Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SHP-1 As a Critical Regulator of Mycoplasma pneumoniae -Induced Inflammation in Human Asthmatic Airway Epithelial Cells

doi: 10.4049/jimmunol.1100573

Figure Lengend Snippet: M. pneumoniae infection induced TLR2–SHP-1 dynamic association. (A) Immunoblot analysis of baseline TLR2 expression in airway epithelial cells from nonasthmatic and asthmatic subjects. The data representing the percentage of densitometry values of nonasthmatic cells are expressed as mean ± SEM (right panel). (B) TLR2-associated SHP-1 levels in airway epithelial cells was measured by coimmunoprecipitation. TLR2 was immunoprecipitated from airway epithelial cell lysates of nonasthmatic and asthmatic subjects without mycoplasma infection (0 h), infected with mycoplasma for 1 and 2 h. TLR2 immunoprecipitates were blotted with anti-TLR2 (top left panel) and anti–SHP-1 (bottom left panel) Ab. TLR2-associated SHP-1 at 2 h after M. pneumoniae infection is expressed as fold change of densitometry values from nonasthmatic cells without mycoplasma treatment (0 h) and was increased in nonasthmatic cells compared with asthmatic cells. The data are presented as mean ± SEM (n = 5). *p < 0.01 compared with asthmatic group.

Article Snippet: The membranes were probed with rabbit anti-human TLR2 Ab (IMGENEX, San Diego, CA), mouse anti-human SHP-1 (Santa Cruz Biotechnology), or a Ser 473 phosphospecific Ab to Akt (Cell Signaling, Danvers, MA) overnight at 4°C.

Techniques: Infection, Western Blot, Expressing, Immunoprecipitation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SHP-1 As a Critical Regulator of Mycoplasma pneumoniae -Induced Inflammation in Human Asthmatic Airway Epithelial Cells

doi: 10.4049/jimmunol.1100573

Figure Lengend Snippet: Proposed model for the mechanism of SHP-1–mediated inhibition of M. pneumoniae-activated TLR2 signaling in normal and asthmatic airway epithelial cells. The ligation of TLR2 by M. pneumoniae initiates TLR2-mediated proinflammatory signaling pathway, resulting in the production of IL-8. M. pneumoniae binding to TLR2 activates and recruits SHP-1, which inhibits the nuclear translocation of NF-κB directly or through inhibition of PI3K/Akt abrogating NF-κB activation and subsequently prevents IL-8 production. In addition, the nuclear SHP-1 may also inhibit NF-κB function by certain nuclear mediators (left panel). In asthmatic airway epithelial cells, M. pneumoniae-induced SHP-1 activation is defective, which contributes to the increased activation of PI3K/Akt and NF-κB, as well as abundant IL-8 production (right panel).

Article Snippet: The membranes were probed with rabbit anti-human TLR2 Ab (IMGENEX, San Diego, CA), mouse anti-human SHP-1 (Santa Cruz Biotechnology), or a Ser 473 phosphospecific Ab to Akt (Cell Signaling, Danvers, MA) overnight at 4°C.

Techniques: Inhibition, Ligation, Binding Assay, Translocation Assay, Activation Assay

Journal: Nature communications

Article Title: Epigenetically regulated digital signaling defines epithelial innate immunity at the tissue level.

doi: 10.1038/s41467-021-22070-x

Figure Lengend Snippet: Fig. 4 Bimodal TLR2 expression limits the fraction of responder cells. a Wild-type MCF10A were stimulated with 1 µg/ml Pam3CSK4 or 100 ng/ml IL-1β. Quantification of nuclear/cytoplasmic p65 immunofluorescent stain is shown in histograms on logarithmic scale. Data represents three replicates. b Cells were transduced with TLR2 under a Tet Responsive Element 3rd Generation (TRE3G) promoter and incubated with or without Doxycycline (2 µg/ml) for 24 h. Cells were then stimulated with 1 µg/ml Pam3CSK4 and immunostained to determine responder status as in Fig. 2a. Representative images of three replicates are shown. Scale bar, 100 µm. c Immunoblot against TLR2 in cells treated with or without DNMT inhibitor (5-AzacytidineC, 500 nM) or HDACi (SAHA 800 nM). HSC70 was used as a loading control. Data represents two replicates. d Dual smRNA FISH-immunofluorescence for TLR2 and NF-κB was done as described in Methods section. Dashed line indicates cell boundaries, yellow squares highlight the TLR2 probes. Representative images of two replicates are shown. Scale bar, 10 µm. Quantification of TLR2 mRNAs in responders and non-responders determined by NF-κB nuclear translocation. N = 354 cells total (148 Responder, 206 Non-Responder), p = 3.8e-19 by χ2 test with 9 degrees of freedom. e Western blot of TRE3G::TLR2 cells treated with a 4-h pulse of Doxycycline (2 µg/ml) and cultured for the indicated time. Relative amounts are normalized to HSC70 loading control. Standard deviation represents two independent experiments for each condition. Swarmplots represent technical replicates of the percentage of responding cells (top) and nucleus/cytoplasm NF-ĸB amplitude (bottom) in the same cells.

Article Snippet: Antibodies used were TLR2 (CST 12276) and HSC70 (Santa Cruz 7298).

Techniques: Expressing, Staining, Transduction, Incubation, Western Blot, Control, Translocation Assay, Cell Culture, Standard Deviation

Journal: Nature communications

Article Title: Epigenetically regulated digital signaling defines epithelial innate immunity at the tissue level.

doi: 10.1038/s41467-021-22070-x

Figure Lengend Snippet: Fig. 7 High steady-state NF-ĸB signaling increases the responder percentage. a WT monolayers were treated with indicated stimulus four times over a 9- day period. Concentrations of inputs were: Poly(I:C) 1 µg/ml, TNFα 100 ng/ml, Flagellin 1 µg/ml, Pam3CSK4 1 µg/ml, IL-1β 100 ng/ml, IFNγ 5 µ/ml, and TGFβ 5 ng/ml. Error bars represent six technical replicates with n > 1000 cells per replicate. Two-sample t test *p < 0.05 and ***p < 0.001. b Schematic indicating experimental workflow for data in panel c. Monolayers were stimulated with inputs every 2 days and washed or not after 30 min. After the last stimulation (day 8), all monolayers were washed for 24 h before determining responder fraction with Pam3CSK4 as in Fig. 2a. c Monolayers treated as detailed in panel b, input concentrations as in panel a. Two biological replicates, five technical replicates for each condition, ***p < 0.001; two-sample t-test. d Summary of model. Epithelial tissue contains cells that are all-or-nothing responsive or non-responsive to lipopeptide agonists. Response status is defined by TLR2 expression, which is controlled by epigenetic modifications to the TLR2 promoter. At steady state the fraction of responders is low; however, high inflammatory signaling involving innate immune cell types (e.g. dendritic cells, macrophages), leads to changes in the rate of epigenetic switching that increase the fraction of responsive cells in the tissue.

Article Snippet: Antibodies used were TLR2 (CST 12276) and HSC70 (Santa Cruz 7298).

Techniques: Expressing