COX-2 inhibition by NS398 treatment reduced Sj infection-drived hepatomegaly, the size of granuloma, and the infiltration of immune cells around granuloma in the liver of mice
We have reported that COX-2/PGE2 axis was involved in the formation of liver fibrosis induced by Sj infection under the controlling of TLR4 pathway [17]. Chronic liver inflammation was supposed to develop into fibrosis. The size of granuloma induced by Sj egg deposition especially at mature and late stage marked as the extent of fibrosis and the amount of early stage granuloma manifested as the extent of inflammation. Herein, we found thatCOX-2 inhibition by NS398 treatment significantly attenuated hepatomegaly (Fig. 1A)(t-test: Sj/DMSO vs Sj/NS398: t(5)=78.17, P=0.046) and the average granuloma size in the liver which including the size of mature and late stage granulomas induced by single egg deposition (Fig. 1C, 1E) (t-test: Sj/DMSO vs Sj/NS398: t(39)=24703.35, P=0.0486). According to H&E staining of the liver sections, Sj infection-induced liver granulomas were classified into different stages (Table 1, Fig. 1B) [1].COX-2 inhibition decreased the percentage of early stage granuloma significantly (Fig. 1D, 1E)(t-test: Sj/DMSO vs Sj/NS398: t(5)=25.74, P=0.047). These supported that NS398 alleviated the extent of both inflammation and fibrosis in the Sj-infected mice liver.
NS398 decreased Sj infection-induced enlargement of mesenteric lymph nodes and splenomegaly of mice
Immune cells in the MLN and spleen tended to migrate or recycle into the liver and promoted hepatic inflammation & fibrosis induced by Sj infection [2]. The diameter of spleen thickness represents the severity of fibrosis. Herein, we showed that the size of MLN and spleen were significantly attenuated(Fig. 2) (t-test: Sj(-)/DMSO vs Sj(+) /DMSO: MSN size: t(5)=11.18, P=0.0001; Spleensize: t(5)=18.77, P<0.0001. Sj(+)/DMSO vs Sj(+)/NS398: MSN size: t(5)=7.13, P=0.0025; Spleen size: t(5)=13.23, P=0.0008), which suggested that NS398 lowered their contribution to liver pathogenesis during Sj infection.
NS398 didn’t affect the percentage of macrophages, neutrophils, and MDSCs in the spleen of Sj-infected mice
To explore the existence of macrophages, neutrophils, and MDSCs in Sj-infected mice spleen at 8wk of Sj infection with or without NS398 treatment, mononuclear cells were isolated from mouse spleen, and the percentage of CD45, CD11b and F4/80 co-expressed macrophage (Mφ), CD11b and Gr-1 co-expressed MDSCs, CD11b and Ly6G co-expressed neutrophils were detected by FACS (Fig. 3A). Sj-infection didn’t change significantly the quantities of macrophages in the murine spleen, but significantly increased the percentage of neutrophils (t-test: Sj(-)/DMSO vs Sj(+) /DMSO: Neu: t(5)=11.80,P=0.0063) and MDSCs (t-test: Sj(-)/DMSO vs Sj(+) /DMSO: t(5)=40.74,P=0.0083), especially polymorphonuclear leucocyte (PMN)-MDSCs (t-test: Sj(-)/DMSO vs Sj(+) /DMSO: t(5)=8.86,P=0.018) (Fig. 2B-E). However, NS398 treatment didn’t significantly change the percentage of all of these cells distributing in the murine spleen (Fig. 3B-E).
COX-2 inhibition reduced Sj infection-drivedTh1, Th2 but not Th17 cells in the spleen
To investigate whether T helper cell subsets in the spleen were involved in the alleviation of mice liver pathogenesis induced by Sj infection during NS398 treatment, mononuclear cells from infected mouse spleen with or without NS398 treatment were stained by fluorescence-labeled anti-CD3, CD4, IFN-γ, IL-4 and IL-17A, and then were detected by FACS (Fig. 4A). Sj-infection significantly increased the percentage of Th1 (t-test: Sj(-)/DMSO vs Sj(+) /DMSO: t(5)=15.76,P=0.022) and Th2 (t-test: Sj(-)/DMSO vs Sj(+) /DMSO: t(5)=8.94,P=0.0032) in the spleen, and these increasing were significantly lowered by NS398 treatment (t-test: Sj/DMSO vs Sj/NS398: Th1: t(5)=8.84, P=0.0095; Th2: t(5)=3.03, P=0.0021) (Fig. 4B, 4C ). However, Sj-infection didn’t increase the percentage of Th17 and NS398 didn’t affect this Th subpopulationin the mice spleen (Fig. 4D). The effect of NS398 on decreasing Th1 and Th2 differentiation explained its implication on alleviating the extent of liver granulomatous inflammation and fibrosis.
NS398 decreased Sj infection-triggered Tfh and Tfr cell generation
Profoundly impaired CD4+ T cell responses are associated with Sj infection. Tfh and Tfr increased in patients with Sj infection [12]. However, the function of Tfr-mediated immune responses to Sj infection and the implication of NS398 to this cell subset is unclear. Flow cytometry was performed to analyze Tfh and Tfr populations within spleen mononuclear cell preparations, as shown in Fig. 5A. As expected, the frequency of Foxp3−CD4+ T cells in CD4+CD3+ T cells was increased in Sj infected mice spleen(t-test: Sj(-)/DMSO vs Sj(+) /DMSO: t(5)=81.96,P=0.051), while Foxp3+ CD4+ T cells (regulatory T cells, Treg) was significantly decreased (t-test: Sj(-)/DMSO vs Sj(+) /DMSO: t(5)=17.54,P=0.039) (Fig. 5B). No significantly increasing of PD-1+CXCR5+ Tfh cells(t-test: Sj(-)/DMSO vs Sj(+) /DMSO: t(5)=31.5,P=0.097) but significantly elevating level of Tfr cells (t-test: Sj(-)/DMSO vs Sj(+) /DMSO: t(5)=30.18,P=0.0032) were shown in the Sj-infected murine spleen (Fig. 5C-D). NS398 treatment didn’t change the amount of Foxp3− and Foxp3+ Th cells (Fig. 5B), but it significantly decreased Tfh cells (Fig. 5C) (t-test: Sj/DMSO vs Sj/NS398:t(5)=26.56,P=0.0087) andTfr populations (Fig. 5D) (t-test: Sj/DMSO vs Sj/NS398:t(5)=16.08, P=0.024). Therefore,PD-1+CXCR5+ Tfh cells and Tfr cells in the spleen might play important role in the shistosomiasis japonica and NS398 will control their role.
NS398 treatment reduced the percentage of GCB cells in the spleen
As reported, during Sj infection, splenic B-cell number was significantly increased [13, 18]. Here, we found that at 8wk of Sj infection, the number of B cells in the mice spleen and GCB cells were increased, especially GCB cells (Fig. 6). NS398 treatment showed no change in B cells, but significantly decreased the amount of GCB cells (Fig. 6B and 6C) (t-test: Sj(-)/DMSO vs Sj(+) /DMSO: t(4)=5.49, P=0.024; Sj/DMSO vs Sj/NS398:t(5)=1.29, P=0.012). The effect of NS398 on GCB cells was consistent to Tfh and Tfr.