The lectin Siglec-G inhibits dendritic cell cross-presentation by impairing MHC class I-peptide complex formation.

CD8α(+) dendritic cells (DCs) are specialized at cross-presenting extracellular antigens on major histocompatibility complex (MHC) class I molecules to initiate cytotoxic T lymphocyte (CTL) responses; however, details of the mechanisms that regulate cross-presentation remain unknown. We found lower expression of the lectin family member Siglec-G in CD8α(+) DCs, and Siglec-G deficient (Siglecg(-/-)) mice generated more antigen-specific CTLs to inhibit intracellular bacterial infection and tumor growth. MHC class I-peptide complexes were more abundant on Siglecg(-/-) CD8α(+) DCs than on Siglecg(+/+) CD8α(+) DCs. Mechanistically, phagosome-expressed Siglec-G recruited the phosphatase SHP-1, which dephosphorylated the NADPH oxidase component p47(phox) and inhibited the activation of NOX2 on phagosomes. This resulted in excessive hydrolysis of exogenous antigens, which led to diminished formation of MHC class I-peptide complexes for cross-presentation. Therefore, Siglec-G inhibited DC cross-presentation by impairing such complex formation, and our results add insight into the regulation of cross-presentation in adaptive immunity.

Alterations of urine microRNA-7977/G6PD level in patients with diabetic kidney disease and its association with dysfunction of albumin-induced autophagy in proximal epithelial tubular cells.

Diabetic kidney disease (DKD) remains as one of the leading long-term complications of type 2 diabetic mellitus (T2DM). Studies have shown that decreased expression of glucose-6-phosphate dehydrogenase (G6PD) plays an important role in DKD. However, the upstream and downstream pathways of G6PD downregulation leading to DKD have not been elucidated. We conducted a series of studies including clinical study, animal studies, and in vitro studies to explore this. First, a total of 90 subjects were evaluated including 30 healthy subjects, 30 patients with T2DM, and 30 patients with DKD. The urinary G6PD activity and its association with the clinical markers were analyzed. Multivariate linear regression analysis was used to analyze the risk factors of urinary G6PD in these patients. Then, microRNAs that were differentially expressed in urine and could bind and degrade G6PD were screened and verified in patients with DKD. After that, high glucose (HG)-cultured human kidney cells (HK-2) and Zucker diabetic fatty (ZDF) rats were used to test the roles of miR-7977/G6PD/albumin-induced autophagy in DKD. Beclin and P62 were used as markers of kidney autophagy indicators. A dual-luciferase reporter assay system was used to test the binding of G6PD by mir-7977. The plasma and urinary G6PD activity were decreased significantly in patients with DKD, accompanied by increased urinary mir-7977 level. The fasting plasma glucose (FPG), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and urinary albumin excretion were independent predictors of urinary G6PD activity, according to multiple linear regression analysis. The increased expression of miR-7977 and decreased expression of G6PD were also found in the kidney of ZDF rats with early renal tubular damage. The correlation analysis showed that beclin protein expression levels were positively correlated with kidney G6PD activity, whereas P62 protein expression was negatively correlated with kidney G6PD activity in rats. In HK-2 cells cultured with normal situation, a low level of albumin could induce autophagy along with the stimulation of G6PD, although this was impaired under high glucose. Overexpression of G6PD reversed albumin-induced autophagy in HK-2 cells under high glucose. Further study revealed that G6PD was a downstream target of miR-7977. Inhibition of miR-7977 expression led to significantly increased expression of G6PD and reversed the effects of high glucose on albumin-induced autophagy. In conclusion, our study supports a new mechanism of G6PD downregulation in DKD. Therapeutic measures targeting the miR-7977/G6PD/autophagy signaling pathway may help in the prevention and treatment of DKD.NEW & NOTEWORTHY This study provides new evidence that reduced glucose-6-phosphate dehydrogenase (G6PD) may damage the endocytosis of renal tubular epithelial cells by reducing albumin-induced autophagy. More importantly, for the first time, our study has provided evidence from humans that the decrease in urinary G6PD activity is positively associated with renal injury, and abnormal glucose and lipid metabolism may be important reasons for reduced G6PD levels. Increased miR-7977 may at least in part explain the downregulation of G6PD.

NK-like CD8 T cell: one potential evolutionary continuum between adaptive memory and innate immunity.

CD8 T cells are crucial adaptive immune cells with cytotoxicity to fight against pathogens or abnormal self-cells via major histocompatibility complex class I-dependent priming pathways. The composition of the memory CD8 T-cell pool is influenced by various factors. Physiological aging, chronic viral infection, and autoimmune diseases promote the accumulation of CD8 T cells with highly differentiated memory phenotypes. Accumulating studies have shown that some of these memory CD8 T cells also exhibit innate-like cytotoxicity and upregulate the expression of receptors associated with natural killer (NK) cells. Further analysis shows that these NK-like CD8 T cells have transcriptional profiles of both NK and CD8 T cells, suggesting the transformation of CD8 T cells into NK cells. However, the specific induction mechanism underlying NK-like transformation and the implications of this process for CD8 T cells are still unclear. This review aimed to deduce the possible differentiation model of NK-like CD8 T cells, summarize the functions of major NK-cell receptors expressed on these cells, and provide a new perspective for exploring the role of these CD8 T cells in health and disease.

Pan-cancer efficacy and safety of anlotinib plus PD-1 inhibitor in refractory solid tumor: A single-arm, open-label, phase II trial.

The combination of immunotherapy and antiangiogenic agents for the treatment of refractory solid tumor has not been well investigated. Thus, our study aimed to evaluate the efficacy and safety of a new regimen of anlotinib plus PD-1 inhibitor to treat refractory solid tumor. APICAL-RST is an investigator-initiated, open-label, single-arm, phase II trial in patients with heavily treated, refractory, metastatic solid tumor. Eligible patients experienced disease progression during prior therapy without further effective regimen. All patients received anlotinib and PD-1 inhibitor. The primary endpoints were objective response and disease control rates. The secondary endpoints included the ratio of progression-free survival 2 (PFS2)/PFS1, overall survival (OS) and safety. Forty-one patients were recruited in our study; 9 patients achieved a confirmed partial response and 21 patients had stable disease. Objective response rate and disease control rate were 22.0% and 73.2% in the intention-to-treat cohort, and 24.3% and 81.1% in the efficacy-evaluable cohort, respectively. A total of 63.4% (95% confidence interval [CI]: 46.9%-77.4%) of the patients (26/41) presented PFS2/PFS1 >1.3. The median OS was 16.8 months (range: 8.23-24.4), and the 12- and 36-month OS rates were 62.8% and 28.9%, respectively. No significant association was observed between concomitant mutation and efficacy. Thirty-one (75.6%) patients experienced at least one treatment-related adverse event. The most common adverse events were hypothyroidism, hand-foot syndrome and malaise. This phase II trial showed that anlotinib plus PD-1 inhibitor exhibits favorable efficacy and tolerability in patients with refractory solid tumor.

Key enzyme in charge of ketone reabsorption of renal tubular SMCT1 may be a new target in diabetic kidney disease.

OBJECTIVE: A ketogenic diet or mildly increased ketone body levels are beneficial for diabetic kidney disease (DKD) patients. Our previous study has found that sodium-coupled monocarboxylate transporter 1 (SMCT1), a key enzyme in charge of ketone reabsorption, possesses beneficial effects on the function of renal tubular epithelial cells (TECs) in energy crisis. Our present study is to investigate whether SMCT1 is important in maintaining the physiological function of renal tubular and plays a role in DKD. METHODS: We tested the expression of SMCT1 in kidney tissues from DKD patients receiving kidney biopsy as well as diabetes mice. We compared the difference of ß-hydroxybutyrate (ß-HB) levels in serum, urine and kidney tissues between diabetic mice and control. Using recombinant adeno-associated viral vector containing SMCT1 (encoded by Slc5a8 gene), we tested the effect of SMCT1 upregulation on microalbuminuria as well as its effects on mitochondrial energy metabolism in diabetic mice. Then we investigated the role of SMCT1 and its ß-HB reabsorption function in maintaining the physiological function of renal tubular using renal tubule-specific Slc5a8 gene knockout mice. Transcriptomes and proteomics analysis were used to explore the underlying mechanism. RESULTS: SMCT1 downregulation was found in DKD patients as well as in diabetic mice. Moreover, diabetic mice had a decreased renal ß-HB level compared with control, and SMCT1 upregulation could improve microalbuminuria and mitochondrial energy metabolism. In renal tubule-specific Slc5a8 gene knockout mice, microalbuminuria occurred early at 24 weeks of age, accompanied by ATP shortage and metabolic reprogramming in the kidney; however, supplementation with ß-HB precursor substance 1,3-butanediol in food alleviated kidney damage as well as energy metabolic reprogramming. CONCLUSIONS: Decreased SMCT1 expression and its ketone reabsorption function play an important role in the occurrence of DKD. SMCT1 may be a new promising target in treating DKD.

Dectin-2 promotes house dust mite-skewed Th2 response through the activation of cDC2s.

The role of Dectin-2 (gene symbol, Clec4n) in house dust mite (HDM) induced Th2 immune response and the exact mechanism remains controversial. In this study, we illustrated that, Clec4n-/- mice had decreased Th2 immune response following HDM challenge, which may ascribe to dramatically reduced type 2 conventional dendritic cells (cDC2s) in lung of Clec4n-/- mice, as cDC2s from lung of Clec4n-/- mice after challenging had less ability to induce Th2 response with decreased production of IL-4/IL-13. Further in vitro experiments showed the activation of Clec4n-/--BMDCs significantly decreased after HDM stimulation accompanied with decreased activation of Syk-NF-κB and Syk-JNK signal pathway. Importantly, Dectin-2 expression in PBMCs from asthmatic patients was significantly higher than that in healthy controls. Taken together, these results demonstrated that Dectin-2 could promote cDC2s activation in lung, which polarizes Th2 immune response outlining a novel mechanism of asthma development.

A phase I/II clinical trial on the efficacy and safety of NKT cells combined with gefitinib for advanced EGFR-mutated non-small-cell lung cancer.

BACKGROUND: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, have achieved good efficacy in EGFR mutation-positive non-small-cell lung cancer (NSCLC) patients, but eventual drug resistance is inevitable. Thus, new TKI-based combination therapies should be urgently explored to extend the overall survival time of these patients. CD8 + CD56+ natural killer T (NKT) cells are a natural and unique subset of lymphocytes in humans that present characteristics of T and NK cells and exert cytotoxicity on tumour cells in a granzyme B-dependent manner. The aim of this trial was to explore the efficacy and safety of CD8 + CD56+ NKT cell immunotherapy combined with gefitinib in patients with advanced EGFR-mutated NSCLC. METHODS: The study was designed as a prospective, randomized, controlled, open-label, phase I/II trial that includes 30 patients with EGFR mutation-positive stage III/IV NSCLC. All patients will be randomized in blocks at a 1:1 ratio and treated with gefitinib 250 mg/day monotherapy or combination therapy with allogeneic CD8 + CD56+ NKT cell infusions twice per month for 12 cycles or until disease progression occurs. The effectiveness of this treatment will be evaluated based on by progression-free survival (PFS), the time to progression (TTP), overall response rate (ORR), disease control rate (DCR) and overall survival (OS). The safety of the trail is being assessed based on adverse events (AEs). Recruitment and data collection, which started in December 2017, are ongoing. DISCUSSION: Although immunotherapy, including programmed death-1/programmed death-1 ligand (PD-1/PD-L1) immunotherapy, has been used for NSCLC treatment with or without EGFR-TKIs, its clear efficacy still has not been shown. Assessing the safety and therapeutic potential of allogeneic CD8 + CD56+ NKT killer cells in combination with EGFR-TKIs in NSCLC will be of great interest. TRIAL REGISTRATION: This trial (Phase I/II Trails of NKT Cell in Combination With Gefitinib For Non Small Cell Lung Cancer) was registered on 21 November 2017 with www.chictr.org.cn , ChiCTR-IIR-17013471 .

Histone lysine methyltransferase Ezh1 promotes TLR-triggered inflammatory cytokine production by suppressing Tollip.

Histone modifications play critical roles in the regulation of gene expression; however, their roles in the regulation of the innate response remain to be fully investigated. Using transcriptome analysis of mouse immature dendritic cells (DCs) and LPS-induced mature DCs, we identified that Ezh1 was the most upregulated histone methyltransferase during DC maturation. In this study, we investigated the role of Ezh1 in regulating the innate immune response. We found that silencing of Ezh1 significantly suppressed TLR-triggered production of cytokines, including IL-6, TNF-α, and IFN-ß, in DCs and macrophages. Accordingly, TLR-activated signaling pathways were impaired in Ezh1-silenced macrophages. By transcriptome analysis of Ezh1-silenced macrophages, we found that Toll-interacting protein (Tollip), one well-known negative regulator of TLR signaling, was upregulated. Silencing of Tollip rescued TLR-triggered cytokine production in Ezh1-silenced macrophages. The SET domain of Ezh1 is essential for its enhancing effect on the TLR-triggered innate immune response and downstream signaling, indicating that Ezh1 promotes a TLR-triggered innate response through its lysine methyltransferase activity. Finally, Ezh1 was found to suppress the transcription of Tollip by directly targeting the proximal promoter of tollip and maintaining the high level of trimethylation of histone H3 lysine 27 there. Therefore, Ezh1 promotes TLR-triggered inflammatory cytokine production by suppressing the TLR negative regulator Tollip, contributing to full activation of the innate immune response against invading pathogens.

Dysfunction of dendritic cells in aged C57BL/6 mice leads to failure of natural killer cell activation and of tumor eradication.

The reciprocal activation of dendritic cells (DCs) and natural killer cells (NKs) plays a key role in both innate and adaptive immunity. The effect of aging on this cross-talk, a critical step in virus disease control and tumor immunology, has not been reported. Splenic DCs and NKs were purified from both young and old C57BL/6 mice and cocultured in the presence of polyinosinic:polycytidylic acid (poly I:C). The resulting activation of NKs was measured as expression of CD69 and secretion of IFN-γ. However, DCs from old mice could not activate NKs from either young or old mice in vitro or in vivo. In contrast, DCs from young mice efficiently activated NKs from both young and old mice. DCs from old mice were deficient in poly I:C-stimulated secretion of IL-15, IL-18, and IFN-α. Gene expression analysis revealed many other differences between DCs of old and young mice. Young mice strongly eradicated MHC class I-negative NK-sensitive RMA-S lymphoma mutant tumor cells, but old mice did not, in concert with the previous report that mousepox kills aged, but not young, C57BL/6 mice. Furthermore, a similar dysfunction of DC and its key role in NK activation was found in 27 out of 55 healthy human donors.

Blockade of CD47 ameliorates autoimmune inflammation in CNS by suppressing IL-1-triggered infiltration of pathogenic Th17 cells.

The migration of Th17 cells into central nervous system (CNS) tissue is the key pathogenic step in experimental autoimmune encephalomyelitis (EAE) model. However, the mechanism underlying the pathogenic Th17 cell migration remains elusive. Here we report that blockade of CD47 with CD47-Fc fusion protein is effective in preventing and curing EAE by impairing infiltration of Th17 cells into CNS. However, CD47 deficiency does not directly impair the migration of Th17 cells. Mechanistic studies showed that CD47 deficiency inhibited degradation of inducible nitric oxide synthase (iNOS) in proteasome of macrophages by Src activation and led to the increased nitric oxide (NO) production. Then NO suppressed inflammasome activation-induced IL-1ß production. This lower IL-1ß reduces the expression of IL-1R1 and migration-related chemokine receptors on CD47(-/-) Th17 cells, inhibiting the ability of Th17 cells to infiltrate into the CNS of CD47(-/-) mice and therefore suppressing EAE development. In vivo administration of exogenous IL-1ß indeed promoted the infiltration CD47(-/-) Th17 cells into CNS and antagonized the protective role of CD47 deficiency in EAE pathogenesis. Our results demonstrate a potential preventive and therapeutic application of CD47 blockade in controlling EAE development.

[Correlation study between obesity and dawn phenomenon in patients with type 2 diabetes].

OBJECTIVE: To investigate the correlation between the frequency of dawn phenomenon and obesity in patients with type 2 diabetes. METHODS: This study was conducted in 98 patients with type 2 diabetes admitted to the Metabolic Disease Hospital of Tianjin Medical University from 2011 to 2014. The subjects were divided into 3 groups according to BMI: the normal weight (BMI 18.5-23.9 kg/m(2), n = 30), the overweight(BMI 24-27.9 kg/m(2), n = 33)and the obesity (BMI ≥ 28.0 kg/m(2), n = 35). All participants underwent continuous glucose monitoring for 72 h. Fasting plasma glucose(FPG), insulin and C-peptide were tested. Frequency of dawn phenomenon among the 3 groups was calculated, and the correlations between dawn phenomenon and its related factors were analyzed. RESULTS: The frequency of dawn phenomenon in type 2 diabetes increased with the increase of BMI in the 3 groups (P < 0.05) with 33.3% in the normal weight, 78.8% in the overweight and 88.6% in the obesity groups, respectively. The dawn phenomenon was positively correlated with BMI (r = 0.424, P < 0.05), Homeostasis model assessment of insulin resistance(HOMA-IR) (r = 0.781, P < 0.05), waist circumference (r = 0.394, P < 0.05), fasting C-peptide (r = 0.254, P < 0.05)and TG (r = 0.220, P < 0.05). It was negatively correlated with the course of diabetes mellitus (r = -0.278, P<0.05) and HDL-C (r = -0.268, P < 0.05). No correlation could be viewed between the dawn phenomenon and age, LDL-C, glycosylated hemoglobin A1c(HbA1c), TC and FPG (P > 0.05). CONCLUSIONS: The dawn phenomenon is closely associated with obesity and insulin resistance. The frequency of dawn phenomenon increases with BMI.

Fas signal promotes the immunosuppressive function of regulatory dendritic cells via the ERK/ß-catenin pathway.

Dendritic cells (DCs) play important roles in the initiation of immune response and also in the maintenance of immune tolerance. Now, many kinds of regulatory DCs with different phenotypes have been identified to suppress immune response and contribute to the control of autoimmune diseases. However, the mechanisms by which regulatory DCs can be regulated to exert the immunosuppressive function in the immune microenvironment remain to be fully investigated. In addition, how T cells, once activated, can feedback affect the function of regulatory DCs during immune response needs to be further identified. We previously identified a unique subset of CD11b(hi)Ia(low) regulatory DCs, differentiated from mature DCs or hematopoietic stem cells under a stromal microenvironment in spleen and liver, which can negatively regulate immune response in a feedback way. Here, we show that CD11b(hi)Ia(low) regulatory DCs expressed high level of Fas, and endothelial stromal cell-derived TGF-ß could induce high expression of Fas on regulatory DCs via ERK activation. Fas ligation could promote regulatory DCs to inhibit CD4(+) T cell proliferation more significantly. Furthermore, Fas ligation preferentially induced regulatory DCs to produce IL-10 and IP-10 via ERK-mediated inactivation of GSK-3 and subsequent up-regulation of ß-catenin. Interestingly, activated T cells could promote regulatory DCs to secrete more IL-10 and IP-10 partially through FasL. Therefore, our results demonstrate that Fas signal, at least from the activated T cells, can promote the immunosuppressive function of Fas-expressing regulatory DCs, providing a new manner for the regulatory DCs to regulate adaptive immunity.

Splenic stroma-educated regulatory dendritic cells induce apoptosis of activated CD4 T cells via Fas ligand-enhanced IFN-γ and nitric oxide.

Stromal microenvironments of bone marrow, lymph nodes, and spleen have been shown to be able to regulate immune cell differentiation and function. Our previous studies demonstrate that splenic stroma could drive mature dendritic cells (DC) to further proliferate and differentiate into regulatory DC subset that could inhibit T cell response via NO. However, how splenic stroma-educated regulatory DC release NO and whether other molecules are involved in the suppression of T cell response remain unclear. In this study, we show that splenic stroma educates regulatory DC to express high level of Fas ligand (FasL) by TGF-ß via ERK activation. The findings, that inhibition of CD4 T cell proliferation by regulatory DC required cell-to-cell contact and FasL deficiency impaired inhibitory effect of regulatory DC, indicate that regulatory DC inhibit CD4 T cell proliferation via FasL. Then, regulatory DC have been found to be able to induce apoptosis of activated CD4 T cells via FasL in caspase 8- and caspase 3-dependent manner. Interestingly, FasL on regulatory DC enhanced IFN-γ production from activated CD4 T cells, and in turn T cell-derived IFN-γ induced NO production from regulatory DC, working jointly to induce apoptosis of activated CD4 T cells. Blockade of IFN-γ and NO could reduce the apoptosis induction. Therefore, our results demonstrated that splenic stroma-educated regulatory DC induced T cell apoptosis via FasL-enhanced T cell IFN-γ and DC NO production, thus outlining a new way for negative regulation of T cell responses and maintenance of immune homeostasis by regulatory DC and splenic stromal microenvironment.

Optimization of polydimethylsiloxane (PDMS) surface chemical modification and formulation for improved T cell activation and expansion.

Adoptive T cell therapy has undergone remarkable advancements in recent decades; nevertheless, the rapid and effective ex vivo expansion of tumor-reactive T cells remains a formidable challenge, limiting their clinical application. Artificial antigen-presenting substrates represent a promising avenue for enhancing the efficiency of adoptive immunotherapy and fostering T cell expansion. These substrates offer significant potential by providing flexibility and modularity in the design of tailored stimulatory environments. Polydimethylsiloxane (PDMS) silicone elastomer stands as a widely utilized biomaterial for exploring the varying sensitivity of T cell activation to substrate properties. This paper explores the optimization of PDMS surface modification and formulation to create customized stimulatory surfaces with the goal of enhancing T cell expansion. By employing soft PDMS elastomer functionalized through silanization and activating agent, coupled with site-directed protein immobilization techniques, a novel T cell stimulatory platform is introduced, facilitating T cell activation and proliferation. Notably, our findings underscore that softer modified elastomers (Young' modulus E∼300 kPa) exhibit superior efficacy in stimulating and activating mouse CD4+ T cells compared to their stiffer counterparts (E∼3 MPa). Furthermore, softened modified PDMS substrates demonstrate enhanced capabilities in T cell expansion and Th1 differentiation, offering promising insights for the advancement of T cell-based immunotherapy.

Surface chemical modification of poly(dimethylsiloxane) for stabilizing antibody immobilization and T cell cultures.

Advances in cell immunotherapy underscore the need for effective methods to produce large populations of effector T cells, driving growing interest in T-cell bioprocessing and immunoengineering. Research suggests that T cells demonstrate enhanced expansion and differentiation on soft matrices in contrast to rigid ones. Nevertheless, the influence of antibody conjugation chemistry on these processes remains largely unexplored. In this study, we examined the effect of antibody conjugation chemistry on T cell activation, expansion and differentiation using a soft and biocompatible polydimethylsiloxane (PDMS) platform. We rigorously evaluated three distinct immobilization methods, beginning with the use of amino-silane (PDMS-NH2-Ab), followed by glutaraldehyde (PDMS-CHO-Ab) or succinic acid anhydride (PDMS-COOH-Ab) activation, in addition to the conventional physical adsorption (PDMS-Ab). By employing both stable amide bonds and reducible Schiff bases, antibody conjugation significantly enhanced antibody loading and density compared to physical adsorption. Furthermore, we discovered that the PDMS-COOH-Ab surface significantly promoted IL-2 secretion, CD69 expression, and T cell expansion compared to the other groups. Moreover, we observed that both PDMS-COOH-Ab and PDMS-NH2-Ab surfaces exhibited a tendency to induce the differentiation of naïve CD4+ T cells into Th1 cells, whereas the PDMS-Ab surface elicited a Th2-biased immunological response. These findings highlight the importance of antibody conjugation chemistry in the design and development of T cell culture biomaterials. They also indicate that PDMS holds promise as a material for constructing culture platforms to modulate T cell activation, proliferation, and differentiation.

Maresin2 negatively regulates DC's maturation via the MAPK/NF-κB pathway in DCs.

OBJECTIVE: To observe the effects and mechanisms of Maresin2 on the function of DCs(Dendritic cells). METHOD: The levels of IL-6, IL-12, TNF-α and IL-1ß secreted by BMDCs (Bone marrow-derived Dendritic cells) after Maresin2 treatment were detected by ELISA. At the same time, the expressions of costimulatory molecules CD40 and CD86 on the surface, the ability of phagocytosis of ovalbumin(OVA) antigen, and antigen presentation function in BMDCs were analyzed by flow cytometry. Finally, MAPK and NF-κB pathway signaling phosphorylation in Maresin2-treated BMDCs were detected by western blot. RESULTS: The secretion levels of IL-6, IL-12, TNF-α and IL-1ß were significantly decreased in the Maresin2 treatment group after LPS treatment (P < 0.05). The expression levels of CD86 and CD40 were significantly decreased after Maresin2 treatment (P < 0.05). Maresin2 enhanced the phagocytosis ability of ovalbumin(OVA) (P < 0.05), but the ability of antigen presentation of BMDCs with the treatment of Maresin2 changed slightly (P > 0.05). Phosphorylation of p38, JNK, p65, ikka/ß and ERK peaked at 15 min in the LPS group, while phosphorylation of p-p38 and p-ERK weakened 30 min and 60 min after treatment with Maresin2. CONCLUSIONS: Maresin2 inhibits inflammatory cytokine secretion but enhances phagocytosis via the MAPK/NF-κB pathway in BMDCs, which may contribute to negatively regulating inflammation.

Regulatory dendritic cells program generation of interleukin-4-producing alternative memory CD4 T cells with suppressive activity.

The heterogeneity and mechanisms for the generation of CD4 memory T (CD4 Tm) cells remain elusive. Distinct subsets of dendritic cells (DCs) have been found to regulate a distinct T-helper (Th)-cell subset differentiation by influencing cytokine cues around CD4 T cells; however, whether and how the regulatory DC subset can regulate Tm-cell differentiation remains unknown. Further, there is no ideal in vitro experimental system with which to mimic the 3 phases of the CD4 T-cell immune response (expansion, contraction, memory generation) and/or to culture CD4 Tm cells for more than a month. By analyzing CD4 T cells programmed by long-term coculture with regulatory DCs, we identified a population of long-lived CD4 T cells with a CD44(hi)CD62L(-)CCR7(-) effector memory phenotype and rapid, preferential secretion of the Th2 cytokines interleukin-4 (IL-4), IL-5, IL-10, and IL-13 after antigenic stimulation. These regulatory DC-programmed Tm cells suppress CD4 T-cell activation and proliferation in vitro via IL-10 and inhibit the delayed-type hypersensitivity response once infused in vivo. We also identify their natural counterpart, which is up-regulated by regulatory DC transfusion and negatively regulates the recall response in vivo. Different from interferon-γ-producing conventional Tm cells, these IL-4-producing CD4 Tm cells act as alternative Tm cells with a regulatory function, suggesting a new way of negative immune regulation by memory T cells.

MicroRNA-148/152 impair innate response and antigen presentation of TLR-triggered dendritic cells by targeting CaMKIIα.

MicroRNAs (miRNAs) are involved in the regulation of immunity, including the lymphocyte development and differentiation, and inflammatory cytokine production. Dendritic cells (DCs) play important roles in linking innate and adaptive immune responses. However, few miRNAs have been found to regulate the innate response and APC function of DCs to date. Calcium/calmodulin-dependent protein kinase II (CaMKII), a major downstream effector of calcium (Ca(2+)), has been shown to be an important regulator of the maturation and function of DCs. Our previous study showed that CaMKIIα could promote TLR-triggered production of proinflammatory cytokines and type I IFN. Inspired by the observations that dicer mutant Drosophila display defect in endogenous miRNA generation and higher CaMKII expression, we wondered whether miRNAs can regulate the innate response and APC function of DCs by targeting CaMKIIα. By predicting with software and confirming with functional experiments, we demonstrate that three members of the miRNA (miR)-148 family, miR-148a, miR-148b, and miR-152, are negative regulators of the innate response and Ag-presenting capacity of DCs. miR-148/152 expression was upregulated, whereas CaMKIIα expression was downregulated in DCs on maturation and activation induced by TLR3, TLR4, and TLR9 agonists. We showed that miR-148/152 in turn inhibited the production of cytokines including IL-12, IL-6, TNF-α, and IFN-ß upregulation of MHC class II expression and DC-initiated Ag-specific T cell proliferation by targeting CaMKIIα. Therefore, miRNA-148/152 can act as fine-tuner in regulating the innate response and Ag-presenting capacity of DCs, which may contribute to the immune homeostasis and immune regulation.

Hyperinsulinemia impairs the metabolic switch to ketone body utilization in proximal renal tubular epithelial cells under energy crisis via the inhibition of the SIRT3/SMCT1 pathway.

Objective: To investigate the effects and mechanism of hyperinsulinemia on the metabolic switch to ß-hydroxybutyrate (BHB) absorption and utilization under a starvation or hypoxic environment in proximal tubular epithelial cells. Methods: A high-fat diet-induced hyperinsulinemia model in ZDF rats was used to test the expression of key enzymes/proteins of ketone body metabolism in the kidney. Notably, 12-week-old renal tubule SMCT1 specific knockout mice (SMCT1 flox/floxCre+) and control mice (SMCT1 flox/floxCre-) were used to confirm the roles of SMCT1 in kidney protection under starvation. The changes of key enzymes/proteins of energy metabolism, mitochondrial function, and albumin endocytosis in HK2 cells under low glucose/hypoxic environments with or without 50 ng/mL insulin were studied. Silent information regulation 2 homolog 3 (SIRT3) was overexpressed to evaluate the effect of hyperinsulinemia on the metabolic switch to BHB absorption and utilization through the SIRT3/SMCT1 pathway in HK2 cells. Results: In ZDF rats, the expression of HMGCS2 increased, the SMCT1 expression decreased, while SCOT remained unchanged. In renal tubule SMCT1 gene-specific knockout mice, starvation for 48 h induced an increase in the levels of urine retinol-binding protein, N-acetyl-ß-glucosaminidase, and transferrin, which reflected tubular damages. In HK2 cells under an environment of starvation and hypoxia, the levels of key enzymes related to fatty acid oxidation and ketone body metabolism were increased, whereas glucose glycolysis did not change. The addition of 2 mmol/l BHB improved ATP production, mitochondrial biosynthesis, and endocytic albumin function, while cell apoptosis was reduced in HK2 cells. The addition of 50 ng/ml insulin resulted in the decreased expression of SMCT1 along with an impaired mitochondrial function, decreased ATP production, and increased apoptosis. The overexpression of SIRT3 or SMCT1 reversed these alterations induced by a high level of insulin both in low-glucose and hypoxic environments. Conclusions: The increased absorption and utilization of BHB is part of the metabolic flexibility of renal tubular epithelial cells under starvation and hypoxic environments, which exhibits a protective effect on renal tubular epithelial cells by improving the mitochondrial function and cell survival. Moreover, hyperinsulinemia inhibits the absorption of BHB through the inhibition of the SIRT3/SMCT1 pathway.

PERK reprograms hematopoietic progenitor cells to direct tumor-promoting myelopoiesis in the spleen.

The spleen is an important site of hematopoietic stem/progenitor cell (HSPC) preconditioning and tumor-promoting myeloid cell generation in cancer, but the regulatory mechanism remains unclear. Here, we found that PKR-like endoplasmic reticulum kinase (PERK) mediated HSPC reprogramming into committed MDSC precursors in the spleen via PERK-ATF4-C/EBPß signaling. Pharmacological and genetic inhibition of this pathway in murine and human HSPCs prevented their myeloid descendant cells from becoming MDSCs even with subsequent exposure to tumor microenvironment (TME) factors. In mice, the selective delivery of PERK antagonists to the spleen was not only sufficient but more effective than the tumor-targeted strategy in preventing MDSC activation in the tumor, leading to profound TME reshaping and tumor regression. Clinically, HSPCs in the spleen of cancer patients exhibit increased PERK signaling correlated with enhanced myelopoiesis. Our findings indicate that PERK-mediated HSPC preconditioning plays a crucial role in MDSC generation, suggesting novel spleen-targeting therapeutic opportunities for restraining the tumor-promoting myeloid response at its source.