Isolation, characterization, and functional analysis of ferret lymphatic endothelial cells.

The lymphatic endothelium (LE) serves as a conduit for transport of immune cells and soluble antigens from peripheral tissues to draining lymph nodes (LNs), contributing to development of host immune responses and possibly dissemination of microbes. Lymphatic endothelial cells (LECs) are major constituents of the lymphatic endothelium. These specialized cells could play important roles in initiation of host innate immune responses through sensing of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including toll-like receptors (TLRs). LECs secrete pro-inflammatory cytokines and chemokines to create local inflammatory conditions for recruitment of naïve antigen presenting cells (APCs) such as dendritic cells (DCs) to sites of infection and/or vaccine administration. In this study, we examined the innate immune potential of primary LEC populations derived from multiple tissues of an animal model for human infectious diseases - the ferret. We generated a total of six primary LEC populations from lung, tracheal, and mesenteric LN tissues from three different ferrets. Standard RT-PCR characterization of these primary LECs showed that they varied in their expression of LEC markers. The ferret LECs were examined for their ability to respond to poly I:C (TLR3 and RIG-I ligand) and other known TLR ligands as measured by production of proinflammatory cytokine (IFNα, IL6, IL10, Mx1, and TNFα) and chemokine (CCL5, CCL20, and CXCL10) mRNAs using real time RT-PCR. Poly I:C exposure induced robust proinflammatory responses by all of the primary ferret LECs. Chemotaxis was performed to determine the functional activity of CCL20 produced by the primary lung LECs and showed that the LEC-derived CCL20 was abundant and functional. Taken together, our results continue to reveal the innate immune potential of primary LECs during pathogen-host interactions and expand our understanding of the roles LECs might play in health and disease in animal models.

Macaque paneth cells express lymphoid chemokine CXCL13 and other antimicrobial peptides not previously described as expressed in intestinal crypts.

CXCL13 is a constitutively expressed chemokine that controls migration of immune cells to lymphoid follicles. Previously, we found CXCL13 mRNA levels increased in rhesus macaque spleen tissues during AIDS. This led us to examine the levels and locations of CXCL13 by detailed in situ methods in cynomolgus macaque lymphoid and intestinal tissues. Our results revealed that there were distinct localization patterns of CXCL13 mRNA compared to protein in germinal centers. These patterns shifted during the course of simian immunodeficiency virus (SIV) infection, with increased mRNA expression within and around follicles during AIDS compared to uninfected or acutely infected animals. Unexpectedly, CXCL13 expression was also found in abundance in Paneth cells in crypts throughout the small intestine. Therefore, we expanded our analyses to include chemokines and antimicrobial peptides (AMPs) not previously demonstrated to be expressed by Paneth cells in intestinal tissues. We examined the expression patterns of multiple chemokines, including CCL25, as well as α-defensin 6 (DEFA6), ß-defensin 2 (BDEF2), rhesus θ-defensin 1 (RTD-1), and Reg3γ in situ in intestinal tissues. Of the 10 chemokines examined, CXCL13 was unique in its expression by Paneth cells. BDEF2, RTD-1, and Reg3γ were also expressed by Paneth cells. BDEF2 and RTD-1 previously have not been shown to be expressed by Paneth cells. These findings expand our understanding of mucosal immunology, innate antimicrobial defenses, homeostatic chemokine function, and host protective mechanisms against microbial translocation.

Simian immunodeficiency virus infection potently modulates chemokine networks and immune environments in hilar lymph nodes of cynomolgus macaques.

BACKGROUND: Chemokines provide critical immune cell homing and activation signals that if altered could affect the inflammatory milieu and cellular composition of lymphoid tissues. During HIV-1 and simian immunodeficiency virus (SIV)-infection, the virus triggers an increase in inflammation or activation, leading to immunodeficiency and development of opportunistic infections, such as in the lungs-a massive interface between the host and the environment. METHODS: Chemokine, cytokine, and chemokine receptor expression profiles were determined using real-time reverse transcriptase-polymerase chain reaction and in situ hybridization in hilar lymph nodes (HiLNs) from cynomolgus macaques at different stages after infection with SIV/DeltaB670. Immunostaining of tissue sections and flow cytometric analysis of cryopreserved cells were used to examine cellular compositions of lymph nodes. RESULTS: Interferon-gamma, type 1 chemokine, and cognate chemokine receptor mRNAs were upregulated, whereas type 2 and homeostatic chemokine and chemokine receptor mRNAs were down-regulated in HiLNs after SIV infection. Local SIV and interferon-gamma levels were positively correlated with type 1 chemokine levels but negatively correlated with type 2 and homeostatic chemokine levels. Using in situ hybridization, Pneumocystis carinii rRNA was detected in lung-draining lymph nodes from animals with P. carinii pneumonia. Changes in the cellular composition of HiLNs included decreased proportions of CD4 cells and dendritic cells and increased proportions of CD8, CXCR3, and CCR5 cells. CONCLUSIONS: SIV infection of cynomolgus macaques dramatically alters the cellular homing signals of lung-draining lymph nodes, which correlated with changes in the immune cellular composition. These changes could contribute to the loss of immune function that defines AIDS.

Simian immunodeficiency virus infection alters chemokine networks in lung tissues of cynomolgus macaques: association with Pneumocystis carinii infection.

Infection by HIV-1 frequently leads to pulmonary complications, including alterations to local immune environments. To better understand these alterations, we have examined in detail the patterns and levels of expression of chemokine, cytokine, and chemokine receptor mRNAs in lung tissues from 16 uninfected or simian immunodeficiency virus (SIV)/DeltaB670 infected cynomolgus macaques at different stages of infection. Among the most up-regulated immune genes were interferon (IFN)-gamma, IFN-gamma-inducible CXCR3 ligands, and CCR5 ligands, as well as the cognate chemokine receptors. These changes were greatest in animals with clear Pneumocystis carinii coinfection. Immunohistochemistry and in situ hybridization revealed monocytes/macrophages to be the predominant type of cell infiltrating into lung tissues and serving as the major cellular source of chemokines. To explore the causes of chemokine alterations, we treated macaque lung cells with IFN-gamma, lipopolysaccharide, Poly(I:C), and P. carinii in vitro, and results revealed that these stimuli can induce the expression of CXCR3 ligand and/or CCR5 ligand mRNAs. Taken together, these studies provide a comprehensive definition of the chemokine networks available to modulate cellular recruitment to lung tissues during SIV infection and implicate both cytokines (IFN-gamma) and pathogens (SIV and P. carinii) as contributors to increased expression of pro-inflammatory chemokines.

Boron-Doped Diamond Microelectrodes Reveal Reduced Serotonin Uptake Rates in Lymphocytes from Adult Rhesus Monkeys Carrying the Short Allele of the 5-HTTLPR.

Uptake resolved by high-speed chronoamperometry on a second-by-second basis has revealed important differences in brain serotonin transporter function associated with genetic variability. Here, we use chronoamperometry to investigate variations in serotonin transport in primary lymphocytes associated with the rhesus serotonin transporter gene-linked polymorphism (rh5-HTTLPR), a promoter polymorphism whose orthologs occur only in higher order primates including humans. Serotonin clearance by lymphocytes is Na(+)-dependent and inhibited by the serotonin-selective reuptake inhibitor paroxetine (Paxil®), indicative of active uptake by serotonin transporters. Moreover, reductions in serotonin uptake rates are evident in lymphocytes from monkeys with one or two copies of the short 's' allele of the rh5-HTTLPR (s/s

Chemokine and cytokine mediated loss of regulatory T cells in lymph nodes during pathogenic simian immunodeficiency virus infection.

Regulatory T cells (T(reg)) play key roles in immune regulation through multiple modes of suppression. The effects of HIV-1 infection on T(reg) levels in lymphoid tissues remain incompletely understood. To explore this issue, we have measured the levels of forkhead box protein 3 (FOXP3)-positive cells and associated immunomodulatory genes in a pathogenic simian immunodeficiency virus/macaque model and found that a loss of T(reg) in lymph nodes occurred following simian immunodeficiency virus infection. Changes in expression of the ligands for CXCR3, CCR4, and CCR7 and the cytokines TGF-beta and IL-2 were all linked to this loss of T(reg), which in turn was linked with increased levels of cellular activation. Our findings identify three mechanisms that likely contribute to SIV-driven loss of T(reg), including reduced levels of cytokines associated with T(reg) differentiation and altered expression of agonist and antagonist chemokines. The loss of T(reg) and the associated cellular activation in lymphoid tissues is consistent with the events in HIV-1-infected individuals and suggest that components of the T(reg) differentiation and trafficking network could be targets for therapeutic intervention.

Association between decreased CXCL12 and CCL25 expression and increased apoptosis in lymphoid tissues of cynomolgus macaques during SIV infection.

BACKGROUND: Chemokines likely play multiple roles in HIV-1 and SIV pathogenesis. To examine potential associations between chemokine expression levels and apoptosis of cells in lymphoid tissues during SIV infection, we measured chemokine and cytokine mRNA levels in multiple lymphoid tissues compartments from uninfected and SIV-infected cynomolgus macaques (Macaca fascicularis). METHODS: Real-time RT-PCR was used to measure host mRNA levels in macaque lymphoid tissues. Proliferating or apoptotic cells were identified in lymphoid tissues by immunohistochemistry. RESULTS: We found that CXCL12 and CCL25 mRNAs in SIV-infected lymphoid tissues were decreased and their levels were negatively correlated with the numbers of proliferating and apoptotic cells. In vitro analyses revealed that CXCL12 and CCL25 were capable of reducing apoptosis induced by SIV infection. CONCLUSIONS: These findings suggest that increased apoptosis in lymphoid tissues due to reduced levels of anti-apoptotic chemokines might be a mechanism that contributes to loss of immune function following pathogenic SIV infection.

Virologic and immunologic events in hilar lymph nodes during simian immunodeficiency virus infection: development of polarized inflammation.

Lymphoid tissues are sites of soluble and cell-associated antigen sampling of peripheral tissues, and they are key compartments for the generation of cellular and humoral immune responses. Hilar lymph nodes (HiLNs), which drain the lungs, were examined to understand the effects of simian immunodeficiency virus (SIV) infection on this compartment of the immune system. Histologic and messenger RNA (mRNA) expression profiling approaches were used to determine the numbers, types, and distributions of SIV viral RNA cells and to identify differentially expressed genes in HiLNs during SIV infection. SIV RNA cells were found to be primarily CD68 and localized to paracortical and medullary regions early in infection, whereas they resided mainly in paracortex during AIDS. As SIV infection progressed, CXCL9, CXCL10, interferon-gamma, and Toll-like receptor 3 levels all increased. In contrast, CCL19 increased early in infection but decreased during AIDS, whereas CCL21 decreased progressively throughout infection. Finally, local levels of cellular activation were increased throughout infection. Taken together, these findings indicate that SIV infection leads to an inflammatory environment in lung-draining lymph nodes that is characterized by type 1 cytokines and chemokines and likely has an impact on the nature and strength of immune responses to pulmonary pathogens.

Productive infection of dendritic cells by simian immunodeficiency virus in macaque intestinal tissues.

Dendritic cells (DCs) are potent antigen-presenting cells that likely play multiple roles in human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) pathogenesis. This paper describes the effects of pathogenic SIV infection on the networks of DCs in rhesus macaque (Macaca mulatta) intestinal tissues. Intestinal tissues were obtained from macaques at different stages of disease following infection with the pathogenic SIV/DeltaB670 isolate. The patterns and levels of expression of SIV and DC-associated mRNAs were examined and quantitated directly in intestinal tissue sections. In situ hybridization was performed for SIV, DC-specific ICAM3-grabbing non-integrin (DC-SIGN), DC-specific lysosome-associated membrane glycoprotein (DC-LAMP), DC-specific C-type lectin 1 (DECTIN-1), CC chemokine receptor 6 (CCR6), CCR7, and macrophage inflammatory protein 3alpha (MIP-3alpha/CCL20) mRNAs and quantitative image analysis was performed to measure mRNA expression levels. To identify the cell types productively infected by SIV, simultaneous in situ hybridization and immunohistochemical staining were performed. The DC networks in macaque intestinal tissues were found to be extensive and although they generally remained intact during the course of SIV infection, there were alterations in the expression of markers for immature DCs. One alteration was an increase in the expression in intestinal submucosa of DC-SIGN, a molecule that binds to HIV-1/SIV and increases its infectivity. Concomitant with this increase, it was found that during AIDS, the population of productively infected cells included DCs, based on co-expression of DC-SIGN and DECTIN-1 mRNAs. These data indicate that SIV infection affects subpopulations of macaque intestinal DCs, including productive infection of DC-SIGN+ DCs, the consequences of which are likely to be ongoing viral propagation and decreased immunostimulatory function.

Simian immunodeficiency virus dramatically alters expression of homeostatic chemokines and dendritic cell markers during infection in vivo.

Dendritic cells (DCs) are potent antigen-presenting cells that likely play multiple roles in human immunodeficiency virus type 1 (HIV-1) pathogenesis. We used the simian immunodeficiency virus (SIV)/macaque model to study the effects of infection on homeostatic chemokine expression and DC localization directly in secondary lymphoid tissues. SIV infection altered the expression of chemokines (CCL19/MIP-3beta, CCL21/ 6Ckine, and CCL20/MIP-3alpha) and of chemokine receptors (CCR7 and CCR6) that drive DC trafficking. CCL19/MIP-3beta, CCL20/MIP-3alpha, CCR6, and CCR7 expression increased in lymph nodes during the early systemic burst of viral replication (acute infection), whereas CCL21/6Ckine expression progressively decreased throughout disease to AIDS. Parallel with the SIV-induced perturbations in chemokine expression were changes in the expression of the DC-associated markers, DC-SIGN, DC-LAMP, and DECTIN-1. During AIDS, DC-LAMP mRNA expression levels were significantly reduced in lymph nodes and spleen, and DC-SIGN levels were significantly reduced in spleen. These findings suggest that the disruption of homeostatic chemokine expression is responsible, in part, for alterations in the networks of antigen-presenting cells in lymphoid tissues, ultimately contributing to systemic immunodeficiency.