RAGE and CCR7 mediate the transmigration of Zika-infected monocytes through the blood-brain barrier.

Details of the "Trojan Horse" mechanism by which Zika virus (ZIKV) crosses the blood-brain barrier (BBB) remain unclear. However, the migration of ZIKV-infected monocytes to the brain is thought to be dependent on both pattern-recognition and chemokine receptors. In this study, we investigated whether the migration of ZIKV-infected MonoMac-1 (MM-1) cells through the BBB is dependent on chemokine receptor 7 (CCR7) and receptor for advanced glycation end (RAGE); we also determined whether high mobility group box protein 1 (HMGB1) could facilitate the permeabilization of endothelial cells. We demonstrated that ZIKV infects MM-1 cells, leading to milieu accumulation of HMGB1. Our results suggest that HMGB1 is involved in the dysregulation of primary human brain microvascular endothelial cell junction markers. Our results also indicate that the migration of ZIKV-infected monocytes is dependent on chemokine ligand 19 (CCL19), the natural ligand of CCR7, in conditions recapitulating inflammation. RAGE-dependent migration of ZIKV-infected cells declined during transmigration assays in the presence of RAGE receptor antagonist FPS-ZM1. Understanding the molecular role of monocyte trafficking during ZIKV infections could facilitate the development of new therapeutic strategies to prevent the deleterious consequences of ZIKV neuroinfection.

Polymorphisms in the interleukin 4 receptor and interleukin 13 genes in immediate allergic reactions to beta-lactam antibiotics: A case-control study.

BACKGROUND: Immediate hypersensitivity reactions to beta-lactams are IgE-mediated and constitute the most common adverse reactions to antibiotics mediated by a specific immunologic mechanism. OBJECTIVE: We investigated the association between four functional polymorphisms of IL13 (R130Q variant) and IL4RA (I50V, S478P and Q551R variants) genes and susceptibility to immediate allergic reactions to beta-lactams in the Algerian population. METHODS: We determined these gene variants in 199 patients and 99 healthy controls from Algeria. In a case-control study using the TaqMan method, we genotyped four single nucleotide polymorphisms (SNPs) including Arg130Gln in IL13, and Ile50Val, Ser478Pro as well as Gln551Arg in IL4RA. RESULTS: IL4RA I50V variant was more significantly connected with the risk of beta-lactam allergy (P = 0.0144) and the total serum IgE level in patients (P = 0.0136). A significant correlation was observed between IL13 R130Q and beta- lactam allergy (P = 0.0384). Also, a significant gene-gene interaction was detected between the predominant allele of the IL13 R130Q polymorphism and the three polymorphisms of IL4RA (P < 0.0001, P = 0.0163, and 0.0301, respectively). Haplotype analysis of IL4RA revealed that GTA haplotype had a significant correlation in patients with beta-lactam allergy (P = 0.0123). CONCLUSIONS: Our results indicate that IL4RA (I50V) and IL13 R130Q are associated with beta-lactam allergy. The combination of IL13 and IL4RA variants markedly increases an individual's susceptibility to beta-lactam allergy in the Algerian population.

TLR4 induces CCR7-dependent monocytes transmigration through the blood-brain barrier.

In this study, we examined whether bacterial pathogen-associated molecular patterns recognized by toll-like receptors (TLRs) can modify the CCR7-dependent migration of human monocytes. MonoMac-1 (MM-1) cells and freshly isolated human monocytes were cultivated in the presence of agonists for TLR4 (which senses lipopolysaccharides from gram-negative bacteria), TLR1/2 (which senses peptidoglycan from gram-positive bacteria), and TLR9 (which recognizes bacterial DNA rich in unmethylated CpG DNA). CCR7 mRNA transcription was measured using quantitative reverse transcription polymerase chain reaction and protein expression was examined using flow cytometry. CCR7 function was monitored using migration and transmigration assays in response to CCL19/CCL21, which are natural ligands for CCR7. Our results show that TLR4 strongly increases monocyte migratory capacity in response to CCL19 in chemotaxis and transmigration assays in a model that mimics the human blood-brain barrier, whereas TLR1/2 and 9 have no effect. Examination of monocyte migration in response to TLRs that are activated by bacterial components would contribute to understanding the excessive monocyte migration that characterizes the pathogenesis of bacterial infections and/or neuroinflammatory diseases.

Optimization of an in vitro human blood-brain barrier model: Application to blood monocyte transmigration assays.

The blood-brain barrier (BBB) is a selectively permeable barrier that separates the circulating blood from the extracellular fluid of the brain and is an essential component in brain homeostasis. In vitro BBB models are valuable supporting tools that can precede and complement animal and human studies of the development and progression of the central nervous system diseases. At present, mono-, co-, and tri-culture models that use porcine, murine, or human cells have been developed. We have optimized a two-dimensional model of the human BBB using primary human brain microvascular endothelial cells and normal human astrocytes. We have validated the effectiveness of our model with transmigration assays of human blood monocytes toward CCL19, a natural ligand of the chemokine receptor CCR7. This model offers the following advantages:•It is simple, convenient, and requires small quantities of material, reagents, and primary cells.•It can be used to monitor cell migration through the BBB.•It can be used to assess brain capillary permeability in the presence of xenobiotic, pro-inflammatory, or other substances.

CCR7 Receptor Expression in Mono-MAC-1 Cells: Modulation by Liver X Receptor α Activation and Prostaglandin E 2.

Cell migration via chemokine receptor CCR7 expression is an essential function of the immune system. We previously showed that prostaglandin E2 (PGE2), an important immunomodulatory molecule, increases CCR7 expression and function in monocytes. Here, we explore the role of the liver X receptor α (LXRα) activation on CCR7 expression in Mono-Mac-1 (MM-1) cells in the presence of PGE2. To do this, MM-1 cells were stimulated with the LXRα synthetic agonist T0901317 in the presence or absence of PGE2. CCR7 mRNA transcription was measured using quantitative RT-PCR and protein expression was examined using flow cytometry. CCR7 function was analyzed using migration assays in response to CCL19/CCL21, which are natural ligands for CCR7. Our results show that agonist-mediated activation of LXRα in the presence of PGE2 increases CCR7 mRNA transcription and MM-1 cell migratory capacity in response to CCL19/21. In addition, our results demonstrate that engagement of the E-prostanoids 2 and 4 (EP2/EP4) receptors present on MM-1 cells is responsible for the observed increase in CCR7 mRNA expression and function during LXRα activation. Examination of monocyte migration in response to lipid derivatives such as PGE2 and oxysterols that are produced at sites of chronic inflammation would contribute to understanding the excessive monocyte migration that characterizes atherosclerosis.

Prostaglandin E 2 Does Not Modulate CCR7 Expression and Functionality after Differentiation of Blood Monocytes into Macrophages.

Previously, we demonstrated that prostaglandin E2 (PGE2) induces C-C chemokine receptor type 7 (CCR7) expression on human monocytes, which stimulates their subsequent migration in response to the CCR7 natural ligands CCL19 and CCL21. In this study, we determined whether PGE2 affects CCR7 expression on macrophages. Flow cytometric analysis and chemotaxis assays were performed on Mono Mac-1-derived macrophage (MDMM-1) as well as unpolarized monocyte-derived macrophages (MDMs) to determine the CCR7 expression and functionality in the presence of PGE2. Data revealed that a MDMM-1 exhibited markedly downregulated CCR7 expression and functionality that were partially restored by treatment with PGE2. In MDMs, we observed a drastic downregulation of CCR7 expression and functionality that were unaffected following PGE2 treatment. Our data indicate that monocyte differentiation induces the loss of CCR7 expression and that PGE2 is unable to modulate CCR7 expression and functionality as shown previously in monocytes.

15-Deoxy-Δ(12,14)-prostaglandin J2 inhibits IL-13 production in T cells via an NF-κB-dependent mechanism.

Interleukin (IL)-13 is a cytokine produced by activated CD4(+) T cells that plays a critical role in promoting allergic responses and tumor cell growth. The 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a natural ligand for the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ), a known regulator of anti-inflammatory activities. We determined the effects of 15d-PGJ(2) on IL-13 expression in the Jurkat E6.1 T-cell line and in peripheral blood mononuclear cells. Semi-quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay revealed that treatment of activated T cells with 15d-PGJ(2) significantly decreased IL-13 mRNA transcription and secretion, respectively. This inhibition by 15d-PGJ(2) was independent of PPAR-γ since treatment with GW9662, an irreversible antagonist of the nuclear receptor, produced no effect. Our data also revealed the involvement of nuclear factor-κB in mediating 15d-PGJ(2)-dependent down regulation of IL-13 expression. Collectively, these results demonstrate the potential of 15d-PGJ(2) in attenuating expression and production of IL-13 in activated T cells.

CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes.

BACKGROUND: HIV latency is an obstacle for the eradication of HIV from infected individuals. Stable post-integration latency is controlled principally at the level of transcription. The HIV trans-activating protein, Tat, plays a key function in enhancing HIV transcriptional elongation. The HIV core promoter is specifically required for Tat-mediated trans-activation of HIV transcription. In addition, the HIV core promoter has been shown to be a potential anti-HIV drug target. Despite the pivotal role of the HIV core promoter in the control of HIV gene expression, the molecular mechanisms that couple Tat function specifically to the HIV core promoter remain unknown. RESULTS: Using electrophoretic mobility shift assays (EMSAs), the TATA box and adjacent sequences of HIV essential for Tat trans-activation were shown to form specific complexes with nuclear extracts from peripheral blood mononuclear cells, as well as from HeLa cells. These complexes, termed pre-initiation complexes of HIV (PICH), were distinct in composition and DNA binding specificity from those of prototypical eukaryotic TATA box regions such as Adenovirus major late promoter (AdMLP) or the hsp70 promoter. PICH contained basal transcription factors including TATA-binding protein and TFIIA. A mutational analysis revealed that CTGC motifs flanking the HIV TATA box are required for Tat trans-activation in living cells and correct PICH formation in vitro. The binding of known core promoter binding proteins AP-4 and USF-1 was found to be dispensable for Tat function. TAR RNA prevented stable binding of PICH-2, a complex that contains the general transcription factor TFIIA, to the HIV core promoter. The impact of TAR on PICH-2 specifically required its bulge sequence that is also known to interact with Tat. CONCLUSION: Our data reveal that CTGC DNA motifs flanking the HIV TATA box are required for correct formation of specific pre-initiation complexes in vitro and that these motifs are also required for Tat trans-activation in living cells. The impact of TAR RNA on PICH-2 stability provides a mechanistic link by which pre-initiation complex dynamics could be coupled to the formation of the nascent transcript by the elongating transcription complex. Together, these findings shed new light on the mechanisms by which the HIV core promoter specifically responds to Tat to activate HIV gene expression.

Involvement of the MAPK and RhoA/ROCK pathways in PGE2-mediated CCR7-dependent monocyte migration.

Prostaglandin E(2) (PGE(2)) induces the expression of C-C chemokine receptor type 7 (CCR7) on human monocytes, thereby enabling their subsequent migration in response to CCL19 and CCL21, the natural ligands for CCR7. To date, important mediators of PGE(2)-mediated monocyte migration remain unknown. In this study, we explored the role of mitogen-activated protein kinases and the RhoA/Rho-associated protein kinase (ROCK) pathway in CCR7-dependent monocyte migration in the presence of PGE(2). Our results indicate that CCL19 binding to CCR7 promotes the activation of p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase and leads to monocyte migration. Moreover, the RhoA/ROCK pathway was essential for PGE(2)-mediated CCR7-dependent monocyte migration.

High sodium butyrate levels induce MDR1 activation in colorectal cells: Impact of 15-deoxy-Δ(12,14)-prostaglandin J(2) on the resistance to saquinavir.

The bioavailability of HIV protease inhibitors is altered by P-glycoproteins (P-gp). The aim of this study was to elucidate the impact of sodium butyrate (NaBut), a unique product of the bacterial fermentation found in elevated concentrations in AIDS patients on P-gp expression. As prostaglandin production is upregulated under inflammatory conditions, we determined the role of 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) in the NaBut-induced P-gp functionality in colonic epithelial cells. Treatment with NaBut significantly increased MDR1 transcription and P-gp expression on the surface of both types of cells. Nevertheless, the addition of 15d-PGJ(2) to NaBut-stimulated cells significantly upregulated MDR1 mRNA expression and P-gp expression and functionality, leading to an important diminution of saquinavir accumulation by these cells. Our data provide evidence that both NaBut and prostaglandins may profoundly affect the intracellular accumulation of saquinavir in AIDS patients with compromised colonic walls.

CCR7-specific migration to CCL19 and CCL21 is induced by PGE(2) stimulation in human monocytes: Involvement of EP(2)/EP(4) receptors activation.

The recent demonstration that newly recruited monocytes do not die at the site of inflammation, but migrate to draining lymph nodes, raises the question on the mechanism involved in this process. In this study, we demonstrate for the first time that prostaglandin E(2) (PGE(2)) regulates the expression and the activity of CCR7 in human blood-isolated monocytes as well as in the MONO-MAC-1 cell lineage. PGE(2) induces intracellular cAMP formation through engagement of the E-prostanoid 2/E-prostanoid 4 (EP(2)/EP(4)) receptors present on monocytes. Migration to chemokines CCL19 and CCL21 in the PGE(2)-stimulated monocytes is mediated through the augmentation of cAMP concentration and furthermore, the cAMP/PKA pathway appears to act as the major inducer of CCR7 transcription in MONO-MAC-1. While p38 MAPK was induced by PGE(2), we observed that PGE(2) can downregulate p42/p44 MAPK phosphorylation. At the transcription level, inhibition of p38 MAPK inhibits CCR7 mRNA expression. Finally, we demonstrated that transcription factors CREB-1 and C/EBPalpha and C/EBPbeta are translocated to the nucleus following PGE(2) stimulation and bind the potent CCR7 promoter region. Our findings may have important implication for HIV-1 migration to the lymph nodes since macrophages and monocytes, particularly CD16 positive subset, are susceptible to HIV-1 infection.

High school intervention for influenza biology and epidemics/pandemics: impact on conceptual understanding among adolescents.

Understanding real-life issues such as influenza epidemiology may be of particular interest to the development of scientific knowledge and initiation of conceptual changes about viruses and their life cycles for high school students. The goal of this research project was to foster the development of adolescents' conceptual understanding of viruses and influenza biology. Thus, the project included two components: 1) pre- and posttests to determine students' conceptions about influenza biology, epidemics/pandemics, and vaccination; and 2) design an intervention that supports conceptual change to promote improvements in influenza knowledge based on these primary conceptions. Thirty-five female students from a high school biology class participated in a series of instructional activities and pre- and posttest assessments. Results from the pretest indicated that high school students exhibit a limited understanding of concepts related to viruses. Six weeks after an intervention that promoted active learning, results from a posttest showed that conceptions about influenza are more accurately related to the provided scientific knowledge. Although adolescents have nonscientific models to explain influenza biology, we showed that a carefully designed intervention can affect students' knowledge as well as influence the implementation of health education programs in secondary schools.

PGJ2 antagonizes NF-kappaB-induced HIV-1 LTR activation in colonic epithelial cells.

Intestinal epithelial cells play an important role in early stages of HIV-1 infection and long-term persistence of the virus. Here we determined the mechanism that regulates HIV-1 activation via prostaglandin J(2) (PGJ(2)) in Caco-2 cells. We showed that treatment of Caco-2 cells with PGJ(2) decreased the infectivity of a luciferase reporter virus, pHXB-luc, as well as HIV production following infection of cells with a X4-tropic virus by antagonizing sodium butyrate, a cellular activator known to induce HIV-1 transcription. Transfection of intestinal epithelial cells such as Caco-2, HT-29 and SW620 cells with full-length HIV-1 LTR (pLTR-luc) revealed that PGJ(2) reduced HIV-1 LTR-mediated reporter gene activity. The involvement of NF-kappaB in the PGJ(2)-dependent down-regulation of HIV-1 transcription was further assessed using the kappaB-regulated luciferase-encoding vectors. In Caco-2 cells, PGJ(2) decreased IKK activity, resulting in reduced NF-kappaB translocation to the nucleus. Since sodium butyrate has been associated with a chronic stress response in AIDS patients, our results suggest that addition of PGJ(2) in the environment of infected intestinal epithelial cells could reduce HIV-1 transcription.

Activation of HTLV-I gene transcription by protein tyrosine phosphatase inhibitors.

Human T-cell leukemia virus type I (HTLV-I) transcription generally depends on the ability of the viral Tax protein to bind the CREB transcription factor and form an active complex by recruiting CBP/p300 coactivators to the long terminal repeat (LTR). Studies have demonstrated that T-cell activating agents that stimulate CREB are potent inducers of HTLV-I transcription. Herein, we demonstrate that bpV[pic], a protein tyrosine phosphatase (PTP) inhibitor activates the HTLV-I LTR in the presence and absence of Tax expression. Optimal activation occurred at 8 h and was synergistic with forskolin or PGE(2). Infected cell lines and cells transfected with HTLV-I proviral DNA were equally responsive to the synergistic effect of bpV and forskolin on HTLV-I gene expression. Activation of the LTR by bpV[pic] was T-cell receptor-independent, but required ZAP70, calcineurin activity and functional calcium entry. Inhibition of the SHP-1 PTP was suggested to be important. Transfection experiments with a CREB dominant-negative mutant and with isolated TRE1- or CREB-responsive reporter constructs and treatment with the MDL-12,330A adenylate cyclase inhibitor all supported the involvement of a CREB/ATF family member in this bpV-dependent activation of the HTLV-I LTR, although CREB itself did not seem to be involved. Analysis of HTLV-I reporter constructs containing mutated CREB-binding sites also implied the involvement of another element in this activation. These results demonstrate for the first time a powerful effect of PTP inhibitors on HTLV-I LTR activity and suggest participation of both CREB-dependent and -independent pathways in this activation.

T-cell receptor/CD28 engagement when combined with prostaglandin E2 treatment leads to potent activation of human T-cell leukemia virus type 1.

Infection with human T-cell leukemia virus type 1 (HTLV-1) is characterized by long latency periods, indicating that viral gene expression is under tight control. There is presently little information available regarding the nature of extracellular stimuli that can transactivate the regulatory elements of HTLV-1 (i.e., long terminal repeat [LTR]). To gain insight into the biological importance of externally induced activation pathways in virus gene expression, primary and established T cells were transfected with HTLV-1-based reporter gene vectors and then were treated with agents that cross-linked the T-cell receptor (TCR) or the costimulatory CD28 molecule with prostaglandin E(2) (PGE(2)). We demonstrated that a potent induction of HTLV-1 LTR-driven reporter gene activity was seen only when the three agents were used in combination. Interestingly, similar observations were made when using C91/PL, a cell line that carries integrated HTLV-1 proviral DNA. This TCR-CD28-PGE(2)-mediated increase in virus transcription was dependent on protein kinase A activation and induction of the cAMP response element binding protein. Experiments with a mutated reporter construct further revealed the importance of the Tax-responsive elements in the HTLV-1 LTR in the observed up regulation of virus gene expression when TCR/CD28 engagement was combined with PGE(2) treatment. The protein tyrosine kinases p56(lck) and the transmembrane tyrosine phosphatase CD45 were all found to be involved in TCR-CD28-PGE(2)-directed increase in HTLV-1 LTR activity. This study presents new information on the possible mechanisms underlying reactivation of this retrovirus.

Mucosal immunization with inactivated human immunodeficiency virus plus CpG oligodeoxynucleotides induces genital immune responses and protection against intravaginal challenge.

Vaccines capable of protecting against sexually transmitted infections, such as human immunodeficiency virus (HIV), will depend on the induction of potent long-lasting mucosal immune responses in the genital tract. We evaluated vaginal and systemic immune responses and protection from vaginal challenge elicited after intranasal immunization of mice with inactivated glycoprotien 120-depleted HIV-1 immunogen alone or in combination with immunostimulatory CpG oligodeoxynucleotides (ODNs). Mice immunized with HIV-1 immunogen plus CpG ODN had significantly enhanced levels of anti-protein 24 immunoglobulin (Ig) G and IgA antibodies in serum and vaginal washes and increased production of beta-chemokines and interferon-gamma, compared with mice immunized with HIV-1 immunogen alone or with control ODN. Furthermore, mice intranasally immunized with HIV-1 immunogen plus CpG were protected against intravaginal challenge with a recombinant vaccinia virus expressing HIV-1 gag. These results indicate that mucosal immunization with whole-killed HIV-1 plus CpG ODN may be an effective means of inducing local immunity and protection against genital infection.

Prostaglandin E(2)-mediated activation of HIV-1 long terminal repeat transcription in human T cells necessitates CCAAT/enhancer binding protein (C/EBP) binding sites in addition to cooperative interactions between C/EBPbeta and cyclic adenosine 5'-monophosphate response element binding protein.

Previous work indicates that treatment of human T cells with PGE(2) results in an increase of HIV-1 long terminal repeat (LTR) transcriptional activity. The noticed PGE(2)-mediated activation of virus gene activity required the participation of specific intracellular second messengers such as calcium and two transcription factors, i.e., NF-kappaB and CREB. We report in this work that the nuclear transcription factor CCAAT/enhancer binding protein (C/EBP) is also important for PGE(2)-dependent up-regulation of HIV-1 LTR-driven gene activity. The implication of C/EBP was shown by using a trans-dominant negative inhibitor of C/EBP (i.e., liver-enriched transcriptional inhibitory protein) and several molecular constructs carrying site-directed mutations in the C/EBP binding sites located within the HIV-1 LTR. Mutated HIV-1 LTR constructs also revealed the involvement of the two most proximal C/EBP binding sites. Data from cotransfection experiments with vectors coding for dominant negative mutants and gel mobility shift assays indicated that PGE(2)-mediated induction of HIV-1 LTR activity results from a cooperative interaction between C/EBPbeta and CREB, two members of the basic leucine zipper family of transcription factors. Altogether these findings indicate that treatment of human T cells with PGE(2) induces HIV-1 LTR activity through a complex interplay between C/EBPbeta and CREB. Such a combinatorial regulation may represent a mechanism that permits a fine regulation of HIV-1 expression by PGE(2) in human T cells.