Differences in neuroinflammation in people who started antiretroviral treatment during primary versus chronic HIV infection: an 18kDa Translocator protein (TSPO) positron emission tomography (PET) study

With the widespread implementation of modern antiretroviral treatment (ART), the prognosis and life expectancy for people with HIV has significantly improved (Hogg et al. 1998; May et al. 2014). However, people with HIV on virologically suppressive ART remain at increased risk of non-AIDS co-morbidities including cognitive impairment (Robertson et al. 2007; Schouten et al. 2014; Simioni et al. 2010). The underlying mechanisms are likely multifactorial; persistent microglial activation (Anthony et al. 2005; Minagar et al. 2002) and neuroinflammation in people with HIV on virological suppressive ART (Edén et al. 2016) is postulated to be a contributor.

The 18kDa Translocator protein (TSPO), located on the outer mitochondrial membrane, is highly expressed in activated microglia (Banati et al. 2004; Liu et al. 2014) and is associated with neuroinflammation (Liu et al. 2014). Synthetic radiolabelled ligands that selectively bind to TSPO have been developed and can be utilised to image dynamic microglial activation in the human brain in vivo, using positron emission tomography (PET) imaging (Stephenson et al. 1995). The first-generation TSPO radiotracer, [¹¹C]-PK11195 was limited by low blood–brain barrier penetration and high nonspecific binding. Second-generation radiotracers such as [¹¹C]PBR28 and [¹¹C]DPA-713 have improved blood–brain barrier penetration and signal-to-noise ratios, however binding affinity is dependent on single-nucleotide polymorphism rs6971, whereby genotypic testing must be carried out to ascertain whether an individual is a low-, medium- or high-affinity binder.

TSPO PET brain imaging has been used to investigate neuroinflammation in people with HIV (Boerwinkle et al. 2020; Coughlin et al. 2014; Garvey et al. 2014; Hammoud et al. 2005; Vera et al. 2016; Wiley et al. 2006), with conflicting results. While higher TSPO binding in people with HIV has generally been reported compared with persons without HIV, the precise anatomical locations have varied between studies. These inconsistencies are likely related to (1) different methodologies used to quantify TSPO radiotracer uptake, (2) cohort differences (inclusion or exclusion of people with HIV with cognitive impairment), (3) differences in criteria for defining cognitive impairment (Nightingale et al. 2021) and (4) small sample sizes.

Strategies to reduce persistent immune activation and inflammation include the early initiation of ART, soon after HIV acquisition (Pace & Frater 2014). Early ART initiation may partly mitigate the effects uncontrolled HIV replication has on the central nervous system and thereby reduce the persistent neuroinflammation which has been observed in ART-treated individuals. Early evidence supports a reduction in cerebrospinal fluid biomarkers of inflammation when ART is initiated during acute HIV infection (Hellmuth et al. 2019; Oliveira et al. 2017), however, long-term data on brain parenchymal inflammation and neurological sequelae are lacking.

The aim of this study was to evaluate neuroinflammation, measured using TSPO [¹¹C]PBR28 radiotracer binding, in people with HIV who initiated ART during acute versus chronic HIV infection, and compared with control individuals, using volume of distribution (V_T) as a primary outcome and distribution volume ratio (DVR) as a secondary outcome.

All participants underwent structural cerebral magnetic resonance imaging (MRI) and cerebral positron emission tomography-computed tomography (PET-CT) imaging with [¹¹C]PBR28 ligand at the Imanova Centre for Imaging Sciences, London, UK.

This study compares neuroinflammation using [¹¹C] PBR28 binding as a proxy in people who initiated ART in acute HIV infection, people who initiated ART in chronic HIV infection and control participants. While no statistically significant differences in [¹¹C] PBR28 binding were observed between the three groups, our analyses consistently demonstrated higher absolute mean [¹¹C] PBR28 binding amongst people who initiated ART during chronic HIV infection compared with people who initiated ART during acute HIV infection and control participants at the majority of brain anatomical regions studied. Our findings should be interpreted with caution due to the small sample size of our pilot study, however this initial signal may herald a potential true signal that warrants further investigation. The trend towards lower absolute [¹¹C] PBR28 binding in participants who initiated ART during acute infection compared with chronic infection corroborates the theory that early ART initiation soon after HIV acquisition may attenuate the neuroinflammatory responses widely reported in persons with HIV who initiated ART during chronic HIV infection (Ulfhammer et al. 2018; Yilmaz et al. 2008).

While some previous TSPO studies in people with HIV have identified statistically significant differences in TSPO binding at certain anatomical locations in people with HIV on ART compared with control participants, we did not observe this in our study. Reasons for the discrepant results between the various TSPO studies in people with HIV include the fact that the previous studies have used different TSPO radiotracers and different methodologies to quantify TSPO binding (Alagaratnam & Winston 2022). Quantification of TSPO binding remains a rapidly evolving field and our study utilised newer models and processes for quantification based on current gold-standard techniques that had not yet been determined at the time when the previous studies were conducted. In a previous study from our group, (Vera et al. 2016) estimated the continuous [¹¹C] PBR28 plasma-to-blood ratio using a constant model in the plasma input function. However, we observed that an exponential-approaching-constant model provided a better fit for the data in the majority of the participants in our study. Brain [¹¹C] PBR28 data for people who initiated ART during chronic HIV was provided from Vera et al.’s study (Vera et al. 2016), but the same control participants’ data were not available for us to use in our analysis, which may also explain the discrepancy seen between the results of our analyses. Additionally, brain [¹¹C] PBR28 signals from grey matter were analysed, as at the time, it was considered the optimal strategy for identifying microglial activation. However, given that HIV disease affects both the grey and white matter throughout the brain, our analyses here included brain [¹¹C] PBR28 signals from both grey and white matter. Furthermore, our study is limited by the sensitivity of [¹¹C]PBR28; although [¹¹C]PBR28 has greater sensitivity than previous TSPO ligands, it may not have sufficient sensitivity to determine differences in ligand binding between the three cohorts of participants in this study.

Whether to use absolute [¹¹C] PBR28 binding (V_T) or [¹¹C] PBR28 V_T binding normalised to a reference region (DVR) to report regional brain [¹¹C] PBR28 binding remains hotly debated. Some studies have suggested a poor test–retest reproducibility with absolute TSPO V_T binding due to intra-individual factors and normalisation to a reference or pseudo-reference region can control for unaccounted physiological factors such as stress responses, hormone-mediated changes in TSPO expression, blood cholesterol changes due to food intake and other genotypic factors that may affect TSPO radioligand uptake in the brain (Coughlin et al. 2014; Drugan 1996; Gavish et al. 1999; Jučaite et al. 2012). These factors may impact cerebral radioligand signal in TSPO PET imaging studies and can also cancel out non-binding effects on the TSPO radioligand signals. A study using [¹¹C] DPA-713, a second-generation TSPO radioligand, demonstrated improved test–retest reproducibility when [¹¹C] DPA-713 VT binding was normalised to a reference region (Coughlin et al. 2014). A major concern with normalising TSPO binding to a reference region is losing the ability to detect changes in the region used as the reference region, by effectively cancelling out the signal in the region of interest by normalising to another region also displaying a signal. Ideally, the reference region chosen should be unaffected by the disease under investigation. However, HIV disease generally affects the whole central nervous system, and a reliable disease-free brain TSPO reference region in people with HIV has not yet been identified. For this reason, we have chosen to use absolute [¹¹C] PBR28 V_T binding as our primary outcome, with [¹¹C] PBR28 DVR binding normalised to cortical grey matter as the secondary outcome. Urgent consensus is required on the optimal methodology for determining [¹¹C] PBR28 binding and the optimal reference region for TSPO studies. Until a gold-standard method of measuring TSPO radiotracer uptake is developed and accepted, interpreting and comparing results from TSPO PET neuroimaging studies will remain challenging.

Strengths of our study include the phenotypically well-described cohorts of participants living with HIV. Participants who initiated ART during acute HIV infection were strictly within 3 months of confirmed primary HIV infection. [¹¹C] PBR28 binding quantification was performed using the gold-standard methodology using continuous plasma input function via arterial sampling.

On the converse, limitations include the small sample size due to the high cost of performing PET neuroimaging studies, and the control participants who were not demographically matched to the people living with HIV. Overall, participants who initiated ART during chronic HIV infection were older and had higher body weight upon enrolment into the study, which may reflect the normal ageing processes. While control participants were on average younger than both groups of HIV-positive participants, the control participants’ body weight were similar to that seen in participants who initiated ART during chronic HIV infection. To date, published findings on the effect of age on TSPO expression have been inconclusive with some studies demonstrating higher TSPO binding with increasing age and no effect in others (Cagnin et al. 2001; Paul et al. 2019; Rissanen et al. 2018; Schuitemaker et al. 2012; Tuisku et al. 2019). Body mass index has been shown to negatively correlate with brain [¹¹C] PBR28 V_T binding (Tuisku et al. 2019), however complete body mass index data was not available for our analysis. We are also limited by a lack of data on alcohol use, recreational drug use and smoking history which could be confounders to our findings.

Astrocytes and microglia play very different roles in the central nervous system but emerging data suggests that reactive astrocytes may also have a contributory role to TSPO expression signals (Lavisse et al. 2012) and it is becoming increasingly recognised that [¹¹C] PBR28 binding can represent pro-, anti- and mixed inflammatory phenotypes (Owen et al. 2017). Thus, TSPO expression signals in this setting should be interpreted with caution and further studies are warranted to determine the phenotype (pro-, anti- and mixed inflammatory) of the signals identified and the cell type contributing to the [¹¹C] PBR28 binding signals (microglia and astrocytes), by correlating brain TSPO binding results with neuropathological specimens with histochemistry and fluid biomarkers.

In summary, this study assessed the effect of early ART initiation soon after HIV acquisition on neuroinflammation using a novel molecular neuroimaging technique. We observed no significant differences in neuroinflammation, using [¹¹C]PBR28 binding as a proxy between people who initiated ART during chronic HIV infection, people who initiated ART during primary HIV infection and control participants. A trend towards higher neuroinflammation in people who initiated ART during chronic infection compared with acute infection and control participants was observed, suggesting early ART initiation may reduce neuroinflammation.