Inflammatory papillomatous hyperplasia and epidermal necrosis in a transgenic rat for HIV-1

Abstract

Background: Skin lesions commonly affect AIDS patients. The pathogenesis of certain dermatologic disorders primarily associated to HIV-1 is unclear, and better forms of therapy for these conditions need to be discovered. Transgenic animal models represent a novel approach for the study of these disorders and for the quest of more effective forms of treatment.

Objective: Characterize this HIV-1 transgenic rat as a model to study skin diseases related to HIV/AIDS.

Methods: A transgenic rat was developed, using an HIV-1 construct with deleted gag and pol genes. Morphological and genotypical evaluations were followed by cytokine profile characterization of the lesions.

Results: We report the characterization of a colony of HIV-1 transgenic rats that developed skin lesions in a frequency of 22.5%. Cutaneous expression of functional HIV-1 transgenes correlated precisely with the severity of the phenotype. In early stages, rats manifested localized areas of xerosis and dispersed papulosquamous lesions. These hyperplastic manifestations were observed in conjunction with an increased epidermal expression of tat protein and a Th1/Th2 profile of cytokines. As the lesions progressed, they formed inflammatory plaques that subsequently ulcerated. Histologically, these lesions displayed a profound lymphocytic infiltrate, epidermal necrosis, and a marked increase of both Th1 and Th2 derived cytokines. Moreover, the presence of circulating IgG antibodies against HIV-1 gp120 was detected.

Conclusion: This animal model as other HIV-1 transgenic mice described in the past, is not able to fully explain the myriad of skin findings that can occur in HIV-infected humans; however, it represents a potential animal model system for the study of immune-mediated inflammatory skin diseases.

Fig. 1

Construct and phenotype of the HIV-1 Tg rat. (a) Genome of the HIV-1 provirus pNL4-3 highlighting the 2 deleted genes: gag and pol, (b) the non-infectious plasmid was generated by removing the 3-kbp SphI-MscI fragment encompassing the 3′ region of the gag and the 5′ region of pol genes, (c) topography of the lesions shown in the phenotype of the HIV-1 Tg rat, (d) tail of a severely affected Tg rat showing areas of epidermal necrosis and (e) opaque cataract manifested by rats with severe phenotype.

Fig. 2

Classification of the skin phenotype according to the body surface area involved. (a) Wild type/non-lesional, (b) mild, (c) moderate and (d) severe. Dorsal fur was shaved for a better exposure of the lesions.

Fig. 3

Histhology and immunohistochemistry for HIV-1 gp120 and tat. Histologic sections of skin biopsy specimens from WT rats were stained with H&E (a), for gp120 (b), and for tat (c). Sections from non-lesional Tg rats were stained with H&E (d), for gp120 (e), and for tat (f). Sections from early lesions were stained with H&E (g), for gp120 (h), and for tat (i). Sections from late lesions were stained with H&E (j), for gp120 (k), and for tat (l). Magnification of epidermal changes seen in late lesions stained in H&E (m), for gp120 (n), and for tat (o). Magnification of dermis of Tg rats stained in H&E (m), for gp120 (n), and for tat (o). Scale bar: 100 μm (a–l) and 50 μm (m–r). Immunohistochemistry sections were counterstained with methyl green.

Fig. 4

Immunohistochemistry for the inflammatory infiltrate. (a) Histologic sections of skin biopsy specimens show abundant CD8+ cells infiltrating the dermis and epidermis. Asterisk indicates a higher magnification on the morphology of the staining (inset). Scale bar: 100 μm, inset (25 μm). (b) Section shows minimal CD4+ cells interspersed in the dermis. Magnification of the dermis showing immunoreactivity (arrow) is shown in the inset. Scale bar: 100 μm, inset (5 μm). All sections counterstained with methyl green.

Fig. 5

Real-time RT-PCR quantitative analysis of the cutaneous expression of the HIV-1 transgenes. Total RNAs were extracted from full-thickness WT rats (n = 3), non-lesional skin (n = 4), early lesions (n = 3), and late lesions (n = 3), and then were reverse transcribed. We used SYBR green mix with transgene-specific primers (env and tat) with identical thermal cycling conditions for all genes. Gene expression was normalized to housekeeping gene 18S. Comparative quantification was performed by Livak Ct method, and results were expressed in fold increase compared early lesions and late lesions. Error bars represent mean values ± S.D. of three independent experiments. (*) Significant increase compared to WT rats; (**) null expression of both transgenes in WT and non-lesional skin was observed.

Fig. 6

Real-time RT-PCR quantitative analysis of the expression of the cytokine milieu in the skin. Total RNAs were extracted from full-thickness WT (n = 3), non-lesional skin (n = 4), early lesions (n = 3), and late lesions (n = 3), and then were reverse transcribed. We used SYBR green mix with cytokine gene-specific primers (IL-4, IL-5, IL-6, IL-10, IFN-γ and TNF-α) using identical thermal cycling conditions for all genes. Gene expression was normalized to housekeeping gene 18S. Comparative quantification was performed by Livak Ct method, and results were expressed in fold increase compared to WT rats and early lesions. Error bars represent mean values ± S.D. of three independent experiments. (*) Significant increase compared to WT; (λ) significant increase compared to early lesions; (&) significant increased compared to WT and early lesions.

Fig. 7

Quantification of circulating antibodies against HIV-1 gp120. Serum samples from fresh drawn blood of the five subsets (wild type rats, n = 3; Tg rats without skin phenotype, n = 3; mild, n = 2; moderate, n = 4; severe, n = 3) were analyzed by ELISA to quantify presence of antibodies against HIV-1 gp120. Error bars represent mean values ± S.D. of three independent experiments. (*) Significant increase compared to the other three subsets (P < 0.01).