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Impaired Cd14 and Cd36 Expression, Bacterial Clearance, and Toll-Like Receptor 4-Myd88 Signaling in Caveolin-1-Deleted Macrophages and Mice

Tsai, Tsung-Huang*†; Chen, Shu-Fen; Huang, Tai-Yu; Tzeng, Chun-Fu; Chiang, Ann-Shyn*; Kou, Yu Ru; Lee, Tzong-Shyuan; Shyue, Song-Kun†§

Author Information
doi: 10.1097/SHK.0b013e3181ea45ca

Abstract

INTRODUCTION

Gram-negative bacterial infection often causes severe sepsis and septic shock, with high patient morbidity and mortality (1). The regulation of immune response such as secretion of cytokines with sepsis plays an important role in the survival of and organ damage in infected patients. Macrophages play a pivotal role in the host innate immune response against microbial infection and subsequent inflammatory cytokine secretion. At the site of infection, macrophages use several membrane receptor systems such as CD36, CD14, and Toll-like receptor 4 (TLR4) to recognize surface components of microbes such as LPS and eradicate the pathogen by phagocytosis and subsequent clearance of internalized contents (2, 3).

Among the identified LPS interaction receptors, CD36 belongs to the B class of scavenger receptors and is expressed by myeloid cells, platelets, endothelial cells, and adipocytes (3, 4). It mediates homeostatic functions, including transport of long-chain fatty acids, inhibition of angiogenesis, and bacterial phagocytosis (4-6). CD14, a glycoprotein preferentially expressed on all mature cells of monocytic lineages, has been shown to mediate LPS-activated signaling in macrophage phagocytosis (7). Upon bacterial infection, LPS first binds to soluble LPS binding protein, and the complex binds to CD14, which then activates TLR4 signaling by recruiting myeloid differentiation factor 88 (MyD88) and many adaptor molecules (8, 9). Activation of TLR4-MyD88 signaling triggers multiple signal pathways such as inhibitory nuclear factor κB (IκB) degradation, which leads to nuclear factor κB (NF-κB) translocation to the nucleus to activate transcription of proinflammatory genes, including TNF-α, IL-1β, IL-6, and iNOS (9).

Caveolin-1 (Cav-1), a major structure protein of caveolae, mediates protein transportation from the endoplasmic reticulum to the cellular membrane and regulates signal transduction and cellular functions by interacting with many proteins (10). Caveolae or lipid rafts are involved in the entry of various pathogens, including Escherichia coli (E. coli), simian virus 40, Leishmania chagasi, and Trypanosoma cruzi (11-15). Deletion of Cav-1 impairs phagocytosis and clearance of apoptotic cells by macrophages (16). Cav-1 is induced during the differentiation of monocytes to macrophages (17). The role of Cav-1 in macrophage functions with regard to phagocytosis and clearance of E. coli and TLR4 signaling activation with bacterial infection remains elusive.

In this study, we addressed the possible role of Cav-1 in the immune response to gram-negative bacteria or LPS using wild-type (WT) and Cav-1−/− macrophages and mice. Our results suggest that deletion of Cav-1 impairs phagocytosis of bacteria and inflammatory cytokine production in macrophages by suppressing CD14 and CD36 expression and TLR4-MyD88-NF-κB signaling. These findings are in good agreement with our in vivo study. Thus, Cav-1 is crucial in macrophage phagocytosis and subsequent clearance of gram-negative bacteria.

MATERIALS AND METHODS

Reagents

Cell culture media, penicillin, streptomycin, oligo-dT, and SuperScript III reverse transcriptase were from Invitrogen (Carlsbad, Calif). Antibodies (Abs) for Cav-1, CD14, CD36, iNOS, NF-κB, and horseradish peroxidase-conjugated secondary Ab were from Santa Cruz Biotechnology (Santa Cruz, Calif). The Ab for F4/80 conjugated with fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-labeled anti-CD36 and CD14 Abs were from BioLegend (San Diego, Calif). The Ab for flotillin 1 was from BD Transduction (San Diego, Calif). Thioglycolate was from Becton Dickinson (San Diego, Calif). TRI reagent, LB medium, ampicillin, LPS, phorbol myristate acetate, methyl-β-cyclodextrin (MβCD), cycloheximide, and Griess reagent were from Sigma-Aldrich (St. Louis, Mo). Enzyme-linked immunosorbent assay (ELISA) kits for the detection of IL-1β, IL-6, and TNF-α were from R&D Systems (Minneapolis, Minn).

Cell culture

LADMAC cells, a transformed cell line derived from mouse bone-marrow cells (18) were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and cultured in minimum essential medium α (MEM-α) containing 10% fetal bovine serum (FBS) (Biological Industries, Kibbutz Beit Haemek, Israel) and 0.1 mM nonessential amino acids supplemented with 64 μg/mL penicillin and 100 μg/mL streptomycin. LADMAC conditioned medium (LCM) was collected and filtered from confluent monolayers of cells as described previously (18). THP-1 cells were induced with 50 nM phorbol myristate acetate for 5 days to differentiate into macrophages.

Mice and bone marrow-derived macrophages

All animal experiments were approved by the Institutional Animal Care and Utilization Committee of Academia Sinica. C57BL/6 mice (The National Laboratory Animal Center, National Science Council, Taiwan) and iNOS-knockout mice (NOS2tm1Lau/J, iNOS−/−) and Cav-1−/− mice (Cav1tm1Mls/J) (The Jackson Laboratory, Bar Harbor, Me) were housed and bred under specific pathogen-free conditions. Femurs and tibiae of mice, 8 to 12 weeks old, were excised, and bone-marrow cells were flushed with RPMI-1640 containing 10% FBS with a 24-gauge needle as described (18). After centrifugation, bone-marrow cells were cultured in MEM-α containing 10% FBS and 30% LCM for 7 days to differentiate into macrophages. Medium was changed every 2 days.

Preparation of peritoneal macrophages

Mice, 8 to 12 weeks old, were intraperitoneally injected with 2 mL of 4% thioglycolate. At 4 days after injection, 10 mL of phosphate-buffered saline (PBS) was injected, and peritoneal macrophages were collected and cultured in MEM-α containing 10% FBS and 10% LCM for 4 days. Medium was changed every 2 days.

Preparation of green fluorescence protein-expressing bacteria

Escherichia coli BL21 (ATCC BBA-1025) carrying green fluorescence protein (GFP) on the pPDT-flag was freshly cultured in LB medium with ampicillin (100 μg/mL) to early logarithmic growth phage (OD600 = 0.6-0.8). Isopropyl-β-d-thiogalactoside (1 mM) (MDbio, Taiwan, ROC) was then added to induce GFP expression at 18°C for 16 h. Escherichia coli was washed and resuspended in PBS. Escherichia coli was aliquoted or heat inactivated by incubation at 65°C for 2 h and then stored at −20°C. Salmonella typhimurium (S. typhimurium) (ATCC 14028) carrying a GFP-expressing plasmid (kindly provided by Dr. H. F. Yang-Yen at Academia Sinica, Taipei, Taiwan) was grown overnight in LB medium without NaCl at 37°C. After a wash, S. typhimurium was resuspended in PBS.

Flow cytometry

Peritoneal macrophages were stained with FITC-labeled anti-F4/80 Ab plus PE-labeled anti-CD14 or anti-CD36 Ab. Protein expression and bacterial phagocytosis in macrophages were analyzed by FACSCalibur (Becton Dickinson) and Cellquest Pro software (San Jose, Calif).

Recombinant adenovirus construction

A recombinant adenovirus encoding Cav-1 was prepared as described previously (19). Briefly, the cDNA fragment containing the human Cav-1 was subcloned into the shuttle plasmid and recombined into adenoviral DNA to generate Ad-Cav-1. The expression of Cav-1 was driven by a human phosphoglycerate kinase (hpgk) promoter.

Reverse transcriptase-polymerase chain reaction

Macrophage RNA was isolated by the TRI reagent. A total of 4 μg of RNA was used for cDNA synthesis with oligo-dT and SuperScript III reverse transcriptase. cDNAs were used for PCR amplification with the primers 5′-GGG TCA AGG AAC AGA AGC AG-3′ and 5′-GCT CAT TTC TCA CCC AGT CC-3′ for TLR4, and 5′-CAC TCG CAG TTT GTT GGA TG-3′ and 5′-TCT GGA AGT CAC ATT CCT TGC-3′ for MyD88. Polymerase chain reaction of β-actin with the primers 5′-CGA CAA CGG CTC CGG CAT GTG-3′ and 5′-GGT CTC AAA CAT GAT CTG GG-3′ was used as a reference. Reverse transcriptase-polymerase chain reaction amplification was 94°C for 3 min, followed by 25 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s.

Preparation of lipid rafts

Detergent-resistant lipid rafts of peritoneal macrophages were prepared by sucrose gradient ultracentrifugation as described previously (20). In brief, cells were resuspended in lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100) containing a mixture of protease inhibitors (Roche Molecular Biochemicals, Indianapolis, Ind) followed by a brief sonication. Sucrose was added to the lysate (final concentration, 40%), and the lysate was placed at the bottom of the ultracentrifuge tube and overlaid with lysis buffer containing 30%, then 5% sucrose. After centrifugation at 200,000g for 24 h, twelve 1-mL fractions from the top to the bottom were collected for Western blot analysis.

Immunoblotting

A total of 30 μg cell lysates or nuclear extracts were resolved on 8% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blotting as described previously (19). Nuclear extracts for NF-κB protein detection were isolated according to the instructions of the NF-κB (p65) transcription factor assay kit (Cayman, Ann Arbor, Mich).

Phagocytosis, bacterial killing, and colony-forming unit assay

Macrophages were infected with E. coli (100 multiplicity of infection [MOI]) in serum-free MEM-α at 37°C for 30 min. Cells were washed with PBS twice to eliminate extracellular bacteria for phagocytosis assay or then incubated in serum-free MEM-α with 100 μg/mL gentamicin for 90 to 360 min for bacterial killing assay. To determine bacterial content in phagocytosis and bacterial killing assay, cells were lysed with 0.1% Triton X-100, serially diluted and plated onto LB agar plates containing 100 μg/mL ampicillin, and incubated at 37°C overnight. Colony-forming units (CFUs) of bacteria engulfed were determined by the number of colonies formed. Relative bacterial content in cells was determined by use of a fluorescence microplate reader (Spectra Max Gemini EM, Sunnyvale, Calif) or flow cytometry. To measure the live bacterial content in blood and organs, WT and Cav-1−/− mice were intraperitoneally injected with viable E. coli (1 × 105) for 24 h. Blood was collected from the lateral tail veins, and the lung (20 mg of left lung), liver (60 mg of left medial lobe), and spleen (100 mg) were dissected, ground, and suspended in 1 mL PBS. After serial dilution and overnight incubation at 37°C on LB agar plates, bacterial CFUs in each sample were counted.

Phagocytosis and clearance of E. coli within the peritoneal cavity

Mice, 8 to 12 weeks old, were intraperitoneally injected with 2 mL of 4% thioglycolate. At 4 days after injection, E. coli (1 × 105) was intraperitoneally injected for 30 or 360 min. Ten milliliters of PBS was then injected, and peritoneal macrophages and fluid were collected for flow cytometry and cytokine quantification.

Determination of NF-κB activity

Nuclear factor κB activity was determined by ELISA measuring the DNA binding activity of p65 by use of the NF-κB (p65) transcription factor assay kit (Cayman) according to the manufacturer's instructions.

Quantification of cytokines

Wild-type and Cav-1−/− mice were intraperitoneally injected with 1 × 105 viable E. coli for 20 h. Plasma from mice and supernatant from cell culture were harvested and stored at −80°C. The total level of IL-1β, IL-6, and TNF-α was measured by ELISA according to the manufacturer's instructions.

Statistical Analysis

Mann-Whitney U test was used for comparisons between treatments, and data are expressed as mean ± SEM. P < 0.05 was considered statistically significant.

RESULTS

Suppressed phagocytosis in Cav-1−/− macrophages in vitro and in vivo

To explore the role of Cav-1 in macrophage phagocytosis, peritoneal macrophages from WT and Cav-1−/− mice were harvested and incubated with GFP-expressing E. coli or S. typhimurium for 30 min for phagocytosis and CFU assay. Deletion of Cav-1 greatly hindered phagocytosis of E. coli by macrophages as detected by CFU assay (Fig. 1A) and intracellular GFP intensity (Fig. 1B). Flow cytometry detected markedly fewer Cav-1−/− than WT macrophages containing GFP-expressing E. coli or S. typhimurium, which suggests reduced phagocytotic ability (Fig. 1, C and D). On the contrary, exogenous expression of Cav-1 by Ad-Cav-1 infection partly restored the phagocytosis ability of Cav-1−/− macrophages (Fig. 1E). The efficiency of phagocytosis in Ad-Cav-1-infected WT macrophages was increased as well. In addition, the expression of activated macrophage markers was attenuated on detecting the expression of CD206 (a mannose receptor) and Mac3 in F4/80-positive macrophages (see Figure, Supplemental Digital Content 1, https://links.lww.com/SHK/A52).

F1-15
Fig. 1:
Deletion of Cav-1 suppresses macrophage phagocytosis. Peritoneal macrophages from WT and Cav-1−/− mice were incubated with GFP-expressing E. coli for 30 min, and bacterial uptake by macrophages was examined by CFU assay (A) and intracellular GFP intensity (B). Green fluorescence protein-expressing E. coli (C) and S. typhimurium (D) were incubated with peritoneal macrophages for 30 min and analyzed with use of a fluorescence microplate reader (B) or flow cytometry (C and D). E, Bone-marrow-derived macrophages were infected with Ad-Cav-1 or Ad-hpgk (a vector control) (20 MOI) for 3 days, then incubated with E. coli for 30 min. Total bacterial content in macrophages was determined by CFU assay. The protein levels of Cav-1 and β-actin were determined by Western blot analysis. Data are means ± SEM (n = 3). *P < 0.05.

Suppressed CD36 and CD14 expression in Cav-1−/− macrophages is related to attenuated phagocytosis

Both CD36 and CD14 are important during bacterial phagocytosis, and CD36 expression is decreased in Cav-1−/− macrophages (21). We examined whether Cav-1 participates in the regulation of CD36 and CD14 expression and the roles of these factors in bacterial phagocytosis. As shown in Fig. 2A, CD36 and CD14 levels were upregulated with increased Cav-1 expression in WT bone marrow-derived macrophages (BMDMs) as compared with their progenitor cells. However, the induction of CD36 and CD14 was markedly reduced in Cav-1−/− BMDMs. Moreover, Cav-1, CD36, and CD14 were cofractionated in the lipid raft fraction (see Figure, Supplemental Digital Content 2, https://links.lww.com/SHK/A53). CD36 and CD14 expression was lower in the lipid rafts of peritoneal macrophages from Cav-1−/− mice than in those from WT mice (Fig. 2B and Supplemental Digital Content 2, https://links.lww.com/SHK/A53). In parallel, flow cytometry revealed that deletion of Cav-1 hindered the expression of CD36 and CD14 in peritoneal macrophages. Moreover, deletion of Cav-1 led to two subsets each, of CD36+ and CD36, and CD14+ and CD14 peritoneal macrophages (Fig. 2, C and D).

F2-15
Fig. 2:
Decreased expression of CD14 and CD36 in Cav-1−/− macrophages is associated with attenuated phagocytosis. A, Bone marrow (BM) was isolated from WT and Cav-1−/− mice and differentiated into BMDMs for 7 days. Cellular lysates of nondifferentiated and differentiated cells underwent Western blot analysis of protein levels of CD14, CD36, Cav-1, or β-actin. B, Detergent-resistant lipid rafts of peritoneal macrophages of WT and Cav-1−/− mice were prepared as described in Materials and Methods. Fractions 5 to 8 were combined and analyzed by Western blot analysis for Cav-1, CD14, CD36, and flotillin-1 (marker of lipid rafts) expression. Relative protein levels were measured by densitometry and indicated as ratios normalized to WT levels. Data are representative of five experiments. C and D, Wild-type and Cav-1−/− peritoneal macrophages were collected and incubated with FITC-labeled anti-F4/80 plus PE-labeled anti-CD36 or anti-CD14 Ab for flow cytometry. E, Wild-type peritoneal macrophages were treated with Abs against CD36, CD14, or both, and the amount of phagocytotic bacteria was determined by CFU assay. Data are means ± SEM (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001.

To investigate whether the suppressed CD36 and CD14 expression was involved in impaired bacterial phagocytosis, peritoneal macrophages were preincubated with Abs against CD36, CD14, or both and then incubated with E. coli. Bacterial phagocytosis was attenuated with anti-CD36 or anti-CD14 treatment (Fig. 2E), and treatment with both anti-CD36 and anti-CD14 produced a synergistic inhibition of bacterial uptake. These data suggest that attenuated bacterial uptake by Cav-1−/− macrophages is associated with downregulated CD36 and CD14 expression. In addition, disruption of lipid rafts, including caveolae, by MβCD hindered bacterial phagocytosis (see Figures, Supplemental Digital Content 3, parts A, C, and D, https://links.lww.com/SHK/A54) and CD14 and CD36 expression (Supplemental Digital Content 3, parts B, E, and F, https://links.lww.com/SHK/A54), which suggests that caveolae play an important role in macrophage phagocytosis.

Cav-1 deletion attenuates bacterial killing in vitro and in vivo

To investigate whether deletion of Cav-1 hampers bacterial killing in vitro, peritoneal macrophages were incubated with GFP-expressing E. coli for 30 min for bacterial killing assay. The content of intracellular bacteria was determined by flow cytometry. Similar to the results shown in Figure 1, E. coli uptake was greatly decreased in Cav-1−/− macrophages (Fig. 3A). The proportion of bacteria-containing macrophages in WT macrophages was greatly decreased, from 64% to 32% at 90 min and to 7% at 360 min. However, bacterial clearance efficiency in Cav-1−/− macrophages was also reduced, with the proportion of bacteria-containing macrophages decreased from 24% to 19% at 90 min and to 12% at 360 min. In parallel, in vivo experiments demonstrated similar results shown in Figures 1 and 3A, that E. coli uptake was markedly attenuated by macrophages in the peritoneal cavity (Fig. 3B). Similarly, bacterial clearance efficiency in the peritoneal cavity of Cav-1−/− mice was also hindered: the proportion of bacterial-containing macrophages was decreased from 50% to 8% in WT mice and from 16% to 12% in Cav-1−/− mice by 360 min (Fig. 3B). Additionally, significantly fewer bacterial colonies were in plasma and tissues, including lung, liver, and spleen, in WT mice than in Cav-1−/− mice intraperitoneally injected with E. coli for 20 h (Fig. 3C). These data suggest an important role of Cav-1 in bacterial clearance in vitro and in vivo.

F3-15
Fig. 3:
Deletion of Cav-1 suppresses bacterial killing in vitro and in vivo. A, Peritoneal macrophages from WT and Cav-1−/− mice were treated with GFP-expressing E. coli for 30 min. After a wash, the number of surviving bacteria in macrophages incubated for another 90 and 360 min was determined by flow cytometry. Data are means ± SEM (n = 3). B, Wild-type and Cav-1−/− mice were intraperitoneally injected with thioglycolate for 4 days. Escherichia coli (1 × 105) was intraperitoneally injected for 30 or 360 min. Macrophages from the peritoneal cavity were isolated for flow cytometry. C, Wild-type and Cav-1−/− mice were intraperitoneally injected with 1 × 105 viable E. coli for 24 h. Living bacteria in blood, lung, liver, and spleen were determined by CFU assay. Data are means ± SEM (n = 6). **P < 0.01 and ***P < 0.001 vs. WT.

Impaired bacterial killing of Cav-1−/− macrophages is implicated in part in impeded iNOS expression

A high level of NO generated by iNOS plays a key role in bacterial killing (22, 23). iNOS expression and NO production were suggested to be reduced in LPS-treated Cav-1−/− mice (24) (Fig. 4, A and B). The decrease in iNOS expression and NO production was further supported in heat-inactivated bacteria-treated macrophages, showing decreased iNOS induction accompanied by greatly lower NO production in Cav-1−/− macrophages than in WT macrophages (Fig. 4, C and D). As well, MβCD treatment significantly inhibited iNOS expression in LPS-treated macrophages (Fig. 4E). To investigate the contribution of decreased iNOS expression to the hindered bacterial killing ability in Cav-1−/− macrophages, bacterial killing efficiency was examined by incubation of E. coli with peritoneal macrophages from WT, iNOS−/−, and Cav-1−/− mice. As shown in Figure 4F, deletion of iNOS attenuated bacterial clearance at 90 and 180 min after bacterial treatment; however, Cav-1 deletion further suppressed bacterial clearance as compared with iNOS deletion in macrophages, which suggests that decreased iNOS expression is responsible in part for the inhibition of bacterial killing in Cav-1−/− macrophages.

F4-15
Fig. 4:
Deletion of Cav-1 impedes LPS- or bacteria-induced iNOS expression and NO production. A-D, Wild-type and Cav-1−/− peritonealmacrophages cultured in serum-free medium were incubated with LPS (1 μg/mL) for 12 h (A and B) or heat-inactivated E. coli (100 MOI) for 4 to 12 h (C and D). The protein levels of Cav-1 and iNOS were determined by Western blot analysis (A and C), and relative NO production was determined by use of Griess reagent (B and D). E, Peritoneal macrophages were incubated with various doses of MβCD and LPS (1 μg/mL) for 12 h, and the expression of iNOS was determined by Western blot analysis. F, Peritoneal macrophages from WT, iNOS−/− and Cav-1−/− mice were incubated with E. coli for 30 min, and the bacterial killing ratio was measured by CFU assay at 90 and 180 min after infection. The Western blot data are representative of three experiments. Data are means ± SEM (n = 6). *P < 0.05 and ***P < 0.001.

Suppressed TLR4 and MyD88 expression and signaling in Cav-1−/− macrophages

TLR4 and MyD88 are involved in bacterial clearance and inflammatory response in macrophages (25). To explore the role of Cav-1 in this function, the expression of TLR4 and MyD88 was evaluated by Western blot analysis in peritoneal macrophages. The protein levels of TLR4 and MyD88 were substantially lower in Cav-1−/− macrophages than in WT macrophages (Fig. 5A). Similar results were obtained in BMDMs (data not shown). To investigate how Cav-1 expression participates in the regulation of TLR4 and MyD88 expression, we examined the mRNA levels and protein stability of both genes. The mRNA level of TLR4 in Cav-1−/− macrophages was comparable to that in the WT (Fig. 5B); however, MyD88 mRNA level was downregulated in Cav-1−/− macrophages. Protein stability assay showed that the half-life of TLR4 protein was longer than 8 h in WT macrophages but decreased to about 4 h in Cav-1−/− macrophages (Fig. 5C). In contrast to TLR4 findings, MyD88 stability was comparable in WT and Cav-1−/− macrophages. Next, we sought to examine whether deletion of Cav-1 affected TLR4-MyD88 signaling. Heat-inactivated bacterial-induced NF-κB binding to target DNA was assayed by ELISA. The binding activity was markedly attenuated in Cav-1−/− macrophages (Fig. 6A). The IκB levels were decreased at 15 min after bacterial treatment and further decreased at 30 min in WT macrophages (Fig. 6B). In Cav-1−/− macrophages, the IκB level was comparable to that in nontreated WT macrophages and did not decrease at 15 and 30 min after treatment (Fig. 6B). Moreover, the level of NF-κB in the nucleus was significantly higher in WT cells than in Cav-1−/− cells after LPS treatment (Fig. 6C).

F5-15
Fig. 5:
Attenuated TLR4 and MyD88 expression in Cav-1−/− macrophages. The protein and mRNA expression of TLR4 and MyD88 was determined by Western blot analysis (A) and reverse transcriptase-polymerase chain reaction (B), respectively, in WT and Cav-1−/− peritoneal macrophages. C, Protein stability of TLR4 and MyD88 in peritoneal macrophages was examined by treatment with cycloheximide for the indicated times on Western blot analysis. *P<0.05.
F6-15
Fig. 6:
Deletion of Cav-1 impairs NF-κB activation. Peritoneal macrophages were treated with LPS (1 μg/mL) or heat-inactivated E. coli (100 MOI). Nuclear factor κB activity was analyzed by ELISA (A). Levels of IκB-α (B) and NF-κB in nucleus (C) were examined by Western blot analysis. The data are representative of three experiments. Relative NF-κB levels in nucleus were determined by densitometry. Data are means ± SEM (n = 3). *P < 0.05.

Cav-1 deletion inhibits inflammatory cytokine production

A number of studies show that activation of TLR4-MyD88-NF-κB signaling results in cytokine production in macrophages (8, 9). The effect of Cav-1 deletion on cytokine production of macrophages induced by LPS was examined by ELISA. The production of IL-1β, IL-6, and TNF-α induced by LPS was markedly reduced in Cav-1−/− peritoneal macrophages (Fig. 7, A-C), and the levels of IL-1β, IL-6, and TNF-α in plasma of mice injected with E. coli were lower in Cav-1−/− mice than in WT mice (Fig. 7, D-F). Additionally, the levels of IL-1β, IL-6, and TNF-α in peritoneal fluid of mice injected with E. coli were lower in Cav-1−/− mice than in WT mice (Fig. 7, G-I). These data suggest that deletion of Cav-1 suppresses TLR4-MyD88-NF-κB signaling and represses relevant cytokine production.

F7-15
Fig. 7:
Decreased production of IL-1β, IL-6, and TNF-α in Cav-1−/− macrophages and mice. A-C, Wild-type and Cav-1−/− peritoneal macrophages were isolated and treated with heat-inactivated E. coli (100 MOI) for 12 (A-C) or 24 h (A and B). The levels of IL-1β, IL-6, and TNF-α in culture media were determined by ELISA. Data are means ± SEM (n = 3). D-F, Wild-type and Cav-1−/− mice were intraperitoneally injected with 1 × 105 viable E. coli for 20 h. Plasma levels of IL-1β, IL-6, and TNF-α were measured by ELISA. Data are means ± SEM (n = 6 to 10). G-I, Wild-type and Cav-1−/− mice were intraperitoneally injected with thioglycolate for 4 days. Escherichia coli (1 × 105) was intraperitoneally injected for 360 min. Peritoneal fluid was isolated for ELISA. Data are means ± SEM (n = 6). **P < 0.01 and ***P < 0.001 vs. WT.

DISCUSSION

Pathogen recognition and internalization play an important role in the response of innate immune system and activation of the downstream inflammatory network. In this study, we demonstrated an important regulatory role of Cav-1 in phagocytosis, bacterial clearance, and related signal transduction in macrophages in response to pathogen infection. Cav-1 expression was increased during monocyte/macrophage differentiation. Cav-1 deletion or destruction of caveolae resulted in macrophages having an impaired capacity for phagocytosis of E. coli and S. typhimurium bacteria (Fig. 1). This result is similar to previous results showing that deletion of Cav-1 hindered clearance of apoptotic cells (16). This notion was further supported by Ad-Cav-1 infection partly restoring the phagocytosis ability of Cav-1−/− macrophages and overexpression of Cav-1 enhancing the phagocytosis of WT macrophages (Fig. 1E).

A number of membrane-associated proteins, including CD14 and CD36, are enriched in caveolae and are required for elimination of infected pathogens (26). In this study, deletion of Cav-1 suppressed phagocytosis by downregulating CD14 and CD36 protein expression. Our results show that deletion of Cav-1 or disruption of lipid rafts by MβCD hindered CD14 and CD36 expression as detected by Western blot analysis in the cell membrane and lipid rafts and by flow cytometry (Fig. 2 and Supplemental Digital Content 3, parts E and F, https://links.lww.com/SHK/A54). Meanwhile, our preliminary data show that deletion of Cav-1 does not alter the mRNA levels of CD14 or CD36 in macrophages (data not shown). Because deletion of Cav-1 abolishes the caveolae formation (26) these results imply that the lipid rafts, especially the caveolae, mediate the expression and membrane targeting of CD14 and CD36. Additionally, CD14 or CD36 Ab alone blocked E. coli uptake and had a synergistic effect on inhibiting E. coli phagocytosis. These data suggest that Cav-1 may play a pivotal role in the expression of CD14 and CD36 during monocyte/macrophage differentiation and in maintaining the normal function of macrophages in bacteria phagocytosis.

In addition to hampering phagocytotic capacity, Cav-1 deletion led to attenuated bacterial clearance in vitro and in vivo, as evidenced by the greatly suppressed bacterial killing ability in Cav-1−/− macrophages and greater survival of E. coli in the blood, lung, liver, and spleen of Cav-1−/− mice than in WT mice (Fig. 3, A-C). The hindered bacterial killing was attributed in part to the suppression of iNOS expression because iNOS deletion reduced bacterial killing efficiency to a lesser extent than did Cav-1 deletion in mice (Fig. 4F). Bacterial killing by macrophages after phagocytosis may be associated with phagosome and phagolysosome formation (2). TLR4, MyD88, and NF-κB activation have been suggested to be crucial in mycobacterial phagosomes and phagolysosome fusion, iNOS induction, and bacterial killing (22, 23, 25, 27). Reduced NF-κB activation has been reported in lungs of Cav-1−/− mice injected with LPS (24). Our results indicate that deletion of Cav-1 downregulated TLR4 and MyD88 protein expression and NF-κB activation in macrophages. Therefore, besides suppressing iNOS expression and NO production, deletion of Cav-1 might also attenuate phagosome and phagolysosome formation via inhibiting TLR4-MyD88-NF-κB signaling, which leads to impeded bacterial killing ability. Recently, Cav-1 was suggested to bind to TLR4 and attenuate LPS-induced proinflammatory cytokine production on carbon monoxide stimulation in murine macrophages (28). These results imply differential regulatory mechanisms of Cav-1 in mediating TLR4 protein expression as well as signaling via protein interaction.

Interestingly, our data indicate that Cav-1 participates in the regulation of TLR4 and MyD88 expression through different pathways: TLR4 was downregulated through accelerated protein degradation, whereas MyD88 was downregulated through the suppression of mRNA expression in Cav-1−/− macrophages (Fig. 5). Impaired activation of TLR4-MyD88 signaling by Cav-1 deletion was further supported by attenuated downstream activation of NF-κB induced by bacteria or LPS (Fig. 6). Meanwhile, LPS-induced production of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α was markedly decreased in Cav-1−/− macrophages and mice (Fig. 7). Decreased cytokine production has also been reported in T. cruzi-infected Cav-1−/− mice (29). In addition, MβCD treatment suppressed CD14 and CD36 expression, phagocytosis, bacterial killing, and iNOS expression, which suggests an important role of the lipid raft in regulating the expression of these proteins and the response to bacterial infection in macrophages.

Gram-negative bacteria have often been implicated in the pathogenesis of severe sepsis and septic shock, which leads to dysregulation of innate immune response and causes multiple organ failure and death (1). Inhibition of monocyte or macrophage function is a major target for suppressing the bacterial-induced inflammatory response. Neutralization or blocking of CD14 or TLR4 affords protection and reduces organ failure and death in animal models of septic shock (30, 31). CD14 Ab attenuates LPS-induced symptoms and inflammatory response in humans (32, 33). Both CD14 and TLR4 inhibitors have been evaluated in clinical studies of patients with sepsis (34). Because our study showed that Cav-1 deletion decreases intrinsic macrophage response to bacterial infection, suppression of Cav-1 or caveolae may serve as a target to attenuate bacteria-induced inflammatory response with severe sepsis by downregulating CD14, CD36, TLR4, and MyD88 expression and subsequent signaling (see Figure, Supplemental Digital Content 4, https://links.lww.com/SHK/A55). However, because suppression of Cav-1 hindered bacterial clearance by macrophages, inhibition of Cav-1 or caveolae should be administered with concurrent antimicrobial therapy in patients with infection.

In conclusion, our results suggest that Cav-1 induction is crucial for induction of CD14 and CD36 and activation of TLR4-MyD88 signaling in response to bacterial infection (see Figure, Supplemental Digital Content 4, https://links.lww.com/SHK/A55). Deletion of Cav-1 suppresses the expression of these proteins, which results in diminished bacterial-induced phagocytosis, bacterial clearance, iNOS expression, NO production, and proinflammatory responses. Our findings provide information on possible targets for therapeutic intervention in the treatment of septic shock.

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Keywords:

Caveolin-1; macrophage; phagocytosis; Toll-like receptor 4; MyD88

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