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Extended Interferon-Alpha Therapy Accelerates Telomere Length Loss in Human Peripheral Blood T Lymphocytes
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PLoS One
August 4, 2011
Joel M. O'Bryan1, James A. Potts1, Herbert L. Bonkovsky2,3, Anuja Mathew1*, Alan L. Rothman1,4, for the HALT-C Trial Group
1 Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 2 Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut, United States of America, 3 Carolinas Medical Center, Charlotte, North Carolina, United States of America, 4 Institute for Immunology and Informatics, University of Rhode Island, Providence, Rhode Island, United States of America
In subjects with cHCV infection, sustained peg-IFNα therapy (90 μg/week) was associated with an increased rate of TL loss in both CD4+ and CD8+ T cell subsets. Additionally, based on a single intermediate time-point, this IFNα therapy-enhanced TL loss was fully concentrated in the initial 21 months. The delineation of TL changes in T cell CD45RA+/-/CD57- subsets shows that declines in the T cell TL are not shifts in naïve (long TL) versus memory (shortened TL) T cell proportions, but true decreases in TL with the greatest impact on the naïve T cell subsets. The lack of an accelerated TL loss effect in naïve T cells in the second interval in the peg-IFNα group suggests that a new homeostasis is reached. Our findings are consistent with the telomere erosion effects of increased lymphocyte turn-over in response to a sustained lymphopenic signal as predicted from the mathematical models of de Boer and Noest [29]. Increased T cell turn-over would also account for our observation that sustained peg-IFNα treatment inhibited the expansion of CD8+ CD45RA+ CD57+ TEMRA cells, a phenomenon observed in the control group.
Enhanced T cell TL loss in subjects who received long-term peg-IFNα therapy suggests that T cells in these subjects have reduced proliferative reserve. Subjects receiving type I IFN therapy are known to be more susceptible to bacterial infections; this has been attributed to neutropenia, but several studies have shown no temporal correlation between neutrophil count and infections [38], [39]. Diminished memory T cells and proliferative reserve related to naïve T cell TL loss, as shown in this study, could contribute to the increased susceptibility to infection and disease while on interferon therapy. Importantly, diminished naive T cell proliferative TL reserve incurred under sustained IFN therapy may persist well beyond the end of therapy. Indeed, as age-related thymic involution severely limits production of new, long telomere, naïve T cells [37], a sustained accelerated TL erosion thus may leave a permanently degraded naïve T cell compartment. Support for this possibility comes from a recent analysis of a subset of patients from the HALT-C cohort prospectively followed for more than 5 years after the trial. That study showed rates of non-liver-related death were significantly higher (p = 0.01) among patients with liver fibrosis who received the 31/2 year peg-IFNα therapy compared to similar patients in the control arm [40].
Abstract
Background
Type I interferons have pleiotropic effects on host cells, including inhibiting telomerase in lymphocytes and antiviral activity. We tested the hypothesis that long-term interferon treatment would result in significant reduction in average telomere length in peripheral blood T lymphocytes.
Methods/Principal Findings
Using a flow cytometry-based telomere length assay on peripheral blood mononuclear cell samples from the Hepatitis-C Antiviral Long-term Treatment against Cirrhosis (HALT-C) study, we measured T cell telomere lengths at screening and at months 21 and 45 in 29 Hepatitis-C virus infected subjects. These subjects had failed to achieve a sustained virologic response following 24 weeks of pegylated-interferon-alpha plus ribavirin treatment and were subsequently randomized to either a no additional therapy group or a maintenance dose pegylated-IFNα group for an additional 3.5 years. Significant telomere loss in naïve T cells occurred in the first 21 months in the interferon-alpha group. Telomere losses were similar in both groups during the final two years. Expansion of CD8+CD45RA+CD57+ memory T cells and an inverse correlation of alanine aminotransferase levels with naïve CD8+ T cell telomere loss were observed in the control group but not in the interferon-alpha group. Telomere length at screening inversely correlated with Hepatitis-C viral load and body mass index.
Conclusions/Significance
Sustained interferon-alpha treatment increased telomere loss in naïve T cells, and inhibited the accumulation of T cell memory expansions. The durability of this effect and consequences for immune senescence need to be defined.
Introduction
Telomeres are repetitive DNA sequences, consisting of hundreds to thousands of double-stranded repeats, found at both ends of every chromosome [1]. A normal cell's replicative potential has been linked to a combination of its telomere length (TL) and the ability to express telomerase. Telomerase assists in TL maintenance and slows telomere erosion during activation-induced proliferation of T lymphocytes [2], [3]. Type I interferons (IFN), in addition to anti-viral and anti-proliferative effects, inhibit expression and activity of telomerase [4]. IFN also commonly causes lymphopenia [5], which is a stimulus for homeostatic proliferation [6]. How these competing IFN responses combine to modulate TL in peripheral naïve and memory T cells is currently unclear.
Combination pegylated-IFNα (peg-IFNα) with ribavirin is the standard therapy for subjects with chronic hepatitis C virus (cHCV) infection. Unfortunately, therapy results in a sustained virologic response (SVR) in less than 50% of HCV subjects [7]. After decades of cHCV, many patients progress to liver cirrhosis and subsequent hepatic failure, and are at risk for developing hepatocellular carcinoma [8]. The Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) trial was a clinical trial designed to assess whether sustained peg-IFNα reduced the progression of liver disease in subjects who did not achieve SVR [9]. All study subjects initially received peg-IFNα plus ribavirin for 24 weeks. Subjects failing to achieve SVR were then randomly assigned either to a control, monitor-only group or to a continued peg-IFNα-treated group at a maintenance dose for an additional 3.5 years [10].
Multiple clinical measures, such as patient age, duration of infection, viral load, obesity, and liver enzyme levels have been noted to correlate to varying degrees with the IFNα treatment virologic response rate in cHCV infection [11], [12], [13], [14], [15], [16]. The role of liver hepatocyte destruction, seen as elevated blood serum levels of alanine aminotransferase (ALT), may reflect cytotoxic killing of virus infected cells by the on-going immune response. Thus serum ALT levels are used in monitoring the progression of liver damage [17]. Additionally, increasing obesity, commonly measured as body mass index (BMI), has been observed to affect telomere length in peripheral blood leukocyte subsets in otherwise healthy adults [18], [19], [20]. The availability of these clinical measures for the HALT-C cohort allowed for additional analyses in this study of their interactions with the measured peripheral blood T cell telomere lengths and telomere length changes from screening to study month 45.
In this study the primary aim was to examine the effects of long-term peg-IFNα therapy on TL in peripheral blood T lymphocytes using a flowFISH (flow cytometric fluorescence in situ hybridization) telomere length assay. Here we report significant associations between changes in TL and treatment group, patient age, serum HCV RNA level, body mass index (BMI) and alanine aminotransferase (ALT) levels.
Discussion
In subjects with cHCV infection, sustained peg-IFNα therapy (90 μg/week) was associated with an increased rate of TL loss in both CD4+ and CD8+ T cell subsets. Additionally, based on a single intermediate time-point, this IFNα therapy-enhanced TL loss was fully concentrated in the initial 21 months. The delineation of TL changes in T cell CD45RA+/-/CD57- subsets shows that declines in the T cell TL are not shifts in naïve (long TL) versus memory (shortened TL) T cell proportions, but true decreases in TL with the greatest impact on the naïve T cell subsets. The lack of an accelerated TL loss effect in naïve T cells in the second interval in the peg-IFNα group suggests that a new homeostasis is reached. Our findings are consistent with the telomere erosion effects of increased lymphocyte turn-over in response to a sustained lymphopenic signal as predicted from the mathematical models of de Boer and Noest [29]. Increased T cell turn-over would also account for our observation that sustained peg-IFNα treatment inhibited the expansion of CD8+ CD45RA+ CD57+ TEMRA cells, a phenomenon observed in the control group.
By HALT-C trial design, subjects in both groups received combination therapy with peg-IFNα (180 μg/week) plus ribavirin for the initial 24 weeks of the study. PBMC were not available from other intermediate time points. Therefore, it is possible that accelerated TL loss occurred in both groups during the initial phase of lead-in therapy. Average TL could then have rebounded in the control group between the end of therapy at week 24 and month 21 whereas ongoing, lower-dose peg-IFNα therapy maintained the lower average TL (or suppressed a TL recovery) in the therapy group. Consistent with this interpretation, the decline in TL was slower during the second interval than in the earlier interval in the therapy group. If this interpretation is correct, it is impossible to determine whether the effect of the initial therapy on TL is attributable to the higher dose of peg-IFNα or to ribavirin. Nevertheless, sustained peg-IFNα was clearly associated with accelerated TL loss during the subsequent 31/2 years in comparison with the control group.
An important finding from our study is the correlation of baseline T cell TL in these cHCV subjects with viremia and BMI. It should be noted that subjects in the randomization phase of the trial had endured HCV viremia for decades (range: 14-51 years) and failed to achieve SVR during combination peg-IFNα-ribavirin therapy. The absence of a negative correlation of baseline T cell TL with age in this cohort may therefore reflect stronger impacts of long-term viremia and the generally high levels of obesity in these subjects.
We speculate that the negative association between serum viral RNA levels and baseline TL reflects chronic elevated T cell activation, cell death, and proliferation due to persistent presentation of HCV antigens in a dose-dependent manner. Alternatively, higher circulating viral RNA levels may also drive greater endogenous type I IFN production. A negative correlation of BMI with baseline TL in naïve T cells could also reflect chronic inflammation in obesity [30]. Chronic inflammation as a result of obesity may lead to decreased thymic output [31], inducing increased homeostatic proliferation of naïve T lymphocytes and thus a decrease in TL through replicative erosion.
Another possible explanation for the lack of an age-dependent TL association in this cohort relates to evidence that short telomeres play a causal role in a variety of age-related diseases [32], [33], [34], [35]. Thus it is plausible that study inclusion criteria biased the enrolled study population away from subjects with very short telomeres.
Elevated ALT levels in cHCV are indicative of hepatocellular inflammation and necrosis [12]. Our finding of a correlation of serum ALT levels with naïve T cell TL loss in the control group likely reflects T cell responses to infected HCV-infected hepatocytes and the extended time frame over which telomere lengths were analyzed. This relationship was possibly obscured in the peg-IFNα group by the effects of continuous therapy. The correlation between TL changes and ALT, which could be due to T cell clonal exhaustion, immunosenescence, or a combination of these and/or other immunological factors, suggests that normalization of liver enzymes levels in the blood may coincide with a reduction in T cell turn-over and thus reduced telomere erosion.
We hypothesized that maintenance peg-IFNα therapy would cause increased TL loss in T cells as a result of inhibition of telomerase activity [4], [36]. However, we did not find a significant difference between the peg-IFNα and control groups in telomerase activity in PBMC stimulated in vitro. It is quite possible that using in vitro-stimulated PBMC the telomerase assay fails to detect an inhibitory effect of peg-IFNα therapy that existed in vivo. In any case, a telomerase inhibition mechanism falls short in explaining the predominance of an accelerated TL loss on naïve T cells in these older adults where thymic output of new, longer telomere naïve T cells is thought to be neglible.
We further found a decline in the peg-IFNα effect on TL loss with increasing age. Increasing age is associated with increased failure rates of IFN therapy for cHCV [14]. This may suggest that elevated TL erosion caused by peg-IFNα is counteracted by replicative senescence deferentially within T cell compartments. The delineation of TL loss effect by CD4+ and CD8+ T cell subset by age group results suggest a hierarchy of age-dependent T cell senescence in chronic HCV patients. Thus senescence may occur in the order: memory CD4+>memory CD8+>naïve CD4+>naïve CD8+ in this cHCV setting. Further, this result of the memory CD4+ T cell compartment becoming refractory to IFN-induced TL decreases as a marker of immunosenescence onset is consistent with the finding by Hoare, et al. where they found TL in CD4+ CD45RO+ memory T cells was a stronger predictor of SVR with IFN therapy than TL in any other T cell subset [37].
Enhanced T cell TL loss in subjects who received long-term peg-IFNα therapy suggests that T cells in these subjects have reduced proliferative reserve. Subjects receiving type I IFN therapy are known to be more susceptible to bacterial infections; this has been attributed to neutropenia, but several studies have shown no temporal correlation between neutrophil count and infections [38], [39]. Diminished memory T cells and proliferative reserve related to naïve T cell TL loss, as shown in this study, could contribute to the increased susceptibility to infection and disease while on interferon therapy. Importantly, diminished naive T cell proliferative TL reserve incurred under sustained IFN therapy may persist well beyond the end of therapy. Indeed, as age-related thymic involution severely limits production of new, long telomere, naïve T cells [37], a sustained accelerated TL erosion thus may leave a permanently degraded naïve T cell compartment. Support for this possibility comes from a recent analysis of a subset of patients from the HALT-C cohort prospectively followed for more than 5 years after the trial. That study showed rates of non-liver-related death were significantly higher (p = 0.01) among patients with liver fibrosis who received the 31/2 year peg-IFNα therapy compared to similar patients in the control arm [40].
Although extended type I interferon therapy beyond 48 weeks in the cHCV settings is not typically warranted, there are additional clinical settings where extended interferon therapy is utilized. Some examples include relapsing-remitting multiple sclerosis and melanoma [41], [42]. Accordingly, as extended type I interferon is clinically practiced, our findings suggest that additional studies of the effects of peg-IFNα therapy on age-related T cell senescence are warranted.
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