Epithelia from many tissues express protease-activated receptors (PARs) that play a major role in several different physiological processes. In this study, we examined their capacity to modulate IL-6, IL-8, and PGE2 production in both the A459 and BEAS-2B cell lines and primary human bronchial epithelial cells (HBECs). All three cell types expressed PAR-1, PAR-2, PAR-3, and PAR-4, as judged by RT-PCR and immunocytochemistry. Agonist peptides corresponding to the nascent N termini of PAR-1, PAR-2, and PAR-4 induced the release of cytokines from A549, BEAS-2B, and HBECs with a rank order of potency of PAR-2 > PAR-4 > PAR-1 at 400 μM. PAR-1, PAR-2, and PAR-4 also caused the release of PGE2 from A549 and HBECs. The PAR-3 agonist peptide was inactive in all systems tested. PAR-1, PAR-2, or PAR-4, in combination, caused additive IL-6 release, but only the PAR-1 and PAR-2 combination resulted in an additive IL-8 response. PAR peptide-induced responses were accompanied by changes in intracellular calcium ion concentrations. However, Ca2+ ion shutoff was ∼2-fold slower with PAR-4 than with PAR-1 or PAR-2, suggesting differential G protein coupling. Combined, these data suggest an important role for PAR in the modulation of inflammation in the lung.

BackgroundDespite the seriousness of dengue-related disease, with an estimated 50–100 million cases of dengue fever and 250,000–500,000 cases of dengue hemorrhagic fever/dengue shock syndrome each year, a clear understanding of dengue pathogenesis remains elusive. Because of the lack of a disease model in animals and the complex immune interaction in dengue infection, the study of host response and immunopathogenesis is difficult. The development of genomics technology, microarray and high throughput quantitative PCR have allowed researchers to study gene expression changes on a much broader scale. We therefore used this approach to investigate the host response in dengue virus-infected cell lines and in patients developing dengue fever.Methodology/Principal FindingsUsing microarray and high throughput quantitative PCR method to monitor the host response to dengue viral replication in cell line infection models and in dengue patient blood samples, we identified differentially expressed genes along three major pathways; NF-κB initiated immune responses, type I interferon (IFN) and the ubiquitin proteasome pathway. Among the most highly upregulated genes were the chemokines IP-10 and I-TAC, both ligands of the CXCR3 receptor. Increased expression of IP-10 and I-TAC in the peripheral blood of ten patients at the early onset of fever was confirmed by ELISA. A highly upregulated gene in the IFN pathway, viperin, was overexpressed in A549 cells resulting in a significant reduction in viral replication. The upregulation of genes in the ubiquitin-proteasome pathway prompted the testing of proteasome inhibitors MG-132 and ALLN, both of which reduced viral replication.Conclusion/SignificanceUnbiased gene expression analysis has identified new host genes associated with dengue infection, which we have validated in functional studies. We showed that some parts of the host response can be used as potential biomarkers for the disease while others can be used to control dengue viral replication, thus representing viable targets for drug therapy.

Protective immunity in tuberculosis is dependent on the coordinated release of cytolytic effector molecules from effector T cells and the subsequent granule-associated killing of infected target cells. In this study, we investigated the expression of cytolytic (perforin and granzyme A) and antimicrobial (granulysin) molecules at the single-cell level in cryopreserved lung tissue from patients with chronic, progressive tuberculosis disease. Quantification of protein-expressing cells was performed by in situ imaging, while mRNA levels in the infected tissue were analyzed by real-time PCR. Persistent inflammation, including excessive expression of inducible nitric oxide synthase in CD68؉ macrophages and significant infiltration of CD3؉ , CD8 ؉ and CD4 ؉ T cells, was evident in tuberculosis lesions in all patients. However, despite the accumulation of CD3 ؉ T cells, perforin-and granulysin-expressing CD3 ؉ T cells were detected at two-to threefold-lower ratios in the tuberculosis lesions than in distal lung parenchyma and uninfected control lungs, respectively. This was evident at both the protein and mRNA levels. Moreover, perforin-and granulysin-expressing CD8 ؉ T cells were scarce in individual granulomas within the tuberculosis lesions. In contrast, significant up-regulation of granzyme A-expressing CD3 ؉ T cells was evident in the lesions from all patients. Confocal microscopy revealed coexpression of perforin and granulysin, primarily in CD8 ؉ T cells; however, this expression was lower in the tuberculosis lesions. These findings suggest that symptomatic, chronic tuberculosis disease is associated with insufficient up-regulation of perforin and granulysin coexpression in CD8؉ T cells at the local site of infection.

Dengue infections are a major cause of morbidity and mortality in the tropical and sub-tropical regions of the world. There is no vaccine for dengue and also there are no anti-viral drugs to treat the infection. Some patients, typically those experiencing a secondary infection with a different dengue serotype, may progress from an acute febrile disease to the more severe forms of disease, dengue haemorrhagic fever and dengue shock syndrome. Here we discuss the significant immunopathological component to severe disease and how T cells, cytokines and cross-reactive antibody combine to contribute to the progression to dengue haemorrhagic fever. These events are thought to lead to vascular leakage, the signature event in dengue haemorrhagic fever, and are addressed in this review by incorporating the concept of heterologous T cell immunity. The need for effective measures against dengue and dengue-related illness is clear. We propose that drugs against dengue virus, or the symptoms of severe dengue disease, are a viable goal.

Immune responses were assessed at the single-cell level in lymph nodes from children with tuberculous lymphadenitis. Tuberculosis infection was associated with tissue remodeling of lymph nodes as well as altered cellular composition. Granulomas were significantly enriched with CD68+ macrophages expressing the M. tuberculosis complex-specific protein antigen MPT64 and inducible nitric oxide synthase. There was a significant increase in CD8+ cytolytic T cells surrounding the granuloma; however, CD8+ T cells expressed low levels of the cytolytic and antimicrobial effector molecules perforin and granulysin in the granulomatous lesions. Quantitative real-time mRNA analysis revealed that interferon-gamma, tumor necrosis factor-alpha, and interleukin-17 were not up-regulated in infected lymph nodes, but there was a significant induction of both transforming growth factor-beta and interleukin-13. In addition, granulomas contained an increased number of CD4+FoxP3+ T cells co-expressing the immunoregulatory cytotoxic T-lymphocyte antigen-4 and glucocorticoid-induced tumor necrosis factor receptor molecules. Low numbers of CD8+ T cells in the lesions correlated with high levels of transforming growth factor-beta and FoxP3+ regulatory T cells, suggesting active immunosuppression at the local infection site. Compartmentalization and skewing of the immune response toward a regulatory phenotype may result in an uncoordinated effector T-cell response that reduces granule-mediated killing of M. tuberculosis-infected cells and subsequent disease control.

Burkholderia cepacia causes pulmonary infection with high mortality in cystic fibrosis (CF) patients which is likely to involve interaction with respiratory epithelium. In this study the pro-inflammatory properties of B. cepacia were examined using a range of respiratory epithelial cell lines. B. cepacia and cell-free culture supernatants were used to stimulate cell lines with (SigmaCFTE29o- and IB3) and without (A549) the CF transmembrane conductance regulator mutation (CFTR), together with corrected cell lines (C38 and S9). Interleukin (IL)-6 and IL-8, but not GM-CSF or IL-1beta, were released from all the cell lines whereas PGE(2) (prostaglandin E(2)) was released from the A549, IB3 and S9 cell lines only. Nuclear factor (NF)-kappaB activation preceded cytokine release and suppression of NF-kappaB activity diminished cytokine release. These studies indicated that B. cepacia secretory products are potent pro-inflammatory agents for respiratory epithelium and suggest functional CFTR is not required for cytokine or prostanoid responses.

The outer membrane proteins of Moraxella catarrhalis, a bacterial pathogen which causes disease in both children and adults, play an important role in its phenotypic properties. However, their proinflammatory potential with regard to respiratory epithelium and macrophages is unclear. To this end, we examined the cytokine- and mediator-inducing capacity of a heat-killed wild-type M. catarrhalis strain and a nonautoagglutinating mutant as well as their outer membrane proteins and secretory/excretory products using the A549 respiratory epithelial cell line. The outer membrane proteins and secretory/excretory products from both isolates as well as the heat-killed bacteria all induced interleukin (IL)-6, IL-8 and prostaglandin E2, but not IL-1beta, from the A549 cell line in a dose- and time-dependent manner. Heat-killed bacteria and secretory/excretory products stimulated the release of IL-1beta, IL-6, IL-8 and prostaglandin E2 from human monocyte-derived macrophages. Both heat-killed isolates also stimulated nuclear translocation and transactivation of nuclear factor-kappaB. The heat-killed wild-type autoagglutinating isolate induced significantly greater amounts of IL-6 and IL-8 from A549 cells than the nonautoagglutinating mutant compared with the monocyte-derived macrophages but no significant differences in the amounts induced by the two strains were observed. These differences were also evident when the respiratory cell line was stimulated with outer membrane proteins as well as in the degree of nuclear factor-kappaB transactivation. There was little difference in the stimulatory activity of the secretory/excretory products. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses revealed some differences in the outer membrane proteins and secretory excretory products between the two isolates. Combined, these data show that M. catarrhalis secretory excretory products and outer membrane proteins are associated with the induction of inflammatory responses in both respiratory epithelium and macrophages.

Dendritic cells (DCs) are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4+ T cells (ApoInf) or apoptotic uninfected activated CD4+ T cells (ApoAct) induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4+ T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1α, MIP-1β, MCP-1, and TNF-α; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-α using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4+ T cells (ApoRest). Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection.