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Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4+CD8+ thymocytes.

Zou W, Denton PW, Watkins RL, Krisko JF, Nochi T, Foster JL, Garcia JV - Retrovirology (2012)

Bottom Line: Both LAI- and LAINefdd-infected mice had about 8% of total peripheral blood CD8+ T cells that were CD38+HLA-DR+ compared <1% for uninfected mice.We conclude Nef is necessary for elevated viral replication and as a result indirectly contributes to CD4+ T cell killing.This depletion of thymic T cell precursors may be a significant factor in the elevated pathogenicity of CXCR4 trophic HIV-1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Infectious Diseases, University of North Carolina, Chapel Hill, NC 27599-7042, USA.

ABSTRACT

Background: The outcome of untreated HIV-1 infection is progression to AIDS and death in nearly all cases. Some important exceptions are the small number of patients infected with HIV-1 deleted for the accessory gene, nef. With these infections, disease progression is entirely suppressed or greatly delayed. Whether Nef is critical for high levels of replication or is directly cytotoxic remains controversial. The major problem in determining the role of Nef in HIV/AIDS has been the lack of tractable in vivo models where Nef's complex pathogenic phenotype can be recapitulated.

Results: Intravenous inoculation (3000 to 600,000 TCIU) of BLT humanized mice with HIV-1LAI reproducibly establishes a systemic infection. HIV-1LAI (LAI) replicates to high levels (peak viral load in blood 8,200,000 ± 1,800,000 copies of viral RNA/ml, range 3,600,000 to 20,400,000; n = 9) and exhaustively depletes CD4+ T cells in blood and tissues. CD4+CD8+ thymocytes were also efficiently depleted but CD4+CD8- thymocytes were partially resistant to cell killing by LAI. Infection with a nef-deleted LAI (LAINefdd) gave lower peak viral loads (1,220,000 ± 330,000, range 27,000 to 4,240,000; n = 17). For fourteen of seventeen LAINefdd-infected mice, there was little to no loss of either CD4+ T cells or thymocytes. Both LAI- and LAINefdd-infected mice had about 8% of total peripheral blood CD8+ T cells that were CD38+HLA-DR+ compared <1% for uninfected mice. Three exceptional LAINefdd-infected mice that lost CD4+ T cells received 600,000 TCIU. All three exhibited peak viral loads over 3,000,000 copies of LAINefdd RNA/ml. Over an extended time course, substantial systemic CD4+ T cell loss was observed for the three mice, but there was no loss of CD4+CD8+ or CD4+CD8- thymocytes.

Conclusion: We conclude Nef is necessary for elevated viral replication and as a result indirectly contributes to CD4+ T cell killing. Further, Nef was not necessary for the activation of peripheral blood CD8+ T cells following infection. However, CD4+CD8+ thymocyte killing was dependent on Nef even in cases of elevated LAINefdd replication and T cell loss. This depletion of thymic T cell precursors may be a significant factor in the elevated pathogenicity of CXCR4 trophic HIV-1.

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Impact of long-term infection with a high dose of LAINefddin BLT humanized mice. The six BLT humanized mice infected with 600,000 TCIU of LAINefdd presented in Figure 6 were harvested for multiple tissue analyses at the last time points depicted in that figure. These mice are referred to here by the panels in which they appear in that figure (6AB or 6CD). Data from these mice are presented alongside data from naïve BLT humanized mice (n = 5 in PB, spleen and HTO or n = 4 in LN, BM, lung and liver) and BLT humanized mice inoculated with 600,000 TCIU of LAI (n = 3) to reveal that the CD4+ T cell loss patterns observed in the peripheral blood are mirrored by the multiple organ analyses performed. Shown are the percentages of human CD4+ T cells present in peripheral blood, lymph nodes, spleen, bone marrow, lung and liver, as well as the percentages of CD4+CD8- and CD4+CD8+ thymocytes in the human thymic organoid. Delta symbols serve to indicate no data are available from bone marrow, lung or liver for LAI at 600,000 TCIU. The percent of CD4+ T cells in peripheral blood or tissues was relative to total CD3+ T cells while the percent of CD4+CD8- and CD4+CD8+ thymocytes was relative to total thymocytes. One-way ANOVA with six Bonferroni multiple comparisons tests was performed to compare the results within each tissue. If no difference was detected, the comparison is unmarked (alpha = 0.05). Comparisons yielding significant differences are represented by a line connecting the arrows above the respective bars (*p < 0.05; **p < 0.01).
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Figure 7: Impact of long-term infection with a high dose of LAINefddin BLT humanized mice. The six BLT humanized mice infected with 600,000 TCIU of LAINefdd presented in Figure 6 were harvested for multiple tissue analyses at the last time points depicted in that figure. These mice are referred to here by the panels in which they appear in that figure (6AB or 6CD). Data from these mice are presented alongside data from naïve BLT humanized mice (n = 5 in PB, spleen and HTO or n = 4 in LN, BM, lung and liver) and BLT humanized mice inoculated with 600,000 TCIU of LAI (n = 3) to reveal that the CD4+ T cell loss patterns observed in the peripheral blood are mirrored by the multiple organ analyses performed. Shown are the percentages of human CD4+ T cells present in peripheral blood, lymph nodes, spleen, bone marrow, lung and liver, as well as the percentages of CD4+CD8- and CD4+CD8+ thymocytes in the human thymic organoid. Delta symbols serve to indicate no data are available from bone marrow, lung or liver for LAI at 600,000 TCIU. The percent of CD4+ T cells in peripheral blood or tissues was relative to total CD3+ T cells while the percent of CD4+CD8- and CD4+CD8+ thymocytes was relative to total thymocytes. One-way ANOVA with six Bonferroni multiple comparisons tests was performed to compare the results within each tissue. If no difference was detected, the comparison is unmarked (alpha = 0.05). Comparisons yielding significant differences are represented by a line connecting the arrows above the respective bars (*p < 0.05; **p < 0.01).

Mentions: Our findings indicated that the dependence on Nef for replication was clearly evident at low and intermediate inoculums, but in some cases of the high initial inoculums of LAINefdd replication approached that of wild-type LAI. At high inoculum, the non-pathogenic phenotype of LAINefdd was still dramatic. We considered the possibility that longer courses of sustained infection may result in substantial CD4+ T cell depletion. For this purpose, we followed six LAINefdd-infected mice infected with 6.0X105 TCIU for up to 170 days. As indicated above, three mice exposed to this high dose of LAINefdd exhibited reductions in CD4+ T cell levels in peripheral blood. These three mice had uncharacteristically high viral loads (>106 copies/ml of plasma) by two weeks (Figure 6A). Associated with this high viral load was a reduction of CD4+ T cells in the peripheral blood (Figure 6B). However, despite viral loads near that of wild-type virus, the time courses of CD4+ T cell depletion were significantly delayed for an average of ten weeks for the nef-defective virus compared to less than three weeks for LAI (Figure 5B and 6B, Mantel-Cox Test, p = 0.025). The reduced levels of CD4+ T cells in peripheral blood were mirrored by similar reductions in the lymph node, spleen, lung and liver but not in bone marrow of these animals (Figure 7, see bars designated 6AB). In contrast, the levels of thymocytes in these mice were not significantly reduced (Figure 7, CD4+CD8-, CD4+CD8+, 6AB). The observations from these three mice suggest that even in the case of clear reductions CD4+ T cells, the nef-deleted virus was still much less cytotoxic than the wild-type virus to thymocytes.


Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4+CD8+ thymocytes.

Zou W, Denton PW, Watkins RL, Krisko JF, Nochi T, Foster JL, Garcia JV - Retrovirology (2012)

Impact of long-term infection with a high dose of LAINefddin BLT humanized mice. The six BLT humanized mice infected with 600,000 TCIU of LAINefdd presented in Figure 6 were harvested for multiple tissue analyses at the last time points depicted in that figure. These mice are referred to here by the panels in which they appear in that figure (6AB or 6CD). Data from these mice are presented alongside data from naïve BLT humanized mice (n = 5 in PB, spleen and HTO or n = 4 in LN, BM, lung and liver) and BLT humanized mice inoculated with 600,000 TCIU of LAI (n = 3) to reveal that the CD4+ T cell loss patterns observed in the peripheral blood are mirrored by the multiple organ analyses performed. Shown are the percentages of human CD4+ T cells present in peripheral blood, lymph nodes, spleen, bone marrow, lung and liver, as well as the percentages of CD4+CD8- and CD4+CD8+ thymocytes in the human thymic organoid. Delta symbols serve to indicate no data are available from bone marrow, lung or liver for LAI at 600,000 TCIU. The percent of CD4+ T cells in peripheral blood or tissues was relative to total CD3+ T cells while the percent of CD4+CD8- and CD4+CD8+ thymocytes was relative to total thymocytes. One-way ANOVA with six Bonferroni multiple comparisons tests was performed to compare the results within each tissue. If no difference was detected, the comparison is unmarked (alpha = 0.05). Comparisons yielding significant differences are represented by a line connecting the arrows above the respective bars (*p < 0.05; **p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 7: Impact of long-term infection with a high dose of LAINefddin BLT humanized mice. The six BLT humanized mice infected with 600,000 TCIU of LAINefdd presented in Figure 6 were harvested for multiple tissue analyses at the last time points depicted in that figure. These mice are referred to here by the panels in which they appear in that figure (6AB or 6CD). Data from these mice are presented alongside data from naïve BLT humanized mice (n = 5 in PB, spleen and HTO or n = 4 in LN, BM, lung and liver) and BLT humanized mice inoculated with 600,000 TCIU of LAI (n = 3) to reveal that the CD4+ T cell loss patterns observed in the peripheral blood are mirrored by the multiple organ analyses performed. Shown are the percentages of human CD4+ T cells present in peripheral blood, lymph nodes, spleen, bone marrow, lung and liver, as well as the percentages of CD4+CD8- and CD4+CD8+ thymocytes in the human thymic organoid. Delta symbols serve to indicate no data are available from bone marrow, lung or liver for LAI at 600,000 TCIU. The percent of CD4+ T cells in peripheral blood or tissues was relative to total CD3+ T cells while the percent of CD4+CD8- and CD4+CD8+ thymocytes was relative to total thymocytes. One-way ANOVA with six Bonferroni multiple comparisons tests was performed to compare the results within each tissue. If no difference was detected, the comparison is unmarked (alpha = 0.05). Comparisons yielding significant differences are represented by a line connecting the arrows above the respective bars (*p < 0.05; **p < 0.01).
Mentions: Our findings indicated that the dependence on Nef for replication was clearly evident at low and intermediate inoculums, but in some cases of the high initial inoculums of LAINefdd replication approached that of wild-type LAI. At high inoculum, the non-pathogenic phenotype of LAINefdd was still dramatic. We considered the possibility that longer courses of sustained infection may result in substantial CD4+ T cell depletion. For this purpose, we followed six LAINefdd-infected mice infected with 6.0X105 TCIU for up to 170 days. As indicated above, three mice exposed to this high dose of LAINefdd exhibited reductions in CD4+ T cell levels in peripheral blood. These three mice had uncharacteristically high viral loads (>106 copies/ml of plasma) by two weeks (Figure 6A). Associated with this high viral load was a reduction of CD4+ T cells in the peripheral blood (Figure 6B). However, despite viral loads near that of wild-type virus, the time courses of CD4+ T cell depletion were significantly delayed for an average of ten weeks for the nef-defective virus compared to less than three weeks for LAI (Figure 5B and 6B, Mantel-Cox Test, p = 0.025). The reduced levels of CD4+ T cells in peripheral blood were mirrored by similar reductions in the lymph node, spleen, lung and liver but not in bone marrow of these animals (Figure 7, see bars designated 6AB). In contrast, the levels of thymocytes in these mice were not significantly reduced (Figure 7, CD4+CD8-, CD4+CD8+, 6AB). The observations from these three mice suggest that even in the case of clear reductions CD4+ T cells, the nef-deleted virus was still much less cytotoxic than the wild-type virus to thymocytes.

Bottom Line: Both LAI- and LAINefdd-infected mice had about 8% of total peripheral blood CD8+ T cells that were CD38+HLA-DR+ compared <1% for uninfected mice.We conclude Nef is necessary for elevated viral replication and as a result indirectly contributes to CD4+ T cell killing.This depletion of thymic T cell precursors may be a significant factor in the elevated pathogenicity of CXCR4 trophic HIV-1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Infectious Diseases, University of North Carolina, Chapel Hill, NC 27599-7042, USA.

ABSTRACT

Background: The outcome of untreated HIV-1 infection is progression to AIDS and death in nearly all cases. Some important exceptions are the small number of patients infected with HIV-1 deleted for the accessory gene, nef. With these infections, disease progression is entirely suppressed or greatly delayed. Whether Nef is critical for high levels of replication or is directly cytotoxic remains controversial. The major problem in determining the role of Nef in HIV/AIDS has been the lack of tractable in vivo models where Nef's complex pathogenic phenotype can be recapitulated.

Results: Intravenous inoculation (3000 to 600,000 TCIU) of BLT humanized mice with HIV-1LAI reproducibly establishes a systemic infection. HIV-1LAI (LAI) replicates to high levels (peak viral load in blood 8,200,000 ± 1,800,000 copies of viral RNA/ml, range 3,600,000 to 20,400,000; n = 9) and exhaustively depletes CD4+ T cells in blood and tissues. CD4+CD8+ thymocytes were also efficiently depleted but CD4+CD8- thymocytes were partially resistant to cell killing by LAI. Infection with a nef-deleted LAI (LAINefdd) gave lower peak viral loads (1,220,000 ± 330,000, range 27,000 to 4,240,000; n = 17). For fourteen of seventeen LAINefdd-infected mice, there was little to no loss of either CD4+ T cells or thymocytes. Both LAI- and LAINefdd-infected mice had about 8% of total peripheral blood CD8+ T cells that were CD38+HLA-DR+ compared <1% for uninfected mice. Three exceptional LAINefdd-infected mice that lost CD4+ T cells received 600,000 TCIU. All three exhibited peak viral loads over 3,000,000 copies of LAINefdd RNA/ml. Over an extended time course, substantial systemic CD4+ T cell loss was observed for the three mice, but there was no loss of CD4+CD8+ or CD4+CD8- thymocytes.

Conclusion: We conclude Nef is necessary for elevated viral replication and as a result indirectly contributes to CD4+ T cell killing. Further, Nef was not necessary for the activation of peripheral blood CD8+ T cells following infection. However, CD4+CD8+ thymocyte killing was dependent on Nef even in cases of elevated LAINefdd replication and T cell loss. This depletion of thymic T cell precursors may be a significant factor in the elevated pathogenicity of CXCR4 trophic HIV-1.

Show MeSH
Related in: MedlinePlus