Src kinase Lyn is crucial for <i>Pseudomonas aeruginosa</i> internalization into lung cells

Kannan, Shibichakravarthy; Audet, Aaron; Knittel, Jessica; Mullegama, Saman; Gao, George F.; Wu, Min

doi:10.1002/eji.200635973

Cited by 53 publications

(73 citation statements)

References 52 publications

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“…Corroborating our in vitro observations, LPS induced augmented production of IL-6 ( Fig. 2A), TNF-a ( a positive role of Lyn in this process has been observed (34). Having seen the negative effects of Lyn on TLR4-mediated cytokine production, we were interested in whether Lyn might play a comparable role in the context of a different TLR-TLR2.…”

Section: Lyn Attenuates Lps Effects In Vivosupporting

confidence: 61%

Activation of Murine Macrophages via TLR2 and TLR4 Is Negatively Regulated by a Lyn/PI3K Module and Promoted by SHIP1

Keck

Freudenberg

Huber

2010

The Journal of Immunology

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Src family kinases are involved in a plethora of aspects of cellular signaling. We demonstrate in this study that the Src family kinase Lyn negatively regulates TLR signaling in murine bone marrow-derived macrophages (BMMΦs) and in vivo. LPS-stimulated Lyn−/− BMMΦs produced significantly more IL-6, TNF-α, and IFN-α/β compared with wild type (WT) BMMΦs, suggesting that Lyn is able to control both MyD88- and TRIF-dependent signaling pathways downstream of TLR4. CD14 was not involved in this type of regulation. Moreover, Lyn attenuated proinflammatory cytokine production in BMMΦs in response to the TLR2 ligand FSL-1, but not to ligands for TLR3 (dsRNA) or TLR9 (CpG 1668). In agreement with these in vitro experiments, Lyn-deficient mice produced higher amounts of proinflammatory cytokines than did WT mice after i. v. injection of LPS or FSL-1. Although Lyn clearly acted as a negative regulator downstream of TLR4 and TLR2, it did not, different from what was proposed previously, prevent the induction of LPS tolerance. Stimulation with a low dose of LPS resulted in reduced production of proinflammatory cytokines after subsequent stimulation with a high dose of LPS in both WT and Lyn−/− BMMΦs, as well as in vivo. Mechanistically, Lyn interacted with PI3K; in correlation, PI3K inhibition resulted in increased LPS-triggered cytokine production. In this line, SHIP1−/− BMMΦs, exerting enhanced PI3K-pathway activation, produced fewer cytokines than did WT BMMΦs. The data suggest that the Lyn-mediated negative regulation of TLR signaling proceeds, at least in part, via PI3K.

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Section: Lyn Attenuates Lps Effects In Vivosupporting

confidence: 61%

Activation of Murine Macrophages via TLR2 and TLR4 Is Negatively Regulated by a Lyn/PI3K Module and Promoted by SHIP1

Keck

Freudenberg

Huber

2010

The Journal of Immunology

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“…These indicate that in cav1 KO mice infection by various bacteria may activate several different pathways to compensate for the loss of this critical protein. Indeed, there is little doubt that Cav-1 is important for regulating lipid raft-mediated endocytosis and various other cellular events (32,64,65). Despite such activation in various pathways, the loss of Cav-1 cannot be completely rescued to combat infections and usually leads to lethal consequences because crucial cellular signaling systems may have been dysregulated.…”

Section: Discussionmentioning

confidence: 99%

“…To determine the function of AM cells, equal amounts of PAO1-GFP, a strain of P. aeruginosa with green fluorescence (31,39), were added into cultured AM cells isolated from cav1 KO mice or WT mice for a 2-h incubation. This allowed us to conveniently quantify phagocytosis by measuring fluorescence intensity using a fluorescence plate reader (BioTek) (29,32). Phagocytosis of cav1 KO AM cells was significantly lower than that of WT AM cells, indicating that Cav-1 may play a role in phagocytosis by AM cells (Fig.…”

Section: Journal Of Biological Chemistry 21817mentioning

confidence: 99%

Elevated Inflammatory Response in Caveolin-1-deficient Mice with Pseudomonas aeruginosa Infection Is Mediated by STAT3 Protein and Nuclear Factor κB (NF-κB)

Yuan

Huang

Fox

et al. 2011

Journal of Biological Chemistry

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Caveolin-1 (Cav-1), an important composition protein within the flask-shaped membrane invaginations termed caveolae, may play a role in host defense against infections. However, the phenotype in Pseudomonas aeruginosa-infected cav1 knock-out (KO) mice is still unresolved, and the mechanism involved is almost entirely unknown. Using a respiratory infection model, we confirmed a crucial role played by Cav-1 in host defense against this pathogen because Cav-1 KO mice showed increased mortality, severe lung injury, and systemic dissemination as compared with wild-type (WT) littermates. In addition, cav1 KO mice exhibited elevated inflammatory cytokines (IL-6, TNF-␣, and IL-12a), decreased phagocytic ability of macrophages, and increased superoxide release in the lung, liver, and kidney. We further studied relevant cellular signaling processes and found that STAT3 and NF-B are markedly activated. Our data revealed that the Cav-1/STAT3/NF-B axis is responsible for a dysregulated cytokine response, which contributes to increased mortality and disease progression. Moreover, downregulating Cav-1 in cell culture with a dominant negative strategy demonstrated that STAT3 activation was essential for the translocation of NF-B into the nucleus, confirming the observations from cav1 KO mice. Collectively, our studies indicate that Cav-1 is critical for inflammatory responses regulating the STAT3/NF-B pathway and thereby impacting P. aeruginosa infection.Pseudomonas aeruginosa accounts for 25% of Gram-negative bacteria isolated from hospitals and is associated with high morbidity and mortality (1). P. aeruginosa frequently infects immunocompromised individuals, such as those affected by ventilator-associated infection, severe burns, and cancer (2). More than 80% of cystic fibrosis patients suffer severe P. aeruginosa infection (3). This bacterium becomes increasingly resistant to various antibiotics. Further understanding the mechanism of the host-pathogen interaction may result in effective approaches to preventing this infection. Thus, P. aeruginosa has been a focus of airway infectious diseases (1, 4 -6).Recent studies suggest that P. aeruginosa also invades the lung epithelial cells through lipid raft-mediated endocytosis (7-9), which is a possible reason why this bacterium develops such formidable resistance to antibiotics (10). The invasion process of P. aeruginosa may involve certain lipid raft-associated proteins, including the caveolin family of proteins (9). Using caveolin-1 (cav1) 3 KO mice, two recent studies investigated the role of Cav-1 during P. aeruginosa infection (11, 12); however, their observations were very different. Thus, the role of Cav-1 in this infection needs to be clearly characterized.Caveolins are a family of integral membrane proteins involved in caveola formation and receptor-dependent endocytosis (13-15). Cav-1 and -2 are co-expressed in various cells, such as endothelial cells, airway epithelial cells, and type I pneumocytes. Cav-1 is the major component of caveolae, flaskshaped plasma membran...

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“…Alveolar epithelial cell lines A549 and murine lung epithelial (MLE)-12, and murine alveolar macrophages, were purchased from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's Modified Eagle's Medium with 10% FCS (HyClone Laboratories, Logan, UT), 100 U/ml of penicillin, and 100 mg/ml of streptomycin (Life Technologies, Rockville, MD) (7,16,17). Mouse AECII cells were isolated using a protocol described previously (18), with some modifications, namely, that we used magnetic spheres to bind and remove lymphocytes (with CD32 and CD45 antigens).…”

Section: Methodsmentioning

confidence: 99%

Lipid-Based Signaling Modulates DNA Repair Response and Survival against Klebsiella pneumoniae Infection in Host Cells and in Mice

Huang

Weaver

et al. 2013

Am J Respir Cell Mol Biol

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Klebsiella pneumoniae causes serious infections in the urinary tract, respiratory tract, and blood. Lipid rafts, also known as membrane microdomains, have been linked to the pathogenesis of bacterial infection. However, whether lipid rafts affect K. pneumoniae internalization into host cells remains unknown. Here, we show for the first time that K. pneumoniae was internalized into lung cells by activating lipid rafts. Disrupting lipid rafts by methyl-b-cyclodextrin inhibited pathogen internalization, impairing host defense. A deficient mutant of capsule polysaccharide (CPS) showed a higher internalization rate than a wild-type strain, indicating that CPS may inhibit bacterial entry to host cells. Furthermore, lipid rafts may affect the function of extracellular regulated kinase (ERK)-1/2, and knocking down ERK1/ 2 via short, interfering RNA increased apoptosis in both alveolar macrophages and epithelial cells after infection. To gain insights into bacterial pathogenesis, we evaluated the impact of lipid rafts on DNA integrity, and showed that raft aggregates also affect DNA damage and DNA repair responses (i.e., 8-oxoguanine DNA glycosylase [Ogg1]) through the regulation of reactive oxygen species. Importantly, cells overexpressing Ogg1 demonstrated reduced cytotoxicity during bacterial infection. Taken together, these results suggest that lipid rafts may modulate bacterial internalization, thereby affecting DNA damage and repair, which is critical to host defense against K. pneumoniae.Keywords: Klebsiella pneumoniae infection; internalization; alveolar epithelial cells; lipid rafts; capsule polysaccharide Klebsiella pneumoniae causes serious infections in multiorgan systems, and is the third most commonly isolated bacterium from the blood of patients with sepsis (1). Because K. pneumoniae rapidly develops multidrug-resistant strains, this "superbug" can also cause outbreaks in intensive care units, imposing significant financial burdens and dangerous health threats (2, 3). Despite intensive research during the past few decades, the pathogenesis of K. pneumoniae, including its internalization of alveolar cells, is incompletely understood. K. pneumoniae expresses two critical antigens on its cell surface, LPS (an O antigen) and capsule polysaccharide (CPS; a K antigen) (4). These two antigens contribute to pathogenicity through interactions with host cells in different manners (5). LPS is a major component of Gramnegative bacterial cell walls with high immunogenicity, but its role in pathogenesis remains elusive. Early studies suggest that LPS-deficient strains failed to show significantly different pathogenesis from the wild-type (WT) strain (5). By contrast, CPS demonstrates the most distinguished characteristic of virulence factors for K. pneumoniae, and is implicated in the internalization of pathogens into epithelial cells (6). The capsule may protect the bacterium from phagocytosis by macrophages, through an escape from bactericidal serum factors (6). Other studies also demonstrated that isogenic CPS mutant...

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Src kinase Lyn is crucial for Pseudomonas aeruginosa internalization into lung cells

Cited by 53 publications

References 52 publications

Activation of Murine Macrophages via TLR2 and TLR4 Is Negatively Regulated by a Lyn/PI3K Module and Promoted by SHIP1

Activation of Murine Macrophages via TLR2 and TLR4 Is Negatively Regulated by a Lyn/PI3K Module and Promoted by SHIP1

Elevated Inflammatory Response in Caveolin-1-deficient Mice with Pseudomonas aeruginosa Infection Is Mediated by STAT3 Protein and Nuclear Factor κB (NF-κB)

Lipid-Based Signaling Modulates DNA Repair Response and Survival against Klebsiella pneumoniae Infection in Host Cells and in Mice

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