Figure 1.
The surface marker molecules expressed by the Passage 5 culture of MSCs were analyzed by flow cytometry. (B) Quantification by image analysis of the positive cells. The numbers in the histograms indicate the mean fluorescence of each MSC markers. (C) Percent of MSCs obtained from culturing bone marrow cells.
Figure 2.
Phenotype analysis of different DCs.
(A) FACS histograms showed the expression of cell surface markers on iDCs, which expressed low levels of CD80, MHCII, OX62, CD86 and CD11b/c. OX62 expressed much higher than other makers, because OX62 is an important marker of rat DCs. (B) LPS- stimulated mature DCs (mDCs) showed about equally up-regulated levels of all markers. Except CD86, three makers showed above 90 percent expression. (C) MSC-DC-LPS-activated DCs in co-culture expressed lower levels of the four markers compared with mDCs, but the expression was slightly higher when compared with iDCs. This result suggests that MSCs inhibit DCs phenotype expression (MSC:DC = 1∶10). Data represent mean fluorescence intensity for the surface density of markers. (D) Quantification by image analysis of the positive cells. The numbers in the histograms indicate the mean fluorescence of each DCs markers. *P<0.05(mDC group versus iDC group), #P<0.05(MSC-DC group versus mDC group).
Figure 3.
IL-10 and IL-12 secretion as assessed by ELISA.
(A) IL-12 secreted in monocultures of MSCs, iDCs and mDCs and in the co-culture of MSC-DC (MSC:DC = 1∶10). (B) IL-10 secreted in monocultures of MSCs, iDCs and mDCs and in the co-culture of MSC-DC (MSC:DC = 1∶10). *P<0.05(mDC group versus iDC group), #P<0.05(MSC-DC group versus MSCs group).
Figure 4.
Immunofluorescence staining of MSCs, DCs and both cells in co-culture plates with IL-10.
(A) Monoculture of MSCs. (B) MSCs in Transwell co-culture with DCs. (C) Monoculture of iDCs-LPS. (D) iDCs-LPS in Transwell co-culture with MSCs (MSC:DC = 1∶10). Red staining shows IL-10 expression; DAPI blue staining of cell nuclei. Scale bar 251658240 = 251658240100 µm.
Figure 5.
Protein expression of JAK1, P-JAK1, STAT3 and P-STAT3 in monocultures of iDCs, mDCs and in co-cultures of MSC-DC (MSC:DC ratio of 1∶10).
(A) Western Blot; (B) Quantification by image analysis of the protein expression. * P<0.05 (MSC-DC group versus mDC group).
Figure 6.
ELISA analysis of co-cultures of MSC-DC supplementated with exogenous IL-10, IL-10NA and JAK inhibitor (AG490).
IL-10 and IL-12 levels in DC–IL-10 and DCs -IL-10 -IL-10NA group by ELISA. (A) IL-12; (B) IL-10.* P<0.05 (IL-10NA group versus MSC-DC group), # P<0.05 (AG490 group versus MSC-DC group), & P<0.05 (DC- IL-10 group versus MSC-DC group), § P<0.05 (DC- IL-10-IL-10NA group versus IL-10NA group),$ P<0.05 (DC- IL-10-IL-10NA group versus DC- IL-10 group).
Figure 7.
DCs surface markers (CD80, CD86, OX62, MHC-II and CD11b/c) in the MSC-DC co-culture system (ratio MSC:DC of 1∶10) supplemented with exogenous: (A) IL-10; (B) IL-10NA and (C) AG490.
(D, E) These makers expression in DC-IL-10 and DC - IL-10 - IL-10NA group by FACS. (F, G) Quantification by image analysis of the positive cells. Numbers in the histograms indicate the mean fluorescence of each DCs markers. *P<0.05(IL-10 group versus MSC-DC group), # P<0.05(IL-10NA group versus MSC-DC group), $ P<0.05(AG490 group versus MSC-DC group).
Figure 8.
Protein expression of JAK1, P-JAK1, STAT3 and P-STAT3 of DCs in co-cultures of MSC-DC (MSC:DC ratio of 1∶10).
(A) Western Blotting of the co-culture, without supplementation, or supplemented with, IL-10 or with IL-10NA (P<0.01); (B) Quantification by image analysis of the protein expression in the same conditions as (A); (C) Western Blotting of the co-culture, without supplementation, or supplemented withAG490 (P<0.01); (D) Quantification by image analysis of the protein expression in the same conditions as in (C). (E) Western Blotting of DC -IL-10 and DC- IL-10 -IL-10NA (P<0.01); (F) Quantification by image analysis of the protein expression in the same conditions as in (E). * P<0.05(IL-10 group versus MSC-DC group), # P<0.05(IL-10NA group versus MSC-DC group), $ P<0.05 (AG490 group versus MSC-DC group), & P<0.05 (DC- IL-10 group versus DC group), § P<0.05 (DCs- IL-10-IL-10NA group versus DC group).
Figure 9.
Newly proposed IL-10 feedback loop.
The figure illustrates the interplay between IL-10 and the JAK1/STAT3 signaling pathway involved in mechanism inhibition of DCs maturation by MSCs. The MSCs facilitate the combination of the cytokine IL-10 with its IL-10 receptor. This complex activates the JAK1/STAT3 signaling pathway that suppresses the DCs maturation. This inhibition of maturation causes a decrease in the expression of IL-10. Thus, the levels of the cytokine IL-10 command this positive feedback loop.