Shao Hung Wang

Toll-Like Receptor 2 Mediates Alveolar Macrophage Response to Pneumocystis murina

ABSTRACT The innate immune response to Pneumocystis infection is not well understood. In this study, normal C57BL/6 mouse alveolar macrophages were found to respond to Pneumocystis murina organisms through Toll-like receptor 2 (TLR2), leading to the nuclear translocation of NF-κB and the production of proinflammatory cytokine tumor necrosis factor alpha (TNF-α) and chemokine macrophage inflammatory protein 2 (MIP-2). P. murina stimulation of normal alveolar macrophages from C57BL/6 mice resulted in increased TLR2 transcription but not increased TLR4 transcription. In gain-of-function studies with HEK293 cells expressing TLR2 or TLR4, only TLR2 was found to stimulate an NF-κB response to P. murina. TNF-α and MIP-2 production in response to P. murina by mouse alveolar macrophages was inhibited by a monoclonal antibody that specifically blocked the ligand-binding ability of TLR2. Alveolar macrophages from TLR2 knockout (TLR2−/−) mice showed little increase in TNF-α and MIP-2 mRNA levels upon P. murina stimulation. An in vivo study showed that TLR2−/− mice challenged with P. murina had reduced cytokine responses. These results indicate that TLR2 plays a major role in the innate immune response to P. murina.

Toll-like Receptor 2 Knockout Reduces Lung Inflammation During Pneumocystis Pneumonia but has No Effect on Phagocytosis of Pneumocystis Organisms by Alveolar Macrophages

THE innate immune system uses a wide variety of pattern recognition receptors to interact with microorganisms, including Toll-like receptors (TLRs), dectin-1, and scavenger receptors. The inflammatory response and phagocytosis are two major components of innate immunity against microbial infection. The innate immune response to Pneumocystis infection is not well understood. During Pneumocystis pneumonia (PcP), there is a chronic inflammatory reaction that results in impaired gas exchange and tissue damage (Wright et al. 1999). Phagocytosis by alveolar macrophages (AMs) is defective in human (Koziel et al. 1998) and rat (Lasbury et al. 2004) hosts during PcP hence there is reduced internalization of organisms by these cells. We have previously reported that TLR2 mediates alveolar macrophage responses to P. murina, leading to NF-kB nuclear translocation and enhanced TNF-a and MIP-2 production (Zhang et al. 2006). This study investigated the role of TLR2 on phagocytosis of Pneumocystis by AMs and in the inflammatory response to Pneumocystis during PcP.

Syndecan‐1 Expression in the Lung during Pneumocystis Infection

SYNDECAN-1 (Sdc-1) is one of the most abundant heparin sulfate proteoglycans in animals. These glycans are ubiquitous receptors or co-receptors of extracellular ligands, such as fibronectin and thrombospondin-1 (Lebakken and Rapraeger 1996). Syndecan-1 has been shown to act as adhesion and internalization receptors for pathogenic microorganisms (Rostand and Esko 1997). It is a membrane protein, and its ectodomain may shed under certain circumstances. This Sdc-1 ectodomain can inhibit the activity of antimicrobial peptides, such as bactenecins 7 and PR-39 (Park et al. 2001). It has been shown that matrix metalloproteinases (MMPs) may be responsible for Sdc-1 shedding (Endo et al. 2003; Fitzgerald et al. 2000; Li et al. 2002). The expression and function of Sdc-1 during Pneumocystis pneumonia (PcP) have not been studied. Our results show that Pneumocystis infection induces the expression and shedding of Sdc-1 and increases mRNA levels of MMP2, MMP12, and MMP14.

Transforming growth factor-β activation and signaling in the alveolar environment during Pneumocystis pneumonia

PNEUMOCYSTIS pneumonia (PcP) is marked by significant inflammatory damage to the alveolus and parenchyma of the lung (Yoneda and Walzer 1981, 1983). While IL-1b and other pro-inflammatory cytokines are known to be increased in the lung during PcP (Perenboom et al. 1996), the mechanisms by which the inflammatory-mediated damage takes place are not fully understood. Transforming growth factor-b (TGF-b) is an immune modulator that can promote inflammatory damage and sclerosis under some circumstances and in some tissues. Transforming growth factor-b is a strong chemotactic agent for inflammatory lymphocytes, macrophages, and neutrophils (Adams et al. 1991; Wahl 1992; Wahl et al. 1987), and for the influx of these cells to the site through up-regulation of integrins (Ignotz and Massague 1986; Wahl et al. 1993). Transforming growth factor-b stimulates sclerosis by promoting fibroblast differentiation and maturation (Ignotz and Massague 1986; Willis, duBois, and Borok 2006) and fibrotic tissue formation (Branton and Kopp 1999; Fine and Goldstein 1987). In other circumstances, TGF-b can serve as an antiapoptotic agent by stimulating activation of the Akt-1 survival pathway (Suzuki et al. 2005), and can also have anti-inflammatory activity in the lung by suppressing pro-inflammatory cytokine production (Brandes et al. 1987; Espevik et al. 1987) and inhibiting T-lymphocyte function (Kuruvilla et al. 1991; Wahl et al. 1988). Transforming growth factor-b must be activated in order to modulate immune responses. The latency-associated peptide (LAP) of TGF-b must be removed after secretion (Gleizes et al. 1997). This activation can be mediated by many substances, including integrins (Sheppard 2005), thrombospondin-1 (SchultzCherry and Murphy-Ullrich 1993), matrix metalloproteases (Yu and Stamenkovic 2000), and plasmin (Yehualaeshet et al. 1999). The role of TGF-b and the level of TGF-b activation in the lung during PcP has not been elucidated. This study sought to determine if TGF-b is present in the lung during PcP and whether it is activated to stimulate downstream smad signaling in alveolar macrophages.

Inflammatory cells are sources of polyamines that induce alveolar macrophage to undergo apoptosis during Pneumocystis pneumonia.

PNEUMOCYSTIS is an opportunistic fungal pathogen that causes pneumonia in immunocompromised individuals such as patients with AIDS. Alveolar macrophages are critical in the clearance of Pneumocystis organisms. However, the number of alveolar macrophages is decreased by approximately 60% during Pneumocystis pneumonia (PcP) (Lasbury, Durant, and Lee 2003). One mechanism that reduces the alveolar macrophage number is apoptosis (Lasbury et al. 2006). High concentrations of polyamines are known to induce apoptosis, and we have found that the levels of some polyamines (spermidine, acetylspermine, and acetylspermidine) are greatly increased in Pneumocystis-infected lungs and that these polyamines can induce normal alveolar macrophages to undergo apoptosis. We also have found that bronchoalveolar lavage (BAL) fluids from Pneumocystis-infected animals can induce apoptosis in normal alveolar macrophages, but this apoptosis-inducing ability of the BAL fluid is lost when the polyamines are depleted. These results suggest that high polyamine levels induce apoptosis in alveolar macrophages during PcP. Pneumocystis organisms produce and excrete polyamines (Chin et al. 1996), but it is unknown whether host cells also produce polyamines during PcP. To identify cells that produce polyamines during Pneumocystis infection, we examined the differential expression of ornithine decarboxylase (ODC), a key enzyme on the production of polyamines. We also investigated the uptake of polyamines by alveolar macrophages from Pneumocystis-infected rats.

GM-CSF Expression in the Lung During Pneumocystis Pneumonia

GRANULOCYTE macrophage-colony stimulating factor (GM-CSF) is an important growth factor that promotes proliferation and maturation of macrophages and monocytes (Shibata et al. 2001). Granulocyte macrophage-colony stimulating factor also mediates cytokine production (Kremlev et al. 1998), stimulates expression of FcgR1 receptors and actin polymerization to increase phagocyte function (Berclaz et al. 2002), inhibits neutrophil apoptosis (Kobayashi et al. 2005), and enhances clearance of surfactant protein by alveolar macrophages in the lung (Ikegami et al. 1996). Granulocyte macrophage-colony stimulating factor present in the alveolar spaces of the lung is produced by alveolar macrophages, T lymphocytes, fibroblasts, and endothelial cells. The expression level of GM-CSF in some cell types is controlled by the ubiquitous calcium binding protein calmodulin through calmodulin-dependent protein kinase II (Liu and Grundstrom 2002). In Pneumocystis pneumonia (PcP), there is a reduction in alveolar macrophage-mediated phagocytosis (Lasbury et al. 2003), a build-up of surfactant protein A (Atochina et al. 2001; Phelps and Rose 1991), and a loss of alveolar macrophages by increased apoptosis (Lasbury et al. 2006). The present study sought to measure the levels of GM-CSF in alveolar macrophages and bronchoalveolar lavage (BAL) fluids from rats with PcP. We also examined the role of calmodulin in controlling GM-CSF expression in alveolar macrophages.

Caspase-9 as a Target for Pneumocystis Pneumonia Therapy

PNEUMOCYSTIS pneumonia (PcP) is a major opportunistic disease in AIDS patients. Although the advent of the highly active anti-retroviral therapy has significantly reduced the incidences of PcP it remains the leading cause of death in patients with AIDS. Several drugs such as trimethoprim/sulfamethoxazole, pentamidine, and clindamycin/primaquine that target Pneumocystis organisms are available. Unfortunately, these drugs have high levels of side effects (Allegra et al. 1988; Jung and Paauw 1994; Raviglione, 1990) and unacceptable rates of treatment failure (Lundberg, Davidson, and Burman 2000; Moorman et al. 1998). Also, Pneumocystis isolates have emerged that exhibit resistance to the most effective drug combination trimethoprim/sulfamethoxazole (Iliades, Meshnick, and Macreadie 2005); therefore, additional drugs are needed to treat the disease. Pneumocystis infection leads to defects in the function (Lasbury et al. 2004) and level (Lasbury et al. 2003) of the innate immune response. One potential PcP therapy is the restoration of the innate immune response to the organism. In this study, we found that alveolar macrophages undergo apoptosis during Pneumocystis infection due to activation of caspase-9 and that inhibition of this caspase-9 activity reduced the severity of PcP, suggesting that caspase-9 can be a target for therapy of PcP.

Survival Pathway Signal Transduction is Reduced in Alveolar Macrophages during Pneumocystis Pneumonia

PNEUMOCYSTIS pneumonia (PcP) causes increased alveolar macrophage apoptosis in humans, rats, and mice (Lasbury et al. 2006). The apoptosis of these cells proceeds via caspase-9 activation, as inhibition of caspase-9 activation increases alveolar macrophage numbers and prolongs survival of rats and mice with PcP (Lasbury et al. 2006). While a pro-apoptotic signal may be present in the lung during Pneumocystis infection, reduced alveolar macrophage resistance to apoptosis may also result in the reduction of alveolar macrophage numbers. Akt-1 is a kinase that acts to promote survival of cells. Akt-1, after it is activated by phosphorylation, is anti-apoptotic. Akt-1 can phosphorylate caspase-9 to prevent its activation (Cardone et al. 1998), inhibit forkhead transcription factor activity (Brunet et al. 1999), phosphorylate glycogen synthase kinase (GSK-3b) to inhibit its pro-apoptotic activities (Pap and Cooper 1998), and inhibit mitochondrial damage by promoting the release of the anti-apoptotic molecules Bcl-xl and Bcl-2 from Bad (del Peso et al. 1997). To begin to determine the role of Akt-1 in the apoptosis of alveolar macrophages during PcP, we assessed the expression and activation of Akt-1.

Alterations in surfactant protein A form and clearance during Pneumocystis pneumonia

Cincinnati) and the secondary antibody was an anti-mouse IgG conjugated with peroxidase. The ECL Advanced reagents (Amersham, Piscataway, NJ) were used to visualize bands by autoradiography. Immunoblot autoradiograms were analyzed using the ImageJ software package (Abramoff, Magelhaes, and Ram 2004). Integrated density was performed to identify summed pixel values (area mean gray value) for all the SpA bands under each condition. The mean gray average was determined for the glyceraldehyde phosphate dehydrogeanse (GAPDH) internal controls, and the alveolar macrophage SpA values were then normalized to GAPDH.

Expression and activation of complement protein and alveolar damage during Pneumocystis pneumonia

Aleading cause of morbidity and mortality during PcP is the inflammatory reaction in the lung alveolus that leads to reduced gas exchange and pulmonary injury (Yoneda and Walzer 1981, 1983). The mechanisms by which this inflammatory damage takes place are not understood and have not been characterized. The complement cascade is one possible source of inflammatory stimulus and tissue damage during PcP. The full array of complement proteins is present in the alveolar lining fluid (Strunk, Eidlen, and Mason 1988). Complement fixation results in the catalytic cleavage of downstream elements of the cascade leading to generation of immune modulating agents, a membrane attack complex, and opsonization of invading organisms. The cleavage products C3a, C4a, and C5a (anaphylatoxins) stimulate a pro-inflammatory response after binding to their cognate receptors on myeloid lineage and tissue cells. Anaphylatoxins induce increased vascular permeability in endothelial cells, stimulate release of pro-inflammatory cytokines, and up-regulate ICAM-1; all of these symptoms are seen in PcP (Benfield et al. 1997; Perenboom et al. 1996; Yu and Limper 1997). The present study sought to determine if activation of the complement cascade takes place in the lung during PcP, as activation of this pathway may contribute to lung inflammation and damage seen during PcP.