Search

Macrophage proinflammatory activation is an important etiologic component of the development of insulin resistance and metabolic dysfunction in obesity. However, the underlying mechanisms are not clearly understood. Here, we demonstrate that a mitochondrial inner membrane protein, adenine nucleotide translocase 2 (ANT2), mediates proinflammatory activation of adipose tissue macrophages (ATMs) in obesity. Ant2 expression was increased in ATMs of obese mice compared with lean mice. Myeloid-specific ANT2-knockout (ANT2-MKO) mice showed decreased adipose tissue inflammation and improved insulin sensitivity and glucose tolerance in HFD/obesity. At the molecular level, we found that ANT2 mediates free fatty acid-induced mitochondrial permeability transition, leading to increased mitochondrial reactive oxygen species production and damage. In turn, this increased HIF-1α expression and NF-κB activation, leading to proinflammatory macrophage activation. Our results provide a previously unknown mechanism for how obesity induces proinflammatory activation of macrophages with propagation of low-grade chronic inflammation (metaflammation).

Metastatic triple negative breast cancer (TNBC) includes poor prognoses and limited effective therapies. Metastasis is the major barrier to durable clinical responses as many patients appear cured of their disease, yet return to clinic with recurrences from dormant micro-metastases. Immune activating agents, such as Interleukin 12 (IL-12) have attracted attention as anti-cancer adjuvants due to their ability to elicit systemic anti-cancer immunity. Although targeting distributed metastases can be accomplished by intravenous (IV) administration, administration of IL-12 into patient vasculature is highly inefficient due to its short half-life and poor toxicity profile. There is a major unmet need to develop biocompatible carriers that improve targeted delivery of IL-12 by specifically homing to metastatic lesions. Mesenchymal stem cells (MSCs) are considered ideal biodelivery vehicles as they display innate tumor-trophism and can deliver biomolecules specifically to systemic metastases. However, major issues need to be addressed before MSCs can be used to deliver IL-12 to patients: MSC tumor homing needs to be improved, and unmodified MSCs can engraft and contribute to tumor growth and metastasis, raising legitimate safety concerns. To address these major limitations, our laboratory developed a MSC-derived biodelivery vehicle with enhanced homing abilities to deliver biomolecules directly to tumors. Our approach involved using scRNA-seq to identify chemoattractant receptor/ligand pairs (CXCR4/CXCL12, CCR2/CCL2) and endothelial adhesion molecules (PSGL-1/P-E-Selectins) that mediate leukocyte homing and extravasation. We then lentivirally expressed these receptors in MSCs and removed nuclei using Ficoll gradient ultracentrifugation. These bioengineered enucleated MSCs (termed Cargocytes™) have a defined lifespan (4 days) and robustly home to TNBC. This is an important breakthrough as TNBC-trophic Cargocytes can be scaled for clinical applications and are safe for in vivo use as described in our recent publication (Nature Biomedical Engineering, in press). By using syngeneic mouse models of metastatic TNBC, we have observed IV-injected Cargocytes robustly extravasate and precisely home to metastatic lung nodules, suggesting Cargocytes act as remarkable biodelivery vehicles. Indeed, the tumor-trophism of Cargocytes includes both integrating into large macro-metastases and homing to broadly distributed micro-metastases. Cargocytes effectively produce and deliver high doses of IL-12 to mouse TNBC, which induces strong immune activation in tumors. This immune activation involved a potent anti-metastatic response, evidencing strong therapeutic efficacy. These findings suggest Cargocyte-delivered IL-12 could constitute a safe, potent, and highly specific anti-metastatic therapy for TNBC and other metastatic cancers sharing similar chemoattractant profiles. Citation Format: Jeffrey White, Huawei Wang, Flavia Franco Da Cunha, Maureen Ruchhoeft, Richard Klemke. Bioengineering enucleated cell vehicles for targeted delivery of Interleukin 12 to metastatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3519.

Metastatic triple negative breast cancer (TNBC) includes poor prognoses and limited effective therapies. The prevalence of systemic micro-metastases is a major barrier to achieving durable clinical outcomes. Oncolytic viruses (OVs) have garnered attention as anti-cancer adjuvants due to their potential for systemic delivery and ability to lyse tumor cells while leaving healthy cells unaffected. Clinical trials have been conducted testing the efficacy of OVs on diverse cancers. Although targeting distributed metastases can be accomplished by intravenous (IV) administration, administration of virus into patient vasculature is inefficient due to the clearance of unprotected viruses by host immunity. There is a major unmet need to develop biocompatible carriers that improve delivery of OVs by circumventing host anti-viral immunity while homing to metastatic lesions. Mesenchymal stem cells (MSCs) are ideal OV carriers as they display innate tumor-trophic properties, propagate OVs, and shield virions from immunity until reaching their target. However, issues need to be addressed before MSCs can deliver OVs to patients: tumor homing needs improved and unmodified MSCs can engraft and contribute to tumor growth and metastasis, raising safety concerns. To address these limitations, our laboratory developed a MSC-derived biodelivery vehicle with enhanced homing abilities to deliver biologics, such as OVs, directly to tumors. Our approach involved using scRNA-seq to identify chemoattractant receptor/ligand pairs (CXCR4/CXCL12, CCR2/CCL2) and endothelial adhesion molecules (PSGL-1/P-E-Selectins) that mediate leukocyte tumor homing and extravasation. We then lentivirally expressed these receptors in MSCs and removed all DNA using ultracentrifugation and Ficoll density gradients. These bioengineered enucleated MSCs (Cargocytes™) have a defined lifespan (4 days) and robustly home to TNBC. This is an important breakthrough as TNBC-trophic Cargocytes can be scaled for clinical applications and are safe for in vivo use as described in our recent publication (Nature Biomedical Engineering, in press). OVs replicate in and lyse interferon-deficient tumor cells while having negligible effects on healthy cells. As the interferon response is absent in enucleated cells, Cargocytes support robust replication of OVs, including oncolytic Herpes Simplex Virus, Measles Virus, and Vesicular Stomatitis Virus (oVSV), allowing IV delivery of high titer OVs to tumors. In in vivo mouse models of metastatic TNBC, carrier-free oVSV fails to infect lung metastases. However, Cargocytes efficiently deliver oVSV to distributed micro-metastases and integrate deep into macro-metastases, evidencing a remarkable improvement in tissue distribution over intra-tumoral injections. Our findings suggest Cargocytes host and target oVSV directly to TNBC, constituting a highly specific OV delivery vehicle. Citation Format: Jeffrey White, Huawei Wang, Flavia Franco Da Cunha, Maureen Ruchhoeft, Richard Klemke. Bioengineering enucleated cell vehicles for tumor targeted intravenous delivery of oncolytic viruses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 378.

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.

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-&gamma, +/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-&beta, 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 TFH cells through targeting multiple genes including TOX, a key transcription factor for TFH cell biology., Follicular helper T (TFH) cells are essential for generating protective humoral immunity. To date, microRNAs (miRNAs) have emerged as important players in regulating TFH cell biology. Here, we show that loss of miR-23~27~24 clusters in T cells resulted in elevated TFH cell frequencies upon different immune challenges, whereas overexpression of this miRNA family led to reduced TFH cell responses. Mechanistically, miR-23~27~24 clusters coordinately control TFH cells through targeting a network of genes that are crucial for TFH cell biology. Among them, thymocyte selection–associated HMG-box protein (TOX) was identified as a central transcription regulator in TFH cell development. TOX is highly up-regulated in both mouse and human TFH cells in a BCL6-dependent manner. In turn, TOX promotes the expression of multiple molecules that play critical roles in TFH cell differentiation and function. Collectively, our results establish a key miRNA regulon that maintains optimal TFH cell responses for resultant humoral immunity.

Organ transplantation is a life-saving procedure, however predicting graft survival is still challenging. Understanding immune-cell pathobiology is critical to the development of effective therapies to prevent rejection. Over the recent years it has become progressively evident that the complex nature of immune cell behavioral dynamics is strongly dependent on cellular metabolism, which in turn, relies on competition for nutrients, oxygen and metabolites with other immune cells and microbiota. Furthermore, the influence of the inflammatory state can lead to substantial changes in conditions within the tissue micro-environment. Considering the context of immunity, alterations in metabolic pathways (glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, the fatty acid oxidation and synthesis, and the amino acid metabolic pathways) will influence the production of different sets of cytokines and affect transplantation outcome. It is now known that naive, resting and effector cells acquire different metabolic profiles and studies have shown that specifically targeting some of these metabolic routes can prevent differentiation of effector T cells in favor of Tregs. Ultimately, to develop effective therapies that will prevent graft loss and understanding how cell metabolism impacts the fate and function of immune cells is now a critical point of discussion. The distinct metabolic features and requirements observed in effector and suppressive cell subsets offer promising opportunities for selective regulation of the immune responses in transplantation and will be discussed in this review.

Inflammatory bowel diseases (IBDs) affect millions of people worldwide and their frequencies in developed countries have increased since the twentieth century. In this context, there is an intensive search for therapies that modulate inflammation and provide tissue regeneration in IBDs. Recently, the immunomodulatory activity of adipose tissue-derived mesenchymal stromal cells (ADMSCs) has been demonstrated to play an important role on several immune cells in different conditions of inflammatory and autoimmune diseases. In this study, we explored the immunomodulatory potential of ADMSC in a classical model of DSS-induced colitis. First, we found that treatment of mice with ADMSC ameliorated the severity of DSS-induced colitis, reducing colitis pathological score and preventing colon shortening. Moreover, a prominent reduction of pro-inflammatory cytokines levels (i.e., IFN-γ, TNF-α, IL-6 and MCP-1) was observed in the colon of animals treated with ADMSC. We also observed a significant reduction in the frequencies of macrophages (F4/80+CD11b+) and dendritic cells (CD11c+CD103+) in the intestinal lamina propria of ADMSC-treated mice. Finally, we detected the up-regulation of immunoregulatory-associated molecules in intestine of mice treated with ADMSCs (i.e., elevated arginase-1 and IL-10). Thus, this present study demonstrated that ADMSC modulates the overall gut inflammation (cell activation and recruitment) in experimental colitis, providing support to the further development of new strategies in the treatment of intestinal diseases.

The evolutionary emergence of an efficient immune system has a fundamental role in our survival against pathogenic attacks. Nevertheless, this same protective mechanism may also establish a negative consequence in the setting of disorders such as autoimmunity and transplant rejection. In light of the latter, although research has long uncovered main concepts of allogeneic recognition, immune rejection is still the main obstacle to long-term graft survival. Therefore, in order to define effective therapies that prolong graft viability, it is essential that we understand the underlying mediators and mechanisms that participate in transplant rejection. This multifaceted process is characterized by diverse cellular and humoral participants with innate and adaptive functions that can determine the type of rejection or promote graft acceptance. Although a number of mediators of graft recognition have been described in traditional immunology, recent studies indicate that defining rigid roles for certain immune cells and factors may be more complicated than originally conceived. Current research has also targeted specific cells and drugs that regulate immune activation and induce tolerance. This review will give a broad view of the most recent understanding of the allogeneic inflammatory/tolerogenic response and current insights into cellular and drug therapies that modulate immune activation that may prove to be useful in the induction of tolerance in the clinical setting.

The antineoplastic drug cisplatin promotes renal injury, which limits its use. Protocols that reduce renal cisplatin toxicity will allow higher doses to be used in cisplatin treatment. Here, we compare physical exercise and caloric restriction (CR) as protocols to reduce cisplatin renal injury in mice. Male C57BL/6 were divided into four groups: Control, cisplatin, exercise + cisplatin, and 30% CR + cisplatin. Animals were injected with a single dose of cisplatin (20 mg/kg i.p.) and sacrificed 96 h after injection. Quantitative real time PCR, histological analyses, immunohistochemistry, and biochemical measurements were performed to investigate renal injury, necrosis, apoptosis, and inflammatory mechanisms. Both protocols protected against cisplatin renal injury, but CR was more effective in reducing uraemia and renal necrosis. The CR + Cisplatin group exhibited reduced serum IL-1β and TNF-α levels. No differences were noted in the renal mRNA expression of cytokines. Both interventions reduced apoptosis, but only the CR + Cisplatin group decreased TNFR2 protein expression. PPAR-α was activated in mice after CR. An antagonist of PPAR-α blocked the protective effect of CR. Both interventions attenuated the nephrotoxicity caused by cisplatin injection, but CR + Cisplatin showed a better response by modulating TNFR2. Moreover, part of the CR benefit depends on PPAR-α activation.

Glioblastoma (GBM) is an infiltrative tumor that is difficult to eradicate. Treating GBM with mesenchymal stem cells (MSCs) that have been modified with the HSV-Tk suicide gene has brought significant advances mainly because MSCs are chemoattracted to GBM and kill tumor cells via a bystander effect. To use this strategy, abundantly present adipose-tissue-derived mesenchymal stem cells (AT-MSCs) were evaluated for the treatment of GBM in mice. AT-MSCs were prepared using a mechanical protocol to avoid contamination with animal protein and transduced with HSV-Tk via a lentiviral vector. The U-87 glioblastoma cells cultured with AT-MSC-HSV-Tk died in the presence of 25 or 50 μM ganciclovir (GCV). U-87 glioblastoma cells injected into the brains of nude mice generated tumors larger than 3.5 mm2 after 4 weeks, but the injection of AT-MSC-HSV-Tk cells one week after the U-87 injection, combined with GCV treatment, drastically reduced tumors to smaller than 0.5 mm2. Immunohistochemical analysis of the tumors showed the presence of AT-MSC-HSV-Tk cells only within the tumor and its vicinity, but not in other areas of the brain, showing chemoattraction between them. The abundance of AT-MSCs and the easier to obtain them mechanically are strong advantages when compared to using MSCs from other tissues.

Background aims Granulocyte macrophage-colony stimulating factor (GM-CSF) promotes vessel formation through several molecular signaling pathways. Mesenchymal stromal cells (MSCs) have an important role in neovasculogenesis during ischemia because they release pro-angiogenic paracrine factors, pro-survival and immunomodulatory substances and can differentiate into endothelial cells. The objective of this study was to evaluate whether there is synergy between GM-CSF and MSCs in recovering ischemic limbs. Methods MSCs from mouse bone marrow were transduced with a lentiviral vector expressing GM-CSF and injected into animals with surgically induced limb ischemia, with unmodified MCSs used as control. The evolution of limb necrosis was evaluated for 1 month. Muscle strength was assessed on the 30th day, and the animals were euthanized to determine the muscle mass and to perform histological analyses to determine the degree of cellular infiltration, capillary and microvessel densities, fibrosis, necrosis and tissue regeneration. Results Both treatments were able to ameliorate ischemia, decrease the areas of fibrosis, necrosis, adipocytes and leukocyte infiltrates and increase the number of capillaries. The addition of GM-CSF promoted the formation of larger vessels, but it also resulted in more fibrosis and less muscle mass without affecting muscle force. Conclusions Both treatments resulted in a remarkable amelioration of ischemia. More fibrosis and less muscle mass produced by the overexpression of GM-CSF did not affect muscle functionality significantly. Importantly, MSCs overexpressing GM-CSF produced larger vessels, which is an important long-term advantage because larger vessels are more efficient in the reperfusion of ischemic tissues physiologically.

Glioblastoma (GBM) is the most common and lethal primary intracranial tumor. Because GBM is highly invasive and diffusely infiltrates the brain, a complete resection of the tumor is unfeasible. The gene encoding thymidine kinase from herpes simplex virus-1 (HSV-Tk) is the most commonly used suicide gene in preclinical and clinical trials against GBM. Treating GBM with mesenchymal stem cells (MSCs) that have been modified with the HSV-Tk suicide gene has brought significant advances mainly because MSCs are chemoattracted to GBM and kill tumor cells via a bystander effect. Aiming at using this strategy in clinical trials, here we evaluated the abundantly present human adipose-tissue-derived mesenchymal stem cells (AT-MSC) to treat GBM in mouse model.AT-MSCs were prepared using a mechanical protocol to avoid contamination with animal proteins and transduced with HSV-Tk via a lentiviral vector. The U-87 glioblastoma cells cultured with AT-MSC-HSV-Tk died in the presence of 25 or 50 μM ganciclovir (GCV). U-87 glioblastoma cells injected into the brains of nude mice generated tumors larger than 3.5 mm2 after 4 weeks, but the injection of AT-MSC-HSV-Tk cells one week after the U-87 injection and treated with GCV drastically reduced tumors to smaller than 0.5 mm2. View Large Image | Download PowerPoint SlideImmunohistochemical analysis of the tumors showed the presence of AT-MSC-HSV-Tk cells only within the tumor and its vicinity, but not in other areas of the brain, indicating specific migration of AT-MSC-HSV-Tk to and interaction with tumours. View Large Image | Download PowerPoint SlideIn conclusion, the AT-MSCs are good carriers of the suicide HSV-Tk gene for the treatment of GBM, and the abundance of AT-MSCs and the easier to obtain them mechanically are strong advantages when compared to using MSCs from other tissues.

O presente experimento foi conduzido com objetivo de avaliar o efeito da adição de níveis crescentes de complexo enzimático em rações com soja integral desativada sobre o consumo de ração (CR), ganho de peso (GP) e conversão alimentar (CA) defrangos de corte no período de 1 a 42 dias de idade. Foram utilizados 480 pintos de corte, machos da linhagem Ross distribuídos em um delineamento inteiramente casualizado, contendo 4 tratamentos, com 6 repetições, cada uma com 20 aves. As aves foram alojadas em galpão convencional, dividido em boxes com piso de maravalha e receberam água e ração àvontade. As rações experimentais foram formuladas à base de milho, farelo de soja e soja integral desativada, obedecendo a níveis nutricionais utilizados pela indústria brasileira. O complexo enzimático (xilanase, âglucanase, mannanase, pectinase e protease) foi adicionado em níveis crescentes de 0; 25; 50 e 75 g/t de ração. A suplementação com 50 g/t do complexo enzimático, em dietas formuladas com soja integral desativada demonstrou aumento de 3% no consumo de ração e 2,5% no ganho de peso em comparação com a dieta sem suplementação. De acordo com a análise de regressão o nível ótimo de adição decomplexo enzimático para ganho de peso e para consumo alimentar (1 – 42 dias de idade) obtido nestas condições experimentais foi de 45,94g/t e 49,30g/t de enzima na ração respectivamente. O nível de inclusão do complexo enzimático foi próximo de 50 g/t

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.