Proliferative vitreoretinopathy (PVR) develops after an unsuccessful or complicated recovery from... more Proliferative vitreoretinopathy (PVR) develops after an unsuccessful or complicated recovery from rhegmatogenous retinal detachment (RRD) surgery. Intraocular scar formation with the contribution of epithelial-mesenchymal transition (EMT) in RPE cells is prominent in the pathology of PVR. In the present study, the EMT process was experimentally induced in human retinal pigment epithelium (RPE; ARPE-19) cells, and the effect of atorvastatin on the process was studied. The mRNA and protein levels of mesenchymal markers actin alpha 2 (ACTA2) / alpha-smooth muscle actin (α-SMA) and fibronectin (FN), and epithelial markers occludin (OCLN) and zonula occludens-1 (ZO-1) were measured using quantitative real-time PCR (qRT-PCR) and western blot methods, respectively. In addition, α-SMA and FN were visualized using immunofluorescence staining. Cells were photographed under a phase contrast light microscope. Changes in the functionality of cells following the EMT process were studied using the IncuCyte scratch wound cell migration assay and the collagen cell invasion assay with confocal microscopy. The induction of EMT in ARPE-19 cells increased the expression of mesenchymal markers ACTA2/α-SMA and fibronectin and reduced the expression of epithelial marker OCLN both at mRNA and protein levels. The mRNA levels of ZO-1 were lower after EMT, as well. Increased levels of α-SMA and FN were confirmed by immunofluorescence staining. Atorvastatin further increased the mRNA levels of mesenchymal markers ACTA2 and FN as well as the protein levels of α-SMA and reduced the mRNA levels of epithelial markers OCLN and ZO-1 under the EMT process. EMT promoted wound closure and cell invasion into the 3D collagen matrix when compared to untreated control cells. These data present cellular changes upon the induction of the EMT process in ARPE-19 cells and the propensity of atorvastatin to complement the effect. More studies are needed to confirm the exact influence of the EMT process and atorvastatin treatment on the PVR development after RRD surgery.
International ophtalmology/International ophthalmology, Mar 26, 2024
simvastatin and amfenac on ARPE-19 cells under inflammatory conditions. Methods ARPE-19 cells wer... more simvastatin and amfenac on ARPE-19 cells under inflammatory conditions. Methods ARPE-19 cells were pre-treated with simvastatin and/or amfenac for 24 h after which interleukin (IL)-1α or IL-1β was added for another 24 h. After treatments, lactate dehydrogenase release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) processing, nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB) activity, prostaglandin E2 (PGE2) level, and extracellular levels of IL-6, IL-8, monocytic chemoattractant protein (MCP-1), vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor, as well as the production of reactive oxygen species (ROS) were determined. Results Pre-treatment of human ARPE-19 cells with simvastatin reduced the production of IL-6, IL-8, and MCP-1 cytokines, PGE2 levels, as well as NF-κB activity upon inflammation, whereas amfenac reduced IL-8 and MCP-1 release but increased ROS production. Together, simvastatin and amfenac reduced the release of IL-6, IL-8, and MCP-1 cytokines as well as NF-κB activity but increased the VEGF release upon inflammation in ARPE-19 cells. Conclusion Our present study supports the antiinflammatory capacity of simvastatin as pre-treatment against inflammation in human RPE cells, and the addition of amfenac complements the effect. The early modulation of local conditions in the retina can prevent inflammation induced PVR formation and subsequent retinal re-detachment.
The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related paramet... more The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related parameters were studied under controlled laboratory conditions using roach (Rutilus rutilus), a cyprinid teleost, as the model fish. In vitro lymphoproliferative responses stimulated with a Tcell-specific mitogen, concanavalin A (ConA), or a Bcell-specific activator, lipopolysaccharide (LPS), were decreased in exposed fish. Also nonstimulated proliferation was lower than in unexposed fish. ConA-activated responses returned to normal levels within 7 days after exposure, but LPS-activated responses were reduced throughout the 14 day follow-up. The capability of UVBexposed fish to produce an antibody response was studied by intraperitoneal immunization with bovine ␥-globulin (BGG). The concentration of anti-BGG antibodies in plasma as well as the number of anti-BGG-specific antibody-secreting cells in the spleen or blood were not decreased in fish exposed either to a single dose of UVB prior to immunization, or to single dose of UVB prior to immunization followed by three additional doses after immunization. Immunoglobulin M (IgM) production, when assayed as plasma IgM level or as the number of IgM-secreting cells in the spleen or blood, was not suppressed after exposure to UVB irradiation. These results indicate that a single dose of UVB or short-term exposure to UVB irradiation has no negative effects on IgM production or reactivity against antigen administered via the intraperitoneal route. However, the suppression of in vitro lymphoproliferative responses suggest that exposure to UVB has the potential to interfere with lymphocyterelated functions in fish.
PurposeInflammasomes are intracellular protein complexes whose activation results in the caspase‐... more PurposeInflammasomes are intracellular protein complexes whose activation results in the caspase‐1‐mediated release of pro‐inflammatory cytokines IL‐1β and IL‐18. We and others have shown that activation of NLRP3 inflammasome is associated with the pathogenesis of age‐related macular degeneration (AMD). Declined proteasomal degradation with concurrent autophagy inhibition is among the risk factors activating the NLRP3 receptor in retinal pigment epithelial (RPE) cells. NLRP3 is known to have various activation mechanisms and in the present study, we have analyzed which of them contribute to the inflammasome activation when protein degradation systems in RPE cells fail.MethodsInflammasome activation was induced in human ARPE‐19 cells by the proteasome inhibitor MG‐132 and the lysosome neutralizer Bafilomycin A. Extracellular ATP was measured, and its receptors were inhibited using a P2X7 inhibitor. Potassium efflux was inhibited using high extracellular ion concentration, and glyburide was added to block the ATP‐mediated potassium efflux. APDC (ammonium pyrrolidine dithiocarbamate) and NAC (N‐acetylcysteine) were used to reduce oxidative stress.ResultsDespite increased extracellular ATP level, the inhibition of P2X7 receptors did not reduce the secretion of IL‐1β. Neither potassium efflux nor cathepsin B contributed to the inflammasome activation in our model, but ROS inhibitors alleviated the IL‐1β production.ConclusionsOur results suggest that oxidative stress plays a role in the inflammasome activation in RPE cells upon declined intracellular clearance.
Ultraviolet B radiation penetrates into water and can affect fish health and the immune system, a... more Ultraviolet B radiation penetrates into water and can affect fish health and the immune system, as is the case with mammals. Teleost fish, the roach, were exposed to UVB irradiation in aquariums and a panel of immune parameters was determined. In addition to altered blood picture and respiratory burst by blood leukocytes, changes were noted also in major lymphatic organs. Respiratory burst and natural cytotoxicity activity of head kidney granulocytes and mitogen-activated proliferation of splenic lymphocytes were suppressed. Although mostly transitory, some parameters remained suppressed for the following 2 weeks. Ultraviolet A radiation had only minor effects. The stress induced by UVB may be involved in the modulation of immune parameters.
AMP-activated protein kinase (AMPK) has a crucial role not only in the regulation of tissue energ... more AMP-activated protein kinase (AMPK) has a crucial role not only in the regulation of tissue energy metabolism but it can also control immune responses through its cooperation with immune signaling pathways, thus affecting immunometabolism and the functions of immune cells. It is known that AMPK signaling inhibits the activity of the NF-κB system and thus suppresses proinflammatory responses. Interestingly, AMPK activation can inhibit several major immune signaling pathways, e.g., the JAK-STAT, NF-κB, C/EBPβ, CHOP, and HIF-1α pathways, which induce the expansion and activation of myeloid-derived suppressor cells (MDSC). MDSCs induce an immunosuppressive microenvironment in tumors and thus allow the escape of tumor cells from immune surveillance. Chronic inflammation has a key role in the expansion and activation of MDSCs in both tumors and inflammatory disorders. The numbers of MDSCs also significantly increase during the aging process concurrently with the immunosenescence associated with chronic low-grade inflammation. Increased fatty acid oxidation and lactate produced by aerobic glycolysis are important immunometabolic enhancers of MDSC functions. However, it seems that AMPK signaling regulates the functions of MDSCs in a context-dependent manner. Currently, the activators of AMPK signaling are promising drug candidates for cancer therapy and possibly for the extension of healthspan and lifespan. We will describe in detail the AMPK-mediated regulation of the signaling pathways controlling the expansion and activation of immunosuppressive MDSCs. We will propose that the beneficial effects mediated by AMPK activation, e.g., in cancers and the aging process, could be induced by the inhibition of MDSC functions.
Highlights FGF21, an endocrine factor, controls energy metabolism and acts as stress hormone ... more Highlights FGF21, an endocrine factor, controls energy metabolism and acts as stress hormone FGF21 regulates energy metabolism and stress responses via interorgan crosstalk FGF21 can increase healthspan and extend lifespan FGF21 regulates longevity via e.g. AMPK signaling and somatotropic axis FGF21 resistance is encountered in metabolic disorders disrupting healthy aging 3
PurposePhloretin is a natural polyphenol that is present e.g. in apple and pear trees. Previous r... more PurposePhloretin is a natural polyphenol that is present e.g. in apple and pear trees. Previous research has demonstrated that phloretin has anti‐inflammatory properties. Inflammation and mitochondrial dysfunction play key roles in the development of age‐related macular degeneration, the most common cause of vision loss amongst the elderly in the developed countries. Here, we assessed the effects of phloretin in a model of mitochondrial damage‐induced inflammation in retinal pigment epithelial (RPE) cells.MethodsARPE‐19 cells were exposed to 25 µM antimycin A for 24 hr to induce mitochondrial dysfunction. Cells were pretreated with 100 µM phloretin for 1 hour and cell viability as well as the release of pro‐inflammatory interleukines (IL)‐6, IL‐8 and the vascular endothelial growth factor (VEGF) was studied. The effect of either high glucose or glucose‐free culture medium was assessed. ELISA measurements of phosphorylated mitogen‐activated protein kinases (MAPK), p38, JNK, and extracellular signal‐regulated protein kinase (ERK1/2) as well as of the cAMP response element binding protein (CREB) were determined alongside the DNA binding efficiency of nuclear factor kB (NF‐kB) subunit p65 and of NFE2L2. Effects on ERK1/2 phosphorylation and NFE2L2 DNA binding were confirmed using the MAPK kinase 1/2 (MEK1/2) inhibitor PD98059, the NFE2L2 activator sulforaphane, and specific NFE2L2 siRNA.ResultsPhloretin significantly reduced the levels of IL‐6, IL‐8, and VEGF in the presence and the absence of antimycin A, without affecting cellular viability. Higher levels or absence of glucose had no effect on the anti‐inflammatory activities of phloretin. Pathway analysis showed that phloretin‐activated ERK1/2 phosphorylation but this was not related to its anti‐inflammatory effects as preventing ERK1/2 phosphorylation using PD98059 decreased inflammation even further. Instead, siRNA experiments revealed that NFE2L2 was involved in phloretin’s ameliorating effect on IL‐8 secretion.ConclusionsPhloretin is a strong anti‐inflammatory agent that is well‐tolerated by RPE cells suffering from mitochondrial dysfunction.
Purposep62/SQSTM1 is a selective autophagy substrate being added to ubiquitinated proteins direct... more Purposep62/SQSTM1 is a selective autophagy substrate being added to ubiquitinated proteins directed to lysosomal degradation. Its levels increase inside the retinal pigment epithelial (RPE) cells when proteasomal activity and autophagic clearance decline during aging. p62/SQSTM1 can also contribute to the activation of pro‐inflammatory nuclear factor kappa B (NF‐κB) or mitogen‐activated protein kinase (MAPK) pathways. According to our previous studies, reduced intracellular clearance promotes the NLRP3 inflammasome‐mediated release of IL‐1β, which subsequently stimulates RPE cells to produce other pro‐inflammatory mediators, such as IL‐8. In the present study, we have investigated, whether increased levels of p62/SQSTM1 play a role in the IL‐1β‐induced response.MethodsUpregulation of p62/SQSTM1 was induced by bafilomycin A1 or the over‐expression vector, or prevented by p62/SQSTM1‐specific small interfering RNA (siRNA) transfection. Thereafter, the cells were exposed to IL‐1β. The release of IL‐6 and IL‐8 were detected using the enzyme‐linked immunosorbent assay (ELISA) method. In addition, the autophagy markers p62/SQSTM1 and LC3 were determined using the western blot technique.ResultsIL‐1β resulted in low IL‐6 and high IL‐8 production in human RPE cells. p62/SQSTM1 and its absence were verified but they did not affect the cytokine profiles.ConclusionsOur data suggest that p62/SQSTM1 does not play a role in the IL‐1β‐induced production of pro‐inflammatory cytokines in human RPE cells.
International Journal of Molecular Sciences, Feb 20, 2019
Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional phagocytes ca... more Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional phagocytes can result in debris formation and development of age-related macular degeneration (AMD). Chronic oxidative stress and inflammation play an important role in AMD pathogenesis. Only a few well-established in vitro phagocytosis assay models exist. We propose human embryonic stem cell-derived-RPE cells as a new model for studying RPE cell removal by professional phagocytes. The characteristics of human embryonic stem cells-derived RPE (hESC-RPE) are similar to native RPEs based on their gene and protein expression profile, integrity, and barrier properties or regarding drug transport. However, no data exist about RPE death modalities and how efficiently dying hESC-RPEs are taken upby macrophages, and whether this process triggers an inflammatory responses. This study demonstrates hESC-RPEs can be induced to undergo anoikis or autophagy-associated cell death due to extracellular matrix detachment or serum deprivation and hydrogen-peroxide co-treatment, respectively, similar to primary human RPEs. Dying hESC-RPEs are efficiently engulfed by macrophages which results in high amounts of IL-6 and IL-8 cytokine release. These findings suggest that the clearance of anoikic and autophagy-associated dying hESC-RPEs can be used as a new model for investigating AMD pathogenesis or for testing the in vivo potential of these cells in stem cell therapy.
Purpose: Dysfunctional intracellular clearance is associated with various diseases, including age... more Purpose: Dysfunctional intracellular clearance is associated with various diseases, including age‐related macular degeneration (AMD). Previously, we reported the inhibition of intracellular clearance by the non‐selective proteasome inhibitor MG‐132 resulted in oxidative stress‐mediated NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and subsequent interleukin‐1β (IL‐1β) secretion in human ARPE‐19 cells. In this study, we used the selective proteasome inhibitor epoxomicin and examined its effect on the inflammasome activation and IL‐1β production.Methods: IL‐1α‐primed human‐derived ARPE‐19 cells were exposed either to MG‐132 or epoxomicin and the autophagy inhibitor bafilomycin A (BafA) and studied using enzyme‐linked immunosorbent assay and cytotoxicity tests.Results: Our data reveal epoxomicin‐induced proteasome inhibition led to NADPH oxidase and mitochondria‐mediated oxidative stress‐related release of mitochondrial DNA to the cytosol, which then resulted in potassium efflux‐dependent absent in melanoma 2 (AIM2) inflammasome activation and subsequent release of inflammatory cytokine IL‐1β in ARPE‐19 cells.Conclusions: Our study emphasizes the role of AIM2 inflammasome in RPE cell degeneration along with the well‐known NLRP3 inflammasome and may represent a novel therapy target.
PurposeCornea is the outermost layer of the eye and mainly responsible for the absorption of ultr... more PurposeCornea is the outermost layer of the eye and mainly responsible for the absorption of ultraviolet B (UVB) radiation. An acute painful inflammatory damage caused by UVB radiation on the corneal epithelium is known as photokeratitis, snow blindness, or welder's burn. We have previously shown that UVB induces inflammation by increasing the production of cytokines IL‐6 and IL‐8 in human corneal epithelial (HCE) cells and now, we have continued this study by exploring the effects of UVB on the inflammasome signaling.MethodsIn vitro‐cultured HCE cells were exposed to UVB radiation after priming with TNF‐α. Thereafter, inflammasome components and cytokines were determined using the ELISA, western blot, and activity assays.ResultsOur results suggest that UVB is capable of activating inflammasome signaling in HCE cells.ConclusionsIn addition to traditional pathways, UVB induces the activation of more recently found pro‐inflammatory signaling system in the human corneal epithelium.
PurposeAge‐related macular degeneration (AMD) is the leading cause of blindness among the elderly... more PurposeAge‐related macular degeneration (AMD) is the leading cause of blindness among the elderly in the western world. It represents not only a dramatic reduction in patients’ quality of life but also a significant burden to the general healthcare system. Yet, despite the severity of the disease much questions regarding the pathways of disease formation and progression are still unanswered and viable treatment options still undiscovered. Here, we evaluate the cytoprotective and anti‐inflammatory potential of fisetin and luteolin in human retinal pigment epithelial cells exposed to increased oxidative stress.MethodsARPE‐19 cells were treated with 4‐Hydroxynonenal (HNE) to simulate high levels of oxidative stress. Thereafter, fisetin or luteolin were added to the culture medium. The MTT and the lactate dehydrogenase assays were used to assess cellular toxicity. Inflammatory cytokines, as well as activation of transcription factors were measured using the ELISA method and a DNA‐binding transcription factor assay. To analyze the importance of SIRT1 and related pathways, the experiments were repeated after specific SIRT1 knock‐out using siRNA. Levels of intracellular SIRT1 were measured using Western Blot.ResultsFisetin and luteolin protected retinal pigment epithelial cells from oxidative stress‐induced cell death and exhibited potent anti‐inflammatory properties even after the initial insult. These effects seemed to be independent of NF‐κB or SIRT1.ConclusionsBioactive polyphenols, fisetin and luteolin are powerful anti‐inflammatory and anti‐oxidant agents and show potential for the development of drugs aimed at specific intracellular pathways that affect inflammation in AMD.
The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related paramet... more The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related parameters were studied under controlled laboratory conditions using roach (Rutilus rutilus), a cyprinid teleost, as the model fish. In vitro lymphoproliferative responses stimulated with a Tcell-specific mitogen, concanavalin A (ConA), or a Bcell-specific activator, lipopolysaccharide (LPS), were decreased in exposed fish. Also nonstimulated proliferation was lower than in unexposed fish. ConA-activated responses returned to normal levels within 7 days after exposure, but LPS-activated responses were reduced throughout the 14 day follow-up. The capability of UVBexposed fish to produce an antibody response was studied by intraperitoneal immunization with bovine ␥-globulin (BGG). The concentration of anti-BGG antibodies in plasma as well as the number of anti-BGG-specific antibody-secreting cells in the spleen or blood were not decreased in fish exposed either to a single dose of UVB prior to immunization, or to single dose of UVB prior to immunization followed by three additional doses after immunization. Immunoglobulin M (IgM) production, when assayed as plasma IgM level or as the number of IgM-secreting cells in the spleen or blood, was not suppressed after exposure to UVB irradiation. These results indicate that a single dose of UVB or short-term exposure to UVB irradiation has no negative effects on IgM production or reactivity against antigen administered via the intraperitoneal route. However, the suppression of in vitro lymphoproliferative responses suggest that exposure to UVB has the potential to interfere with lymphocyterelated functions in fish.
Proliferative vitreoretinopathy (PVR) develops after an unsuccessful or complicated recovery from... more Proliferative vitreoretinopathy (PVR) develops after an unsuccessful or complicated recovery from rhegmatogenous retinal detachment (RRD) surgery. Intraocular scar formation with the contribution of epithelial-mesenchymal transition (EMT) in RPE cells is prominent in the pathology of PVR. In the present study, the EMT process was experimentally induced in human retinal pigment epithelium (RPE; ARPE-19) cells, and the effect of atorvastatin on the process was studied. The mRNA and protein levels of mesenchymal markers actin alpha 2 (ACTA2) / alpha-smooth muscle actin (α-SMA) and fibronectin (FN), and epithelial markers occludin (OCLN) and zonula occludens-1 (ZO-1) were measured using quantitative real-time PCR (qRT-PCR) and western blot methods, respectively. In addition, α-SMA and FN were visualized using immunofluorescence staining. Cells were photographed under a phase contrast light microscope. Changes in the functionality of cells following the EMT process were studied using the IncuCyte scratch wound cell migration assay and the collagen cell invasion assay with confocal microscopy. The induction of EMT in ARPE-19 cells increased the expression of mesenchymal markers ACTA2/α-SMA and fibronectin and reduced the expression of epithelial marker OCLN both at mRNA and protein levels. The mRNA levels of ZO-1 were lower after EMT, as well. Increased levels of α-SMA and FN were confirmed by immunofluorescence staining. Atorvastatin further increased the mRNA levels of mesenchymal markers ACTA2 and FN as well as the protein levels of α-SMA and reduced the mRNA levels of epithelial markers OCLN and ZO-1 under the EMT process. EMT promoted wound closure and cell invasion into the 3D collagen matrix when compared to untreated control cells. These data present cellular changes upon the induction of the EMT process in ARPE-19 cells and the propensity of atorvastatin to complement the effect. More studies are needed to confirm the exact influence of the EMT process and atorvastatin treatment on the PVR development after RRD surgery.
International ophtalmology/International ophthalmology, Mar 26, 2024
simvastatin and amfenac on ARPE-19 cells under inflammatory conditions. Methods ARPE-19 cells wer... more simvastatin and amfenac on ARPE-19 cells under inflammatory conditions. Methods ARPE-19 cells were pre-treated with simvastatin and/or amfenac for 24 h after which interleukin (IL)-1α or IL-1β was added for another 24 h. After treatments, lactate dehydrogenase release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) processing, nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB) activity, prostaglandin E2 (PGE2) level, and extracellular levels of IL-6, IL-8, monocytic chemoattractant protein (MCP-1), vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor, as well as the production of reactive oxygen species (ROS) were determined. Results Pre-treatment of human ARPE-19 cells with simvastatin reduced the production of IL-6, IL-8, and MCP-1 cytokines, PGE2 levels, as well as NF-κB activity upon inflammation, whereas amfenac reduced IL-8 and MCP-1 release but increased ROS production. Together, simvastatin and amfenac reduced the release of IL-6, IL-8, and MCP-1 cytokines as well as NF-κB activity but increased the VEGF release upon inflammation in ARPE-19 cells. Conclusion Our present study supports the antiinflammatory capacity of simvastatin as pre-treatment against inflammation in human RPE cells, and the addition of amfenac complements the effect. The early modulation of local conditions in the retina can prevent inflammation induced PVR formation and subsequent retinal re-detachment.
The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related paramet... more The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related parameters were studied under controlled laboratory conditions using roach (Rutilus rutilus), a cyprinid teleost, as the model fish. In vitro lymphoproliferative responses stimulated with a Tcell-specific mitogen, concanavalin A (ConA), or a Bcell-specific activator, lipopolysaccharide (LPS), were decreased in exposed fish. Also nonstimulated proliferation was lower than in unexposed fish. ConA-activated responses returned to normal levels within 7 days after exposure, but LPS-activated responses were reduced throughout the 14 day follow-up. The capability of UVBexposed fish to produce an antibody response was studied by intraperitoneal immunization with bovine ␥-globulin (BGG). The concentration of anti-BGG antibodies in plasma as well as the number of anti-BGG-specific antibody-secreting cells in the spleen or blood were not decreased in fish exposed either to a single dose of UVB prior to immunization, or to single dose of UVB prior to immunization followed by three additional doses after immunization. Immunoglobulin M (IgM) production, when assayed as plasma IgM level or as the number of IgM-secreting cells in the spleen or blood, was not suppressed after exposure to UVB irradiation. These results indicate that a single dose of UVB or short-term exposure to UVB irradiation has no negative effects on IgM production or reactivity against antigen administered via the intraperitoneal route. However, the suppression of in vitro lymphoproliferative responses suggest that exposure to UVB has the potential to interfere with lymphocyterelated functions in fish.
PurposeInflammasomes are intracellular protein complexes whose activation results in the caspase‐... more PurposeInflammasomes are intracellular protein complexes whose activation results in the caspase‐1‐mediated release of pro‐inflammatory cytokines IL‐1β and IL‐18. We and others have shown that activation of NLRP3 inflammasome is associated with the pathogenesis of age‐related macular degeneration (AMD). Declined proteasomal degradation with concurrent autophagy inhibition is among the risk factors activating the NLRP3 receptor in retinal pigment epithelial (RPE) cells. NLRP3 is known to have various activation mechanisms and in the present study, we have analyzed which of them contribute to the inflammasome activation when protein degradation systems in RPE cells fail.MethodsInflammasome activation was induced in human ARPE‐19 cells by the proteasome inhibitor MG‐132 and the lysosome neutralizer Bafilomycin A. Extracellular ATP was measured, and its receptors were inhibited using a P2X7 inhibitor. Potassium efflux was inhibited using high extracellular ion concentration, and glyburide was added to block the ATP‐mediated potassium efflux. APDC (ammonium pyrrolidine dithiocarbamate) and NAC (N‐acetylcysteine) were used to reduce oxidative stress.ResultsDespite increased extracellular ATP level, the inhibition of P2X7 receptors did not reduce the secretion of IL‐1β. Neither potassium efflux nor cathepsin B contributed to the inflammasome activation in our model, but ROS inhibitors alleviated the IL‐1β production.ConclusionsOur results suggest that oxidative stress plays a role in the inflammasome activation in RPE cells upon declined intracellular clearance.
Ultraviolet B radiation penetrates into water and can affect fish health and the immune system, a... more Ultraviolet B radiation penetrates into water and can affect fish health and the immune system, as is the case with mammals. Teleost fish, the roach, were exposed to UVB irradiation in aquariums and a panel of immune parameters was determined. In addition to altered blood picture and respiratory burst by blood leukocytes, changes were noted also in major lymphatic organs. Respiratory burst and natural cytotoxicity activity of head kidney granulocytes and mitogen-activated proliferation of splenic lymphocytes were suppressed. Although mostly transitory, some parameters remained suppressed for the following 2 weeks. Ultraviolet A radiation had only minor effects. The stress induced by UVB may be involved in the modulation of immune parameters.
AMP-activated protein kinase (AMPK) has a crucial role not only in the regulation of tissue energ... more AMP-activated protein kinase (AMPK) has a crucial role not only in the regulation of tissue energy metabolism but it can also control immune responses through its cooperation with immune signaling pathways, thus affecting immunometabolism and the functions of immune cells. It is known that AMPK signaling inhibits the activity of the NF-κB system and thus suppresses proinflammatory responses. Interestingly, AMPK activation can inhibit several major immune signaling pathways, e.g., the JAK-STAT, NF-κB, C/EBPβ, CHOP, and HIF-1α pathways, which induce the expansion and activation of myeloid-derived suppressor cells (MDSC). MDSCs induce an immunosuppressive microenvironment in tumors and thus allow the escape of tumor cells from immune surveillance. Chronic inflammation has a key role in the expansion and activation of MDSCs in both tumors and inflammatory disorders. The numbers of MDSCs also significantly increase during the aging process concurrently with the immunosenescence associated with chronic low-grade inflammation. Increased fatty acid oxidation and lactate produced by aerobic glycolysis are important immunometabolic enhancers of MDSC functions. However, it seems that AMPK signaling regulates the functions of MDSCs in a context-dependent manner. Currently, the activators of AMPK signaling are promising drug candidates for cancer therapy and possibly for the extension of healthspan and lifespan. We will describe in detail the AMPK-mediated regulation of the signaling pathways controlling the expansion and activation of immunosuppressive MDSCs. We will propose that the beneficial effects mediated by AMPK activation, e.g., in cancers and the aging process, could be induced by the inhibition of MDSC functions.
Highlights FGF21, an endocrine factor, controls energy metabolism and acts as stress hormone ... more Highlights FGF21, an endocrine factor, controls energy metabolism and acts as stress hormone FGF21 regulates energy metabolism and stress responses via interorgan crosstalk FGF21 can increase healthspan and extend lifespan FGF21 regulates longevity via e.g. AMPK signaling and somatotropic axis FGF21 resistance is encountered in metabolic disorders disrupting healthy aging 3
PurposePhloretin is a natural polyphenol that is present e.g. in apple and pear trees. Previous r... more PurposePhloretin is a natural polyphenol that is present e.g. in apple and pear trees. Previous research has demonstrated that phloretin has anti‐inflammatory properties. Inflammation and mitochondrial dysfunction play key roles in the development of age‐related macular degeneration, the most common cause of vision loss amongst the elderly in the developed countries. Here, we assessed the effects of phloretin in a model of mitochondrial damage‐induced inflammation in retinal pigment epithelial (RPE) cells.MethodsARPE‐19 cells were exposed to 25 µM antimycin A for 24 hr to induce mitochondrial dysfunction. Cells were pretreated with 100 µM phloretin for 1 hour and cell viability as well as the release of pro‐inflammatory interleukines (IL)‐6, IL‐8 and the vascular endothelial growth factor (VEGF) was studied. The effect of either high glucose or glucose‐free culture medium was assessed. ELISA measurements of phosphorylated mitogen‐activated protein kinases (MAPK), p38, JNK, and extracellular signal‐regulated protein kinase (ERK1/2) as well as of the cAMP response element binding protein (CREB) were determined alongside the DNA binding efficiency of nuclear factor kB (NF‐kB) subunit p65 and of NFE2L2. Effects on ERK1/2 phosphorylation and NFE2L2 DNA binding were confirmed using the MAPK kinase 1/2 (MEK1/2) inhibitor PD98059, the NFE2L2 activator sulforaphane, and specific NFE2L2 siRNA.ResultsPhloretin significantly reduced the levels of IL‐6, IL‐8, and VEGF in the presence and the absence of antimycin A, without affecting cellular viability. Higher levels or absence of glucose had no effect on the anti‐inflammatory activities of phloretin. Pathway analysis showed that phloretin‐activated ERK1/2 phosphorylation but this was not related to its anti‐inflammatory effects as preventing ERK1/2 phosphorylation using PD98059 decreased inflammation even further. Instead, siRNA experiments revealed that NFE2L2 was involved in phloretin’s ameliorating effect on IL‐8 secretion.ConclusionsPhloretin is a strong anti‐inflammatory agent that is well‐tolerated by RPE cells suffering from mitochondrial dysfunction.
Purposep62/SQSTM1 is a selective autophagy substrate being added to ubiquitinated proteins direct... more Purposep62/SQSTM1 is a selective autophagy substrate being added to ubiquitinated proteins directed to lysosomal degradation. Its levels increase inside the retinal pigment epithelial (RPE) cells when proteasomal activity and autophagic clearance decline during aging. p62/SQSTM1 can also contribute to the activation of pro‐inflammatory nuclear factor kappa B (NF‐κB) or mitogen‐activated protein kinase (MAPK) pathways. According to our previous studies, reduced intracellular clearance promotes the NLRP3 inflammasome‐mediated release of IL‐1β, which subsequently stimulates RPE cells to produce other pro‐inflammatory mediators, such as IL‐8. In the present study, we have investigated, whether increased levels of p62/SQSTM1 play a role in the IL‐1β‐induced response.MethodsUpregulation of p62/SQSTM1 was induced by bafilomycin A1 or the over‐expression vector, or prevented by p62/SQSTM1‐specific small interfering RNA (siRNA) transfection. Thereafter, the cells were exposed to IL‐1β. The release of IL‐6 and IL‐8 were detected using the enzyme‐linked immunosorbent assay (ELISA) method. In addition, the autophagy markers p62/SQSTM1 and LC3 were determined using the western blot technique.ResultsIL‐1β resulted in low IL‐6 and high IL‐8 production in human RPE cells. p62/SQSTM1 and its absence were verified but they did not affect the cytokine profiles.ConclusionsOur data suggest that p62/SQSTM1 does not play a role in the IL‐1β‐induced production of pro‐inflammatory cytokines in human RPE cells.
International Journal of Molecular Sciences, Feb 20, 2019
Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional phagocytes ca... more Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional phagocytes can result in debris formation and development of age-related macular degeneration (AMD). Chronic oxidative stress and inflammation play an important role in AMD pathogenesis. Only a few well-established in vitro phagocytosis assay models exist. We propose human embryonic stem cell-derived-RPE cells as a new model for studying RPE cell removal by professional phagocytes. The characteristics of human embryonic stem cells-derived RPE (hESC-RPE) are similar to native RPEs based on their gene and protein expression profile, integrity, and barrier properties or regarding drug transport. However, no data exist about RPE death modalities and how efficiently dying hESC-RPEs are taken upby macrophages, and whether this process triggers an inflammatory responses. This study demonstrates hESC-RPEs can be induced to undergo anoikis or autophagy-associated cell death due to extracellular matrix detachment or serum deprivation and hydrogen-peroxide co-treatment, respectively, similar to primary human RPEs. Dying hESC-RPEs are efficiently engulfed by macrophages which results in high amounts of IL-6 and IL-8 cytokine release. These findings suggest that the clearance of anoikic and autophagy-associated dying hESC-RPEs can be used as a new model for investigating AMD pathogenesis or for testing the in vivo potential of these cells in stem cell therapy.
Purpose: Dysfunctional intracellular clearance is associated with various diseases, including age... more Purpose: Dysfunctional intracellular clearance is associated with various diseases, including age‐related macular degeneration (AMD). Previously, we reported the inhibition of intracellular clearance by the non‐selective proteasome inhibitor MG‐132 resulted in oxidative stress‐mediated NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and subsequent interleukin‐1β (IL‐1β) secretion in human ARPE‐19 cells. In this study, we used the selective proteasome inhibitor epoxomicin and examined its effect on the inflammasome activation and IL‐1β production.Methods: IL‐1α‐primed human‐derived ARPE‐19 cells were exposed either to MG‐132 or epoxomicin and the autophagy inhibitor bafilomycin A (BafA) and studied using enzyme‐linked immunosorbent assay and cytotoxicity tests.Results: Our data reveal epoxomicin‐induced proteasome inhibition led to NADPH oxidase and mitochondria‐mediated oxidative stress‐related release of mitochondrial DNA to the cytosol, which then resulted in potassium efflux‐dependent absent in melanoma 2 (AIM2) inflammasome activation and subsequent release of inflammatory cytokine IL‐1β in ARPE‐19 cells.Conclusions: Our study emphasizes the role of AIM2 inflammasome in RPE cell degeneration along with the well‐known NLRP3 inflammasome and may represent a novel therapy target.
PurposeCornea is the outermost layer of the eye and mainly responsible for the absorption of ultr... more PurposeCornea is the outermost layer of the eye and mainly responsible for the absorption of ultraviolet B (UVB) radiation. An acute painful inflammatory damage caused by UVB radiation on the corneal epithelium is known as photokeratitis, snow blindness, or welder's burn. We have previously shown that UVB induces inflammation by increasing the production of cytokines IL‐6 and IL‐8 in human corneal epithelial (HCE) cells and now, we have continued this study by exploring the effects of UVB on the inflammasome signaling.MethodsIn vitro‐cultured HCE cells were exposed to UVB radiation after priming with TNF‐α. Thereafter, inflammasome components and cytokines were determined using the ELISA, western blot, and activity assays.ResultsOur results suggest that UVB is capable of activating inflammasome signaling in HCE cells.ConclusionsIn addition to traditional pathways, UVB induces the activation of more recently found pro‐inflammatory signaling system in the human corneal epithelium.
PurposeAge‐related macular degeneration (AMD) is the leading cause of blindness among the elderly... more PurposeAge‐related macular degeneration (AMD) is the leading cause of blindness among the elderly in the western world. It represents not only a dramatic reduction in patients’ quality of life but also a significant burden to the general healthcare system. Yet, despite the severity of the disease much questions regarding the pathways of disease formation and progression are still unanswered and viable treatment options still undiscovered. Here, we evaluate the cytoprotective and anti‐inflammatory potential of fisetin and luteolin in human retinal pigment epithelial cells exposed to increased oxidative stress.MethodsARPE‐19 cells were treated with 4‐Hydroxynonenal (HNE) to simulate high levels of oxidative stress. Thereafter, fisetin or luteolin were added to the culture medium. The MTT and the lactate dehydrogenase assays were used to assess cellular toxicity. Inflammatory cytokines, as well as activation of transcription factors were measured using the ELISA method and a DNA‐binding transcription factor assay. To analyze the importance of SIRT1 and related pathways, the experiments were repeated after specific SIRT1 knock‐out using siRNA. Levels of intracellular SIRT1 were measured using Western Blot.ResultsFisetin and luteolin protected retinal pigment epithelial cells from oxidative stress‐induced cell death and exhibited potent anti‐inflammatory properties even after the initial insult. These effects seemed to be independent of NF‐κB or SIRT1.ConclusionsBioactive polyphenols, fisetin and luteolin are powerful anti‐inflammatory and anti‐oxidant agents and show potential for the development of drugs aimed at specific intracellular pathways that affect inflammation in AMD.
The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related paramet... more The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related parameters were studied under controlled laboratory conditions using roach (Rutilus rutilus), a cyprinid teleost, as the model fish. In vitro lymphoproliferative responses stimulated with a Tcell-specific mitogen, concanavalin A (ConA), or a Bcell-specific activator, lipopolysaccharide (LPS), were decreased in exposed fish. Also nonstimulated proliferation was lower than in unexposed fish. ConA-activated responses returned to normal levels within 7 days after exposure, but LPS-activated responses were reduced throughout the 14 day follow-up. The capability of UVBexposed fish to produce an antibody response was studied by intraperitoneal immunization with bovine ␥-globulin (BGG). The concentration of anti-BGG antibodies in plasma as well as the number of anti-BGG-specific antibody-secreting cells in the spleen or blood were not decreased in fish exposed either to a single dose of UVB prior to immunization, or to single dose of UVB prior to immunization followed by three additional doses after immunization. Immunoglobulin M (IgM) production, when assayed as plasma IgM level or as the number of IgM-secreting cells in the spleen or blood, was not suppressed after exposure to UVB irradiation. These results indicate that a single dose of UVB or short-term exposure to UVB irradiation has no negative effects on IgM production or reactivity against antigen administered via the intraperitoneal route. However, the suppression of in vitro lymphoproliferative responses suggest that exposure to UVB has the potential to interfere with lymphocyterelated functions in fish.
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Papers by Anu Kauppinen