1. Correlation between Th9 and SSc
Compared to normal subjects, IL-9R and IL-17R were highly expressed in the skin lesions of SSc patients
(Fig. 1). IL-9 and IL-9R mRNA
levels of PBMCs and IL-9 levels in SSc serum were increased (Fig. 2). Flow cytometry showed that the proportion of CD4+
IL-9+ T cells in PBMCs of SSc patients was significantly higher (Fig. 3). The proportion of Th9 was associated with SSc.
2. Crosstalk between Th17 and Th9 cells in vitro and regulation of tanshinone IIA
2.1 IL-9 and SSc serum promote Th17 differentiation and the reversal effect of tanshinone IIA
The results showed that the proportion of Th17 cells in IL-9 and SSc serum groups was significantly higher than
that in the control group; however, the proportion of Th17 cells in the IL-9 neutralizing antibody group was
significantly lower than that in the control group, and IL-9 neutralizing antibody decreased the promoting
effect of serum on Th17 cells (Fig. 4A). These
findings indicated that IL-9 stimulates the differentiation of immature T lymphocytes into Th17 cells in vitro,
as well as the differentiation of immature T lymphocytes into Th17 cells. The data showed that tanshinone IIA
decreases the proportion of Th17 and inhibits the effect of SSc serum on Th17 cells as compared to the control
(Fig. 4B). Moreover, the content of IL-17 in
the culture supernatant of each group was detected by ELISA. These results showed that IL-9 and SSc serum
significantly promotes the secretion of IL-17 by immature T lymphocytes, while IL-9 neutralizing antibody and
tanshinone IIA significantly inhibits the secretion of IL-17 by immature T lymphocytes and reverses the
triggering effect of IL-9 and SSc serum (Fig. 4C).
2.2 IL-17 and SSc serum promote Th9 differentiation and the reversal effect of tanshinone IIA
The results showed that the proportion of Th9 cells in the IL-17 and SSc serum groups was significantly higher
than that in the control group; however, the proportion of Th9 cells in IL-17 neutralizing antibody group was
significantly lower than that in the control group and IL-17 neutralizing antibody decreased the promoting
effect of serum on Th9 cells (Fig. 5A). These
findings indicated that IL-17 stimulates the differentiation of immature T lymphocytes into Th9 cells in vitro,
and IL-17 in the serum of SSc patients stimulates the differentiation of immature T lymphocytes into Th9 cells.
In addition, tanshinone IIA decreases the proportion of Th9 and inhibits the promoting effect of SSc serum on
Th9 cells as compared to the control (Fig. 5B).
Moreover, the content of IL-9 in the culture supernatant of each group was detected by ELISA. The results showed
that IL-17 and SSc serum significantly promoted the secretion of IL-9 by immature T lymphocytes, while IL-17
neutralizing antibody and tanshinone IIA significantly inhibited the secretion of IL-9 by immature T lymphocytes
and reversed the promoting effect of IL-17 and SSc serum (Fig. 5C).
3. Effects of IL-17 and IL-9 on the functional activation of DVSMCs and regulation of tanshinone
IIA
3.1 IL-9 and SSc serum promote the expression of IL-17R in DVSMCs, and tanshinone IIA reverses
this effect
The DVSMCs of SSc patients were isolated and incubated with IL-9, IL-9 neutralizing antibody, SSc serum, SSc
serum and IL-9 neutralizing antibody, tanshinone IIA, IL-9 and tanshinone IIA, and SSc serum and tanshinone IIA
for 3 days. The expression of IL-17R was detected by immunofluorescence. The results showed that IL-9 and SSc
serum significantly promote the expression of IL-17R, and IL-9 neutralizing antibody and tanshinone IIA reversed
the promoting effect of IL-9 and SSc serum (Fig. 6).
3.2 IL-17 and SSc serum promote the expression of IL-9R in DVSMCs, and tanshinone IIA reverses this
effect
The DVSMCs were incubated with IL-17, IL-17 neutralizing antibody, SSc serum, SSc serum and IL-17 neutralizing
antibody, tanshinone IIA, IL-17 and tanshinone IIA, and SSc serum and tanshinone IIA for 3 days. The expression
of IL-9R was detected by immunofluorescence. The results showed that IL-17 and SSc serum significantly promote
the expression of IL-9R, and IL-17 neutralizing antibody and tanshinone IIA reversed the promoting effect of
IL-17 and SSc serum (Fig. 7).
3.3 IL-9 and SSc serum promote the functional activation of DVSMCs, and tanshinone IIA reverses this
effect
The DVSMCs were incubated with IL-9, IL-9 and IL-9 neutralizing antibodies, IL-9 and tanshinone IIA, SSc serum,
SSc serum and IL-9 neutralizing antibodies, and SSc serum and tanshinone IIA for 3 days. Collagen I, collagen
III, α-SMA, P-P38, P38, P-ERK, and ERK were detected by WB. The results showed that IL-9 and SSc serum
significantly promote the expression of collagen I, collagen III, α-SMA, P-P38, and P-ERK. Moreover, IL-9
neutralizing antibody and tanshinone IIA reversed the effects of IL-9 and SSc serum (Fig. 8).
3.4 IL-17 and SSc serum promote the functional activation of DVSMCs, and tanshinone IIA reverse this
effect
The DVSMCs were incubated with IL-17, IL-17 and IL-17 neutralizing antibodies, IL-17 and tanshinone IIA, SSc
serum, SSc serum and IL-17 neutralizing antibodies, and SSc serum and tanshinone IIA for 3 days. Collagen I,
collagen III, α-SMA, P-P38, P38, P-ERK, and ERK were detected by WB. The results showed that IL-17 and SSc serum
significantly promote the expression of collagen I, collagen III, α-SMA, P-P38, and P-ERK. IL-17 neutralizing
antibody and tanshinone IIA reversed the effects of IL-17 and SSc serum (Fig. 9).
3.5 Tanshinone IIA inhibits the synergistic effect of IL-9 and IL-17 on functional activation of DVSMCs
DVSMCs were treated with IL-17, IL-9, IL-17 and IL-9, IL-17 neutralizing antibody and IL-9 neutralizing antibody,
IL-17 and IL-9 and tanshinone IIA, SSc serum, SSc serum/IL-17 neutralizing antibody/IL-9 neutralizing antibody,
and SSc serum and tanshinone IIA for 3 days. The proteins were collected for WB detection of collagen I,
collagen III, α-SMA, P-P38, P38, P-ERK, and ERK. The results showed that IL-17, IL-9, and SSc serum
significantly promote the expression of collagen I, collagen III, α-SMA, P-P38, and P-ERK. In addition, IL-9 and
IL-17 neutralizing antibodies and tanshinone IIA inhibited the synergistic effect of IL-9 and IL-17
(Fig. 10).