Evidence of the innate antiviral and neuroprotective properties of progranulin

Abstract

Background: Compelling data exist that show that normal levels of progranulin (PGRN) are required for successful CNS aging. PGRN production is also modulated by inflammation and infection, but no data are available on the production and role of PGRN during CNS HIV infection.

Methods: To determine the relationships between PGRN and HIV disease, neurocognition, and inflammation, we analyzed 107 matched CSF and plasma samples from CHARTER, a well-characterized HIV cohort. Levels of PGRN were determined by ELISA and compared to levels of several inflammatory mediators (IFNγ, IL-6, IL-10, IP-10, MCP-1, TNFα, IL-1β, IL-4 and IL-13), as well as clinical, virologic and demographic parameters. The relationship between HIV infection and PGRN was also examined in HIV-infected primary human microglial cultures.

Results: In plasma, PGRN levels correlated with the viral load (VL, p<0.001). In the CSF of subjects with undetectable VL, lower PGRN was associated with neurocognitive impairment (p = 0.046). CSF PGRN correlated with CSF IP-10, TNFα and IL-10, and plasma PGRN correlated with plasma IP-10. In vitro, microglial HIV infection increased PGRN production and PGRN knockdown increased HIV replication, demonstrating that PGRN is an innate antiviral protein.

Conclusions: We propose that PGRN plays dual roles in people living with HIV disease. With active HIV replication, PGRN is induced in infected macrophages and microglia and functions as an antiviral protein. In individuals without active viral replication, decreased PGRN production contributes to neurocognitive dysfunction, probably through a diminution of its neurotrophic functions. Our results have implications for the pathogenesis, biomarker studies and therapy for HIV diseases including HIV-associated neurocognitive dysfunction (HAND).

Figure 1. HIV infection induces PGRN production in microglia.

Primary human microglial cells were inoculated with VSVg env HIV or HIV_ADA and PGRN in culture supernatants were determined by ELISA as described in the Materials and Methods. Control cultures were treated with VSVg env protein or mock infected. (A) Results with VSVg env HIV are shown. Culture supernatants were collected at 2 D and 4 D with complete change of medium (mean ± SD from triplicate cultures) (B) Microglia were incubated with AZT (10 µg/ml) or vehicle for 1 h, then exposed to VSVg env HIV as in A. PGRN was measured at 7 D and 14 D with complete change of medium. Data shown are accumulation between 7D–14D (mean ± SD, n = 3). (C) HIV_ADA (HIV env bearing virus) was used to determine PGRN production as described in the Materials and Methods. Data shown are PGRN accumulation in culture supernatants between 7D–14D (mean ± SD, n = 3) (D) Microglial cultures infected with VSVg env HIV in B showing cell viability, gag p24 expression, and complete suppression of p24 by AZT treatment. **P<0.01, ***P<0.001 by ANOVA followed by Bonferroni correction.

Figure 2. PGRN is an endogenous anti-HIV factor.

Microglial PGRN was knocked down using RNAi 2–4 days prior to VSVg env HIV exposure as described in the Materials and Methods. Control cultures were treated with control, irrelevant siRNA (Ctr). The amounts of HIV (p24) and PGRN expression were determined by western blot analyses. (A) A representative western blot showing suppression of PGRN and increase of p24 following PGRN siRNA treatment. (B) Pooled densitometry data from four independent experiments showing significant inhibition of PGRN and increase of HIV (gag p24 express) in microglial cells treated with PGRN siRNA (vs. control siRNA). **P<0.01, ***P<0.001 by paired t-test.

Figure 3. Hypothetical scenario linking HIV, inflammation, PGRN and neurocognition based on our data and literature.

(A) under normal homeostatic conditions, PGRN is produced by neurons and microglia and contributes to neuroprotection and immune balance. (B) In HIV+ individuals with detectable HIV replication, viral infection contributes to M2 predominant macrophage/microglial cytokine environment (M2>M1) in part due to IL-10 production and CD4+ T cell loss. This environment favors PGRN overproduction. IP-10 and TNFα are also co-regulated with PGRN, possibly downstream of PGRN, though the mechanisms are unclear. PGRN contributes to innate antiviral immunity in HIV-infected macrophages. (C) In HIV+ individuals with undetectable virus, macrophages are under M1 predominant (M1>M2) conditions which decrease the production of PGRN. Reduction of PGRN in the CSF/CNS compartment in which the amount of PGRN is limiting, neuronal function and survival is further compromised contributing to neurocognitive impairment (“Type II HAND”) (See text for detail).