Sigma-1 Receptor Agonist Fluvoxamine Ameliorates Fibrotic Response of Trabecular Meshwork Cells.

Primary open-angle glaucoma remains a global issue, lacking a definitive treatment. Increased intraocular pressure (IOP) is considered the primary risk factor of the disease and it can be caused by fibrotic-like changes in the trabecular meshwork (TM) such as increased tissue stiffness and outflow resistance. Previously, we demonstrated that the sigma-1 receptor (S1R) agonist fluvoxamine (FLU) has anti-fibrotic properties in the kidney and lung. In this study, the localization of the S1R in TM cells was determined, and the anti-fibrotic efficacy of FLU was examined in both mouse and human TM cells. Treatment with FLU reduced the F-actin rearrangement, inhibited cell proliferation and migration induced by the platelet-derived growth factor and decreased the levels of fibrotic proteins. The protective role of the S1R in fibrosis was confirmed by a more pronounced increase in alpha smooth muscle actin and F-actin bundle and clump formation in primary mouse S1R knockout TM cells. Furthermore, FLU demonstrated its protective effects by increasing the production of nitric oxide and facilitating the degradation of the extracellular matrix through the elevation of cathepsin K. These findings suggest that the S1R could be a novel target for the development of anti-fibrotic drugs and offer a new therapeutic approach for glaucoma.

Lyophilization and homogenization of biological samples improves reproducibility and reduces standard deviation in molecular biology techniques.

Lyophilization is a cost-effective method for biological specimen preservation but detailed tissue-specific reference protocols are still lacking. Moreover, data are limited on the long-term stability of proteins and nucleic acids in lyophilized samples.Here, we offer lyophilization protocols for various rat and mouse tissues (kidney, heart, liver, lung, aorta, and skin) coupled with technical hints for optimal sample preparation. We demonstrate that lyophilized samples stored at 4 °C for 20 months can yield protein and RNA of similar quantity and quality to -80 °C storage, while phosphorylated proteins are preserved as well. Freeze-dried and subsequently pulverized samples can provide more consistent, more reliable data especially when investigating focal injuries, such as fibrosis. We developed a protocol for the concentration of biological solutions and achieved 20-times concentration in human peritoneal dialysis effluent solution which enables the previously unattainable detection of proteins in these samples. We established a method for water removal as well as accurate water content measurement of fecal samples, which can be valuable for gut metabolome analysis.Taken together, lyophilization is a valuable tool for the preservation of biological samples with many advantages. We aim to draw attention to the wide range of possibilities offered by freeze drying in pre-clinical or basic research.

Reduced O-GlcNAcylation and tubular hypoxia contribute to the antifibrotic effect of SGLT2 inhibitor dapagliflozin in the diabetic kidney.

Diabetic kidney disease is a worldwide epidemic, and therapies are incomplete. Clinical data suggest that improved renal outcomes by Na+-glucose cotransporter 2 inhibitor (SGLT2i) are partly beyond their antihyperglycemic effects; however, the mechanisms are still elusive. Here, we investigated the effect of the SGLT2i dapagliflozin (DAPA) in the prevention of elevated O-GlcNAcylation and tubular hypoxia as contributors of renal fibrosis. Type 1 diabetes was induced by streptozotocin in adult male Wistar rats. After the onset of diabetes, rats were treated for 6 wk with DAPA or DAPA combined with losartan (LOS). The effect of hyperglycemia was tested in HK-2 cells kept under normal or high glucose conditions. To test the effect of hypoxia, cells were kept in 1% O2 for 2 h. Cells were treated with DAPA or DAPA combined with LOS. DAPA slowed the loss of renal function, mitigated renal tubular injury markers (kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin), and reduced tubulointerstitial fibrosis. DAPA diminished high glucose-induced protein O-GlcNAcylation and moderated the tubular response to hypoxia through the hypoxia-inducible factor pathway. DAPA alone was as effective as combined treatment with LOS in all outcome parameters. These data highlight the role of ameliorated O-GlcNAcylation and diminished tubular hypoxia as important benefits of SGLT2i treatment. Our results support the link between glucose toxicity, tubular hypoxia, and fibrosis, a vicious trio that could be targeted by SGLT2i in kidney diseases of other origins as well.

Novel therapeutic potential of angiotensin receptor 1 blockade in a rat model of diabetes-associated depression parallels altered BDNF signalling.

AIMS/HYPOTHESIS: Diabetes is a worldwide epidemic linked with diverse diseases of the nervous system, including depression. A few studies suggested a connection between renin-angiotensin-aldosterone system blockers and reduced depressive symptoms, although underlying mechanisms are unclear. Here we investigated the antidepressant effect and the mechanisms of action of the angiotensin receptor 1 blocker (ARB) losartan in an experiential model of diabetes-associated depression. METHODS: Experimental diabetes was induced by streptozotocin in adult male Wistar rats. After 5 weeks of diabetes, rats were treated for 2 weeks with a non-pressor oral dose of losartan (20 mg/kg). In protocol 1, cerebrovascular perfusion and glial activation were evaluated by single-photon emission computed tomography-MRI and immunohistochemistry. In protocol 2, behaviour studies were performed (forced swim test and open field test). Hippocampal proinflammatory response and brain-derived neurotrophic factor (BDNF) signalling were also assessed. RESULTS: Here, we show that diabetic rats exhibit depression-like behaviour, which can be therapeutically reversed by losartan. This action of losartan occurs via changes in diabetes-induced neuroinflammatory responses rather than altered cerebral perfusion. We also show that as a part of its protective effect losartan restores BDNF production in astrocytes and facilitates BDNF-tropomyosin receptor kinase B-cAMP response element-binding protein signalling in the diabetic brain. CONCLUSIONS/INTERPRETATION: We identified a novel effect of losartan in the nervous system that may be implemented to alleviate symptoms of diabetes-associated depression. These findings explore a new therapeutic horizon for ARBs as possible antidepressants and suggest that BDNF could be a target of future drug development in diabetes-induced complications.

RAAS inhibitors directly reduce diabetes-induced renal fibrosis via growth factor inhibition.

KEY POINTS: Increased activation of the renin-angiotensin-aldosterone system (RAAS) and elevated growth factor production are of crucial importance in the development of renal fibrosis leading to diabetic kidney disease. The aim of this study was to provide evidence for the antifibrotic potential of RAAS inhibitor (RAASi) treatment and to explore the exact mechanism of this protective effect. We found that RAASi ameliorate diabetes-induced renal interstitial fibrosis and decrease profibrotic growth factor production. RAASi prevents fibrosis by acting directly on proximal tubular cells, and inhibits hyperglycaemia-induced growth factor production and thereby fibroblast activation. These results suggest a novel therapeutic indication and potential of RAASi in the treatment of renal fibrosis. ABSTRACT: In diabetic kidney disease (DKD) increased activation of renin-angiotensin-aldosterone system (RAAS) contributes to renal fibrosis. Although RAAS inhibitors (RAASi) are the gold standard therapy in DKD, the mechanism of their antifibrotic effect is not yet clarified. Here we tested the antifibrotic and renoprotective action of RAASi in a rat model of streptozotocin-induced DKD. In vitro studies on proximal tubular cells and renal fibroblasts were also performed to further clarify the signal transduction pathways that are directly altered by hyperglycaemia. After 5 weeks of diabetes, male Wistar rats were treated for two more weeks per os with the RAASi ramipril, losartan, spironolactone or eplerenone. Proximal tubular cells were cultured in normal or high glucose (HG) medium and treated with RAASi. Platelet-derived growth factor (PDGF) or connective tissue growth factor (CTGF/CCN2)-induced renal fibroblasts were also treated with various RAASi. In diabetic rats, reduced renal function and interstitial fibrosis were ameliorated and elevated renal profibrotic factors (TGFß1, PDGF, CTGF/CCN2, MMP2, TIMP1) and alpha-smooth muscle actin (αSMA) levels were decreased by RAASi. HG increased growth factor production of HK-2 cells, which in turn induced activation and αSMA production of fibroblasts. RAASi decreased tubular PDGF and CTGF expression and reduced production of extracellular matrix (ECM) components in fibroblasts. In proximal tubular cells, hyperglycaemia-induced growth factor production increased renal fibroblast transformation, contributing to the development of fibrosis. RAASi, even in non-antihypertensive doses, decreased the production of profibrotic factors and directly prevented fibroblast activation. All these findings suggest a novel therapeutic role for RAASi in the treatment of renal fibrosis.

The role of Sigma-1 receptor in sex-specific heat shock response in an experimental rat model of renal ischaemia/reperfusion injury.

We previously showed that female rats are more protected against renal ischaemia/reperfusion (I/R) injury than males, which is partly attributed to their more pronounced heat shock response. We recently described that Sigma-1 receptor (S1R) activation improves postischaemic survival and renal function. 17ß-estradiol activates S1R, thus here we investigated the role of sex-specific S1R activation and heat shock response in severe renal I/R injury. Proximal tubular cells were treated with 17ß-estradiol, which caused direct S1R activation and subsequent induction of heat shock response. Uninephrectomized female, male and ovariectomized female (Ovx) Wistar rats were subjected to 50-min renal ischaemia followed by 2 (T2) and 24 (T24) hours of reperfusion. At T24 renal functional, impairment was less severe and structural damage was less prominent in females versus males or Ovx. Postischaemic increase in S1R, pAkt, HSF-1, HSP72 levels were detected as early as at T2, while pHSP27 was elevated later at T24. Abundance of heat shock proteins was higher in healthy female rats and remained higher at T2 and T24 (female versus male or Ovx; resp.). We propose a S1R-dependent mechanism, which contributes to the relative renoprotection of females after I/R injury by enhancing the heat shock response.

σ1-Receptor Agonism Protects against Renal Ischemia-Reperfusion Injury.

Mechanisms of renal ischemia-reperfusion injury remain unresolved, and effective therapies are lacking. We previously showed that dehydroepiandrosterone protects against renal ischemia-reperfusion injury in male rats. Here, we investigated the potential role of σ1-receptor activation in mediating this protection. In rats, pretreatment with either dehydroepiandrosterone or fluvoxamine, a high-affinity σ1-receptor agonist, improved survival, renal function and structure, and the inflammatory response after sublethal renal ischemia-reperfusion injury. In human proximal tubular epithelial cells, stimulation by fluvoxamine or oxidative stress caused the σ1-receptor to translocate from the endoplasmic reticulum to the cytosol and nucleus. Fluvoxamine stimulation in these cells also activated nitric oxide production that was blocked by σ1-receptor knockdown or Akt inhibition. Similarly, in the postischemic rat kidney, σ1-receptor activation by fluvoxamine triggered the Akt-nitric oxide synthase signaling pathway, resulting in time- and isoform-specific endothelial and neuronal nitric oxide synthase activation and nitric oxide production. Concurrently, intravital two-photon imaging revealed prompt peritubular vasodilation after fluvoxamine treatment, which was blocked by the σ1-receptor antagonist or various nitric oxide synthase blockers. In conclusion, in this rat model of ischemia-reperfusion injury, σ1-receptor agonists improved postischemic survival and renal function via activation of Akt-mediated nitric oxide signaling in the kidney. Thus, σ1-receptor activation might provide a therapeutic option for renoprotective therapy.

Role of O-linked N-acetylglucosamine modification in diabetic nephropathy.

Increased O-linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) is a known contributor to diabetes; however, its relevance in diabetic nephropathy (DN) is poorly elucidated. Here, we studied the process and enzymes of O-GlcNAcylation with a special emphasis on Akt-endothelial nitric oxide synthase (eNOS) and heat shock protein (HSP)72 signaling. Since tubular injury is the prominent site of DN, the effect of hyperglycemia was first measured in proximal tubular (HK2) cells cultured in high glucose. In vivo O-GlcNAcylation and protein levels of O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), phosphorylated (p)Akt/Akt, peNOS/eNOS, and HSP72 were assessed in the kidney cortex of streptozotocin-induced diabetic rats. The effects of various renin-angiotensin-aldosterone system (RAAS) inhibitors were also evaluated. In proximal tubular cells, hyperglycemia-induced OGT expression led to increased O-GlcNAcylation, which was followed by a compensatory increase of OGA. In parallel, peNOS and pAkt levels decreased, whereas HSP72 increased. In diabetic rats, elevated O-GlcNAcylation was accompanied by decreased OGT and OGA. RAAS inhibitors ameliorated diabetes-induced kidney damage and prevented the elevation of O-GlcNAcylation and the decrement of pAkt, peNOS, and HSP72. In conclusion, hyperglycemia-induced elevation of O-GlcNAcylation contributes to the progression of DN via inhibition of Akt/eNOS phosphorylation and HSP72 induction. RAAS blockers successfully inhibit this process, suggesting a novel pathomechanism of their renoprotective action in the treatment of DN.

Hyperthymic affective temperament and hypertension are independent determinants of serum brain-derived neurotrophic factor level.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) has neuroprotective, proangiogenic and myogenic effects and, therefore, possibly acts as a psychosomatic mediator. Here, we measured serum BDNF (seBDNF) level in hypertensive patients (HT) and healthy controls (CONT) and its relation to affective temperaments, depression and anxiety scales, and arterial stiffness parameters. METHODS: In this cross-sectional study, affective temperaments, anxiety, and depression were studied with questionnaires (TEMPS-A, HAM-A, and BDI, respectively). SeBDNF level and routine laboratory parameters were measured as well. Arterial stiffness was evaluated with a tonometric method. RESULTS: Allover, 151 HT, and 32 CONT subjects were involved in the study. SeBDNF level was significantly higher in HT compared to CONT (24880 ± 8279 vs 21202.6 ± 6045.5 pg/mL, p < 0.05). In the final model of regression analysis, hyperthymic temperament score (Beta = 405.8, p = 0.004) and the presence of hypertension (Beta = 6121.2, p = 0.001) were independent determinants of seBDNF. In interaction analysis, it was found that in HT, a unit increase in hyperthymic score was associated with a 533.3 (95 %CI 241.3-825.3) pg/mL higher seBDNF. This interaction was missing in CONT. CONCLUSIONS: Our results suggest a complex psychosomatic involvement of BDNF in the pathophysiology of hypertension, where hyperthymic affective temperament may have a protective role. BDNF is not likely to have an effect on large arteries.

Identification of hypertensive patients with dominant affective temperaments might improve the psychopathological and cardiovascular risk stratification: a pilot, case-control study.

BACKGROUND: Although mood disorders and cardiovascular diseases have widely studied psychosomatic connections, data concerning the influence of the psychopathologically important affective temperaments in hypertension are scarce. To define a possibly higher cardiovascular risk subpopulation we investigated in well-treated hypertensive patients with dominant affective temperaments (DOM) and in well-treated hypertensive patients without dominant temperaments the level of depression and anxiety, arterial stiffness and serum Brain-derived Neurotrophic Factor (seBDNF). METHODS: 175 hypertensive patients, free of the history of psychiatric diseases, completed the TEMPS-A, Beck Depression Inventory and Hamilton Anxiety Scale questionnaires in two primary care practices. Of those 175 patients, 24 DOM patients and 24 hypertensive controls (matched in age, sex and the presence of diabetes) were selected for measurements of arterial stiffness and seBDNF level. RESULTS: Beck and Hamilton scores in DOM patients were higher compared with controls. Pulse wave velocity and augmentation index did not differ between the groups while in the DOM patients decreased brachial systolic and diastolic and central diastolic blood pressures were found compared with controls. SeBDNF was lower in the DOM group than in the controls (22.4 ± 7.2 vs. 27.3 ± 7.8 ng/mL, p < 0.05). CONCLUSIONS: Although similar arterial stiffness parameters were found in DOM patients, their increased depression and anxiety scores, the decreased brachial and central diastolic blood pressures as well as the decreased seBDNF might refer to their higher vulnerability regarding the development not only of major mood disorders, but also of cardiovascular complications. These data suggest that the evaluation of affective temperaments should get more attention both with regard to psychopathology and cardiovascular health management.

Sigma-1 Receptor Activation Is Protective against TGFß2-Induced Extracellular Matrix Changes in Human Trabecular Meshwork Cells.

The trabecular meshwork (TM) route is the principal outflow egress of the aqueous humor. Actin cytoskeletal remodeling in the TM and extracellular matrix (ECM) deposition increase TM stiffness, outflow resistance, and elevate intraocular pressure (IOP). These alterations are strongly linked to transforming growth factor-ß2 (TGFß2), a known profibrotic cytokine that is markedly elevated in the aqueous humor of glaucomatous eyes. Sigma-1 receptor (S1R) has been shown to have neuroprotective effects in the retina, but data are lacking about its role in the TM. In this study, we identified the presence of S1R in mouse TM tissue and investigated the effect of an S1R agonist fluvoxamine (FLU) on TGFß2-induced human TM cells regarding cell proliferation; ECM-related functions, including F-actin reorganization; and the accumulation of ECM elements. TGFß2 increased the proliferation, cytoskeletal remodeling, and protein levels of fibronectin, collagen type IV, and connective tissue growth factor, and decreased the level of matrix metalloproteinase-2. Most importantly, FLU reversed all these effects of TGFß2, suggesting that S1R agonists could be potential candidates for preserving TM function and thus maintaining normal IOP.

SGLT2 inhibitor dapagliflozin prevents atherosclerotic and cardiac complications in experimental type 1 diabetes.

INTRODUCTION: Cardiovascular disease (CVD) is two to five times more prevalent in diabetic patients and is the leading cause of death. Therefore, identification of novel therapeutic strategies that reduce the risk of CVD is a research priority. Clinical trials showed that reduction in the relative risk of heart failure by sodium-glucose cotransporter 2 inhibitors (SGLT2i) are partly beyond their glucose lowering effects, however, the molecular mechanisms are still elusive. Here we investigated the role of SGLT2i dapagliflozin (DAPA) in the prevention of diabetes-induced cardiovascular complications. METHODS: Type 1 diabetes was induced with streptozotocin (65 mg/bwkg, ip.) in adult, male Wistar rats. Following the onset of diabetes rats were treated for six weeks with DAPA (1 mg/bwkg/day, po.). RESULTS: DAPA decreased blood glucose levels (D: 37±2.7 vs. D+DAPA: 18±5.6 mmol/L; p<0.05) and prevented metabolic decline. Aortic intima-media thickening was mitigated by DAPA. DAPA abolished cardiac hypertrophy, and myocardial damage. Cardiac inflammation and fibrosis were also moderated after DAPA treatment. CONCLUSIONS: These data support the preventive and protective role of SGLT2i in diabetes-associated cardiovascular disease. SGLT2i may provide novel therapeutic strategy to hinder the development of cardiovascular diseases in type 1 diabetes, thereby improve the outcomes.

Antidepressant effect in diabetes-associated depression: A novel potential of RAAS inhibition.

The incidence of depression doubles in diabetic patients and is associated with poor outcomes. Studies indicate that renin-angiotensin-aldosterone system inhibitors (RAASi) might relieve depression, however the mechanism of action is not well understood. We recently showed that angiotensin receptor blockers have antidepressant effects in experimental diabetes comorbid depression. Here we investigated whether all types of RAASi exhibit antidepressant and neuroprotective properties. Diabetes was induced by streptozotocin in adult male Wistar rats. After 5 weeks of diabetes, rats were treated per os with non-pressor doses of enalapril, ramipril, spironolactone or eplerenone for 2 weeks. Behavior was evaluated using forced swim test and open field test. Inflammatory response and brain-derived neurotrophic factor (BDNF) signaling were investigated in the hippocampus. Both ACEi and MR antagonists reversed diabetes-induced behavioral despair confirming their antidepressant-like effect. This may occur via alterations in hippocampal cytokine-mediated inflammatory response. Repressed BDNF production was restored by RAASi. Both ACEi and MR antagonists facilitated the BDNF-tropomyosin receptor kinase B-cAMP response element-binding protein signaling pathway as part of their neuroprotective effect. These data highlight the important benefits of ACEi and MR antagonists in the treatment of diabetes-associated depressive symptoms. Our novel findings support the link between diabetes comorbid depression, inflammation and repressed BDNF signaling. RAASi could provide new therapeutic options to improve the outcomes of both disorders.

Autologous apoptotic neutrophils inhibit inflammatory cytokine secretion by human dendritic cells, but enhance Th1 responses.

Neutrophils represent the most abundant cell type in peripheral blood and exhibit a remarkably brief (6-8 h) half-life in circulation. The fundamental role of these professional phagocytes has been established in acute inflammation, based on their potential to both initiate and receive inflammatory signals. Furthermore, neutrophils also take part in maintaining chronic inflammatory processes, such as in various autoimmune diseases. Here, we demonstrate that human autologous apoptotic neutrophils are readily engulfed by immature monocyte-derived dendritic cells (moDCs) with similar efficiency as allogeneic apoptotic neutrophils [Majai G et al. (2010) J Leukoc Biol 88, 981-991]. Interestingly, in contrast to the allogeneic system, exposure of moDCs to autologous apoptotic neutrophils inhibits LPS + IFN-γ-induced production of inflammatory cytokines in a phagocytosis-independent manner. Autologous apoptotic neutrophil-primed DCs are able to modulate T-cell responses by inducing the generation of IFN-γ-secreting cells while hampering that of IL-17A-producing cells. Our observations indicate that capture of autologous apoptotic neutrophils by immature DCs may impede further neutrophil-mediated phagocytosis and tissue damage, and allow increased clearance of dying cells by macrophages.

The role of sigma-1 receptor and brain-derived neurotrophic factor in the development of diabetes and comorbid depression in streptozotocin-induced diabetic rats.

RATIONALE: Depression is highly prevalent in diabetes (DM). Brain-derived neurotrophic factor (BDNF) which is mainly regulated by the endoplasmic reticulum chaperon sigma-1 receptor (S1R) plays a relevant role in the development of depression. OBJECTIVES: We studied the dose-dependent efficacy of S1R agonist fluvoxamine (FLU) in the prevention of DM-induced depression and investigated the significance of the S1R-BDNF pathway. METHODS: We used streptozotocin to induce DM in adult male rats that were treated for 2 weeks p.o. with either different doses of FLU (2 or 20 mg/bwkg) or FLU + S1R antagonist NE100 (1 mg/bwkg) or vehicle. Healthy controls were also enrolled. Metabolic, behaviour, and neuroendocrine changes were determined, and S1R and BDNF levels were measured in the different brain regions. RESULTS: In DM rats, immobility time was increased, adrenal glands were enlarged, and thymuses were involuted. FLU in 20 mg/bwkg, but not in 2 mg/bwkg dosage, ameliorated depression-like behaviour. S1R and BDNF protein levels were decreased in DM, while FLU induced SIR-BDNF production. NE100 suspended all effects of FLU. CONCLUSIONS: We suggest that disturbed S1R-BDNF signaling in the brain plays a relevant role in DM-induced depression. The activation of this cascade serves as an additional target in the prevention of DM-associated depression.

Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps.

Neutrophil extracellular trap (NET) ejected from activated dying neutrophils is a highly ordered structure of DNA and selected proteins capable to eliminate pathogenic microorganisms. Biochemical determinants of the non-randomly formed stable NETs have not been revealed so far. Studying the formation of human NETs we have observed that polyamines were incorporated into the NET. Inhibition of myeloperoxidase, which is essential for NET formation and can generate reactive chlorinated polyamines through hypochlorous acid, decreased polyamine incorporation. Addition of exogenous primary amines that similarly to polyamines inhibit reactions catalyzed by the protein cross-linker transglutaminases (TGases) has similar effect. Proteomic analysis of the highly reproducible pattern of NET components revealed cross-linking of NET proteins through chlorinated polyamines and ɛ(γ-glutamyl)lysine as well as bis-γ-glutamyl polyamine bonds catalyzed by the TGases detected in neutrophils. Competitive inhibition of protein cross-linking by monoamines disturbed the cross-linking pattern of NET proteins, which resulted in the loss of the ordered structure of the NET and significantly reduced capacity to trap bacteria. Our findings provide explanation of how NETs are formed in a reproducible and ordered manner to efficiently neutralize microorganisms at the first defense line of the innate immune system.

The glucocorticoid dexamethasone programs human dendritic cells for enhanced phagocytosis of apoptotic neutrophils and inflammatory response.

GCs are powerful anti-inflammatory compounds inhibiting inflammatory cell recruitment and production of proinflammatory cytokines. We have recently found that DCs, the key players of T cell priming and polarization, respond to allogeneic apoptotic neutrophils with proinflammatory cytokine release and Th1 cell activation. Here, we show that monocyte-derived human DCs develop their capacity to engulf apoptotic cells by up-regulating a set of apoptophagocytic genes. This gene expression pattern was reprogrammed when differentiation took place in the presence of the synthetic GC Dex, which increased the expression of phagocytosis receptors MERTK and CD14, the bridging molecule C1QA, DNASE2, and ADORA3. The increased phagocytosis was attenuated by the addition of ADORA3 antagonist and could not be observed when bone marrow-derived DCs of ADORA3 KO mice were treated with Dex. The GC-treated human DCs loaded with allogeneic apoptotic neutrophils secreted, in response to LPS and IFN-γ, the inflammatory cytokine TNF-α. Furthermore, the Dex-treated DCs could activate autologous T lymphocytes toward Th1 effector cells, and this was enhanced by their exposure to allogeneic apoptotic neutrophils.

Phagocytosis of cells dying through autophagy induces inflammasome activation and IL-1ß release in human macrophages.

Phagocytosis of naturally dying cells usually blocks inflammatory reactions in host cells. We have recently observed that clearance of cells dying through autophagy leads to a pro-inflammatory response in human macrophages. Investigating this response further, we found that during engulfment of MCF-7 or 293T cells undergoing autophagic death, but not apoptotic or anoikic ones, caspase-1 was activated and IL-1ß was processed, then secreted in a MyD88-independent manner. Autophagic dying cells were capable of preventing some LPS-induced pro-inflammatory responses, such as TNFα, IL-6 and IL-8 induction, but synergized with LPS for IL-1ß production. Caspase-1 inhibition prevented macrophage IL-1ß release triggered by the dying cells and also other pro-inflammatory cytokines which were not formed in the presence of IL-1 receptor antagonist anakinra either. IL-1ß secretion was also observed using calreticulin knock down or necrostatin treated autophagic MCF-7 cells and it required phagocytosis of the dying cells which led to ATP secretion from macrophages. Blocking K (+) efflux during phagocytosis, the presence of apyrase, adding an antagonist of the P2X7 receptor or silencing the NOD-like receptor protein NALP3 inhibited IL-1ß secretion. These data suggest that during phagocytosis of autophagic dying cells ATP, acting through its receptor, initiates K (+) efflux, inflammasome activation and secretion of IL-1ß, which initiates further pro-inflammatory events. Thus, autophagic death of malignant cells and their clearance may lead to immunogenic response.

PPARγ modulated inflammatory response of human dendritic cell subsets to engulfed apoptotic neutrophils.

The means of how phagocytes handle apoptotic cells has a great impact on the outcome of immune responses. Here, we show that phagocytosis of allogeneic, apoptotic neutrophils by human monocyte-derived DCs is slow and less efficient than that of macrophages, and CD1a(-) DCs are more active in the engulfment of apoptotic neutrophils than CD1a(+) DCs. Blocking DC-SIGN function partially interferes with the uptake of apoptotic cells, and long-term interaction of apoptotic neutrophils with DCs makes them prone to proinflammatory cytokine responses. Engulfment of apoptotic cells sensitizes CD1a(-) DCs for high IL-8, TNF-α, IL-6, and CD1a(+) cells for IL-12 and IL-10 cytokine secretion elicited by additional inflammatory stimuli, which also result in the polarization of autologous T lymphocytes to Th1 effector cells. Ligand-induced activation of PPARγ by RSG results in enhanced phagocytosis, but the proinflammatory response and the capacity to trigger Th1 cell activation of CD1a(-) DCs are not enhanced. These results demonstrate that DCs are able to respond to allogeneic, apoptotic neutrophils with inflammatory cytokines and T cell responses in a subtype-specific manner that is modulated by the anti-inflammatory effects of PPARγ.

Transglutaminase 2 is expressed and active on the surface of human monocyte-derived dendritic cells and macrophages.

The multifunctional enzyme, transglutaminase 2 (TG2), can be found intracellularly, in the extracellular matrix and on the cell surface. Cell surface TG2 (csTG2) could not be detected by TG2-specific antibodies or autoantibodies on immunocompetent cells. A supposedly csTG2-specific antibody, 6B9, was recently shown to actually react with CD44. Though the importance of TG2-mediated deamidation of gluten in the pathogenesis of celiac disease has been well recognized, it is not known in which intestinal cells or cell compartment the deamidation occurs. Duodenal dendritic cells (DCs) can be directly involved in gluten-reactive T-cell activation. Here we use blood monocyte-derived dendritic cells (iDC) and macrophages (MPhi) as a model for intestinal antigen-presenting cells (APCs) and show that they contain large amounts of TG2. We found that TG100, a commercial TG2-specific monoclonal antibody can recognize TG2 on the surface of these cells, that is monocyte-derived APCs express surface-associated TG2. TG2 expression was found on the surface of individual tunica propria cells in frozen small bowel tissue sections from both normal and celiac subjects. We also demonstrate that the pool of TG2 on the surface of iDCs can be catalytically active, hence it might directly be involved in the deamidation of gliadin peptides. Bacterial lipopolysaccharide (LPS) increased the level of TG2 on the surface of maturing DCs, supporting the hypothesis that an unspecific inflammatory process in the gut may expose more transglutaminase activity.