Assessment of dermal sensitization by nickel salts in a novel humanized TLR-4 mouse model

Abstract

The fundamental goal of this study was to determine the potential utility of a novel humanized Toll-like receptor-4 (hTLR-4) mouse model for future in vivo studies of nickel allergy. First, mice of both sexes and hTLR-4 expression profiles were incorporated into a Local Lymph Node Assay (LLNA) to assess skin sensitization. Next, a set of hTLR-4 hTLR-4-positive mice (female and male groups) was similarly exposed to vehicle control (VC) or 10% NiSO₄ on Days 1, 2, and 3. Mice were euthanized on Day 10, lymph node (LN) cellularity was assessed, LN and spleen cells were phenotyped, and serum was collected to quantify circulating cytokine and IgE levels. In the LLNA, hTLR-4-positive mice of both sexes exhibited enhanced responsivity to nickel. NiSO₄ (10%) had a stimulation index (SI) of 3.7 (females) and 3.8 (males) in hTLR-4-positive animals, and an SI of 0.5 (females) and 0.8 (males) in hTLR-4 hTLR-4-negative mice. In the 10d study, hTLR-4-positive mice exposed to 10% NiSO₄ exhibited increased LN cellularity (6.0× increase in females, 3.2× in males) and significantly higher concentrations of circulating IgE (4.1× increase in females, 3.4× in males). Significant increases in serum interferon (IFN)-γ, interleukin (IL)-4, and IL-5 levels were seen in female mice, while altered concentrations of IL-4 and IL-10 were detected in male mice. The results of this study ultimately demonstrate that murine expression of hTLR-4 confers enhanced susceptibility to dermal sensitization by nickel, and consequently, the hTLR-4 mouse model represents a viable approach for future studies of nickel allergy in vivo.

Keywords: Nickel; allergy model; hypersensitivity; local lymph node assay; sensitization; toll-like receptor-4.

Figure 1.

A summary of the study designs for the Local Lymph Node Assay and 10-day study are shown. (A) For the LLNA, hTLR-4-positive and -negative female (F) and male (M) mice (n = 3–4) were topically exposed to mineral oil (vehicle control, VC) or NiSO₄ (2.5, 5, or 10% w/v) on days 1–3. Mice were rested for two days, then injected intravenously with tritiated-thymidine on day 6, and euthanized by CO₂ asphyxiation 5 h later. Auricular lymph nodes were harvested from each animal, processed, and radio-isotope incorporation was assessed. (B) For the 10-d study, male and female hTLR-4-positive mice (n = 6–10) were topically exposed to mineral oil (vehicle control, VC) or 10% NiSO₄ for three consecutive days, similar to the exposure scheme used in the LLNA study. Mice were rested until day 10, then euthanized by intraperitoneal injection of sodium pentobarbital euthanasia solution. The auricular lymph nodes (left and right) were harvested, total cellularity was assessed, and phenotypic analysis was performed on lymph node cell subsets. The spleen was isolated, weighed, and phenotypic analysis was performed on spleen cell subpopulations. Whole blood was collected to perform differential analyses on circulating leukocyte subsets and serum was isolated to measure circulating total IgE and cytokine concentrations.

Figure 2.

Results from the LLNA study using female (A, B) and male (C, D) hTLR-4-negative (A, C) and hTLR-4-positive (B, D) mice. Lymph node radioisotope incorporation [expressed as disintegrations per minute (DPM)] is shown for each animal. Stimulation index (SI) was calculated for each group (value listed above corresponding data points) by dividing the group average DPM by that of the respective control group. In the LLNA, an SI of ≥ 3 was considered indicative of allergic sensitization. n = 4, p < 0.05, *statistically different from same-sex control group and SI of ≥ 3 (dermal sensitization).

Figure 3.

Auricular lymph node cellularity in female (A) and male (B) hTLR-4-positive mice at 10-d timepoint. In left panel (a), total cell number is shown for female mice exposed to vehicle (gray dots) or 10% NiSO₄ (red squares). In right panel (B), total cell number is shown for male mice exposed to vehicle (gray dots) or 10% NiSO₄ (blue squares). n = 6–10, p < 0.05, *statistically different from same-sex control group.

Figure 4.

Total IgE levels in female (A) and male (B) hTLR-4-positive mice at the 10-d timepoint. In left panel (a), circulating IgE concentration (ng/ml serum) is shown for female mice exposed to vehicle (gray dots) or 10% NiSO₄ (red squares). In right panel (B), concentrations are shown for male mice exposed to vehicle (gray dots) or 10% NiSO₄ (blue squares). n = 6–10, p < 0.05, *statistically different from same-sex control group.

Figure 5.

Serum cytokine concentrations in female (A) and male (B) hTLR-4-positive mice at the 10-d timepoint. Cytokines quantified include prototypical T_H1 (IFNg, IL-2, IL-12) and T_H2 (IL-4, IL-5, IL-9, IL-13) cytokines, as well as two miscellaneous cytokines (IL-6, IL-10). In left panel (a), cytokine concen-trations (pg/ml serum) are shown for female mice exposed to vehicle (hatched red bars) or 10% NiSO₄ (solid red bars). In right panel (B), concentrations are shown for male mice exposed to vehicle (hatched blue bars) or 10% NiSO₄ (solid blue bars). n = 6–10, p < 0.05, *statistically different from same-sex control group.

Figure 6.

Nickel-induced alterations in serum cytokine profiles of hTLR-4-positive mice at the 10-d timepoint. Changes in circulating cytokine concentrations (expressed as fold-change over same-sex vehicle control animals) are listed in (a) for NiSO₄-exposed mice of both sexes. A radar plot illustrating these changes in female (red line) and male (blue line) animals is shown in B. n = 6–10, p < 0.05, *statistically different from same-sex control group.