OBJECTIVE: To investigate the molecular alterations of splenocytes associated with anti-factor Ⅷ (FⅧ) immune response and the underlying mechanisms based on hemophilia A (HA) murine model via RNA sequencing (RNA-seq) technology. METHODS: Severe HA mice were immunized with recombinant human factor Ⅷ (rhF8) weekly for 4 weeks to establish an FⅧ inhibitor model. High quality raw data were obtained by using bulk RNA-seq and CASAVA base identification technology, and the differentially expressed genes (DEGs) were identified. The DEGs were statistically classified by gene ontology (GO) annotation to obtain information on the major signaling pathways and biological processes involved in anti-FⅧ immune response in HA mouse splenocytes. The cell clusters, genes, and signaling pathway datasets were comprehensively analyzed by GO, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and single cell RNA-seq (ScRNA-seq) analysis, respectively. Flow cytometry analysis was used to verify the changes in T follicular helper cells (Tfh) and regulatory T cells (Treg). RESULTS: A total of 3731 DEGs was identified, including 2275 genes with up-regulated expression and 1456 genes with down-regulated expression. The DEGs were enriched in helper T cell differentiation, cytokine receptor, T cell receptor signaling pathway, ferroptosis, etc. Uniform Manifold Approximation and Project (UMAP) downscaling and visualization analysis yielded a total number of 11 T/NK cell subsets, visualizing the overall expression distribution of C-X-C chemokine-specific receptor gene cxcr5 among these T/NK cell subsets. Higher expression of cxcr5 was found in activated Tfh from FⅧ inhibitor mice, in comparison to the control group. The visualization using Upset plot R language showed a close interaction between Tfh and Treg. Moreover, the increased frequencies of Tfh and the decreased frequencies of Treg in inhibitor mouse splenocytes were further verified by flow cytometry analysis. CONCLUSION Multiple immune cell subsets, signaling pathways, and characteristic genes may be involved in the process of anti-FⅧ immune response in HA mouse splenocytes. The molecules involved in the regulation of Tfh/Treg may play key roles, which provide potential biological targets and therapeutic strategies for HA patients with inhibitors in the future.
Animals ; Hemophilia A/genetics* ; Mice ; Sequence Analysis, RNA ; Disease Models, Animal ; Spleen/cytology* ; T-Lymphocytes, Regulatory/immunology* ; Humans ; Signal Transduction ; Factor VIII/immunology* ; T-Lymphocytes, Helper-Inducer/immunology*

Immune thrombocytopenia (ITP) is a hemorrhagic autoimmune disease characterized by antibody-mediated platelet injury. ITP has complicated immunopathological mechanisms that need further elucidation. It is well known that the costimulatory molecules OX40 ligand (OX40L) and OX40 play essential roles in the immunological mechanisms of autoimmune diseases. Previously, we discovered that the expression of OX40L and OX40 is significantly increased in the peripheral blood mononuclear cells (PBMCs) of ITP patients. In our present study, OX40L-induced follicular helper T (Tfh) cells exhibited an activated phenotype with elevated expression of inducible T-cell costimulator (ICOS), programmed cell death protein-1 (PD-1), and cluster of differentiation 40 ligand (CD40L) in vitro. Moreover, aberrant OX40L‒OX40 expression might promote the Tfh1-to-Tfh2 shift in vivo, inducing the generation of autoantibodies by enhancing the helper function of Tfh cells for B lymphocytes in a mouse model, which might accelerate the progression of ITP. Additionally, signal transduction through the OX40L‒OX40 axis might be related to the activation of tumor necrosis factor receptor-associated factor (TRAF)‒nuclear factor-κB (NF-κB) and Janus kinase (JAK)‒signal transducer and activator of transcription (STAT) signaling pathways. Overall, OX40L‒OX40 signaling is proposed as a potential novel therapeutic target for ITP.
Animals ; OX40 Ligand/physiology* ; Purpura, Thrombocytopenic, Idiopathic/immunology* ; Cell Differentiation ; Mice ; T-Lymphocytes, Helper-Inducer/cytology* ; T Follicular Helper Cells/cytology* ; Signal Transduction ; Receptors, OX40 ; Mice, Inbred C57BL ; Humans ; Female

OBJECTIVETo investigate the regulation of adipose-derived mesenchymal stem cells (ADSC) on helper T cells and regulatory T cells in allergic rhinitis(AR) mouse model and the underlying mechanisms.

METHODSUsing random number table, 60 Balb/c mice were divided into 6 groups (represented by: sensitized/challenged/treated ), they were the experimental group 1(OVA/OVA/high dose ADSC), the experimental group 2(OVA/OVA/low dose ADSC), the experimental group 3(OVA/OVA/PBS), the experimental group 4(OVA/OVA/0), the control group 1(PBS/PBS/0) and the control group 2(0/0/0). The mouse ADSC were isolated and cultured through conventional method, and AR mouse model was built with OVA and aluminum. The mice were injected with high (3×10(6)), low (1×10(6)) ADSC respectively labeled by CM-Dil for 3 consecutive days via tail-vein injection and sacrificed 48 hours later. Finally, levels of IL-4, IL-6, IL-10 and IFN -γ in serum were examined by ELISA; expressions of the four cytokines in spleen were examined by q RT-PCR; migration of ADSC to mouse model nasal mucosa were observed through fluorescence microscope; eosinophil infiltration were observed by the nasal HE staining.

RESULTSMouse ADSC was isolated, cultured and identified successfully. There was significant difference in symptom scores of AR models (compared with 0/0/0 group, P<0.01). The IL-4 and IL-6 levels of OVA/OVA/high ADSC group were significantly lower than OVA/OVA/0 group (group 1: (17.95±7.78), (27.51±5.93) pg/ml; group 4: (56.82±9.12), (70.03±7.22) pg/ml), the IFN-γ and IL-10 levels increased significantly (group 1: (367.74±13.79), (417.10±72.40) pg/ml; group 4: (199.46±11.25), (122.50±15.57) pg/ml) in serum. These differences were statistically significant(P<0.01). Compared with OVA/OVA/low ADSC group, the IL-4 and IL-6 levels decreased significantly (group 1: (17.95±7.78), (27.51±5.93) pg/ml; group 2: (41.57±12.27), (56.21±9.23)pg/ml) of OVA / OVA / high ADSC group, and the IFN-γ and IL-10 increased significantly (group 1: (367.74±13.79), (417.10±72.40)pg/ml; group 2: (281.77±30.41), (203.45±87.10) pg/ml). These differences were statistically significant(P<0.01). At the same time, the corresponding changes observed at the levels of the cytokines' mRNA. ADSC labeled by CM-Dil could migrate to the mouse nasal mucosa. OVA/OVA/high ADSC group showed the more red fluorescence than the OVA/OVA/low ADSC group. The eosinophils in nasal mucosa of the two groups reduced compared with the normal control.

CONCLUSIONADSC injected via tail-vein can migrate to nasal mucosa and play non-specific immune effects, that may to effect the releases of some cytokines then to regulate the Th1/Th2 imbalance and the function of Treg cell, finally that be dose-related in a certain extent.

Adipose Tissue ; cytology ; Animals ; Cytokines ; blood ; Disease Models, Animal ; Eosinophils ; immunology ; Inflammation ; Mesenchymal Stem Cell Transplantation ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa ; immunology ; Rhinitis, Allergic ; immunology ; therapy ; T-Lymphocytes, Helper-Inducer ; immunology ; T-Lymphocytes, Regulatory ; immunology

This study was purposed to detect the quantity and function of bone marrow (BM) T follicular helper (Tfh) cells of patients with immune thrombocytopenia, and to explore the role of Tfh cells in the pathogenesis of ITP. Twenty-one newly diagnosed ITP patients, twenty ITP patients in recovery stage and eighteen normal controls were enrolled in this study. The percentages of Tfh cells, Tfh-related molecules ICOS, CD40L, IL-21 in BM were detected by flow cytometry (FCM), and the mRNA expression of BCL-6 in BMMNC was determined by semi-quantitive RT-PCR. Correlation of Tfh cell level with the disease severity of ITP patients was analysed. The results showed that the ratio of CD4(+)CXCR5(+)/CD4(+) cells in newly diagnosed ITP patients [(5.532 ± 2.599)%] was significantly higher than that in ITP patients with recovery stage [(4.064 ± 2.026)%] and controls [(4.048 ± 1.413)%] (P < 0.05). The ratio of CD4(+)CXCR5(+)ICOS(+)/CD4(+) CXCR5(+) cells in newly diagnosed ITP patients [(14.586 ± 8.561)%] was higher than that in recovery stage ITP patients [(12.884 ± 10.161)%] and controls [(7.487 ± 5.176)%]. The differences be-tween newly diagnosed ITP patients and controls were statistically significant (P < 0.05). The ratio of CD4(+)CXCR5(+) CD40L(+)/CD4(+) CXCR5(+) cells in newly diagnosed ITP patients [(15.309 ± 10.756)%] and in ITP patients with recovery stage [(18.242 ± 12.243)%] were significantly higher than that in controls [(8.618 ± 5.719) %] (P < 0.05). The ratio of intracytoplasm CD4(+) CXCR5(+) IL-21(+)/CD4(+)CXCR5(+) cells in newly diagnosed ITP patients [(58.560 ± 26.285)%] and in ITP patients with recovery stage [(57.035 ± 30.936)%] were significantly higher than that in controls [(36.289 ± 24.868)%] (P < 0.05). The relative expression levels of BCL-6 mRNA in BMMNC of three groups were (1.407 ± 0.264), (1.149 ± 0.217) and (0.846 ± 0.157), respectively. The differences between 3 groups were significant(P < 0.05). It is concluded that the quantity and function of Tfh cells in ITP patients increase, which may play an important role in the pathogenesis of ITP.
Adolescent ; Adult ; Aged ; Bone Marrow ; immunology ; Bone Marrow Cells ; cytology ; Case-Control Studies ; Child ; Female ; Flow Cytometry ; Humans ; Lymphocyte Count ; Male ; Middle Aged ; T-Lymphocytes, Helper-Inducer ; cytology ; immunology ; Thrombocytopenia ; immunology ; Young Adult

Dendritic cells (DCs) are key modulators that shape the immune system. In mucosal tissues, DCs act as surveillance systems to sense infection and also function as professional antigen-presenting cells that stimulate the differentiation of naive T and B cells. On the basis of their molecular expression, DCs can be divided into several subsets with unique functions. In this review, we focus on intestinal DC subsets and their function in bridging the innate signaling and adaptive immune systems to maintain the homeostasis of the intestinal immune environment. We also review the current strategies for manipulating mucosal DCs for the development of efficient mucosal vaccines to protect against infectious diseases.
Animals ; Dendritic Cells/*immunology/metabolism ; Humans ; Immunity, Mucosal ; Intestinal Mucosa/cytology/*immunology ; T-Lymphocytes, Helper-Inducer/immunology

OBJECTIVETo study the quantity and function of bone marrow (BM) T follicular helper (Tfh) cells of the cytopenia patients with positive bone marrow mononuclear cells (BMMNC)- Coombs test (also known as immuno-related pancytopenia, IRP), and explore the role of Tfh cells in the pathogenesis of IRP.

METHODSForty- three untreated IRP patients, 47 recovered IRP patients and 25 healthy donors were enrolled in this study. The percentages of Tfh cells, Tfh-related molecules ICOS, CD40L, IL-21 and Bcl-6 in BM were investigated by flow cytometry and semiquantitive RT-PCR.

RESULTSThe ratio of CD4⁺CXCR5⁺/CD4⁺ cells of untreated IRP patients [(28.79 ± 19.70)%] was significantly higher than that of recovered IRP patients [(21.15 ± 12.81)% ] and normal controls ([ 13.42 ± 6.72)% ](P<0.05). The ratio of CD4⁺CXCR5⁺ICOS⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(5.05 ± 4.71)% ] was significantly higher than that of recovered IRP patients [(2.96 ± 2.89)% ] and normal controls [(2.99 ± 2.23)% ] (P<0.05). The ratio of CD4⁺CXCR5⁺CD40L⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(5.87 ± 4.14)%] and recovered IRP patients [(6.52±5.47)%] were significantly higher than that of normal controls [(2.93 ± 2.92)%] (P<0.05). The ratio of intracytoplasmic CD4⁺CXCR5⁺IL-21⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(8.20 ± 7.41)% ] and recovered IRP patients [(6.30 ± 6.03)% ] were significantly higher than that of normal controls [(3.43 ± 3.40)%] (P<0.05). The relative expressions of Bcl-6 mRNA in BMMNC were 0.625 ± 0.248, 0.485 ± 0.253, 0.306 ± 0.210 in three groups, respectively. The differences between untreated IRP patients, recovered IRP patients and normal controls were significant (P<0.05).

CONCLUSIONThere exists increased quantity and hyperfunction of Tfh cells in the IRP patients, they may play important role in the pathogenesis of IRP. Tfh cells and their related effector molecules could be a potential therapeutic target for the disease.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Child ; Child, Preschool ; Coombs Test ; Female ; Flow Cytometry ; Humans ; Interleukins ; metabolism ; Lymphocyte Count ; Male ; Middle Aged ; Pancytopenia ; blood ; diagnosis ; etiology ; T-Lymphocytes, Helper-Inducer ; cytology ; metabolism ; Young Adult

BACKGROUNDSurgical stress causes a helper T-cell type 2 (Th2)-dominant status and disturbs the Th1/Th2 cytokine balance. Anesthesia can suppress the stress response to surgery, therefore it may inhibit the imbalance in the Th1/Th2 ratio. In this study, we assessed if propofol anesthesia and sevoflurane anesthesia influence the Th1/Th2 cytokine balance, and which anesthesia method better attenuates this ratio.

METHODSTwenty-eight patients with an American Society of Anesthesiologists (ASA) physical status of I undergoing laparoscopic cholecystectomy were selected. They were randomly allocated into two groups of 14. Group 1 received propofol anesthesia by a target-controlled-infusion (TCI) pump and group 2 received sevoflurane anesthesia. Non-invasive blood pressure, heart rate, and end-expiration CO2 partial pressure were monitored during anesthesia. The depth of anesthesia was measured using the bispectral index (BIS), and maintained between 50 and 60. During surgery we adjusted the doses of propofol and sevoflurane according to the BIS. Samples of peripheral blood were taken before the induction of anesthesia (T1), after the induction of anesthesia (T2), at the beginning of surgery (T3), at the end of surgery (T4) and on the first day after surgery (D1). Blood samples were analyzed to give the Th1/Th2 ratio and plasma level of cortisol.

RESULTSNon-invasive blood pressure, heart rate and end-expiration CO2 partial pressure were not notably different in the two groups. At T4, the percentage of T1 cells was higher in group 1 and had statistical significance (P < 0.05). The percentage of T2 cells was not significantly different in the two groups. At T4, the difference in the Th1/Th2 ratio was significantly different. At T3, T4, and D1, the plasma level of cortisol was lower in group 1 (P < 0.05).

CONCLUSIONCompared with sevoflurane, propofol can preferably promote Th cells to differentiate into Th1 cells and inhibit surgical stress. Propofol may therefore be immunoprotective for such patients.

Adult ; Cell Differentiation ; drug effects ; Female ; Flow Cytometry ; Humans ; Hydrocortisone ; blood ; Male ; Methyl Ethers ; pharmacology ; therapeutic use ; Middle Aged ; Propofol ; pharmacology ; therapeutic use ; T-Lymphocytes, Helper-Inducer ; cytology ; drug effects ; Th1 Cells ; cytology ; drug effects ; Th2 Cells ; cytology ; drug effects

OBJECTIVETo establish a new animal model of resorption of lumbar disc herniation.

METHODSTwenty 3-month-old Sprague Dawley (SD) male rats were randomly divided into experimental group and control group. The caudal discs of the experimental rats were surgically removed and were implanted in the epidural. The animals were killed at 30 days, and the implanted nucleus pulposus were took out for HE staining,flow cytometry and immunohistochemistry. In the control group, coils were implanted in the back muscles, and the animals were killed at 30 days after the operation for the above test.

RESULTSIn the experimental group, immunohistochemical staining of TNF-alpha, VEGF were positive at 30 days. The Th cells and B cells in the experimental group were more than that in the control group with statistically significant differences.

CONCLUSIONThe model can well reveal the re-absorption process the of the ruptured disc, and provide a new re-absorption disc animal model for the further study.

Absorption ; Animals ; B-Lymphocytes ; cytology ; immunology ; Disease Models, Animal ; Flow Cytometry ; Immunohistochemistry ; Intervertebral Disc Displacement ; immunology ; Lumbar Vertebrae ; Male ; Rats ; Rats, Sprague-Dawley ; T-Lymphocytes, Helper-Inducer ; cytology ; immunology

The nomenclature "embryonic lymphoid tissue inducer (LTi) cell" reflects the fundamental role of the cell in secondary lymphoid tissue organization. In addition, it is equally important in primary lymphoid tissue development as it regulates central tolerance to self-antigens in the thymus. An adult LTi cell constitutively expresses two sets of tumor necrosis factor (TNF) family members, whereas its embryonic counterpart expresses only one. The first set is lymphotoxin (LT)alpha, LTbeta, and TNFalpha, which are essential for the secondary lymphoid organogenesis during embryogenesis and for maintaining an organized secondary lymphoid structure during adulthood. The second set is OX40- and CD30-ligands, which are critical for memory T cell generation. Adult LTi cells regulate adaptive immune responses by providing LTbetaR signals to stromal cells to maintain secondary lymphoid tissue structure, and determine adaptive immune responses by providing OX40 and CD30 survival signals to activated T cells in memory T cell generation. Along with the consideration of the roles of embryonic LTi cells in primary and secondary lymphoid tissues, this review highlights the roles of adult LTi cells in secondary lymphoid tissue function.
Adult ; Animals ; Humans ; Lymphoid Tissue/cytology/embryology/*immunology ; Lymphokines/immunology/metabolism ; T-Lymphocytes, Helper-Inducer/cytology/*immunology/metabolism ; Thymus Gland/cytology/embryology/*immunology

Th17(T helper 17 cell), a newly discovered subset of T cells is associated with IL-23 and characterized by production of IL-17, the functions of which are distinct from those of Th1, Th2 and Treg subsets. The development of Th17 cells can be promoted by TGF-beta1, IL-6, and IL-23; but inhibited by IFN-gamma, IL-4 and Socs3. It is clear that Th17 cells have protective effects on body by facilitating the pro-inflammatory responses. On the other hand, the role of Th17 cells in the pathophysiology of autoimmune diseases has been described.
Autoimmune Diseases ; immunology ; physiopathology ; Interleukin-17 ; biosynthesis ; immunology ; Interleukin-23 ; biosynthesis ; genetics ; T-Lymphocyte Subsets ; immunology ; physiology ; T-Lymphocytes, Helper-Inducer ; classification ; cytology ; immunology ; Transforming Growth Factor beta1 ; biosynthesis ; genetics