Blocking of CD44 receptors on human pDCs cell-line GEN2.2 using BRIC 235 would eliminate the suppressive effect of rhTSG-6 in both CpG-A (Figure 6(a)) and R837 (Figure 6(b)) stimulated GEN2.2.
Taken together, not only did we observe an induction of CD44 expression by TSG-6 in human pDCs stimulated with CpG-A or R837, but more importantly, the regulatory effect ofTSG-6 seen in the human pDCs was mediated by CD44.
Suppressive Effect of TSG-6 on Human Peripheral Blood pDCs Stimulated with CpG-A or R837. Since we observed the immune-regulatory role of TSG-6 in GEN 2.2, we would like to see if the same phenomenon could also be observed in pDCs in peripheral blood from healthy donors.
Our results unfold a novel mechanism in which rhTSG-6 abated the surge of inflammatory cytokine TNF-[alpha] and IFN-[alpha] produced by human pDCs when stimulated with CpG-A or R837. These data not only extend our knowledge of the suppressive effect of TSG-6 mediated by downregulation of IRF7 phosphorylation via TLR7 or TLR9 activation in human pDCs but also confirm Choi and colleagues' observation  that TSG-6 induced a negative feedback loop to reduce the inflammatory response.
Our finding, for the first time, reveals that CpG-A and R837 can induce TSG-6 expression in human pDCs.
The induction of TSG-6 by TLR7 and TLR9 specific agonists CpG-A and R837 in GEN2.2 may provide evidence that TSG-6 is an important regulator to restore immune homeostasis in the cellular microenvironment.
TSG-6 was more effective in decreasing R837 induced IFN-[alpha] in human pDC and additionally, the suppressive effect was more evident on IFN-[alpha] than on TNF-[alpha] in both CpG-A or R837 activated human pDCs.
Our findings showed that TSG-6 reduced IRF7 phosphorylation in CpG-A or R837 stimulated GEN2.2.
First, we found that human pDCs pretreated with TSG-6 followed by CpG-A or R837 stimulation had a higher transcriptional level of CD44 and surface expression compared with no TSG-6 pretreatment.
Furthermore, exogenous addition of TSG-6 was able to downregulate CpG-A or R837 primed pDCs by decreasing IRF7 phosphorylation.
ELISA analysis of TNF-[alpha] (a) and IFN-[alpha] (b) in GEN2.2 cells treated with TSG-6 (1000 ng/mL) overnight, with or without stimulation with CpG-A at 2 [micro]M or R837 at 10 ng/mL for 6 hours.
Real-time PCR analysis of IRF7, IFN-[alpha]2, TNF-[alpha], and IL-1[beta] in GEN2.2 cells treated with TSG-6 (1000 ng/mL) overnight, followed by stimulation with CpG-A at 2 [micro]M (a) or R837 at 10 ng/mL (b) for 3 hours.