Rapid inflammasome activation in microglia contributes to brain disease in HIV/AIDS

Abstract

Background: Human immunodeficiency virus type 1(HIV-1) infects and activates innate immune cells in the brain resulting in inflammation and neuronal death with accompanying neurological deficits. Induction of inflammasomes causes cleavage and release of IL-1β and IL-18, representing pathogenic processes that underlie inflammatory diseases although their contribution HIV-associated brain disease is unknown.

Results: Investigation of inflammasome-associated genes revealed that IL-1β, IL-18 and caspase-1 were induced in brains of HIV-infected persons and detected in brain microglial cells. HIV-1 infection induced pro-IL-1β in human microglia at 4 hr post-infection with peak IL-1β release at 24 hr, which was accompanied by intracellular ASC translocation and caspase-1 activation. HIV-dependent release of IL-1β from a human macrophage cell line, THP-1, was inhibited by NLRP3 deficiency and high extracellular [K+]. Exposure of microglia to HIV-1 gp120 caused IL-1β production and similarly, HIV-1 envelope pseudotyped viral particles induced IL-1β release, unlike VSV-G pseudotyped particles. Infection of cultured feline macrophages by the related lentivirus, feline immunodeficiency virus (FIV), also resulted in the prompt induction of IL-1β. In vivo FIV infection activated multiple inflammasome-associated genes in microglia, which was accompanied by neuronal loss in cerebral cortex and neurological deficits. Multivariate analyses of data from FIV-infected and uninfected animals disclosed that IL-1β, NLRP3 and caspase-1 expression in cerebral cortex represented key molecular determinants of neurological deficits.

Conclusions: NLRP3 inflammasome activation was an early and integral aspect of lentivirus infection of microglia, which was associated with lentivirus-induced brain disease. Inflammasome activation in the brain might represent a potential target for therapeutic interventions in HIV/AIDS.

Figure 1

Expression of inflammasome components and substrates in the HIV-1 infected human brain. A. Relative fold change (RFC) in mRNA expression in the white matter of persons with HIV-1 infection (HIV [+]) (n = 12) compared to other disease controls (HIV [-]) (n = 12), as measured by semi-quantitative real-time PCR. Mean values are reported. Bars indicate standard error. *indicate p-value of <0.05. B. Immunohistochemistry staining of cerebral white matter sections from other disease control (HIV [-]) and HIV-infected (HIV [+]) (200X view). Inset 'i’ represents double immunostaining for MHC II and IL-1β. Insets 'ii’, 'iii’ and 'iv’ are magnified images of the indicated cells within the HIV [+] section.

Figure 2

Expression and activation of the inflammasome in primary human CNS cells. Isolated primary human cells including microglia (A), astrocytes (B) and neurons (C) were immunolabelled with antibodies to Iba-1, GFAP and MAP-2, respectively, to verify the purity of cell cultures. D. Qualitative RT-PCR showing transcript expression of caspase-1 as well as various inflammasome-forming cytoplasmic pattern recognition receptors in primary cultured human microglia, astrocytes and neurons. E. Semi-quantitative real-time PCR showing the relative fold change in inflammasome-associated gene expression in cultured primary human microglia and astrocytes. Bars indicate standard error. F. Protein expression of IL-1β and caspase-1 in primary human microglia but not astrocytes. G. IL-1β release from Mock-or LPS-primed (10 ng/mL, 6 hr) primary human microglia followed by ATP stimulation (5 mM, 1 hr). Mean values are reported. Bars indicate standard error.

Figure 3

IL-1β expression and release from primary human microglia following HIV-1 infection or exposure. A. Viral p24 and B. IL-1β release from microglia at days 4, 7 and 10 following infection with HIV-1_SF162.C. IL-1β induction and D. release from microglia cultures over 24 hr following exposure to HIV-1_SF162. E. Induction, processing and F. release of IL-1β following 24 hr exposure to HIV-1_SF162 +/- the caspase inhibitor YVAD-fmk (80 μM). G. Induction and H. release of IL-1β from microglia cultures exposed to recombinant gp120 (80 nM) for 24 hr +/- YVAD-fmk. Mean levels are reported for each ELISA. Bars indicate standard error. We compared the effect of HIV-1gp120 exposure on IL-1β release +/- YVAD-fmk using two-tailed Student t-test (*p < 0.05).

Figure 4

IL-1β release in response to HIV-1_SF162 exposure is dependent on the NLRP3 inflammasome but not viral fusion or CCR5. A. Induction, processing and B. release of IL-1β from PMA-differentiated THP-1 cells following 4 hr exposure to HIV-1_SF162. C. IL-1β release from PMA-differentiated THP-1 cells exposed to HIV-1_SF162 for 4 hr +/- extracellular KCl (50 mM) or YVAD-fmk (80 μM). D. Release of IL-1β from PMA-differentiated THP-1 cells following 18 hr exposure to HIV-1_SF162 +/- the inhibitor of viral fusion T20 (20 μg/mL) or the CCR5 antagonist maraviroc (100nM). E. IL-1β release from PMA-differentiated THP-1 cells or THP1-defNLRP3 cells exposed to HIV-1_SF162 or poly dA:dT for 4 hr (poly dA:dT was transfected using Lipofectamine 2000). F. IL-1β release from PMA-differentiated THP-1 cells exposed to HIV-1_YU2, HxBRuR⁺E^- + VSVG, or HxBRuR⁺E^- + HIV-1_Env3098 for 24 hours. Mean levels are reported for each ELISA. Bars indicate standard error. (*p < 0.05). “N.D.” indicates that IL-1β was “Not Detected”.

Figure 5

HIV-1_SF162 infection of human microglia induces rapid ASC intracellular translocation. A. Mock-infected microglia exhibit minimal ASC immunoreactivity. B. At 1 hr post-infection ASC immunoreactivity (red) was detected in the cytoplasm. C. At 2 hr post-infection, punctate ASC immunoreactivity was evident in the cytoplasm of microglia. D. At 3 hr post-infection, aggregates of ASC immunoreactivity was readily apparent in microglia. (Original magnification 400X).

Figure 6

HIV-1_SF162 infection of human microglia activates caspase-1. A. Caspase-1 activity was not detected in mock-infected microglial cultures that were labeled with Hoescht nuclear stain. B. In contrast, microglia infected by HIV-1 showed caspase-1 activity at 24 hr post-infection (Original magnification 400X). C. Quantitation of caspase-1 activity in microglia at 4 and 24 hr post-infection showed an increased in caspase-1 activity induced by HIV-1 infection and also over time. (Mean values reported. Bars indicate standard error. Student t-test, *p < 0.05).

Figure 7

IL-1β and caspase-1 expression in the brains of FIV-infected animals with neurologic defects. A. IL-1β immunoreactivity was evident in feline monocyte-derived macrophages at 8 and 24 hr following FIV infection while caspase-1 expression was unaffected. B. CD4+ T cell count in blood of FIV-infected (n = 10) and uninfected animals (n = 6) at weeks 8 and 12 post-infection. C. Neuronal counts per high power field (400X) from FIV [+] animals versus FIV [-] controls at week 12 D. Immunohistochemical detection of Iba-1, IL-1β and caspase-1 in both striatum and cortex (CTX) from FIV [-] and FIV [+] animals (original magnification: Iba-1 and IL-1β X200; caspase-1 X600; Nissl stain, X100). Inset 'i’ shows an IL-1β immunoreactive microglial nodule. Insets 'ii’ and 'iii’ display IL-1β and Iba-1 co-immunolabeling. E. Detection of FIV pol RNA in the cortex and striatum of FIV [-] (n = 6) and FIV [+] animals (n = 10) at week 12. Performance of FIV [-] (n = 6) and FIV [+] (n = 10) animals in neurobehavioral tests of gait (F) and memory (G) at week 12 post-infection. “N.D.” indicates that virus was “Not Detected”.

Figure 8

In vivo association of inflammasome-related genes with neurological disease in FIV [+] animals. A. Relative fold change (RFC) in mRNA expression of inflammasome- related and immune genes in the cortex and striatum of FIV [+] (n = 6) and FIV [-] (n = 10) animals at 12 weeks post-infection. Mean RFC values are reported. Bars indicate standard error. *indicate p-value of <0.05. B. Principal component analysis (PCA) comparing 49 clinical, neurobehavioral and molecular variables in FIV [-] and FIV [+] animals. PC1 represents the separation of the FIV [-] and FIV [+] animals while PC2 represents intra-group variance. C. Hierarchical Cluster Analysis of multivariate similarities between variables and animals. Only variables that significantly contributed to PC1 (infection status) are presented in the heat map.