Proinflammatory hepatic macrophage activation plays a key role in the development of nonalcoholic steatohepatitis (NASH). This involves increased embryonic hepatic Kupffer cell (KC) death, facilitating the replacement of KCs with bone marrow–derived recruited hepatic macrophages (RHMs) that highly express proinflammatory genes. Moreover, phago/efferocytic activity of KCs is diminished in NASH, enhancing liver inflammation. However, the molecular mechanisms underlying these changes in KCs are not known. Here, we show that hypoxia-inducible factor 2α (HIF-2α) mediates NASH-associated decreased KC growth and efferocytosis by enhancing lysosomal stress. At the molecular level, HIF-2α stimulated mammalian target of rapamycin (mTOR)– and extracellular signal–regulated kinase-dependent inhibitory transcription factor EB (TFEB) phosphorylation, leading to decreased lysosomal and phagocytic gene expression. With increased metabolic stress and phago/efferocytic burden in NASH, these changes were sufficient to increase lysosomal stress, causing decreased efferocytosis and lysosomal cell death. Of interest, HIF-2α–dependent TFEB regulation only occurred in KCs but not RHMs. Instead, in RHMs, HIF-2α promoted mitochondrial reactive oxygen species production and proinflammatory activation by increasing ANT2 expression and mitochondrial permeability transition. Consequently, myeloid lineage–specific or KC-specific HIF-2α depletion or the inhibition of mTOR-dependent TFEB inhibition using antisense oligonucleotide treatment protected against the development of NASH in mice. Moreover, treatment with an HIF-2α–specific inhibitor reduced inflammatory and fibrogenic gene expression in human liver spheroids cultured under a NASH-like condition. Together, our results suggest that macrophage subtype–specific effects of HIF-2α collectively contribute to the proinflammatory activation of liver macrophages, leading to the development of NASH.

Mesenchymal stromal cells (MSCs) can generate immunological tolerance due to their regulatory activity in many immune cells. Extracellular vesicles (EVs) release is a pivotal mechanism by which MSCs exert their actions. In this study, we evaluate whether mesenchymal stromal cell extracellular vesicles (MSC-EVs) can modulate T cell response. MSCs were expanded and EVs were obtained by differential ultracentrifugation of the supernatant. The incorporation of MSC-EVs by T cells was detected by confocal microscopy. Expression of surface markers was detected by flow cytometry or CytoFLEX and cytokines were detected by RT-PCR, FACS and confocal microscopy and a miRNA PCR array was performed. We demonstrated that MSC-EVs were incorporated by lymphocytes in vitro and decreased T cell proliferation and Th1 differentiation. Interestingly, in Th1 polarization, MSC-EVs increased Foxp3 expression and generated a subpopulation of IFN-γ⁺/Foxp3⁺T cells with suppressive capacity. A differential expression profile of miRNAs in MSC-EVs-treated Th1 cells was seen, and also a modulation of one of their target genes, TGFbR2. MSC-EVs altered the metabolism of Th1-differentiated T cells, suggesting the involvement of the TGF-β pathway in this metabolic modulation. The addition of MSC-EVs in vivo, in an OVA immunization model, generated cells Foxp3⁺. Thus, our findings suggest that MSC-EVs are able to specifically modulate activated T cells at an alternative regulatory profile by miRNAs and metabolism shifting.

MiR-23~27~24 regulates T _FH cells through targeting multiple genes including TOX, a key transcription factor for T _FH cell biology.

Introduction: BALB/c mice and C57/BL6 mice have different abilities to recover from ischemia. C57/BL6 mice display increased vessel collateralization and vascular endothelial growth factor expression with a consequent rapid recovery from ischemia compared with BALB/c mice. Mesenchymal stem cells (MSCs) are one of the main cell types that contribute to the recovery from ischemia because, among their biological activities, they produce several proangiogenic paracrine factors and differentiate into endothelial cells. The objective of this study was to evaluate whether the MSCs of these two mouse strains have different inductive capacities for recovering ischemic limbs. Methods: MSCs from these two strains were obtained from the bone marrow, purified and characterized before being used for in vivo experiments. Limb ischemia was surgically induced in BALB/c mice, and MSCs were injected on the fifth day. The evolution of limb necrosis was evaluated over the subsequent month. Muscle strength was assessed on the 30th day after the injection, and then the animals were sacrificed to determine the muscle mass and perform histological analyses to detect cellular infiltration, capillary and microvessel densities, fibrosis, necrosis and tissue regeneration. Results: The MSCs from both strains promoted high level of angiogenesis similarly, resulting in good recovery from ischemia. However, BALB/c MSCs promoted more muscle regeneration (57%) than C57/BL6 MSCs (44%), which was reflected in the increased muscle strength (0.79 N versus 0.45 N). Conclusion: The different genetic background of MSCs from BALB/c mice and C57/BL6 mice was not a relevant factor in promoting angiogenesis of limb ischemia, because both cells showed a similar angiogenic activity. These cells also showed a potential myogenic effect, but the stronger effect promoted by BALB/c MSCs indicates that the different genetic background of MSCs was more relevant in myogenesis than angiogesis.

Sepsis is a complex infectious syndrome in which neutrophil participation is crucial for patient survival. Neutrophils quickly sense and eliminate the pathogen by using different effector mechanisms controlled by metabolic processes. The mammalian target of rapamycin (mTOR) pathway is an important route for metabolic regulation, and its role in neutrophil metabolism has not been fully understood yet, especially the importance of mTOR complex 2 (mTORC2) in the neutrophil effector functions. In this study, we observed that the loss of Rictor (mTORC2 scaffold protein) in primary mouse-derived neutrophils affects their chemotaxis by fMLF and their microbial killing capacity, but not the phagocytic capacity. We found that the microbicidal capacity was impaired in Rictor-deleted neutrophils because of an improper fusion of granules, reducing the hypochlorous acid production. The loss of Rictor also led to metabolic alterations in isolated neutrophils, increasing aerobic glycolysis. Finally, myeloid-Rictor–deleted mice (LysMRic Δ/Δ) also showed an impairment of the microbicidal capacity, increasing the bacterial burden in the Escherichia coli sepsis model. Overall, our results highlight the importance of proper mTORC2 activation for neutrophil effector functions and metabolism during sepsis.