[Effects of herbal cake separated moxibustion on Tim-4 and ubiquitination of PD-1 in rabbits with immunosuppression].

OBJECTIVE: To observe the effects of herbal cake separated moxibustion on macrophage effector molecule T-cell immunoglobulin and mucin-domain containing-4 (Tim-4) and ubiquitination of programmed cell death protein 1 (PD-1) in rabbits with immunosuppression, and to explore the possible mechanism on herbal cake separated moxibustion in improving immunosuppression. METHODS: Thirty-two big-ear white rabbits were randomly divided into a normal group, a model group, a moxa stick moxibustion group and a herbal cake separated moxibustion group, 8 rabbits in each group. Except the normal group, the immunosuppression model was established by intraperitoneal injection of cyclophosphamide of60 mg/kg in the other 3 groups. "Shenque" (CV 8), "Shenshu" (BL 23), "Zusanli" (ST 36), etc. were selected in both the moxa stick moxibustion group and the herbal cake separated moxibustion group. Moxa stick moxibustion was applied in the moxa stick moxibustion group, one cone at each acupoint; herbal cake separated moxibustion was applied in the herbal cake separated moxibustion group, 5 cones at each acupoint. The intervention was given once every other day for 10 times in both groups. Leukocyte content in peripheral blood was detected by blood cell analyzer; the positive expression of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood was measured by flow cytometry, the serum levels of interleukin 2 (IL-2), CD8, CD68 and Tim-4 were detected by ELISA, and the expression of Tim-4 and F-box only protein 38 (FBXO38) in the liver and spleen tissues was measured by immunohistochemistry. RESULTS: Compared with the normal group, in the model group, white blood cell count (WBC) and percentage of neutrophils (NEU%) were decreased while percentage of lymphocyte (LYM%) was increased (P<0.01) in peripheral blood; the positive expression rates of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood were increased (P<0.01); the serum levels of IL-2, CD68 and Tim-4 were increased (P<0.01), the serum level of CD8 was decreased (P<0.01); the average optical density (AOD) of Tim-4 in the liver tissue and FBXO38 in the liver and spleen tissues was increased (P<0.01). Compared with the model group, in the moxa stick moxibustion group and the herbal cake separated moxibustion group, WBC and NEU% were increased (P<0.01); the positive expression rates of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood were decreased (P<0.01); the serum levels of IL-2, CD68 and Tim-4 were decreased (P<0.01), the serum levels of CD8 were increased (P<0.01); the AOD of Tim-4 and FBXO38 in the liver tissue and FBXO38 in the spleen tissue was decreased (P<0.01, P<0.05). Compared with the moxa stick moxibustion group, in the herbal cake separated moxibustion group, the positive expression rate of PD-1 in CD+68 macrophages in peripheral blood was increased (P<0.05); serum level of Tim-4 was increased (P<0.01); AOD of Tim-4 in the liver tissue was decreased (P<0.05). CONCLUSION: Herbal cake separated moxibustion can improve immunosuppression by regulating the expression of macrophage effector molecule Tim-4 and the FBXO38 mediated ubiquitination of PD-1, Tim-4 may be one of the specific indexes of immunomodulation involving with herbal cake separated moxibustion.

Inflammatory Macrophage Interleukin-1ß Mediates High-Fat Diet-Induced Heart Failure With Preserved Ejection Fraction.

Diabetes mellitus (DM) is a main risk factor for diastolic dysfunction (DD) and heart failure with preserved ejection fraction. High-fat diet (HFD) mice presented with diabetes mellitus, DD, higher cardiac interleukin (IL)-1ß levels, and proinflammatory cardiac macrophage accumulation. DD was significantly ameliorated by suppressing IL-1ß signaling or depleting macrophages. Mice with macrophages unable to adopt a proinflammatory phenotype were low in cardiac IL-1ß levels and were resistant to HFD-induced DD. IL-1ß enhanced mitochondrial reactive oxygen species (mitoROS) in cardiomyocytes, and scavenging mitoROS improved HFD-induced DD. In conclusion, macrophage-mediated inflammation contributed to HFD-associated DD through IL-1ß and mitoROS production.

Effects of herbal cake separated moxibustion on Tim-4 and ubiquitination of PD-1 in rabbits with immunosuppression / 中国针灸

OBJECTIVE@#To observe the effects of herbal cake separated moxibustion on macrophage effector molecule T-cell immunoglobulin and mucin-domain containing-4 (Tim-4) and ubiquitination of programmed cell death protein 1 (PD-1) in rabbits with immunosuppression, and to explore the possible mechanism on herbal cake separated moxibustion in improving immunosuppression.@*METHODS@#Thirty-two big-ear white rabbits were randomly divided into a normal group, a model group, a moxa stick moxibustion group and a herbal cake separated moxibustion group, 8 rabbits in each group. Except the normal group, the immunosuppression model was established by intraperitoneal injection of cyclophosphamide of60 mg/kg in the other 3 groups. "Shenque" (CV 8), "Shenshu" (BL 23), "Zusanli" (ST 36), etc. were selected in both the moxa stick moxibustion group and the herbal cake separated moxibustion group. Moxa stick moxibustion was applied in the moxa stick moxibustion group, one cone at each acupoint; herbal cake separated moxibustion was applied in the herbal cake separated moxibustion group, 5 cones at each acupoint. The intervention was given once every other day for 10 times in both groups. Leukocyte content in peripheral blood was detected by blood cell analyzer; the positive expression of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood was measured by flow cytometry, the serum levels of interleukin 2 (IL-2), CD8, CD68 and Tim-4 were detected by ELISA, and the expression of Tim-4 and F-box only protein 38 (FBXO38) in the liver and spleen tissues was measured by immunohistochemistry.@*RESULTS@#Compared with the normal group, in the model group, white blood cell count (WBC) and percentage of neutrophils (NEU%) were decreased while percentage of lymphocyte (LYM%) was increased (P<0.01) in peripheral blood; the positive expression rates of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood were increased (P<0.01); the serum levels of IL-2, CD68 and Tim-4 were increased (P<0.01), the serum level of CD8 was decreased (P<0.01); the average optical density (AOD) of Tim-4 in the liver tissue and FBXO38 in the liver and spleen tissues was increased (P<0.01). Compared with the model group, in the moxa stick moxibustion group and the herbal cake separated moxibustion group, WBC and NEU% were increased (P<0.01); the positive expression rates of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood were decreased (P<0.01); the serum levels of IL-2, CD68 and Tim-4 were decreased (P<0.01), the serum levels of CD8 were increased (P<0.01); the AOD of Tim-4 and FBXO38 in the liver tissue and FBXO38 in the spleen tissue was decreased (P<0.01, P<0.05). Compared with the moxa stick moxibustion group, in the herbal cake separated moxibustion group, the positive expression rate of PD-1 in CD+68 macrophages in peripheral blood was increased (P<0.05); serum level of Tim-4 was increased (P<0.01); AOD of Tim-4 in the liver tissue was decreased (P<0.05).@*CONCLUSION@#Herbal cake separated moxibustion can improve immunosuppression by regulating the expression of macrophage effector molecule Tim-4 and the FBXO38 mediated ubiquitination of PD-1, Tim-4 may be one of the specific indexes of immunomodulation involving with herbal cake separated moxibustion.

Recipient TIM4 signaling regulates ischemia reperfusion-induced ER stress and metabolic responses in liver transplantation: from mouse-to-human.

T-cell immunoglobulin and mucin (Tim)4 is expressed on APCs, including macrophages, as one of the main amplifiers in the mechanism of liver ischemia-reperfusion injury (IRI) following orthotopic liver transplantation (OLT). Though donor Tim4 selectively expressed on Kupffer cells serves as a checkpoint regulator of innate immune-driven IRI cascades, its role on cells outside the OLT remains unclear. To dissect the role of donor vs. recipient-specific Tim4 signaling in IR-induced stress and hepatocellular function, we employed a murine OLT model utilizing Tim4-knockout (KO) mice as either donor or recipient (WT → WT, WT → Tim4-KO, Tim4-KO → WT). In the experimental arm, disruption of donor Tim4 attenuated IRI-OLT damage, while recipient Tim4-null mutation aggravated hepatic IRI concomitant with disturbed lipid metabolism, enhanced endoplasmic reticulum stress, and activated pro-apoptotic signaling in the grafts. In the in vitro study, murine hepatocytes co-cultured with Tim4-null adipose tissue showed enhanced C/EBP homologous protein (CHOP) expression pattern and susceptibility to hepatocellular death accompanied by activated caspase cascade in response to TNF-α stimulation. In the clinical arm, liver grafts from forty-one transplant patients with enhanced TIM4 expression showed higher body mass index, augmented hepatic endoplasmic reticulum stress, enhanced pro-apoptotic markers, upregulated innate/adaptive immune responses, exacerbated hepatocellular damage, and inferior graft survival. In conclusion, although TIM4 is considered a principal villain in peri-transplant early tissue injury, recipient TIM4 signaling may serve as a savior of IR-triggered metabolic stress in mouse and human OLT recipients.

TIM4/NLRP3 axis participates in the effects of M1 macrophages on inflammatory factor release, apoptosis and cell adhesion in thyroid follicular cells.

Hashimoto's thyroiditis (HT) is an autoimmune thyroid disorder that predominantly affects women. The role of the T-cell immunoglobulin and mucin domain-containing 4 (TIM4)/ NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) signaling pathway in macrophages has previously been studied, but its effects on macrophage-mediated HT has not yet been reported. Therefore, the aim of the current study was to explore the regulatory role of TIM4/NLRP3 in the effects of M1 macrophages on the inflammation, apoptosis and cell adhesion of thyroid follicular cells. To induce M1 macrophage, 10 ng/ml of LPS and 20 ng/ml IFN-γ were applied for the administration of THP-1 cells for 24 h. After induction, the mRNA expressions of M1 macrophage markers were assessed utilizing reverse transcription-quantitative (RT-q)PCR. Western blotting and immunofluorescence assay were adopted for the appraisement of inducible nitric oxide synthase. Additionally, the expression levels of TIM4 and NLRP3 before or after transfection were tested using RT-qPCR and western blotting. The release of inflammatory cytokines (TNF-α, IL-1ß and IL-6) were estimated using RT-qPCR and western blotting was adopted for the estimation of phosphorylated (p)-p65, p65, I-κB and p-I-κB. Furthermore, the apoptosis level as well as the accompanied proteins was appraised via TUNEL and western blotting. The mRNA and protein expressions of αvß3 were evaluated employing RT-qPCR and western blotting. The results demonstrated that TIM4 silencing decreased NLRP3 expression level in M1 macrophages. Moreover, TIM4 silencing in M1 macrophages reduced the expression levels of TNF-α, IL-6 and IL-1ß, as well as the phosphorylation levels of p65 and IκB in M1 macrophages co-cultured with Nthy-ori 3-1 cells, whereas NLRP3 overexpression significantly reversed these effects. Furthermore, NLRP3 overexpression reversed the decreased apoptotic rate and cell adhesion of Nthy-ori 3-1 cells induced by TIM4 silencing. In summary, the present study demonstrated that TIM4-silencing alleviated the inflammatory damage, apoptosis and cell adhesion of M1 macrophages co-cultured with Nthy-ori 3-1 cells through downregulation of NLRP3. Therefore, the regulation of M1 macrophages via the TIM4/NLRP3 axis may be a potential therapeutic approach for the treatment of patients with HT.

Increased T cell immunoglobulin and mucin domain containing 4 (TIM-4) is negatively correlated with serum concentrations of interleukin-1ß in type 2 diabetes.

BACKGROUND: T cell immunoglobulin and mucin domain containing 4 (TIM-4), a novel immune regulator, is selectively expressed on antigen-presenting cells, especially macrophages and mature dendritic cells. Although TIM-4 plays key roles in mutiple immune diseases, whether it is involved in type 2 diabetes mellitus (T2D) remains unknown. The aim of the present study was to investigate the expression of TIM-4 in T2D and determine its significance in disease progression. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from T2D patients and healthy controls to measure TIM-4 mRNA expression by real-time polymerase chain reaction (PCR), and sera were collected to determine interleukin (IL)-1ß concentrations and other clinical indicators (high-sensitivity C-reactive protein [hsCRP], total cholesterol, low-density lipoprotein cholesterol [LDL-C], high-density lipoprotein cholesterol, triglyceride, fasting glucose, HbA1c, aspartate aminotransferase, and alanine aminotransferase). RESULTS: Expression of TIM-4 mRNA was increased significantly in PBMCs from T2D patients compared with healthy controls. There was a positive correlation between TIM-4 mRNA expression and serum concentrations of hsCRP. However, there was a negative correlation between TIM-4 mRNA expression and IL-1ß concentrations, indicating the potential role for TIM-4 to negatively regulate IL-1ß production. In addition, TIM-4 mRNA expression was negatively correlated with lowLDL-C, and there was a tendency for a negative relationship between TIM-4 mRNA expression and HbA1c. CONCLUSIONS: The results of the present study indicate that TIM-4 contributes, at least in part, to the pathogenesis of T2D, possibly by regulating IL-1ß.