Table 1.
TLR3 stimulation alters the expression of JAK/STAT-related genes.
Figure 1.
TLR3 stimulation activates a JAK2/STAT1 pathway in hMSC.
(A) Human MSC gene induction, following 6 hours of poly(I:C) treatment, was analyzed by qPCR using 5 separate donors. Data are presented as mean ± SEM. Significance was determined by comparing the poly(I:C) treated samples to the untreated control. *p<0.05. (B-C) JAK2/STAT1 phosphorylation was analyzed by Western blot following TLR3 stimulation for 0–6 hours. Of note, the monoclonal STAT1 antibody preferentially binds total STAT1, which is why total STAT1 expression appears inversely proportional to phosphorylated STAT1. Densitometry was determined by subtracting overall background, then each experimental band was normalized to the actin loading control band within its lane, and fold change was calculated based upon the untreated control band. Density values below each band are representative of results from 3 separate donors.
Figure 2.
SOCS overexpression disrupts STAT1 activation.
(A-B) SOCS1 and SOCS3 expression following TLR3 activation in hMSC by Western blot analysis. (C) STAT1 activation in untransfected, mock-transfected, SOCS1- or SOCS3-overexpressing hMSC after 4 hours of poly(I:C) treatment. (D) IRF1 expression in untransfected, mock-transfected, SOCS1- or SOCS3-overexpressing hMSC after 2 hours of poly(I:C) treatment. Density values below each band are representative of results from 3 separate donors.
Figure 3.
TLR3-mediated internalization and degradation of CXCR4 and CXCR7.
(A, C) TLR3-stimulated, non-permeabilized hMSC were stained for cell-surface expression of CXCR4, CXCR7 or isotype control and analyzed by flow cytometry. Statistical significance was determined by comparing the experimental mean fold change in MFI to the untreated control (black, p<0.05; aqua, p<0.01). (B, D) Western blot analysis of total CXCR4 and CXCR7 expression following poly(I:C) treatment. Density values below each band are representative of results from 3 separate donors.
Figure 4.
CXCR4 and CXCR7 are internalized following TLR3 activation.
Human MSC were plated on chamber slides and treated for 0, 4, or 6 hours with poly(I:C) and then stained with an Alexa-488 conjugated secondary only control, Alexa-488 labeled α-CXCR4 (A-C) or α-CXCR7 (D-F) and DAPI. 40X. Scale bar represents 10 µm. Photomicrographs are representative of results from 5 separate donors.
Figure 5.
SOCS3 modulates the subcellular location of CXCR4 in hMSC.
Mock-transfected (A-C), SOC1-overexpressing (D-F) or SOCS3-overexpressing (G-I) hMSC were plated onto chamber slides and treated for 0, 4, or 6 hours with poly(I:C) and then stained with an Alexa-488 conjugated secondary only control or Alexa-488 labeled α-CXCR4 and DAPI. 40X. Scale bar represents 10 µm. Photomicrographs are representative of results from 3 separate donors.
Figure 6.
SOCS1 and SOCS3 both modulate the subcellular location of CXCR7 in hMSC.
Mock-transfected (A-C), SOC1-overexpressing (D-F) or SOCS3-overexpressing (G-I) hMSC were plated onto chamber slides and treated for 0, 4, or 6 hours with poly(I:C) and then stained with an Alexa-488 conjugated secondary only control or Alexa-488 labeled α-CXCR7 and DAPI. 40X. Scale bar represents 10 µm. Photomicrographs are representative of results from 3 separate donors.
Figure 7.
SOCS1 and SOCS3 inhibit TLR3 signaling in hMSC.
TLR3 signaling induces cytokine expression and mediates the internalization of both CXCR4 and CXCR7, as well as the degradation of CXCR4. A JAK2/STAT1 pathway is also activated, and subsequent upregulation of SOCS1 and SOCS3 inhibits TLR3-mediated signaling by targeting different branches of the signaling pathways. SOCS1 inhibits STAT1 phosphorylation and CXCR7 internalization, while SOCS3 inhibits IRF1 upregulation and the internalization of both CXCR4 and CXCR7.