Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and p38 regulate the expression of chemokine CXCL8 in human astrocytes.

CXCL8, one of the first chemokines found in the brain, is upregulated in the brains and cerebrospinal fluid of HIV-1 infected individuals suggesting its potential role in human immune deficiency virus (HIV)-associated neuroinflammation. Astrocytes are known to be the major contributors to the CXCL8 pool. Interleukin (IL)-1β activated astrocytes exhibit significant upregulation of CXCL8. In order to determine the signaling pathways involved in CXCL8 regulation in astrocytes, we employed pharmacological inhibitors for non-receptor Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and mitogen-activated protein kinases (MAPK) pathway and observed reduced expression of CXCL8 following IL-1β stimulation. Overexpression of SHP2 and p38 enzymes in astrocytes led to elevated CXCL8 expression; however, inactivating SHP2 and p38 with dominant negative mutants abrogated CXCL8 induction. Furthermore, SHP2 overexpression resulted in higher SHP2 and p38 enzyme activity whereas p38 overexpression resulted in higher p38 but not SHP2 enzyme activity. Phosphorylation of SHP2 was important for phosphorylation of p38, which in turn was critical for phosphorylation of extracellular signal regulated kinase (ERK). Thus, our findings suggest an important role for SHP2 in CXCL8 expression in astrocytes during inflammation, as SHP2, directly or indirectly, modulates p38 and ERK MAPK in the signaling cascade leading to CXCL8 production. This study provides detailed understanding of the mechanisms involved in CXCL8 production during neuroinflammation

Chemokine CXCL8 promotes HIV-1 replication in human monocyte-derived macrophages and primary microglia via nuclear factor-κB pathway.

Chemokine CXCL8 is an important neutrophil chemoattractant implicated in various neurodegenerative disorders. Cytokine/chemokine imbalance, with an increase in proinflammatory cytokines like interleukin-1β and tumor necrosis factor-α within the central nervous system, is a hallmark of human immunodeficiency virus (HIV)-1 infection. We previously reported that HIV-1 infection is linked to upregulation of CXCL8 in brain tissues and human astrocytes. Chemokines play crucial roles in trafficking of leukocytes and trafficking of HIV-1-infected across the blood-brain barrier play an important role in HIV-1 central nervous system disease. In the post-antiretroviral therapy era, low level of productive replication of HIV-1 in brain is a critical component of neuropathogenesis regulation. The present study investigated the effect of CXCL8 on productive infection of HIV-1 in human monocytes-derived macrophages (MDM) and primary human microglia.Human MDM and microglia were infected with the blood or brain derived HIV-1 isolates, HIV-1ADA or HIV-1JRFL. Treatment with CXCL8 significantly upregulated HIV-1p24 levels in supernatants of both HIV-1-infected MDM as well as microglia. In addition, the formation of 2-long terminal repeat (LTR) circles, a measure of viral genome integration, was significantly higher in CXCL8-treated, HIV-1-infected MDM and microglia. Transient transfection of U937 cells with HIV-1 LTR luciferase reporter construct resulted in increased promoter activity when treated with CXCL8. Moreover, increased nuclear translocation of nuclear factor-κB was seen in HIV-1-infected MDM following CXCL8 treatment. Blocking CXCL8 receptors CXCR1 and CXCR2 abrogated the CXCL8-mediated enhanced HIV-1 replication.Our results show that CXCL8 mediates productive infection of HIV-1 in MDM and microglia via receptors CXCR1 and CXCR2. These results demonstrate that CXCL8 exerts its downstream effects by increasing translocation of nuclear factor-κB into the nucleus, thereby promoting HIV-1 LTR activity

SHP2, p38 and ERK phosphorylation following IL-1β stimulation of SHP2WT-, SHP2CS-, p38- and p38agf-transfected astrocytes.

<p>Astrocytes were transfected with SHP2WT, SHP2CS, p38 or p38agf plasmids and stimulated with or without IL-1β for 5, 15 and 25 min. Whole cell protein lysates were collected and equivalent amounts were resolved by SDS-PAGE, transferred and immunoblotted for P-SHP2, SHP2, P-p38, p38, P-ERK and ERK. β-actin was used as loading control. <b>A)</b> Immunoblot of SHP2WT- and SHP2CS-transfected astrocytes following stimulation with IL-1β. <b>B)</b> Immunoblot of p38- and p38agf-transfected astrocytes following stimulation with IL-1β.</p

CXCL8 upregulation by SHP2 and p38 overexpression in astrocytes.

<p>Primary human astrocytes were transfected with SHP2WT and p38 overexpression plasmids with corresponding dominant negative mutants SHP2CS and p38agf. Twenty-four h post-transfection supernatants were analyzed for CXCL8 protein levels by ELISA. 24 h post-transfection astrocytes were treated with IL-1β (20 ng/ml) for 24 h, protein lysates and supernatants were collected and analyzed for SHP2 and p38 enzyme activity. <b>A)</b> CXCL8 protein levels assayed by ELISA normalized to MTT. <b>B)</b> SHP2 <i>in vitro</i> phosphatase assay showing phosphate released as a measure of SHP2 phosphatase activity. Results are representative of three independent experiments performed in triplicate and expressed as mean ± SEM, analyzed by one-way ANOVA and Newman-Keuls post-test for multiple comparisons. <b>C)</b><i>In vitro</i> p38 kinase assay based on immunoprecipitation of p38, followed by incubation with ATF-2 as substrate. P-ATF-2 band at 40 kD, which is a function of higher p38 kinase activity, is shown with densitometry. Results are representative of three independent experiments.</p

CXCL8 increases 2-LTR circle formation in HIV-1-infected MDM.

<p>Human MDM were plated in 6-well plates and infected with HIV-1_ADA or HIV-1_JRFL (MOI 0.1) overnight. Following day, MDM were treated with/without CXCL8 (50 ng/ml). DNA was extracted 1 week post-infection and 2-LTR circle junctions were amplified by real-time PCR. (<b>A</b>) Standard curve generated using pTA2LTR/CCR5 plasmid with 2-LTR copy numbers at X-axis and real-time PCR threshold counts at Y-axis. (<b>B</b>) Comparison of 2-LTR circle copy numbers in HIV-1_ADA- or HIV-1_JRFL-infected MDM. Results are expressed as mean ± SEM, analyzed by one-way ANOVA and Newman-Keuls post-test for multiple comparisons.</p

Endogenous CXCL8 stimulates HIV-1<i>p24</i> levels in HIV-1-infected MDM.

<p>Human MDM were infected with HIV-1_ADA or HIV-1_JRFL (MOI 0.1) overnight, followed by treatment with/without anti-CXCL8 IgG (1.2 μg/ml). Mouse IgG (1.2 μg/ml) was used as a control. Cell culture supernatants were collected at indicated time points. CXCL8 levels were determined by ELISA in MDM infected with (<b>A</b>) HIV-1_ADA or (<b>B</b>) HIV-1_JRFL (<b>C</b>) HIV-1<i>p24</i> levels as determined by ELISA in MDM infected with HIV-1_ADA or (<b>D</b>) HIV-1_JRFL. Results are expressed as mean ± SEM, analyzed by one-way ANOVA and Newman-Keuls post-test for multiple comparisons.</p

CXCL8 increases HIV-1<i>p24</i> and 2-LTR circles in HIV-1-infected primary human microglia.

<p>Primary human microglia were infected with HIV-1_ADA or HIV-1_JRFL (MOI 0.1) overnight, followed by treatment with 50 ng/ml CXCL8. (<b>A</b>) Supernatants were collected 2 weeks post-infection and HIV-1<i>p24</i> levels were measured by ELISA. (<b>B</b>) DNA was isolated and 2-LTR circle junctions were amplified by real-time PCR. (<b>C–E</b>) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent was added to microglia and incubated for 15 minutes at 37°C. Results are expressed as mean ± SEM, analyzed by one-way ANOVA and Newman-Keuls post-test for multiple comparisons. Phase contrast pictures showing (<b>C</b>) cell morphology of uninfected primary human microglia. (<b>D</b>) Multinucleated giant cells formed with HIV-1_ADA infection (<b>E</b>) HIV-1_ADA infection and CXCL8 treatment. (<b>F-H</b>) Expression of CD68 (green) and HIV-1<i>p24</i> (red) was measured by immunocytochemistry 2 weeks post-infection with HIV-1_ADA. Nuclei were stained in blue by DAPI. (<b>F</b>) Uninfected primary human microglia (<b>G</b>) co-localization (yellow) of CD68 and HIV-1<i>p24</i> in HIV-1_ADA-infected microglia. (<b>H</b>) HIV-1<i>p24</i> expression and multinucleated giant cells in HIV-1_ADA-infected microglia. Arrows represent multinucleated giant cells. Original magnification 200X.</p

CXCL8 levels are elevated in brain lysates of HIV-1 infected patients.

<p>Human brain mRNA and protein lysates collected from frontal cortex of HIV-1 infected individuals and age matched controls were analyzed for CXCL8 levels. <b>A)</b> CXCL8 mRNA expression analyzed by real-time PCR normalized to GAPDH. <b>B)</b> CXCL8 protein levels assayed by ELISA shown as ng/µg total protein. Results are expressed as mean ± SEM of indicated number of donors. Statistical analyses were performed using unpaired student’s t-test.</p

Effect of p38 overexpression and SHP2 inhibition on CXCL8 levels.

<p>p38- and p38agf-transfected astrocytes were treated with PTP inhibitor, Na₃VO₄; p38 inhibitor, SB203580, or negative control inhibitor, SB202474. CXCL8 levels measured by ELISA in cellular supernatants collected 24 h post-treatment with <b>A)</b> Na₃VO₄<b>B)</b> SB203580 <b>C)</b> SB202474. ND denotes not detectable. Results are representative of three independent experiments performed in triplicate and expressed as mean ± SEM, analyzed by student’s t-test.</p

CXCL8 induces a dose-dependent upregulation of HIV-1<i>p24</i> in HIV-1-infected MDM.

<p>Human MDM were infected with HIV-1_ADA or HIV-1_JRFL (MOI 0.1) overnight, followed by treatment with indicated concentrations of CXCL8. Cell culture supernatants were collected 1 week post-infection and HIV-1<i>p24</i> levels were measured by ELISA in MDM infected with (<b>A</b>) HIV-1_ADA or (<b>B</b>) HIV-1_JRFL. Expression of macrophage marker CD68 and HIV-1<i>p24</i> was measured by immunocytochemistry in HIV-1_ADA-infected MDM 1 week post-infection. Nuclei were stained in blue by DAPI. (<b>C</b>) Control, CD68-positive cells (green) (<b>D</b>) Co-localization (yellow) of CD68 and HIV-1<i>p24</i> (red) in HIV-1_ADA-infected MDM (<b>E, F</b>) HIV-1<i>p24</i> expression and multinucleated giant cells (represented by arrowhead) in CXCL8-treated, HIV-1_ADA-infected MDM. Original magnification 200X. Results are expressed as mean ± SEM, analyzed by one-way ANOVA and Newman-Keuls post-test for multiple comparisons.</p