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AIDS:
doi: 10.1097/QAD.0b013e32833c3268
Clinical Science

Factors associated with collagen deposition in lymphoid tissue in long-term treated HIV-infected patients

Diaz, Albaa; Alós, Llúciaa; León, Agatheb; Mozos, Annaa; Caballero, Miguelc; Martinez, Antonioa; Plana, Montserratd; Gallart, Teresae; Gil, Cristinad; Leal, Manuelf; Gatell, Jose Mb; García, Felipeb; for the Study Group of Lymphoid Tissue immunopathogenesis in HIV infection

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Author Information

aDepartment of Pathology, Spain

bDepartment of Infectious Diseases, Spain

cDepartment of ENT, Spain

dRetrovirology and Viral Immunopathology Laboratories, Spain

eImmunology Laboratory, Hospital Clínic, IDIBAPS, University of Barcelona, Barcelona, Spain

fDepartment of Infectious Diseases, Hospital Virgen del Rocío, Sevilla, Spain.

Received 27 January, 2010

Revised 6 May, 2010

Accepted 14 May, 2010

Correspondence to Dr Felipe García, Department of Infectious Diseases, Hospital Clínic, IDIBAPS, University of Barcelona, Villarroel 170, 08036 Barcelona, Spain. Tel: +34 93 2275586; fax: +34934514438; e-mail: fgarcia@clinic.ub.es

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Abstract

Objective: The factors associated with fibrosis in lymphoid tissue in long-term treated HIV-infected patients and their correlation with immune reconstitution were assessed.

Methods: Tonsillar biopsies were performed in seven antiretroviral-naive patients and 29 successfully treated patients (median time on treatment, 61 months). Twenty patients received protease inhibitors-sparing regimens and nine protease inhibitor-containing regimens. Five tonsillar resections of HIV-negative individuals were used as controls. Lymphoid tissue architecture, collagen deposition (fibrosis) and the mean interfollicular CD4+ cell count per μm2 were assessed.

Results: Naive and long-term treated HIV-infected patients had a higher proportion of fibrosis than did HIV-uninfected persons (P < 0.001). Patients with greater collagen deposition showed lower levels of CD4+ cells in lymphoid tissue (P = 0.03) and smaller increase in peripheral CD4+ T cells (r = −0.40, P = 0.05). The factors independently associated with fibrosis in lymphoid tissue were age (P < 0.0001), treated patients with detectable lymphoid tissue viral load when compared with patients with undetectable lymphoid tissue viral load (median 5 vs. 12%, respectively, P = 0.017) and patients receiving a protease inhibitor-sparing vs. a protease inhibitor-containing regimen (median 8 vs. 2.5%, respectively, P = 0.04).

Conclusion: Fibrosis in lymphoid tissue was associated with a poor reconstitution of CD4+ T cells and long-term antiretroviral therapy did not reverse this abnormality. HIV infection, older age, a detectable level of lymphoid tissue viral load in treated patients and protease inhibitor-sparing regimens seem to favour fibrosis in lymphoid tissue.

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Introduction

There are a number of studies [1–6] suggesting that, regardless of the baseline CD4+ T-cell count, CD4+ T cells reach a plateau or slow the rate of immune recovery after 4–5 years on antiretroviral therapy (ART). Recent studies [7,8] demonstrate that the recovery of CD4+ T cells could be incomplete even after 10 years of ART. The reasons for this incomplete recovery of CD4+ T cells are unclear [6,7]. Although plasma viral load (PVL) and peripheral CD4+ T-cell counts are used to monitor HIV infection, key pathogenesis events and most viral replication occur in lymphoid tissues, where the pathological hallmark of HIV infection (i.e. depletion of CD4+ T cells) is manifest [9,10]. Lymphoid tissue indemnity, especially that of secondary lymphoid tissue, is crucial for immunological recovery in HIV-infected patients receiving ART, and damage to this tissue may limit its ability to restore normal function despite treatment [10–13].

There are a number of pathogenic events in lymph nodes that could prevent correct immune restoration in treated HIV-1-infected patients. Some authors have found persistence of viral replication [14,15] or of HIV-1 structural proteins and glycoproteins [16] in lymph nodes of successfully treated patients (defined as an undetectable level of PVL). These findings could be partially explained by data suggesting that lymphoid follicles may be immune-privileged sites. Connick et al. [17] found that HIV-1-specific cytotoxic T lymphocytes were abundant within lymphoid tissues, but failed to accumulate within lymphoid follicles where HIV-1 replication is concentrated. Our group has reported that expression of the intercellular adhesion molecule-1 (an adhesion molecule that plays an important role in the transmission of HIV-1 to CD4+ target cells and in the decrease of these cells in lymphoid tissue) is upregulated in lymphoid tissue, and also that its level was associated with a marked effacement of lymphoid tissue architecture. All these factors favour abnormal activation and cytokine spectra in lymph nodes [18]. Thus, activated effector cells are inappropriately attracted, retained or both, whereas the levels of regulatory cells (which could reduce activation) are decreased in lymphoid tissue [19].

Inflammation and immune activation appear to be the driving stimulus for collagen deposition. It has been hypothesized that collagen deposition contributes to CD4+ T-cell decline and limits CD4+ T-cell repopulation with ART [20,21]. The loss of lymphoid tissue organizing structure has been related to the deposition of collagen as well as to the consequent fibrotic damage to the lymphatic tissue T-cell compartment [21]. The amount of lymphatic tissue fibrosis in HIV infection has been correlated with the extent of immune reconstitution observed during treatment [22,23]. Moreover, fibrosis of lymphoid tissue could explain the incomplete immune reconstitution observed in the gut [24] and the drop of CD4+ T-cell counts in patients receiving successfully suppressive ART [25].

All these data suggest that collagen deposition is pivotal in explaining the partial immune recovery in HIV-infected patients. However, the factors that are associated with this collagen deposition or the influence of long periods on ART on collagen deposition are incompletely understood. The current study analysed these factors in 36 chronic HIV-infected patients in whom tonsillar biopsies were performed, the sample including 29 patients with a median of 61 months on ART.

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Patients and methods

Patient population and lymphoid tissue collection

Thirty-six tonsillar biopsies were performed in chronic HIV-infected patients. Inclusion criteria were chronic HIV-1-infected patients with tonsillar tissue, followed-up during at least 12 months in our centre. Furthermore, in the group of treated patients, all determinations of PVL had to be below detectable levels 6 months after initiation of ART. Seven biopsies were performed in treatment-naive patients and 29 in successfully treated patients with an undetectable level of PVL (median time on treatment, 61 months; range, 12–112 months). Twenty patients were receiving protease inhibitor-sparing regimens and nine protease inhibitor-containing-regimens (Table 1). Clinical data including CD4 T-cell counts and PVL were obtained from each patient. The purpose of the study was explained in detail to all patients, and all patients gave written informed consent. The study was approved by the institutional ethical review board.

Table 1
Table 1
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Each sample of tonsillar tissue was split into two parts: one half was frozen immediately in liquid nitrogen and used to determine tonsillar tissue viral load, whereas the other half was formalin-fixed and paraffin-embedded for histological and immunohistochemical studies. All samples had an adequate amount of lymphoid tissue. The tonsillar therapeutic resections of five non-HIV-infected persons with sleep apnoea syndrome (four men, median age of 42 years) were used as controls.

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Tonsillar tissue viral load

Total RNA was extracted from 50 μl of tonsil samples treated with guanidine thiocyanate (mean 11.5 mg) using RNeasy Kit (Qiagen, Venlo, The Netherlands) and eluted in 50 μl of water and quantified. Two micrograms of total RNA in 50 μl of water was treated with 2 μl rDNase I (DNA-free; Ambion, Inc., Austin, Texas, USA) at 37°C for 60 min following the kit instructions. The final sample was RNA free of residual DNA. COBAS TaqMan HIV-1 Test (Roche, Basel, Switzerland) procedure was previously modified and validated to amplify HIV-1 RNA obtained from cells and from lymphoid tissue. Briefly, 50 μl of final sample containing an input of 1 μg of total RNA and 4 μl of added HIV-1 quantitation standard were mixed with 50 μl of HIV-1 master mix plus manganase solution Mn2+ to initiate the amplification reaction by COBAS TaqMan HIV-1. The quantification results of each PCR were outputted by the automatic system as copies of HIV-1 RNA/ml of plasma, the system's detection limit being set at 40 HIV-1 RNA copies/ml. For tissue RNA, the output results were considered as HIV-1 RNA copies/real-time PCR reaction and were normalized to the sample input. The adjusted results were expressed as HIV-1 RNA copies/μg of total RNA.

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Lymphoid tissue architecture, collagen deposition and quantitative assessment of CD4+ cells in lymphoid tissue

From the formalin-fixed and paraffin-embedded material, the histological and immunohistochemical studies were carried out. A haematoxylin–eosin stain was used in each case to assess overall morphology. In addition, a trichrome stain, using the Masson method, was used to identify collagen fibres. The CD4 immunohistochemical technique used both the automated immunohistochemical system TechMate 500 (Dako, Carpinteria, California, USA) and the EnVision system (Dako). Slides were incubated with the primary antibody for 30 min (CD4: clone 1F6, dilution 1: 5; Novocastra Laboratories, Newcastle, UK).

Lymphoid tissue architecture, collagen deposition and number of CD4+ cells in lymphoid tissue were assessed using the Olympus Cell-B Basic Imaging Software (Olympus Corporation, Shinzuku, Tokyo, Japan). From each specimen, the follicular area, defined as the proportion of follicular areas with respect to the total amount of lymphoid tissue, was evaluated by morphometry from low-power fields (×10 objective). In order to quantify collagen fibres, 10 images from 10 high-power fields (HPFs) (×40 objective) from interfollicular areas were captured on trichrome-stained slides and the percentage of lymphoid tissue occupied by collagen was determined. CD4+ cells were counted in the interfollicular areas through the capture of 10 images from 10 HPFs and the mean value per μm2 was obtained.

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Serum markers related to pathogenesis of fibrosis (D-dimer and hyaluronic acid) and inflammation (ultrasensitive C-reactive protein and interlukin-6)

D-dimer was measured by a turbidimetric method (Innovance; Siemens Diagnostics, Marburg, Germany) in an automated coagulation system BCS XP (Siemens Diagnostics). Serum levels of hyaluronic acid were determined by ELISA using a kit from Corgenix Medical Corporation (Westminster, Colorado, USA) and following the instructions of the manufacturer. High-sensitive C-reactive protein (hsCRP) was measured using a latex-enhanced immunoturbidimetric method (Advia 2400; Siemens Diagnostics). The assay used for the measurement of interlukin-6 (IL-6) was a solid-phase enzyme-amplified sensitivity immunoassay performed on microtitre plate (Diasource, Nivelles, Belgium). The assay uses mAbs directed against distinct epitopes of IL-6. An incubation period is needed for the formation of a sandwich between coated MAb 1, human IL-6 and MAb 2– HRPAn. Thereafter, the microtitre plate is washed to remove unbound enzyme-labelled antibody. Bound enzyme-labelled antibody is measured through a chromogenic reaction. The amount of substrate turnover is determined colourimetrically by measuring the absorbance, which is proportional to the IL-6 concentration. A calibration curve is plotted and IL-6 concentration in samples is determined by interpolation from the calibration curve. The normal cutoff level is 5 pg/ml. The analytical sensitivity is 2 pg/ml.

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Statistical analysis

Quantitative data were compared between groups (uninfected individuals, antiretroviral-naive patients and antiretroviral-treated patients) using either a Student's t-test, for variables with a normal distribution and similar variances, or a Mann–Whitney U test for variables without a normal distribution. When data from the three groups were compared, a one-way analysis of variance or Kruskal–Wallis test was performed, correcting P values for multiple comparisons using the Bonferroni test. A multivariate analysis (multiple regression) was performed to detect those factors that were independently associated with collagen deposition in lymphoid tissue.

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Results

Patient characteristics

Patient characteristics are shown in Table 1. The median age was 38 years and most of the patients (85%) were men. In 66% of patients, the risk for HIV infection was MSM. Overall, 6% of patients had a hepatitis B and 9% a hepatitis C coinfection. Current and nadir (before any treatment) CD4+ T-cell count were high [median (interquartile range or IQR) 787 (536–982) and 477 (361–606) cells/μl, respectively]. Twenty-nine patients (81%) had been on ART for a long time (median, 61 months; range, 12–112 months). Twenty patients were receiving a protease inhibitor-sparing regimen [six with three nucleoside reverse transcriptase inhibitors (NRTIs), five with two NRTIs plus nevirapine and nine with two NRTIs plus efavirenz]. Nine patients were receiving a protease inhibitor-containing regimen with two NRTIs plus a ritonavir-boosted protease inhibitor. Age, nadir of CD4+ T-cell count and viral load peak were significantly different between naive and antiretroviral-treated patients (Table 1).

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Lymphoid tissue viral load

Seven out of 29 patients on treatment had a detectable level of lymphoid tissue viral load (LTVL), despite having an undetectable level of PVL (<50 copies/ml). Only one out of nine patients (11%) on a protease inhibitor-containing regimen had a detectable level of LTVL (708 copies/μg of total RNA), whereas LTVL was detectable in six out of 20 patients (30%) taking a protease inhibitor-sparing regimen (median, 12 950; range, 9300–368 550 copies/μg of total RNA; P = 0.27 for the difference). These six patients received the following treatments: three NRTIs (n = 3), two NRTIs plus nevirapine (n = 2) and two NRTIs plus efavirenz (n = 1).

Patients on ART with detectable LTVL had a shorter period with an undetectable level of PVL than did patients with an undetectable level of LTVL [median (IQR) months with undetectable PVL of 19 (13–20) vs. 52 (18–77), respectively, P < 0.0001] and a lower increase in peripheral blood CD4+ T-cell count after ART initiation (as measured by the difference between current and nadir CD4+ T cells) [median (IQR) 127 (7–224) vs. 333 (145–615), patients with detectable vs. undetectable LTVL, respectively, P = 0.001]. However, if the increase in peripheral blood CD4+ T-cell count was normalized for the duration of complete suppression of PVL, the differences between the two groups were not significant [the median (IQR) increase per month of treatment with an undetectable level of PVL was 6.92 (0.29–11) vs. 7.42 (5.04–10) cells/μl, respectively, P = 0.34].

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Lymphoid tissue architecture and lymphoid tissue CD4+ cell count

Compared with patients on ART, antiretroviral-naive patients showed effacement of lymphoid tissue architecture, with absent or small follicle structures [median (IQR) for follicular areas was 5% (0–8) and 12% (6–17.5) in naive and treated patients, respectively, P = 0.005] (Fig. 1). Those patients with greater follicular areas also showed higher counts of lymphoid tissue interfollicular CD4+ cells (r = 0.63, P < 0.0001).

Fig. 1
Fig. 1
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Antiretroviral-naive patients also had lower counts of lymphoid tissue interfollicular CD4+ cells than did patients on ART [median (IQR) 1.74 × 10−3 (1.38 × 10−3–2.21 × 10−3) vs. 2.63 × 10−3 (2.18 × 10−3–3.30 × 10−3) cells/μm2, respectively, P = 0.02] (Fig. 2), despite both groups having a similar peripheral blood CD4+ T-cell count (Table 1). CD4+ cells measured in interfollicular areas in patients on ART correlated positively with the number of months of undetectable PVL (r = 0.39, P = 0.04) and months on ART (r = 0.53, P = 0.002). Even after long periods (median, 61 months) on ART, the lymphoid tissue CD4+ cell count did not reach the level of HIV-uninfected individuals [median (IQR) HIV-uninfected 3.92 × 10−3 (2.79 × 10−3–4.04 × 10−3) vs. 2.63 × 10−3 (2.18 × 10−3–3.30 × 10−3) cells/μm2 in treated HIV-infected patients, P = 0.05] (Fig. 2a), even if only patients with an undetectable level of LTVL were analysed (data not shown).

Fig. 2
Fig. 2
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Lymphoid tissue collagen deposition and the association with architecture and lymphoid tissue CD4+ cell count

Collagen deposition was similar in naive and treated patients [median (IQR) of 3% (1–13%) and 5.5% (2–12.5%), respectively, P = 0.42]. Despite long periods on ART (median, 61 months), treated HIV-infected patients showed a higher proportion of collagen deposition than did HIV-uninfected individuals [median (IQR) 5.5% (2–12.5%) vs. 0.75% (0.62–0.80%)] (P < 0.001) (Fig. 3). All HIV-uninfected persons had a collagen deposition level below 1%. Only one out of 29 treated patients and none of the seven antiretroviral-naive patients showed a collagen deposition level below 1%.

Fig. 3
Fig. 3
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The group of naive HIV-infected patients with higher collagen deposition showed smaller follicular areas (r = −0.80, P = 0.02), lower levels of CD4+ T cells in lymphoid tissue (r = −0.81, P = 0.03) (Fig. 4a) and higher levels of CD8+ T cells in lymphoid tissue (r = 0.88, P = 0.009). The level of current peripheral blood CD4+ and CD8+ T cells, nadir of CD4+ T cells and peak of PVL were not associated with the proportion of collagen in lymphoid tissue (data not shown).

Fig. 4
Fig. 4
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Conversely, in treated patients, there was no correlation between collagen deposition and architecture or lymphoid tissue CD4/CD8 cell count (data not shown). However, there was a significant inverse correlation between the proportion of collagen deposition and the increase in peripheral blood CD4+ T-cell count after treatment initiation (as defined by the difference between current and nadir CD4+ T cells) (r = −0.40, P = 0.05) (Fig. 4b). This correlation was confirmed when the change in peripheral blood CD4+ T-cell count was normalized for the duration of ART (r = −0.41, P = 0.04) or for the duration of complete suppression of PVL (r = −0.43, P = 0.04).

As lymph node biopsies are not feasible in clinical practice, we did some measurements from serum from contemporaneous time points to measure some markers that could be related to pathogenesis of fibrosis (D-dimer and hyaluronic acid) and inflammation (hsCRP and IL-6) and analyse whether these serum values correlated with the histological findings. We did not find any correlation between D-dimer, hyaluronic acid or hsCRP with follicular areas, levels of T cells in lymphoid tissue or collagen deposition (data not shown). However, those patients with higher levels of IL-6 in serum showed higher levels of collagen deposition (r = 0.42, P = 0.02) and CD8+ T cells in lymphoid tissue (r = 0.40, P = 0.03) and lower levels of CD4+ T cells in lymphoid tissue (r = −0.35, P = 0.06).

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Predictors of lymphoid tissue collagen deposition

A significant positive correlation between age and collagen deposition was observed (r = 0.43, P = 0.01). However, sex, risk group of HIV infection and coinfection of hepatitis B and C were not associated with collagen deposition (data not shown). Antiretroviral-treated HIV-1-infected patients with an undetectable level of LTVL had a lower proportion of collagen deposition than did treated patients with a detectable LTVL [median (IQR) 5 (2–8.5) vs. 12 (3.5–19), respectively, P = 0.051]. When different types of therapy were analysed, a higher collagen deposition in patients receiving protease inhibitor-sparing regimens [median (IQR) 8% (2.2–13.7%)] as compared with those receiving protease inhibitor-containing regimens [median (IQR) 2.5% (1.5–4.75%)] (P = 0.029) was observed. This difference in collagen deposition in treated patients according to the type of treatment was maintained when this group was split into three parts based on the type of previous regimen, as follows: patients who had always had protease inhibitor-containing regimens (n = 5), patients who had always taken protease inhibitor-sparing regimens (n = 12) and patients who had received both types of regimens (n = 12) [median (IQR) proportion of collagen deposition of 3 (1.6–5.5) vs. 11 (4–15) vs. 2.2 (2–8), respectively, P = 0.02].

A multivariate analysis was then performed including all the HIV-infected patients and introducing the following variables: age, detectable level of LTVL, presence of ART and IL-6. Age (P = 0.004) and a detectable level of LTVL (P = 0.014) were the factors independently associated with collagen deposition in lymphoid tissue (R2 = 0.39).

To assess further the influence of current ART regimen on collagen deposition, a second multivariate analysis was then performed, including only patients on ART. The variables introduced into this model were the same as those included in the first multivariate analysis, with the addition of type of ART, previous ART, duration of ART and change in CD4+ T-cell count, normalized for duration of ART and complete suppression of PVL. Age (P = 0.003), a detectable level of LTVL (P < 0.0001) and type of treatment (P = 0.04) were the factors independently associated with collagen deposition in lymphoid tissue (R2 = 0.77).

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Discussion

Persistent abnormalities in immunoarchitecture and T-cell subset changes have been found in lymphoid tissue of HIV-infected patients successfully treated with ART for a year, and these findings have been correlated with viral persistence [10,12]. It is known that a worse virological response in lymphoid tissue cannot be explained by local selection of resistance, and that it is associated with a less durable virological response in plasma [14]. To our knowledge, there are no data about changes in architecture and T-cell subsets in lymphoid tissue after long periods on ART. Here, we found that abnormalities in immunoarchitecture and T-cell subsets in lymphoid tissue persist despite a median of 5 years on successful ART, even in patients with an undetectable level of LTVL. Although CD4+ cells measured in lymphoid tissue in patients on ART correlated positively with the number of months on ART, patients with 5 years on ART did not recover their lymphoid tissue CD4+ cell count to the level of HIV-uninfected individuals (Fig. 2a). A limitation of the study is that the HIV-uninfected control group is very small and it is hard to draw definitive conclusions, particularly as the resection was done for a clinical indication (sleep apnoea). Therefore, the differences in lymphoid tissue CD4 cell counts between HIV-infected and uninfected groups should be interpreted with caution. A further limitation is that the analysis is cross-sectional and there was no paired sample of the same patients. Nevertheless, the data would appear to confirm in lymphoid tissue the findings of a limited recovery of CD4+ T-cell count in peripheral blood in long-term antiretroviral-treated HIV-1-infected patients [7,8]. Although the clinical significance of this finding is unknown, it suggests a limited functional recovery of the immune system [26]. If, in turn, it implies a shorter life span or higher risk of comorbidities, then it merits further investigation.

Some patients had a detectable level of LTVL despite a median of 61 months on treatment. Although most patients were taking a protease inhibitor-sparing regimen, as has been reported with shorter periods on ART [11,14,27], the duration of complete suppression of PVL in patients on a protease inhibitor-sparing regimen was significantly shorter than in patients on protease inhibitor-containing regimens, suggesting that the duration and not the type of regimen is responsible for the incomplete viral suppression.

In a series of seven HIV-1-infected patients treated with ART and four individuals deferring therapy, Schacker et al. [21] reported evidence of significant paracortical T-cell zone damage associated with deposition of collagen, the extent of which was inversely correlated with both the size of the lymphoid tissue CD4+ T-cell population and the change in peripheral CD4+ T-cell count with anti-HIV therapy. It is acknowledged that larger scale studies that assess changes in the structure of the lymphoid tissue after prolonged HIV-1 therapy would be required to determine whether the niche damage is reversible and at what level ART alone improves lymphoid tissue immune reconstitution [20,21]. Here, we studied tonsillar biopsies of 29 patients with a median of 61 months on ART. It seems that sustained ART did not reverse fibrosis in lymphoid tissue. In fact, the current data indicate that after 5 years on ART, collagen deposition is associated with a limited immune reconstitution in both lymphoid tissue and peripheral blood, and also show that the recovery of lymphoid tissue architecture was worse in those patients with the highest collagen deposition. Although collagen deposition was found to be similar in naive and treated patients, it should be noted that antiretroviral-naive patients were younger and that age is positively correlated with fibrosis. Therefore, the fact that the treated (and older) group had similar collagen deposition to the naive (and younger) patients could mean that ART has some effect on collagen deposition, which is not detected due to this bias.

As collagen deposition could play a pivotal role in limiting immunological reconstitution, it is essential to learn more about the factors associated with lymphoid tissue fibrosis so as to understand better this process and investigate adjuvant therapies to ART in order to improve immune reconstitution. As suggested by other authors [28,29] who have analysed fibrosis in other disorders, collagen deposition in lymphoid tissue was associated with age. Taken together with other factors such as poor thymic output, this fact could partially explain why immune reconstitution is poorer in older patients [4]. Other factors such as sex, risk group of HIV infection and coinfection of hepatitis B and C were not associated with collagen deposition. The data on the influence of hepatitis B and C coinfection in lymphoid tissue fibrosis should be interpreted with caution, given the low number of patients included. As reported by other authors [21], there was no association between the percentage area of collagen and the level of current peripheral blood CD4+ and CD8+ T cells, the nadir of CD4+ T cells and the peak of PVL, which are the markers of disease severity.

It has been reported previously that although the LTVL and the immunoexpression of p24 antigen decreased significantly after treatment with ART, LTVL or HIV-1 structural proteins and glycoproteins [16] did not disappear in all cases, despite showing an undetectable PVL [10,11,14,15,27,30–33]. These virus or viral proteins contribute to the persistence of inflammation and immune activation, which appear to be the driving stimulus for collagen deposition. In support of this hypothesis, we found that a detectable level of LTVL in treated patients was a factor independently associated with collagen deposition in lymphoid tissue. It has been suggested that adjunctive anti-inflammatory therapy would provide substantial benefit by limiting the damage to the lymphoid tissue [21]. Our data suggest that in addition to anti-inflammatory therapy, it would be useful to reduce LTVL to an undetectable level so as to minimize activation and reverse the damage in lymphoid tissue. Clinical trials of ART with adjunctive anti-inflammatory therapy, with the aim of reducing LTVL to an undetectable level and diminishing inflammation, are, therefore, warranted.

As discussed above, in this study, most of the patients on successful treatment and with detectable LTVL were taking a protease inhibitor-sparing regimen. The type of treatment also seems important with respect to the level of CD4+ T-cell recovery. Cohort studies suggest that immunological recovery of patients on ART is highly variable and depends partially on the type of antiretroviral used in the combination [34]. In the AIDS Clinical Trials Group 5142 trial, the mean increase of CD4+ T cells after 96 weeks of treatment was significantly greater in the lopinavir–ritonavir group than in the efavirenz group [35]. The reasons for this difference in immunological recovery with a similar (or even worse) virological response are not clear. It has been suggested that protease inhibitors could have a direct effect on host cells that is not mediated by the decrease in viral replication or changes in viral fitness [36–39]. Some authors [36,40–46] have hypothesized that protease inhibitors could modulate the activation of peripheral blood CD4(+) T cells and decrease their susceptibility to apoptosis in vitro and in vivo. Conversely, it is known that efavirenz induces caspase and mitochondrion-dependent apoptosis of Jurkat T cells and human peripheral blood mononuclear cells [47]. We found a higher collagen deposition in lymphoid tissue in patients receiving protease inhibitor-sparing regimens compared with those receiving protease inhibitor-containing regimens, even when previous ART regimens were considered. However, this association should be interpreted with caution, as the number of individuals on specific antiretroviral regimens is too small to draw any definitive conclusion in this regard. As such, this finding should be confirmed and further research is required to clarify whether protease inhibitors limit collagen deposition and, ultimately, improve immune recovery through antiviral or other anti-inflammatory effects.

In summary, a number of studies [1–6] have reported that immunological reconstitution is limited in some patients, even after long-term ART [7,8]. There is some evidence to suggest that collagen deposition and fibrotic damage in lymphoid tissue are contributing mechanisms in CD4+ T-cell loss and in limiting reconstitution [20–25]. The current study confirmed that fibrosis in lymphoid tissue was associated with a poorer reconstitution in both lymphoid tissue and peripheral blood, and showed that long-term ART for 5 years did not reverse fibrosis in lymphoid tissue. ART did improve lymphoid tissue architecture and a significant increase in CD4+ T cells in lymphoid tissue was observed in patients on ART. However, the CD4+ cell count in the lymphoid tissue of HIV-infected patients remained lower than in noninfected individuals, despite long periods on ART and a high nadir of CD4+ T cells. We found that in addition to HIV infection, older age, a detectable level of LTVL in patients who were successfully treated with antiretrovirals and protease inhibitor-sparing regimens seem to favour fibrosis in lymphoid tissue. Additional measures (i.e. anti-inflammatory therapy) should be used to test whether diminishing lymphoid tissue fibrosis could improve immunological reconstitution.

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Acknowledgements

This study was supported in part by grants: Red Temática Cooperativa de Grupos de Investigación en Sida del Fondo de Investigación Sanitaria (RIS), Center for Research and Development of HIV Vaccines in Catalonia (HIVACAT), SAF 2006-26667-E, FIS PI050058, FIS PI 040503, FIS PI 070291, FIT 090100-2005-9 and SAF 2008-04359.

F.G. and J.M.M. were recipients of a research grant from Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain. M.P. was supported by contract FIS 03/00072 from the Fundació Privada Clínic per la Recerca Biomèdica in collaboration with the Spanish Health Department.

Study Group of Lymphoid Tissue Immunopathogenesis in HIV Infection: Alba Diaz, Llúcia Alós, Anna Mozos, Antonio Martinez, Department of Pathology, Hospital Clínic, IDIBAPS, University of Barcelona, Spain. Agathe León, Borja Mora, Diaz-Brito, Vicens José L. Blanco, Esteban Martínez, Josep Mallolas, Josep M. Miró, María Larrousse, Monserrat Laguno, Ana Milinkovic, Jose M. Gatell, Felipe García, Department of Infectious Diseases, Hospital Clínic, IDIBAPS, University of Barcelona, Spain. Miguel Caballero, Department of ENT, Hospital Clínic, IDIBAPS, University of Barcelona, Spain. Montserrat Plana, Cristina Gil, Retrovirology and Viral Immunopathology Laboratories, Hospital Clínic, IDIBAPS, University of Barcelona, Spain. Teresa Gallart, Immunology Laboratory, Hospital Clínic, IDIBAPS, University of Barcelona, Spain. Manuel Leal, Department of Infectious Diseases, Hospital Virgen del Rocío, Sevilla, Spain. Ramón Deulofeu, Xavier Filella, Centro de Diagnóstico Biomédico, Hospital Clínic, IDIBAPS, University of Barcelona, Spain.

A.D., L.A. and F.G. conceived and designed the study, undertook the statistical analyses and drafted the manuscript. A.L., M.C., M.P., T.G., C.G. and M.L. contributed to the study design and data management. A.D., L.A., A. M. and A.M. analysed lymphoid tissue architecture and collagen deposition and performed the quantitative assessment of CD4+ cells in lymphoid tissue. J.M.G. participated in study analyses and manuscript preparation. All authors reviewed and approved the final version of the manuscript.

There are no conflicts of interest.

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Cited By:

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Immunological Reviews
Pathobiology of HIV/SIV-associated changes in secondary lymphoid tissues
Estes, JD
Immunological Reviews, 254(): 65-77.
10.1111/imr.12070
CrossRef
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Keywords:

collagen deposition; HIV infection; immune reconstitution

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