Tuberculous meningitis (TB meningitis) is the most severe form of tuberculosis (TB), requiring 12 months of multidrug treatment for cure, and is associated with high morbidity and mortality. High-dose rifampin (35 mg/kg/d) is safe and improves the bactericidal activity of the standard-dose (10 mg/kg/d) rifampin-containing TB regimen in pulmonary TB. However, there are conflicting clinical data regarding its benefit for TB meningitis, where outcomes may also be associated with intracerebral inflammation. We conducted cross-species studies in mice and rabbits, demonstrating that an intensified high-dose rifampin-containing regimen has significantly improved bactericidal activity for TB meningitis over the first-line, standard-dose rifampin regimen, without an increase in intracerebral inflammation. Positron emission tomography in live animals demonstrated spatially compartmentalized, lesion-specific pathology, with postmortem analyses showing discordant brain tissue and cerebrospinal fluid rifampin levels and inflammatory markers. Longitudinal multimodal imaging in the same cohort of animals during TB treatment as well as imaging studies in two cohorts of TB patients demonstrated that spatiotemporal changes in localized blood-brain barrier disruption in TB meningitis are an important driver of rifampin brain exposure. These data provide unique insights into the mechanisms underlying high-dose rifampin in TB meningitis with important implications for developing new antibiotic treatments for infections.

Background: Osteopontin (OPN) as a secreted signaling protein is dramatically induced in response to cellular injury and neurodegeneration. Microglial inflammatory responses in the brain are tightly associated with the neuropathologic hallmarks of neurodegenerative disease, but understanding of the molecular mechanisms remains in several contexts poorly understood.Methods: Micro-positron emission tomography (PET) neuroimaging using radioligands to detect increased expression of the translocator protein (TSPO) receptor in the brain is a non-invasive tool used to track neuroinflammation in living mammals.Results: In humanized, chronically HIV-infected female mice in which OPN expression was knocked down with functional aptamers, uptake of TSPO radioligand DPA-713 was markedly upregulated in the cortex, olfactory bulb, basal forebrain, hypothalamus, and central grey matter compared to controls. Microglia immunoreactive for Iba-1 were more abundant in some HIV-infected mice, but overall, the differences were not significant between groups. TSPO⁺microglia were readily detected by immunolabeling of post-mortem brain tissue and unexpectedly, two types of neurons also selectively stained positive for TSPO. The reactive cells were the specialized neurons of the cerebellum, Purkinje cells, and a subset of tyrosine hydroxylase-positive neurons of the substantia nigra.Conclusions: In female mice with wild-type levels of osteopontin, increased levels of TSPO ligand uptake in the brain was seen in animals with the highest levels of persistent HIV replication. In contrast, in mice with lower levels of osteopontin, the highest levels of TSPO uptake was seen, in mice with relatively low levels of persistent infection. These findings suggest that osteopontin may act as a molecular brake regulating in the brain, the inflammatory response to HIV infection.

The advent of Human Immunodeficiency Virus (HIV) antiretrovirals have reduced the severity of HIV related neurological comorbidities but they nevertheless remain prevalent. Synaptic degeneration due to the action of several viral factors released from infected brain myeloid and glia cells and inflammatory cytokines has been attributed to the manifestation of a range of cognitive and behavioral deficits. The contributions of specific pro-inflammatory factors and their interplay with viral factors in the setting of treatment and persistence are incompletely understood. Exposure of neurons to chemokine receptor-4(CXCR4)-tropic HIV-1 envelope glycoprotein (Env) can lead to post-synaptic degradation of dendritic spines. The contribution of members of the extracellular matrix (ECM) and specifically, of perineuronal nets (PNN) toward synaptic degeneration, is not fully known, even though these structures are found to be disrupted in post-mortem HIV-infected brains. Osteopontin (Opn, gene name SPP1), a cytokine-like protein, is found in abundance in the HIV-infected brain. In this study, we investigated the role of Opn and its ECM integrin receptors, β1- and β3 integrin in modifying neuronal synaptic sculpting. We found that in hippocampal neurons incubated with HIV-1 Env protein and recombinant Opn, post-synaptic-95 (PSD-95) puncta were significantly increased and distributed to dendritic spines when compared to Env-only treated neurons. This effect was mediated through β3 integrin, as silencing of this receptor abrogated the increase in post-synaptic spines. Silencing of β1 integrin, however, did not block the increase of post-synaptic spines in hippocampal cultures treated with Opn. However, a decrease in the PNN to βIII-tubulin ratio was found, indicating an increased capacity to support spine growth. From these results, we conclude that one of the mechanisms by which Opn counters the damaging impact of the HIV Env protein on hippocampal post-synaptic plasticity is through complex interactions between Opn and components of the ECM which activate downstream protective signaling pathways that help maintain the potential for effective post-synaptic plasticity.

TB meningitis is the most severe form of tuberculosis (TB), associated with high morbidity and mortality. High-dose rifampin (35mg/kg/day) is safe in adults and substantially improves the bactericidal activity of standard TB regimen. However, there is conflicting data regarding its benefit in TB meningitis where outcomes may also be associated with intracerebral inflammatory responses. A novel mouse and a validated rabbit model of TB meningitis utilizing intracranial Mycobacterium tuberculosis infections were used for these studies (Fig. 1). Animals received high-dose (35 mg/kg/day) or standard-dose (10 mg/kg/day) rifampin in combination with isoniazid, pyrazinamide and dexamethasone at human equipotent dosing. Bacterial burden, multi-modality positron emission tomography (PET) imaging, tissue drug concentrations, markers of neuroinflammation, and vascular leak were measured. Imaging data from a patient with TB meningitis was analyzed and correlated with the findings in animals. Figure 1. Mouse model of TB meningitis replicates human histopathology hallmarks. (A) Scheme of infection. (B) Histopathology hematoxylin-eosin (H&E) and acid-fast bacilli (AFB) staining in a representative M. tuberculosis-infected mouse shows regions of meningitis, ventriculitis, choroiditis, necrotizing and non-necrotizing granulomas. The bar represents 100µm. (C) Images show immunofluorescence of microglia activation in red (Iba-1) and nuclear stain in blue (DAPI). The rabbit model of TB meningitis has been described previously (Tucker et al. Dis Model Mech. 2016 and Tucker et al. Sci Transl Med. 2018). Animal studies were approved by the Johns Hopkins Animal Care and Use Committee. Administration of the high-dose rifampin regimen achieved four times higher brain concentration than the standard-dose regimen and displayed higher bactericidal activity in both mice and rabbits (P < 0.01) (Fig. 2). There were no differences in intracerebral microglial activation (¹²⁴I-DPA-713 PET and iDISCO) and pro-inflammatory cytokines during treatment in animals receiving high- or standard-dose rifampin regimens (Fig. 3). Whole-brain PET and immunolabeling demonstrated spatially compartmentalized inflammation, vascular leak and rifampin exposures (Fig. 4). Longitudinal imaging in the same animals showed a 40% decrease in vascular leak after two weeks of TB treatment. Spatially compartmentalized brain rifampin exposures and decreases in vascular edema over TB treatment were also noted in the TB meningitis patient. Figure 2. High-dose rifampin treatment in mouse and rabbit models of TB meningitis. (A) Experimental scheme. R10 (rifampin 10mg/kg), R35 (rifampin 35mg/kg), H (isoniazid), Z (pyrazinamide), D (dexamethasone). (B) Bactericidal activity of high-dose rifampin (n = 60 animals) and standard-dose rifampin (n = 60 animals) regimens in mice. (C) Grouped colony forming units (CFU) and (D) rifampin brain concentration in mice after 42 days of TB treatments. (E) Bactericidal activity of high-dose rifampin (n = 4 animals) and standard-dose rifampin (n = 3 animals) regimens in rabbits. Data from untreated rabbits (n = 2 animals) is also shown. (F) Grouped CFU and (G) rifampin brain concentration in rabbits after 14 days of TB treatments. Tissues were assayed using validated ultra-high-performance liquid chromatography and tandem mass spectrometry (LC-MS/MS) for rifampin at the Infectious Diseases Pharmacokinetics Laboratory of the University of Florida. The bacterial burden is represented as CFU per gram of tissue and presented on a logarithmic scale. CFU and mass spectrometry data are represented as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001 calculated using a two-way ANOVA test. Figure 3. Neuroinflammatory responses during TB treatment. (A) Iba-1 and DAPI immunofluorescence in a representative untreated, high-dose and low-dose-treated mouse. (B) Quantification of Iba-1 immunofluorescence before treatment and after 6 weeks of treatment (n = 3 mice per group). Sections were imaged at 40X with Nikon A1+ confocal microscope. HALO was used for visualization and quantification. Quantification of intraparenchymal (C) INF-γ, (D) TNF and (E) MCP-1 in untreated and treated mice (Milliplex Multiplex Luminex assay). (F) Coronal CT and 124I-DPA-713 PET/CT of a representative mouse with TB meningitis before treatment initiation. (G) 124I-DPA-713 PET quantification before treatment (n = 15 animals) and after 2 (n = 5 animals per group) and 6 (n = 5 animals per group) weeks of TB treatment. PET data is represented as median ± IQ. *P < 0.05, **P < 0.01 and ***P < 0.001 were calculated using a two-way ANOVA test. Figure 4. Changes in vascular leakage and rifampin penetration during TB treatment. (A) Experimental scheme in mice. (B) Whole-brain immunolabeling (iDISCO) of a representative M. tuberculosis-infected mouse prior to treatment initiation. Images show immunolabeling of α-smooth muscle actin in grey and microglia activation in red (Iba-1). Asterix represents the areas of vascular amputation. (C) Coronal 18F-py-Albumin PET/CT and quantification (tissue to plasma ratio) in untreated and 2 weeks-treated mice (n = 10 animals at each time-point). (D) Serial imaging in a patient with TB meningitis. (E) Transverse T2 post-contrast section and maximal intensity projection (MIP) showing vasogenic edema. (F) Co-registered T2 post-contrast MIP with transverse 11C-rifampin area under the curve (AUC) heat-map, and 11C-rifampin tissue to plasma ratio quantification of the areas with and without vasogenic edema, and unaffected brain. (G) T2 post-contrast MIP at 3 and 45 months after treatment initiation. The patient with TB meningitis was recruited as part of a 11C-rifampin PET research study performed under the U.S. FDA Radioactive Drug Research Committee program for investigational drugs (Tucker et al. Sci Transl Med. 2018; Ordonez et al. Nat Med. 2020). Human studies were approved by the Johns Hopkins University Institutional Review Board Committee. M = moxifloxacin. PET data is represented as median ± IQ. *P < 0.05, **P < 0.01 and ***P < 0.001 calculated using a two-way ANOVA test. Our cross-species findings suggest that an intensified high-dose rifampin regimen is more efficacious than the standard treatment for TB meningitis without an increase in neuroinflammation. Vascular leak decreases during TB treatment and may account for decreases in rifampin permeability over time. These studies have important implications for antimicrobial development for TB meningitis. Charles A. Peloquin, Pharm.D., Nothing to disclose Alvaro A. Ordonez, MD, Cubresa (Consultant)Fujirebio Diagnostics (Research Grant or Support) Sanjay K. Jain, MD, Fujirebio Diagnostics, Inc., USA (Research Grant or Support)Novobiotic LLC, USA (Research Grant or Support)T3 Pharma, Switzerland (Research Grant or Support) Sanjay K. Jain, MD, Fujirebio Diagnostics, Inc., USA (Individual(s) Involved: Self): Research Grant or Support; Novobiotic LLC, USA (Individual(s) Involved: Self): Research Grant or Support; T3 Pharma, Switzerland (Individual(s) Involved: Self): Research Grant or Support