Souvenir D. Tachado

Negative regulatory role of mannose receptors on human alveolar macrophage proinflammatory cytokine release in vitro.

Alveolar macrophages (AM) are critical components of lung innate immunity and contribute to an effective host response to Pneumocystis pneumonia. Recognition of unopsonized Pneumocystis organisms by human AM is mediated predominantly via mannose receptors and results in phagocytosis, release of reactive oxygen species, and activation of the nuclear transcription factor (NF)‐κB. However, the AM host defense genes activated by Pneumocystis have not been defined. In the present study, incubation of AM with unopsonized Pneumocystis organisms was not associated with release of interleukin (IL)‐1β, IL‐6, or tumor necrosis factor (TNF)‐α (important cytokines in the host response to Pneumocystis) and did not induce IL‐1β, IL‐6, or TNF‐α mRNA transcripts. These findings were not attributed to Pneumocystis‐induced cytopathic changes, as these same AM released IL‐8 and matrix metalloproteinase‐9 in response to Pneumocystis. NF‐κB‐mediated IL‐8 release was independent of Pneumocystis phagocytosis. The observed response was specific, as IL‐1β, IL‐6, and TNF‐α release and mRNA induction were preserved in response to lipopolysaccharide or serum‐opsonized Pneumocystis. The absence of IL‐1β, IL‐6, and TNF‐α release in response to Pneumocystis was predominately influenced by AM mannose receptors, as blocking mannose receptors or targeted mannose receptor small interfering RNA functional gene silencing resulted in TNF‐α release in response to unopsonized Pneumocystis organisms. Furthermore, ligation of AM mannose receptors by unopsonized Pneumocystis organisms reduced Toll‐like receptor 4‐mediated TNF‐α release. Taken together, these data suggest that mannose receptors on human AM may suppress select proinflammatory cytokine release and may serve to regulate the innate inflammatory responses to infectious challenge in the lungs.

HIV Impairs TNF-α Mediated Macrophage Apoptotic Response to Mycobacterium tuberculosis

The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MTb) disease in HIV+ persons are poorly understood. Macrophage apoptosis represents a critical innate host cell response to control MTb infection and limit disease. In the current study, virulent live or irradiated MTb (iMTbRv) induced apoptosis of differentiated human U937 macrophages in vitro, in part dependent on TNF-α. In contrast, apoptosis of differentiated HIV+ human U1 macrophages (HIV+ U937 subclone) was markedly reduced in response to iMTbRv and associated with significantly reduced TNF-α release, whereas apoptosis and TNF-α release were intact to TLR-independent stimuli. Furthermore, reduced macrophage apoptosis and TNF-α release were independent of MTb phagocytosis. Whereas surface expression of macrophage TLR2 and TLR4 was preserved, IL-1 receptor associated kinase-1 phosphorylation and NF-κB nuclear translocation were reduced in HIV+ U1 macrophages in response to iMTbRv. These findings were confirmed using clinically relevant human alveolar macrophages (AM) from healthy persons and asymptomatic HIV+ persons at clinical risk for MTb infection. Furthermore, in vitro HIV infection of AM from healthy persons reduced both TNF-α release and AM apoptosis in response to iMTbRv. These data identify an intrinsic specific defect in a critical macrophage cellular response to MTb that may contribute to disease pathogenesis in HIV+ persons.

Pneumocystis-mediated IL-8 release by macrophages requires coexpression of mannose receptors and TLR2.

Interaction with the unique fungus Pneumocystis (Pc) promotes IL‐8 release by human alveolar macrophages (AM), although the receptor(s) mediating IL‐8 release have not been identified. TLR2 recognizes fungal components and mediates release of host defense cytokines and chemokines, although whether TLR2 mediates signaling in response to Pc is not known. In the current study, Pc induced IL‐8 release by human AM, and AM pretreatment with anti‐TLR2 neutralizing antibody reduced IL‐8 release. However, in nonphagocytic human embryonic kidney (HEK)293 cells transfected with human TLR2 cDNA, incubation with Pc did not induce IL‐8 release, whereas these same cells released IL‐8 in response to the TLR2 agonist lipoteichoic acid. Targeted gene silencing of AM mannose receptors (MR; phagocytic receptors for Pc) using small interfering RNA also reduced Pc‐mediated IL‐8 release in human AM. However, HEK293 cells transfected with human MR cDNA alone did not release IL‐8 in response to Pc. In contrast, HEK293 cells cotransfected with human TLR2 and human MR cDNA released IL‐8 in response to Pc. In human AM, Pc promoted direct interaction of MR and TLR2, IL‐8 release was reduced markedly upon simultaneous blocking of TLR2 and gene silencing of MR, and IL‐8 release was dependent in part on transcription factor NF‐κB and ERK1/2 and JNK MAPKs. These studies demonstrate that Pc‐mediated IL‐8 release by human AM requires the coexpression of MR and TLR2 and further supports the concept that combinatorial interactions of macrophage innate receptors provide specificity of host defense cell responses to infectious challenge.

Cannabinoids Inhibit HIV-1 Gp120-Mediated Insults in Brain Microvascular Endothelial Cells

HIV-1 infection has significant effect on the immune system as well as on the nervous system. Breakdown of the blood-brain barrier (BBB) is frequently observed in patients with HIV-associated dementia (HAD) despite lack of productive infection of human brain microvascular endothelial cells (HBMEC). Cellular products and viral proteins secreted by HIV-1 infected cells, such as the HIV-1 Gp120 envelope glycoprotein, play important roles in BBB impairment and HIV-associated dementia development. HBMEC are a major component of the BBB. Using cocultures of HBMEC and human astrocytes as a model system for human BBB as well as in vivo model, we show for the first time that cannabinoid agonists inhibited HIV-1 Gp120-induced calcium influx mediated by substance P and significantly decreased the permeability of HBMEC as well as prevented tight junction protein down-regulation of ZO-1, claudin-5, and JAM-1 in HBMEC. Furthermore, cannabinoid agonists inhibited the transmigration of human monocytes across the BBB and blocked the BBB permeability in vivo. These results demonstrate that cannabinoid agonists are able to restore the integrity of HBMEC and the BBB following insults by HIV-1 Gp120. These studies may lead to better strategies for treatment modalities targeted to the BBB following HIV-1 infection of the brain based on cannabinoid pharmacotherapies.

Dependence of regulatory volume decrease on transient receptor potential vanilloid 4 (TRPV4) expression in human corneal epithelial cells

TRPV4 is a non-selective cation channel with moderate calcium permeability, which is activated by exposure to hypotonicity. Such a stress induces regulatory volume decrease (RVD) behavior in human corneal epithelial cells (HCEC). We hypothesize that TRPV4 channel mediates RVD in HCEC. Immunohistochemistry revealed centrally and superficially concentrated TRPV4 localization in the corneal tissue. Immunocytochemical and fluorescence activated cell sorter (FACS) analyses identified TRPV4 membrane surface and cytosolic expression. RT-PCR and Western blot analyses identified TRPV4 gene and protein expression in HCEC, respectively. In addition, 4alpha-PDD or a 50% hypotonic medium induced up to threefold transient intracellular Ca2+ ([Ca2+]i) increases. Following TRPV4 siRNA HCEC transfection, its protein expression level declined by 64%, which abrogated these [Ca2+]i transients. Similarly, exposure to either ruthenium red or Ca(2+)-free Ringers solution also eliminated this response. In these transfected cells, RVD declined by 51% whereas in the non-transfected counterpart, ruthenium red and Ca(2+)-free solution inhibited RVD by 54 and 64%, respectively. In contrast, capsazepine, a TRPV1 antagonist, failed to suppress [Ca2+]i transients and RVD. TRPV4 activation contributes to RVD since declines in TRPV4 expression and activity are associated with suppression of this response. In conclusion, there is TRPV4 functional expression in HCEC.

Specificity in signal transduction among glycosylphosphatidylinositols of Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp.*

Glycosylphosphatidylinositols (GPIs) and related glycoconjugates of parasite origin have been shown to regulate both the innate and acquired immune systems of the host. This is achieved through the activation of novel GPI‐dependent signalling pathways in macrophages, lymphocytes and other cell types. Parasite GPIs impart at least two distinct signals to host cells through the structurally distinct inositolphosphoglycan (IPG) and fatty acid domains. Binding of IPG to as yet uncharacterized cell surface receptor(s) leads to activation of src‐family protein tyrosine kinases: depending upon structure, GPI‐derived fatty acids can either activate or antagonize protein kinase C, and may enter the sphingo‐myelinase pathway. The degree of fatty acid saturation may also contribute to signalling activity. Thus, variation in structure of parasite GPIs imparts different properties of signal transduction upon this class of glycolipid. The divergent activities of GPIs from various protozoal taxa reflect global aspects of the host/parasite relationship, suggesting that GPI signalling is a central determinant of disease in malaria, leishmaniasis and both American and African trypanosomiases.

REGULATION OF HOST CELL FUNCTION BY GLYCOSYLPHOSPHATIDYLINOSITOLS OF THE PARASITIC PROTOZOA

Antigenic variation, antigenic drift, molecular mimicry, intracellular localization and sequestration in privileged sites are important mechanisms of immune evasion by infectious organisms. Added to this however is the phenomenon by which pathogens deliberately regulate host cell function by the production of glycolipids with agonistic or antagonistic signal transduction capacity. Such pro‐active glycolipids are often pathogenicity factors, but they also serve as immunomodulators and immunosuppressants, and these activities may serve as mechanisms of immune evasion. Here we review glycosylphosphatidylinositols and related structures, a novel class of glycolipid common to eukaryotic parasites and their hosts, which recent studies suggest may play a role in immune evasion and immunosuppression by regulating host cell function via the activation or suppression of endogenous host signalling pathways.

Impaired M. tuberculosis-mediated apoptosis in alveolar macrophages from HIV+ persons: potential role of IL-10 and BCL-3

The mechanism of increased MTb disease susceptibility in HIV+ persons remains poorly understood. Apoptosis of macrophages in response to MTb represents a critical host defense response, and decreased apoptosis may represent a mechanism of increased susceptibility to MTb in HIV. In the current study, MTb‐mediated apoptosis of human AM was reduced in HIV+ subjects compared with healthy subjects in a TNF‐α‐dependent manner. IL‐10 levels in BALF from HIV+ persons were significantly elevated compared with HIV– persons, and exogenous IL‐10 reduced MTb‐mediated apoptosis in healthy AM, suggesting that IL‐10 could mediate decreased apoptosis observed in HIV. Further study showed that IL‐10 reduced TNF release in response to MTb in AM through a reduction in TNF mRNA levels, and exogenous TNF could partially reverse IL‐10‐associated effects on AM apoptosis. IL‐10 did not influence p‐IRAK, IκB degradation, or NF‐κB p65 nuclear translocation in response to MTb, but IL‐10 did increase levels of AM BCL‐3, an inhibitor of NF‐κB nuclear activity. BCL‐3 knockdown in human macrophages increased MTb‐mediated TNF release. Importantly, BCL‐3 levels in AM from HIV+ subjects were higher compared with healthy subjects. Taken together, these data suggest that elevated lung levels of IL‐10 may impair MTb‐mediated AM apoptosis in HIV through a BCL‐3‐dependent mechanism. BCL‐3 may represent a potential therapeutic target to treat or prevent MTb disease in HIV+ persons.

Regulation of tumor invasion and metastasis in protein kinase C epsilon‐transformed NIH3T3 fibroblasts

Protein kinase C epsilon is an oncogenic, actin nucleating protein that coordinately regulates changes in cell growth and shape. Cells constitutively expressing PKCϵ spontaneously acquire a polarized morphology and extend long cellular membrane protrusions. Here we report that the regulatory C1 domain of PKCϵ contains an actin binding site that is essential for the formation of elongate invadopodial‐like structures, increased pericellular metalloproteinase activity, in vitro invasion of a Matrigel barrier, and the invasion and metastasis of tumors grown in vivo by PKCϵ‐transformed NIH3T3 fibroblasts in nude mice. While removing this small actin binding motif caused a dramatic reversion of tumor invasion, the deletion mutant of PKCϵ remained oncogenic and tumorigenic in this experimental system. We propose that PKCϵ directly interacts with actin to stimulate polymerization and the extension of membrane protrusions that transformed NIH3T3 cells use in vivo to penetrate and degrade surrounding tissue boundaries. J. Cell. Biochem. 85: 785–797, 2002.

Constitutive Activation of Phosphatidylinositol 3-Kinase Signaling Pathway Down-regulates TLR4-mediated Tumor Necrosis Factor-α Release in Alveolar Macrophages from Asymptomatic HIV-positive Persons in Vitro

Alveolar macrophages represent critical effector cells of innate immunity to infectious challenge in the lungs and recognize bacterial pathogens through pattern recognition receptors such as Toll-like receptors (TLRs). Phosphatidylinositol 3-kinse (PI3K) regulates TLR-mediated cytokine release, but whether HIV infection influences PI3K signaling pathway and alters TLR4-mediated macrophage response has not been investigated. In the current study, surface TLR4 expression were similar but TLR4 activation (lipid A, 10 μg/ml) resulted in lower TNF-α release by HIV+ human macrophages compared with healthy cells. Pharmacological inhibition of PI3K (LY294002) normalized TNF-α release in HIV+ macrophages and augments ERK1/2 mitogen-activated protein kinase phosphorylation in response to lipid A. Importantly, HIV+ macrophages demonstrated increased constitutive phosphatidylinositol 3,4,5-trisphosphate formation, increased phosphorylation of downstream signaling molecules Akt and glycogen synthase kinase-3β (GSK-3β) at Ser9, and reduced PTEN protein expression. As a functional assessment of GSK-3β phosphorylation, TLR4-mediated interleukin-10 release was significantly higher in HIV+ human macrophages compared with healthy cells. Incubation of human macrophages with exogenous HIV Nef protein induced phosphorylation of Akt and GSK-3β (whereas phosphorylation was reduced by PI3K inhibition) and promoted interleukin-10 release. Taken together, these data demonstrate increased constitutive activation of the PI3K signaling pathway in HIV+ macrophages and support the concept that PI3K activation (by HIV proteins such as Nef) may contribute to reduced TLR4-mediated TNF-α release in HIV+ human macrophages and impair host cell response to infectious challenge.