SERPIN-Derived Small Peptide (SP16) as a Potential Therapeutic Agent against HIV-Induced Inflammatory Molecules and Viral Replication in Cells of the Central Nervous System

Abstract

Despite the success of combined antiretroviral therapy (cART) increasing the survival rate in human immunodeficiency virus (HIV) patients, low levels of viremia persist in the brain of patients leading to glia (microglia and astrocytes)-induced neuroinflammation and consequently, the reactivation of HIV and neuronal injury. Here, we tested the therapeutic efficacy of a Low-Density Lipoprotein Receptor-Related Protein 1 (LRP-1) agonistic small peptide drug (SP16) in attenuating HIV replication and the secretion of inflammatory molecules in brain reservoirs. SP16 was developed by Serpin Pharma and is derived from the pentapeptide sequence of the serine protease inhibitor alpha-1-antitrypsin (A1AT). The SP16 peptide sequence was subsequently modified to improve the stability, bioavailability, efficacy, and binding to LRP-1; a scavenger regulatory receptor that internalizes ligands to induce anti-viral, anti-inflammatory, and pro-survival signals. Using glial cells infected with HIV, we showed that: (i) SP16 attenuated viral-induced secretion of pro-inflammatory molecules; and (ii) SP16 attenuated viral replication. Using an artificial 3D blood-brain barrier (BBB) system, we showed that: (i) SP16 was transported across the BBB; and (ii) restored the permeability of the BBB compromised by HIV. Mechanistically, we showed that SP16 interaction with LRP-1 and binding lead to: (i) down-regulation in the expression levels of nuclear factor-kappa beta (NF-κB); and (ii) up-regulation in the expression levels of Akt. Using an in vivo mouse model, we showed that SP16 was transported across the BBB after intranasal delivery, while animals infected with EcoHIV undergo a reduction in (i) viral replication and (ii) viral secreted inflammatory molecules, after exposure to SP16 and antiretrovirals. Overall, these studies confirm a therapeutic response of SP16 against HIV-associated inflammatory effects in the brain.

Keywords: blood-brain barrier; intranasal delivery; low-density lipoprotein receptor related-protein 1; serine protease inhibitors; therapeutic efficacy.

Figure 1

Anti-inflammatory and anti-viral responses of SP16 in microglia. SP16 at increasing concentrations was directly added to cells (B,D,F,H,J) or to the luminal surface of the BBB inserts (A,C,E,G,I). Supernatant was collected after 12 h and inflammatory molecules (A–H) and p24 (I,J) were measured by ELISA. Gray bars = non-infected cells, black bars = infected cells. Results are reported as the mean ± SEM of 3 independent experiments. Data were analyzed using one or two-way ANOVA analysis followed by Dunnett multiple comparisons. A value of p < 0.05 was considered significant. * vs. 0 SP16 (media); ^$ vs. 1 μg/mL SP16.

Figure 2

Movement of SP16 across an artificial blood-brain barrier (BBB). Supernatant was collected after 6 h (A) and 12 h (B). MALDI-TOF-MS analysis was conducted on the Burker AutoFlex III instrument to test the transportation of different SP16 concentrations across the BBB at the indicated time points. Integrity of the BBB was measured using a Millicell ERS microelectrode (C–H) and by a FITC-dextran Assay (I,J). Results are reported as the mean ± SEM of 3 independent experiments. Data was analyzed using one or two-way ANOVA analysis followed by Dunnett multiple comparisons A value of p < 0.05 was considered significant. * vs. media; ^$ vs. 25 μg/mL SP16.

Figure 3

Anti-inflammatory and anti-viral responses of SP16 in astrocytes and endothelial cells. Supernatant was collected after 6 (astrocytes) and 12 (astrocytes and endothelial cells) hours and used to measure inflammatory molecules (A–H) in astrocytes, (K–N) in endothelial cells, and viral protein p24 (I,J) in astrocytes, (O) in endothelial cells, by ELISA. Gray bars = non-infected cells, black bars = infected cells. Results are reported as the mean ± SEM of 3 independent experiments. Data were analyzed using one or two-way ANOVA analysis followed by Dunnett multiple comparisons. A value of p < 0.05 was considered significant. * vs. media; ^$ vs. 1 μg/mL SP16; ^# vs. 25 μg/mL SP16 (in astrocytes); ^$ vs. 25 μg/mL SP16 (in endothelial cells); ^& vs. 100 μg/mL SP16.

Figure 4

SP16 does not affect cell viability in microglia, astrocytes, or endothelial cells. SP16 at increasing concentrations was added to microglia (A–D), astrocytes (E–H), and endothelial cells (I–L). MTT (A,C,E,G,I,K); a Trypan Blue Staining (B,D,F,H,J,L) assay was used to assess cell viability at 24 h (gray bars) and 96 h (black bars). Results are reported as the mean ± SEM of three independent experiments. Data were analyzed using one or two-way ANOVA analysis followed by Dunnett multiple comparisons. A value of p < 0.05 was considered significant. * vs. media.

Figure 5

LRP-1 is expressed in brain cells. Protein expression levels of LRP-1 was quantitatively measured by flow cytometry in microglia (A) and astrocytes (B), and by immunoblotting in astrocytes (C,D). LRP-1 was detected by immunofluorescence staining and imaged by fluorescence microscopy (G,H), and quantitatively by immunoblotting in endothelial cells (E,F). Data was analyzed using one or two-way ANOVA analysis followed by Dunnett multiple comparisons. A value of p < 0.05 was considered significant. * vs. media.

Figure 6

SP16 increases the expression levels of Akt in astrocytes and endothelial cells. Cell lysates were collected after 12 h and used to measure NF-κB (A,B,E,F) and total Akt (C,D,G,H) expression levels by immunoblotting. Gray bars = non-infected cells, black bars = infected cells. Results are reported as the mean ± SEM of three independent experiments. Data was analyzed using one or two-way ANOVA analysis followed by Dunnett multiple comparisons. A value of p < 0.05 was considered significant. * vs. media.

Figure 7

Silencing the LRP-1 gene leads to increased expression levels of NF-κB and decreased levels of Akt in astrocytes and endothelial cells. Cell lysates from astrocytes (A,C,E) and endothelial cells (B,D,F) were collected after 12 h and LRP-1 (A,B), NF-κB (C,D), and Akt (E,F) expression were measured by immunoblotting. Results are reported as the mean ± SEM of three independent experiments. Data were analyzed using one or two-way ANOVA analysis followed by Dunnett multiple comparisons. A value of p < 0.05 was considered significant. * vs. SP16/-siLRP-1; ^& vs. 25 μg/mL SP16; ^$ vs. 100 μg/mL SP16/-siLRP-1; ^# vs. SP16.

Figure 8

LRP-1 mediates the anti-inflammatory action of SP16 in astrocytes and endothelial cells. Supernatant from astrocytes (A–C) and endothelial cells (D–F) collected after 12 h was used to measure inflammatory molecules by ELISA. Results are reported as the mean ± SEM of three independent experiments. Data were analyzed using one or two-way ANOVA analysis followed by Dunnett multiple comparisons. A value of p < 0.05 was considered significant. * vs. SP16/-siLRP-1; ^& vs. 25 μg/mL SP16; ^$ vs. 100 μg/mL SP16/-siLRP-1; ^# vs. SP16.

Figure 9

SP16 induces anti-inflammatory and anti-viral responses in EcoHIV-infected mouse brain. Supernatant was collected after 24 h post treatment and used to measure the secretion of inflammatory molecules (A,B) and viral titer (C) by ELISA. LRP-1 (D), NF-κB (E), and Akt (F) protein expression levels were measured by immunoblotting. Results are reported as the mean ± SEM of 3 independent experiments. Data were analyzed using one or two-way ANOVA analysis followed by Dunnett multiple comparisons. A value of p < 0.05 was considered significant. * vs. media; ^$ vs. 25 μg/mL SP16. Brains of adult mice (N = 3–5/treatment) were collected at necropsy. EcoHIV Infected animals received cART alone and in combination with SP16 (as illustrated in schematic diagram G). Homogenized brain lysates were used to measure secretion of inflammatory molecules (H–J) and viral titer (K) by ELISA. LRP-1 (L), NF-κB (M), and Akt (N) protein expression levels were measured by immunoblotting. Data were analyzed using one or two-way ANOVA analysis followed by Dunnett multiple comparisons. A value of p < 0.05 was considered significant. * vs. PBS control; ^$ vs. EcoHIV; # vs. cART.