Abstract Schistosomiasis morbidity and mortality are primarily driven by egg-triggered granulomas with subsequent fibrosis. However, the dynamics of cellular composition within these granulomas, particularly the identity and function of key immune cells that orchestrate their formation and evolution, remain poorly understood. Here, we combine single-cell RNA sequencing with multiplex immunofluorescence to characterize the hepatic immune landscape in Schistosoma japonicum -infected mice and further validate the functional roles of two neutrophil subsets. We find that eggs alter hepatic immune cell composition, which is characterized by extensive neutrophil recruitment and differentiation. Neutrophil recruitment correlates with CXCL2 derived from both an autocrine loop and paracrine signaling from monocytes. Cellular localization analysis reveals that granulomas progress through three distinct developmental stages: early Ly6G hi F4/80 lo Desmin lo , developing Ly6G mid F4/80 hi Desmin mid , and advanced Ly6G lo F4/80 mid Desmin hi . Neutrophil subsets display a zonal distribution within granulomas, with CD177 + neutrophils surrounding the eggs and Ltf + neutrophils localizing to the mid-outer layer. Cd177 knockdown reduces granuloma size and fibrosis, whereas Ltf suppression increases these pathological features, indicating that CD177 and LTF exert opposite effects on granuloma formation. Our findings provide new insights into the cellular complexity of S. japonicum egg-induced granulomas and may help guide the development of novel therapies for liver fibrosis.

Schistosomiasis morbidity and mortality are primarily driven by hepatic granulomas triggered by parasite eggs with subsequent periportal fibrosis. As a common pathological consequence of various chronic liver injuries, hepatic fibrosis is well-documented. However, the dynamics of cellular composition within the schistosome egg-induced granuloma, particularly the identity and function of key immune cell subsets that orchestrate its formation and evolution, remain poorly resolved. To address this, we combined single-cell RNA sequencing with multiplex immunofluorescence, histopathological analysis, and serological profiling to comprehensively characterize the hepatic immune landscape in a murine model of Schistosoma japonicum infection, and further validated the functional roles of two neutrophil subsets through targeted marker gene knockdown. We found that egg deposition altered hepatic immune cell composition, characterized by extensive neutrophil recruitment and differentiation. Neutrophil recruitment correlated with CXCL2 derived from both an autocrine loop and paracrine signaling by monocytes. Cellular localization analysis of the developmental trajectory of granulomas showed that they progressed from the early Ly6G hi F4/80 lo Desmin lo stage, through the developing Ly6G mid F4/80 hi Desmin mid stage, to the advanced Ly6G lo F4/80 mid Desmin hi stage. In addition, neutrophil subsets display zonal distribution within early granulomas: CD177 + neutrophils surrounded the eggs, while Ltf + neutrophils localized to the mid-outer layer. Cd177 knockdown reduced granuloma size and fibrosis, while Ltf suppression increased these pathological features, indicating that CD177 + and LTF + neutrophils have opposite effects on granuloma formation. Our findings provide new insight into the cellular complexity of S. japonicum egg-induced granulomas and could help guide the development of novel treatments for liver fibrosis.

FcεRIα is responsible for high-affinity binding with the Fc portion of IgE, which is critical for IgE-dependent disease responses such as allergy responses and anti-parasite immunity. FcεRIα is expressed on a few cell types, including mast cells and basophils.

Functioned as α-subunit of the high-affinity immunoglobulin E receptor (Fcε RIα), Fcε RIα plays a central role in the pathogenesis of Ig-E-mediated allergy and other IgE-related disorders. Fcε RIα is normally expressed only in limited spectrum of cells like basophils and mast cells, but the mechanism of controlling Fcε RIα expression in these cells is less well understood. In this study, we found fully overlapped natural antisense transcript (NATs) of Fcε RIα ( FCER1A -AS) is co-expressed with cognate sense transcript ( FCER1A -S) in IL-3 induced Fcε RIα-expressing cells or in high Fcε RIα-expressing cell line MC/9. When FCER1A -AS is selectively knocked down by CRISPR/RfxCas13d (CasRx) approach in MC/9, expression of mRNA and proteins of FCER1A -S is also markedly decreased. Furthermore, deficiency of FCER1A -AS along with lack of FCER1A -S expression is found in two different lines of gene-targeted mice in which FCER1A locus is disturbed at different sites. More importantly, the FCER1A -AS-deficient homozygous mice display similarly diminished anaphylactic reaction as FCER1A gene knockout mice. Thus, we uncovered in this investigation that the expression of FCER1A -S is positively regulated by co-expressed fully overlapped antisense transcript.

目的 探究细胞因子IL-4抑制骨髓细胞分化产生嗜碱性粒细胞的能力及机制.方法 采用IL-3诱导培养小鼠骨髓细胞,流式细胞术检测FcεRIα+ckit-CD49b+嗜碱性粒细胞以及FcεRIα+ckit-CD49b-新发现的单阳细胞群,并用Ki67检测其增殖状态.流式细胞术分选单阳细胞群后继续用IL-3诱导培养,以确定其后续分化能力.结果 经IL-3诱导培养的骨髓细胞,3～4d后开始有嗜碱性粒细胞出现,至第9天细胞比例达到高峰,然后开始下降.与野生型相比,IL-4缺陷的骨髓细胞诱导出的嗜碱性粒细胞,无论是在细胞比例或数目还是该细胞表达FcεRIα的平均荧光强度都显著增加.在IL-4缺陷型骨髓细胞诱导过程中加入外源重组IL-4可抑制嗜碱性粒细胞的产生;而在野生型骨髓细胞诱导过程中加入抗IL-4抗体则可增加嗜碱性粒细胞的产生,提示诱导过程中产生的IL-4具有负向调控嗜碱性粒细胞形成的作用.与野生型相比,IL-4缺陷鼠骨髓细胞经诱导可产生更多未曾报道过的表型为FcεRIα+ckit-CD49b-的单阳细胞群,且该细胞群的Ki67阳性率更高.单阳细胞群经IL-3刺激后可继续分化出嗜碱性粒细胞.结论 IL-3诱导培养骨髓细胞过程中,内源产生的IL-4可抑制嗜碱性粒细胞的产生及FcεRIα 的表达水平.

Guided by the educational concept of outcomes based education ( OBE), based on the bridge professional knowledge of pathology, medical microbiology, medical immunology as well as the pathogenesis and outcome of diseases in almost all organs and systems of human body in the teaching of basic medicine courses, aiming at their characteristics of response between knowledge points, broad contents and close connection with clinical practice, an English integrated teaching module of "Injury, Infection, Immunity and Repair" in basic medicine was constructed. The modular teaching proceeds from knowledge structure to reverse curriculum design, implements student-centered and diversified teaching and learning methods, and continuously improves teaching evaluation and guarantee system. It optimizes the shortcomings of traditional subject teaching system and organ system integration curriculum system, further improves the quality of medical education, promotes the construction of basic medicine, advances the globalization process of medical education, and cultivates more medical professionals for our country to adapt to the global development in the future.

Autoimmune hepatitis is a worldwide health problem and significant cause of mortality. However, the disease etiology is largely unknown, which accounts for ineffective treatment and uncontrolled disease progression. In this study, we demonstrated the functional importance of the IL-17C/IL-17RE axis in Con A-induced hepatitis. Elevated IL-17C expression was detected in liver samples of both human and mouse autoimmune hepatitis. IL-17C, produced by hepatocytes, and its specific receptor IL-17RE on liver-resident T cells were both found to be required in Con A-induced liver damage. Mechanistically, IL-17C augmented the expression of IL-2 by intrahepatic CD4+ T cells to promote NK cell activation and liver damage. To our knowledge, our findings thus for the first time defined the indispensable role of IL-17C/IL-17RE in autoimmune hepatitis; this axis may serve as a novel drug target for the treatment of this disease.

Aluminium hydroxide (alum), the most widely used adjuvant in human and animal vaccines, has long been known to promote T helper type 2 (Th2) responses and Th2-associated humoral responses, but the mechanisms have remained poorly understood. In this study, we explored whether alum is able to directly modulate antigen-presenting cells to enhance their potency for Th2 polarization. We found that alum treatment of dendritic cells failed to show any Th2-promoting activities. In contrast, alum was able to enhance the capacity of basophils to induce Th2 cells. When basophils from interleukin-4 (IL-4) knockout mice were examined, the intrinsic Th2-promoting activities by basophils were largely abrogated, but the alum-enhanced Th2-promoting activities on basophils were still detectable. More importantly, Th2-promoting adjuvant activities by alum found in IL-4 knockout mice were also largely reduced when basophils were depleted by antibody administration. Therefore, basophils can mediate Th2-promoting activities by alum both in vitro and in vivo through IL-4-independent mechanisms. Further studies revealed that secreted soluble molecules from alum-treated basophils were able to confer the Th2-promoting activities, and neutralization of thymic stromal lymphopoietin or IL-25 attenuated the IL-4-independent development of Th2 cells elicited by alum-treated basophils. Finally, alum was able to activate NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in murine basophils in the same way as alum in professional antigen-presenting cells, but NLRP3 was not required for Th2-promoting activities on basophils by alum in vitro. These results demonstrated that alum can enhance the capacities of basophils to polarize Th2 cells via IL-4- and NLRP3-independent pathways.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/imm.12636 This article is protected by copyright. All rights reserved. Received Date : 31-Oct-2015 Revised Date : 11-Jun-2016 Accepted Date : 12-Jun-2016 Article type : Original Article

Although dendritic cells (DCs) have been widely demonstrated to play essential roles in initiation of Th2 responses in helminth infections and allergic reactions, the mechanisms remain uncertain largely because DCs do not produce IL-4. In present investigation, we have uncovered a novel subset of DCs from mice infected with Th2-provoking pathogens Schistosoma japonica, which independently promoted Th2 cells via IL-4–dependent pathway. These DCs contained similar levels of IL-4 mRNA and higher levels of IL-12p40 mRNA comparing to basophils, correlating to their Th2-promoting and Th1-promoting dual polarization capacities. Characterized by expression of FcεRI+, these DCs were induced independent of T cells. Further investigations revealed that Th2-promoting FcεRI+ DCs were monocyte-derived inflammatory DCs, which were sufficient to induce Th2 cells in vivo. Egg Ags together with GM-CSF or IL-3 alone were able to stimulate the generation of Th2-promoting FcεRI+ DCs from bone marrow cells in vitro. To our knowledge, our data for the first time demonstrate that IL-4–producing DCs are induced under some Th2-provoking situations, and they should play important roles in initiation of Th2 response.

BACKGROUND:It has been well accepted that glycans present in schistosomes are highly antigenic. However, it is not clear what kind of worm glycans can affect the infected host to mount IgG responses and whether mounted anti-glycan IgG responses are protective.METHODS:The contribution of antigenicity by glycans was measured by using competitive ELISA assay in sera from infected mice and humans. Monoclonal antibodies towards soluble Schistosoma japonicum egg antigens (SjEA) were generated from SjEA immunizated mice. The expression of glycans on surfaces of cercaria or young worm and their distributions were examined by immunofluorescence assay. The protective roles of glycans-specific mAbs were assayed by determination of the worm and egg burden in infected mice.RESULTS:Both periodate-resistant glycans and periodate-sensitive glycans are antigenic in schistosome infections. When monoclonal antibodies against either periodate-sensitive or periodate-resistant glycans were administered prior to schistosome infections in mice, both kinds of anti-glycan antibodies were found to successfully provide protective immunity to infected mice.CONCLUSIONS:Both periodate-resistant and periodate-sensitive glycans are antigenic, and dominant anti-glycan IgG responses can play important roles in protective immunity in schistosome infected hosts.

Cell-mediated immunity plays a crucial role in the development of host resistance to asexual blood-stage malaria infection. However, little is known of the regulatory factors involved in this process. In this study, we investigated the impact of MAPK phosphotase 5 (MKP5) on protective immunity against a lethal Plasmodium yoelii 17XL blood-stage infection using MKP5 knockout C57BL/6 mice. Compared with wild-type control mice, MKP5 knockout mice developed significantly lower parasite burdens with prolonged survival times. We found that this phenomenon correlated with a rapid and strong IFN-γ-dependent cellular immune response during the acute phase of infection. Inactivation of IFN-γ by the administration of a neutralizing Ab significantly reduced the protective effects in MKP5 knockout mice. By analyzing IFN-γ production in innate and adaptive lymphocyte subsets, we observed that MKP5 deficiency specifically enhanced the IFN-γ response mediated by CD4+ T cells, which was attributable to the increased stimulatory capacity of splenic CD11c+ dendritic cells. Furthermore, following vaccination with whole blood-stage soluble plasmodial Ag, MKP5 knockout mice acquired strongly enhanced Ag-specific immune responses and a higher level of protection against subsequent P. yoelii 17XL challenge. Finally, we found the enhanced response mediated by MKP5 deficiency resulted in a lethal consequence in mice when infected with nonlethal P. yoelii 17XNL. Thus, our data indicate that MKP5 is a potential regulator of immune resistance against Plasmodium infection in mice, and that an understanding of the role of MKP5 in manipulating anti-malaria immunity may provide valuable information on the development of better control strategies for human malaria.

目的 探讨炎症性细胞因子IL-17A及免疫抑制细胞(myeloid-derived suppressor cells,MDSC)在二乙基亚硝铵(1,2-diethylnirtosamine,DEN)诱导的小鼠肝癌模型中的作用.方法 建立小鼠肝癌模型,取肝脏,实时荧光定量RT-PCR法检测IL-17A的mRNA表达;通过免疫组化法检测IL-17A的蛋白水平表达;胞内细胞因子染色法检测产生IL-17A的细胞来源;细胞表面分子染色法检测MDSC的浸润.结果 肝癌小鼠中瘤内和瘤周IL-17A的mRNA表达显著高于正常小鼠,分别是正常小鼠的7.3倍和2.8倍;在肝脏局部中CD4+T细胞、γδT细胞能产生大量的IL-17A,而CD8+T细胞也能产生少量的IL-17A;肝癌小鼠肝脏局部浸润的MDSC要明显高于正常小鼠,MDSC在正常小鼠肝脏局部免疫细胞中分别约占1.7％,而在肝癌小鼠中达到了15.8％.结论 炎症细胞因子IL-17A以及免疫抑制细胞MDSC在肝癌的病理进程中均起着非常重要的作用.

UNLABELLEDOBJECTIVE; To observe the changes in various dendritic cell (DC) subsets, macrophages, basophils, eosinophils and mast cells in mouse spleen before and after Schistosoma japonicum infection induced Th2 response.METHODSC57BL6 mice were infected with 20 S. japonicum cercariae via abdominal skin. Before infection and at 2, 4, and 6 weeks post-infection, the mice were sacrificed and spleen was removed. The frequencies of non-T, non-B basophils (NTNB), eosinophils, mast cells, subsets of DC and macrophages in the spleen were measured by flow cytometry.RESULTSAt 4 weeks after infection, when Th2 cells started to occur, the proportion of CD11c+CD8+DC and CD11c+CD4+DC in B220CD11c+DC increased from 7.4% and 7.9% before infection to 17.1% and 12.0%, respectively. During the infection, CD11c+CD4-CD8-DC, the majority of B220CD11c+DC, remained on a nearly constant level (70%); the percentage of B220+ CD11c+ DC in NTNB decreased. The macrophages were subdivided into two subsets: F4/80+CD11b(int) and F4/80+CD11b(high). The percentage of F4/80+CD11b(int) and F4/80+CD11b(high) in NTNB dropped from 15.4% and 13.7% before infection to 2.7% and 8.6% at 4 weeks post-infection. The proportion of CD11b high macrophages in F4/80+ cells significantly increased from 47.1% before infection to 75.5% at 4 weeks after infection. During S. japonicum infection, eosinophil percentage in the spleen gradually increased, while the frequency of basophils and mast cells in NTNB greatly decreased.CONCLUSIONAt the time when Th2 response starts to occur, the frequency of CD11c+CD8+DC and CD11c+CD4+DC in CD11c+ dendritic cells increases. Once Th2 immune response established, the eosinophil frequency increases.

SR-PSOX can function as a scavenger receptor, a chemokine and an adhesion molecule, and it could be an interesting player in the formation of atherosclerotic lesions. Our previous studies demonstrated that basic amino acid residues in the chemokine domain of SR-PSOX are critical for its functions. In this study the combinations of the key basic amino acids in the chemokine domain of SR-PSOX have been identified. Five combinations of basic amino acid residues that may form conformational motif for SR-PSOX functions were selected for multi-point mutants. The double mutants of K61AR62A, R76AK79A, R82AH85A, and treble mutants of R76AR78AK79A, R78AR82AH85A were successfully constructed by replacing the combinations of two or three basic amino acid residues with alanine. After successful expression of these mutants on the cells, the functional studies showed that the cells expressing R76AK79A and R82AH85A mutants significantly increased the activity of oxLDL uptake compared with that of wild-type SR-PSOX. Meanwhile, the cells expressing R76AK79A mutant also dramatically enhanced the phagocytotic activity of SR-PSOX. However, the cells expressing the construct of combination of R78A mutation in R76AK79A or R82AH85A could abolish these effects. More interestingly, the adhesive activities were remarkably down regulated in the cells expressing the multi-point mutants respectively. This study revealed that some conformational motifs of basic amino acid residues, especially R76 with K79 in SR-PSOX, may form a common functional motif for its critical functions. R78 in SR-PSOX has the potential action to stabilize the function of oxLDL uptake and bacterial phagocytosis. The results obtained may provide new insight for the development of drug target of atherosclerosis.

Objective To investigate the effects of thymus activation regulated chemokine (TARC)and macrophage-derived chemokine (MDC) on Th2 responses and liver fibrosis in Schistosoma japonicum infection.Methods Mice were infected by percutaneous infection with 20 cercariae of S.japonicum.The Th2 response in mesentenic lymph node(MLN) and liver was determined by intracellular cytokine staining (ICC)and ELISA.TARC and MDC mRNA level in MLN and liver were measured by quantitative real time PCR.The fibrosis was measured by the content of hepatic hydroxyproline.Results The hepatic Th2 responses were observed with similar time kinetics as those in the MLN,whereas IL-13-producing Th2 cells in the liver (5.3％) were more detectable than those in the MLN (about 3％).Additionally,liver fibrosis was aggravated during the S.japonicum infection.The hepatic hydroxyproline was 2.9μg/mg,5.1μg/mg and 8.3μg/mg at 5,7 and 10 weeks post infection.The mRNA levels of TARC and MDC in the MLN were 0.5 and 0.4 fold of uninfected mice while those levels in liver were significantly higher and 12.8 and 8.2 fold of those in uninfected mice at 7 weeks after infection.Meanwhile TARC and MDC in the liver maintained high level of mRNA in the chronic stage and were 3.8 and 4.4 fold of those in uninfected mice,respectively.Conclusion As specific chemokines for tissue recruitment of Th2 cells,TARC and MDC may play an important role in recruiting Th2 cells to liver to cause inflammatory responses and liver fibrosis.

Aim To study the effect of interleukin-10(IL-10) on the expression of adhesion molecules and scavenger receptor lectin-like oxidized low density lipoprotein receptor-1(LOX-1) in human vascular endothelial cells,and to approach the function of IL-10 on anti-atherosclerosis. Methods Human umbilical vein endothelial cells were randomly divided into four groups: treated with or without oxidized low density lipoprotein(ox-LDL),treated with IL-10 plus or minus ox-LDL for 24 h.Flow cytometry technique was used to detect the expressions of E-selectin and intercellular adhesion molecule-1(ICAM-1) on the surface of the cells.Real-time PCR and Western Blotting analysis were used to detect mRNA and protein expression levels of LOX-1 in the cells. Results After 24 h stimulation,the expression level of E-selectin and LOX-1 on the surface of cells were significantly increased by 20% and 25% respectively in the ox-LDL treated group compared with untreated group.However these remarkable increments were revoked when IL-10 was present at the same time with ox-LDL.But,IL-10 alone had no such effect.The expression levels of ICAM-1 on the surface of cell was not affected by ox-LDL. Conclusion IL-10 has a protective effect on endothelial cells injury induced by ox-LDL,which may be one of the mechanisms of IL-10 in anti-atherosclerosis.The protective effect of IL-10 is probably by down-regulating the expression of LOX-1 and E-selectin on the surface of endothelial cells and inhibiting the adherence of monocytes to endothelial cells,reducing the cell activation.

Background Atherosclerosis (AS) is a common cardiovascular disease. Transformation of macrophages to form foam cells by internalizing modified low density-lipoprotein (LDL) via scavenger receptor (SR) is a key pathogenic process in the onset of AS. It has been demonstrated that SR-PSOX functions as either a scavenger receptor for uptake of atherogenic lipoproteins and bacteria or a membrane-anchored chemokine for adhesion of macrophages and T-cells to the endothelium. Therefore, SR-PSOX plays an important role in the development of AS. In this study the key basic amino acids in the chemokine domain of SR-PSOX have been identified for its functions. Results A cell model to study the functions of SR-PSOX was successfully established. Based on the cell model, a series of mutants of human SR-PSOX were constructed by replacing the single basic amino acid residue in the non-conservative region of the chemokine domain (arginine 62, arginine 78, histidine 80, arginine 82, histidine 85, lysine 105, lysine 119, histidine 123) with alanine (designated as R62A, R78A, H80A, R82A, H85A, K105A, K119A and H123A, respectively). Functional studies showed that the mutants with H80A, H85A, and K105A significantly increased the activities of oxLDL uptake and bacterial phagocytosis compared with the wild-type SR-PSOX. In addition, we have also found that mutagenesis of either of those amino acids strongly reduced the adhesive activity of SR-PSOX by using a highly non-overlapping set of basic amino acid residues. Conclusion Our study demonstrates that basic amino acid residues in the non-conservative region of the chemokine domain of SR-PSOX are critical for its functions. Mutation of H80, H85, and K105 is responsible for increasing SR-PSOX binding with oxLDL and bacteria. All the basic amino acids in this region are important in the cells adhesion via SR-PSOX. These findings suggest that mutagenesis of the basic amino acids in the chemokine domain of SR-PSOX may contribute to atherogenesis.

Background A long-term existing schistosome infection can aid in maintaining immuno-homeostasis, thus providing protection against various types of autoimmune diseases to the infected host. Such benefits have often been associated with acute or egg stage infection and with the egg-induced Th2 response. However, since schistosome infection undergoes different stages, each associated with a specific induction of Th responses, the requirements for the ability of the different stages of schistosome infection to protect against autoimmune disease has not been elucidated. The present study was designed to study whether different stages of schistosome infection offer unique protection in collagen-induced arthritis and its mechanisms. Results Arthritis susceptible strain DBA/1 male mice were infected with Schistosoma japonicum for either 2 weeks resulting in early stage infection or for 7 weeks resulting in acute or egg stage infection. Following Schistosoma japonicum infection, collagen II was administered to induce collagen-induced arthritis, an animal model for human rheumatoid arthritis. Infection by Schistosoma japonicum significantly reduced the severity and the incidence of experimental autoimmune collagen-induced arthritis. However, this beneficial effect can only be provided by a pre-established acute stage of infection but not by a pre-established early stage of the infection. The protection against collagen-induced arthritis correlated with reduced levels of anti-collagen II IgG, especially the subclass of IgG2a. Moreover, in protected mice increased levels of IL-4 were present at the time of collagen II injection together with sustained higher IL-4 levels during the course of arthritis development. In contrast, in unprotected mice minimal levels of IL-4 were present at the initial stage of collagen II challenge together with lack of IL-4 induction following Schistosoma japonicum infection. Conclusion The protective effect against collagen-induced arthritis provided by Schistosoma japonicum infection is infection stage-dependent. Furthermore, the ability of schistosomiasis to negatively regulate the onset of collagen-induced arthritis is associated with a dominant as well as long-lasting Th2 response at the initiation and development of autoimmune joint and systemic inflammation.