Dennis Tschang

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.

Suppression of Alveolar Macrophage Apoptosis Prolongs Survival of Rats and Mice with Pneumocystis Pneumonia

The number of alveolar macrophages is decreased in patients or animals with Pneumocystis pneumonia (Pcp). This loss of alveolar macrophages is in part due to apoptosis caused by Pneumocystis infection. The mechanism of apoptosis induction is unknown. Cell-free bronchoalveolar lavage fluids from Pneumocystis-infected rats or mice have the ability to induce apoptosis in normal alveolar macrophages. To characterize the mechanisms by which apoptosis proceeds in alveolar macrophages during Pcp, specific caspase inhibitors are tested for their ability to suppress the apoptosis. In vitro induction of apoptosis can be inhibited by the caspase-9 inhibitor (Z-LEHD-FMK) but not by the inhibitor to caspase-8 or -10. The caspase-9 inhibitor can also inhibit apoptosis of alveolar macrophages in vivo when it is intranasally instilled into dexamethasone-immunosuppressed, Pneumocystis-infected rats or L3T4 cell-depleted, Pneumocystis-infected mice. The number of alveolar macrophages rebounds in caspase-9 inhibitor-treated Pcp animals. Phagocytic activity of alveolar macrophages in treated animals is also recovered, and organism burden in these animals is reduced. Administration of caspase-9 inhibitor also clears the exudate that normally fills the alveoli during Pcp and decreases lung inflammation. Furthermore, caspase-9-treated Pcp animals survive for the entire 70-day period of the study, whereas nontreated Pcp animals die 40–60 days after initiation of infection. Depletion of recovered alveolar macrophages by intranasal administration of clodronate-containing liposomes in caspase-9 inhibitor-treated animals abrogates the effects of the inhibitor. Together, these results indicate that immunomodulation of the host response may be an alternative to current treatments for Pcp.

Polyamine-mediated apoptosis of alveolar macrophages during Pneumocystis pneumonia

The number of alveolar macrophages is decreased during Pneumocystis pneumonia (Pcp), partly because of activation of apoptosis in these cells. This apoptosis occurs in both rat and mouse models of Pcp. Bronchoalveolar lavage (BAL) fluids from Pneumocystis-infected animals were found to contain high levels of polyamines, including spermidine, N1-acetylspermine, and N1-acetylspermidine. These BAL fluids and exogenous polyamines were able to induce apoptosis in alveolar macrophages. Apoptosis of alveolar macrophages during infection, after incubation with BAL fluids from Pneumocystis-infected animals, or after incubation with polyamines was marked by an increase in intracellular reactive oxygen species, activation of caspases-3 and -9, DNA fragmentation, and leakage of mitochondrial cytochrome c into the cytoplasm. When polyamines were depleted from the BAL fluids of infected animals, the ability of these BAL fluids to induce apoptosis was lost. Interestingly, the apoptosis inducing activity of the polyamine-depleted BAL fluids was restored when polyamines were added back. The results of this study suggested that Pneumocystis infection results in accumulation of high levels of polyamines in the lung. These polyamines activate apoptosis of alveolar macrophages, perhaps because of the ROS that are produced during polyamine metabolism.

Effect of Bronchoalveolar Lavage Fluid from Pneumocystis carinii- Infected Hosts on Phagocytic Activity of Alveolar Macrophages

ABSTRACT Alveolar macrophages from Pneumocystis carinii-infected rats are defective in phagocytosis. To investigate whether this defect is due to a certain factor present in P. carinii-infected lungs, alveolar macrophages from uninfected rats were incubated with bronchoalveolar lavage (BAL) fluid samples from P. carinii-infected rats. Alveolar macrophages treated with these BAL fluid samples became defective in phagocytosis but remained normal when treated with BAL fluid samples from noninfected or Toxoplasma gondii-infected rats. The suppressive activity of the BAL fluid samples from P. carinii-infected rats on phagocytosis was retained when the BAL fluid samples were passed through a filter with a pore size of 0.45 μm but was lost when the BAL fluid samples were digested with proteases such as trypsin, pepsin, papain, or endopeptidase Gly-C. Lipid fractions of these BAL fluid samples had no suppressive activity on phagocytosis. The suppressive activity of these BAL fluid samples was also lost when they were incubated with concanavalin A-agarose beads, suggesting that the inhibitor is a glycoprotein. The inhibitor was estimated to be larger than 100,000 Da by exclusion filtration. After binding to the concanavalin A-agarose beads, the inhibitor in BAL fluid samples and P. carinii lysate could be eluted with 200 mM methylmannose. Treatment of both the crude BAL fluid samples and P. carinii lysate and the 200 mM methylmannose eluate with antibody against the major surface glycoprotein of P. carinii eliminated their suppressive activity. These results suggest that the factor capable of suppressing the phagocytic activity of alveolar macrophages is P. carinii major surface glycoprotein or one or more of its derivatives.

Decreased inflammatory response in Toll-like receptor 2 knockout mice is associated with exacerbated Pneumocystis pneumonia.

Pneumocystis pneumonia (PcP) is marked by substantial inflammatory damage to the lung. We have found that Toll-like receptor 2 (TLR2) mediates macrophage inflammatory responses to Pneumocystis and hypothesized that TLR2 deficiency would lead to less severe inflammation and milder lung injury during PcP. Histopathology examination showed that TLR2-/- mice with PcP indeed exhibited milder pulmonary inflammation. TLR2-/- mouse lungs contained less TNF-alpha and displayed lower levels of NF-kappaB activation during PcP. However, TLR2-/- mice with PcP displayed increased severity in symptoms and organism burden. The increased organism burden is likely due to defects in protective mechanisms in TLR2-/- mice. mRNA levels of the inducible nitric oxide synthase and NADPH oxidase p47phox, as well as nitric oxide levels in the lungs, were decreased in TLR2-/- PcP mice. Taken together, this study shows that TLR2-mediated inflammatory responses contribute to a certain degree to the clearance of Pneumocystis organism in mice.

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.

Defective Nitric Oxide Production by Alveolar Macrophages during Pneumocystis Pneumonia

The effect of nitric oxide (NO) on Pneumocystis (Pc) organisms, the role of NO in the defense against infection with Pc, and the production of NO by alveolar macrophages (AMs) during Pneumocystis pneumonia (PCP) were investigated. The results indicate that NO was toxic to Pc organisms and inhibited their proliferation in culture. When the production of NO was inhibited by intraperitoneal injection of rats with the nitric oxide synthase inhibitor L-N(5)-(1-iminoethyl) ornithine, progression of Pc infection in immunocompetent rats was enhanced. Concentrations of NO in bronchoalveolar lavage fluids from immunosuppressed, Pc-infected rats and mice were greatly reduced, compared with those from uninfected animals, and AMs from these animals were defective in NO production. However, inducible nitric oxide synthase (iNOS) mRNA and protein concentrations were high in AMs from Pc-infected rats and mice. Immunoblot analysis showed that iNOS in AMs from Pc-infected rats existed primarily as a monomer, but the homo-dimerization of iNOS monomers was required for the production of NO. When iNOS dimerization cofactors, including calmodulin, were added to macrophage lysates, iNOS dimerization increased, whereas incubation of the same lysates with all cofactors except calmodulin did not rescue iNOS dimer formation. These data suggest that NO is important in the defense against Pc infection, but that the production of NO in AMs during PCP is defective because of the reduced dimerization of iNOS.

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.