pathogenesis. Results: Although NOD.Zbtb32-/- male NOD mice showed a trend towards increased diabetes incidence compared to littermate controls, the difference was not significant. Furthermore, no significant alteration in lymphocyte number or function was observed. Importantly, in vitro stimulation of lymphocytes from NOD.Zbtb32-/- mice did not produce the expected hypersensitive phenotype observed in other genetic strains, potentially due to compensation by homologous genes. Conclusions: The loss of Zbtb32 in the NOD background does not result in the expected T cell activation phenotype

Repeated hapten exposure induces persistent tactile sensitivity in mice modeling localized provoked vulvodynia.

BACKGROUND:Vulvodynia is a remarkably prevalent chronic pain condition of unknown etiology. Epidemiologic studies associate the risk of vulvodynia with a history of atopic disease. We used an established model of hapten-driven contact hypersensitivity to investigate the underlying mechanisms of allergy-provoked prolonged sensitivity to pressure. METHODS:We sensitized female ND4 Swiss mice to the hapten oxazolone on their flanks, and subsequently challenged them four days later with oxazolone or vehicle for ten consecutive days on the labia. We evaluated labiar sensitivity to touch, local mast cell accumulation, and hyperinnervation after ten challenges. RESULTS:Oxazolone-challenged mice developed significant tactile sensitivity that persisted for over three weeks after labiar allergen exposures ceased. Allergic sites were characterized by mast cell accumulation, sensory hyper-innervation and infiltration of regulatory CD4+CD25+FoxP3+ T cells as well as localized early increases in transcripts encoding Nerve Growth Factor and nerve-mast cell synapse marker Cell Adhesion Molecule 1. Local depletion of mast cells by intra-labiar administration of secretagogue compound 48/80 led to a reduction in both nerve density and tactile sensitivity. CONCLUSIONS:Mast cells regulate allergy-provoked persistent sensitivity to touch. Mast cell-targeted therapeutic strategies may provide novel means to manage and limit chronic pain conditions associated with atopic disease

Repeated hapten exposure induces persistent tactile sensitivity in mice modeling localized provoked vulvodynia - Fig 1

<p><b>Timeline of oxazolone sensitization, challenge, and post-challenge outcome measures</b> (A) To measure oxazolone-driven vulvar tactile sensitivity, mice were topically sensitized with 2% Ox on the shaved flank (day 1) and subsequently challenged on the shaved labiar skin (days 5–14) with 1% Ox or EtOH vehicle for a total of 10 challenges. Tactile sensitivity was assessed in the ano-genital ridge area 1, 21, and 42 days after challenge cessation. Labiar skin was harvested at these time points from a separate cohort of mice for assessing molecular and cellular changes in the tissue. (B) To characterize T cell infiltration in Ox-challenged skin, mice were topically sensitized on their shaved back with 2% Ox (day 1) and challenged on both shaved flanks with 1% Ox or EtOH (days 5–14). Flank skin was harvested from both sides 1 day after challenge cessation for flow cytometric analysis of T cell infiltration. (C) To assess the effects of local mast cell depletion on Ox-induced tactile sensitivity and hyperinnervation, mice were topically sensitized with 2% Ox on the shaved flank (day 1), challenged on the shaved labia with 1% Ox or EtOH (days 5–14) and treated with intralabiar injection of saline or c48/80 (days 5–8 after Ox challenge cessation). Tactile sensitivity, mast cell levels and innervation were assessed 9, 21, and 35 days after the final Ox challenge.</p

Injection of c48/80 after challenges depletes mast cells and reduces CGRP⁺ nerve density and sensitivity.

<p>Density of Avidin⁺ mast cells (A) and CGRP⁺ cutaneous nerves (D) on day 9 after 4 treatments with c48/80 or saline (administered on days 5–8 after cessation of 10 Ox challenges) in 10 μm labiar cryo-sections, displayed as mean ± SEM (n = 2-3/treatment group). Representative images for mast cells (B-C) and nerves (E-F); 20x magnification; scale bar represents 50 μm. Means are compared to Ox/EtOH (** = p<0.01, *** = p<0.001); significance determined using one-way ANOVA and Tukey Kramer <i>post hoc</i> analysis. (G) Tactile sensitivity in mice treated with either saline or c48/80 (n = 6–9 mice per treatment group; two independent experiments). Means are compared to Ox/Ox/Saline (* = p<0.05, ** = p<0.01, *** = p<0.001); significance determined using an unpaired Student’s T test at each time point.</p

Injection of c48/80 after challenges depletes mast cells and reduces CGRP⁺ nerve density and sensitivity.

<p>Density of Avidin⁺ mast cells (A) and CGRP⁺ cutaneous nerves (D) on day 9 after 4 treatments with c48/80 or saline (administered on days 5–8 after cessation of 10 Ox challenges) in 10 μm labiar cryo-sections, displayed as mean ± SEM (n = 2-3/treatment group). Representative images for mast cells (B-C) and nerves (E-F); 20x magnification; scale bar represents 50 μm. Means are compared to Ox/EtOH (** = p<0.01, *** = p<0.001); significance determined using one-way ANOVA and Tukey Kramer <i>post hoc</i> analysis. (G) Tactile sensitivity in mice treated with either saline or c48/80 (n = 6–9 mice per treatment group; two independent experiments). Means are compared to Ox/Ox/Saline (* = p<0.05, ** = p<0.01, *** = p<0.001); significance determined using an unpaired Student’s T test at each time point.</p