Effects of taurocholic acid on immunoregulation in mice

ContextCurrently, there is a dramatically growing interest in Chinese traditional medicines, especially in the therapy of inflammatory diseases. Taurocholic acid (TCA), as a kind of natural bioactive substance of animal bile acid, has medicinal applications to treat a wide range of inflammatory diseases.ObjectiveThe study was designed to evaluate the effects of TCA on cytokine secretion, such as TNF-α and IL-1β and on the ratio of CD4⁺/CD8⁺, which is beneficial for understanding the mechanism of TCA on immunoregulation preliminarily, and also will benefit our further research.Materials and methodsThe gene and protein expressions of TNF-α and IL-1β were measured by real time RT-PCR and ELISA in serum, spleen and lymphocytes respectively. The ratio of CD4⁺/CD8⁺ in peripheral blood and lymphocytes was measured by flow cytometry.ResultsOur present study has shown that lipopolysaccharide (LPS) and cyclosporin A (CsA) could increase or decrease the gene and protein expressions of TNF-α and IL-1β respectively. TCA (0.25 g/kg, 0.125 g/kg) could recover the suppressed expressions of TNF-α and IL-1β and increase the ratio of CD4⁺/CD8⁺. In vitro, TCA (15 μg/mL) could inhibit the increased production of TNF-α and IL-1β; TCA (0.15 μg/mL–15 μg/mL) could inhibit the increased gene expressions of IL-1β and TNF-α. TCA (0.15 μg/mL) could recover the suppressed expressions of TNF-α and IL-1β.ConclusionThe function of immunoregulation of TCA may be accomplished through modulating the gene and protein expressions of TNF-α and IL-1β and elevating CD4⁺/CD8⁺ T-cell ratio.     

CXCR5+ CD8+ T cells could induce the death of tumor cells in HBV-related hepatocellular carcinoma

The follicular CXCR5⁺ CD8⁺ T cells have recently emerged as a critical cell type in mediating peripheral tolerance as well as antiviral immune responses during chronic infections. In this study, we investigated the function of CXCR5⁺ CD8⁺ T cells in HBV-related hepatocellular carcinoma patients. Compared to CXCR5⁻ CD8⁺ T cells, CXCR5⁺ CD8⁺ T cells presented elevated PD-1 expression but reduced Tim-3 and CTLA-4 expression. Upon anti-CD3/CD28 stimulation, CXCR5⁺ CD8⁺ T cells demonstrated higher proliferation potency than CXCR5⁻ CD8⁺ T cells, especially after PD-1 blockade. CXCR5⁺ CD8⁺ T cells also demonstrated significantly higher granzyme B synthesis and release, as well as higher level of degranulation. Tumor cells were more readily eliminated by CXCR5⁺ CD8⁺ T cells than by CXCR5⁻ CD8⁺ T cells. Interestingly, we found that B cells were more resistant to CXCR5⁺ CD8⁺ T cell-mediated killing than tumor cells, possibly through IL-10-mediated protection. In addition, the CXCR5⁺ CD8⁺ T cell-mediated cytotoxic effects on tumor cells could be significantly enhanced by PD-L1 blockade. Together, we presented that in patients with in HBV-related hepatocellular carcinoma, CXCR5⁺ CD8⁺ T cells could mediate tumor cell death more potently than the CXCR5⁻ CD8⁺ T cells in vitro while the autologous B cells were protected.     

CD163+ M2-type tumor-associated macrophage support the suppression of tumor-infiltrating T cells in osteosarcoma

Osteosarcoma is one of the most common childhood cancers with high numbers of cancer-related deaths. Progress in conventional therapies is showing limited improvement. An adaptive T cell-based immunotherapy represents a promising new therapeutic option, but to improve its efficacy, regulatory mechanisms in osteosarcoma need further elucidation. Here, to evaluate the regulatory effect of tumor microenvironment of T cells in osteosarcoma, we examined the peripheral blood (PB) and tumor infiltrating (TI) T cells, and their correlations with PB and tumor immune characteristics. We found that TI T cells contained significantly higher levels of TIM-3⁺ PD-1⁻ and TIM-3⁺ PD-1⁺ cells than their PB counterparts. Similar to that in chronic HIV and HCV infections, these TIM-3⁺ PD-1⁻ and TIM-3⁺ PD-1⁺ T cells presented reduced proliferation and proinflammatory cytokine secretion in response to stimulation. Presence of M2-type (CD163⁺) macrophages exacerbated T cell immunosuppression, since frequencies of CD163⁺ tumor-associated macrophages were directly correlated with the frequencies of suppressed TIM-3⁺ PD-1⁺ T cells. Moreover, depletion of CD163⁺ macrophages significantly improved T cell proliferation and proinflammatory cytokine production. Overall, our data presented an intratumoral T cell-specific immunosuppression that was amplified by M2-type tumor-associated macrophages.     

Enhanced effect of κ-carrageenan on TNBS-induced inflammation in mice

BackgroundAs a sulfated polysaccharide, carrageenan has been widely used as common food additive.MethodsIn the present study, we investigated the effects of κ-carrageenan on TNBS-induced gut inflammation in mice. BALB/c mice were pretreated with κ-carrageenan for 14 days prior to the administration of TNBS.ResultsOur results showed that κ-carrageenan pretreatment aggravated the loss of body weight and further increased the mortality rate. Histological and morphological analyses revealed that the TNBS-induced colonic inflammation was deteriorated by the κ-carrageenan administration. The ratio of CD4⁺ CD25⁺ CD127dim/CD4⁺ of the κ-carrageenan + TNBS groups was significantly lower than that of the TNBS group. The expression of IL-2, TNF-α and IL-6 was significantly increased, whereas the expression of IL-10 was significantly decreased in the κ-carrageenan + TNBS groups. In addition, κ-carrageenan, together with TNBS, decreased the enzyme activity of SOD and GSH-px and up-regulated the expression of TLR4, NF-κB, p-ERK, p-JNK, p-Jun., IL-8 and MDA in the colonic mucosa.Conclusionsκ-Carrageenan aggravated the TNBS-induced intestinal inflammation, and such an effect could be associated with the oxidative stress and activation of TLR4-NF-κB and MAPK/ERK1/2 pathway.     

A higher frequency of CD4+ CXCR5+ T follicular helper cells in patients with newly diagnosed Henoch–Schönlein purpura nephritis

T follicular helper (TFH) cells play an important role in the humoral immune responses. The aim of this study was to examine the frequency of different subsets of CD4⁺ CXCR5⁺ TFH cells and B cells in patients with new-onset Henoch–Schönlein purpura nephritis (HSPN). The numbers of different subsets of CD4⁺ CXCR5⁺ TFH cells, B cells and the constituents of serum cytokines were detected in a total of 25 patients with newly diagnosed HSPN before and after treatment, and in 14 healthy controls (HC). The potential connection of these cells with the clinical characteristics in HSPN patients was analyzed. The numbers of circulating CD4⁺ CXCR5⁺, CD4⁺ CXCR5⁺ ICOS⁺ and CD4⁺ CXCR5⁺ PD-1⁺ TFH cells, CD86⁺ CD19⁺, CD38⁺ CD19⁺ B cells and serum IL-2, IL-4, IL-17A, IL-21 and IFN-γ were significantly higher in HSPN patients (p < 0.05) than in HC. Before and after treatment the numbers of CD4⁺ CXCR5⁺ TFH cells were negatively correlated with the values of eGFR (r = − 0.7162, p < 0.05; r = − 0.732, p < 0.05, respectively). Similarly the numbers of CD4⁺ CXCR5⁺ PD-1⁺ TFH cells were negatively correlated with 24-h urinary proteins (r = − 0.4013, p < 0.05; r = − 0.7857, p < 0.05, respectively), and the numbers of CD4⁺ CXCR5⁺ ICOS⁺ TFH cells were positively correlated with the levels of serum IL-21 (r = 0.5186, p < 0.05; r = 0.8503, p < 0.05, respectively) and 24-h urinary protein (r = 0.6045, p < 0.05; r = 0.833, p < 0.05, respectively) in these patients, regardless of treatment. Following treatment the numbers of CD4⁺ CXCR5⁺, CD4⁺ CXCR5⁺ PD-1⁺, and CD4⁺ CXCR5⁺ ICOS⁺ TFH cells, as well as serum levels of IL-21 were significantly reduced, however IL-4 levels were noticeably increased (p < 0.05). A higher frequency of circulating CD4⁺ CXCR5⁺ TFH cells existed in patients with HSPN and may be a viable therapeutic target.     

Gabapentin-induced changes of plasma cortisol level and immune status in hysterectomized women

AimWe have examined the effects of gabapentin (GBP) on stress-related changes of cortisol and catecholamines in patients who underwent hysterectomy because of uterine fibrinoids. Additionally, we have observed the effect of GBP on the immune status in the acute stress response to surgery.MethodsSixty patients scheduled for an abdominal hysterectomy were randomly assigned to the GBP administration 1 h before surgery (n = 30 pts), or to the placebo group (n = 30 pts). Blood samples were collected before and 24 h after the surgery. The intensity of pain was assessed by a visual analogue scale (VAS) every 8 h at rest. Immunomodulatory effects of GBP were determined by flow cytometry. We followed the total proportion of CD3⁺ lymphocytes, CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺ B lymphocytes, CD16⁺CD56⁺CD3⁻NK cells and CD16⁺CD56⁺CD3⁺ NKT cells before and 24 h after hysterectomy. The plasma cortisol and catecholamines concentration was used to estimate the level of the stress response.ResultsVAS pain score at rest was significantly lower in the GBP group than in the placebo group (P = 0.003). Application of GBP significantly decreased the plasma cortisol level 24 h after the operation in comparison to the placebo group (P < 0,001). We found significant positive correlation between the VAS pain score and concentration of cortisol in all patients (P = 0.025). GBP reduced the concentration of catecholamines (p < 0.05). The proportion of CD3⁺ (P = 0.027) and CD3⁺CD4⁺cells (P = 0.006) was significantly lower in the GBP group 24 h after operation, while the contribution of CD19⁺ (P = 0.033) was significantly higher.ConclusionPreoperative administration of GBP reduced the pain scores at rest in patients at 0, 16 and 24 h after abdominal hysterectomy. Additionally, GBP reduced the stress response and changed immune parameters in the reaction to surgery.     

Donor pre-treatment with tacrolimus reduces transplant vasculopathy

We tested whether transplant arteriosclerosis can be reduced by pre-treatment of the donor with immunosuppressive agents, using a rat allogeneic aorta transplantation model.Donor rats received no pre-treatment, or tacrolimus, methylprednisolone, rapamycin, or mycofenolate mofetil (MMF) 16 and 2 h before explantation of the grafts. Eight weeks after transplantation, aorta allografts were harvested. Percent intima area/intima + media area (I/I + M), inflammatory cells and in situ MMP-2 and -9 activity were determined. In pre-transplantation biopsies, MMP-2 and -9 ratio, and mRNA levels for genes of interest were determined.In pre-transplantation biopsies we found no differences in MMP-2/9 ratio, and Bcl-2, Bax, TGF-β, HO-1, p21, and HIF-1α mRNA expression between the groups.Aorta allografts, pre-treated with tacrolimus, showed significantly lower I/I + M ratio compared to untreated controls (p < 0.01). Pre-treatment with methylprednisolone, rapamycin or MMF did not significantly reduce I/I + M ratio. In situ MMP-2/MMP-9 activity was significantly reduced in grafts treated with tacrolimus and rapamycin compared to controls (p < 0.05). Immunohistochemistry revealed a high number of CD4+ cells and high CD4/CD8 ratio in grafts pre-treated with tacrolimus.Donor pre-treatment with tacrolimus significantly reduces transplant arteriosclerosis and is associated with reduced in situ MMP-2/MMP-9 activity and increased number of CD4+ cells.     

CXCR5+ CD8+ T cells present elevated capacity in mediating cytotoxicity toward autologous tumor cells through interleukin 10 in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a common and aggressive subtype of non-Hodgkin's lymphomas, with limited treatment options in refractory and relapsed patients. Growing evidence supports the notion that CD8⁺ T cell immunity could be utilized to eliminate B cell lymphomas. CXCR5⁺ CD8⁺ T cell is a novel cell subtype and share CXCR5 expression with CD19⁺ tumor cells. In this study, we investigated the frequency and function of existing CXCR5⁺ CD8⁺ T cells in DLBCL patients. We found that DLBCL patients as a group demonstrated significantly higher level of CXCR5⁺ CD8⁺ T cells than healthy individuals, with huge variability in each patient. Using anti-CD3/CD28-stimulated CD8⁺ T cells as effector (E) cells and autologous CD19⁺ tumor cells as target (T) cells, at high E:T ratio, no difference between the intensities of CXCR5⁺ CD8⁺ T cell- and CXCR5⁻ CD8⁺ T cell-mediated cytotoxicity were observed. However, at intermediate and low E:T ratios, the CXCR5⁺ CD8⁺ T cells presented stronger cytotoxicity than CXCR5⁻ CD8⁺ T cells. The expressions of granzyme A, granzyme B, and perforin were significantly higher in CXCR5⁺ CD8⁺ T cells than in CXCR5⁻ CD8⁺ T cells, with no significant difference in the level of degranulation. Tumor cells in DLBCL were known to secrete high level of interleukin 10 (IL-10). We therefore blocked the IL-10/IL-10R pathway, and found that the expressions of granzyme A, granzyme B, and perforin by CXCR5⁺ CD8⁺ T cells were significantly elevated. Together, these results suggest that CXCR5⁺ CD8⁺ T cells are potential candidates of CD8⁺ T cell-based immunotherapies, could mediate elimination of autologous tumor cells in DLBCL patients, but are also susceptible to IL-10-mediated suppression.     

Mesenchymal stem cells upregulate Treg cells via sHLA-G in SLE patients

BackgroundSoluble human leukocyte antigen-G (sHLA-G) is a non-classical HLA class I molecule, exhibiting strong immunosuppressive properties by inducing the differentiation of T regulatory cells (Treg). Mesenchymal stem cells (MSCs) transplantation alleviates disease progression in systemic lupus erythematosus (SLE) patients. However, the underlying mechanisms are largely unknown.ObjectivesTo explore whether sHLA-G is involved in upregulating effects of MSCs on Treg, which contributes to therapeutic effects of MSCs transplantation in SLE.MethodsThe serum sHLA-G levels of SLE patients and healthy controls were detected by ELISA. The percentages of peripheral blood CD4 + ILT2 +, CD8 + ILT2 +, CD19 + ILT2 + cells and Treg cells were examined by flow cytometry. Ten patients with active SLE, refractory to conventional therapies, were infused with umbilical cord derived MSCs (UC-MSCs) and serum sHLA-G was measured 24 h and 1 month after infusion. The mice were divided into three groups: C57BL/6 mice, B6.MRL-Fas^lpr mice infused with phosphate buffer saline (PBS), and B6.MRL-Fas^lpr mice infused with bone marrow MSCs (BM-MSCs). Then, the concentrations of serum Qa-2 were detected. Peripheral blood mononuclear cells (PBMCs) were isolated from SLE patients and co-cultured with UC-MSCs for 3 days at different ratios (50:1, 10:1, and 2:1) with or without HLA-G antibody, and the frequencies of CD4 + CD25 + Foxp3 + T cells were then determined by flow cytometry.ResultsThe concentrations of serum sHLA-G were comparable between SLE patients and healthy controls. However, there was a negative correlation between sHLA-G levels and SLE disease activity index (SLEDAI) scores in active SLE patients (SLEDAI > 4). We found that serum sHLA-G levels were negatively correlated with blood urea nitrogen, serum creatinine and 24-hour urine protein in SLE patients. The sHLA-G levels were significantly lower in SLE patients with renal involvement than those without renal involvement. The expression of ILT2 on CD4 + T cells from SLE patients decreased significantly compared to that of healthy controls. A positive correlation between the frequencies of Treg and CD4 + ILT2 + T cells was found in SLE patients. The levels of sHLA-G increased 24 h post UC-MSCs transplantation. The concentrations of Qa-2 in BM-MSCs transplanted mice were significantly higher than those of control group. In vitro studies showed that MSCs increased the frequency of Treg cells in SLE patients in a dose-dependent manner, which was partly abrogated by the anti-HLA-G antibody.ConclusionsOur results suggested that MSCs may alleviate SLE through upregulating Treg cells, which was partly dependent on sHLA-G.     

Vitamin D3 enhances bactericidal activity of macrophage against Pseudomonas aeruginosa

BackgroundThe bioactive form of vitamin D3, i.e.1,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) vitamin D has been shown to modulate monocytes/macrophages physiology and its response against bacterial infections. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic bacterial pathogen that can most frequently be fatal in immunocompromised infected people.MethodsWe investigated the ex vivo effect of 1,25(OH)₂D₃ on monocyte-derived macrophages function against P. aeruginosa infection.ResultsRelative vitamin D receptor (VDR) mRNA expression was significantly increased in infected and 1,25(OH)₂D₃-treated macrophages compared to controls (p < 0.01). Treatment with 1,25(OH)₂D₃ markedly resulted in up-regulation of nitric oxide (NO) and IL-1β production and down-regulation of IL-10 levels (respectively, p = 0.029, p = 0.048 and p = 0.008). Additionally, 1,25(OH)₂D₃ significantly increased M1/M2 macrophage ratio (p < 0.05) and slightly reduced intracellular bacterial development. Furthermore, the arginase activity, P. aeruginosa phagocytosis and killing were significantly increased in cells that were both infected and 1,25(OH)₂D₃-treated compared to the infected, but not 1,25(OH)₂D₃-treated macrophages (respectively, p < 0.001, p < 0.01 and p < 0.001).ConclusionsWe show in this study that bioactive from of vitamin D [1,25-dihydroxyvitamin D3 (1,25D3)] can enhance M1 macrophage polarization and their bactericidal protective activity against P. aeruginosa. Future works would involve improving the treatment response through dose-dependent effect studies, both in ex vivo and in vivo models.     

Chicken egg yolk antibodies (IgY) modulate the intestinal mucosal immune response in a mouse model of Salmonella typhimurium infection

This study determined the effects of chicken egg yolk antibodies (IgY) on immune responses in the intestinal mucosal of mice infected with Salmonella typhimurium. Sixty, 28-day-old mice were divided into 4 groups and treated with streptomycin or sterile water for 2 days followed by 1 day without treatment. The control group was unchallenged whereas the mice in the other three groups were treated twice with 10⁹ CFU mL^− 1 S. typhimurium. For the next 3 days, control mice continued to receive no treatment whereas the mice in the remaining three groups were orally administered with 20 mg mL^− 1 of specific IgY, 20 mg mL^− 1 of nonspecific IgY or PBS. S. typhimurium activated gut-associated lymphoid tissue, increasing the release of IFN-γ and TNF-α in the mucosa and increased the number of activated T-lymphocytes and cytotoxic T-γδ. Specific IgY attenuated the increase in IFN-γ and TNF-α and the decrease in IL-10. S. typhimurium induced mobilization of CD8⁺ and CD8⁺ TCRγδ T cells in the epithelium and CD4⁺ and CD8⁺ T cells in the lamina propria reflecting an inflammatory process that was attenuated by IgY. These results suggest that specific IgY modulates intestinal mucosal immune responses during a S. typhimurium infection.     

Interferon gamma induced by resveratrol analog,HS-1793, reverses the properties of tumor associated macrophages

Macrophages are capable of both inhibiting and promoting the growth and spread of cancers, depending on their activation state. Tumor-associated macrophages (TAM) are a kind of alternatively activated M2 macrophage, which may contribute to tumor progression. Following our previous study to evaluate the anti-tumor effect of a synthetic resveratrol analog HS-1793, the current study demonstrated that HS-1793 treatment significantly increased IFN-γ secreting cells in splenocytes and decreased CD206 + macrophage infiltration compared to CD68 + cells in the tumor site with a higher expression of IFN-γ. As these results suggested that IFN-γ increased locally at the tumor sites could modulate the status of TAM, we designed an in vitro model to study macrophage morphology and functions in relation to the tumor microenvironment. Human monocytic cell line THP-1 cells stimulated with phorbol-12-myristate-13-acetate (PMA) differentiated to macrophages with M2-like phenotypes. TAM-like properties of CD206^high, CD204^high, IL-10^high, TGF-β^high, IL-6^low, IL-12^low, VEGF^high, and MMP-9^high and promotion of tumor cell invasion were more pronounced in M-2-polarized THP-1 macrophages generated by differentiating THP-1 cells with PMA and subsequently polarizing them with Th2 cytokines (IL-4/IL-13). Upon IFN-γ exposure, THP-1-derived TAM changed their phenotypes to the M-1-like morphology and intracellular granular pattern with an expression of an increased level of proinflammatory and immunostimulatory cytokines and a reduced level of immunosuppressive and tumor progressive mediators. These results explain the underlying mechanism of the anti-tumor activity of HS-1793. The elevated level of IFN-γ production after HS-1793 treatment evoked reprogramming of M-2 phenotype TAM, which efficiently countered the immunosuppressive and tumor progressive influences of TAM.     

Compact bone-derived mesenchymal stem cells attenuate nonalcoholic steatohepatitis in a mouse model by modulation of CD4 cells differentiation

Increasing evidence has accrued which indicates that mesenchymal stem cells (MSCs) have a potential clinical value in the treatment of certain diseases. Globally, nonalcoholic steatohepatitis (NASH) is a widespread disorder. In the present study, MSCs were isolated successfully from compact bone and a mouse model of NASH was established as achieved with use of a methionine-choline deficient (MCD) diet. Compact bone-derived MSCs transplantation reduced MCD diet-induced weight loss, hepatic lipid peroxidation, steatosis, ballooning, lobular inflammation and fibrogenesis. It was shown that MSCs treatment hampered MCD diet-induced proliferation of CD4⁺ IFN-γ⁺ and CD4⁺ IL-6⁺ T spleen cells. In addition, CD4⁺ IL-17⁺ lymphocytes that associated with anti-inflammation show little change in MCD as well as in MCD + MSCs splenocytes. We conclude that MSCs may have a potential clinical value upon NASH, through their capacity to suppress activation of CD4⁺ IFN-γ⁺ and CD4⁺ IL-6⁺ lymphocytes.     

Air pollution-related metals induce differential cytokine responses in bronchial epithelial cells

Different transition metals have been shown to induce inflammatory responses in lung. We have compared eight different metal ions with regard to cytokine responses, cytotoxicity and signalling mechanisms in a human lung epithelial cell model (BEAS-2B). Among the metal ions tested, there were large differences with respect to pro-inflammatory potential. Exposure to Cd^2 +, Zn^2 + and As^3 + induced CXCL8 and IL-6 release at concentrations below 100 μM, and Mn^2 + and Ni^2 + at concentrations above 200 μM. In contrast, VO₄^3 −, Cu^2 + and Fe^2 + did not induce any significant increase of these cytokines. An expression array of 20 inflammatory relevant genes also showed a marked up-regulation of CXCL10, IL-10, IL-13 and CSF2 by one or more of the metal ions. The most potent metals, Cd^2 +, Zn^2 + and As^3 + induced highest levels of oxidative activity, and ROS appeared to be central in their CXCL8 and IL-6 responses. Activation of the MAPK p38 seemed to be a critical mediator. However, the NF-κB pathway appeared predominately to be involved only in Zn^2 +- and As^3 +-induced CXCL8 and IL-6 responses. Thus, the most potent metals Cd^2 +, Zn^2 + and As^3 + seemed to induce a similar pattern for the cytokine responses, and with some exceptions, via similar signalling mechanisms.     

P-glycoprotein function in peripheral T lymphocyte subsets of myasthenia gravis patients: Clinical implications and influence of glucocorticoid administration

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder with a chronic clinical course that requires long-term glucocorticoid (GC) therapy. A drug efflux pump, P-glycoprotein (P-gp), actively transports GC out of target cells, thereby reducing its efficacy. We evaluated the P-gp function of peripheral-blood mononuclear cells in 59 MG patients. P-gp function was estimated from a decrease in fluorescent P-gp substrate Rhodamine 123 and its inhibition by the conformation-sensitive UIC2 monoclonal antibody. P-gp function on CD8⁺ T cells in 21 MG patients having experienced GC therapy was higher than that in 19 MG patients having no history of GC therapy (p = 0.026). There was a significant correlation between P-gp function in CD3⁺ (r = 0.55, p = 0.014) or CD4⁺ (r = 0.48, p = 0.034) T cells and the total dose of prednisolone for treatment. P-gp function on CD4⁺ T cells in MG patients who showed low responses to prednisolone therapy (n = 8) was higher than that in patients who showed relatively high responses to prednisolone therapy (n = 10) (p = 0.045). These results suggest that higher P-glycoprotein activity on CD3⁺ or CD4⁺ cells necessitated treatment with higher steroid doses in order to achieve a clinical response. The measurement of P-gp function on CD4⁺ T cells is useful in the assessment of clinical response to GC therapy.     

Circulating follicular helper T cells presented distinctively different responses toward bacterial antigens in primary biliary cholangitis

Primary biliary cholangitis (PBC) is a chronic and progressive cholestatic liver disease with unknown causes. The initiation of PBC is associated with bacterial infections and abnormal immune correlates, such as the presence of self-reactive anti-mitochondrial antibodies and shifted balance of T cell subsets. In particular, the CD4⁺ CXCR5⁺ follicular helper T (Tfh) cells are highly activated in PBC patients and are significantly associated with PBC severity, but the underlying reasons are unknown. In this study, we found that the circulating CD4⁺ CXCR5⁺ T cells were enriched with the interferon (IFN)-γ-secreting Th1-subtype and the interleukin (IL)-17-secreting Th17-subtype, but not the IL-4-secreting Th2 subtype. We further demonstrated that a host of microbial motifs, including Pam3CSK4, poly(I:C), LPS, imiquimod, and CpG, could significantly stimulate IFN-γ, IL-17, and/or IL-21 from circulating CD4⁺ CXCR5⁺ T cells in PBC patients, especially in the presence of monocytes and B cells. Whole bacterial cells of Escherichia coli, Novosphingobium aromaticivorans, and Mycobacterium gordonae, could also potently stimulate IFN-γ, IL-17, and/or IL-21 production from circulating CD4⁺ CXCR5⁺ T cells. But interestingly, while the whole cell could potently stimulate circulating CD4⁺ CXCR5⁺ T cells from both healthy controls and PBC patients, the cell protein lysate could only potently stimulate circulating CD4⁺ CXCR5⁺ T cells from PBC patients, but not those from healthy controls, suggesting that circulating CD4⁺ CXCR5⁺ T cells in PBC patients had distinctive antigen-specificity from those in healthy individuals. Together, these data demonstrated that bacterial antigen stimulation is a potential source of aberrant Tfh cell activation in PBC patients.     

Characterization and structure-activity relationship of natural flavonoids as hERG K+ channel modulators

ObjectivesFlavonoids are present in varying concentrations in plant foods and have been reported to have numerous pharmacological activities, such as anti-cancer, antioxidant, anti-inflammatory, hepatoprotective, and vasodilator effects. We found that quercetin, fisetin, and some related flavonoid derivatives could inhibit human ether-à-go-go-related gene (hERG) K⁺ channels.Key findingsIn this study, we tested the effects of a series of flavonoids on the hERG K⁺ channel expressed in HEK293 cells. For the first time, we demonstrate that quercetin and fisetin (Fise) are potent hERG current blockers. The 50% inhibiting concentration (IC₅₀) and maximum efficacy (E_max) of quercetin were 11.8 ± 0.9 μM and 82 ± 2%, while those of fisetin were 38.4 ± 6 μM and 100 ± 6%, respectively. Luteolin (Lute) was a less potent inhibitor of hERG current (48 ± 1% at 100 μM). Galangin, kaempferol, and isorhamnetin (100 μM) showed weaker activity on the hERG currents.ConclusionThese results suggest that quercetin, fisetin, and luteolin are potent hERG K⁺ channel inhibitors and reveal the structure-activity relationship of natural flavonoids.     

Circulating CD4+ CXCR5+ T cells contribute to proinflammatory responses in multiple ways in coronary artery disease

Coronary artery disease (CAD) is a common subtype of cardiovascular disease. The major contributing event is atherosclerosis, which is a progressive inflammatory condition resulting in the thickening of the arterial wall and the formation of atheromatous plaques. Recent evidence suggests that circulating CD4⁺ CXCR5⁺ T cells can contribute to inflammatory reactions. In this study, the frequency, phenotype, and function of circulating CD4⁺ CXCR5⁺ T cells in CAD patients were examined. Data showed that circulating CD4⁺ CXCR5⁺ T cells in CAD patients were enriched with a PD-1⁺ CCR7⁻ subset, which was previously identified as the most potent in B cell help. The CD4⁺ CXCR5⁺ T cells in CAD patients also secreted significantly higher levels of IFN-γ, IL-17A, and IL-21 than those from healthy controls. Depleting the PD-1⁺ population significantly reduced the cytokine secretion. Interestingly, the CD4⁺ CXCR5⁺ PD-1⁻ T cells significantly upregulated PD-1 following anti-CD3/CD28 or SEB stimulation. CD4⁺ CXCR5⁺ T cells from CAD patients also demonstrated more potent capacity to stimulate B cell inflammation than those from healthy individuals. The phosphorylation of STAT1 and STAT3 were significantly higher in B cells incubated with CD4⁺ CXCR5⁺ T cells from CAD than controls. The IL-6 and IFN-γ expression were also significantly higher in B cells incubated with CD4⁺ CXCR5⁺ T cells from CAD. Together, this study demonstrated that CAD patients presented a highly activated CD4⁺ CXCR5⁺ T cell subset that could contribute to proinflammatory responses in multiple ways. The possibility of using CD4⁺ CXCR5⁺ T cells as a therapeutic target should therefore be examined in CAD patients.