IFNγ and TNFα induce IL-6 expression synergistically in MSCs
Previous studies have shown that cytokines such as IFNγ and TNFα can markedly affect the physiological functions of bone marrow-derived MSCs (6
). Recently, IL-6 also has been shown to play an important role in MSC function (20
). To investigate whether IFNγ and TNFα affect the expression of IL-6, we added recombinant cytokines to cultures of cloned murine MSCs, cultured the cells for ?? days, and assayed for IL-6 mRNA by real-time PCR. We found that, when added concomitantly to MSCs in vitro,
IFNγ and TNFα acted synergistically to dramatically increase IL-6 expression (), while either cytokine was much less effective individually. This effect was concentration-dependent and occurred as early as three hours, and endured for at least three days ().
IFNγ and TNFα induce IL-6 expression synergistically in MSCs
C/EBPβ mediates the synergistic induction of IL-6 by IFNγ and TNFα
The transcription factor C/EBPβ is well-known to regulate IL-6 expression (14
). To determine whether C/EBPβ is involved in the synergistic effect of IFNγ and TNFα on IL-6 expression in MSCs, we used siRNA technology to inhibit C/EBPβ production. This knockdown resulted in >95% decrease in C/EBPβ mRNA in MSCs in vitro
(), a result that was verified at the protein level by western blotting analysis (). We found that knockdown of C/EBPβ resulted in markedly reduced IL-6 upregulation in MSCs in response to IFNγ and TNFα, added alone or in combination (). These results suggest that C/EBPβ is critical in the synergistic induction of IL-6 by IFNγ and TNFα.
C/EBPβ is involved in the IL-6 expression induced by IFNγ and TNFα
To further verify the role of C/EBPβ in the production of IL-6 induced by TNFα and IFNγ, we examined the expression of C/EBPβ mRNA and protein in MSCs. IFNγ and TNFα each induced respectable levels of C/EBPβ expression, and much greater expression occurred when both cytokines were present (). Therefore, the synergistic induction of IL-6 in MSCs in response to IFNγ and TNFα is likely to occur through the upregulation of C/EBPβ expression.
IFNγ and TNFα induce C/EBPβ expression synergistically in MSCs
Synergistic regulation of other genes by IFNγ and TNFα is also C/EBPβ-dependent
IFNγ and TNFα also synergistically induced the expression of additional genes, such as iNOS, CCL5, and CXCL1, as shown in . We therefore asked if upregulation of these genes is also C/EBPβ-dependent. We knocked down C/EBPβ in MSCs, and measured the expression of iNOS, CCL5, and CXCL1 in response to IFNγ and TNFα. C/EBPβ knockdown crippled the normal upregulation of iNOS, CCL5 and CXCL1 (), suggesting that C/EBPβ is a key molecule in the synergistic effects of IFNγ and TNFα on gene expression in MSCs.
C/EBPβ is involved in the synergistic upregulation of several genes byIFNγ and TNFα
C/EBPβ also mediates the synergistic induction of IL-6 by IFNγ combined with IL-1β or LPS
Previous experiments have shown that IFNγ can synergize with IL-1β or LPS, as well as TNFα, to induce gene expression in various cell types (6
). To examine the possible role of C/EBPβ in the response of MSCs to IFNγ and IL-1β or LPS, we measured C/EBPβ expression in cytokine-stimulated MSCs and found that both IL-1β and LPS act synergistically with IFNγ to induce C/EBPβ (). To determine whether the increased levels of IL-6 observed after concomitant stimulation with IFNγ and IL-1β or IFNγ and LPS is mediated by C/EBPβ, we measured IL-6 mRNA by real time PCR in MSCs with or without C/EBPβ knockdown after culture with these cytokine pairs. Both IL-1β and LPS displayed dramatic synergy with IFNγ in the induction high levels of IL-6 expression. Furthermore, C/EBPβ knockdown severely curtailed this effect (). Therefore, C/EBPβ is a critical mediator of the synergy between IFNγ and several other proinflammatory stimuli in upregulating gene expression in MSCs.
C/EBPβ is involved in the synergistic upregulation of IL-6 by IFNγ in combination with IL-1β or LPS
STAT1 is not involved in the induction of IL-6 expression in MSCs by IFNγ and TNFα
It is well known that IFNγ activates the JAK1/2-STAT1 signaling pathway in a variety of cell types, and this pathway accounts for the majority of IFNγ-mediated biological effects. Consistent with previous reports, we also found that IFNγ induces STAT1 expression in MSCs (). To better understand the molecular mechanism by which IFNγ synergizes with TNFα to induce gene expression, we knocked down STAT1. Transfection of MSCs with STAT1-specific siRNA resulted in > 95% elimination of STAT1 mRNA () and similar prevention of STAT1 protein production (). Interestingly, however, STAT1 knockdown did not inhibit the upregulation of IL-6 or CXCL1 induced by IFNγ + TNFα, while iNOS and CCL5 were greatly reduced (). This strongly suggests that while STAT1 is induced by IFNγ in MSCs, it regulates only some genes (iNOS and CCL5, but not IL-6 or CXCL1), unlike C/EBPβ, which upregulates all of these genes.
The role of STAT1 in the synergistic induction of gene expression by IFNγ and TNFα
STAT1 does not affect C/EBPβ expression in MSCs
As described above, knockdown of C/EBPβ, but not STAT1, inhibited the expression of IL-6 and CXCL1. This suggests that STAT1 does not affect C/EBPβ expression. To further verify this, we measured C/EBPβ levels in MSCs and found indeed that knockdown of STAT1 had no effect on C/EBPβ expression (). Therefore, even for STAT1-dependent genes such as iNOS and CCL5, transcription factors STAT1 and C/EBPβ are likely to act in parallel rather than by regulating each other.
Knockdown of STAT1 does not affect C/EBPβ expression
Knockdown of C/EBPβ partly inhibited the immunosuppression by MSCs
We have shown that the same cytokine combinations that induce the immunomodulatory function of MSCs also synergistically upregulate C/EBPβ. We have also reported that chemokines and iNOS play important roles in effecting immunosuppression by MSCs (6
). Since C/EBPβ mediates the synergistic expression of iNOS and chemokines, we predicted that knockdown of C/EBPβ would reverse the immunosuppressive function of MSCs. To test this, siC/EBPβ-transfected MSCs were cultured with splenocytes and the effect on proliferation in response to anti-CD3 was determined. As shown in the supplemental figure
, knockdown of C/EBPβ in MSCs inhibited their ability to suppress splenocyte proliferation, indicating that the immunosuppressive function is at least in part dependent on C/EBPβ.