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Association of Functional Impairment with Inflammation and Immune Activation in HIV-1-Infected Adults on Effective Antiretroviral Therapy
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Journal of Infectious Diseases Advance Access published April 4, 2013
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Kristine M Erlandson1,2, Amanda A Allshouse3, Catherine M Jankowski2, Eric J
Lee1, Kevin M Rufner4, Brent E Palmer5, Cara C Wilson1, Samantha MaWhinney3, Wendy M Kohrt2, Thomas B Campbell1
"we hypothesized that functional impairment in HIV-1 infection would be associated with markers of immune activation and inflammation...... [we found] Functional impairment during successful antiretroviral therapy was associated with higher CD8+ T cell activation and interleukin-6 levels. Interventions to decrease immune activation and inflammation should be evaluated for effects on physical function and frailty.....We also detected higher monocyte activation, as measured by sCD14, although the differences were not statistically significant after adjusting for smoking status. Monocyte activation is associated with frailty and disability in HIV-uninfected persons [8,33] and with mortality among HIV-infected persons.......Functional status incorporates the concomitant processes of aging, inflammation, HIV-1 infection, comorbid diseases and lifestyle. Using a case-control design, we demonstrated significant independent associations of functional impairment with IL-6 and CD8+ T-cell activation in patients on effective combination antiretroviral therapy. Our findings add to a growing body of evidence for the association of chronic immune activation and inflammation to the development of non-AIDS morbidity and mortality [30-32]. To our knowledge, this is the first evidence linking physical function and frailty to immune activation and inflammation among persons with HIV-infection. .......Similar to prior studies of frailty and disability among HIV-uninfected elderly, we detected an association between high IL-6 and low function and a trend towards higher hs-CRP and TNF-alpha, suggesting an overall inflammatory milieu among functionally impaired persons. Moreover, increased CD8+ T-cell activation was also associated with functional impairment among HIV-1 infected persons. Higher IL-6 is closely linked to frailty, disability and mortality among HIV-uninfected elderly [3,7], while CD8 T-cell activation is associated with disease progression and poor immune recovery in HIV-1 infected persons [17]. However, the role of CD8+ T-cell activation in physical function impairment is largely unexplored in HIV- infected or -uninfected populations [8]."
"369 consented and 359 completed functional testing. 33 (9%) were identified as low-function, 186 (52%) as moderate and 140 (39%) as high-function......Telomere length from our cohort (mean 64.8 kb/diploid genome, SD 22) was similar to an uninfected cohort 15 years older (mean 69.5 kb/diploid genome, SD 37) [26] suggesting an increase in cell turnover unrelated to functional capacity......We identified a distinct clinical phenotype based on physical function and frailty assessment that is associated with markers of inflammation and immune activation that predict poor prognosis in HIV-1 infected persons. A measurement of physical function can be easy, fast and inexpensive. Future interventions targeted at decreasing immune activation and inflammation in HIV-1 infected persons should assess the clinical impact of therapy through measures of physical function......Low-function cases had lower nadir CD4+ T-cell count, a greater number of comorbid conditions, higher Veterans Aging Cohort Study Index Scores and were prescribed a greater number of non-antiretroviral medications"
Abstract
Background. The relationships of inflammation, immune activation and immune senescence markers with functional impairment in aging HIV-1-infected persons are unknown.
Methods. HIV-1-infected persons, aged 45 to 65 years, with plasma HIV-1 RNA <48 copies/mL on antiretroviral therapy underwent standardized functional testing. In a nested case-control analysis, low-function cases were matched (1:1.5) by age, gender and HIV-1 diagnosis date to high-function controls. Markers of inflammation, T-cell activation, microbial translocation, immune senescence and immune recovery estimated functional status in conditional logistic regression. Primary analyses were adjusted for CD4 count, smoking, and hepatitis.
Results. 31 low-function cases were compared to 49 high-function controls. After adjusting, lower proportions of CD4+ T-cells and higher proportion of CD8+ T-cells, higher CD38/HLA-DR expression on CD8+ T-cells, and higher interleukin-6 were associated with a significantly greater odds of low-function (all OR≥1.1; P≤0.03). Other inflammatory, senescence and microbial translocation markers were not significantly different (all P ≥0.11) between low and high-function groups.
Conclusion. Functional impairment during successful antiretroviral therapy was associated with higher CD8+ T cell activation and interleukin-6 levels.
Interventions to decrease immune activation and inflammation should be evaluated for effects on physical function and frailty.
Study Population
All individuals receiving care for HIV-1 infection in the Infectious Diseases
Group
Practice at the University of Colorado were evaluated for participation.
Individuals
meeting the following criteria were eligible: 1) 45 to 65 years of age; 2) able to consent and participate in study procedures; and 3) taking a minimum of two antiretroviral drugs for at least six months with one undetectable plasma HIV-1 RNA (<48 copies/mL) and no plasma HIV-1 RNA >200 copies/mL in the prior six months. Approval was obtained by the Colorado Multiple Institutional Review Board and informed consent was obtained from all participants.
Functional Assessments
A frailty phenotype was assessed as previously described by Fried, et al [20]. Shrinking was defined as unintentional weight loss of ≥ 10 pounds, or decrease of 5% of body weight in the last year (self-reported and verified by records when available). Exhaustion was defined by three to four times per week of feeling "everything I do is an effort" or "sometimes I just cannot get going" [11,12,14,20]. Low activity was defined as self-report of being "limited a lot" in vigorous physical activities from the Short-Form (SF)-36 ® [11,12,14].
Weakness was assessed by the average of three dominant hand grip measurements using a single Lafayette dynamometer, applying previously defined gender and body mass index (BMI) cutoffs [20]. Slowness was defined by 4.5-m walk time: men ≤173 cm and women ≤159 cm in height requiring ≥7 seconds or men >173 cm and women >159 cm requiring ≥6 seconds met a criterion [20]. One point was given for each abnormality. Three to five points were considered low function, one or two points moderate function, and zero points high function [20].
The Short Physical Performance Battery assessed tandem stand, walking speed, and sit-stand test time, with zero points indicating inability to complete a task and four points indicating performance within the expected range [21]. Tandem stand was measured by ability to stand heel-to-toe for ten seconds, walking speed by the faster of two 4-m walks at usual pace, and sit-stand test time by five repetitions of sit-to-stand without use of the arms [21]. A score of less than 9 is highly predictive for subsequent disability and was considered low function, 9-11 points moderate function, and 12 points (no deficits) high function [21,22].
400-m walk time was measured on a set walking course by asking the participant to walk as quickly as possible to complete the distance [23,24]. We defined high-function as ability to complete a 400-m walk and no deficits on either the frailty phenotype or the Short Physical Performance Battery. Low-function was defined as a score of 3 or more on the frailty phenotype or a score of less than 9 on the Short Physical Performance Battery with at least 1 deficit on the opposing test. Moderate function was defined as at least 1 deficit on the frailty phenotype or Short Physical Performance Battery but not meeting the definition of low-function.
RESULTS
Study Population
Between February and November, 2010, 542 HIV-1-infected persons met eligibility requirements and were asked to participate: 171 either did not respond to correspondence or were not interested; 2 died prior to obtaining consent; 369 consented and 359 completed functional testing. 33 (9%) were identified as lowfunction, 186 (52%) as moderate and 140 (39%) as high-function. One low-function person moved from the area and the other could not be contacted. 31 low-function cases were matched to 49 high-function controls and underwent laboratory evaluations at the second study visit. Overall, participants were 85% male and 74% Caucasian with a median age of 50.8 years (interquartile range 47.7-55.7 years), median CD4+ lymphocyte count of 551 cells/μL (interquartile range 361-768 cells/μL), and HIV-1 RNA below limits of detection in 95% of subjects [29].
The low- and high-function groups were similar by baseline demographic characteristics with the exception that low-function cases were more likely to be current smokers (Table 1). Low-function cases had lower nadir CD4+ T-cell count, a greater number of comorbid conditions, higher Veterans Aging Cohort Study Index Scores and were prescribed a greater number of non-antiretroviral medications (Table 1). Three subjects had detectable plasma HIV-1 RNA on routine laboratory monitoring after the screening visit (1 low-function case had 491 copies/mL, 2 high-function controls had 78 and 1670 copies/mL). One low-function case had a prior liver transplant and was receiving cyclosporine and sirolimus at the time of evaluation. Although total CD4+ T-cell count was not significantly different between low-function and high-function groups (Table 1), lower CD4%, higher CD8%, and lower CD4/8 ratio were associated with greater odds of low-function status (Table 2). Significant differences were detected between low- and high-function groups in CD4+ T-cell percentage (Figure 1A), CD8+ Tcell percentage (Figure 1B), and CD4/CD8 ratio (Figure 1C).
Markers of Immune Activation and Inflammation
To determine variability in CD38/HLA-DR expression on CD8+ T-cells, 20 subjects had a repeat blood draw two to five days after the initial blood draw. Among these 20 subjects, interclass correlation was 0.88 (95% CI 0.73, 0.95). CD8+ but not CD4+ T-cell expression of CD38/HLA-DR was associated with a greater odds of being low-function, and remained significant after adjusting for CD4+ T-cell count, smoking, and hepatitis B or C (Table 2). Low-function persons had higher percentages of CD8+ and CD4+ T-cells co-expressing CD38 and HLA-DR compared to high-function persons (Figures 1D and 1E). In multivariate analyses adjusting for CD4+ T-cell count, smoking, and hepatitis, CD38/HLA-DR on CD8+ T-cells remained significantly higher among low-function persons (adjusted difference = 4.8, 95% CI 1.4, 8.3; adjusted P = 0.02) but significant differences in CD4+ T-cell activation were no longer detected (adjusted-difference = 2.7, 95% CI -0.09, 5.4; adjusted P = 0.07).
Among the three subjects with detectable plasma HIV-1 RNA (Table 1), CD8+ T-cell expression of CD38/HLA-DR increased with increasing plasma HIV-1 RNA concentration (7% in a control with 78 copies/mL, 17% in a case with 491 copies/mL, and 25% in a control with 1670 copies/mL). CD8+ T-cell expression of CD38/HLA-DR remained higher in low-function cases (17.6 ± 1.4%) than high-function controls (12.6 ±1.2%) when the three subjects with detectable plasma HIV-1 RNA were excluded (P =0.003). Among all subjects, higher CD4+ and CD8+ T-cell activation were moderately inversely correlated with CD4+ T-cell count (r = -0.41, P < 0.001 and r = -0.38, P =0.002, respectively).
Higher plasma sCD14 was associated with greater odds of being low-function, however differences were not robust after adjustment (Table 2). Similarly, plasma sCD14 concentration was higher among low- compared to high-function persons in univariate (Figure 1F) analysis but was not significant after multivariate analysis (adjusted difference= 0.3; 95% CI 0.5, 6.0; adjusted P = 0.09).
Although each log increase in IL-6 increased the odds of low- function classification by 1.2 (95% CI 1.02, 1.51, p=0.03), TNF-alpha and hs-CRP were not associated with a greater odds of low-function (Table 2). In mixed model regression, plasma IL-6 and TNF-alpha concentrations were significantly higher among low- than high-function persons in univariate analyses (Figure 2A and 2B). IL-6 but not TNF-alpha concentration remained significantly higher in the adjusted model (IL-6 log10 unadjusted difference = 0.36, 95% CI -0.54, -0.18, log10 adjusted difference = 0.31, 95% CI -0.50, -0.12, adjusted P = 0.002 and TNF-alpha adjusted difference = 0.5, 95% CI -0.02, 1.0; adjusted P = 0.06). There was a trend towards higher hs-CRP concentration in low-function cases (Figure 2C).
Markers of Microbial Translocation
Markers of microbial translocation were not significantly associated with a greater odds of being low-function (Table 2) and in secondary analyses were not significantly different between low- and high-function groups in unadjusted or adjusted analyses: 16S rDNA, LPS, LBP, i-FABP, or endoCAB (Figure 3A-E).
Markers of Immunosenescence
Lack of CD28 expression on CD4+ or CD8+ T-cells was not associated with greater odds of low-function (Table 2) and was not significantly different between functional groups (Figure 4A and 4B). Quadruplicate measurements of telomere length had an interclass correlation of 0.89 (95% CI 0.86, 0.91). Mean leukocyte telomere length was not associated with greater odds of low-function (Table 2) and was not significantly different between functional groups (Figure 4C). Among all subjects, there was no significant correlation between age and percentage of CD8+ T-cells without CD28 expression (r = 0.21, P = 0.06) or telomere length (r = 0.03, P = 0.71).
DISCUSSION
Functional status incorporates the concomitant processes of aging, inflammation, HIV-1 infection, comorbid diseases and lifestyle. Using a case-control design, we demonstrated significant independent associations of functional impairment with IL-6 and CD8+ T-cell activation in patients on effective combination antiretroviral therapy. Our findings add to a growing body of evidence for the association of chronic immune activation and inflammation to the development of non-AIDS morbidity and mortality [30-32]. To our knowledge, this is the first evidence linking physical function and frailty to immune activation and inflammation among persons with HIV-infection.
Similar to prior studies of frailty and disability among HIV-uninfected elderly, we detected an association between high IL-6 and low function and a trend towards higher hs-CRP and TNF-alpha, suggesting an overall inflammatory milieu among functionally impaired persons. Moreover, increased CD8+ T-cell activation was also associated with functional impairment among HIV-1 infected persons. Higher IL-6 is closely linked to frailty, disability and mortality among HIV-uninfected elderly [3,7], while CD8 T-cell activation is associated with disease progression and poor immune recovery in HIV-1 infected persons [17]. However, the role of CD8+ T-cell activation in physical function impairment is largely unexplored in HIV- infected or -uninfected populations [8].
We also detected higher monocyte activation, as measured by sCD14, although the differences were not statistically significant after adjusting for smoking status. Monocyte activation is associated with frailty and disability in HIV-uninfected persons [8,33] and with mortality among HIV-infected persons [36]. Given that sCD14 is released from monocytes upon LPS stimulation, we expected to find evidence of microbial translocation among low-functioning persons. Contrary to our hypothesis, differences in microbial translocation markers were not detected. Given that all subjects were treated with effective antiretroviral therapy, we suspect immune reconstitution from antiretroviral therapy attenuated any potential differences between low- and high-functioning individuals. Furthermore, variability in the assays and transient fluctuation in microbial translocation products may have impacted our ability to detect differences.
In HIV-uninfected persons, chronic inflammation, antigen stimulation, and age lead to immune remodeling manifest by decline in CD4+ T-lymphocytes, increase in CD8+ T-lymphocytes, and expansion of CD28(-)T-lymphocytes with shortened telomeres [3]. These changes have been associated with frailty and disability in HIV-uninfected elderly [3-5,34,35]. Although the median number of CD4 lymphocytes for our subjects was greater than 500 cells/μL and current CD4 T cell count was similar between cases and controls, low-functioning cases had lower nadir CD4 T cell count and lower percent of CD4+ T cells. Thus, our findings suggest that lower nadir CD4 count prior to initiation of antiretroviral therapy and continued perturbations in CD4+ T cell proportions during antiretroviral therapy place HIV-infected patients at risk for functional impairment through chronic immune activation and inflammation.
Although HIV-uninfected elderly with low-functional status have increased markers of immune senescence, we did not detect significant differences in CD8+ CD28(-) T-cells, CD4+CD28(-) T-cells, or telomere length between low- and high-functioning groups. Although telomere length does appear to shorten with time and with several disease processes, considerable inter-individual variability is due to an array of genetic, environmental, and lifestyle factors, which complicates between-group comparisons [36]. Telomere length from our cohort (mean 64.8 kb/diploid genome, SD 22) was similar to an uninfected cohort 15 years older (mean 69.5 kb/diploid genome, SD 37) [26] suggesting an increase in cell turnover unrelated to functional capacity.
The strengths of our study were the careful matching of low- and high-functioning participants to control for potential effects of age, gender, and duration of HIV infection on inflammatory and activation markers. A relatively narrow age range provided focus on the relationships between inflammation and physical impairment in early aging, rather than age-related changes in the elderly. Limitations to our study include the cross-sectional and observational design. Longitudinal studies are needed to investigate whether increased activation and inflammation are associated with a decline in functional capacity, or whether interventions to decrease inflammation and immune activation improve physical function. We did not control for differences in comorbid conditions or medications and larger studies are needed to study the effects of specific comorbidities or medications (including statins) on physical function, immune activation and inflammation.
Our study was the first comparison of markers of immune activation, immune senescence, and microbial translocation in HIV-infected persons with phenotypes defined by functional capacity. We identified a distinct clinical phenotype based on physical function and frailty assessment that is associated with markers of inflammation and immune activation that predict poor prognosis in HIV-1 infected persons. A measurement of physical function can be easy, fast and inexpensive. Future interventions targeted at decreasing immune activation and inflammation in HIV-1 infected persons should assess the clinical impact of therapy through measures of physical function.
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