Granulocyte colony-stimulating factor: Its role in gut-homing macrophage generation and colitis, and production by probiotics

The pleiotropic cytokine granulocyte-colony stimulatory factor (G-CSF) is mainly required for the generation of neutrophils, but its role in macrophage generation has also been reported. In addition, G-CSF is effective for the down-regulation of inflammatory cytokines and ameliorating gut disorders, such as colitis. However, the G-CSF function in macrophage generation and gut immunity remains unclear. The first focus of this thesis was to assess the role of G-CSF in macrophage generation and its contribution to gut immunity. G-CSF was found to promote the generation of Gr-1high/F4/80+ macrophages in macrophage (M)-CSF-treated bone marrow cells, most likely through suppressing cell death. Gr-1high macrophages showed anti- inflammatory/regulatory macrophage (M2)-like cytokine and surface marker profiles. G-CSF receptor deficient (G-CSFR-/-) mice harbored less gut macrophages, but had a similar number of neutrophils in the gut. In addition, adoptive transfer of G-CSF- treated bone marrow-derived macrophages (G-BMDM) showed a dominant gut- homing phenotype. G-CSFR-/- mice were also more susceptible to dextran sulfate sodium (DSS)-induced colitis than wild-type mice and adoptive transfer of G-BMDM protected these mice from DSS-induced colitis. The second focus of this thesis was to explore the signaling mechanism(s) controlling the preferential G-CSF production over inflammatory cytokines in probiotic bacteria-exposed BMDM. Lactobacillus rhamnosus GR-1 (GR-1) renders several immunomodulatory effects, at least in part, through preferentially inducing G-CSF in macrophages. However, the mechanism(s) by which GR-1 induces preferential G-CSF production in macrophages is still unknown. Among 84 genes tested, G-CSF was the cytokine induced at highest levels in GR-1-treated BMDM, but the induction of inflammatory cytokines, such as TNF-α, was minimal. The signaling pathway of GR-1-preferential G-CSF production was TLR2-, NF-κB-, ERKs- and PI3K/Akt-dependent. A secreted protein-like molecule(s) was found to be responsible for GR-1-preferential G-CSF production. Collectively, these results demonstrated the immune regulatory function of G-CSF on macrophages in gut immunity and a potential mechanism of action of certain probiotics

Preferential production of G-CSF by a protein-like Lactobacillus rhamnosus GR-1 secretory factor through activating TLR2-dependent signaling events without activation of JNKs

Different species and strains of probiotic bacteria confer distinct immunological responses on immune cells. Lactobacillus rhamnosus GR-1 (GR-1) is a probiotic bacterial strain found in both the intestinal and urogenital tracts, and has immunomodulatory effects on several cell types including macrophages. However, detailed immunological responses and the signaling mechanism involved in the response are largely unknown

Additional file 1: Table S1. of Preferential production of G-CSF by a protein-like Lactobacillus rhamnosus GR-1 secretory factor through activating TLR2-dependent signaling events without activation of JNKs

Expression of cytokines and chemokines in immortalized BMDMs treated with GR-1 and TLR ligands. (DOC 105 kb

p62/SQSTM1 Enhances NOD2-Mediated Signaling and Cytokine Production through Stabilizing NOD2 Oligomerization

<div><p>NOD2 is a cytosolic pattern-recognition receptor that senses muramyl dipeptide of peptidoglycan that constitutes the bacterial cell wall, and plays an important role in maintaining immunological homeostasis in the intestine. To date, multiple molecules have shown to be involved in regulating NOD2 signaling cascades. p62 (sequestosome-1; SQSTM1) is a multifaceted scaffolding protein involved in trafficking molecules to autophagy, and regulating signal cascades activated by Toll-like receptors, inflammasomes and several cytokine receptors. Here, we show that p62 positively regulates NOD2-induced NF-κB activation and p38 MAPK, and subsequent production of cytokines IL-1β and TNF-α. p62 associated with the nucleotide binding domain of NOD2 through a bi-directional interaction mediated by either TRAF6-binding or ubiquitin-associated domains. NOD2 formed a large complex with p62 in an electron-dense area of the cytoplasm, which increased its signaling cascade likely through preventing its degradation. This study for the first time demonstrates a novel role of p62 in enhancing NOD2 signaling effects.</p> </div

p62 co-localizes with NOD2 through the NBD domain of NOD2.

<p><b>A</b>. HEK293T cells were transfected for 24 h with scramble siRNA (left top panel), si-p62 (right top panel), and GFP-NOD2. GFP-NOD2 was visualized using confocal microscopy as described in “Methods”. <b>B</b>. Similarly, DsRed-NBD domain, LRR region or full-length NOD2 and GFP-p62 expression vectors were transfected in HEK293T cells and co-localization of these proteins was examined using confocal microscopy. <b>C</b>. Immunogold staining of co-localized pCMV-HA-p62 (18 nm colloidal gold) and GFP-NOD2 (10 nm colloidal gold) in HEK293T cells. Cells on grids were viewed using a transmission electron microscope. Scale bars: 500 nm (left bottom), 100 nm (middle, right bottom). <b>D</b>. HEK293T cells were transfected with DsRed-NOD2 and GFP-LC3 plasmids. Cells were observed by confocal microscopy and images were acquired using ZEN software.</p

NOD2 physically interacts with p62.

<p><b>A–C</b>. HEK293T cells were transiently transfected with expression vectors encoding GFP-tagged p62 (GFP-p62) and/or Myc-tagged NOD2 (Myc-NOD2). After 24 h, total cell lysates were subjected to immunoprecipitation using anti-GFP (A) or anti-Myc (B) antibodies and the immune complexes were resolved by SDS-PAGE followed by immunoblotting against GFP and HA. <b>C</b>. Similar experiments as A-B were performed but with or without N-glycorylated muramyldipeptide (gMDP: 5 µg/mL) treatments for 4 h. Data shown are representative images of 3 independent experiments.</p

p62 enhances pro-IL-1β expression and TNF-α production in macrophages.

<p><b>A</b>. RAW 264.7 cells were transfected with scrambled (si-Scramble) or p62 targeting (si-p62) small interference RNAs using Lipofectamine™ 2000. Twenty four hours post-transfection, cells were treated with a low dose of LPS (50 ng/mL) for 4 h, rinsed with complete media twice, and then incubated with gMDP (5 µg/mL) for another 4 h. Expression of pro-IL-1β was detected using an antibody against IL-1β and p38 was used as a loading control. Densitometric analysis of blots was done using ImageJ (NIH). Data are expressed as mean ± S.D. (n = 3). N.S., not significant; * p<0.05 (Student t-test). <b>B</b>. RAW 264.7 cells were transfected with scramble siRNA or p62-siRNA as above A, and cells were treated with LPS (20 ng/mL) for 4 h. After two washes with complete media, cells were further incubated with gMDP (5 µg/mL) for another 4 h and TNF-α concentrations in cell culture media were measured by ELISA according to the manufacturer's instructions (eBioscience). Data are expressed as mean ± S.D. (n = 4); * p<0.05 (Tukey's Multiple Comparison Test). <b>C</b>. THP-1 cells were stably transfected with sh-scramble (sh-Scramble) or p62 targeting (sh-p62) small-hairpin RNA producing constructs as described in “Methods”. Three cell clones stably knocked down in p62, pooled sh-Scramble control clones and non-treated wild-type cells were treated with gMDP (5 µg/mL) for 4 h and TNF-α production in the cell culture media was measured using TNF-α bioassay as described in “Methods”.</p

The NBD of NOD2 interacts with both TRAF6 and UBA domains of p62.

<p><b>A</b>. NOD2 (left top panel) and p62 (right top panel) structures, and their mutant constructs are schematically presented. <b>B</b>. HEK293T cells were transfected with GFP-p62 and Myc-NBD (left middle panel), GFP-p62 and Myc-CARD (center middle panel) or GFP-p62 and Myc-ΔLRR (LRR region-deleted NOD2) (right middle panel). <b>C</b>. Similarly, HEK293T cells were transiently transfected with GFP-TRAF6 domain of p62 and Myc-ΔLRR (left bottom panel), GFP-UBA domain of p62 and Myc-ΔLRR (middle bottom panel), and GFP-PB1 domain of p62 and Myc-ΔLRR (right bottom panel); co-immunoprecipitation assays were performed as described in legend to Fig. 2. Data shown are representative images of 3 independent experiments.</p

p62 is required for the activation of NF-κB and p38 MAPK, and ubiquitination of RIP2 and TRAF6.

<p><b>A</b>. HEK293T cells were transfected with pCMV-Myc-NOD2 and NF-κB luciferase reporter constructs in the presence of scrambled or p62-targeting small interference RNAs (si-p62). Cells were then treated with gMDP (5 µg/mL) for 4 h and NF-κB activity was measured. Data are expressed as the fold of luciferase activity ± SD (n = 3). <b>B-C</b>. HEK293T cells stably expressing NOD2 were first treated with scramble siRNA or si-p62 for 24 h, and then transfected with expression vectors for HA-ubiquitin (HA-Ub) and pcDNA3-Myc-RIP2 or pCMV-Myc-TRAF6 for another 24 h. After treating the cells with gMDP (5 µg/mL) for 4 h, RIP2 (B) or TRAF6 (C) was immunoprecipitated with Myc antibodies from total cell lysates and the immune complexes were resolved by SDS-PAGE followed by immunoblotting against HA. Myc-RIP2 or Myc-TRAF6, NOD2 and HA-ubiquitin were analyzed by immunoblot as the inputs (bottom panels). p62 protein levels were also measured by immunoblot. Data shown are representative images of 3 independent experiments. <b>D</b>, HEK293T cells stably expressing NOD2 were first treated with scramble siRNA or si-p62 for 24 h, and then treated with gMDP (5 µg/ml) for the times indicated. Activation of p38 was analysed through immunoblotting against tyrosine phosphoryled p38. The ImageJ (NIH) program was used for densitometry analysis of phosphor-p83 bands and data were expressed as mean ± S.D. (n = 3).</p