Potential roles of microglial cell progranulin in HIV-associated CNS pathologies and neurocognitive impairment

Abstract

Progranulin (PGRN) is a highly unusual molecule with both neuronal and microglial expression with two seemingly unrelated functions, i.e., as a neuronal growth factor and a modulator of neuroinflammation. Haploinsufficiency due to loss of function mutations lead to a fatal presenile dementing illness (frontotemporal lobar degeneration), indicating that adequate expression of PGRN is essential for successful aging. PGRN might be a particularly relevant factor in the pathogenesis of HIVencephalitis (HIVE) and HIV-associated neurocognitive disorders (HAND). We present emerging data and a review of the literature which show that cells of myeloid lineage such as macrophages and microglia are the primary sources of PGRN and that PGRN expression contributes to pathogenesis of CNS diseases. We also present evidence that PGRN is a macrophage antiviral cytokine. For example, PGRN mRNA and protein expression are significantly upregulated in brain specimens with HIVE, and in HIV infected microglia in vitro. Paradoxically, our preliminary CHARTER data analyses indicate that lower PGRN levels in CSF trended towards an association with HAND, particularly in those without detectable virus. Based upon these findings, we introduce the hypothesis that PGRN plays dual roles in modulating antiviral immunity and neuronal dysfunction in the context of HIV infection. In the presence of active viral replication, PGRN expression is increased functioning as an anti-viral factor as well as a neuroprotectant. In the absence of active HIV replication, ongoing inflammation or other stressors suppress PGRN production from macrophages/microglia contributing to neurocognitive dysfunction. We propose.

Figure 1. PGRN expression in the mouse

PGRN expression is highly organ- and cell type-specific. (A) In the brain, neurons show fine punctate cytoplasmic staining (arrows) while perivascular macrophages (asterisk) and microglia (not shown) also show immunoreactivity (X 200). (B) In injured brain, such as following intracerebral AAV vector injection as shown here, ameboid microglia show strong PGRN immunoreactivity (X 400). (C) In other organs such as lung, PGRN immunoreactivity is minimal, limited to scattered macrophages (X 400). Asterisk indicates a bronchial lumen with little PGRN expression in the epithelium. (D) Lymphoid organs such as spleen had numerous PGRN+ cells that are consistent with macrophages and dendritic cells (X 100). (W = white pulp) (E) Normal liver shows punctate granular staining of the hepatocytes (X 200). (F) However, strong PGRN immunoreactivity emerges in liver macrophages (Kupffer cells) following systemic AAV injection (X 100). (C = central vein) (G) In the kidney, PGRN immunoreactivity is limited to select proximal tubular epithelial cells, accentuated in their luminal border (X 400). Asterisks mark PGRN-negative glomeruli. (F) Macrophages associated with visceral adipose tissue throughout the body show strongly PGRN positivity (X 400). Methods: Paraffin-embedded sections of mouse organs were subjected to PGRN immunohistochemistry using antigen retrieval, goat anti-PGRN IgG (R&D Systems, 1:80 dilution) and the micropolymer-based secondary antibody (ImmPress) methods, as previously described (Tarassishin et al., 2013).

Figure 2. PGRN expression in mouse spleen and human tonsil

Double label immunofluorescence studies were performed for PGRN (green) and macrophage marker (Iba-1: red) or with lymphocyte marker (CD45: red). In mouse spleen, PGRN immunoreactivity (predominantly in red pulp) co-localizes with Iba-1. In human tonsil, PGRN immunoreactivity co-localizes with Iba-1 but little with CD45 (lymphocyte common antigen). PGRN is intracellular localized in endosomal reticulum, Golgi or lysosome (Hu et al., 2010). Iba-1 is localized predominantly on the cell membrane. Methods: paraffin-embedded sections of mouse spleen or human tonsillectomy specimens were subjected to antigen retrieval, incubation with primary antibodies (goat anti-PGRN from R&D Systems at 1:80, rabbit anti-Iba-1 from WACO at 1:500 or mouse anti-human CD45 from DAKO at 1:50) followed by Alexa 488 chicken anti-goat, Alexa 555 donkey anti-rabbit or Alexa 555 anti mouse IgG at a 1:1000 dilution.

Figure 3. PGRN expression in HIV encephalitis and control brains

Formalin-fixed, paraffin-embedded postmortem human brain sections from HIVE or control brains from NNTC were subjected to PGRN immunohistochemistry as described in Figure 1 legend. (A) A white matter section from a HIV-negative brain shows numerous ramified microglial cells with fine processes showing PGRN immunoreactivity. (B) A section from HIVE shows a microglial nodule containing multinucleated giant cells with strong PGRN immunoreactivity. This contrasts with weak staining of the neurons and reactive astrocytes in the background. (C) A section from a HIV-negative control brain shows a vessel with PGRN+ intravascular leukocytes. Methods: See Figure 1 legend. All sections were lightly counterstained with hematoxylin to visualize all nuclei.

Figure 4. PGRN expression in HIVE by double-label immunofluorescence

(A) HIVE brain showing co-localization of PGRN (green) with HIV gag antigen p24 (red). (B) HIVE brain showing colocalization of PGRN (green) with the macrophage and microglial marker CD68 (red). Methods: Double immunofluorescence study was performed as described previously (Cosenza-Nashat et al., 2011). Also see Figure 2 legend. All sections were counterstained with DAPI for nuclear stain.

Figure 5. Gene arrays studies demonstrate increase of PGRN mRNA in HIVE

(A) Data from the NNTC brain gene array (Gelman et al., 2012a) demonstrate that GRN expression is significantly elevated in the white matter and basal ganglia but not in frontal cortex of HIVE cases compared to controls. Control groups included A: brains from uninfected individuals (CON), B: brains from HIV-seropositive individuals without HIVE (HIV), and C: brains from HIV+ individuals with neurocognitive impairment but without HIVE (DEM). Mean values from 5–8 brains (horizontal bar) are shown. P<0.05 by ANOVA and Bonferroni post-hoc analysis, n.s. = not significant. (B) Real-time reverse transcription PCR (qPCR) was performed to confirm the gene array data. * p<0.05 vs. control

Figure 6. Hypothesis

Based on all available data, we hypothesize that PGRN plays a dual role in the pathogenesis of HIV-associated neurocognitive disorders. (A) In normal brain, constitutive PGRN expression maintains normal neuronal survival in part through promoting neurotrophic and antioxidant gene expression in a paracrine and cell-cell interactions involving microglia, astrocytes and neurons. (B) In the CNS of individuals with active HIV replication (such as in HIVE), macrophage and microglial PGRN expression is upregulated which is reflected in the elevated levels of PGRN. PGRN expressed in the HIV-infected cells mainly functions as an innate antiviral factor, as well as to protect neurons from damaging viral and host products, via mechanisms described above. We speculate that unlike in murine cells, PGRN’s role in human microglia is to promote production of cytokines and chemokines that function as antimicrobial factors during acute infection. (C) In most HIV-associated neurocognitive disorders that are not associated with active viral replication (Type II HAND), PGRN production from macrophages and microglia is reduced by proinflammatory mediators and other unknown stressor contributing to neuronal dysfunction. We propose CSF PGRN as a surrogate disease marker for HAND not associated with active viral replication.