HIV infection of thymocytes inhibits IL-7 activity without altering CD127 expression
© Young and Angel; licensee BioMed Central Ltd. 2011
Received: 15 March 2011
Accepted: 16 September 2011
Published: 16 September 2011
Thymic function is altered in HIV infection and characterized by dysregulation of the thymic epithelial network, reduced thymic output and ultimately an impaired naïve T-cell pool. The IL-7/IL-7 receptor (IL-7R) signalling pathway is critical for the maturation and differentiation of thymocytes. HIV infection is associated with a decrease in IL-7Rα (CD127) expression and impaired CD127 signalling in circulating CD8+ T-cells; however, little is known about the effect of HIV on CD127 expression and IL-7 activity in the thymus. Therefore, the effect of in vitro HIV infection on CD127 expression and IL-7-mediated function in thymocytes was investigated.
In vitro HIV infection of thymocytes did not affect CD127 expression on either total thymocytes or on single positive CD4 or single positive CD8 subsets. However, HIV infection resulted in a decrease in the level of IL-7-induced STAT-5 phosphorylation and Bcl-2 expression in unfractionated thymocytes.
These findings indicate that HIV infection alters IL-7 responsiveness of thymocytes by a mechanism other than CD127 downregulation and potentially explain the disruption in thymopoiesis observed in HIV infection.
Human immunodeficiency virus (HIV) infection is characterized by a loss of CD4+ T-cells and a progressive loss in cytotoxic T-cell lymphocyte (CTL) function resulting in immunodeficiency. HIV infection has also been associated with impaired thymic output . Examination of the thymus of HIV-infected pediatric patients reveals selective thymocyte depletion and disruption of the thymic microenvironment, which is thought to contribute to more rapid progression to AIDS [2–4]. In HIV-1 infected SCID-hu Thy/Liv mouse models, there is a depletion of intrathymic progenitor T-cells which precedes the loss of infected CD4+CD8+ thymocytes, suggesting that HIV infection interrupts thymocyte development at an early stage . However, the mechanisms of disrupted thymic development by HIV have yet to be fully elucidated.
Interleukin-7 (IL-7) is a pleiotropic cytokine that is critical for several stages of thymopoiesis, maintains mature T-cell homeostasis, enhances CTL function and increases T-cell survival [6–14]. IL-7 signals through the IL-7 receptor complex (IL-7R), which is composed of two subunits: the IL-7Rα chain (CD127), that is also shared by TSLP , and the IL-2Rγ chain which is shared by a number of other cytokines including IL-2, IL-4, IL-9, IL-15 and IL-21 [7, 8]. The role of IL-7 in thymopoiesis is multifaceted, as it is critical for early stages of T-cell development in allowing chromatin accessibility to enable T-cell receptor VDJ gene rearrangement, inducing thymocyte proliferation and maintaining thymocyte survival by upregulating the anti-apoptotic protein Bcl-2 and downregulating the pro-apoptotic protein Bax [12, 16–18]. Disrupting IL-7 signalling can result in profoundly impaired immunity as seen in patients with T-B+NK+ Severe Combined Immunodeficiency (SCID), a genetic defect that results in inactivation of the IL-7Rα signalling pathway . The importance of the IL-7 signalling complex in thymic development was confirmed in knock-out mice for both IL-7 and IL-7R. IL-7-/- mice have a 20 fold decrease in thymic cellularity and an increase in triple negative (TN) cells, indicative of a developmental block at the TN stage . The phenotype with IL-7R-/- knockout mice is much more severe with a 90-99.99% decrease in thymic cellularity .
We and others have previously demonstrated that HIV infection is associated with decreased CD127 expression on circulating CD8+ T-cells, and with effective antiretroviral therapy CD127 expression on T-cells is partially restored [21–23]. The regulation of CD127 by HIV may play a role in disease pathogenesis since the expression of CD127 has been correlated with measures of disease progression (decreased CD4 count, increased viral load, increased immune activation) [24, 25]. In addition to decreased CD127 expression on T-cells, we and others have also shown that CD127 signalling is impaired in HIV infection [26–28]. Given the importance of the role of IL-7 in HIV pathogenesis and the current development of IL-7 as a therapeutic agent for HIV infection and other conditions, understanding the mechanism by which HIV impairs IL-7 activity within the thymus is of the greatest importance. The aim of this study is to evaluate the effects of HIV infection on CD127 expression and IL-7 activity in primary human thymocytes.
IL-7 also signals through the PI3K pathway leading to cell proliferation and glucose uptake . Thymocytes were infected for up to 96 hours, serum starved for 2 hours and stimulated with IL-7 (10 ng/ml) for 1 hour. Cells were lysed, and proteins were separated on an 8% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Activation of the PI3K pathway was visualised by probing the membranes with antibodies for phosphorylated AKT (Cell Signalling, Danvers, MA). In contrast to its effect on STAT-5 and Bcl-2, HIV infection did not affect the ability of IL-7 to induce PI3K posphorylation (data not shown).
The importance of IL-7 and its effect on thymopoiesis are unequivocal. Disrupting this pathway leads to a block in thymopoiesis and the arrest of T-cell development. IL-7 signals through both the JAK/STAT and PI3K pathways to mediate cell survival, proliferation and differentiation [35, 36]. HIV infection both in vitro and in vivo is associated with reduced CD127 on CD4+ T-cells and CD8+ T cells [21–25]. We have, however, demonstrated that in vitro HIV infection of thymocytes does not affect the surface expression of CD127 on thymocytes. The decreased CD127 expression on CD8+ T-cells following in vitro HIV infection appears to be due to soluble factors released in the culture microenvironment by PBMCs . Any such factors present in PBMC cultures may not be present in thymocyte/TEC co-cultures, potentially accounting for the differential effect of HIV on CD127 expression.
Although decreased IL-7 activity can result from decreased receptor expression, a block in the IL-7 signalling pathway may also result in altered IL-7 activity. This phenomenon has been reported for IL-2 activity where CD4+ T-cells and CD8+ T-cells from HIV+ individuals are less responsive to IL-2 compared to those from healthy controls which has been attributed to a block in the JAK/STAT pathway [37, 38]. The results in this report indicate that IL-7-induced STAT-5 phosphorylation and Bcl-2 expression are impaired in thymocyte cultures infected with HIVcs204 while no effect on CD127 expression was observed. This suggests that HIV infection results in a block in the IL-7 pathway that occurs independent of its effect on CD127 expression. These data support the findings by Vranjkovic et al., which demonstrated reduced IL-7 responsiveness in CD127-expressing CD8+ T-cells from HIV+ patients. In that study, isolated CD8+CD127+ cells from HIV+ individuals had lower levels of STAT-5 phosphorylation following IL-7 stimulation when compared to those from uninfected controls . Such a block in IL-7 signalling has also been observed in other disease states. For example, CD4+ and CD8+ T-cells isolated from breast cancer patients are less responsive to IL-7, as measured by STAT-5 phosphorylation .
HIV may affect thymocyte function by altering the viability of the cells, consequently lowering the output of functional T-cells from the thymus [2, 3, 40, 41]. In support of this hypothesis, our data show that HIV infection interferes with the ability of IL-7 to induce Bcl-2 expression. A similar block in the ability of IL-7 to upregulate Bcl-2 expression was reported in a study in which CD4+ T-cells from HIV+ individuals had lower levels of Bcl-2 expression following IL-7 stimulation when compared to those from healthy controls. That study found no correlation between CD127 expression of CD4+ T cells and IL-7 responsiveness, suggesting that the block in IL-7 activity was independent of the level of CD127 expression .
The exact mechanism by which HIV interferes with the IL-7 signalling pathway has yet to be determined, however our results indicate that binding of HIV to the cell surface is likely insufficient to mediate this effect since there was no impact of HIV on IL-7 activity within the first 24 hours of infection. Rather, our data demonstrated that the cells need to be infected for longer periods of time (72-96 hours) for the effect of HIV to be observed, suggesting that the mechanism of inhibition might require the production of specific cellular or viral factors.
In summary, we demonstrated that HIV infection alters IL-7 activity in thymocytes independent of CD127 expression suggesting a potential mechanism by which HIV infection interrupts thymic output and contributes to immune deficiency.
We appreciatively acknowledge Dr. G. Maharajh and staff at the Children's Hospital of Eastern Ontario for providing the thymus samples. We are grateful to Dr. Angela Crawley for critical review of the manuscript. This research was supported by grants to J.B.A from the Ontario HIV Treatment Network (Grant #ROGB131), the Canadian Institutes of Health Research (Grant #HOP84649) and the Canadian Foundation for AIDS Research (Grant # 019014). C.Y. is a recipient of a CIHR studentship and J.B.A. is an OHTN Career Scientist.
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