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Type I interferon-dependent gene MxA in perinatal HIV-infected patients under antiretroviral therapy as marker for therapy failure and blood plasmacytoid dendritic cells depletion.

Badolato R, Ghidini C, Facchetti F, Serana F, Sottini A, Chiarini M, Spinelli E, Lonardi S, Plebani A, Caimi L, Imberti L - J Transl Med (2008)

Bottom Line: However, in a group of patients with long disease duration, having a low number of both pDC and CD4+ lymphocytes and a significant increase of serum interferon-alpha, MxA mRNA was produced at high level and its expression directly correlated with HIV RNA copy numbers.HIV replication unresponsive to antiretroviral treatment in perinatal-infected patients with advanced disease and pDC depletion may lead to interferon-alpha expression and subsequent induction of MxA mRNA.Thus, the latter measurement may represent a valuable marker to monitor the clinical response to therapy in HIV patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Istituto di Medicina Molecolare Angelo Nocivelli, Department of Pediatrics, University of Brescia, Brescia 25123, Italy. badolato@med.unibs.it

ABSTRACT

Background: To determine the role of interferon-alpha in controlling HIV infection we phenotypically and functionally analyzed circulating plasmacytoid dendritic cells (pDC), which are known to be the highest interferon-alpha producing cells, in 33 perinatally infected HIV+ patients undergoing standard antiretroviral therapy.

Methods: Circulating pDC were identified by flow cytometry using anti-BDCA-2 monoclonal antibody and by measuring BDCA-2 mRNA by real-time PCR, while tissue-resident pDC were identified by immunohistochemistry. mRNA for interferon-alpha and MxA, a gene that is specifically induced by interferon-alpha, was quantified in peripheral blood cells by real-time PCR, while serum interferon-alpha protein was measured by ELISA.

Results: While median values of pDC, both in terms of percentage and absolute number, were not statistically different from age-matched controls, interferon-alpha mRNA was increased in HIV-infected patients. However, in a group of patients with long disease duration, having a low number of both pDC and CD4+ lymphocytes and a significant increase of serum interferon-alpha, MxA mRNA was produced at high level and its expression directly correlated with HIV RNA copy numbers. Furthermore in patients displaying a low CD4+ blood cell count, a severe depletion of pDC in the tonsils could be documented.

Conclusion: HIV replication unresponsive to antiretroviral treatment in perinatal-infected patients with advanced disease and pDC depletion may lead to interferon-alpha expression and subsequent induction of MxA mRNA. Thus, the latter measurement may represent a valuable marker to monitor the clinical response to therapy in HIV patients.

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(A) MxA mRNA levels in ART-treated HIV+ children and controls (CTRL). Correlation between MxA mRNA levels and HIV viral load (B) and pDC number (C). NR: normalization ratio.
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Figure 2: (A) MxA mRNA levels in ART-treated HIV+ children and controls (CTRL). Correlation between MxA mRNA levels and HIV viral load (B) and pDC number (C). NR: normalization ratio.

Mentions: Since pDC are specialized cells that produce large amounts of IFN-α in response to viral infections, we sought to determine whether HIV infection induces the expression of this cytokine. We observed a significant increase of IFN-α mRNA in PBMC in HIV-infected patients as compared to healthy control subjects (Figure 1D), suggesting that the chronic infection induces IFN-α production. Next, we analyzed the expression of MxA mRNA, which is selectively induced after type-I IFN receptor binding and is a marker for cell responsiveness to IFN-α [14,35]. We found that MxA mRNA levels were significantly increased in HIV-infected patients with abnormal HIV viral load RNA. Indeed, we observed a direct correlation between MxA expression and HIV RNA copies in HIV infected patients (Figure 2B), suggesting that a high number of viral copies may directly influence the induction of MxA mRNA. Due to the fact that pDC are one of the principal producers of IFN-α [9] and that MxA is a marker of type I IFN bioactivity [29,35,36], one might predict that patients with the largest number of circulating pDC might express more MxA as compared to age-matched controls (Figure 2A). On the contrary, we have observed that higher levels of MxA mRNA are found in those patients with lower number of circulating pDC, as indicated by the strong negative correlation between these two parameters (Figure 2C). In particular, we observed that 15 patients displayed MxA mRNA levels above the cut-off, established as the 99th percentile of the distribution of MxA in healthy control subjects (NR = 2.7, shown as dotted line in the figure), while the remaining presented normal MxA mRNA expression. Surprisingly, patients with MxA mRNA levels above the cut-off are those with lower numbers of pDC (Figure 3A) and significantly higher serum levels of IFN-α (Figure 3B). It is of note that the median level of this cytokine is low in both groups, at levels observed in control children, but it has been demonstrated that circulating IFN-α is elevated in the sera just at the moment of HIV antigen appearance [37]. Moreover, we have also observed that children with low MxA mRNA (<2.7) below the cut-off show an increased number of CD4+ lymphocyte (Figure 3C) and that children with increased MxA mRNA (>2.7) are of older age (Figure 3D). Since MxA levels do not vary among ages [29,36], we speculate that increased levels of MxA mRNA are associated with the length of HIV infection. Finally, since pDC are present within the thymus [15] and immune-reconstitution is related to thymic function [38], we utilized real-time PCR to determine the levels of DNA episomes created in the thymus during T-cell receptor rearrangement process, known as TRECS. We observed that TREC values were comparable in HIV-infected patients and age-matched controls (63,345 TRECs/1 × 106 cells, range: 39,470–173,194 vs. 54,560 TRECs/1 × 106, range: 7,279–299,500), without any detectable difference between patients with normal or increased MxA mRNA values (median: 65,925 TRECs/1 × 106 cells, range: 15,259–155,003 vs. 51,125 TRECs/1 × 106 cells, range: 17,586–128,900). Similarly, there was no significant correlation between TRECs and pDC cell number (data not shown).


Type I interferon-dependent gene MxA in perinatal HIV-infected patients under antiretroviral therapy as marker for therapy failure and blood plasmacytoid dendritic cells depletion.

Badolato R, Ghidini C, Facchetti F, Serana F, Sottini A, Chiarini M, Spinelli E, Lonardi S, Plebani A, Caimi L, Imberti L - J Transl Med (2008)

(A) MxA mRNA levels in ART-treated HIV+ children and controls (CTRL). Correlation between MxA mRNA levels and HIV viral load (B) and pDC number (C). NR: normalization ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2542353&req=5

Figure 2: (A) MxA mRNA levels in ART-treated HIV+ children and controls (CTRL). Correlation between MxA mRNA levels and HIV viral load (B) and pDC number (C). NR: normalization ratio.
Mentions: Since pDC are specialized cells that produce large amounts of IFN-α in response to viral infections, we sought to determine whether HIV infection induces the expression of this cytokine. We observed a significant increase of IFN-α mRNA in PBMC in HIV-infected patients as compared to healthy control subjects (Figure 1D), suggesting that the chronic infection induces IFN-α production. Next, we analyzed the expression of MxA mRNA, which is selectively induced after type-I IFN receptor binding and is a marker for cell responsiveness to IFN-α [14,35]. We found that MxA mRNA levels were significantly increased in HIV-infected patients with abnormal HIV viral load RNA. Indeed, we observed a direct correlation between MxA expression and HIV RNA copies in HIV infected patients (Figure 2B), suggesting that a high number of viral copies may directly influence the induction of MxA mRNA. Due to the fact that pDC are one of the principal producers of IFN-α [9] and that MxA is a marker of type I IFN bioactivity [29,35,36], one might predict that patients with the largest number of circulating pDC might express more MxA as compared to age-matched controls (Figure 2A). On the contrary, we have observed that higher levels of MxA mRNA are found in those patients with lower number of circulating pDC, as indicated by the strong negative correlation between these two parameters (Figure 2C). In particular, we observed that 15 patients displayed MxA mRNA levels above the cut-off, established as the 99th percentile of the distribution of MxA in healthy control subjects (NR = 2.7, shown as dotted line in the figure), while the remaining presented normal MxA mRNA expression. Surprisingly, patients with MxA mRNA levels above the cut-off are those with lower numbers of pDC (Figure 3A) and significantly higher serum levels of IFN-α (Figure 3B). It is of note that the median level of this cytokine is low in both groups, at levels observed in control children, but it has been demonstrated that circulating IFN-α is elevated in the sera just at the moment of HIV antigen appearance [37]. Moreover, we have also observed that children with low MxA mRNA (<2.7) below the cut-off show an increased number of CD4+ lymphocyte (Figure 3C) and that children with increased MxA mRNA (>2.7) are of older age (Figure 3D). Since MxA levels do not vary among ages [29,36], we speculate that increased levels of MxA mRNA are associated with the length of HIV infection. Finally, since pDC are present within the thymus [15] and immune-reconstitution is related to thymic function [38], we utilized real-time PCR to determine the levels of DNA episomes created in the thymus during T-cell receptor rearrangement process, known as TRECS. We observed that TREC values were comparable in HIV-infected patients and age-matched controls (63,345 TRECs/1 × 106 cells, range: 39,470–173,194 vs. 54,560 TRECs/1 × 106, range: 7,279–299,500), without any detectable difference between patients with normal or increased MxA mRNA values (median: 65,925 TRECs/1 × 106 cells, range: 15,259–155,003 vs. 51,125 TRECs/1 × 106 cells, range: 17,586–128,900). Similarly, there was no significant correlation between TRECs and pDC cell number (data not shown).

Bottom Line: However, in a group of patients with long disease duration, having a low number of both pDC and CD4+ lymphocytes and a significant increase of serum interferon-alpha, MxA mRNA was produced at high level and its expression directly correlated with HIV RNA copy numbers.HIV replication unresponsive to antiretroviral treatment in perinatal-infected patients with advanced disease and pDC depletion may lead to interferon-alpha expression and subsequent induction of MxA mRNA.Thus, the latter measurement may represent a valuable marker to monitor the clinical response to therapy in HIV patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Istituto di Medicina Molecolare Angelo Nocivelli, Department of Pediatrics, University of Brescia, Brescia 25123, Italy. badolato@med.unibs.it

ABSTRACT

Background: To determine the role of interferon-alpha in controlling HIV infection we phenotypically and functionally analyzed circulating plasmacytoid dendritic cells (pDC), which are known to be the highest interferon-alpha producing cells, in 33 perinatally infected HIV+ patients undergoing standard antiretroviral therapy.

Methods: Circulating pDC were identified by flow cytometry using anti-BDCA-2 monoclonal antibody and by measuring BDCA-2 mRNA by real-time PCR, while tissue-resident pDC were identified by immunohistochemistry. mRNA for interferon-alpha and MxA, a gene that is specifically induced by interferon-alpha, was quantified in peripheral blood cells by real-time PCR, while serum interferon-alpha protein was measured by ELISA.

Results: While median values of pDC, both in terms of percentage and absolute number, were not statistically different from age-matched controls, interferon-alpha mRNA was increased in HIV-infected patients. However, in a group of patients with long disease duration, having a low number of both pDC and CD4+ lymphocytes and a significant increase of serum interferon-alpha, MxA mRNA was produced at high level and its expression directly correlated with HIV RNA copy numbers. Furthermore in patients displaying a low CD4+ blood cell count, a severe depletion of pDC in the tonsils could be documented.

Conclusion: HIV replication unresponsive to antiretroviral treatment in perinatal-infected patients with advanced disease and pDC depletion may lead to interferon-alpha expression and subsequent induction of MxA mRNA. Thus, the latter measurement may represent a valuable marker to monitor the clinical response to therapy in HIV patients.

Show MeSH
Related in: MedlinePlus