Role of CD44 in Regulating TLR2 Activation of Human Macrophages and Downstream Expression of Proinflammatory Cytokines

Qadri, Marwa; Almadani, Sara; Jay, Gregory D.; Elsaid, Khaled A.

doi:10.4049/jimmunol.1700713

Cited by 57 publications

(53 citation statements)

References 56 publications

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“…Macrophage CD44 receptor was shown to mediate complement-dependent and independent phagocytosis [ 48 , 49 ]. In addition to its direct phagocytic role, CD44 was shown to negatively regulate TLR stimulation [ 32 , 50 , 51 ]. Neutralization of CD44 using a CD44-specific antibody was shown to reduce NFκB nuclear translocation and proinflammatory cytokine expression and production by macrophages in response to TLR2 ligand stimulation [ 32 ].…”

Section: Discussionmentioning

confidence: 99%

“…In addition to its direct phagocytic role, CD44 was shown to negatively regulate TLR stimulation [ 32 , 50 , 51 ]. Neutralization of CD44 using a CD44-specific antibody was shown to reduce NFκB nuclear translocation and proinflammatory cytokine expression and production by macrophages in response to TLR2 ligand stimulation [ 32 ]. The anti-phagocytic activity of CD44 receptor neutralization, shown in our murine macrophage experiments, provides evidence that CD44 may act as a regulator of MSU induced inflammation in macrophages.…”

Section: Discussionmentioning

confidence: 99%

“…NFκB p65 subunit translocation studies were performed as previously described [ 32 ]. THP-1 macrophages (600,000 cells per well) were treated with MSU (100μg/ml) ± rhPRG4 (50 and 100μg/ml) for 1 h followed by cell harvest and nuclear protein extraction.…”

Section: Methodsmentioning

confidence: 99%

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Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

et al. 2018

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BackgroundGout is an inflammatory arthritis caused by monosodium urate monohydrate (MSU) crystals’ joint deposition. MSU phagocytosis by resident macrophages is a key step in gout pathogenesis. MSU phagocytosis triggers nuclear factor kappa B (NFκB) activation and production of cytokines and chemokines. Proteoglycan-4 (PRG4) is a glycoprotein produced by synovial fibroblasts and exerts an anti-inflammatory effect in the joint mediated by its interaction with cell surface receptor CD44. PRG4 also binds and antagonizes TLR2 and TLR4. The objective of this study is to evaluate the efficacy of recombinant human PRG4 (rhPRG4) in suppressing MSU-induced inflammation and mechanical allodynia in vitro and in vivo.MethodsTHP-1 macrophages were incubated with MSU crystals ± rhPRG4 or bovine submaxillary mucin (BSM), and crystal phagocytosis, cytokines and chemokines expression and production were determined. NFκB p65 subunit nuclear translocation, NLRP3 induction, caspase-1 activation and conversion of proIL-1β to mature IL-1β were studied. MSU phagocytosis by Prg4+/+ and Prg4−/− peritoneal macrophages was determined in the absence or presence of rhPRG4, BSM, anti-CD44, anti-TLR2, anti-TLR4 and isotype control antibodies. Rhodamine-labeled rhPRG4 was incubated with murine macrophages and receptor colocalization studies were performed. Lewis rats underwent intra-articular injection of MSU crystals followed by intra-articular treatment with PBS or rhPRG4. Weight bearing and SF myeloperoxidase activities were determined.ResultsrhPRG4 reduced MSU crystal phagocytosis at 4 h (p < 0.01) and IL-1β, TNF-α, IL-8 and MCP-1 expression and production at 6 h (p < 0.05). BSM did not alter MSU phagocytosis or IL-1β production in human and murine macrophages. rhPRG4 treatment reduced NFκB nuclear translocation, NLRP3 expression, caspase-1 activation and generation of mature IL-1β (p < 0.05). MSU-stimulated IL-1β production was higher in Prg4−/− macrophages compared to Prg4+/+ macrophages (p < 0.001). rhPRG4, anti-CD44, anti-TLR2 and anti-TLR4 antibody treatments reduced MSU phagocytosis and IL-1β production in murine macrophages (p < 0.05). rhPRG4 preferentially colocalized with CD44 on Prg4−/− peritoneal macrophages compared to TLR2 or TLR4 (p < 0.01). rhPRG4 normalized weight bearing and reduced SF myeloperoxidase activity compared to PBS in vivo.ConclusionrhPRG4 inhibits MSU crystal phagocytosis and exhibits an anti-inflammatory and anti-nociceptive activity in vitro and in vivo. rhPRG4’s anti-inflammatory mechanism may be due to targeting CD44 on macrophages.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

et al. 2018

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“…in the present study, the inhibition of cd44 expression may be caused by the absence of cd44-related ligands on the cell wall surface of A. fumigatus. Qadri et al (34) demonstrated that cd44-knockdown reduces nF-κB nuclear translocation and downstream proinflammatory cytokines production. Therefore, the present study assayed the expression levels of proinflammatory cytokines following transfection with cd44 sirna.…”

Section: Discussionmentioning

confidence: 99%

iTRAQ‑based proteomic analysis of the interaction of A549 human lung epithelial cells with Aspergillus fumigatus conidia

Zhang

Gao

et al. 2020

Mol Med Rep

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Severe invasive aspergillosis infection occurs when human immune function is impaired. The interaction between Aspergillus fumigatus (A. fumigatus) conidia and type ii lung epithelial cells serves an important role in disease progression. The present study compared the proteomes of a549 human lung epithelial cells with and without A. fumigatus infection. Gene ontology (Go), Kyoto encyclopedia of Genes and Genomes (KeGG) and protein interaction analyses were performed, and differential protein expression was verified by western blotting and reverse transcription-quantitative Pcr (rT-qPcr). in addition, the rna interference method, an internalization assay and eliSa were performed. isobaric tags for relative and absolute quantification analysis detected a total of 1,582 proteins, from which 111 proteins with differential expression were obtained (fold change >1.5 or <0.75). among them, 18 proteins were upregulated and 93 proteins were downregulated in a549 cells challenged with A. fumigatus. Go and KeGG analyses revealed that the altered proteins were mainly involved in biological functions, such as cell metabolism, synthesis, the cellular stress response, metabolic pathways and pyruvate metabolism. n-myc downstream-regulated gene 1 (ndrG1) expression was upregulated 1.88-fold, while cd44 expression was downregulated 0.47-fold following A. fumigatus infection. The expression levels of specific proteins were verified by western blotting and rT-qPcr. The internalization efficiency was affected by NDRG1 gene silencing. The secretion of il-6 and il-8 was affected when cd44 was inhibited. These results indicated that A. fumigatus affects lung epithelial cell metabolism and biological synthetic functions. a number of novel molecules, including ndrG1 and cd44, were found to be related to A. fumigatus infection.

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“…Cd44 +/+ and Cd44 −/− male mice (12-14 weeks old, n = 2 in each group) were euthanized, and isolation and culture of bone marrow derived macrophages (BMDMs) were performed as previously described using 10 ng/mL M-CSF [52]. BMDMs were seeded onto cover slips in 6-well plates (500,000 cells per well) and incubated with MSU crystals (100 µg/mL) for 4 h, followed by cell staining using fluoroshield-mounting medium with DAPI and visualized under a microscope.…”

Section: Synthesis Of Fluorescein-labeled P6 Peptidementioning

confidence: 99%

Design and Biological Evaluation of Colchicine-CD44-Targeted Peptide Conjugate in an In Vitro Model of Crystal Induced Inflammation

et al. 2019

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Gout is an inflammatory arthritis due to the joint deposition of monosodium urate (MSU) crystals. Phagocytosis of MSU crystals by tissue macrophages results in the generation of reactive oxygen species (ROS) and production of inflammatory cytokines and chemokines. Colchicine use in gout is limited by severe toxicity. CD44 is a transmembrane glycoprotein that is highly expressed in tissue macrophages and may be involved in gout pathogenesis. The P6 peptide is a 20-amino acid residue peptide that binds to CD44. We hypothesized that the conjugation of colchicine to the P6 peptide would reduce its off-target cytotoxicity while preserving its anti-inflammatory effect. A modified version of P6 peptide and colchicine-P6 peptide conjugate were synthesized using Fmoc/tBu solid-phase and solution-phase chemistry, respectively. A glutaryl amide was used as a linker. The P6 peptide was evaluated for its binding to CD44, association, and internalization by macrophages. Cytotoxic effects of P6 peptide, colchicine, and colchicine-P6 peptide on macrophages were compared and the inhibition of ROS generation and interleukin-8 (IL-8) secretion in MSU-stimulated macrophages treated with P6 peptide, colchicine, or colchicine-P6 peptide was studied. We confirmed that the P6 peptide binds to CD44 and its association and internalization by macrophages were CD44-dependent. Colchicine (1, 10, and 25 µM) demonstrated a significant cytotoxic effect on macrophages while the P6 peptide and colchicine-P6 peptide conjugate (1, 10 and 25 µM) did not alter the viability of the macrophages. The P6 peptide (10 and 25 µM) reduced ROS generation and IL-8 secretion mediated by a reduction in MSU phagocytosis by macrophages. The colchicine-P6 peptide significantly reduced ROS generation and IL-8 secretion compared to the P6 peptide alone at 1 and 10 µM concentrations. Conjugation of colchicine to the P6 peptide reduced the cytotoxic effect of colchicine while preserving its anti-inflammatory activity.

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Role of CD44 in Regulating TLR2 Activation of Human Macrophages and Downstream Expression of Proinflammatory Cytokines

Cited by 57 publications

References 56 publications

Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

iTRAQ‑based proteomic analysis of the interaction of A549 human lung epithelial cells with Aspergillus fumigatus conidia

Design and Biological Evaluation of Colchicine-CD44-Targeted Peptide Conjugate in an In Vitro Model of Crystal Induced Inflammation

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