Table 1.
Papilloma incidence in UVB-irradiated FVB/NJ mice.
Fig 1.
MmuPV1-induced papillomas in UVB-irradiated FVB/NJ mice.
(A) Sequence of manipulations to FVB/NJ mice in the MmuPV1-UVB infection model system. Ear sites were scarified and exposed to 108 VGE of MmuPV1 either 24 hours before (FVB+UVB 24h.b.i.+MmuPV) or 24 hours after (FVB+MmuPV+UVB 24h.p.i.) 300mJ/cm2 UVB whole-body irradiation. Sites were then scored weekly for presence of papillomas. (B) Kaplan-Meyer plots of the fraction of papilloma-free infected ear sites with respect to time. There is no significant difference in the temporal onset of disease between the two experimental groups (FVB+UVB24h.p.i.+MmuPV vs FVB+MmuPV+UVB 24h.b.i.; P = 0.814, Wilcoxon log-rank test, two-sided). (C) Percentage of sites with overt papillomas over a 6-month observation period. In both the cohorts of mice, a subset of papillomas completely regressed by 6 months post-infection. (D) Distribution of ear sites of UVB-treated FVBN/J mice (FVB+UVB24h.p.i.+MmuPV group) infected that developed papillomas that completely regressed (red), partially regressed (blue) or continued to grow (green) over the 6 month monitoring period. In grey is the fraction of sites that did not develop papillomas. (E) Growth profiles of individual papillomas arising in the FVB+UVB24h.p.i.+MmuPV group during the 6-month monitoring period. Individual lines represent the geometric mean diameter of each papilloma as a function of time. Red lines are papillomas that completed regressed. Blue lines are papillomas that partially regressed. Green lines are papillomas that continued to grow.
Fig 2.
Viral dose-dependent incidence of papillomas in UVB-treated FVB/NJ mice and in FoxN1nu/nu mice.
Immunocompetent FVB/NJ mice were infected at ear sites with 108, 107 or 106 VGE of MmuPV1 virions following scarification. Twenty-four hours post-infection (h.p.i.), mice were irradiated with 300mJ/cm2 UVB and scored weekly for presence of ear papillomas up to 6 months. In parallel, T-cell deficient FoxN1nu/nu mice not treated with UVB were infected at ear sites with the same stock of virus at designated doses. Graphs for each strain are shown separately. (A) Kaplan-Meyer plot of the fraction of papilloma-free infected sites over the initial four-month period following infection. There was no significant difference between incidences of disease between UVB-irradiated FVB mice treated with 107 versus 108 VGE of MmuPV1 (P = 0.21, Wilcoxon log-rank test, two-sided). FVB/NJ mice infected with 106 VGE of MmuPV1 did not develop any papillomas. (B) Percentage of sites with overt papillomas over a 6-month observation period. Note that in both groups, UVB-irradiated FVB mice infected with 107 and 108 VGE of MmuPV1, a subset of papillomas completely regressed by 6 months post-infection (left). There was no regression of papillomas in FoxN1nu/nu mice (right).
Fig 3.
Histopathological analysis of MmuPV1-induced papillomas in UVB –irradiated mice.
(A) H&E staining of ear tissue indicating presence of a sessile ear papilloma caused by MmuPV1 in FVB mice following UVB irradiation (left). Presence of koilocytes is indicated (middle). L1-immunofluorescent staining of papillomas (right). (B) Sessile papilloma with areas indicating frank malignant transformation (inset;i) and foci of inflammation (inset;ii). K14 staining of papilloma and associated epithelial invasion into underlying dermis. Papillomas on the tail of an immunodeficient BALB/c FoxN1nu mice (right, top). These papillomas were formed as a result of infection with viral extracts prepared from papillomas that arose in immunocompetent FVB mice infected with MmuPV1 followed by UVB irradiation. (C) L1 (green)-MmuPV1 FISH (red) co-staining of MmuPV1-induced ear papillomas in UVB irradiated FVB/NJ mice. The nuclei were counterstained with DAPI (blue). Scale bars represent 100μm.
Fig 4.
Systemic effect of UVB assists in MmuPV-dependent papillomatosis.
(A) Schematic illustrating the experimental design. Twenty-four hours post infection with 108 VGE, groups of mice were either whole body UVB irradiated (FVB+MmuPV+UVB 24h.p.i.) or infected sites were shielded from UVB (FVB+MmuPV+UVB 24h.p.i. shielded) and scored weekly for presence of ear papillomas up to 6 months. UVB dose was 300mJ/cm2. Scoring data is compared to the FVB+MmuPV+UVB 24h.p.i. group also shown in Fig 2, as both experiments were performed at the same time. (B) Kaplan-Meyer plots of the fraction of papilloma-free infected sites over the first 12 weeks post-infection. There was no significant difference between the two experimental groups (shielded versus unshielded ears) (P = 0.938) as assessed by Wilcoxon log-rank analysis. (C) Percentage of sites with overt papillomas over a 6-month observation period. Some papillomas completely regressed in both groups. Control animals i.e. non-UVB irradiated FVB mice infected with MmuPV1 (FVB+MmuPV-UVB) did not develop any papillomas over the 6 month observation period.
Fig 5.
UVB causes systemic immunosuppression of host.
Three groups of mice were treated as follows: a control group that was not UVB irradiated (FVB-UVB, White bars, n = 3), UVB-irradiated group (FVB+UVB, Striped bars, n = 6) and UVB-irradiated group in which ears were shielded from UVB exposure [FVB+UVB (ears shielded), Black bars]. Ear thickness reported as the average of the difference between DNCB-challenged (left) and unchallenged (right) ears four days post challenge. SEM refers to standard error in measurement computed by measuring standard deviation. DTH measured by the change in thickness of the ear (ΔEar) was found to be statistically significant between UVB-treated mice and non-irradiated control mice (student t-test, p<0.005, two-sided) for both UVB-treated groups.
Fig 6.
Long-lived immune suppression in UVB-irradiated mice correlates with papilloma incidence.
Mice were either infected with MmuPV1 or vehicle followed by UVB irradiation (300mJ/cm2) in the right ear. Mice were sensitized with DNCB 10 days post UVB exposure and were challenged DNCB in the left ear 3 months following infection. Ear swelling was measured by means of a Vernier calipers. Ear thickness is reported as the average of the difference between ear thickness 0 hrs post challenge and 72 hrs post challenge. Ear thickness for each mouse is shown as a dot plot. The black lines represent the mean reading for each group. Wilcoxon rank-sum test was used to analyze difference between several groups. There was significant difference between UVB-irradiated (UV) and control (no UV) groups (*p = 0.021, two-sided). There was significant difference between the UVB irradiated animals infected with MmuPV1 that developed warts and those that did not develop warts (**p = 0.016, two-sided). There was no significant difference between control (no UV) and UVB-irradiated mice infected with MmuPV1 that did not develop warts (MmuPV1+ UV without warts).