Anterior Chamber Flare as a Non-Invasive Assessment of Intraocular Immune Status and Ocular Complications in Proliferative Diabetic Retinopathy.

Proliferative diabetic retinopathy (PDR) is a vision-threatening complication of diabetes mellitus (DM). Anterior chamber (AC) flare and intraocular cytokines are potent biomarkers reflecting the intraocular immune status in PDR. This study aimed to elucidate the complex interrelationship between AC flare and intraocular cytokines in PDR eyes. A retrospective observational study was conducted on 19 PDR eyes of 19 patients with type 2 DM, and on 19 eyes of 19 patients with idiopathic macular hole or epiretinal membrane as controls. AC flare was measured before pars plana vitrectomy (PPV). Aqueous humor (AH) and vitreous fluid (VF) samples were collected at the time of PPV, and the quantities of 27 cytokines in both intraocular fluids were analyzed. In the PDR and control groups, Spearman's rank correlation analysis revealed a positive correlation between AC flare and IL-8 level in both AH and VF. Additionally, IL-8 levels in AH correlated positively with IL-8 levels in VF. In the PDR group, receiver operating characteristic curve analysis identified IL-8 level in AH as a significant predictor for both diabetic macular edema (DME) and vitreous hemorrhage (VH) complications. The cut-off values of IL-8 were established at ≥26.6 pg/mL for DME and ≥7.96 pg/mL for VH. Given the positive correlation between AC flare and AH IL-8 level, the present findings suggest that AC flare value may potentially be a non-invasive biomarker for predicting DME.

Programmed Cell Death-1 Pathway Deficiency Enhances Autoimmunity Leading to Dacryoadenitis of Mice.

Programmed cell death protein (PD)-1 is a coinhibitory molecule that suppresses immune response and maintains immune homeostasis. Moreover, the PD-1 pathway blocks cancers from being attacked by immune cells. Anti-PD-1 antibody therapy such as nivolumab improves survival in cancer patients. However, the occurrence of autoimmune inflammatory disorders in various organs has been increasingly reported as an adverse effect of nivolumab. Of the disorders associated with nivolumab, Sicca syndrome occurs in 3% to 11% of cases and has unknown pathologic mechanisms. Whether the absence of the PD-1 pathway causes functional and morphologic disorders in lacrimal glands was determined by analyzing PD-1 gene-knockout (Pdcd1-/-) mice. Histopathologic analysis showed that Pdcd1-/- mice developed dacryoadenitis beginning at 3 to 4 months of age, and deteriorated with age. Flow-cytometric analysis confirmed that cells infiltrating the affected lacrimal glands consisted mainly of CD3+ T cells and only a small proportion of CD19+ B cells. Among infiltrating T cells, the CD4+ Th-cell subset consisted of Th1 cells producing interferon-γ in an early stage of dacryoadenitis in Pdcd1-/- mice. Experiments of lymphocyte transfer from Pdcd1-/- into irradiated wild-type mice confirmed that CD4+ T cells from Pdcd1-/- mice induced dacryoadenitis. These results indicate that PD-1 plays an important role in the prevention of autoimmune inflammatory disorders in lacrimal glands caused by activated CD4+ Th1 cells.

Osteopontin-induced vascular hyperpermeability through tight junction disruption in diabetic retina.

Diabetic retinopathy is a major cause of blindness in developed countries, and is characterized by deterioration of barrier function causing vascular hyperpermeability and retinal edema. Vascular endothelial growth factor (VEGF) is a major mediator of diabetic macular edema. Although anti-VEGF drugs are the first-line treatment for diabetic macular edema, some cases are refractory to anti-VEGF therapy. Osteopontin (OPN) is a phosphoglycoprotein with diverse functions and expressed in various cells and tissues. Elevated OPN level has been implicated in diabetic retinopathy, but whether OPN is involved in hyperpermeability remains unclear. Using streptozotocin-induced diabetic mice (STZ mice) and human retinal endothelial cells (HRECs), we tested the hypothesis that up-regulated OPN causes tight junction disruption, leading to vascular hyperpermeability. The serum and retinal OPN concentrations were elevated in STZ mice compared to controls. Intravitreal injection of anti-OPN neutralizing antibody (anti-OPN Ab) suppressed vascular hyperpermeability and prevented decreases in claudin-5 and ZO-1 gene expression levels in the retina of STZ mice. Immunohistochemical staining of retinal vessels in STZ mice revealed claudin-5 immunoreactivity with punctate distribution and attenuated ZO-1 immunoreactivity, and these changes were prevented by anti-OPN Ab. Intravitreal injection of anti-OPN Ab did not change VEGF gene expression or protein concentration in retina of STZ mice. In an in vitro study, HRECs were exposed to normal glucose or high glucose with or without OPN for 48 h, and barrier function was evaluated by transendothelial electrical resistance and Evans blue permeation. Barrier function deteriorated under high glucose condition, and was further exacerbated by the addition of OPN. Immunofluorescence localization of claudin-5 and ZO-1 demonstrated punctate appearance with discontinuous junction in HRECs exposed to high glucose and OPN. There were no changes in VEGF and VEGF receptor-2 expression levels in HRECs by exposure to OPN. Our results suggest that OPN induces tight junction disruption and vascular hyperpermeability under diabetic conditions. Targeting OPN may be an effective approach to manage diabetic retinopathy.

Retinal changes in mice spontaneously developing diabetes by Th17-cell deviation.

Elevated level of interleukin (IL)-17, predominantly produced by T helper (Th) 17 cells, has been implicated in diabetic retinopathy (DR), but it remains unclear whether IL-17 is involved in the pathogenesis of DR. Ins2Akita (Akita) mice spontaneously develop diabetes, and show early pathophysiological changes in diabetic complications. On the other hand, interferon-γ knock out (GKO) mice exhibit high differentiation and activation of Th2 and Th17 cells as a result of Th1 cell inhibition. In this study, Ins2Akita IFN-γ-deficient (Akita-GKO) mice were established by crossbreeding Akita mice with GKO mice, and Th17-mediated immune responses on DR were investigated. Blood glucose levels (BGL) of Akita mice and Akita-GKO mice were significantly higher than those of age-matched wild type (WT) or GKO mice, and there was no significant difference in BGL between Akita and Akita-GKO mice. Relative mRNA expression of ROR-γt that is a transcriptional factor of Th17 cells but not GATA-3 that is for Th2 cells was significantly upregulated only in Akita-GKO mice compared with WT mice, and the proportions of IL-17 and IL-22-producing splenic CD4+ cells were significantly higher in Akita-GKO mice than in wild type (WT), Akita, or GKO mice. In the retina, mRNA expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) were increased in Akita-GKO mice more than in Akita or GKO mice, and statistically significant differences were observed between Akita-GKO mice and WT mice. Leukostasis in retinal vessels and ocular level of VEGF protein increased significantly in Akita-GKO mice compared with the other groups. Edematous change in the retinal surface layer, retinal exudative lesions depicted as areas of hyperfluorescence in fluorescein angiography (FA), and vascular basement membrane thickening in all layers of the retina were also observed in Akita-GKO mice at 9-week-old but not in age-matched Akita or GKO mice. These results suggested that Th17 cell-mediated immune responses might be involved in promotion of functional and morphological changes in the retina of mice spontaneously developing diabetes.

5-Aminolevulinic acid with sodium ferrous citrate induces autophagy and protects cardiomyocytes from hypoxia-induced cellular injury through MAPK-Nrf-2-HO-1 signaling cascade.

BACKGROUND: Hypoxia causes cardiac disease via oxidative stress and mitochondrial dysfunction. 5-Aminolevulinic acid in combination with sodium ferrous citrate (ALA/SFC) has been shown to up-regulate heme oxygenase-1 (HO-1) and decrease macrophage infiltration and renal cell apoptosis in renal ischemia injury mice. However, its underlying mechanism remains largely unknown. The aim of this study was to investigate whether ALA/SFC could protect cardiomyocytes from hypoxia-induced apoptosis by autophagy via HO-1 signaling. MATERIALS & METHODS: Murine atrial cardiomyocyte HL-1 cells were pretreated with ALA/SFC and then exposed to hypoxia. RESULTS: ALA/SFC pretreatment significantly attenuated hypoxia-induced cardiomyocyte apoptosis, reactive oxygen species production, and mitochondrial injury, while it increased cell viability and autophagy levels. HO-1 expression by ALA/SFC was associated with up-regulation and nuclear translocation of Nrf-2, whereas Nrf-2 siRNA dramatically reduced HO-1 expression. ERK1/2, p38, and SAPK/JNK pathways were activated by ALA/SFC and their specific inhibitors significantly reduced ALA/SFC-mediated HO-1 upregulation. Silencing of either Nrf-2 or HO-1and LY294002, inhibitor of autophagy, abolished the protective ability of ALA/AFC against hypoxia-induced injury and reduced ALA/SFC-induced autophagy. CONCLUSION: Taken together, our data suggest that ALA/SFC induces autophagy via activation of MAPK/Nrf-2/HO-1 signaling pathway to protect cardiomyocytes from hypoxia-induced apoptosis.

Autoimmune uveitis attenuated in diabetic mice through imbalance of Th1/Th17 differentiation via suppression of AP-1 signaling pathway in Th cells.

Purpose: Inflammation is involved in the pathogenesis of diabetes, however the impact of diabetes on organ-specific autoimmune diseases remains unexplored. Experimental autoimmune uveoretinitis (EAU) is a widely accepted animal model of human endogenous uveitis. In this study, we investigated the effects of diabetic conditions on the development of EAU using a mouse diabetes model. Methods: EAU was induced in wild-type C57BL/6 (WT) mice and Ins2Akita (Akita) mice with spontaneous diabetes by immunization with IRBP peptide. Clinical and histopathological examinations, and analysis of T cell activation state were conducted. In addition, alternations in the composition of immune cell types and gene expression profiles of relevant immune functions were identified using single-cell RNA sequencing. Results: The development of EAU was significantly attenuated in immunized Akita (Akita-EAU) mice compared with immunized WT (WT-EAU) mice, although T cells were fully activated in Akita-EAU mice, and the differentiation into Th17 cells and regulatory T (Treg) cells was promoted. However, Th1 cell differentiation was inhibited in Akita-EAU mice, and single-cell analysis indicated that gene expression associated AP-1 signaling pathway (JUN, FOS, and FOSB) was downregulated not only in Th1 cells but also in Th17, and Treg cells in Akita-EAU mice at the onset of EAU. Conclusions: In diabetic mice, EAU was significantly attenuated. This was related to selective inhibition of Th1 cell differentiation and downregulated AP-1 signaling pathway in both Th1 and Th17 cells.

Classification of Peripheral Blood Leukocyte Phenotypes and Serum Cytokines in Vogt-Koyanagi-Harada Disease before and after Glucocorticoid Therapy.

Vogt-Koyanagi-Harada disease (VKH) is an autoimmune disease, and glucocorticoid therapy (GC) is widely used for VKH. We provided a profile of leukocyte populations and serum cytokines in VKH patients under GC. A prospective observational study was conducted on three treatment-naïve VKH patients. Peripheral blood samples were collected from the patients before GC (VKH-acute) and after 6 months (VKH-remission), and healthy individuals were used as controls. Proportions of 37-type leukocytes and levels of 27-kind cytokines were measured by mass cytometry and multiplex bead analysis. Property similarity was analyzed using hierarchical cluster analysis. The leukocytes and cytokines were broadly classified into four and three clusters: (1) a cluster with high intensity in VKH-acute consisting of B cells, Th2-like, Th17-like, basophils, and IL-7 and IP-10; (2) a cluster with high intensity in VKH-remission composed of monocytes, neutrophils, IL-4, and TNFα; in leukocytes, (3) a cluster with low intensity in VKH-acute and -remission consisting of CD8+ T cells, Th1-like, and NKT cells; (4) a cluster with low intensity in VKH-remission composed of NK cells, Tregs, and DCs; and in cytokines, (5) a cluster with high intensities in VKH-acute and -remission comprising G-CSF, MCP-1, eotaxin, and IL-17A. These findings suggest that inflammatory composition in blood during the acute phase of VKH represents complex hyperimmune responses dominantly driven by Th and B cells.

Association between Systemic Factors and Vitreous Fluid Cytokines in Proliferative Diabetic Retinopathy.

Proliferative diabetic retinopathy (PDR) is a vision-threatening complication of diabetes mellitus (DM). Systemic and intraocular factors are intricately related to PDR, and vitreous fluid (VF) cytokines are representative intraocular biomarkers. However, the associations between systemic factors and VF cytokines and their influence on PDR pathology are unclear. This study aimed to examine the correlation between systemic factors and VF cytokines and analyze their contributions to the pathology of PDR using multivariate analyses. We conducted a retrospective observational study on 26 PDR eyes of 25 patients with type 2 DM, and 30 eyes of 30 patients with idiopathic macular hole or epiretinal membrane as controls. Fifteen systemic and laboratory tests including blood pressure (BP) and body mass index (BMI), and 27 cytokines in VF were analyzed. BP and BMI correlated positively with VF levels of IL-6 and IP-10 in PDR patients, while no significant correlation was found between systemic factors and VF cytokines in controls. MCP-1 and VEGF-A in VF separately clustered with different systemic factors in controls, but these cytokines lost the property similarity with systemic factors and acquired property similarity with each other in PDR. Systemic factors contributed to only 10.4%, whereas VF cytokines contributed to 42.3% out of 52.7% variance of the whole PDR dataset. Our results suggest that intraocular factors play a major role in the pathology of PDR, whereas systemic factors may have limited effects, and that BP and BMI control in PDR could be useful interventions to improve intraocular immune condition.

Case report: Bilateral panuveitis resembling Vogt-Koyanagi-Harada disease after second dose of BNT162b2 mRNA COVID-19 vaccine.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a serious pandemic. COVID-19 vaccination is urgent needed for limiting SARS-CoV-2 outbreaks by herd immunity. Simultaneously, post-marketing surveillance to assess vaccine safety is important, and collection of vaccine-related adverse events has been in progress. Vision-threatening ophthalmic adverse events of COVID-19 vaccines are rare but are a matter of concern. We report a 45-year-old Japanese male with positive for HLA-DR4/HLA-DRB1*0405, who developed bilateral panuveitis resembling Vogt-Koyanagi-Harada (VKH) disease after the second dose of Pfizer-BioNTech COVID-19 mRNA (BNT162b2) vaccine. Glucocorticosteroid (GC) therapy combined with cyclosporine A (CsA) readily improved the panuveitis. The immune profile at the time of onset was analyzed using CyTOF technology, which revealed activations of innate immunity mainly consisting of natural killer cells, and acquired immunity predominantly composed of B cells and CD8+ T cells. On the other hand, the immune profile in the remission phase was altered by GC therapy with CsA to a profile composed primarily of CD4+ cells, which was considerably similar to that of the healthy control before the vaccination. Our results indicate that BNT162b2 vaccine may trigger an accidental immune cross-reactivity to melanocyte epitopes in the choroid, resulting in the onset of panuveitis resembling VKH disease.

Photodynamic therapy with 5-aminolevulinic acid: A new diagnostic, therapeutic, and surgical aid for neuroblastoma.

BACKGROUND: 5-Aminolevulinic acid (ALA)-based photodynamic therapy (PDT) is widely used in cancer therapy because of the tumor-specific accumulation of photosensitizing protoporphyrin IX (PpIX). We aimed to assess the susceptibility of human neuroblastoma cell lines to ALA-PDT and determine the mechanism of PDT. METHODS: We used four human neuroblastoma cell lines (GOTO, NB9, IMR32, and NB1) and a gastric cancer cell line (MKN45) as a positive control. Cells were treated with increasing concentrations of ALA, and the ALA-induced production of PpIX in tumor cells was quantified using fluorescence spectrophotometry. PDT photocytotoxicity was measured by exposing the cells to a 630-nm irradiation for 10 min, and apoptotic cells stained with phosphatidylserine (PS) and propidium iodide (PI) were detected through flow cytometry. RESULTS: ALA cytotoxicity was not observed in any cell line. The intracellular concentration of PpIX increased in an ALA dose-dependent manner, and intracellular fluorescence of PpIX increased in a time-dependent manner. The viability of NB-1 cells treated with 250 µM 5-ALA rapidly decreased to 5%. Photocytotoxicity was observed in the following order: NB1, IMR32, NB-9, and GOTO. Photocytotoxicity was positively correlated with intracellular PpIX concentrations. PS+/PI- cells increased up to 21% after 12 h, and PS+/PI+ cells accounted for 35% of all cells after 24 h, which suggests that ALA-PDT induced apoptotic cell death. CONCLUSION: This study shows that neuroblastoma cell lines were susceptible to 5-ALA-PDT, resulting in persistent apoptotic cell death. LEVELS OF EVIDENCE: N/A for basic study.

Dimethyl Fumarate Protects Retinal Pigment Epithelium from Blue Light-Induced Oxidative Damage via the Nrf2 Pathway.

The purpose of this study is to investigate the protective effect of dimethyl fumarate (DMF), the methyl-ester of fumaric acid, against blue-light (BL) exposure in retinal pigment epithelial (RPE) cells. ARPE-19 cells, a human RPE cell line, were cultured with DMF followed by exposure to BL. Reactive oxygen species (ROS) generation, cell viability, and cell death rate were determined. Real-time polymerase chain reaction and Western blotting were performed to determine the change in nuclear factor (erythroid-derived)-like 2 (NRF2) expression. Twenty-seven inflammatory cytokines in the supernatant of culture medium were measured. BL exposure induced ROS generation in ARPE-19 cells, which DMF alleviated in a concentration-dependent manner. BL exposure increased the ARPE-19 cell death rate, which DMF alleviated. BL exposure induced ARPE-19 cell apoptosis, again alleviated by DMF. Under BL exposure, DMF increased the NRF2 mRNA level and promoted NRF2 expression in the nucleus. BL also strongly increased interleukin (IL)-1ß and fibroblast growth factor (FGF) expression. BL strongly induced RPE cell damage with apoptotic change while DMF mainly reduced inflammation in BL-induced RPE damage, resulting in blockade of cell death. DMF has a protective effect in RPE cells against BL exposure via activation of the NRF2 pathway.

Oral administration of 5-aminolevulinic acid induces heme oxygenase-1 expression in peripheral blood mononuclear cells of healthy human subjects in combination with ferrous iron.

Heme oxygenase-1 (HO-1) is a major anti-inflammatory enzyme and a key regulator that induces immune tolerance through affecting the differentiation of dendritic cells. The aim of this study is to determine whether the combination of 5-aminolevulinic acid (ALA) and iron induces HO-1 expression in healthy human peripheral blood mononuclear cells (PBMC). The study was an open labeled, non-randomized, non-placebo-controlled trial using healthy male adults and consisted of three parts. Study A aimed to find the peak HO-1 expression at 0, 1, 2, 3, 4, 6, 8, 12, 16, and 24 h after administration. Study B aimed to examine HO-1 dose dependency at 150, 300, and 600 mg of ALA and the need for iron supplementation. Study C aimed to investigate HO-1 changes during a three-day, repetitive administration of ALA and iron. The combination of ALA 600 mg and sodium ferrous citrate (SFC) 942 mg upregulated HO-1 in PBMC at 8 h after administration while sole administration of ALA or SFC was unable to induce HO-1. HO-1 in blood myeloid and plasmacytoid dendritic cells was also upregulated with ALA+SFC. Clear dose dependency of ALA+SFC was not detected, and a slight tendency towards a cumulative effect of HO-1 after three-day, repetitive administration was observed. ALA, which is already approved for use in several countries as a diagnosis agent for cancer, has the potential to become a novel therapeutic drug for diseases stemming from unwanted immune response such as autoimmune diseases and the rejection response following organ transplantation.

5-Aminolevulinic acid regulates the inflammatory response and alloimmune reaction.

5-Aminolevulinic acid (5-ALA) is a naturally occurring amino acid and precursor of heme and protoporphyrin IX (PpIX). Exogenously administrated 5-ALA increases the accumulation of PpIX in tumor cells specifically due to the compromised metabolism of 5-ALA to heme in mitochondria. PpIX emits red fluorescence by the irradiation of blue light and the formation of reactive oxygen species and singlet oxygen. Thus, performing a photodynamic diagnosis (PDD) and photodynamic therapy (PDT) using 5-ALA have given rise to a new strategy for tumor diagnosis and therapy. In addition to the field of tumor therapy, 5-ALA has been implicated in the treatment of inflammatory disease, autoimmune disease and transplantation due to the anti-inflammation and immunoregulation properties that are elicited with the expression of heme oxygenase (HO)-1, an inducible enzyme that catalyzes the rate-limiting step in the oxidative degradation of heme to free iron, biliverdin and carbon monoxide (CO), in combination with sodium ferrous citrate (SFC), because an inhibitor of HO-1 abolishes the effects of 5-ALA. Furthermore, NF-E2-related factor 2 (Nrf2), mitogen-activated protein kinase (MAPK), and heme are involved in the HO-1 expression. Biliverdin and CO are also known to have anti-apoptotic, anti-inflammatory and immunoregulatory functions. We herein review the current use of 5-ALA in inflammatory diseases, transplantation medicine, and tumor therapy.

MicroRNAs Involved in Acute Rejection and Tolerance in Murine Cardiac Allografts.

OBJECTIVES: Induction of immunologic tolerance is the ultimate goal of organ transplant. To investigate the involvement of microRNA in tolerance induction after organ transplant, murine cardiac allografts were performed and the expression of microRNA in the grafts was analyzed. MATERIALS AND METHODS: Cardiac allografts were performed using C57BL/10 (H2-Kb) to CBA/N (H2-Kk) fully mismatched combination with or without eicosapentaenoic acid for tolerance induction. Ten microRNA, mir-146a, 15b, 223, 23a, 27a, 34a, 451, 101a, 101b, 148a, discovered in hepatic grafts were examined by quantitative reverse transcription polymerase chain reaction using RNA from the cardiac allografts. RESULTS: The administration of eicosapentaenoic acid markedly prolonged the cardiac allograft survival (median survival time > 100 days) and decreased the pathological score. Quantitative reverse transcription polymerase chain reaction revealed that mir-223 was up-regulated in accordance with pathological deterioration as compared with the expression observed in the syngeneic grafts. In contrast, the other microRNA was down-regulated. Pearson product moment correlation analysis demonstrated that the expression patterns of mir-223 and mir-146a had high or moderate positive associations between the cardiac and haptic allografts in mice. CONCLUSIONS: The change in the microRNA expression in the allografts suggests that microRNA plays a role in the induction and/or maintenance of tolerance after allograft transplant. Our findings suggest that mir-223 may be associated with rejection while mir-146a, -15b, -23a, -27a, -34a, -451, -101a, -101b, -148a may be involved in tolerance. A superior grasp of the mechanism for rejection and tolerance observed in the murine heart allotransplant model may provide a better curative treatment strategy to mitigate allograft rejection.

Impaired CD98 signaling protects against graft-versus-host disease by increasing regulatory T cells.

Graft-versus-host disease (GvHD) is a major barrier to the broader use of allogenic hematopoietic stem cell transplantation for non-malignant clinical applications. A murine model of C57BL/6 to B6D2F1 acute GvHD was employed with T lymphocytes harboring a deletion of the CD98 heavy chain (CD98hc(-/-)) as donor cells. The CD98hc(-/-) resulted in lower responses to alloantigen stimulation in a mixed leukocyte reaction assay, and prevented the mortality associated with disease progression. The percentage of donor CD8 T lymphocytes was significantly decreased, while the percentage of Foxp3-positive regulatory T cells (Tregs) in recipients was increased by CD98hc(-/-). Decreased expression of FAS, FASL, ICOS, ICOSL, PD-1 and PD-L1 by donor CD8 T cells, and mRNA expression of cytotoxic T cell-related cytokines in the recipients were shown in those with CD98hc(-/-). Fewer infiltrated cells are found in the lungs, liver, tongue and skin of recipients with CD98hc(-/-) compared with the wild type recipients. Taken together, our data indicate that T cell-specific deletion of CD98hc can contribute to the prevention of GvHD development due to the attenuation of lymphocyte migration and by increasing the generation of Treg cells. These findings are expected to make it possible to develop novel approaches for the prevention of GvHD.

5-Aminolevulinic acid combined with ferrous iron enhances the expression of heme oxygenase-1.

5-Aminolevulinic acid (5-ALA) is the naturally occurring metabolic precursor of heme. Heme negatively regulates the Maf recognition element (MARE) binding- and repressing-activity of the Bach1 transcription factor through its direct binding to Bach1. Heme oxygenase (HO)-1 is an inducible enzyme that catalyzes the rate-limiting step in the oxidative degradation of heme to free iron, biliverdin and carbon monoxide. These metabolites of heme protect against apoptosis, inflammation and oxidative stress. Monocytes and macrophages play a critical role in the initiation, maintenance and resolution of inflammation. Therefore, the regulation of inflammation in macrophages is an important target under various pathophysiological conditions. In order to address the question of what is responsible for the anti-inflammatory effects of 5-ALA, the induction of HO-1 expression by 5-ALA and sodium ferrous citrate (SFC) was examined in macrophage cell line (RAW264 cells). HO-1 expression induced by 5-ALA combined with SFC (5-ALA/SFC) was partially inhibited by MEK/ERK and p38 MAPK inhibitor. The NF-E2-related factor 2 (Nrf2) was activated and translocated from the cytosol to the nucleus in response to 5-ALA/SFC. Nrf2-specific siRNA reduced the HO-1 expression. In addition, 5-ALA/SFC increased the intracellular levels of heme in cells. The increased heme indicated that the inactivation of Bach1 by heme supports the upregulation of HO-1 expression. Taken together, our data suggest that the exposure of 5-ALA/SFC to RAW264 cells enhances the HO-1 expression via MAPK activation along with the negative regulation of Bach1.

Rokitamycin induces a mitochondrial defect and caspase-dependent apoptosis in human T-cell leukemia Jurkat cells.

Macrolides are a well-known family of oral antibiotics whose antibacterial spectrum of activity covers most relevant bacterial species responsible for respiratory infectious disease. In recent years, it has been reported that macrolides have not only bactericidal activity but also direct immunomodulating activity in mammals. In this study, we observed new physiological activity of macrolides and examined whether various macrolides induce apoptosis in human leukemia cell lines. We investigated the effects of 13 different macrolides on the viability of Jurkat and HL-60 cells. Among all the macrolides used in this study, rokitamycin, a semisynthetic macrolide with a 16-member ring, effectively induced cell death. Rokitamycin induced DNA fragmentation and caspase activation, resembling the progression of apoptosis. Moreover, rokitamycin reduced the mitochondrial transmembrane potential and released cytochrome c from mitochondria to the cytosol, suggesting that mitochondrial perturbation is involved in rokitamycin-induced apoptosis. These results suggest that rokitamycin possesses not only bactericidal activity but also pro-apoptotic activity in human leukemia cells.