Role of anti-inflammatory compounds in human immunodeficiency virus-1 glycoprotein120-mediated brain inflammation

Abstract

Background: Neuroinflammation is a common immune response associated with brain human immunodeficiency virus-1 (HIV-1) infection. Identifying therapeutic compounds that exhibit better brain permeability and can target signaling pathways involved in inflammation may benefit treatment of HIV-associated neurological complications. The objective of this study was to implement an in vivo model of brain inflammation by intracerebroventricular administration of the HIV-1 viral coat protein gp120 in rats and to examine anti-inflammatory properties of HIV adjuvant therapies such as minocycline, chloroquine and simvastatin.

Methods: Male Wistar rats were administered a single dose of gp120ADA (500 ng) daily for seven consecutive days, intracerebroventricularly, with or without prior intraperitoneal administration of minocycline, chloroquine or simvastatin. Maraviroc, a CCR5 antagonist, was administered intracerebroventricularly prior to gp120 administration for seven days as control. Real-time qPCR was used to assess gene expression of inflammatory markers in the frontal cortex, hippocampus and striatum. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) secretion in cerebrospinal fluid (CSF) was measured applying ELISA. Protein expression of mitogen-activated protein kinases (MAPKs) (extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNKs) and P38 kinases (P38Ks)) was detected using immunoblot analysis. Student's t-test and ANOVA were applied to determine statistical significance.

Results: In gp120ADA-injected rats, mRNA transcripts of interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) were significantly elevated in the frontal cortex, striatum and hippocampus compared to saline or heat-inactivated gp120-injected controls. In CSF, a significant increase in TNF-α and IL-1β was detected. Maraviroc reduced upregulation of these markers suggesting that the interaction of R5-tropic gp120 to CCR5 chemokine receptor is critical for induction of an inflammatory response. Minocycline, chloroquine or simvastatin attenuated upregulation of IL-1β and iNOS transcripts in different brain regions. In CSF, minocycline suppressed TNF-α and IL-1β secretion, whereas chloroquine attenuated IL-1β secretion. In gp120-injected animals, activation of ERK1/2 and JNKs was observed in the hippocampus and ERK1/2 activation was significantly reduced by the anti-inflammatory agents.

Conclusions: Our data demonstrate that anti-inflammatory compounds can completely or partially reverse gp120-associated brain inflammation through an interaction with MAPK signaling pathways and suggest their potential role in contributing towards the prevention and treatment of HIV-associated neurological complications.

Figure 1

Effect of gp120 on the mRNA levels of (A) IL-1β, (B) iNOS and (C) TNF-α in different brain regions of ICV-administered gp120 (500 ng) rats along with a CCR5 antagonist, maraviroc (MVC). Wild-type (no surgery), control (saline) and heat-inactivated (HI) gp120-injected animals were also analyzed. Results are expressed as mean ± SEM (n = 10 for saline and gp120 groups; n = 3 for wild-type, HI gp120 and maraviroc-treated groups). Asterisks represent data points significantly different from saline-administered animals (***P <0.001; **P <0.01; *P <0.05). (gp120 = glycoprotein120; IL-1β = interleukin-1β; iNOS = inducible nitric oxide synthase; TNFα = tumor necrosis factor-α; ICV = intracerebroventricular; MVC = Maraviroc; HI = heat inactivated).

Figure 2

ELISA analysis of (A) IL-1β and (B) TNF-α secretion in the CSF of gp120 (500 ng) administered rats in the presence of maraviroc (MVC). CSF collected from saline- and heat-injected animals were used as control. Results are expressed as mean ± SEM (n = 10 for saline and gp120 groups; n ≥3 for wild-type, HI gp120 and maraviroc-treated groups). Asterisks represent data points significantly different from saline-administered animals (***P <0.001; **P <0.01). (ELISA = Enzyme linked immunoabsorbant assay; IL-1β = interleukin-1β; TNFα = tumor necrosis factor-α; CSF = cerebrospinal fluid; gp120 = glycoprotein120; MVC = Maraviroc; HI = heat inactivated).

Figure 3

GFAP expression in the hippocampus of ICV-administered gp120 rats. (A) Immunohistochemical, (B) immunoblotting (upper panel) and densitometric analysis (lower panel) of GFAP in the hippocampus of ICV-administered gp120 rats are compared to saline-treated animals. A representative blot is shown. Results are expressed as mean ± SEM. For immunoblotting, samples obtained from five different animals were used per group. Asterisk represents a data point significantly different from saline-administered animals (*P <0.05). (GFAP = glial fibrillary acidic protein; ICV = intracerebroventricular; gp120 = glycoprotein120).

Figure 4

Immunoblot (upper panel) and densitometric analysis (lower panel) of drug efflux transporters (P-gp, Mrp1 and Bcrp) in (A) frontal cortex, (B) hippocampus and (C) striatum of ICV-administered gp120 rats. Transporter over-expressing cell lines were used as positive controls. Representative blots are shown. Results are expressed as mean ± SEM. Samples obtained from six different animals were used per group. Asterisks represent data points significantly different from saline-administered animals (**P <0.01; * P <0.05). (P-gp = P-glycoprotein; Mrp1 = multidrug resistance protein 1; Bcrp = breast cancer resistance protein; ICV = intracerebroventricular; gp120 = glycoprotein120).

Figure 5

Effect of gp120 on the mRNA levels of (A) IL-1β, (B) iNOS and (C) TNF-α in different brain regions of ICV-administered gp120 rats receiving simultaneous intraperitoneal injection of chloroquine or minocycline or simvastatin. Saline-injected animals were used as control. Results are expressed as mean ± SEM. Samples obtained from at least 10 different animals were used per group. Asterisks represent data points significantly different from saline-administered animals (***P <0.001; **P <0.01; *P <0.05). Diamonds represent data points significantly different from gp120 treatment (♦♦♦P <0.001; ♦♦P <0.01). (gp120 = glycoprotein120; IL-1β = interleukin-1β; iNOS = inducible nitric oxide synthase; TNFα = tumor necrosis factor-α; ICV = intracerebroventricular; CQ = chloroquine; Mino = minocycline; Sim = simvastatin).

Figure 6

ELISA analysis of (A) IL-1β and (B) TNF-α secretion in the CSF of gp120 (500 ng) administered rats receiving simultaneous intraperitoneal injection of chloroquine or minocycline or simvastatin. CSF collected from saline-injected animals was used as a control. Results are expressed as mean ± SEM. Samples obtained from at least 10 different animals were used per group. Asterisks represent data points significantly different from saline-administered animals (***P <0.001; **P <0.01). Diamonds represent data points significantly different from gp120 treatment (♦♦♦P <0.001). (ELISA = Enzyme linked immunoabsorbant assay; IL-1β = interleukin 1β; TNFα = tumor necrosis factorα; CSF = cerebrospinal fluid; gp120 = glycoprotein120; CQ = chloroquine; Mino = minocycline; Sim = simvastatin).

Figure 7

Protein (upper panel) and densitometric analysis (lower panel) of (A) ERK1/2 and phospho-ERK1/2; JNK and phospho-JNK; P38K and phospho-P38K, (B) ERK1/2 and phospho-ERK1/2 in the presence of chloroquine, minocycline and simvastatin, (C) ERK1/2 and phospho-ERK1/2 in the presence of ERK1/2 inhibitor U0126; (D) JNK and phospho-JNK in the presence of SP600125 in the hippocampal tissue of gp120-administered rats. Representative blots are shown. Tissue collected from saline-administered animals was used as control. Results are expressed as mean ± SEM. Samples obtained from three to six different animals per group were used. Asterisks represent data points significantly different from saline-administered animals (***P <0.001; *P <0.05). Diamonds represent data points significantly different from gp120-treatment (♦P <0.05). (ERK1/2 = extracellular signal-related kinase 1/2; p-ERK1/2 = phospho- ERK1/2; JNKs = c-Jun N-terminal kinases; p-JNKs = phospho-JNKs; P38Ks = P38 kinases; p-P38Ks = phosphor-p38Ks; gp120 = glycoprotein120).