T cell fate decisions during memory cell generation with aging.

The defense against infectious diseases, either through natural immunity or after vaccinations, relies on the generation and maintenance of protective T cell memory. Naïve T cells are at the center of memory T cell generation during primary responses. Upon activation, they undergo a complex, highly regulated differentiation process towards different functional states. Naïve T cells maintained into older age have undergone epigenetic adaptations that influence their fate decisions during differentiation. We review age-sensitive, molecular pathways and gene regulatory networks that bias naïve T cell differentiation towards effector cell generation at the expense of memory and Tfh cells. As a result, T cell differentiation in older adults is associated with release of bioactive waste products into the microenvironment, higher stress sensitivity as well as skewing towards pro-inflammatory signatures and shorter life spans. These maladaptations not only contribute to poor vaccine responses in older adults but also fuel a more inflammatory state.

A Lipid-Based Nanocarrier Containing Active Vitamin D3 Ameliorates NASH in Mice via Direct and Intestine-Mediated Effects on Liver Inflammation.

The gut-liver axis may be involved in non-alcoholic steatohepatitis (NASH) progression. Pathogen-associated molecular patterns leak through the intestinal barrier to the liver via the portal vein to contribute to NASH development. Active vitamin D3 (1,25(OH)2D3) is a potential therapeutic agent to enhance the intestinal barrier. Active vitamin D3 also suppresses inflammation and fibrosis in the liver. However, the adverse effects of active vitamin D3 such as hypercalcemia limit its clinical use. We created a nano-structured lipid carrier (NLC) containing active vitamin D3 to deliver active vitamin D3 to the intestine and liver to elicit NASH treatment. We found a suppressive effect of the NLC on the lipopolysaccharide-induced increase in permeability of an epithelial layer in vitro. Using mice in which NASH was induced by a methionine and choline-deficient diet, we discovered that oral application of the NLC ameliorated the permeability increase in the intestinal barrier and attenuated steatosis, inflammation and fibrosis in liver at a safe dose of active vitamin D3 at which the free form of active vitamin D3 did not show a therapeutic effect. These data suggest that the NLC is a novel therapeutic agent for NASH.

Improvement of multilineage hematopoiesis in hematopoietic stem cell-transferred c-kit mutant NOG-EXL humanized mice.

Human hematopoietic stem cell (HSC)-transferred humanized mice are valuable models for exploring human hematology and immunology. However, sufficient recapitulation of human hematopoiesis in mice requires large quantities of enriched human CD34+ HSCs and total-body irradiation for adequate engraftment. Recently, we generated a NOG mouse strain with a point mutation in the c-kit tyrosine kinase domain (W41 mutant; NOGW mice). In this study, we examined the ability of NOGW mice to reconstitute human hematopoietic cells. Irradiated NOGW mice exhibited high engraftment levels of human CD45+ cells in the peripheral blood, even when only 5,000-10,000 CD34+ HSCs were transferred. Efficient engraftment of human CD45+ cells was also observed in non-irradiated NOGW mice transferred with 20,000-40,000 HSCs. The bone marrow (BM) of NOGW mice exhibited significantly more engrafted human HSCs or progenitor cells (CD34+CD38- or CD34+CD38+ cells) than the BM of NOG mice. Furthermore, we generated a human cytokine (interleukin-3 and granulocyte-macrophage colony-stimulating factor) transgenic NOG-W41 (NOGW-EXL) mouse to achieve multilineage reconstitution with sufficient engraftment of human hematopoietic cells. Non-irradiated NOGW-EXL mice showed significantly higher engraftment levels of human CD45+ and myeloid lineage cells, particularly granulocytes and platelets/megakaryocytes, than non-irradiated NOGW or irradiated NOG-EXL mice after human CD34+ cell transplantation. Serial BM transplantation experiments revealed that NOGW mice exhibited the highest potential for long-term HSC compared with other strains. Consequently, c-kit mutant NOGW-EXL humanized mice represent an advanced model for HSC-transferred humanized mice and hold promise for widespread applications owing to their high versatility.

CISH impairs lysosomal function in activated T cells resulting in mitochondrial DNA release and inflammaging.

Chronic systemic inflammation is one of the hallmarks of the aging immune system. Here we show that activated T cells from older adults contribute to inflammaging by releasing mitochondrial DNA (mtDNA) into their environment due to an increased expression of the cytokine-inducible SH2-containing protein (CISH). CISH targets ATP6V1A, an essential component of the proton pump V-ATPase, for proteasomal degradation, thereby impairing lysosomal function. Impaired lysosomal activity caused intracellular accumulation of multivesicular bodies and amphisomes and the export of their cargos, including mtDNA. CISH silencing in T cells from older adults restored lysosomal activity and prevented amphisomal release. In antigen-specific responses in vivo, CISH-deficient CD4+ T cells released less mtDNA and induced fewer inflammatory cytokines. Attenuating CISH expression may present a promising strategy to reduce inflammation in an immune response of older individuals.

IL-4 prevents adenosine-mediated immunoregulation by inhibiting CD39 expression.

The ectonucleotidase CD39 functions as a checkpoint in purinergic signaling on effector T cells. By depleting eATP and initiating the generation of adenosine, it impairs memory cell development and contributes to T cell exhaustion, thereby causing defective tumor immunity and deficient T cell responses in older adults who have increased CD39 expression. Tuning enzymatic activity of CD39 and targeting the transcriptional regulation of ENTPD1 can be used to modulate purinergic signaling. Here, we describe that STAT6 phosphorylation downstream of IL-4 signaling represses CD39 expression on activated T cells by inducing a transcription factor network including GATA3, GFI1, and YY1. GATA3 suppresses ENTPD1 transcription through prevention of RUNX3 recruitment to the ENTPD1 promoter. Conversely, pharmacological STAT6 inhibition decreases T cell effector functions via increased CD39 expression, resulting in the defective signaling of P2X receptors by ATP and stimulation of A2A receptors by adenosine. Our studies suggest that inhibiting the STAT6 pathway to increase CD39 expression has the potential to treat autoimmune disease while stimulation of the pathway could improve T cell immunity.

Synergistic effects of trans-p-coumaric acid isolated from the ethanol extract of Gynura procumbens in promoting intestinal absorption of chlorogenic acid and reversing alcoholic fatty liver disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Our previous studies found that the ethanol extract of Gynura procumbens (EEGS) reduced hepatic steatosis in alcoholic fatty liver disease (AFLD). AIM OF THE STUDY: To explore the active ingredients from EEGS and their relevant mechanism of action in alleviating alcoholic liver injuries. AIM OF THE STUDY: To explore the active ingredients from EEGS and their intestinal absorption characteristics as an approach for understanding mechanism of action in alleviating alcoholic liver injuries. MATERIALS AND METHODS: Monitored by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), chemical constituents from the prepared EEGS were isolated by means of solvent extraction, repeated column chromatography, preparative HPLC and other methods, and their structures were identified based on spectroscopic methods. The in vivo intestinal absorption rate of chlorogenic acid (CA), the active component of the EEGS, both in a single form and in the EEGS were monitored by the single-pass intestinal perfusion (SPIP) method in rats. The protective effect of EEGS and its active components on alcoholic liver injuries was evaluated in the alcoholic liver injury model of C57BL/6J male mice induced by Lieber-DeCarli alcohol liquid feed. RESULTS: Three noncaffeoyl quinic acid components were isolated and identified from the EEGS, namely, 3-trans-p-coumaroyl quinic acid (0.9%), 3-cis-p-coumaroyl quinic acid (2.7%), and trans-p-coumaric acid (0.6%). In vivo intestinal absorption of CA decreased with the increase of pH value of perfusion solution in the range of 5.5-7.8. The maximum absorption percentage of CA alone was 6.7 ± 2.4%, while the maximum absorption percentage of CA in the EEGS was 16.0 ± 2.2%, which was 2.4 times higher than that of CA alone. The results of animal experiments showed that the degree of fatty liver of mice treated with EEGS was significantly lower than that of the CA, trans-p-coumaric acid, and the combination group of CA and trans-p-coumaric acid alone. CONCLUSION: The above results indicated that trans-p-coumaric acid isolated from the dried stems of Gynura procumbens assisted CA being absorbed into the body and worked together with CA to improve the function of liver lipid metabolism, reduce hepatic lipid accumulation in a mouse model of AFLD and effectively counteract alcohol-induced fatty liver disease.

Efficient differentiation of human neutrophils with recapitulation of emergency granulopoiesis in human G-CSF knockin humanized mice.

Neutrophils are critical mediators during the early stages of innate inflammation in response to bacterial or fungal infections. A human hematopoietic system reconstituted in humanized mice aids in the study of human hematology and immunology. However, the poor development of human neutrophils is a well-known limitation of humanized mice. Here, we generate a human granulocyte colony-stimulating factor (hG-CSF) knockin (KI) NOD/Shi-scid-IL2rgnull (NOG) mouse in which hG-CSF is systemically expressed while the mouse G-CSF receptor is disrupted. These mice generate high numbers of mature human neutrophils, which can be readily mobilized into the periphery, compared with conventional NOG mice. Moreover, these neutrophils exhibit infection-mediated emergency granulopoiesis and are capable of efficient phagocytosis and reactive oxygen species production. Thus, hG-CSF KI mice provide a useful model for studying the development of human neutrophils, emergency granulopoiesis, and a potential therapeutic model for sepsis.

Effect of Size and Loading of Retinoic Acid in Polyvinyl Butyrate Nanoparticles on Amelioration of Colitis.

Butyrate has been used in the treatment of inflammatory bowel diseases (IBD). However, the controlled release of butyrate has been indicated to be necessary in order to avoid the side effects verified at high concentrations. We previously developed nanoparticles (NPs) of polyvinyl butyrate (PVBu) as an oral butyrate donor for the controlled release of butyrate for the treatment of colitis. To examine the effect of the size of NPs on the therapeutic effect of colitis, here we prepared PVBu NPs with different sizes (100 nm and 200 nm). Both sizes of PVBu NPs significantly suppressed the inflammatory response in macrophages in vitro. PVBu NPs with 200 nm showed better effects on the amelioration of colitis compared with the 100 nm-NPs. We found unexpectedly that 200 nm-NP incorporated with all-trans retinoic acid (ATRA) showed a much better therapeutic effect than those with unloaded 200 nm-NPs, although ATRA alone was reported to worsen the inflammation. The synergistic effect of ATRA with butyrate shows evidence of being a promising approach for IBD treatment.

Polyvinyl Butyrate Nanoparticles as Butyrate Donors for Colitis Treatment.

Butyrate has been attracting attention for the suppression of inflammatory bowel disease (IBD). However, clinical trials of butyrate for IBD treatment have resulted in controversial outcomes, likely owing to the adverse effect of butyrate on the intestinal epithelium that was observed at high butyrate concentrations. Herein, we propose polyvinyl butyrate (PVBu) nanoparticles (NPs) as butyrate donors for delivery to the lower part of the intestine for the treatment of colitis. The PVBu NPs suppressed the inflammatory activation of macrophages in vitro, although sodium butyrate inversely further activated macrophages. Oral administration of NPs did not change the luminal concentration of free butyrate; however, NPs showed a therapeutic effect on a colitis mouse model. In addition, incorporation of vitamin D3 into the NPs enhanced the therapeutic effect on colitis. Hence, PVBu NPs are a promising therapeutic for IBD treatment, not only as a butyrate donor but also as a carrier for hydrophobic drugs like vitamin D3.

Chronic high-dosage fish oil exacerbates gut-liver axis injury in alcoholic steatohepatitis in mice: the roles of endotoxin and IL-4 in Kupffer cell polarization imbalance.

In the present study, intestinal tight junctions (TJs) and Kupffer cell polarization were investigated in an alcoholic steatohepatitis (ASH) mouse model to uncover the potential side effects of overexposure to fish oil or omega-3 fatty acids. The mice were fed ad libitum with a liquid diet containing ethanol and fish oil. In the meantime, ethanol was given every 5-7 days by gavage to simulate binge drinking. After the 7th binge, steatosis, necrosis, inflammatory infiltration, and bridging fibrosis were observed in the liver by histological staining. After the 13th binge, the inducers, markers and other downstream genes/proteins of the Kupffer cell M1/M2 phenotype in the liver, serum, and small intestine were analysed. The results suggested that a chronic high dosage of fish oil alone reduced the mRNA levels of most genes tested and showed a tendency to damage the intestinal zonula occludens-1 localization and reduce the number of M2 Kupffer cells. Meanwhile, the combination of fish oil and ethanol damaged the intestinal TJs, resulting in an increased endotoxin level in the liver. Gut-derived endotoxin polarized Kupffer cells to the M1 phenotype, whereas the number of cells with the M2 phenotype (markers: CD163 and CD206) was decreased. Interleukin-4 (IL-4), an M2 Kupffer cell inducer, was also decreased. Moreover, in vitro experiments showed that IL-4 reversed eicosapentaenoic acid-induced CD163 and CD206 mRNA suppression in RAW 264.7 cells. Overall, our results showed that a chronic high dosage of fish oil exacerbated gut-liver axis injury in alcoholic liver disease in mice, and endotoxin/IL-4-induced Kupffer cell polarization imbalance might play an important role in that process.

Gynura procumbens Reverses Acute and Chronic Ethanol-Induced Liver Steatosis through MAPK/SREBP-1c-Dependent and -Independent Pathways.

The present study aimed to evaluate the hepatoprotective effect and mechanism of action of Gynura procumbens on acute and chronic ethanol-induced liver injuries. Ethanol extract from G. procumbens stems (EEGS) attenuated acute ethanol-induced serum alanine aminotransferase levels and hepatic lipid accumulation. Therefore, EEGS was successively extracted by petroleum, ethyl acetate, and n-butyl alcohol. The results showed that the n-butyl alcohol extract was the active fraction of EEGS, and hence it was further fractionated on a polyamide glass column. The 60% ethanol-eluted fraction that contained 13.6% chlorogenic acid was the most active fraction, and its effect was further evaluated using a chronic model. Both the n-butyl alcohol extract and the 60% ethanol-eluted fraction inhibited chronic ethanol-induced hepatic lipid accumulation by modulating lipid metabolism-related regulators through MAPK/SREBP-1c-dependent and -independent signaling pathways and ameliorated liver steatosis. Our findings suggest that EEGS and one of its active ingredients, chlorogenic acid, may be developed as potential effective agents for ethanol-induced liver injury.