CSF1R inhibition depletes brain macrophages and reduces brain virus burden in SIV-infected macaques.

Perivascular macrophages (PVMs) and, to a lesser degree, microglia are targets and reservoirs of HIV and simian immunodeficiency virus (SIV) in the brain. Previously, we demonstrated that colony-stimulating factor 1 receptor (CSF1R) in PVMs was upregulated and activated in chronically SIV-infected rhesus macaques with encephalitis, correlating with SIV infection of PVMs. Herein, we investigated the role of CSF1R in the brain during acute SIV infection using BLZ945, a brain-penetrant CSF1R kinase inhibitor. Apart from three uninfected historic controls, nine Indian rhesus macaques were infected acutely with SIVmac251 and divided into three groups (n = 3 each): an untreated control and two groups treated for 20-30 days with low- (10 mg/kg/day) or high- (30 mg/kg/day) dose BLZ945. With the high-dose BLZ945 treatment, there was a significant reduction in cells expressing CD163 and CD206 across all four brain areas examined, compared with the low-dose treatment and control groups. In 9 of 11 tested regions, tissue viral DNA (vDNA) loads were reduced by 95%-99% following at least one of the two doses, and even to undetectable levels in some instances. Decreased numbers of CD163+ and CD206+ cells correlated significantly with lower levels of vDNA in all four corresponding brain areas. In contrast, BLZ945 treatment did not significantly affect the number of microglia. Our results indicate that doses as low as 10 mg/kg/day of BLZ945 are sufficient to reduce the tissue vDNA loads in the brain with no apparent adverse effect. This study provides evidence that infected PVMs are highly sensitive to CSF1R inhibition, opening new possibilities to achieve viral clearance.

Stressor control and regional inflammatory responses in the brain: regulation by the basolateral amygdala.

Increasing evidence has connected the development of certain neuropsychiatric disorders, as well as neurodegenerative diseases, to stress-induced dysregulation of the immune system. We have shown that escapable (ES) and inescapable (IS) footshock stress, and memories associated with ES or IS, can differentially alter inflammatory-related gene expression in brain in a region dependent manner. We have also demonstrated that the basolateral amygdala (BLA) regulates stress- and fear memory-induced alterations in sleep, and that differential sleep and immune responses in the brain to ES and IS appear to be integrated during fear conditioning and then reproduced by fear memory recall. In this study, we investigated the role of BLA in influencing regional inflammatory responses within the hippocampus (HPC) and medial prefrontal cortex (mPFC) by optogenetically stimulating or inhibiting BLA in male C57BL/6 mice during footshock stress in our yoked shuttlebox paradigm based on ES and IS. Then, mice were immediately euthanized and RNA extracted from brain regions of interest and loaded into NanoString® Mouse Neuroinflammation Panels for compilation of gene expression profiles. Results showed differential regional effects in gene expression and activated pathways involved in inflammatory-related signaling following ES and IS, and these differences were altered depending on amygdalar excitation or inhibition. These findings demonstrate that the stress-induced immune response, or "parainflammation", is affected by stressor controllability and that BLA influences regional parainflammation to ES or IS in HPC and mPFC. The study illustrates how stress-induced parainflammation can be regulated at the neurocircuit level and suggests that this approach can be useful for uncovering circuit and immune interactions in mediating differential stress outcomes.

Reversing neural circuit and behavior deficit in mice exposed to maternal inflammation by Zika virus.

Zika virus (ZIKV) infection during pregnancy is linked to various developmental brain disorders. Infants who are asymptomatic at birth might have postnatal neurocognitive complications. However, animal models recapitulating these neurocognitive phenotypes are lacking, and the circuit mechanism underlying behavioral abnormalities is unknown. Here, we show that ZIKV infection during mouse pregnancy induces maternal immune activation (MIA) and leads to autistic-like behaviors including repetitive self-grooming and impaired social memory in offspring. In the medial prefrontal cortex (mPFC), ZIKV-affected offspring mice exhibit excitation and inhibition imbalance and increased cortical activity. This could be explained by dysregulation of inhibitory neurons and synapses, and elevated neural activity input from mPFC-projecting ventral hippocampus (vHIP) neurons. We find structure alterations in the synaptic connections and pattern of vHIP innervation of mPFC neurons, leading to hyperconnectivity of the vHIP-mPFC pathway. Decreasing the activity of mPFC-projecting vHIP neurons with a chemogenetic strategy rescues social memory deficits in ZIKV offspring mice. Our studies reveal a hyperconnectivity of vHIP to mPFC projection driving social memory deficits in mice exposed to maternal inflammation by ZIKV.

Activation of cannabinoid-2 receptor protects against Pseudomonas aeruginosa induced acute lung injury and inflammation.

BACKGROUND: Bacterial pneumonia is a major risk factor for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Pseudomonas aeruginosa (PA), an opportunistic pathogen with an increasing resistance acquired against multiple drugs, is one of the main causative agents of ALI and ARDS in diverse clinical settings. Given the anti-inflammatory role of the cannabinoid-2 receptor (CB2R), the effect of CB2R activation in the regulation of PA-induced ALI and inflammation was tested in a mouse model as an alternative to conventional antibiotic therapy. METHODS: In order to activate CB2R, a selective synthetic agonist, JWH133, was administered intraperitoneally (i.p.) to C57BL/6J mice. Furthermore, SR144528 (a selective CB2R antagonist) was administered in combination with JWH133 to test the specificity of the CB2R-mediated effect. PA was administered intratracheally (i.t.) for induction of pneumonia in mice. At 24 h after PA exposure, lung mechanics were measured using the FlexiVent system. The total cell number, protein content, and neutrophil population in the bronchoalveolar lavage fluid (BALF) were determined. The bacterial load in the whole lung was also measured. Lung injury was evaluated by histological examination and PA-induced inflammation was assessed by measuring the levels of BALF cytokines and chemokines. Neutrophil activation (examined by immunofluorescence and immunoblot) and PA-induced inflammatory signaling (analyzed by immunoblot) were also studied. RESULTS: CB2R activation by JWH133 was found to significantly reduce PA-induced ALI and the bacterial burden. CB2R activation also suppressed the PA-induced increase in immune cell infiltration, neutrophil population, and inflammatory cytokines. These effects were abrogated by a CB2R antagonist, SR144528, further confirming the specificity of the CB2R-mediated effects. CB2R-knock out (CB2RKO) mice had a significantly higher level of PA-induced inflammation as compared to that in WT mice. CB2R activation diminished the excess activation of neutrophils, whereas mice lacking CB2R had elevated neutrophil activation. Pharmacological activation of CB2R significantly reduced the PA-induced NF-κB and NLRP3 inflammasome activation, whereas CB2KO mice had elevated NLRP3 inflammasome. CONCLUSION: Our findings indicate that CB2R activation ameliorates PA-induced lung injury and inflammation, thus paving the path for new therapeutic avenues against PA pneumonia.

Shifting Dynamics of Intestinal Macrophages during Simian Immunodeficiency Virus Infection in Adult Rhesus Macaques.

The intestinal tract is a primary barrier to invading pathogens and contains immune cells, including lymphocytes and macrophages. We previously reported that CD163+CD206- (single-positive [SP]) interstitial macrophages of the lung are short-lived and succumb early to SIV infection. Conversely, CD163+CD206+ (double-positive [DP]) alveolar macrophages are long-lived, survive after SIV infection, and may contribute to the virus reservoir. This report characterizes analogous populations of macrophages in the intestinal tract of rhesus macaques (Macaca mulatta) with SIV/AIDS. By flow cytometry analysis, immunofluorescence staining, and confocal microscopy, CD163+CD206+ DP macrophages predominated in the lamina propria of uninfected animals, compared with CD163+CD206- SP macrophages, which predominated in the lamina propria in animals with SIV infection that were exhibiting AIDS. In submucosal areas, CD163+CD206+ DP macrophages predominated in both SIV-infected and uninfected macaques. Furthermore, BrdU-labeled CD163+CD206+ DP and CD163+CD206- SP macrophages recently arriving in the colon, which are both presumed to be shorter-lived, were observed to localize only in the lamina propria. Conversely, longer-lived CD163+CD206+ DP macrophages that retained dextran at least 2 mo after in vivo administration localized exclusively in the submucosa. This suggests that CD163+CD206+ DP intestinal macrophages of the lamina propria were destroyed after SIV infection and replaced by immature CD163+CD206- SP macrophages, whereas longer-lived CD163+CD206+ DP macrophages remained in the submucosa, supporting their potential role as an SIV/HIV tissue reservoir. Moreover, the DP macrophages in the submucosa, which differ from lamina propria DP macrophages, may be missed from pinch biopsy sampling, which may preclude detecting virus reservoirs for monitoring HIV cure.

Rapid Turnover and High Production Rate of Myeloid Cells in Adult Rhesus Macaques with Compensations during Aging.

Neutrophils, basophils, and monocytes are continuously produced in bone marrow via myelopoiesis, circulate in blood, and are eventually removed from circulation to maintain homeostasis. To quantitate the kinetics of myeloid cell movement during homeostasis, we applied 5-bromo-2'-deoxyuridine pulse labeling in healthy rhesus macaques (Macaca mulatta) followed by hematology and flow cytometry analyses. Results were applied to a mathematical model, and the blood circulating half-life and daily production, respectively, of each cell type from macaques aged 5-10 y old were calculated for neutrophils (1.63 ± 0.16 d, 1.42 × 109 cells/l/d), basophils (1.78 ± 0.30 d, 5.89 × 106 cells/l/d), and CD14+CD16- classical monocytes (1.01 ± 0.15 d, 3.09 × 108 cells/l/d). Classical monocytes were released into the blood circulation as early as 1 d after dividing, whereas neutrophils remained in bone marrow 4-5 d before being released. Among granulocytes, neutrophils and basophils exhibited distinct kinetics in bone marrow maturation time and blood circulation. With increasing chronological age, there was a significant decrease in daily production of neutrophils and basophils, but the half-life of these granulocytes remained unchanged between 3 and 19 y of age. In contrast, daily production of classical monocytes remained stable through 19 y of age but exhibited a significant decline in half-life. These results demonstrated relatively short half-lives and continuous replenishment of neutrophils, basophils, and classical monocytes during homeostasis in adult rhesus macaques with compensations observed during increasing chronological age.

Dysregulation of sonic hedgehog pathway and pericytes in the brain after lentiviral infection.

BACKGROUND: Impairment of the blood-brain barrier (BBB) has been associated with cognitive decline in many CNS diseases, including HIV-associated neurocognitive disorders (HAND). Recent research suggests an important role for the Sonic hedgehog (Shh) signaling pathway in the maintenance of BBB integrity under both physiological and pathological conditions. METHODS: In the present study, we sought to examine the expression of Shh and its downstream effectors in relation to brain pericytes and BBB integrity in HIV-infected humans and rhesus macaques infected with simian immunodeficiency virus (SIV), an animal model of HIV infection and CNS disease. Cortical brain tissues from uninfected (n = 4) and SIV-infected macaques with (SIVE, n = 6) or without encephalitis (SIVnoE, n = 4) were examined using multi-label, semi-quantitative immunofluorescence microscopy of Shh, netrin-1, tight junction protein zona occludens 1 (ZO1), glial fibrillary acidic protein, CD163, platelet-derived growth factor receptor b (PDGFRB), glucose transporter 1, fibrinogen, and SIV Gag p28. RESULTS: While Shh presence in the brain persisted during HIV/SIV infection, both netrin-1 immunoreactivity and the size of PDGFRB+ pericytes, a cellular source of netrin-1, were increased around non-lesion-associated vessels in encephalitis compared to uninfected brain or brain without encephalitis, but were completely absent in encephalitic lesions. Hypertrophied pericytes were strongly localized in areas of fibrinogen extravasation and showed the presence of intracellular SIVp28 and HIVp24 by immunofluorescence in all SIV and HIV encephalitis cases examined, respectively. CONCLUSIONS: The lack of pericytes and netrin-1 in encephalitic lesions, in line with downregulation of ZO1 on the fenestrated endothelium, suggests that pericyte loss, despite the strong presence of Shh, contributes to HIV/SIV-induced BBB disruption and neuropathogenesis in HAND.

HIV-1 Tat and opioids act independently to limit antiretroviral brain concentrations and reduce blood-brain barrier integrity.

Poor antiretroviral penetration may contribute to human immunodeficiency virus (HIV) persistence within the brain and to neurocognitive deficits in opiate abusers. To investigate this problem, HIV-1 Tat protein and morphine effects on blood-brain barrier (BBB) permeability and drug brain penetration were explored using a conditional HIV-1 Tat transgenic mouse model. Tat and morphine effects on the leakage of fluorescently labeled dextrans (10-, 40-, and 70-kDa) into the brain were assessed. To evaluate effects on antiretroviral brain penetration, Tat+ and Tat- mice received three antiretroviral drugs (dolutegravir, abacavir, and lamivudine) with or without concurrent morphine exposure. Antiretroviral and morphine brain and plasma concentrations were determined by LC-MS/MS. Morphine exposure, and, to a lesser extent, Tat, significantly increased tracer leakage from the vasculature into the brain. Despite enhanced BBB breakdown evidenced by increased tracer leakiness, morphine exposure led to significantly lower abacavir concentrations within the striatum and significantly less dolutegravir within the hippocampus and striatum (normalized to plasma). P-glycoprotein, an efflux transporter for which these drugs are substrates, expression and function were significantly increased in the brains of morphine-exposed mice compared to mice not exposed to morphine. These findings were consistent with lower antiretroviral concentrations in brain tissues examined. Lamivudine concentrations were unaffected by Tat or morphine exposure. Collectively, our investigations indicate that Tat and morphine differentially alter BBB integrity. Morphine decreased brain concentrations of specific antiretroviral drugs, perhaps via increased expression of the drug efflux transporter, P-glycoprotein.

Lack of susceptibility in neonatally infected rhesus macaques to simian immunodeficiency virus-induced encephalitis.

Despite combination antiretroviral therapies making HIV a chronic rather than terminal condition for many people, the prevalence of HIV-associated neurocognitive disorders (HAND) is increasing. This is especially problematic for children living with HIV. Children diagnosed HAND rarely display the hallmark pathology of HIV encephalitis in adults, namely infected macrophages and multinucleated giant cells in the brain. This finding has also been documented in rhesus macaques infected perinatally with simian immunodeficiency virus (SIV). However, the extent and mechanisms of lack of susceptibility to encephalitis in perinatally HIV-infected children remain unclear. In the current study, we compared brains of macaques infected with pathogenic strains of SIV at different ages to determine neuropathology, correlates of neuroinflammation, and potential underlying mechanisms. Encephalitis was not found in the macaques infected within 24 h of birth despite similar high plasma viral load and high monocyte turnover. Macaques developed encephalitis only when they were infected after 4 months of age. Lower numbers of CCR5-positive cells in the brain, combined with a less leaky blood-brain barrier, may be responsible for the decreased virus infection in the brain and consequently the absence of encephalitis in newborn macaques infected with SIV.

Critical Role for Monocytes/Macrophages in Rapid Progression to AIDS in Pediatric Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Infant humans and rhesus macaques infected with the human or simian immunodeficiency virus (HIV or SIV), respectively, express higher viral loads and progress more rapidly to AIDS than infected adults. Activated memory CD4+ T cells in intestinal tissues are major primary target cells for SIV/HIV infection, and massive depletion of these cells is considered a major cause of immunodeficiency. Monocytes and macrophages are important cells of innate immunity and also are targets of HIV/SIV infection. We reported previously that a high peripheral blood monocyte turnover rate was predictive for the onset of disease progression to AIDS in SIV-infected adult macaques. The purpose of this study was to determine if earlier or higher infection of monocytes/macrophages contributes to the more rapid progression to AIDS in infants. We observed that uninfected infant rhesus macaques exhibited higher physiologic baseline monocyte turnover than adults. Early after SIV infection, the monocyte turnover further increased, and it remained high during progression to AIDS. A high percentage of terminal deoxynucleotidyltransferase dUTP nick end label (TUNEL)-positive macrophages in the lymph nodes (LNs) and intestine corresponded with an increasing number of macrophages derived from circulating monocytes (bromodeoxyuridine positive [BrdU+] CD163+), suggesting that the increased blood monocyte turnover was required to rapidly replenish destroyed tissue macrophages. Immunofluorescence analysis further demonstrated that macrophages were a significant portion of the virus-producing cells found in LNs, intestinal tissues, and lungs. The higher baseline monocyte turnover in infant macaques and subsequent macrophage damage by SIV infection may help explain the basis of more rapid disease progression to AIDS in infants.IMPORTANCE HIV infection progresses much more rapidly in pediatric cases than in adults; however, the mechanism for this difference is unclear. Using the rhesus macaque model, this work was performed to address why infants infected with SIV progress more quickly to AIDS than do adults. Earlier we reported that in adult rhesus macaques, increasing monocyte turnover reflected tissue macrophage damage by SIV and was predictive of terminal disease progression to AIDS. Here we report that uninfected infant rhesus macaques exhibited a higher physiological baseline monocyte turnover rate than adults. Furthermore, once infected with SIV, infants displayed further increased monocyte turnover that may have facilitated the accelerated progression to AIDS. These results support a role for monocytes and macrophages in the pathogenesis of SIV/HIV and begin to explain why infants are more prone to rapid disease progression.