PKCλ/ι deficiency could not alter the amounts of adult schistosomes and schistosome eggs in the infected mice
The mice were dissected 7 weeks after infection; the number of adult schistosomes and eggs in the liver tissue were counted. The results showed that the average worm burdens in the WTSJ and KOSJ groups were 8.40 ± 1.70 and 7.40 ± 1.30 per mouse, respectively (Fig. 1a); hence, no statistical significance was observed (P > 0.05). The number of egg burdens in the liver in WTSJ and KOSJ groups was 46,117.00 ± 5399.00 vs. 43,995.00 ± 4506.00 eggs per gram, respectively (Fig. 1b), indicating no statistically significant difference (P > 0.05).
PKCλ/ι deficiency reduces serum levels of ALT and HMGB1 in mice infected with S. japonicum
HMGB1 level in the serum of the murine host was determined by conducting ELISA; the serum levels of ALT were determined using the microplate method. The results showed that the ALT level in the WT group, WTSJ group, KO group and KOSJ group was 7.10 ± 0.70, 19.32 ± 3.10, 9.35 ± 1.00, and 11.90 ± 2.50 U/l, respectively (Fig. 2a). Schistosoma japonicum infection obviously elevated the content of ALT in serum in WTSJ and KOSJ mice, yet the increasing rate was lower in KOSJ group than that in WTSJ group. HMGB1 in WT group and WTSJ group was 1164.00 ± 201.00 and 27,820.00 ± 8612.00 pg/ml, respectively, indicating a 23.9-fold increase after infection. Consistently, HMGB1 concentrations showed a 5.3-fold increase in the KOSJ group compared with that in the KO group (36,441 ± 5491 vs. 6858 ± 4749 pg/ml). As illustrated in Fig. 2b, PKCλ/ι deficiency obviously suppressed the increasing proportion of serum HMGB1 levels induced by schistosome infection.
PKCλ/ι deficiency alleviates liver granuloma and fibrosis in mice infected with S. japonicum
Seven weeks after infection, the mice showed obvious formation of egg granuloma in the liver and indication of hepatic fibrosis (Fig. 3a, d). The average area of single liver granuloma in the WTSJ was significantly higher than that in KOSJ group (1,433,702.04 ± 16,294.01 vs. 85,295.10 ± 5399.30 μm2, P < 0.001) (Fig. 3b). The area of single egg granuloma collagen in WTSJ group was significantly higher than that in KOSJ group (163,103.01 ± 11,103.20 vs. 93,778.20 ± 8949.05 μm2, P < 0.001) (Fig. 3c). These results suggest that PKCλ/ι loss-of-function mutation can significantly suppress egg granuloma and diminish fibrotic lesions.
As seen in Fig. 3e, the content of HYP was 0.11 μg/mg, 0.29 μg/mg, 0.07 μg/mg and 0.23 μg/mg, respectively. KO group exhibited significantly lower levels of HYP than WT group (P < 0.01). The content of HYP was significantly higher in WTSJ group compared to that in WT group (P < 0.001). Consistently, the content of HYP was significantly enhanced in KOSJ group compared to that in KO group (P < 0.01).
In addition, we examined the protein expression of α-SMA and Col-1 in liver tissue by immunoblotting. The results showed that α-SMA and Col-1 expression was reduced in KOSJ compared with that in WTSJ group (Fig. 3f).
PKCλ/ι deficiency thwarts Th2 immune response and inhibits the secretion of hepatic fibrosis-related factors in liver of mice with S. japonicum infection
Compared with WT group, the level of IFN-γ was not obviously reduced in KO group. Although IFN-γ level in KOSJ group decreased as opposed to WTSJ group, the magnitude of the decrease did not reach statistical significance (Fig. 4a).
IL-4, which functions to induce the characteristic Th2 immune response, was significantly increased in both WTSJ group and KOSJ group after infection in mRNA and cytokine level (Fig. 4b, h). Of note, the increasing magnitude of IL-4 in the KOSJ group was significantly lower than that in the WTSJ group (P < 0.05). These results collectively indicate that schistosome infection acts to polarize immune response and facilitate the shift toward Th2-type response, with or without PKCλ/ι loss of function (KO group or WT group). Intriguingly, the conditional loss of PKCλ/ι gene effectively diminished Th2 polarization induced by schistosome infection.
The expression levels of fibrosis-related factors, such as TGF-β1 (Fig. 4d), Col-1(Fig. 4f) and Col-3 (Fig. 4g), in KOSJ group were notably lower than those in WTSJ group (3.98 vs. 5.61; 47.17 vs. 58.77; 16.59 vs. 18.54, respectively). Intriguingly, the levels of these fibrosis-related factors decreased after PKCλ/ι loss of function. The level of HMGB1, which signifies the extent of cell necrosis, in the KOSJ group was significantly lower than that in the WTSJ group (1.30 vs. 1.00, P < 0.05) (Fig. 4e).
PKCλ/ι deficiency inhibits the polarization of CD4+T cells to Th2 induced by S. japonicum
The intracellular cytokine profile of CD4+T in the spleen was determined by performing flow cytometry analysis. The proportions of CD4+IFN-γ+ cells in WT group, WTSJ group, KO group and KOSJ group were 8.60 ± 0.15%, 13.80 ± 0.13%, 9.40 ± 0.05% and 14.55 ± 0.38%, respectively (Fig. 5a, b). Our data indicate that schistosome infection could induce the increase of Th1 cell proportion in spleen; nevertheless, no statistical significance between WTSJ and KOSJ could be determined (P > 0.05). The levels of CD4+IL-4+ T cells in WT group, WTSJ group, KO group and KOSJ group were 6.00 ± 0.17%, 10.20 ± 0.17%, 5.50 ± 0.06% and 8.70 ± 0.14%, respectively (Fig. 5c, d). Hence, we observed that the Th2-skewed immune response induced by schistosome infection was significantly diminished because of the effect of PKCλ/ι knockout, as indicated by notably reduced Th2 cell proportions. These above results concur with the mRNA level of cytokines in the liver, collectively demonstrating that PKCλ/ι knockout could prevent the polarization of Th2-type immune response induced by schistosome infection.