Novel Identification of Myeloid-Derived Suppressor Cells in Children With Septic Shock.

OBJECTIVES: Immunoparalysis in children with septic shock is associated with increased risk of nosocomial infections and death. Myeloid-derived suppressor cells (MDSCs) potently suppress T cell function and may perpetuate immunoparalysis. Our goal was to test the hypothesis that children with septic shock would demonstrate increased proportions of MDSCs and impaired immune function compared with healthy controls. DESIGN: Prospective observational study. SETTING: Fifty-four bed PICU in a quaternary-care children's hospital. PATIENTS: Eighteen children with septic shock and thirty age-matched healthy children. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and stained for cell surface markers to identify MDSCs by flow cytometric analysis, including granulocytic and monocytic subsets. Adaptive and innate immune function was measured by ex vivo stimulation of whole blood with phytohemagglutinin-induced interferon (IFN) γ production and lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production, respectively. Prolonged organ dysfunction (OD) was defined as greater than 7 days. Children with septic shock had a higher percentage of circulating MDSCs, along with lower LPS-induced TNFα and phytohemagglutinin-induced IFNγ production capacities, compared with healthy controls. A cut-off of 25.2% MDSCs of total PBMCs in initial samples was optimal to discriminate children with septic shock who went on to have prolonged OD, area under the curve equal to 0.86. Children with prolonged OD also had decreased TNFα production capacity over time compared with those who recovered more quickly ( p = 0.02). CONCLUSIONS: This article is the first to describe increased MDSCs in children with septic shock, along with an association between early increase in MDSCs and adverse OD outcomes in this population. It remains unclear if MDSCs play a causative role in sepsis-induced immune suppression in children. Additional studies are warranted to establish MDSC as a potential therapeutic target.

Splenic macrophage phagocytosis of intravenously infused mesenchymal stromal cells attenuates tumor localization.

Mesenchymal stromal cells (MSCs) possess remarkable tumor tropism, making them ideal vehicles to deliver tumor-targeted therapeutic agents; however, their value in clinical medicine has yet to be realized. A barrier to clinical utilization is that only a small fraction of infused MSCs ultimately localize to the tumor. In an effort to overcome this obstacle, we sought to enhance MSC trafficking by focusing on the factors that govern MSC arrival within the tumor microenvironment. Our findings show that MSC chemoattraction is only present in select tumors, including osteosarcoma, and that the chemotactic potency among similar tumors varies substantially. Using an osteosarcoma xenograft model, we show that human MSCs traffic to the tumor within several hours of infusion. After arrival, MSCs are observed to localize in clusters near blood vessels and MSC-associated bioluminescence signal intensity is increased, suggesting that the seeded cells expand after engraftment. However, our studies reveal that a significant portion of MSCs are eliminated en route by splenic macrophage phagocytosis, effectively limiting the number of cells available for tumor engraftment. To increase MSC survival, we transiently depleted macrophages with liposomal clodronate, which resulted in increased tumor localization without substantial reduction in tumor-associated macrophages. Our data suggest that transient macrophage depletion will significantly increase the number of MSCs in the spleen and thus improve MSC localization within a tumor, theoretically increasing the effective dose of an anti-cancer agent. This strategy may subsequently improve the clinical efficacy of MSCs as vehicles for the tumor-directed delivery of therapeutic agents.

Endoplasmic reticulum stress is induced in growth plate hypertrophic chondrocytes in G610C mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a hereditary bone disorder most commonly caused by autosomal dominant mutations in genes encoding type I collagen. In addition to bone fragility, patients suffer from impaired longitudinal bone growth. It has been demonstrated that in OI, an accumulation of mutated type I collagen in the endoplasmic reticulum (ER) induces ER stress in osteoblasts, causing osteoblast dysfunction leading to bone fragility. We hypothesize that ER stress is also induced in the growth plate where bone growth is initiated, and examined a mouse model of dominant OI that carries a G610C mutation in the procollagen α2 chain. The results demonstrated that G610C OI mice had significantly shorter long bones with growth plate abnormalities including elongated total height and hypertrophic zone. Moreover, we found that mature hypertrophic chondrocytes expressed type I collagen and ER dilation was more pronounced compared to wild type littermates. The results from in vitro chondrocyte cultures demonstrated that the maturation of G610C OI hypertrophic chondrocytes was significantly suppressed and ER stress related genes were upregulated. Given that the alteration of hypertrophic chondrocyte activity often causes dwarfism, our findings suggest that hypertrophic chondrocyte dysfunction induced by ER stress may be an underlying cause of growth deficiency in G610C OI mice.

Intratumoral Delivery of Interferonγ-Secreting Mesenchymal Stromal Cells Repolarizes Tumor-Associated Macrophages and Suppresses Neuroblastoma Proliferation In Vivo.

Neuroblastoma, the most common extracranial solid tumor in childhood, remains a therapeutic challenge. However, one promising patient treatment strategy is the delivery of anti-tumor therapeutic agents via mesenchymal stromal cell (MSC) therapy. MSCs have been safely used to treat genetic bone diseases such as osteogenesis imperfecta, cardiovascular diseases, autoimmune diseases, and cancer. The pro-inflammatory cytokine interferon-gamma (IFNγ) has been shown to decrease tumor proliferation by altering the tumor microenvironment (TME). Despite this, clinical trials of systemic IFNγ therapy have failed due to the high blood concentration required and associated systemic toxicities. Here, we developed an intra-adrenal model of neuroblastoma, characterized by liver and lung metastases. We then engineered MSCs to deliver IFNγ directly to the TME. In vitro, these MSCs polarized murine macrophages to the M1 phenotype. In vivo, we attained a therapeutically active TME concentration of IFNγ without increased systemic concentration or toxicity. The TME-specific IFNγ reduced tumor growth rate and increased survival in two models of T cell deficient athymic nude mice. Absence of this benefit in NOD SCID gamma (NSG) immunodeficient mouse model indicates a mechanism dependent on the innate immune system. IL-17 and IL-23p19, both uniquely M1 polarization markers, transiently increased in the tumor interstitial fluid. Finally, the MSC vehicle did not promote tumor growth. These findings reveal that MSCs can deliver effective cytokine therapy directly to the tumor while avoiding systemic toxicity. This method transiently induces inflammatory M1 macrophage polarization, which reduces tumor burden in our novel neuroblastoma murine model. Stem Cells 2018;36:915-924.

Apolipoprotein E plays crucial roles in maintaining bone mass by promoting osteoblast differentiation via ERK1/2 pathway and by suppressing osteoclast differentiation via c-Fos, NFATc1, and NF-κB pathway.

Apolipoprotein E (ApoE) plays crucial roles not only in lipid metabolism but also in bone metabolism. Specifically ApoE4, one of major ApoE isoforms, has been demonstrated to be associated with increased risk of developing osteoporosis compared to another major isoform ApoE3. However, the detailed mechanism of how the different ApoE isoforms affect bone metabolism remains unclear. Micro-CT analyses of distal femora demonstrated severely decreased bone mass in 48-week-old female homozygous ApoE-knockout (ApoE-KO) mice compared to age- and gender-matched wild type C57BL/6 J (WT) mice. Physiological levels of either ApoE3 or ApoE4 protein (1-20 µg/ml) significantly increased the expression of osteoblast-related genes and alkaline phosphatase (ALP) activity of primary calvarial osteoblasts by inhibiting extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in a dose-dependent manner, and ApoE3 showed greater osteoblastic induction compared to ApoE4. Furthermore, both ApoE3 and ApoE4 protein inhibited osteoclastogenesis and the expression of osteoclast-related genes of mouse bone marrow derived macrophages (BMDM) via down regulation of c-Fos, nuclear factor of activated T-cells 1 (NFATc1) and nuclear factor-kappa B (NF-κB) pathway. Moreover, ApoE3 showed greater inhibition of c-Fos, dendritic cell-specific transmembrane protein (DC-STAMP), and Cathepsin K gene expression compared to ApoE4. Collectively, ApoE plays crucial roles in preserving bone mass, suggesting that targeting ApoE and its isoforms as a promising treatment candidate of both osteoporosis and hyperlipidemia.

Extracellular vesicles released from mesenchymal stromal cells stimulate bone growth in osteogenesis imperfecta.

BACKGROUND: Systemic infusion of mesenchymal stromal cells (MSCs) has been shown to induce acute acceleration of growth velocity in children with osteogenesis imperfecta (OI) despite minimal engraftment of infused MSCs in bones. Using an animal model of OI we have previously shown that MSC infusion stimulates chondrocyte proliferation in the growth plate and that this enhanced proliferation is also observed with infusion of MSC conditioned medium in lieu of MSCs, suggesting that bone growth is due to trophic effects of MSCs. Here we sought to identify the trophic factor secreted by MSCs that mediates this therapeutic activity. METHODS: To examine whether extracellular vesicles (EVs) released from MSCs have therapeutic activity, EVs were isolated from MSC conditioned medium by ultracentrifugation. To further characterize the trophic factor, RNA or microRNA (miRNA) within EVs was depleted by either ribonuclease (RNase) treatment or suppressing miRNA biogenesis in MSCs. The functional activity of these modified EVs was evaluated using an in vitro chondrocyte proliferation assay. Finally, bone growth was evaluated in an animal model of OI treated with EVs. RESULTS: We found that infusion of MSC-derived EVs stimulated chondrocyte proliferation in the growth plate, resulting in improved bone growth in a mouse model of OI. However, infusion of neither RNase-treated EVs nor miRNA-depleted EVs enhanced chondrocyte proliferation. CONCLUSION: MSCs exert therapeutic effects in OI by secreting EVs containing miRNA, and EV therapy has the potential to become a novel cell-free therapy for OI that will overcome some of the current limitations in MSC therapy.

Genomic and functional comparison of mesenchymal stromal cells prepared using two isolation methods.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have been applied to patients in cell therapy for various diseases. Recently, we introduced a novel MSC separation filter device which could yield approximately 2.5-fold more MSCs from bone marrow in a closed system compared with the conventional open density gradient centrifugation method. MSCs isolated with these two methods were phenotypically similar and met the criteria defining human MSC proposed by the International Society for Cellular Therapy. However, these criteria do not reflect the functional capacity of MSCs. It has been shown that the donor, source, isolation method, culture condition and cryopreservation of MSCs have potential to alter their therapeutic efficacy. To determine the equivalency of MSCs isolated by these two methods, we compared their genomic profiles as an index of their biologic potential and evaluated their growth promoting potential as an index of function. METHODS: The gene expression profiles of human MSCs isolated from 5 healthy donors with two distinct methods were obtained from microarray analyses. The functional activity of freshly expanded/cryopreserved MSCs from these two isolation methods was evaluated using an in vitro chondrocyte proliferation assay. RESULTS: Freshly expanded MSCs isolated by these two methods were found to exhibit similar gene expression profiles and equivalent therapeutic effects, while freshly thawed, cryopreserved MSCs lacked all measureable therapeutic activity. CONCLUSIONS: The MSC separation device generates genomically and functionally equivalent MSCs compared with the conventionally isolated MSCs, although freshly thawed, cryopreserved MSCs, isolated by either method, are devoid of activity in our bioassay.

Albumin-induced podocyte injury and protection are associated with regulation of COX-2.

Albuminuria is both a hallmark and a risk factor for progressive glomerular disease, and results in increased exposure of podocytes to serum albumin with its associated factors. Here in vivo and in vitro models of serum albumin-overload were used to test the hypothesis that albumin-induced proteinuria and podocyte injury directly correlate with COX-2 induction. Albumin induced COX-2, MCP-1, CXCL1, and the stress protein HSP25 in both rat glomeruli and cultured podocytes, whereas B7-1 and HSP70i were also induced in podocytes. Podocyte exposure to albumin induced both mRNA and protein and enhanced the mRNA stability of COX-2, a key regulator of renal hemodynamics and inflammation, which renders podocytes susceptible to injury. Podocyte exposure to albumin also stimulated several kinases (p38 MAPK, MK2, JNK/SAPK, and ERK1/2), inhibitors of which (except JNK/SAPK) downregulated albumin-induced COX-2. Inhibition of AMPK, PKC, and NFκB also downregulated albumin-induced COX-2. Critically, albumin-induced COX-2 was also inhibited by glucocorticoids and thiazolidinediones, both of which directly protect podocytes against injury. Furthermore, specific albumin-associated fatty acids were identified as important contributors to COX-2 induction, podocyte injury, and proteinuria. Thus, COX-2 is associated with podocyte injury during albuminuria, as well as with the known podocyte protection imparted by glucocorticoids and thiazolidinediones. Moreover, COX-2 induction, podocyte damage, and albuminuria appear mediated largely by serum albumin-associated fatty acids.

Comparison of direct action of thiazolidinediones and glucocorticoids on renal podocytes: protection from injury and molecular effects.

The U.S. Food and Drug Administration-approved thiazolidinediones pioglitazone and rosiglitazone are peroxisome proliferator-activated receptor-γ (PPARγ) agonists developed to control serum glucose in patients with diabetes. They have been found to reduce proteinuria and microalbuminuria in both diabetic nephropathy and nondiabetic glomerulosclerosis. We hypothesized that the renal protective effects of thiazolidinediones result, at least in part, from their direct action on podocytes, similar to glucocorticoids. Treatment with pioglitazone, rosiglitazone, or dexamethasone significantly protected podocytes against puromycin aminonucleoside-induced injury (designed to mimic nephrotic syndrome-related injury), as determined by both cell survival and actin cytoskeletal integrity. Furthermore, we compared the ability of these drugs to modulate key signaling pathways in podocytes that may be critical to their protective effects. Rosiglitazone deactivated the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1/2, p38 MAPK, and stress-activated protein kinase/c-Jun NH2-terminal kinase, whereas pioglitazone did not, and dexamethasone deactivated to some extent. Similar to dexamethasone, both thiazolidinediones increased the glucocorticoid receptor phosphorylation, and this response to rosiglitazone and possibly to pioglitazone was PPARγ-dependent. Furthermore, both drugs mimicked or enhanced the effects of dexamethasone on glucocorticoid-responsive genes in a PPARγ- and glucocorticoid receptor-dependent manner. In addition, both thiazolidinediones mimicked dexamethasone-induced effects on calcineurin activity. In summary, thiazolidinediones are able to modulate the glucocorticoid pathway and exert direct protective effects on podocytes, similar to glucocorticoids. This suggests that thiazolidinediones may have potential clinical utility as either primary or adjunctive therapy for nephrotic syndrome or other diseases treated with glucocorticoids. These findings may also lend mechanistic insight into the well established but poorly understood renal protective effects of thiazolidinediones in diabetic nephropathy.

Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury.

While mitogen-activated protein kinase (MAPK) activation has been implicated in the pathogenesis of various glomerular diseases, including nephrotic syndrome (NS), its specific role in podocyte injury is not known. We hypothesized that MK-2, a downstream substrate of p38 MAPK, mediates the adverse effects of this pathway and that inhibition of MK-2 would protect podocytes from NS-related injury. Using cultured podocytes, we analyzed 1) the roles of MK-2 and p38 MAPK in puromycin aminonucleoside (PAN)-induced podocyte injury; 2) the ability of specific MK-2 and p38 MAPK inhibitors to protect podocytes against injury; 3) the role of serum albumin, known to induce podocyte injury, in activating p38 MAPK/MK-2 signaling; and 4) the role of p38 MAPK/MK-2 signaling in the expression of Cox-2, an enzyme associated with podocyte injury. Treatment with protein kinase inhibitors specific for both MK-2 (C23, a pyrrolopyridine-type compound) or p38 MAPK (SB203580) reduced PAN-induced podocyte injury and actin cytoskeletal disruption. Both inhibitors reduced baseline podocyte p38 MAPK/MK-2 signaling, as measured by the degree of phosphorylation of HSPB1, a downstream substrate of MK-2, but exhibited disparate effects on upstream signaling. Serum albumin activated p38 MAPK/MK-2 signaling and induced Cox-2 expression, and these responses were blocked by both inhibitors. Given the critical importance of podocyte injury to both NS and other progressive glomerular diseases, these data suggest an important role for p38 MAPK/MK-2 signaling in podocyte injury and identify MK-2 inhibition as a promising potential therapeutic strategy to protect podocytes in various glomerular diseases.