Fig 1.
IgG deposition in the transgenic ear tissue increases with phenotypic stage and age.
Protein (50μg/track) was extracted from L2LMP1 transgenic ears (stages 1 to 5, upper blot as indicated) and control (NSC lower blot) of mice of increasing age and western blotted (age in days, as indicated above or below (respectively)). The blots were probed with antibodies to actin (42kD) and GAPDH (37kD), using anti-mouse IgG as secondary, also detecting tissue immunoglobulin-G: IgH and IgL, as indicated. Protein size markers in kD are indicated to the left of each panel. Below: the normalised values (IgH/actin) are shown graphically for transgenic (L2LMP1) and NSC samples, plotted against age.
Fig 2.
IgG, IgM, IgA, IgE and complement C3c levels are increased in the transgenic tissue.
(A) The phenotypic stage was monitored in a cohort of LMP1 transgenic mice in a wild type background (LMP1CD40WT: n = 17) compared to LMP1 transgenic mice in a CD40 null background (LMP1CD40KO: n = 15) and plotted over time; error bars show SEM. (B) Ear tissue protein samples from mice of the following genotypes: L2LMP1 transgene (LMP1tg) positive (+) or negative (-), in either a CD40 null background (ko), heterozygous (het) or wild type for CD40 (wt) were western blotted. Age in days and phenotypic stage are indicated. The blots were probed with antibodies to mouse IgG, IgM, IgA, IgE and complement C3 and re-probed with antibody to GAPDH (37kD). Protein size markers in kD are indicated to the left of each panel. Note: IgG, IgE and C3c are not produced in CD40 null mice, however, IgM and IgA are induced in the LMP1 transgenic tissues.
Fig 3.
NAC treatment delays phenotypic progression.
A to G photographs of ear skin phenotype: A: The phenotype of a transgenic mouse (right) at 42 days old, referred to as stage 1 (St1), compared to NSC (left). (B-G): The phenotypic effect of systemic treatment of transgenic mice with NAC (right) from 38 days old, compared to untreated (left: UT) at progressive ages and phenotypic stages. Age and stage details: B: 42 days old: left: untreated stage 1, right: 4 days NAC. C: 60 days old: left: untreated stage 2, right: 22 days NAC. D: 84 days old: left: untreated stage 3, right: 46 days NAC. E: 100 days old: left: untreated stage 3, right: 56 days NAC. F: 140 days old: left: untreated stage 4, right: 96 days NAC. G: 160 days old: left: untreated stage 5, right: 116 days NAC treatment. (H) The mean age of progression to the next phenotypic stage is shown graphically for untreated transgenic mice (n = 57, error bars show SD). (I) The average phenotypic stage with age is plotted for a cohort of transgenic mice treated with NAC (total n = 34, with at least n = 7 for each time point) compared to untreated (total n = 188, with at least n = 10 for each time point). Stage progression is significantly different between the two groups (P<0.0001) from 7 weeks of age onwards. Error bars show SE. Note: drops in the curve for untreated mice do not reflect phenotype reversal but are due to removal of mice from study.
Fig 4.
NAC treatment reduces the quantifiable levels of chronic inflammation in the L2LMP1 transgenic mice.
Top: typical epifluorescent image taken 24 hours after IV injection of p680 into a transgenic L2LMP1 mouse at phenotypic stage 5 (St5) and negative sibling control (NSC); colour scale shows radiant efficiency range displayed ((photons/sec/cm2/sr)/(μW/cm2)). Below: graphs depicting the mean (with SD error bars) radiant efficiency (Y axis) observed in mice in increasing age groups (X axis) imaged at 24 and 30 hours (as indicated) post injection of 1nmol/mouse p680. Four groups were examined, transgenic (Tg) and NSC mice, either untreated or treated with NAC-water. N = 2 to N = 8 for each age and group (as detailed in Table A in S2 File).
Fig 5.
NAC treatment reduces the number of leukocytes in the inflamed tissue and their oxidative status.
Cell suspensions were generated from ear tissue from mice aged approximately 105 days old. Samples were taken from L2LMP1 transgenic mice (Tg) treated with NAC (n = 6) or untreated (U: n = 7) and NSC treated with NAC (n = 3) or untreated (n = 3) (as indicated) and analysed by flow cytometry. Live cells were gated (staining negative for 7AAD) and the proportion of leukocytes (CD45+) assayed and examined for evidence of intracellular ROS through DCFH-DA. Top panels show representative dot plots obtained for each category. The percentage of CD45+ cells, as quantified using the quadrant values (top right and top left quadrants combined) is graphed below. The difference between Tg U and Tg NAC and the difference between Tg U and NSC U is statistically significant (P<0.0001).
Fig 6.
NAC treatment does not impact a set of proteins deregulated by LMP1.
Ear tissue protein samples, taken from different mice were analysed by western blotting for expression of the indicated proteins. (A) Samples from L2LMP1 transgene positive mice of increasing age and stage are shown. (B) Samples were taken from NAC treated and control mice at 100 days old. LMP1 transgenic status (+ or -) and NAC treatment (+ or -) are indicated above. Actin or GAPDH was used as a loading control. Protein size markers in kD are indicated to the left of each panel.
Fig 7.
Leukocyte recruitment to the inflamed tissue correlates with phenotypic stage, which is ameliorated by NAC treatment.
CPD stained leukocytes collected from inflamed L2LMP1 transgenic stage 5 ears were injected into six L2LMP1 transgenic mice (aged 103 days old), three of which had received NAC treated water (NAC) and three were not treated (UT). Mice were imaged at 3, 24 and 48 hours post injection (hpi). Top: typical epifluorescent image taken at 3 and 24 hpi. Below: the average radiant efficiency of the ROI (taken around the ear) is plotted (n = 6 for each, error bars show SEM). The average of each of the readings taken (6 repeats) for un-injected control mouse VII (Table 1), is plotted as the base line. The difference between NAC and UT is significant at each time point (p<0.0001, 0.0018, 0.0002 respectively).
Table 1.
L2LMP1 transgenic mice were designated with numbers I to VII (as indicated). Treatment with NAC (from 48 days old) or untreated is indicated (+,—respectively) and ear stage at age 103 days. Mice I to VI were recipients for leukocytes (cells inj) collected from donor L2LMP1 stage 5 ears.