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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3191-3198
IMMUNOBIOLOGY
Alteration of tumor necrosis factor- T-cell homeostasis
following potent antiretroviral therapy: contribution to the
development of human immunodeficiency virus-associated lipodystrophy
syndrome
Eric Ledru,
Névéna Christeff,
Olivier Patey,
Pierre de
Truchis,
Jean-Claude Melchior, and
Marie-Lise Gougeon
From the Unité d'Oncologie Virale, URA CNRS 1930, Département SIDA et Rétrovirus, Institut Pasteur, Paris,
France; the Service des Maladies Infectieuses et Tropicales, Centre
Hospitalier Intercommunal, Villeneuve Saint Georges, France; and the
Service des Maladies Infectieuses et Tropicales, Hôpital Raymond
Poincaré, Garches, France.
 |
Abstract |
Highly-active antiretroviral therapy (HAART) has lead to a dramatic
decrease in the morbidity of patients infected with the human
immunodeficiency virus (HIV). However, metabolic side effects, including lipodystrophy-associated (LD-associated) dyslipidemia, have
been reported in patients treated with antiretroviral therapy. This
study was designed to determine whether successful HAART was
responsible for a dysregulation in the homeostasis of tumor necrosis
factor- (TNF-), a cytokine involved in lipid metabolism. Cytokine
production was assessed at the single cell level by flow cytometry
after a short-term stimulation of peripheral blood T cells from
HIV-infected (HIV+) patients who were followed during 18 months of HAART. A dramatic polarization to TNF- synthesis of both
CD4 and CD8 T cells was observed in all patients. Because it was
previously shown that TNF- synthesis by T cells was highly
controlled by apoptosis, concomitant synthesis of TNF- and priming
for apoptosis were also analyzed. The accumulation of T cells primed
for TNF- synthesis is related to their escape from
activation-induced apoptosis, partly due to the cosynthesis of
interleukin-2 (IL-2) and TNF-. Interestingly, we observed that LD is
associated with a more dramatic TNF- dysregulation, and positive
correlations were found between the absolute number of TNF- CD8
T-cell precursors and lipid parameters usually altered in LD including
cholesterol, triglycerides, and the atherogenic ratio apolipoprotein B
(apoB)/apoA1. Observations from the study indicate that HAART
dysregulates homeostasis of TNF- synthesis and suggest that this
proinflammatory response induced by efficient antiretroviral therapy is
a risk factor of LD development in HIV+ patients.
(Blood. 2000;95:3191-3198)
© 2000 by The American Society of Hematology.
| |
Introduction |
Highly-active antiretroviral therapy (HAART) combines 1 protease inhibitor (PI) and 2 nucleoside analogue reverse transcriptase inhibitors (bi-RTI). It is highly efficient at reducing plasma human
immunodeficiency virus-1 (HIV-1) RNA to undetectable concentrations and increasing CD4 T-cell numbers. Recent studies of the immune system
reconstitution during antiretroviral therapy have pointed out the
complexity of the mechanisms involved. The increase in CD4 T cells
after HAART could result from a combination of several mechanisms
including the release of sequestered cells from lymphoid compartments
to peripheral blood,1 increased peripheral cell survival,2-4 increased peripheral
proliferation,5 and central renewal of
lymphocytes.6,7 Apart from these changes in CD4 T-cell
numbers, in vitro improvement in the proliferative response of T cells
to recall antigens,8 partial restoration of the frequency
of cytomegalovirus-specific CD4 memory T lymphocytes,9,10 and evolution of the T-cell receptor
repertoire11,12 have also been demonstrated, all of which
suggest at least a partial restoration of the immune system in response
to HAART. However, standard HAART regimens are not able to eradicate
HIV-1 infection,13 and specific immunity to HIV-1 antigens
is not preserved by antiretroviral therapy.10 As a result,
prolonged intensive therapeutic regimens might be required to maintain
suppression of viral replication.
Long-term HAART has been associated with a unique and unexpected
syndrome consisting of metabolic abnormalities and body fat redistribution, called lipodystrophy (LD), which associates various levels of peripheral fat wasting in face and limbs; hyperlipidemia; insulin resistance; and central adiposity (accumulation of visceral fat, breast hypertrophy, and cervical fat-pads).14-16
Development of this syndrome is linked to new potent antiretroviral
regimens, and although it was first recognized in patients submitted to PI therapy,15-17 it was recently reported in patients
receiving only RTIs.18-20 The pathogenesis of this
syndrome, which may be linked to premature coronary artery disease
recently reported in HAART-treated HIV-infected (HIV+)
patients,21,22 remains unknown, although direct effects of antiretroviral drugs, such as PIs and RTIs, on lipid metabolism have
been hypothesized.23,24 Proinflammatory cytokines, such as
tumor necrosis factor-alpha (TNF- ), induce a number of metabolic alterations including hyperlipidemia and insulin
resistance.25,26 At the single cell level we analyzed the
influence of HAART on TNF- synthesis in an 18-month longitudinal
study and asked whether modifications in the frequency of TNF-
producers may contribute to alterations in lipid metabolism observed in
patients with LD.
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Patients, materials, and methods |
Patients studied
We conducted a longitudinal 18-month follow-up study of 15 HIV+ patients naïve of PI therapy. Peripheral blood
samples were obtained the day before initiation of PI, at month 0 (M0),
and then at regular intervals (M1, M2, M6, M9, M12, M15, and M18). Clinical and biological characteristics of these patients are given in
Table 1. In parallel, a cross-sectional
study was performed on HIV+ patients treated for at least 9 months with PI (n = 41). Of these patients, 17 presented with
LD+, assessed according to a standardized clinical score
(Table 2). Points were assigned to the
following symptoms: peripheral facial loss (Bichat fat-pad lipoatrophy)
(2 points), peripheral fat loss with skin-fold thickness less than 6 mm
on arms or legs (1 point), prominent superficial veins on arms or legs
(1 point), abdominal obesity with waist:hip ratio greater than 0.9 (2 points), cervical fat-pad enlargement ("buffalo hump"), or
adipomasty (2 points).20 We also included 32 HIV-1+ bi-RTI persons receiving 2 RTIs but no PI therapy
and 19 HIV healthy blood donors (Centre National de
Transfusion Sanguine, Paris, France). Informed consent was obtained
from all the patients.
Monoclonal antibodies
The following monoclonal antibodies (mAbs) specific for
human surface antigens were directly coupled either to fluorescein isothiocyanate (FITC) or peridinin chlorophyll protein (PerCP): anti-CD3 immunoglobulin G1 (IgG1, clone SK7); anti-CD4 (IgG1, clone SK3); and anti-CD8 (IgG1, clone SK1) (Becton Dickinson, Pont
de Claix, France). Control antibodies were IgG1 or IgG2a FITC- or
PerCP-conjugated (Becton Dickinson). For intracellular detection of
cytokines, we used phycoerythrin-conjugated (PE-conjugated) or
FITC-conjugated anti-TNF- (clone Mab11) and PE-conjugated anti-IL-2 mAb (clone MQ1-17H12) (PharMingen, La Jolla, CA).
Cytokine production by flow cytometry
Peripheral blood mononuclear cells (PBMCs) from healthy donors or
HIV+ patients were isolated from heparinized blood by
centrifugation on a Ficoll-Hypaque density gradient (Pharmacia Biotech,
Uppsala, Sweden). The cells were then resuspended in Roswell Park
Memorial Institute-1640 medium (RPMI-1640) (Bio-Whittaker, Verviers,
Belgium) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Institut Jacques Boy, Reims, France), 10 IU/mL penicillin, 10 mg/mL
streptomycin, 20 mmol/L 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 2 mmol/L L-glutamine. Next the cells were stimulated for 16 hours with 50 ng/mL PMA (Sigma Aldrich, France), 100 ng/mL PHA
(Murex Diagnostic, Paris, France), and 300 ng/mL ionomycin (Sigma
Aldrich). Brefeldin A (Sigma Aldrich) was added at the concentration of
10 µg/mL during the last 12 hours to inhibit cytokine release.
Enumeration at the single cell level of cytokine-producing peripheral T
cells was performed as previously described.27 Briefly,
cells were stained externally with FITC-conjugated anti-CD3 or anti-CD8
mAbs, washed in phosphate-buffered serum-bovine serum albumin-sodium nitrogen (PBS-BSA-NaN3), and fixed in
PBS-BSA-NaN3 containing 1% paraformaldehyde (PFA) for 15 minutes at 4°C. Intracellular cytokine staining was then performed
with PE-conjugated anticytokine mAbs in saponin buffer (Sigma Aldrich)
for 30 minutes at 4°C. Double-stained cells were washed in
PBS-BSA-NaN3 and fixed with 1% PFA in
PBS-BSA-NaN3. Immediate acquisition of 20 000 cells on a
fluorescence-activated cell sorter (FACS) flow cytometer (FACScan,
Becton Dickinson, San Jose, CA) was followed by analysis with CellQuest
software (Becton Dickinson). Due to the down-regulation of the CD4
molecule following PMA stimulation, CD4+ T cells were
determined as CD3+ CD8 cells.
Cytokine production by enzyme-linked immunosorbent assays
TNF- and TNF-RII concentrations were determined by
enzyme-linked immunosorbent assays (QUANTIKINE ELISA; R&D Systems,
Minneapolis, MN) on plasma previously stored at  80°C.
Thresholds of detection were 4 pg/mL for TNF- and 1 pg/mL for
TNF-RII.
Susceptibility to apoptosis of cytokine-producing T cells
The relationship between the propensity to undergo apoptosis, the
synthesis of a given cytokine, and the membrane phenotyping was
performed as previously described.27,28 Briefly, [PMA + PHA + ionomycin]-stimulated PBMCs were dual-stained with
FITC-conjugated anti-CD3 or anti-CD8 mAbs and with 20 µg/mL nuclear
dye 7-amino-actinomycin D (7-AAD) (Sigma-Aldrich). Cytokine staining
was then performed as described above, and 20 000 events were
immediately analyzed on a FACScan flow cytometer. Apoptotic cells were
quantified among cytokine-producing cells according to their 7-AAD staining.
Lipid measurements
Total cholesterol and triglyceride plasmatic concentrations were
determined in the fasted state by the usual enzymatic method (Kone
Optima; Kone Instruments, Espoo, Finland). After precipitation of other
lipid components with dextran sulfate-magnesium, high-density lipoprotein (HDL) cholesterol was measured enzymatically.
Apolipoprotein A1 (apoA1) and apoB concentrations were measured by
enzyme-linked immunonephelometric assay (BNA; Behring, Marburg,
Germany). Calibration standards from the National Federation of French
Clinical Biochemistry were used.
Statistical analyses
The impact of PI therapy on immunological parameters was evaluated
with intention to treat. Nonparametric measures of associations were
used including the Mann-Whitney U test, Wilcoxon signed
rank test, and Spearman rank correlation. P < .05 was
considered significant.
| |
Results |
Accumulation of TNF--producing T cells during HAART
The HIV+ patients who participated in this study had CD4
T-lymphocyte counts of at least 50 cells per µL (mean CD4 T cells 306 ± 167 cells per µL) and plasmatic HIV RNA levels ranging
from 2.3-5.5 log10 copies per mL (mean viral load
4.2 ± 1.2 log10 copies per mL). Upon entering the
study, all patients were naive of PIs, although two-thirds of these
patients had previously received a bi-RTI combination (Table 1). All
patients in the study received triple combination therapy with bi-RTI
and the PI indinavir (IDV). Using this combined therapy, plasma HIV RNA
was decreased to levels below the limit of detection in a large
majority of patients (73% and 87% of patients at M3 and M18,
respectively), the mean reduction being 1.8 log10
copies per mL. A concomitant increase in the CD4 T-cell count was
detected in the blood of all patients, and the mean increase was
206 ± 142 CD4 T cells per µL at M12
(P < .02 vs baseline) (Figure
1). The mean absolute number of CD8 T cells
progressively decreased by a mean of 448 ± 231 CD8 T cells per
µL at M12 (P < .05 vs baseline) (Table 1). The increase
in CD4 counts was correlated to the baseline viral load
(r = 0.65, P < .02) and to the decrease in viral
load (r = 0.59, P < .04). None of these
patients developed LD. The body mass index (BMI) of patients was
unchanged during the follow-up (M0, 22.5 ± 2.4 kg/m2,
and M18, 22.8 ± 1.9 kg/m2).
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| Fig 1.
Accumulation of TNF--producing T cells following
HAART.
(A) Evolution of plasmatic viral load and ex vivo CD4 T-cell counts is
depicted in treated patients. Boxes and Whisker plots
indicate mean values, SEM and SD, respectively; vertical bars, SEM; and
shaded rectangle, threshold of viral load detection. (B) TNF- and
TNF-RII plasmatic concentrations are given, with open box indicating
HIV controls; the shaded box, HIV+
PI patients; and the solid box, HIV+
PI+ patients after 9 months of treatment including PIs. (C)
PBMCs were stimulated 16 hours with PMA, PHA, and ionomycin. Dot-plots
from a representative patient are given before and after 18 months of
treatment (left panel); percentages and absolute counts (middle and
right panel, respectively) of TNF--producing T cells following
HAART. Asterisk indicates P < .05 vs baseline level
(Wilcoxon signed rank test); paragraph symbol, P < .05 vs
HIV controls (Mann-Whitney U test).
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TNF- is a potent inducer of HIV replication and thus may be involved
in controlling the pool of latently infected cells.29 As a
result, it was important to determine the evolution of TNF- production during HAART. We previously reported that the chronic phase
of HIV infection is associated with a moderate decrease in the
proportion of T cells producing TNF-, determined by single cell
analysis of intracellular cytokine expression following PMA + PHA + ionomycin activation, and correlated with progression of HIV
disease.27 We confirmed that baseline proportions of CD3+TNF-+ T cells in HIV+
patients were lower than the baseline proportions of control donors
(23.2% ± 16.4% vs 37.4% ± 23.4%, respectively). This
same decrease in the proportion of TNF-+ T cells was
observed in both CD4 and CD8 subsets. A reduction in the absolute
number of CD4 T cells producing TNF- was detected in untreated
patients compared with healthy donors (121 ± 98 cells per
µL vs 374 ± 201 cells per µL
[P < .01]), whereas the absolute number of
CD8+TNF-+ T cells was not
altered (Figure 1C). In parallel, baseline plasma levels of TNF- and
TNF-RII were increased in patients compared with controls (Figure 1B),
and single cell analysis of TNF- production by monocytes suggested
that in patients not receiving PIs, plasma TNF- originated from
monocytes (M. Bocchino et al, unpublished data, May 1999).
Following PI administration, a dramatic increase in the percentage and
absolute number of T cells synthesizing TNF- was observed in all the
patients. Proportions of CD3+TNF-+ T cells
increased from 23.2% ± 16.4% to 46.1% ± 17.9% at M12 (P < .01 vs control donors). Analysis of CD4 and CD8 T
cells revealed that both subsets were highly polarized to TNF-
synthesis. Indeed, the proportions of CD4 T cells producing TNF-
increased progressively and became statistically significant compared
with baseline values following M12, and the absolute number of T cells
increased from 121 ± 98 cells per µL at M12 to 376 ± 206
cells per µL at M18, thereby reaching the range of normal
values (Figure 1C). A similar increase was observed for
TNF--synthesizing CD8 T cells, which became over-represented
because their absolute number increased more than 3-fold compared with
control donors and increased 2-fold compared with baseline values: M18,
431 ± 460 cells per µL vs 147 ± 79 cells per µL for
controls (P < .01) and 206 ± 239 cells per µL
at M0 (P < .03) (Figure 1C). This polarization of T
cells to TNF- synthesis was associated with increased concentrations
of TNF- and TNF-RII in the sera of PI-treated patients (Figure 1B).
The contribution of monocytes to the rise in plasma TNF- was
excluded following single cell analysis because the proportion of
monocytes producing TNF- was not increased as the result of HAART
(M. Bocchino et al, unpublished data, May 1999). It is noteworthy that
this accumulation of TNF- T-cell producers occurred in the context
of efficient suppression of HIV RNA in plasma (Figure 1A), and no
correlation was found between the viral load (VL) at baseline or during
the follow-up and the percentage or the absolute number of CD4 or CD8
TNF- producers. In contrast, the change in the number of
CD8+ TNF-+ T cells correlated to the change
in VL (r = 0.56, P < .05). Plasmatic
TNF-RII concentrations and viral load were correlated at baseline
(r = 0.81, P < .02), but there were no further
correlations following PI administration. Altogether these data suggest
that the regulation of the TNF- system is altered following
successful antiretroviral therapy, which leads to the dramatic
accumulation of T cells polarized for the synthesis of this
proinflammatory cytokine.
Defective control by apoptosis on TNF-+ T cells
following HAART
We previously reported that the decreased proportion of
TNF--producing T cells in untreated or RTI-treated patients was
correlated with an increased rate of apoptosis in this
subset.27 Because apoptosis, physiological
anti-inflammatory process,30 is dramatically reduced
following HAART,2-4 we asked whether the accumulation of
TNF- producers in patients receiving HAART was related to their
increased survival. We first confirmed that HAART is associated with a
significant decrease in activation-induced apoptosis in CD3+ T cells and noted 18.6% ± 11.6% at M12 vs
39.0% ± 17.9% at M0 (P < .01), leading to values
close to those of control donors (21.8% ± 13.5%). This drop in
apoptosis concerned all T-cell subsets including TNF--synthesizing
CD3 T cells. Indeed, analysis of apoptosis at the single cell level
within TNF- CD3 T-cell producers, as shown in Figure
2A, indicates a remarkable increased
survival of this subset following HAART. In this representative
patient, the proportion of apoptotic cells within
CD3+TNF-+ T cells decreased from 58% at M0
to 16% at M12, whereas the percentage of TNF-+ cells
within CD3+ T cells increased from 26% to 49% (Figure
2A).
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| Fig 2.
The peripheral accumulation of TNF--producing T cells
is linked to their escape from apoptosis.
Apoptosis susceptibility of TNF-+ T cells was determined
by concomitant intracellular TNF-+and 7-AAD stainings.
(A) Dot-plots of gated CD3+ T cells from a representative
HIV+ patient before HAART and at M12. (B) Follow-up of
susceptibility to activation-induced apoptosis of TNF-+
T cells during HAART. Vertical bars indicate SEM. (C) Scatter-plots of
apoptosis rates in TNF-+ T cells and percentages of
TNF-+ peripheral cells in 16 patients who received at
least 9 months of HAART (Spearman rank correlations). Values are given
as the mean plus or minus SD. Shaded box indicates HIV+
PI patients; open circle, CD4 T cells; and closed
circle, CD8 T cells. Asterisk indicates P < .05 vs baseline
values (Wilcoxon signed rank test); paragraph symbol,
P < .05 vs HIV controls (Mann-Whitney
U test).
|
|
The 18-month follow-up of susceptibility to apoptosis of CD4 and CD8
TNF--producing T cells revealed that the initially high level of
apoptosis in both subsets declined progressively during HAART:
19.3% ± 11.6% in CD4+TNF-+ T cells
at M18 vs 41.4% ± 16.7% at baseline (P < .003) and
58.4% ± 18.7% in CD8+TNF-+ T cells
at M18 vs 30.5% ± 10.0% at baseline (P < .001). At
M18, values were below those of controls (Figure 2B). Furthermore, the
accumulation of TNF--producing T cells within both CD4 and CD8
subsets was inversely correlated with the level of activation-induced apoptosis in these cells (Figure 2C), which supports an important role
of apoptosis in the homeostasis of TNF-+ T cells. It is
noteworthy that only patient No. 10, who showed a persistent high rate
of T-cell apoptosis in TNF--producers (60% and more than 80% in
CD4 and CD8 T cells, respectively), had a concomitant very low
proportion (less than 10%) of TNF- T cells and was not efficiently
controlled at the viral level (Figure 2C and Table 1). From these data,
we conclude that peripheral accumulation of TNF--synthesizing T
cells in patients receiving HAART is the direct consequence of a
defective physiological control by apoptosis.
Escape from activation-induced apoptosis of
TNF--producing cells partly related to cosynthesis of IL-2
Regulation by endogenous cytokines of lymphocyte survival can be
studied by flow cytometry, which allows the simultaneous analysis at
the single cell level of the coproduction of several cytokines and the
susceptibility to activation-induced apoptosis. Because IL-2 is able to
prevent ex vivo apoptosis of T cells from HIV+
patients,31 we tested whether increased survival of TNF-
producers in HAART-treated patients was related to the cosynthesis of
IL-2 by these cells. We show that a significant fraction of
TNF-+ cells coproduce IL-2 in both control donors and
HIV+ patients (Figure 3A).
Indeed, the proportion of double-positive CD3 T cells in these
representative donors was 55% of the total T cells in the control
donor, and the proportion was reduced to 15% in the
PI patient and restored to 48% in the
PI+ patient. The decrease in the proportion of
IL-2+TNF-+ cells in the
PI patient was mainly due to an alteration within
the CD8 subset (21.5% ± 11.1%
CD8+TNF-+ T cells in PI
patients vs 48.8% ± 16.9% cells in controls
(P < .001), and it was restored during PI therapy
(38.5% ± 15.2% cells, nonsignificant vs controls).
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| Fig 3.
Escape from apoptosis of TNF-+ T cells
is associated with cosynthesis of IL-2.
Following 16 hours of PMA+PHA+ionomycin stimulation, dual intracellular
staining of PBMCs was performed with anti-IL-2-PE and
anti-TNF--FITC mAbs. (A) Dot-plots, gated on CD3+ T
cells, from a representative HIV control, an
HIV+ PI patient (M0), and an
HIV+ PI+ patient treated for 12 months (M12).
(B) Differential susceptibility to activation-induced apoptosis of
IL-2+TNF-,
IL-2+TNF-+, and
IL-2TNF-+ lymphocytes. (C) Relative
contribution of IL-2+TNF-+ and
IL-2TNF-+ cells to the expansion of
total TNF-+ T cells following HAART. The letter C
indicates healthy subjects (n = 7); PI,
HIV+ PI patients naïve of PI
therapy (n = 12); and PI+, HIV+ PI+
patients with at least 9 months of PI therapy (n = 13).
Paragraph symbol indicates P < .05 vs
HIV controls; asterisk, P < .05 vs
HIV+ PI patients (Mann-Whitney U
test).
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Concomitant analysis of apoptosis within cytokine producers indicated
the existence of a gradient of susceptibility related to the cytokine
synthesized: IL-2 TNF-+ cells were
more susceptible to apoptosis than
IL-2+TNF-+ cells, which were more
susceptible than IL-2+TNF- cells
(Figure 3B). This gradient of apoptosis was observed in lymphocytes
from control donors and in HIV+ PI
patients, although the rate of apoptosis was higher in all 3 subsets
(Figure 3B). A decreased susceptibility to activation-induced apoptosis
was observed in the 3 subsets in PI+ patients, and in spite
of this, the IL-2+TNF-+ subset was still
less susceptible to apoptosis than the single TNF-+
subset (Figure 3B). A relationship between susceptibility to apoptosis
of TNF- producers and their absolute number in the blood was
observed. Indeed, in PI patients, a dramatic
reduction in the number of double-positive cells within
both the CD4 subset (110 ± 37 cells per µL in
PI patients vs 244 ± 131 cells per µL in
controls [P < .03]) and the CD8 subset (44 ± 12
cells per µL in PI patients vs 73 ± 30 cells
per µL in controls, NS) was observed (Figure 3B). With PI therapy,
the enrichment in TNF- T-cell producers was associated with an
increase in the number of both CD4 and CD8 double-positive
cells (171 ± 82 cells per µL
IL-2+TNF-+ CD4 T cells (NS vs
M0) and 105 ± 55 cells per µL
IL-2+TNF-+ CD8 T cells (P < .02
vs M0) and also an increase in the number of single positive cells in
both subsets (P < .01 vs controls for the CD8 subset)
(Figure 3B).We conclude from these data that accumulation of CD4 and
CD8 T cells producing TNF- during HAART results from the escape from
physiological apoptosis of TNF- T-cell producers, partly due
to an increased proportion of apoptosis-resistant IL-2+TNF-+ T cells.
Lipid alterations in PI and PI+
HIV+ patients
LD, recently reported in HIV-treated patients, is
characterized by abnormal fat redistribution (peripheral loss of fatty
tissue and central fat accumulation) together with insulin resistance and dyslipidemia.15,16 In a cross-sectional study, we
analyzed the lipid parameters in 20 healthy HIV-negative
(HIV) controls, 10 HIV+ patients
receiving bi-RTI (zidovudine [ZDV] and didanosine [ddI] or ZDV and
zalcitabine [ddC]) for 3-6 months, and 30 HIV+ men
receiving HAART including PIs for at least 9 months. LD was assessed by
a clinical score of facial and limb fat wasting and central fat
deposition as previously reported.20 Clinical
characteristics of patients are given in Table 2. In patients receiving
bi-RTI, a decrease in the concentration of HDL cholesterol and an
increase in the concentration of triglycerides, as compared with
HIV healthy controls, was observed. Nevertheless, in
these patients the apolipoprotein fractions were not significantly
altered (Table 3). In
PI+LD patients, no alterations were
observed in total cholesterol, HDL cholesterol, and triglycerides, but
a trend toward an increase in the atherogenic ratio apoB/apoA1 was
observed. In PI+LD+ patients, dyslipidemia
consists of dramatically increased concentrations of cholesterol and
triglycerides together with a strong increase in the atherogenic
fraction apoB and in the apoB/apoA1 ratio,20 which is
significantly different compared with other groups of donors (Table 3).
Relation between cytokine alterations and
HAART-associated LD
The precise mechanisms involved in the LD syndrome still
remain unclear. Because lipid metabolism is under the control of some
cytokines, including TNF-,25 we asked whether
dysregulation in TNF- synthesis during HAART was related to this
syndrome. In a cross-sectional study, we analyzed 41 HIV+
men who were taking HAART including PIs for at least 9 months. The LD
score20 of the 17 LD+ patients was between 4 and 10. The 24 LD patients did not present any of
the scoring symptoms. We also included 19 HIV
controls in the study. Clinical and laboratory characteristics of
LD+ and LD groups are given in Table 2.
BMI did not significantly differ between the male patients from the 2 groups (22.2 ± 2.4 kg/m2 and 23.8 ± 3.5
kg/m2 in LD+ and LD
patients, respectively). LD+ and LD
patients had similar CD4 T-cell numbers and a similar CD4 increase under HAART at the time of the study. It is noteworthy that the number
of CD8 lymphocytes and the increase in CD8 T cells were significantly higher in LD+ patients, and the mean duration
of PI therapy was longer in LD+ patients compared with
LD patients. The viral load was efficiently
controlled in both groups (Table 2).
Determination of the frequency of TNF- producers revealed that the
accumulation of CD8+TNF-+ T cells detected
in HAART-treated LD patients also occurred in
LD+ patients but to levels higher than seen in
LD patients (mean percentage, 42.2% ± 11.1%
in LD+ vs 36.7% ± 15.5% in LD,
NS; mean absolute count, 516 ± 265 in LD+ vs
350 ± 353 in LD [P < .003])
(Figure 4A). The mean percentage of
apoptotic cells in CD8+TNF-+ T cells was
decreased in both LD patients
(29.4% ± 15.0%) and LD+ patients
(42.2% ± 22.1%) vs PI patients
(58.7% ± 15.0%) (P < .01 vs LD,
and P < .05 vs LD+). The absolute numbers of
CD4+TNF-+T cells were similar in
LD+ vs LD patients (data not shown), but
a spectacular reduction in the mean number of IL-2-producing CD4
T cells was observed (151 ± 138 cells per µL in
LD+ patients vs 328 ± 265 cells per µL in
LD patients [P < .02]). The decreased
capacity of IL-2 production was also observed at the CD8 T-cell level
(data not shown), but the absolute number was not altered because of a
compensation due to the CD8 lymphocytosis observed in LD+
patients. The concomitant alteration in IL-2 production by CD4 T cells
and the TNF- polarization of CD8 T cells in LD+ patients
resulted in a significant decrease in the
IL-2+/TNF-+ CD3 ratio detected in
LD+ vs LD patients. Strikingly, lipid
alterations in HAART-treated patients correlated with cytokine
perturbations. Indeed, the absolute numbers of
CD8+TNF-+ T cells were positively correlated
with total cholesterol (P < .005) (Figure 4), total
triglyceride (P < .002), and apoB concentrations (P < .001) (Figure 4) and the apoB/apoA1 ratio
(P < .0001) and inversely correlated with the HDL
concentration (P < .04). The absolute number of
CD4+IL-2+ T cells was negatively correlated
with the total triglyceride level (P < .02) and apoB
concentration (P < .03) and positively correlated with HDL
concentration (P < .05). Consequently, the CD3+IL-2+/CD3+TNF-+
ratio was found to be a good marker of dyslipidemia and atherogenic risks because it was negatively correlated with total cholesterol (P < .01) (Figure 4), total triglyceride
(P < .009), and apoB (P < .001) concentrations
(Figure 4) and the apoB/apoA1 ratio (P < .04).
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| Fig 4.
Cytokine alterations during HAART are correlated to
LD-associated dyslipidemia.
(A) Absolute numbers of CD4+IL-2+ T cells,
CD8+TNF-+ T cells (upper panel), and
CD3+IL-2+/CD3+TNF-+
ratio (lower panel) in HIV+ PI+ patients
treated for at least 9 months. Shaded box indicates
LD patients (n = 24), and darkened box,
LD+ patients (n = 17). Histograms show mean values and
SEM. (B) Correlations between cytokine and lipid alterations are given
for 30 HIV+ PI+ patients with at least 9 months
of PI therapy. The patient population includes 13 LD patients, 17 LD+ 13 LD, and 17 LD+ (Spearman rank
correlation).
|
|
| |
Discussion |
Recent studies have reported unusual clinical inflammatory
syndromes, such as immune recovery vitritis (IRV) in CMV retinitis patients32 or focal mycobacterial
lymphadenitis,33 after initiation of HAART. It was
suggested that these syndromes represent immunopathological responses
which occur as a result of the recovery of specific immune reactivity
against microbial pathogens that are subclinically present at the time
HAART is initiated.34 Our observations demonstrate that
HAART is associated with the progressive accumulation of T cells
producing TNF-+ thereby creating a proinflammatory
environment that might contribute to the development of these immune
restoration inflammatory diseases. In addition our data suggest that
this T-cell polarization to TNF- synthesis is favoring the
development of the LD syndrome by contributing to lipid metabolism alteration.
A wide number of TNF- inducers, including HIV
proteins,35 have been described. Although HIV viral load is
highly suppressed by HAART, viral reservoirs still
persist,29 and the release of early viral proteins, such as
Tat, together with increased concentrations of soluble
TNF-RII, could lead to NF-B activation36 and subsequent
induction of TNF- production.35 Stringent control mechanisms are therefore necessary to prevent chronic production of
TNF- and subsequent adverse consequences. At the macrophage level,
this control is provided by anti-inflammatory cytokines such as
interleukin-4 (IL-4), IL-10, and transforming growth factor- (TGF-).37 We did not observe an increase in Th2 cytokine
production in HIV+ patients,27 even in those
patients receiving HAART. Our data argue for a role of apoptosis in the
physiological regulation of TNF-. The anti-inflammatory function of
apoptosis has been reported at the macrophage level,30 and
it is exploited by bacterial pathogens to suppress TNF-
production.38 Apoptosis was also shown to contribute to the
resolution of inflammatory processes involved in the delayed-type
hypersensitivity response.39 This study and our previous
study using combined detection of intracellular cytokine synthesis and
apoptosis on lymphocytes from untreated patients27 suggest
that apoptosis plays an essential role in the negative regulation of
TNF- synthesis by T cells. Indeed, even in healthy donors, TNF-
producers are highly sensitive to apoptosis, and this is partly related
to the decreased expression of the Bcl-2 molecule.27 During
the natural progression of HIV infection, susceptibility of TNF-
producers to apoptosis progressively increases, and this is correlated
to their decreased representation in patient blood
samples.27 The present study shows that during combined
therapy, including indinavir, apoptosis in TNF- T-cell producers is
highly suppressed and leads to their progressive accumulation.
Suppression of activation-induced apoptosis with HAART 2-4
is likely to be a multifactorial process. Indeed, the mechanisms
involved in apoptosis susceptibility of patients' lymphocytes during
the chronic phase of HIV infection, ie, the in vivo immune activation
state of these lymphocytes,40 is down-regulated during HAART,8 and the production of HIV proteins with
proapoptotic effects, such as gp120,41,42 is decreased,
thereby restoring the global survival of patients' lymphocytes. In
addition, our data show for the first time that regulation of TNF-
synthesis can occur at the single cell level through the coproduction
of a survival factor. This regulation occurs because increased survival of TNF- T-cell producers during HAART is associated with the cosynthesis of IL-2. Finally, we cannot exclude a direct
immunomodulatory effect of PIs on T cells. PIs suppress physiological
apoptosis by inhibition of cellular proteases4 and exert a
direct impact on cytokine production.43 However, these in
vitro effects of PIs were mainly observed in patients taking ritonavir.
Recently, a series of reports have associated HAART with a syndrome
that includes peripheral fat wasting, dyslipidemia, insulin resistance,
and central fat accumulation.15,16 The pathophysiology of
LD remains unknown. The amino-acid sequence homologies
between HIV protease and the low density lipid receptor-related
protein or cytoplasmic retinoic-acid binding protein type 1 resulted in the suggestion that LD results from a possible interaction between PIs
and these molecules.23 However, this hypothesis cannot
account for the occurrence of LD in patients who never received PIs,
recently reported in several studies.18-20 Our data show
that hyperlipidemia and atherogenic alterations observed in
LD+ patients are related to dysregulation in cytokine
synthesis. Indeed, increased concentrations in circulating lipids,
including cholesterol, triglycerides, and apoB, were positively
correlated with the absolute number of TNF--producing CD8 T cells
in LD+ patients. Although these associations do not prove
causality, our study suggests that the progressive accumulation of
TNF- producers during HAART is a factor involved in dyslipidemia.
TNF- inhibits the intake of free fatty acids (FFA) by adipocytes via
the inhibition of lipoprotein lipase, which leads to fat wasting, and
increases lipogenesis via the stimulation of the hepatic triglyceride
synthetase, which leads to hyperlipidemia.25 Increased
TNF- production may also indirectly contribute to fat wasting
by triggering the production of leptin, a hormone that depletes adipocyte fat content via down-regulation of the lipogenic enzymes.44 Accumulation of TNF- could also account for
insulin resistance, both indirectly via FFA increase and
directly via the inhibition of signal transduction through
the insulin receptor.26 Whereas the number of
T-cell-producing TNF- was strongly correlated with lipid parameters
in PI-treated patients, no direct association could be
observed between these lipid parameters and plasmatic TNF indices
(Christeff et al, unpublished data, October 1999). These data suggest
that lipid alterations may involve local cleavage of cell-borne TNF in
target tissues rather than systemic diffusion of the
cytokine.45-47 According to that hypothesis, Domingo et al48 recently reported that subcutaneous fat atrophy in
LD+ patients is associated with focal lipogranuloma
formation and adipocyte apoptosis, which is compatible with an
excessive local production of TNF-.49 It remains to be
determined whether subsets other than lymphocytes, including
adipocytes,50 are involved in the increased potentiality of
TNF- production following HAART. In addition, persistent high
plasmatic levels of TNF-RII following HAART may also be involved in
insulin resistance and dyslipidemia, as was recently proposed for
another metabolic disease.51
Although prospective studies are needed, we propose that the
development of the LD syndrome is a sequential and multifactorial process for the following reasons: First, as previously reported by
Zangerle et al52 and reported in Table 3, lipid
alterations, such as decreased HDL cholesterol and increased
triglycerides, occur in HIV+ patients who received bi-RTI,
while the apoB/apoA1 ratio remains normal. This later parameter is
progressively altered after PI administration, in the context of
increased TNF- production by T cells, while the production of this
cytokine is low in patients receiving bi-RTI.27 In
addition, increased cortisol secretion and an imbalance in the
cortisol/DHEA (dehydroepiandrosterone) ratio may contribute to the
altered equilibrium between lipolysis and lipogenesis in
LD+ patients.20,53 Interestingly, decreased
serum DHEA concentration detected in LD+ patients compared
with LD patients20 may be related to the
alteration in IL-2 production54 that we observed in
LD+ patients. Additional mechanisms involved in central fat
deposition remain to be elucidated. Although we did not observe
modifications in BMI during PI therapy, including in LD+
patients,20 it is possible that fat redistribution in
LD+ patients masks a residual muscle wasting,55
which is compatible with an excess in both TNF-
production and the catabolic/anabolic cortisol/DHEA
ratio.56
Alteration in TNF--producing T-cell homeostasis may have other
consequences, particularly at the viral level. It could favor the HIV
rebound rapidly observed after interruption of HAART.29 On
the other hand, it may contribute to eradication of a long decay
half-life HIV reservoir from resting CD4 lymphocytes.13,29 It remains to be determined whether this proinflammatory response is
triggered by specific antiretroviral therapy and/or is a consequence of
the sudden suppression of HIV production, which would perturb a
preexisting equilibrium between the immune system and the virus. Altogether our data indicate that HAART is associated with the progressive accumulation of proinflammatory T cells, which are committed to TNF- synthesis, due to a dramatic down-regulation of
physiological apoptosis. These findings give new insights into strategies to prevent the development of LD-associated dyslipidemia in
treated HIV+ patients.
| |
Acknowledgments |
The authors wish to acknowledge the nurses of Centre
Hospitalier Intercommunal, Villeneuve-St-Georges, France, and
Hôpital Raymond Poincaré, Garches, France, for patient care
and for collecting the samples. We are indebted to Dr Ahmed Chater,
Centre Hospitalier, Villeneuve-St Georges, and to Mrs Françoise
Rives, Pasteur Institute, Paris, France, for their efficient help in
the capture of clinical data. We particularly acknowledge Dr Luc
Montagnier for his interest in this study.
| |
Footnotes |
Supported by grants from the Agence Nationale de Recherche
sur le SIDA; the Fondation pour la Recherche Médicale (FRM); the CNRS; the Institut Pasteur, Paris, France; and by contracts BMH4-CT 97-2055 and ERB-IC15-CT97-0901 from the Union Européenne. E. L. was supported by the FRM.
Submitted October 21, 1999; accepted January 19, 2000.
Reprints: Marie-Lise Gougeon, Unité d'Oncologie Virale,
Département SIDA et Rétrovirus, Institut Pasteur, 28 rue du
Dr Roux, 75724 Paris, France; e-mail: mlgougeo{at}pasteur.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Am J Physiol Endocrinol Metab,
July 1, 2002;
283(1):
E138 - E145.
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J. Mayo, J. Collazos, E. Martinez, and S. Ibarra
Adrenal Function in the Human Immunodeficiency Virus-Infected Patient
Arch Intern Med,
May 27, 2002;
162(10):
1095 - 1098.
[Abstract]
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L. M. de Oliveira Pinto, S. Garcia, H. Lecoeur, C. Rapp, and M.-L. Gougeon
Increased sensitivity of T lymphocytes to tumor necrosis factor receptor 1 (TNFR1)- and TNFR2-mediated apoptosis in HIV infection: relation to expression of Bcl-2 and active caspase-8 and caspase-3
Blood,
March 1, 2002;
99(5):
1666 - 1675.
[Abstract]
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D Goldmeier, G Scullard, M Kapembwa, H Lamba, and G Frize
Does increased aromatase activity in adipose fibroblasts cause low sexual desire in patients with HIV lipodystrophy?
Sex Transm Inf,
February 1, 2002;
78(1):
64 - 66.
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R. FAGGIONI, K. R. FEINGOLD, and C. GRUNFELD
Leptin regulation of the immune response and the immunodeficiency of malnutrition
FASEB J,
December 1, 2001;
15(14):
2565 - 2571.
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Top
Abstract
Introduction