Blood Brain Barrier Impairment in HIV-positive Naïve and Effectively Treated Patients: Immune Activation versus Astrocytosis
Calcagno A, MD1,Romito A, MD2, Atzori C, BSc3, Ghisetti V, BSc4, Cardellino C, MD1, Audagnotto S, MD1, Scarvaglieri E, MD1, Lipani F, MD1, Imperiale D, MD3, Di Perri G, MD, PhD1 and Bonora S, MD1.
1Unit of Infectious Diseases, Department of Medical Sciences, University of Torino; 2Laboratory of
Immunology and3Unit of Neurology, Ospedale Maria Vittoria, ASLTO2; 4Laboratory of Microbiology and
Molecular Biology, Ospedale Amedeo di Savoia, ASL TO2, Torino, Italy.
Running Head: Astrocytosis and BBB in HIV+ patients Type of article: Original article
Key words: blood brain barrier; albumin ratio; biomarkers; S-100 beta; astrocytosis. Word count: 2115 (Abstract 248)
Figures: 1 Tables: 3
Corresponding Author:
Andrea Calcagno
Unit of Infectious Diseases Department of Medical Sciences University of Torino
Amedeo di Savoia Hospital C.so Svizzera 164
10149 Torino Italy
Conflicts of Interest and Source of Funding: AC has received honoraria from Abbvie, BMS, Gilead, Janssen-Cilag, MSD, Viiv and he is currently receiving research grants from BMS, Gilead and Viiv. GDP and SB have received honoraria from Abbvie, BMS, Gilead, Janssen-Cilag, MSD, Viiv. The remaining authors have no conflict of interest to declare.
Background: Blood brain barrier (BBB) damage is a common feature in central nervous system infections by HIV and it may persist despite effective antiretroviral therapy. Astrocyte involvement has not been studied in this setting.
Methods: Patients were enrolled in an ongoing prospective study and subjects with central nervous system-affecting disorders were excluded. Patients were divided into two groups: treated subjects with cerebrospinal fluid (CSF) HIV RNA <50 copies/mL (CSF-controllers) and in late-presenters CD4+ T lymphocytes <100/uL. CSF biomarkers of neuronal or astrocyte damage were measured and compared to CSF serum-to-albumin ratio.
Results: 134 patients were included; 67 subjects in each group (50%) with similar demographic characteristics (with the exception of older age in CSF controllers). CD4 (cells/uL), plasma and CSF HIV RNA (Log10 copies/mL) were 43 (20-96), 5.6 (5.2-6) and 3.9 (3.2-4.7) in LPs and 439 (245-615), <1.69 (9 patients <2.6) and <1.69 in CSFc. BBB impairment was observed in 17 late-presenters (25.4%) and in 9 CSF-controllers (13.4%). CSF biomarkers were similar but for higher CSF neopterin values in late-presenters (2.3 vs. 0.6 ng/mL, p<0.001). CSARs were associated with CSF neopterin (rho=0.31, p=0.03) and HIV RNA (rho=0.24, p=0.05) in late-presenters and with CSF tau (rho=0.51, p<0.001), p-tau (rho=0.47, p<0.001) and S100beta (rho=0.33, p=0.009) in CSF-controllers.
Conclusion: In HAART-treated subjects with suppressed CSF HIV RNA, BBB altered permeability was associated with markers of neuronal damage and astrocytosis. Additional treatment targeting astrocytosis and/or viral protein production might be needed in order to reduce HIV effects in the central nervous system.
HIV affects the central nervous system (CNS) by infecting microglia, perivascular macrophages and astrocytes; neuronal damage is thought to be caused by cytokines and mediators produced by such cells.1 Although several factors contribute to the incidence of neurocognitive impairment it has been shown that uncontrolled cerebrospinal fluid (CSF) HIV replication may lead to severe neurological disorders.2
A restricted low-level infection of astrocytes has been observed with the potential to substantially affect blood brain barrier (BBB) permeability given their involvement in the neurovascular unit.3 Although only 5% of astrocytes may be HIV-infected this event leads to intense modifications in BBB both in in vitro models and in SIV-infected macaques. Impairment in BBB (BBBi) has been observed in several HIV-positive patients and it has been constantly reported in those presenting with HIV-associated dementia.4
S 100 Beta (S100B) is a member of the S100 family of calcium binding proteins and it is produced by glial cells (primarily by astrocytes) although several other cellular sources have been identified.5 However it is only expressed by mature astrocytes that ensheath blood vessels and by Neuronal/Glial antigen 2-expressing cells (now referred to as oligodendrocyte precursor cells). S100B is involved in several cellular processes including neurite extension, astrocytosis and axonal proliferation. It has been used as a marker of different CNS processes; plasma levels, reflecting BBBi, have been proposed as early markers of brain damage including neurodegenerative disorders, while plasma and CSF concentrations were reported to increase after traumatic brain injury and seizures.6-8 Few studies have investigated the role of S100B during HIV infection; however S100B-defined astrocytosis has been associated with uncontrolled HIV replication in the CSF, chronic pain, impairment in verbal fluency and executive dysfunction and with severe, rapidly progressing dementia.9,10 Histopathological studies suggested a significant involvement of astrocytes in patients with dementia.11
After describing an association between BBBi and markers of neuronal damage we aimed at better clarifying such association and at studying astrocyte involvement in the course of HIV infection.12 Therefore we compared two “extreme” groups of HIV-infected patients: those subjects presenting with less than 100 CD4+ T lymphocyte/uL with those ones with controlled CSF HIV RNA under HAART.
Methods:
Adult HIV-positive patients undergoing lumbar punctures for clinical reasons were included: patients with central nervous system infectious, autoimmune or neoplastic disorders were excluded. Written informed
consent was obtained from all study participants and the study was approved by the local Ethics Committee (“Comitato Etico Interaziendale Ospedale San Luigi di Orbassano”). Demographic, immunovirological and therapeutic data were recorded.
HIV-positive late-presenters with less than 100 CD4+ T lymphocytes/uL (Late Presenters, LPs) were compared to HAART-treated patients showing a CSF HIV RNA below 50 copies/mL (CSF-controllers – CSFc); 55 patients from this group were included in the aforementioned paper.12
Quantitative determination of albumin in serum and CSF was measured by Immunoturbidimetric methods (AU 5800, Beckman Coulter, Brea, CA, USA). CSAR, calculated as CSF albumin (mg/L)/serum albumin (g/L), was used to evaluate BBB function. BBB damage definition was derived from age-adjusted Reibergrams (normal if below 6.5 in patients aged <40 years and below 8 in patients >40 years).13
CSF total tau (t-tau), phosphorylated tau (p-tau) and β-amyloid1-42 (Aβ1-42) were measured by immunoenzymatic methods (Fujirebio diagnostics, Malvern, U.S.A.) with limits of detection respectively of 57, 20 and 225 pg/ml. Neopterin and S100B were measured through validated ELISA methods [DRG Diagnostics (Marburg, Germany) and DIAMETRA S.r.l. (Spello, Italy), respectively]. Reference values were as follows: t-tau [<300 pg/mL (in patients aged 21-50), <450 pg/mL (in patients aged 51-70) or <500 pg/mL in older patients], p-tau (<61 pg/mL), 1-42 beta amyloid (>500 pg/mL), neopterin (<1.5 ng/mL) and S100B (<380 pg/mL).14 HIV RNA was measured through the real time Polymerase Chain Reaction (PCR) assay CAP/CTM HIV-1 vs. 2.0 (CAP/CTM, Roche Molecular System, Branchburg, NJ, detection limit: 20 copies/mL of HIV-1 RNA).
Neurocognitive tests and Instrumental Activity of Daily Living (IADL) questionnaire were administered by a trained neuropshycologist exploring 8 cognitive areas: diagnosis of HAND was performed according to the Frascati criteria.15
Data were analysed using standard statistical methods: variables were described with medians [interquartile ranges (IQR) or ranges (minimum-maximum)] and they were compared using non-parametric tests (Mann-Whitney, Chi-square and Spearman’s tests as specified in the text). Data analysis was performed using SPSS software for Mac (version 22.0, IBM Corp).
134 patients were included; 67 in each group. Baseline characteristics are shown in table 1.
Patients in the LP group showed no neurological complaint and brain magnetic resonance imaging (MRI) was either within normality or showing bilateral periventricular FLAIR-hyperintensities (19, 28.3%). 7 (10.5%) were AIDS-presenters; all of them had a Pneumocystis jirovecii pneumonia. Lumbar punctures were performed before starting HAART. Patients in the CSFc group received spinal taps for excluding CSF escape in patients with cognitive complaints (20, 29.8%), white matter abnormalities on MRIs (18, 26.9%), headache (7, 10.5%) or in the differential diagnosis of other disorders. Antiretroviral regimens included 1 (2, 3%), 2 (10, 14.9%), 3 (53, 79.1%) or 4 drugs (2, 3%): protease inhibitors were the most used third-agents (46, 68.6%) followed by integrase strand transfer inhibitors (10, 14.9%) and non-nucleoside reverse transcriptase inhibitors (9, 13.4%). HAART duration was 21 months (16-31) in this group of patients.
The results of CSF analysis are shown in Table 2. The two groups differed significantly as for number of CSF cells and neopterin concentrations; BBBi was marginally more prevalent in the LPs group (25.4% vs. 13.4%, p=0.081). Biomarkers outside reference ranges were observed (in LPs and CSFc respectively) in 7.8% vs. 0% (t-tau, p=0.048), 7.7% vs. 1.7% (p-tau, p=0.184), 7.8% vs. 5.1% (Aβ1-42, p=0.702), 64.6% vs. 8.1% (neopterin, p<0.001) and 6.8% vs. 11.3% (S100B, p=0.438).
CSF neuronal damage biomarkers (t-tau, p-tau and Aβ1-42) showed a high correlation among them in both groups as shown in table 3. Interestingly neopterin correlated with neuronal damage biomarkers in LPs while S100B was associated with such damage in CSFc. Specifically CSAR was associated with neopterin (rho=0.308, p=0.033) and CSF HIV RNA levels (rho=0.24, p=0.05) in LPs while it was associated with S100B (rho=0.328, p=0.009) and duration of viral suppression (rho=-0.472, p=0.001) in CSFc (Fig.1). In patients in the CSFc group the presence of BBBi was associated with higher concentrations of several biomarkers including t-tau (201.6 vs. 86.6 pg/mL, p=0.006), p-tau (35.3 vs. 32.1 pg/mL, p=0.040) and S100B (241.1 vs. 138.15 pg/mL, p=0.016).
Neurocognitive tests were available in 21 LPs and in 29 in CSFc: HAND was diagnosed in 9 (42.9%) and 10 patients (34.5%), respectively; International HIV Dementia score (IHDS) and Mini Mental State Examination (MMSE) were 10.7 (9.1-11.5) and 29.5 (29-30) in LPs and 6 (3.9-8.6) and 26.4 (21.8-28.5) in CSFc. CSF tau was higher in patients with HAND (147 vs. 92 pg/mL, p=0.037) and it was inversely correlated with IHDS (rho=-0.552, p=0.0013) and scores. An inverse correlation was observed between CSF S100Beta and IHDS (rho=-0.423, p=0.027) and MMSE (rho=-0.414, p=0.032) scores (Figure 2).
Discussion:
In this analysis we observed that BBB impairment is present in several HIV-positive patients. It is more common in those who present late for diagnosis and that have been exposed to uncontrolled viral replication for years: in this group the degree of permeability of the barrier was directly related to the degree of compartmental viral replication and immune activation. BBB impairment was less frequent in HAART-treated patients that reached CSF viral suppression (13.4% in this group); however CSF serum albumin ratios were directly related to markers of neuronal damage (total and phosphorylated tau) and astrocytosis (S100Beta). Interestingly such markers of neuronal damage seem to be associated with neopterin (and thus immune activation) in late presenters and with S100Beta (and thus astrocytosis) in those on efficacious treatment.
BBB alterations have been observed in asymptomatic and demented HIV-patients with prevalence rates ranging from 2 to 100% (in the severely affected group). 4,12,17 The leading mechanism is not known but it
may involve the secretion of pro-inflammatory cytokines by infected glial cells, the direct effect of HIV viral proteins on the BBB, astrocyte podocytes abnormalities following cellular infection and the direct involvement of endothelial cells.18 Higher CSF HIV RNA have been observed in patients with neurological symptoms and they have also been associated with BBB damage.12,19 In treated patients CSF escape (with CSF HIV RNA 1 Log10 higher than plasma levels or detectable in case of plasma undetectable HIV RNA) has been associated with severe neurological symptoms that resolved upon the CSF-tailored change in antiretroviral treatment.20 However even if CSF escape might be an isolated and non progressive event observed in 10% of treated patients it has been shown that very low level but detectable CSF HIV RNA are associated with higher CSF levels of neopterin thus suggesting a direct link between residual replication and immune activation.12,21,22 Our data might support this association in patients with several years of uncontrolled HIV replication:since damage appear to be virus-related antiretroviral treatment seems to be the best intervention as it has been recently shown in patients with acute infections.23 However in some patients BBB impairment and intrathecal inflammation may persist: it would be important to predict such event by baseline clinical or cerebrospinal fluid features.24
The introduction of HAART is effective in controlling HIV replication in the CSF; nevertheless the high prevalence of HAND challenges the idea of a complete compartmental efficacy. Several factors may influence CNS response to antiretrovirals including their penetration/effectiveness, CSF exposure, pharmacogenetics, adherence to medications, drug abuse, HIV DNA as well as demographic factors such as
age and ethnicity. Antiretroviral efficacy might be different according to target cells and monocyte-derived HIV-infected cells (microglia, perivascular macrophages and astrocytes) have shown variable responses to different compounds and residual microglial activation despite efficacious HAART have been demonstrated
in vivo.25,26 Brain tissue micro array studies suggested that while HIV replication might be relevant in approximately 10% of patients, 35% of the included subjects presented markers of vascular damage.27
Being the neurovascular unit a complex entity including endothelial cells, astrocytes and neurons it appears that the role of infected astrocytes (although a minority of such cells is infected by HIV and mature virions have not been observed) could be a key factor in the impairment of BBB. As neuro-inflammation persists astrocytes may lose their ability to regulate BBB integrity, and neurotransmission in tripartite synapses is
impaired: reactive oxygen species have been shown to be increased under chronic inflammatory conditions.28
Eventually, this may lead to impaired neuronal function and survival following insufficient support and indirect toxicity. However more studies are needed in order to understand the role of activated astrocytes in the pathogenesis of HAND and to clarify the meaning of S100beta in HIV-infected patients. Of note several cells are able to secrete S100beta (including adipocytes, chondrocytes, lymphocytes, bone marrow cells and melanoma cells) and low concentrations of this protein have been shown to be neurotrophic and even beneficial.5,29
In this analysis we may support the hypothesis that astrocytosis can be associated with persistent BBB impairment and markers of neuronal damage; having several patients in this group been diagnosed with
HAND, it further supports earlier observations of a link between astrocytosis and executive functioning.7
Such persistence despite effective antiretroviral treatment might have three possible explanations: incomplete cellular efficacy, a mechanism that does not rely on viral transcription and the possible direct toxic effect of antiretrovirals. Each of these hypothesis has some supportive evidence but different targeted interventions. 30 The observed association of lower CSAR in patients with long-lasting HIV RNA suppression supports the efficacy of HAART in the majority of patients. However some subjects may require longer treatment in order to normalize central nervous system immune activation and BBBi; the association of HIV DNA (a
recognized marker of reservoires size) with HAND confirms the need for individualized treatments.31 The
association of HIV DNA and CD4 nadir with failure and CSF escape under protease inhibitor monotherapy
further confirms this hypothesis.32 Du Pasquier et al. showed markedly increased S100Beta in the CSF of
patients treated with such strategy suggesting that NRTI removal was detrimental for compartmental effectiveness: CSF S100B levels here observed were between those observed in the aforementioned study
Although it was beyond the aim of this analysis we had 50 clinically collected neurocognitive tests available. The significant correlation between CSF tau and S100Beta and cognitive performance further support the biological relevance of these markers in the process of neuronal damage.34
Several limitations of this analysis must be clearly mentioned including the cross-sectional design, the imperfect matching of the two groups (different nadir CD4 and age), the lack of neurocognitive tests for all patients, variable and short duration of HIV RNA suppression in the CSFc group. Furthermore we cannot exclude that BBB impairment and inflammation may be transient and resulting from concomitant opportunistic infections (such as Pneumocystis jirovecii pneumonia).
In conclusion we observed different patterns of biomarkers in the CSF of HIV-positive patients: in late-presenters we observed that BBB impairment was associated with viral replication and immune-activation while in CSF controllers it was associated, along with markers of neuronal damage, with astrocytosis.
Late Presenters CSF controllers P value n 67 67 Age: years 43 (37-49) 49 (43-56) 0.001 Male/Female 49/18 48/19 -BMI: Kg/m2 22 (19-23) (20-24)23 0.158 CD4: Cells/uL (20-96)46 (245-615)439 0.0001 CD4 nadir: Cells/uL 46 (20-96) 108 (24-243) 0.003
plasma VL: Log10 copies/mL 5.6
(5.2-5.9) - -*
CSF VL: Log10 copies/mL (3.2-4.7)3.9 - -*
Table 1. Baseline characteristics. “CSF”, cerebrospinal fluid; “BMI”, Body Mass Index; “VL”, viral load; “HAART”, Highly Active Antiretroviral Treatment. Comparisons were made through Chi-square (categorical variable) and Mann-Whitney (continuous variables) tests. * inclusion criteria. p-values were calculated using the Mann-Whitney test.
Late Presenters CSF controllers P value
(range 0-25) (range 0-5) CSAR (3.8-7.1)5.7 (3.8-6.4)5 0.333 BBBi (25.4%)17 (13.4%)9 0.081 t-tau: pg/mL 102 (37-211) 88 (37-167.5) 0.269 p-tau: pg/mL (21-40)28.4 (23-39)32.7 0.408 Aβ1-42: ng/mL 789 (620-1022) 851 (635-974) 0.804 Neopterin: : ng/mL (1.1-4.7)2.3 (<0.5-1)0.6 0.0003 S100B: pg/mL (92-265)140 (93-208)139 0.972
Table 2. Cerebrospinal fluid biomarkers in both study groups. “CSF”, cerebrospinal fluid; “CSAR”, Cerebrospinal fluid Serum Albumin Ratio; “BBBi”, Blood Brain Barrier impairment; “t-tau”, total tau; “p-tau”, phosphorylated tau; “Aβ1-42”, 1-42 beta amyloid; “S100B”, S100 Beta. Comparisons were made through Chi-square (categorical variable) and Mann-Whitney (continuous variables) tests. p-values were calculated using the Mann-Whitney test.
CSAR t-tau p-tau Aβ1-42 Neop S100B CSAR t-tau p-tau Aβ1-42 Neop S100B CSAR - 0.142 0.015 0.022 0.308* -0.040 0.515** 0.475** 0.295** 0.020 0.328** t-tau 0.639** 0.613** 0.470** 0.079 0.600** 0.379** 0.271* 0.389** p-tau 0.601** 0.238 -0.085 0.472** 0.221 0.275** Aβ1-42 0.404** 0.139 0.000 0.285* Neopterin -0.017 0.118 S100B -
-Table 3. Correlation between cerebrospinal fluid biomarkers in both groups. Spearman’s rho are shown for each bivariate comparison. “CSF”, cerebrospinal fluid; “CSAR”, Cerebrospinal fluid Serum Albumin Ratio; “BBBi”, Blood Brain Barrier impairment; “t-tau”, total tau; “p-tau”, phosphorylated tau; “Aβ42”, 1-42 beta amyloid; “S100B”, S100 Beta. *p-value<0.05 level, ** p-value<0.01. p-values were calculated using the Spearman’s test.
Figures
rho=0.020 p=0.875 rho=0.308
Figure 1. Correlation between Cerebrospinal fluid Serum Albumin Ratios (CSAR) with neopterin (above) and S100Beta (below) in the two groups (LPs left, light gray and CSFc right, dark grey). Continuous lines represent fit-line with 95% confidence intervals (dotted lines). Rho and p-values were calculated using the Spearman’s test.
rho=0.328 p=0.009 rho=-0.040
Figure 2. Correlation between Cerebrospinal fluid tau (left) and S100Beta (right) and International HIV Dementia Scores. Continuous lines represent fit-line with 90% confidence intervals (grey lines). Rho and p-values were calculated using the Spearman’s test.
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