Inhibition of COX-2 Ameliorates Murine Liver Schistosomiasis Japonica Through Splenic Cellular Immunoregulation

We have reported the positive association of cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) axis with liver brosis induced by Schistosoma japonicum (Sj) infection, and TLR4 signaling controlled this axis. However, how COX-2 regulated immune response during Sj infection is still unclear. Here we further studied the effect of COX-2 specic inhibitor-NS398 on liver granulomatous inammation, brosis and explored the mechanisms via evaluating different immune cell during Sj infection.The results showed that NS398 signicantly reduced the size of liver granuloma, spleen and mesenteric lymph node (MLN) and alleviated chronic granulomatous inammation. Mechanically, it might be via decreasing the numbers of Sj-induced T helper type 1 (Th1), Th2, T follicular helper (Tfh), T follicular regulatory cells (Tfr) and germinal center B (GCB) cells to alleviate the liver inammation and brosis. In addition, there were no difference of the numbers of macrophages, neutrophils, MDSCs,Th17 and total B cells in the spleen of the mice with or without NS-398 treatment. Our above data suggests that COX-2/PGE2 inhibition may represent a potential therapeutic approach to schistosomiasis japonica.


Introduction
Schistosomiasis is a chronic helminthic disease affecting over 250 million people in over 78 countries [(WHO 2018, https://www.who.int/news-room/fact-sheets/detail/schistosomiasis). The three major schistosomes infecting humans are Schistosoma mansoni (Sm), Schistosoma haematobium (Sh), and Schistosoma japonicum (Sj). Sj causes a hepato-intestinal form of the disease and is endemic in China, the Philippines, Indonesia, and the Mekong Delta. Sj resides in the mesenteric veins and hepatic portal vein, where they release eggs that induce a dramatic immune response in the intestines and liver, and then granuloma formation which characterized as eggs encapsulation within layers of immune cells embedded in extracellular matrix (ECM). Schisotosomiasis japonica is divided into acute and chronic phases.The acute phase in murine occurs in the rst 6 weeks after exposure, which manifested mainly as early liver granuloma stage [1]. In the chronic phase, the hepatointestinal or the hepatosplenic disease may occur [2]. Splenomegaly is a consequence and an important clinical indicator of portal hypertension [3]. Spleen is composed of three areas-white pulp, red pulp, and a transitional zone, and plays a role as a lter of the blood and as one of the major peripheral immune organs. In its red pulp area, eticuloendothelial cells such as macrophages will clear away abnormal red blood cells or pathogens.  [4]. The recruitment of neutrophils to the liver by IL-17A have been associated with the development of brosis in many chronic liver diseases including schistosomiasis japonica [5]. T lymphocytes are classi ed into CD4 + T helper (Th) cells and CD8 + cytotoxic T lymphocytes (CTLs). The roles of Th cells in the immune-pathogenesis of schistosomiasis have been intensely reviewed [6]. Moderate Th1 responses are included in the acute schistosomiasis and early granuloma formation, while excessive Th1 response will develop easily severe acute cachexia followed by death and is detrimental to the host. Th2 immunity acts as a double-edged sword: on the one side, it exerts anti-in ammatory effects and suppress Th1-mediated immunopathology, but on the other side, it drives liver immunopathological damage especially liver brosis. Therefore, Th1/Th2 balance maintaining is important to control the excessive pathology of schistosomiasis.
Th17/IL-17 exacerbate the egg-induced liver immunopathology in schistosomiasis. Follicular helper T cells (Tfh) are mainly located in the periphery of B cell follicles in secondary lymphoid organs, which regulate antigen-speci c B cells to become professional antibody producers, and help the formation of germinal centers (GC), a nity maturation of antibodies, somatic hypermutation and for the production of memory B cells. T follicular regulatory cells (Tfr) also exist in GCs, where they play a inhibitory role in GC reactions [7][8]. Tfh promoted liver granulomas and brogenesis in Sjinfected mice [9,10,11]. B cell lymphoma 6 (Bcl6)&programmed death-1 (PD-1) positive Tfh in the GC of murine spleen correlate with progression of liver brosis [10]. Tfh and Tfr increased in patients with schistosomiasis japonica [12].
Humoral immunity requires cross-talk between Tfh, Tfr and B cells. Several studies have demonstrated that the B-cell number in the lymph nodes and spleen signi cantly increase during the schistosome infection [13]. Sj recombinant fusion proteinSjGST-32combined with tacrolimus (FK506) immunization augmented the induction of Tfh cells, and the expression of IL-21R on GCB cells and memory B cells increased in these immunized mice [14].
Cyclooxygenase (COX)-1 and COX-2 catalyze the rst step in prostanoid biosynthesis. COX-1 is constitutively expressed whereas COX-2 is induced by some stimuli. High COX-2 expression has been detected in several liver pathologies, while the implication of COX-2inmany liver diseases is a matter of controversy [15]. Soluble egg antigen (SEA) from Sm drove potent Th2 responses through triggering dendritic cells to produce COX-1, COX-2 and then prostaglandin E2 (PGE2) [16]. We have reported that COX-2 inhibitor-NS398 protected mice from hepatic brosis induced by Sj infection [17].However, there is a lack of studies on the effects of hepatic COX-2 on immune cells during Sj-induced liver brosis.
Herein, we will demonstrate the effect of NS398 as the COX-2 & PGE2 synthesis inhibitoron macrophages, neutrophils, MDSCs and Th cell subsets, B cells and in the spleen during Sj infection. Mice, parasite infection and NS398 treatment 6-8-week-old female C57BL/6 mice were obtained from the SPF Biotechnology Co. Ltd (Beijing) and were maintained according to institutional guidelines. All mice experiments were approved to be humane by the Institutional Animal Care and Use Committee at South China Agricultural University (2019-1013). Mice were infected by 20 ± 3 Sj cercariae of the Chinese mainland strain through abdomen skin penetration. NS398 (3mg/kg body weight) in 2%DMSO was medicated into mice by intraperitoneal injection 3 times a week from week 5 to week 7 with Sj infection (n=8), while the infection control grouponly received 2%DMSO (n=7). Two non-infected control mice groups were treated with NS398 (n=5) and 2%DMSO (n=9) respectively. Mice were sacri ced at week 8 (8wks) after Sj infection. Spleens, MSN and liver tissues were collected for further analysis.

HE staining
Fresh hepatic tissues were xed in 4% paraformaldehyde for 24 h, and then were embedded with para n. The 4µm liver sections were prepared and stained with hematoxylin & eosin (HE) staining for the granuloma size and the extent of liver granulomatous in ammation analysis. The severity of liver granulomatous in ammation was evaluated according to calibrated criteria (Table 1), Fig. 1Band reference [1]. Table 1 Schistosoma japonicum-induced liver granuloma stages and in ammation Mice were anaesthetized, and sterile normal saline was injected into left ventricle to remove blood from organs. Then, the spleens were used to harvest cell suspensions by pressing these tissue pieces through a 100-mm cell strainer (BD Falcon) and then suspending in Hanks' balanced salt solution (HBSS). Red blood cells were lysed with ammonium chloride (NH4Cl) for 10 min. Cell suspensions were incubated with LIVE/DEAD Zombie NIR™ Fixable Viability Kit(Biolegend) for 20 min, and then resuspended at 2-

Statistical analysis
The results are presented as the standard deviation (±SD) of the indicated number of replicates/experiments. Data from each group were analyzed using SPSS (v11.0). Statistical evaluation of the difference between means was performed by one or two-tailed, paired or unpaired, Student's t test.
Ap-value of ≤0.05 was considered statistically signi cant.
Results COX-2 inhibition by NS398 treatment reduced Sj infection-drived hepatomegaly, the size of granuloma, and the in ltration 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 brosis induced by Sj infection under the controlling of TLR4 pathway [17]. Chronic liver in ammation was supposed to develop into brosis. The size of granuloma induced by Sj egg deposition especially at mature and late stage marked as the extent of brosis and the amount of early stage granuloma manifested as the extent of in ammation. Herein, we found thatCOX-2 inhibition by NS398 treatment signi cantly attenuated hepatomegaly (Fig. 1A) Fig. 1B) [1].COX-2 inhibition decreased the percentage of early stage granuloma signi cantly (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 in ammation and brosis 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 in ammation & brosis induced by Sj infection [2]. The diameter of spleen thickness represents the severity of brosis. Herein, we showed that the size of MLN and spleen were signi cantly attenuated(  (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 in ammation and brosis.

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: 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 signi cantly 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 signi cantly 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 signi cantly decreased the amount of GCB cells (Fig. 6B and 6C

Discussion
In this study, we investigated the in ltration of multiple immune cells into the mice spleen during 8wks of Sj infection, and the relationship between NS398 (COX-2 speci c inhibitor) treatment and the amount of these in ltrating cells and the extent of schisotsomiasis japonica. We found that NS398 decreased Sj infection-drived hepatomegaly, the size of granuloma, and the extent of liver brosis and granulomatous in ammation, which was consistent to our last study [17]. The involved mechanisms might include the NS398's signi cantly decreasing Sj infection-induced enlargement of mesenteric lymph nodes and splenomegaly, and Th1, Th2, Tfh, Tfr differentiation and GCB maturation in the mice spleen. We rstly identi ed that 8wk of Sj infection signi cantly increased the amount of MDSCs, Tfr and GCB cells in the spleen. These suggested that liver pathogenesis induced by Sj infection might be through the activation of COX-2/PGE2 axis in the liver and then the induction ofTh1, Th2, Tfh, Tfr differentiation and GCB maturation in the spleen.
In terms of the effect of Sj-infection on the cellular immune regulation, Zheng L et al [5] showed that neutrophils in the spleen of C57BL/6 mice increased gradually from 6, 8 and 12 wks of 20 cercariae infection. The tendency between neutrophils and Th17 cells was consistent in the mice with Schistosomiasis [19][20]. Yang Q et al identi ed that MDSCs in the spleen of C57BL/6 mice with 5-6wks of 40 ± 5 Sj cercariae infection were strikingly increased [4].Huang P et al [21] showed that Th2, Th17 and CD25 + Foxp3 + CD4 + T cells (Treg) but not Th1 signi cantly increased in the BALB/c mice spleen post 7wks of 30 ± 2 Sj cercariae infection. Tebeje BM et al [22]identi ed that 5wks of 34 Sj cercariae-infected splenic Th1 cells responded more highly to Sj adult worm antigen preparation (SWAP)compared to SEA. Th2 immune response to SEA was dominant at week 6, and Treg response was high in the CBA mice spleen at week 5 followed by a decline at week 6. Su C's group [23] identi ed the dynamics of Th1, Th2, Th17 and Treg cells and their role in 12 Sjcercariae infection in C57BL/6 mice and showed that all of these T cell subsets increased gradually in the infected mice spleen at week 5, 8 and 13.Elevated frequencies of Th17 cells have been shown in the Sm-infected C57BL/6 mice spleen at week 6, but not at week 4 or 8 [24]. In contrast to C57BL/c, CBA mice develop more severe lesions driven by Th17 cells [25].Tfh cells in the spleen of C57BL/6 mice with both 5 and 6 weeks of 40 ± 5 Sj cercariae infection was signi cantly increased, especially at week 5 [11]. High frequency of Tfh and Tfr cells were signi cantly increased in the peripheral blood mononuclear cells (PBMCs) of patients with schistosomiasis japonica [26]. Infection with Sj induced TGF-β& IL-10 producing B cells while decreased CR5 + B1a cells [13,18,27].
Herein, we showed the consistent increasing of neutrophils, MDSCs, Th1, Th2, Tfh, Tfr cells in the C57BL/6 mice with 8wks of 20 ± 3 Sj cercariae infection. However, the amount of Th17 didn't increased as reported. Different dose of Sjcercariae infection and different mice strain and infecting duration might explain.
COX-2 plays an important role in the progression of liver brosis[28] [17]. We have found that the COX-2/PGE2 axis was positively associated with the extent of liver brosis induced by Sj infection [17]. Herein, we showed that NS398, the COX-2 speci c inhibitor lowered the granuloma size, and ameliorated splenomegaly and the size of MSN, which supported its effect on liver in ammation and brosis. Macrophages in the Sj-infected mice spleen were almost signi cantly decreased by NS398 (p=0.055).
Macrophage deletion with chlodronate signi cantly attenuated granuloma formation in the liver of mice induced by Sj infection [29]. COX-2 blockage by NS398 inhibited accumulation and function of MDSCs and promoted proliferation and inhibited apoptosis of CD4 + T cells in the spleen and bone marrow of mice with traumatic stress [30]. Triggering the COX-2-PGE2/EP2 pathway resulted on the induction of Th2 immune response [31]. Septic rats given NS398 showed amelioration of IL-6, tumor necrosis factor alpha (TNF-α)and CD4 + /CD8 + T cells' imbalance in the liver and decreased liver injury [32]. NS398 signi cantly increased IL-4 secretion while decreased IFN-γsecretion by splenocytes after ovalbumin stimulation in mice with allergic skin in ammation [33].

Conclusions
Our study rstly outlined the reciprocal relationships between the COX-2/PGE2 axis and the size of liver, spleen, MSN and liver granuloma, and multiplex immune cell in ltration in the spleen. We provided the evidence of COX-2 inhibition ameliorated liver in ammation and brosis induced by Sj infection through suppressing Th1, Th2, Tfh, Tfr and GCB cells accumulation in the spleen. COX-2/PGE2 inhibition may represent a potential therapeutic approach to schistosomiasis japonica.

Declarations
Six to eight-week-old female C57BL/6 wild-type mice were obtained from the speci c-pathogen-free (SPF) Biotechnology Co. Ltd (Beijing