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. 2024 Feb 22;21(2):e00329. doi: 10.1016/j.neurot.2024.e00329

Honokiol hexafluoro confers reversal of neuropathological markers of HIV infection in a murine SCID model

Zhan Zhang a,b,c, Aaron Scanlan a,b, Rajeth Koneru a,b, Chelsea Richardson Morrell a,b, Monica D Reece c, Emily Edwards c, Sebastian Roa c, Christina Gavegnano b,c,d,e,f, Heather Bimonte-Nelson g, Jack Arbiser h,i, William Tyor a,b,∗
PMCID: PMC10943487  PMID: 38388224

Abstract

Cognitive impairment remains a persistent challenge in people living with HIV (PWLH) despite antiretroviral therapy (ART) due to ART's inability to eliminate brain HIV. HIV-induced cognitive dysfunction results from immune dysregulation, ongoing neuroinflammation, and the continuous virus presence, collectively contributing to cognitive deficits. Therefore, adjunctive therapies are needed to reduce cerebral HIV reservoirs, mitigate neuroinflammation, and impede cognitive dysfunction progression. Our study focused on Honokiol, known for its anti-inflammatory and neuroprotective properties, in an experimental mouse model simulating HIV-induced cognitive dysfunction. Using Honokiol Hexafluoro (HH), a synthetic analogue, we comprehensively evaluated its potential to ameliorate cognitive dysfunction and cerebral pathology in HIV-associated cognitive dysfunction. Our findings showed that HH treatment effectively reversed HIV-induced cognitive dysfunction, concurrently suppressing astrocyte activation, restoring neuronal dendritic arborization, and reducing microglial activation. Furthermore, HH remodeled the metabolic profile of HIV-infected human monocyte-derived macrophages, resulting in decreased activation and the promotion of a quiescent state in vitro.

Keywords: Honokiol, HIV, Cognitive dysfunction, Mouse model

Introduction

Over the past three decades, the field of HIV treatment has witnessed significant advancements, leading to a remarkable reduction in morbidity and mortality among people living with HIV (PLWH) [1]. A pivotal breakthrough in HIV management has been the introduction of combined antiretroviral therapy (ART), which has revolutionized the approach to treating HIV infection. Notably, the implementation of ART has demonstrated substantial effectiveness in mitigating the risk and delaying the progression of severe HIV-associated cognitive dysfunction (CD). In PLWH on ART the incidence of HIV dementia has dramatically declined, but mild CD remains highly prevalent [2]. Elucidating the intricate pathogenic mechanisms underlying the onset of HIV-associated CD presents a complex challenge due to its multifactorial nature [[2], [3], [4]]. This inflammatory immune environment in the brain includes viral protein production, release of viral nucleic acids, and secretion of proinflammatory cytokines/chemokines within the central nervous system (CNS). These processes collectively contribute to the activation of bystander microglia and astrocytes, as well as the recruitment of monocytes across the blood-brain barrier (BBB) to the site of infection [5]. Individuals with HIV-associated CD consistently exhibit elevated levels of pro-inflammatory cytokines in their cerebrospinal fluid (CSF), including TNF-α, IL-1β, and IFN-α [6,7]. Notably, TNF-α has been shown to directly induce neuronal toxicity [8], while both TNF-α and IL-1β can contribute to neuronal injury [9]. Collectively, these mechanisms confer a feedback loop that is not addressed by direct acting antivirals. Notably, although ART has demonstrated efficacy in suppressing HIV replication and reducing certain markers of HIV-induced immune activation, ART may not result in normal immune status occurring in the absence of CNS inflammatory disease [[10], [11], [12]]. Consequently, PLWH receiving ART often experience persistent chronic systemic inflammation, which continues to exert detrimental effects on the body, despite the presence of treatment. Furthermore, it has been observed that viral proteins, including gp120, Tat, and Vpr, possess the capability to directly impair neuronal function, thus suggesting a potential link between persistent viral expression and neuroinflammation [8,13,14]. This implies that the ongoing presence of viral proteins may contribute to the maintenance of a neuroinflammatory state. These compelling findings strongly support the tenet that the continued presence of neuroinflammation, despite successful viral suppression, may play a significant role in the etiology and progression of HIV associated CD. Despite considerable progress, there still exists a substantial knowledge gap concerning the fundamental virological and neuroimmunological factors contributing to the development of HIV associated CD.

Despite the considerable impact of ART, the persistence of cognitive impairment highlights the limitations of current ART regimens [15]. Consequently, our objective is to identify a novel therapy capable of possibly reducing viral burden within the CNS while concurrently reducing brain inflammation, thereby mitigating the phenotype of CD in PLWH, without inducing neurotoxicity. To achieve this, we have established a murine model of HIV induced CD (aka HIV associated neurocognitive disorders or HAND) by intracranially inoculating HIV-1ADA-infected monocyte-derived macrophages (MDM). This model exhibits CD in HIV-infected mice, as measured by object recognition testing and water radial-arm maze testing [16,17]. We further characterized neuropathological markers in neurons-Microtubule-associated protein 2 (MAP2), glial cells-Glial fibrillary acidic protein (GFAP), and mononuclear phagocytes (Major Histocompatibility Complex Class II (MHC II)/CD11b). This experimental model is designed to detect milder forms of HIV CD and facilitate early treatment intervention [18]. Indeed, it is crucial to identify and intervene in the early stages, as emerging evidence suggests that severe forms of HIV CD may be irreversible [2]. By utilizing this murine HIV CD model, we aim to explore novel therapeutic interventions that can effectively address the challenges associated with cognitive impairment in HIV-infected individuals.

Despite the successful suppression of plasma viruses to undetectable levels through ART, the detection of viral RNA in postmortem brain tissue and cerebrospinal fluid (CSF) of individuals with aviremic status and suppressed viral loads indicates that the brain is a reservoir for HIV, presenting a formidable challenge in the pursuit of HIV eradication [[19], [20], [21], [22]]. Honokiol Hexafluoro (HH) is a chemically analogous compound to Honokiol (HNK), a biphenolic compound derived from Magnolia officinalis, which has gained attention due to its notable neuroprotective properties, primarily attributed to its direct safeguarding effects on neuronal function [23,24]. Studies have indicated that HNK can potentially ameliorate CD in mouse models of Alzheimer's disease by suppressing the activation of microglial cells and astrocytes [25], suggesting potential anti-inflammatory effects on glial cells [26]. Considering the pleiotropic effects of HNK in neuroprotection, it is reasonable to hypothesize that HH may also exert influences on HIV-1-infected MDM. Therefore, the purpose of this study is to examine the therapeutic potential of HH in effectively mitigating HIV-associated CD. Specifically, we treated HIV-infected MDM with HH in vitro and also treated mice in our HIV CD model.

Materials and Methods

Mice

Five-week-old B6. CB17-Prkdc/Szj (SCID) male mice were purchased from the Jackson Laboratory. HIV-1ADA-infected mice were housed in an animal biosafety level 2 facility under institutionally approved animal use protocols (IACUC #V003-22).

Human monocyte-derived macrophages (MDM) infection with HIV-1ADA

Human monocytes were obtained from the University of Nebraska Medical Center in Omaha, NE, and differentiated into monocyte-derived macrophages (MDMs) using well-established, published methodologies [27,28]. Briefly, the cells were cultured in Teflon flasks and exposed to 10 ​ng/mL of Macrophage Colony Stimulating Factor (M-CSF) (Sigma) for a period of 7 days, then approximately 50 million monocyte-derived macrophages (MDMs) were exposed to 1 ​mL of HIV-1ADA virus at a TCID50 of 10ˆ5.62. The infection was allowed to progress, and on Day 7 post-intracranial (IC) inoculation (p.i.), the monocyte-derived macrophages (MDMs) were subjected to immunostaining for p24 antigen to estimate the infection rate as previously reported [28]. The infection rate was estimated by assessing the proportion of cells exhibiting the presence of the p24-positive antigen, relative to the total number of cells observed under the microscope. Typically, the infection rate ranges between 70% and 80%. Uninfected human MDM were designated for the control group of mice (see below).

Viral inoculation

Five-week-old mice (n ​= ​24) were IC injected with 100,000 HIV-1ADA-infected MDMs resuspended in ∼30 ​μL of PBS, directly into the right frontal lobe, while the control group (n ​= ​11) were only injected with uninfected MDM. The depth of the injection was 4 ​mm stopped right before the basal ganglia. More detailed descriptions of experimental procedures are previously described [28].

ORT and HH treatment

The ORT procedure was similar to our previously published protocols [27]. On day 4 p.i., all mice were acclimated to the ORT (Object Recognition Test) chamber, with no objects present, for 5 ​min. On day 5 p.i., a training phase, a delay of 5 ​min, and then a preference test after the delay was performed on each mouse. In the training phase, two identical objects were placed near two corners at the opposite sides of the chamber (Fig. 1a). The animal was placed into the chamber and allowed a total of 5 ​min of exploration of the two identical objects, and then the animal was removed from the chamber. One of the identical objects was then replaced by a novel object (Fig. 1b). After a 5 ​min delay, the preference phase for novel object testing was initiated; for this phase, the mouse was placed back into the chamber and allowed another 5 ​min of exploration of the two objects. This procedure was repeated on day 6 p.i. as well, except with a 2 ​h delay before the mouse was placed back into the chamber for the preference phase for novel object testing. HH was provided by Dr. Arbiser. It is 99% pure as assessed by nuclear magnetic resonance and is pharmaceutical grade. Treatment with HH (50 ​mg/kg daily, intraperitoneally injected) started after ORT on day 6, and was given consecutively for 7 days (Fig. 1). On day 12, mice underwent ORT which was similar to the ORT on day 5 where there was a 5 ​min delay between the training phase and the preference phase. ORT testing on day 13 was similar to the ORT on day 6 where there was a 2 ​h delay between the training phase and the preference phase. Exploration times were recorded and used to calculate a discrimination index, which was [time spent with original object minus time spent with new object] / [total time exploring both objects].

Fig. 1.

Fig. 1

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Object Recognition Test (ORT). On day 5 post-injection, a training phase followed by a preference test after a delay of 5 ​min was performed on each mouse. In the training phase (a), two identical objects were placed near the two corners at the opposite sides of the chamber. The animal was placed into the chamber and allowed a total of 5 ​min of exploration of the two identical objects, and then removed from the chamber. (b) One of the identical objects was replaced by a novel object. And after 5 ​min delay, we started the preference phase or novel object testing; the mouse was placed back into the chamber, allowed another total of 5 ​min of exploration of the two objects. This procedure was repeated on day 6 post-injection as well, except with a 2-hr delay before the mouse was placed back into the chamber for novel object testing. Treatment with CH started after ORT on day 6 consecutively for 7 days. On day 12, mice underwent ORT which was similar to the ORT on day 5, meanwhile, the ORT on day 13 was similar to the ORT on day 6. Exploration times were recorded and used to calculate a discrimination index.

Microscopic assessment of neuropathological markers

Fourteen days after IC injection of human MDM, the mice were sacrificed, and their brains were extracted and frozen in embedding medium for subsequent pathological analysis. Coronal brain sections, 8 ​μm thick, were collected starting from the rostral end (frontal lobe) with approximately 250 ​μm intervals in the antero-posterior direction. The injection site was approximately located in the anterior to bregma. The collected brain sections were immediately fixed in 95% ethanol and immunohistochemically stained for astrocytes using a 1:500 dilution of glial fibrillary acidic protein (GFAP) antibody (Millipore Sigma) and for neurons using a 1:200 dilution of microtubule-associated protein-2 (MAP2) antibody (Millipore Sigma). The brain pathology was initially assessed using an Olympus microscope, and slides containing the regions predominantly affected after inoculation were selected for densitometry evaluation. For example, the slide with the highest intensity of astrogliosis signal was chosen first and scored for the density of the GFAP signal, following by scoring another four adjacent anatomical section slides. The densitometry scoring procedure has been described in previous publications [[28], [29], [30]]. To capture images, an Olympus DP80 digital camera attached to an Olympus BX51 microscope was used, ensuring that parameters such as resolution, magnification, and exposure time were consistent for images displaying the same marker staining. The threshold for each marker staining can be adjusted to eliminate background staining. Captured images were then analyzed using Image J 1.45S software (NIH) for densitometry analysis (The analyst was blinded to treatment during the data scoring process). For more detailed procedures regarding densitometry scoring analysis for each specific marker staining, please refer to the previous publications [[28], [29], [30]].

Single Cell Suspension and Flow Cytometry

In vivo study

The brains were harvested and preserved in MACS Tissue Storage Solution (Miltenyi) before proceeding with single cell isolation. A commercial kit (Miltenyi, murine brain dissociation kit for live cells) and the GentleMacs dissociator (Miltenyi) were used for the isolation process. The isolated cells were then stained for specific mouse cell markers, including GFAP (GFAP-APC, Miltenyi; 1:400 dilution), MHC Class II (I-A/I-E) Monoclonal Antibody (M5/114.15.2), APC, eBioscience), and CD11b (Monoclonal Antibody (M5/114.15.2), PE, eBioscience). Total events were collected using forward scatter (FSC) and side scatter (SSC) parameters, followed by discrimination of single cells based on FSC-area versus FSC-height, with gating strategies to eliminate any doublet cell formations. The gating strategy for GFAP+ cells and total mouse MHCII+/CD11b+ double-positive cells (mouse mononuclear phagocytes) was determined using fluorescence-minus-one (FMO) controls with isotype-matched immunoglobulins. The gating strategy for MHCIIintermediate/high and CD11bhigh populations was referenced from a previous publication [31,32].

Invitro studies

See Mouse Microglia Activation and Honokiol Hexafluoro (HH) Treatment section below for details of mouse microglial culture. Once the mouse microglial cells reached confluency, their activation markers were measured following stimulation with various doses of lipopolysaccharide (LPS) and treatment with different concentrations of HH (15 ​μM, 75 ​μM). We have previously used HH at a concentration of 20 ​μM to treat BV2 cells (mouse microglial cell line) in vitro to show that SIRT 3 is stimulated resulting in improved mitochondrial respiration [33] To prepare for flow cytometry staining, the cells were detached from the culture plates using CellStripper (Corning) and gently trypsinized using 0.25% Trypsin, 0.1% EDTA in HBSS with Calcium and Magnesium (Corning, Burlington MA, USA) to obtain a single-cell suspension. For flow cytometry staining, the cells were first stained with a Live/Dead cell dye (LIVE/DEAD™ Fixable Dead Cell Stain Sampler Kit, ThermoFisher) to distinguish live and dead cells. Subsequently, the cells were stained for activation markers, including CD45, MHC II, and CD11b. The same gating strategies discussed earlier [31,32] were employed for the analysis.

Correlation matrices between ORT behavior test and brain mononuclear phagocytes activation marker

Bivariate scatter plots of correlation data were created using GraphPad statistically assess the correlations between the percentage of MHCIIintermediate/high/CD11bhigh-double positive cell populations from each mouse, correlated with their respective object discrimination index on Day 13. Fitted trendlines were displayed to demonstrate the degree and pattern of the relationships. The coefficient of determination R2 and p-values were calculated and displayed on the plots.

Mouse Microglia Activation and Honokiol Hexafluoro (HH) Treatment

The mouse microglia cell line (IMG) was purchased from Kerafast. The cells were initially seeded in 24-well plates and allowed to reach confluence, followed by LPS-induced activation for 4 ​h. Subsequently, the culture medium containing LPS was replaced with fresh culture medium supplemented with 75 ​μM HH. This concentration of HH was determined through cell viability testing of MDM (see Supplemental Figure 2) and literature that supports the use of HNK at this concentration for in vitro studies [34]. The cells underwent further incubation for 12 ​h before harvesting for flow cytometry. To examine the preventive effect of HH on cell activation, another set of cells was pretreated with 75 ​μM HH for 8 ​h before the addition of LPS to the culture medium. These concentrations were chosen given previous literature demonstrating that efficacy without apparent toxicity was observed in other studies [35].

Enzyme-linked immunosorbent assay (ELISA) for cytokine

HIV-1ADA-infected monocyte-derived macrophages (MDM) were seeded in 24-well plates and treated with HH (15 ​μM, 75 ​μM) for 24 and 48 ​h. After the respective treatment periods, culture supernatant was collected. The concentration of cytokines (i.e., IFN-α, TGF-β, Interleukin 10, TNF-α, IL-1β, etc.) was measured using commercial ELISA kits (ThermoFisher) following the manufacturer's instructions.

Measurement of viral expression in vitro

Human monocytes were infected with HIV-1ADA as described previously in the “Human monocyte-derived macrophages (MDM) infection with HIV-1ADA” section above. On Day 7 post-infection, MDM were treated with different concentrations of HH (15 ​μM, 75 ​μM) for 24 ​h in Teflon flasks. After the treatment, the cells were lysed for total RNA extraction using the RNeasy kit (Qiagen, Germantown MD, USA) following the manufacturer's instructions. First strand cDNA synthesis was performed using DNase-treated RNA, random primers, and the SuperScript™ IV First-Strand Synthesis System (ThermoFisher) at 55 ​°C for 20 ​min, according to the manufacturer's instructions. The cDNA was diluted 1:10 before being quantified using droplet digital PCR on the Bio-Rad QX200 Digital Droplet PCR system.

For the ddPCR reaction, 5 ​μL of cDNA was added to the master mix, which contained 2x ddPCR supermix (no dUTP, Bio-rad), primers (final concentration 900 ​nM), probes (final concentration 250 ​nM), and nuclease-free water. The primer and probe sequences (5′ and 3′) are listed below. To ensure consistent PCR results, each sample was quantified in six replicates. The PCR reaction droplets were prepared using the QX200 Droplet Generator. The thermal cycling program specified below was used for ddPCR reactions. The ddPCR plate was kept at 4 ​°C for at least 4 ​h prior to transfer to the QX200 droplet reader, a step that improves the quality metrics of droplets and achieves a higher number of accepted droplets per reaction. The data were normalized using an endogenous control, Human HPRT1 (HGPRT) (FAM™/MGB probe, non-primer limited) purchased from ThermoFisher, Waltham, MA, USA.

Primer and Probe Sequences:

Tat-Rev F CTTAGGCATCTCCTATGGCAGGAA.

Tat-Rev R GGATCTGTCTCTGTCTCTCTCTCCACC.

Tat-Rev Probe ACCCGACAGGCC (HEX)

Cell Toxicity Assay

Human MDM were differentiated as described above, and cultured until they reached confluency in a 24-well plate before being treated with different doses of HH. The cell viability was assessed after 24 or 48 ​h of HH treatment using a colorimetric-based assay called the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, as discussed previously [27]. The objective of this experiment was to ensure that the statistically significant findings obtained from the standardized pharmacological cytotoxicity measurement in vitro within the 48 ​h timeframe were specifically attributed to the efficacy of HH rather than non-specific toxicity observed during this timeframe. The 48 ​h timeline was selected specifically for tandem assessment of toxicity within the data collected for in vivo efficacy, and no apparent toxicity was observed in our animal model across the 12-day treatment course of HH (Fig. S2).

Blood-brain Barrier Penetration Assay

In order to quantitatively assess the membrane permeability of HH across the blood-brain barrier (BBB), we utilized the Parallel Artificial Membrane Permeability Assay-BBB Kit from BioAssay Systems (Hayward, CA, USA) as per the manufacturer's instructions. The kit provided three control samples representing high, medium, and low permeability. Controls and HH were incubated for 18 ​h with a membrane coated in BBB lipids in well culture inserts after which optical density of the receiving well was measured and permeability rate was calculated. By comparing the relative permeability rate of HH to the in vitro permeability control, we estimated its membrane permeability across the BBB [31,36,37]. This assay was selected specifically to measure permeability rates, and dovetails with findings in our animal model that demonstrate peripheral administration of HH confers reduction of cellular inflammatory cells in resident CNS cells, underscoring ability of HH to cross the BBB.

Quantification and statistical analysis

Significance was determined using the one-way analysis of variance (ANOVA) (Tukey's multiple comparisons test), or student's t-test (one-tailed), (GraphPad Prism software, version 6.04). Differences were noted as significant ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001, ∗∗∗∗P ​< ​0.0001.

Results

Honokiol hexafluoro (HH) treatment successfully reversed Object Recognition Test (ORT) abnormalities

The HIV associated CD mouse model enables a short-term analysis of cognitive and brain pathology relationships. Importantly, the model presents an invaluable preclinical platform for evaluating novel therapeutic interventions [18]. Here, our objective was to ascertain whether treatment with HH could effectively ameliorate neurocognitive deficits associated with HIV infection.

On day 5 post-intracranial inoculation (p.i.), there was no statistically significant difference observed in the ORT data when comparing a 5 ​min delay task between the groups, although pooled data suggests a difference in controls and HIV infected mice in ORT (Fig. 2a and b). On day 6 p.i., pooled data indicates that the control group of mice outperformed their HIV-infected counterparts, which were untreated at this point (Fig. 2b). After administering seven days of HH treatment, commencing on Day 6 p.i. (Fig. 1), on day 12 HIV-infected but untreated mice (HIV only in Fig. 2c) do not perform as well as controls on ORT after a 5-min delay (Fig. 2c). Interestingly, the difference between controls and HH treated HIV-infected mice is not significant (Fig. 2c). Nevertheless, on day 13, the ORT results obtained from a more challenging 2-h task revealed a reversal of the abnormalities observed in HH-treated HIV-associated CD mice compared to the HIV-infected, untreated group (Fig. 2d). Notably, the uninfected mice consistently displayed the highest object discrimination index score across all time points, underscoring the detrimental impact of HIV infection on cognitive performance. Overall, these findings suggest that HH effectively mitigated the typical cognitive abnormalities observed in HAND mice. Furthermore, using a blood-brain permeability penetration assay (Fig. S1) that mimics the lipid composition found in the BBB, allows quantification of HH permeability. These data support previously published in vivo data indicating that HH exhibits the ability to traverse the BBB [38]. It also supports our data reported herein that shows that HH administered to HIV associated CD mice confers reversal of cognitive deficits and improvement of pathological parameters.

Fig. 2.

Fig. 2

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Honokiol Hexafluoro (HH) reversed ORT abnormalities. Discrimination index for novel object testing (a) Day 5, (b) Day 6, (c) Day 12, (d) day 13. HH treatment on day 13 suggested reversal of ORT abnormalities. ∗P < 0.05.

Honokiol hexafluoro improved HIV-associated neuropathology in HIV infected mice

Honokiol hexafluoro significantly reduced astrogliosis

Next, our aim was to elucidate the mechanisms underlying the amelioration of HIV-associated neuropathology by HH in our HIV induced CD mouse model. Following HIV infection, astrocytes become actively involved in the inflammatory response and play a role in the progression of neurodegenerative diseases [39,40]. Notably, activated astrocytes upregulate the expression of glial fibrillary acidic protein (GFAP), a key marker associated with astrocyte activation. Through immunostaining (Fig. 3a) of brain tissue sections and flow cytometry analyses of dissociated brains (Fig. 3b), we demonstrated that HIV infection led to a significant increase in GFAP signal. The administration of HH to HIV-infected mice resulted in a significant reduction in GFAP signal in both tissue section stained for GFAP and flow analysis (Fig. 3a and b), indicating that HH effectively inhibits astrocyte activation.

Fig. 3.

Fig. 3

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Both densitometry (a) and Flow cytometry (b) data revealed that activated (GFAP) astrocytes were significantly decreased in HH-treated HAND mice compared to untreated HAND mice. ∗P < 0.05, ∗∗∗∗P < 0.0001.

Honokiol hexafluoro largely restored the changes of neuronal dendritic arborization

Previous studies have utilized the extent of disruption in neuronal MAP2 expression as a pathological correlate for cognitive function in this mouse model [29,41]. MAP2 serves as a marker that reflects the intricate complexity of dendritic arborization, which plays a crucial role in cognition. Fig. 4a and b demonstrate that HIV-induced neuro-inflammation reduces MAP2 expression, compared to the control group. Treatment with HH largely restored the alterations in neuronal dendritic arborization, underscoring its therapeutic potential in mitigating the effects of HIV on neuronal morphology. These findings further support the cognitive data, suggesting that HH effectively reverses behavioral deficits associated with HIV infection.

Fig. 4.

Fig. 4

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(a) Immunostaining for MAP2; (b) Densitometry data suggested that HH recovered MAP2 expression in neurons. ∗P < 0.05.

Honokiol hexafluoro ameliorated mouse brain mononuclear phagocyte activation

In this study, we provide evidence suggesting that IC inoculation of HIV-infected human MDM leads to an increased number of activated mouse mononuclear phagocytes within the brain (Fig. 5a). These findings align with previously published studies demonstrating elevated levels of mononuclear phagocytes in the brains of HIV-infected mice. Our data suggests that the HIV untreated group exhibited heightened frequencies of MHCII+/CD11b+ mononuclear phagocytes compared to the control group. A statistically significant difference was observed between the control group and the HIV-infected, HH treated group (Fig. 5a). To evaluate the extent of mononuclear phagocyte activation following HIV infection in the three groups, we assessed the expression levels of MHCII, a crucial marker indicative of cellular activation. Flow cytometry of dissociated brains from HIV-infected mice treated with HH demonstrates increased total numbers of MHCII and CD11b double positive mouse mononuclear phagocytes compared to control mice (Fig. 5a), suggesting that HH facilitates the recruitment of these cells. However, HH simultaneously decreases the intermediate/high MHCII expression of these mouse mononuclear phagocytes (Fig. 5b), suggesting that mice treated with HH have mouse mononuclear phagocytes with reduced activation. This suggests an important therapeutic effect of HH because the literature strongly suggests that activated brain mononuclear phagocytes are involved in the pathogenesis of HIV associated CD [42,43]. To further substantiate our hypothesis regarding the inhibitory effect of HH on mononuclear phagocyte activation, we conducted a correlation analysis between inflammatory markers and the ORT discrimination index score to better understand if activation markers in the CNS that are upregulated due to CNS infection by HIV-1 are correlated with worse cognition. Regression analysis revealed a statistically significant correlation between markers of mononuclear phagocyte activation (e.g., MHCII) within the mouse brain and the severity of CD in HIV-infected mice (Fig. 5c). These results provide additional evidence supporting the potential involvement of mononuclear phagocyte activation in HIV-related cognitive impairment and further emphasize the therapeutic implications of HH treatment in this context.

Fig. 5.

Fig. 5

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(a) HIV infection suggested an increased frequency of mononuclear phagocytes in mouse brain. “∗” indicates P ​< ​0.05. (b) Flow cytometry data suggested that HH inhibited MHC II expression in mononuclear phagocytes. (c) Correlation analysis. Linear regression plot shows significant correlations of discrimination index with mononuclear phagocytes activation markers.

Honokiol hexafluoro prevented mouse microglial activation in vitro

The intricate mechanisms that underlie HIV-induced neuroinflammation involve a complex interplay of cellular and molecular interactions. Given the pivotal role of microglia in maintaining cerebral homeostasis, and considering that microglial dysfunction resulting from persistent HIV infection is postulated to have a significant impact on CNS functionality, our research strategy prioritizes an initial investigation into the potential protective effects of HH in mitigating microglial activation. To achieve this goal, we opted to use LPS as a direct stimulant for murine microglia in an in vitro experimental context. Our selection of LPS is well-documented in the scientific literature, thus affirming its suitability as an optimized model for studying microglial activation and its potential implications in the context of persistent neuroinflammation [34]. To support our in vivo findings, we performed in vitro experiments to investigate the influence of HH on microglial activation. We utilized a mouse microglia cell line and induced activation through LPS stimulation. This approach enabled us to assess the potential protective effects of HH by attenuating microglial activation. The experiments were conducted to provide an in vitro evaluation of the ability of HH to block LPS induced activation, thereby allowing direct evaluation of the ability of the agent to block activation in microglia, to provide additional mechanistic rationale to observed in vivo findings.

To assess the potential of HH in preventing LPS-induced cell activation, cells were pre-treated with 75 ​μM HH for 8 ​h before subjecting them to a 12-h LPS stimulation. Interestingly, we found that HH significantly reduced the frequency of MHC IIinter/hi/CD11bhi-positive cells compared to the LPS-only group, at both 20 ​ng/mL and 50 ​ng/mL LPS concentrations. This observation indicates that HH promotes a state of cellular quiescence in the context of microglial activation (Fig. 6a).

Fig. 6.

Fig. 6

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(a) Pre-treatment with 75 ​μM HH prevented activation of mouse microglia. (b) Even with higher dosages of LPS HH significantly downregulated activation markers of microglia. ∗∗∗∗P < 0.0001.

Due to the limited ability of current ARTs to effectively cross the BBB and eliminate HIV in the central nervous system (CNS), numerous studies have confirmed the presence of HIV DNA and RNA in the human brain and cerebrospinal fluid (CSF) [19,44] Consequently, microglia become activated in response to the presence of HIV [45,46]. To simulate this scenario, mouse microglia were pre-treated with different doses of LPS (250 ​ng/mL and 100 ​ng/mL) for 4 ​h before administering HH. The administration of HH significantly reduced the expression of the activation marker MHCIIint/hi/CD11bhi, suggesting that underscoring that HH has potential efficacy towards reduction in LPS-induced microglial activation (Fig. 6b). Our data also demonstrates a significant downregulation of the activation marker after 48 ​h of LPS-induced activation using a concentration of 400 ​ng/mL (data not shown).

Honokiol hexafluoro remodels human monocyte derived macrophages (MDM) metabolism by promoting cell quiescence and suppressing HIV expression

(Monocytes latently infected with HIV have the potential for reactivation within the CNS, transitioning from a dormant state to actively expressing viral transcripts. Our subsequent focus has shifted towards unraveling the intricate mechanisms underlying HH-induced reshaping of the immune profile in HIV-latently infected MDMs.) Treatment with 75 ​μM HH for 48 ​h significantly increased the expression of TGF-β in HIV-infected MDMs, implying a potential role for HH in promoting a protective quiescent state within these cells (Fig. S3a). Particularly after 48 ​h of HH treatment, a significant inhibitory effect was observed on IFN-alpha production associated with HIV infection (see Fig. 7a). Furthermore, HH exhibited suppressed the HIV-associated upregulation of TNF-α expression in MDMs 24 ​h after treatment (Fig. 7b). Although viral infections often trigger inflammasome activation and the subsequent production of active cytokines like IL-1β, we did not observe an increase in IL-1β expression in our in vitro MDM model (Fig. S3c).

Fig. 7.

Fig. 7

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HH exerts multiple beneficial effects, including (a) the inhibition of neurotoxic IFN-alpha and (b) pro-inflammatory TNF-alpha expression, as well as (c) the suppression of HIV transcript levels. ∗∗P < 0.01, ∗∗∗∗P < 0.0001.

Cytokines that generally have inhibitory effects (IL-10 and TGF-β) were examined. HH inhibited the HIV-associated upregulation of IL-10 48 ​h after treatment (Fig. S3b). On the other hand, high dose HH increased TGF-β expression in HIV-infected MDM 48 ​h after treatment (Fig. S3a). Lastly, we investigated the influence of HH treatment on HIV expression in MDM. Our results demonstrated a significant reduction in the levels of HIV Tat-Rev transcripts following HH treatment (Fig. 7c). This is a potentially important and unexpected effect of honokiol that if corroborated, would further increase its potential as an adjunctive treatment for HIV brain infection.

Discussion

The common occurrence of cognitive decline among PLWH, despite the advent of ART, highlights the enduring risk of HIV-associated CD. The extension of life expectancy facilitated by ART increases the susceptibility to progressive cognitive impairment over time due to accruing comorbidities in aging PLWH [[47], [48], [49]]. Chronic neuroinflammation and persistent viral reservoirs within the CNS have been associated with cognitive decline [2]. The limited ability of ART to penetrate the BBB, coupled with the mechanistic neuroinflammatory component of HIV-associated CD underscores the critical imperative to identify innovative therapeutic strategies capable of eradicating viral persistence within the CNS while concurrently mitigating sustained neuroinflammatory responses.

To address this critical unmet need for the development of novel treatments for HIV associated CD, we evaluated the impact of HH on key markers of neuroinflammation that drive CD in PLWH, along with quantification of the impact of HH on cognitive function via the ORT. Collectively, we found that HH confers mitigation of key markers of CD across neurons, astrocytes, and phagocytic cells within the CNS compartment. Furthermore, we found HH capable of BBB penetration via a BBB permeability assay, substantiating observations of neuroinflammation mitigation here.

The ORT data reported herein strongly suggest that HH reverses abnormalities observed in HIV-infected mice (Fig. 2b) by day 13 (p.i.) (Fig. 2d). This finding is supported by histopathological data suggesting MAP2 decreases are improved after HH treatment (Fig. 4). HIV associated CD represents a major unmet clinical need, as existing ART cannot mitigate the inflammatory components that drive this phenotype, and this phenotype exists in up to half of PLWH, underscoring the global need for agents to target CD. Neuronal dendritic synaptic shortening, quantified with MAP-2 staining (Fig. 4), provides a measure of neuronal integrity [41]. When neuronal integrity is compromised, this is associated with CD and specifically across our model, correlates with poor ORT scores [18]. As well, postmortem brains of subjects diagnosed with HIV associated CD exhibit loss of dendrites [50]. Importantly, a neuropathological hallmark of HIV-associated cognitive decline is characterized by the loss of dendrites, which coincides with a significant reduction in MAP2 expression-a critical component of dendrite length and branching, highlighting the mechanistic findings for MAP-2, neuronal integrity, and relationship to ORT and cognition also observed in our murine model. The data suggest that HH reduces MAP2 loss associated with HIV infection of the brain (Fig. 4). Therefore, the MAP2 data suggesting that HH improves dendritic integrity is consistent with the ORT data suggesting cognitive improvement with HH treatment. These effects in combination, along with HH effects on mononuclear phagocytes (see below), strongly suggest that HH holds promise as a treatment for HIV associated CD. We also observed that HH effectively suppresses the activation of mononuclear phagocytes induced by the presence of HIV-infected human MDM in the brain (Fig. 5b), a contributing factor to neuroinflammation and ensuing CD in PLWH. Furthermore, the administration of HH effectively inhibits astrocyte activation, as indicated by the downregulation of glial fibrillary acidic protein (GFAP) expression (Fig. 3). Astrocytes are a predominant glial cell type in the CNS and contribute to neuroinflammation and neurodegeneration during HIV infection. These findings strongly suggest that HH may exert its effects by attenuating astrocyte-mediated neuroinflammation, in addition to mononuclear phagocyte activation and modulation of neuronal integrity (MAP-2).

The SCID mouse model of HIV CD has important merits, which include: 1) the ability to measure CD before and after novel treatments (18), which is particularly important when considering critical clinical features of HIV brain infection in humans, 2) correlation of brain histopathological features of inflammation and neuronal disruption with CD and treatment effects, which enhances the understanding of potential mechanisms of action involved with novel treatments, and 3) a relatively rapid system to evaluate or screen potential novel treatments compared to other animal models. However, this model has limitations, as do all animal models of human disease. These include: 1) the short period of time to assess novel treatments. Consequently, the long-term effects of treatment cannot be assessed. 2) relatively brief life of human xenografts (macrophages) and limited viral replication and HIV protein production. This limits the long-term assessment of treatment on viral load and the evaluation of potential effects of HIV protein neurotoxicities. 3) limitations of determining mechanisms of action of treatments that are inherent in all animal models.

In order to address some of the limitations of our in vivo system and to provide further mechanistic insights, we employed an in vitro model using LPS-induced activation of mouse microglia [51,52] to understand the impact of HH on inflammation. The treatment of HIV-infected mice with honokiol suggests that it results in a decrease in the expression of MHCII on mononuclear phagocytes (Fig. 5). Correlation of MHCII expression on brain mononuclear phagocytes (which include microglia and brain macrophages) with ORT further suggests that decreasing the activation of these cells is responsible for improvement in cognition in the mice. In order to strengthen this in vivo observation, we used a microglial cell line in vitro and LPS treatment to mimic inflammatory conditions induced by HIV infection in the brain. LPS induces microglial activation, as evidenced by increased expression of CD11b and MHCII (Fig. 6); importantly, these markers were downregulated by HH treatment, providing a mechanistic link between HH and ability to mitigate a pro-inflammatory response induced in microglia. Next, we sought to understand if HH can block HIV-induced inflammatory cytokine production, using primary human macrophages as a model representing mononuclear phagocytes permissive to HIV-1 infection that contribute to the CNS HIV reservoir. We observed that HH inhibits the induction of TNF-α and IFN-α in these cells while promoting the production of TGF-β during the early stages of viral infection (Fig. 7a–b and Fig. S3a), again highlighting that HH treatment could possibly serve as a therapeutic intervention to reverse or prevent HIV-induced inflammation that drives CD. In addition, the data suggest that honokiol treatment of HIV-infected macrophages in vitro inhibits gene expression of Tat-Rev. This suggests that by reducing the activation of mononuclear phagocytes, HIV production could consequently be reduced by honokiol. This provides additional rationale for the ameliorating effects of honokiol on cognitive function and brain inflammation. The relevance of these findings to the human condition is that most NeuroHIV investigators believe that neuroinflammation is primarily driven by the presence of HIV, which activates brain mononuclear phagocytes leading to cognitive dysfunction (15). Considering our data and the literature in toto, reduction of mononuclear phagocyte activation by honokiol is likely the reason our treated mice show improvement in cognitive function on day 13 (Fig. 2, Fig. 5c).

Overall, the findings presented here in this manuscript provide evidence for the potential therapeutic benefits of HH in reversing behavioral abnormalities and pathological features associated with HIV-associated CD. Further research is warranted to explore additional underlying mechanisms of these effects. Moreover, studies to validate the efficacy of HH utilizing in vivo models are an important future direction of this work, including chronic HIV infection of humanized mice as well as in vitro models to further investigate the mechanism of action of HH in reducing inflammatory activities and HIV expression.

Author Contributions

Zhan Zhang was responsible for designing and implementation of most of the experiments, interpretation of data and manuscript preparation. Aaron Scanlan was responsible for contributing to experimental procedures and manuscript preparation. Rajeth Koneru helped with experimental design and data interpretation. Chelsea Richardson contributed to experimental procedures and manuscript preparation. Monica Reece carried out the blood brain barrier experiment (Supplemental Fig. 1) and contributed to manuscript preparation. Emily Edwards also carried out the blood brain barrier experiment (Supplemental Fig. 1) and contributed to manuscript preparation. Sebastian Roa helped with the HH cell toxicity assay and contributed to writing the manuscript particularly with citations. Christina Gavegnano helped with the design and interpretation of the flow cytometry experiments. Heather Bimonte-Nelson contributed to design and interpretation of the object recognition testing. Jack Arbiser provided honokiol hexafluoro, contributed to the design of the animal treatment experiment and manuscript preparation. William Tyor contributed to experimental design, data interpretation and manuscript preparation.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

William Tyor reports financial support was provided by Emory University School of Medicine. William Tyor reports a relationship with National Institutes of Health that includes: funding grants. Jack Arbiser has patent #US8889744B2 licensed to Jack L. Arbiser. None to my knowledge If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The study was supported by VA Merit awards BX001506 and BX005402 and R01 MH116695.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.neurot.2024.e00329.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

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