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BackgroundEpidermolytic ichthyosis (EI) is a major form of nonsyndromic inherited ichthyosis, characterized by erythroderma, marked hyperkeratosis and scale, bulla and erosion at birth, associated with KRT1/KRT10 mutations. The cytokine and chemokine profiles in EI are poorly understood, and specific treatment options have not been established.AimTo explore novel biomarkers and therapeutic targets in patients with EI.MethodsWe analysed cytokine levels in serum and skin samples from 10 patients with inherited ichthyosis, including seven patients with EI. Wild-type and mutant KRT1 constructs were established and transfected into HaCaT cells, an immortalized keratinocyte cell line, for in vitro immunoblotting and immunocytochemistry analyses.ResultsMultiplex cytokine/chemokine analysis revealed that 10 cytokines/chemokines [interleukin (IL)-1β, IL-4, IL-17A, IL-16, IL-18, IL-1 receptor-α, macrophage colony-stimulating factor, interferon-α2, basic fibroblast growth factor and monocyte chemotactic protein-3] were significantly increased in patients with EI. Furthermore, IL-18 levels were significantly higher in patients with EI [n = 7; 2714.1 (1438.0) pg mL−1] than in healthy controls [n = 11; 218.4 (28.4) pg mL−1, P < 0.01]. Immunohistochemical analyses showed that IL-18 expression was elevated in skin samples from patients with EI. Serum IL-18 levels correlated with the severity of ichthyosis, as measured by the Ichthyosis Scoring System. Immunoblotting analysis revealed that mature IL-18 levels were increased in the supernatant of mutant KRT1 expressing HaCaT cells. Additionally, these cells showed NLRP3 aggregation in the cytoplasm and ASC clustered around mutant keratin aggregations. These findings suggest that mutant keratin might promote the activation of the NLRP3 inflammasome and its downstream caspase-1-mediated IL-18 release in keratinocytes from patients with EI.ConclusionsOur results suggest that serum IL-18 is a severity marker released from the skin of patients with EI. Blockade of IL-18 may be a useful novel therapeutic option for patients with EI.

Tuberculosis (TB) is the archetypical chronic infection, with patients having months of symptoms before diagnosis. In the two years after successful therapy, survivors of TB have a three-fold increased risk of death.Guinea pigs were infected withGuinea pigs with TB demonstrated DNA hypermethylation and showed system-level similarity to humans with TB (In both guinea pigs and humans, TB perturbs epigenetic processes, promoting premature cellular aging and inflammation, a plausible means to explain the long-term detrimental health outcomes after TB.

Contains fulltext : 245732.pdf (Publisher’s version ) (Closed access) There is hope that host-directed therapy (HDT) for Tuberculosis (TB) can either shorten treatment duration, help cure drug resistant disease or limit the immunopathology. Many candidate HDT drugs have been proposed, however solid evidence only exists for a few select patient groups. The clinical presentation of TB is variable, with differences in severity, tissue pathology, and bacillary burden. TB clinical phenotypes likely determine the potential benefit of HDT. Underlying TB clinical phenotypes, there are TB "endotypes," defined as distinct molecular profiles, with specific metabolic, epigenetic, transcriptional, and immune phenotypes. TB endotypes can be characterized by either immunodeficiency or pathologic excessive inflammation. Additional factors, like comorbidities (HIV, diabetes, helminth infection), structural lung disease or Mycobacterial virulence also drive TB endotypes. Precise disease phenotyping, combined with in-depth immunologic and molecular profiling and multimodal omics integration, can identify TB endotypes, guide endotype-specific HDT, and improve TB outcomes, similar to advances in cancer medicine.

G protein-coupled receptors (GPCRs) transduce extracellular signals into cells by interacting with G proteins and arrestins. Emerging evidence suggests that GPCRs on the plasma membrane are in a dynamic equilibrium among monomers, dimers, and larger oligomers. Nevertheless, the role of the oligomer formation in the GPCR signal transduction remains unclear. Using multicolor single-molecule live-cell imaging, we show a dynamic interconversion between small and large oligomer states of a chemoattractant GPCR, Formyl Peptide Receptor 1 (FPR1), and its binding affinity with G protein. Full agonist stimulation increased a fraction of large FPR1 oligomers, which allowed for prolonged FPR1-G protein interaction. The G protein interaction with FPR1 was most stabilized at the full agonist-bound large FPR1 oligomers. Based on these results, we propose that G protein-mediated signal transduction may be regulated synergistically by the ligand-binding and FPR1 oligomerization. Cooperative signal control induced by receptor oligomerization is anticipated as a target for drug discovery.

In vitro, animal model and anecdotal clinical evidence suggests that tuberculosis (TB) is not a monomorphic disease, and that host response to TB is protean with multiple distinct molecular pathways and pathologies (endotypes). We used unbiased clustering to identify separate TB endotypes with distinctive gene expression patterns and different clinical outcomes. A discovery cohort that included 443 TB patients and 527 asymptomatic controls identified two TB endotypes exhibiting distinct gene expression in proliferation, metabolism, and immune pathways. Specifically, TB patient endotype A was characterized by increased expression of genes related to inflammation and immunity and decreased metabolism and proliferation; in contrast, endotype B expressed increased pathways related to metabolism and proliferation. The endotype signatures were validated using an independent RNA-seq validation cohort consisting of two studies that included 118 TB patients and 208 controls. A second validation cohort confirmed differential pathway activity between TB endotypes and demonstrated that TB patients in endotype A had slower time to culture conversion, increased risk of death, and increased risk of poor clinical outcomes. These observations suggest that endotypes reflect different functional immunity; this postulate was further supported by the observation that TB patients with the hyperinflammatory endotype showed hyporesponsive cytokine and chemokine responses. Collectively, these findings provide further evidence that metabolic and immune profiling could inform and optimize targeted endotype-specific therapies for TB.

Background Severe and chronic infections, such as sepsis and Tuberculosis, result in macrophage immune tolerance in order to prevent pathologic inflammation. Immune tolerance is believed to be a result of metabolic dysregulation, especially of the TCA cycle. Metabolites play a critical role in regulating epigenetic mechanism, but many aspects remain poorly understood. Methods An in-vitro LPS tolerance model was used to evaluate if manipulating the TCA cycle could mitigate immune tolerance. Intracellular signaling, DNA methylation, and nuclear localization were measured using RPPA, Illumina EPIC, and confocal microscopy, respectively. Results Metabolic inhibitors of glycolysis (metformin), the TCA cycle (BAY 1436032), and mTOR (rapamycin and everolimus) blocked LPS-induced immune tolerance. LPS treatment changed the methylome with hypermethylation of key genes of the TCA cycle including PDP2, NDUFS8, TRAP1 and PDK2 and lead to increased nuclear localization of the TCA enzymes, which could be ameliorated with inhibitors. Increased levels of key mediators of the TCA by LPS treatment including p-mTOR, pAKT, LDHA, IDH1, LC3A and epigenetic regulator p300 was significantly downregulated by pre-treatment with metformin, everolimus and BAY 1436032. BAY 1436032 also significantly reduced the LPS induced upregulation of epigenetic enzymes including histone deacetylases (HDAC) 3,4, and EZH2. Conclusion Previous studies demonstrated that LPS induces epigenetic-mediated immune tolerance. Here we show this can be mitigated by inhibiting glycolysis, mTOR pathway and TCA cycle. This could have clinical implications in treatment of sepsis and Tuberculosis among other diseases. Supported by NIAID K23 AI141681-02

The development of disseminated cryptococcosis has historically occurred in patients living with advanced human immunodeficiency virus or other immunosuppressive conditions affecting T-cell function. Recently, patients with anti-cytokine neutralising autoantibodies have been recognised to be at risk for disseminated infections by opportunistic intracellular pathogens, including Cryptococcus species. Herein, we present a previously healthy 26-year-old man who was evaluated with disseminated cryptococcosis involving the bone, lung, mediastinum and brain. The patient's serum cryptococcal antigen titres were >1:1,100,000, and evaluation for an underlying immunodeficiency revealed high titres for anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies. We also review the literature of all published cases of disseminated cryptococcosis associated with the presence of anti-GM-CSF autoantibodies. Clinicians should have a heightened awareness of anti-cytokine autoantibodies in patients without a known immunodeficiency and development disseminated infections by opportunistic intracellular pathogens.

Contains fulltext : 220633.pdf (Publisher’s version ) (Closed access) Mycobacterium tuberculosis (M. tuberculosis) has coevolved with humans for millennia and developed multiple mechanisms to evade host immunity. Restoring host immunity in order to improve outcomes and potentially shorten existing therapy will require identification of the full complement by which host immunity is inhibited. Perturbation of host DNA methylation is a mechanism induced by chronic infections such as HIV, HPV, lymphocytic choriomeningitis virus (LCMV), and schistosomiasis to evade host immunity. Here, we evaluated the DNA methylation status of patients with tuberculosis (TB) and their asymptomatic household contacts and found that the patients with TB have DNA hypermethylation of the IL-2/STAT5, TNF/NF-kappaB, and IFN-gamma signaling pathways. We performed methylation-sensitive restriction enzyme-quantitative PCR (MSRE-qPCR) and observed that multiple genes of the IL-12/IFN-gamma signaling pathway (IL12B, IL12RB2, TYK2, IFNGR1, JAK1, and JAK2) were hypermethylated in patients with TB. The DNA hypermethylation of these pathways was associated with decreased immune responsiveness with decreased mitogen-induced upregulation of IFN-gamma, TNF, IL-6, CXCL9, CXCL10, and IL-1beta production. The DNA hypermethylation of the IL-12/IFN-gamma pathway was associated with decreased IFN-gamma-induced gene expression and decreased IL-12-inducible upregulation of IFN-gamma. This study demonstrates that immune cells from patients with TB are characterized by DNA hypermethylation of genes critical to mycobacterial immunity resulting in decreased mycobacteria-specific and nonspecific immune responsiveness.

Safe and efficient therapeutic agents for bone diseases are required in natural sources. We previously found that edible seaweed-derived polysaccharide porphyran exhibited anti-inflammatory effects through the down regulation of nuclear factor-κB. The aim of this study was to investigate the availability of porphyran as a therapeutic agent for bone diseases. The effects of porphyran on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells were examined. Porphyran suppressed RANKL-induced osteoclast formation in a concentration-dependent manner (6.25?50 μg/ml) without any cytotoxic effects. Furthermore, real-time polymerase chain reaction analyses indicated that porphyran at 50 μg/ml significantly attenuated the RANKL-induced increase in the mRNA levels of osteoclastogenesis-related marker genes such as nuclear factor of activated T cells, tartrate-resistant acid phosphatase, cathepsin K, and matrix metalloproteinase-9 in RAW264.7 cells. To our knowledge, this is the first report showing that edible-seaweed-derived polysaccharide porphyran can suppress RANKL-induced osteoclastogenesis. Our results suggest that porphyran can be used as a safe therapeutic agent to improve osteoclast-related pathological conditions., Phytotherapy Research, 32(3), pp.452-458; 2018

Enzymatically prepared alginate oligomer (AO) promoted the growth of Chlamydomonas reinhardtii in a concentration-dependent manner. AO at 2.5 mg/mL induced increase in expression levels of cyclin A, cyclin B, and cyclin D in C. reinhardtii. CuSO4 at 100 μM suppressed the growth of C. reinhardtiin, and AO at 2.5 mg/mL significantly alleviated the toxicity of CuSO4. Increased intracellular reactive oxygen species level in C. reinhardtii induced by CuSO4 was reduced by AO. After cultivation with CuSO4 at 100 μM, expression levels of ascorbate peroxidase and superoxide dismutase in C. reinhardtii were increased, and AO reduced the increased levels of these enzymes. These results suggest that AO exhibits beneficial effects on C. reinhardtii through influencing the expression of various genes not only at normal growth condition but also under CuSO4 stress.

Porphyran, a sulfated polysaccharide, isolated from discolored nori (Porphyra yezoensis) (dc-porphyran) and one fraction (F1) purified from dc-porphyran by DEAE-chromatography showed the protective effects on LPS-induced endotoxin shock in mice. Intraperitoneal (i.p.) treatment with dc-porphyran or F1 (100 mg/kg) 60 min prior to i.p. injection of LPS (30 mg/kg) completely protected mice from LPS lethality. At 10 mg/kg concentration, F1 demonstrated more protection than dc-porphyran. Intravenous (i.v.) challenge of LPS, even at 20 mg/kg, was more lethal than i.p. administration; i.v. injection of F1 (100 mg/kg) with LPS significantly improved the survival rate. However, i.v. dc-porphyran (100 mg/kg) produced an even lower survival rate than that of LPS alone. We examined pro-inflammatory mediators such as NO and TNF-α in serum. F1 significantly reduced the levels of these markers. Additionally, F1 significantly decreased the malondialdehyde level in the liver, a marker of oxidative stress, while dc-porphyran had almost no effect. Furthermore, F1 significantly decreased the production of TNF-α and NO in peritoneal exudate cells harvested from LPS-challenged mice, while dc-porphyran treatment showed a lesser decrease. Our results suggest that porphyran isolated from discolored nori, especially F1, is capable of suppressing LPS-induced endotoxin shock in vivo., International Journal of Biological Macromolecules, 93(Part A), pp.1273-1278; 2016

Mycobacterium tuberculosis (Mtb) has co-evolved with humans for millennia and developed multiple mechanisms to evade host immunity. Restoring host immunity in order to shorten existing therapy and improve outcomes will require identifying the full complement by which host immunity is inhibited. Perturbing host DNA methylation is a mechanism induced by chronic infections such as HIV, HPV, LCMV and schistosomiasis to evade host immunity. Here, we evaluated the DNA methylation status of TB patients and their asymptomatic household contacts demonstrating that TB patients have DNA hyper-methylation of the IL-2-STAT5, TNF-NF-ϰB and IFN-γ signaling pathways. By MSRE-qPCR, multiple genes of the IL-12-IFN-γ signaling pathway (IL12B, IL12RB2, TYK2, IFNGR1, JAK1 and JAK2) were hyper-methylated in TB patients. The DNA hyper-methylation of these pathways is associated with decreased immune responsiveness with decreased mitogen induced upregulation of IFN-γ, TNF, IL-6 and IL-1β production. The DNA hyper-methylation of the IL-12-IFN-γ pathway was associated with decreased IFN-γ induced gene expression and decreased IL-12 inducible up-regulation of IFN-γ. This work demonstrates that immune cells from TB patients are characterized by DNA hyper-methylation of genes critical to mycobacterial immunity resulting in decreased mycobacteria-specific and non-specific immune responsiveness.

Mortality associated with Staphylococcus aureus infection remains high during the sub-acute phase of burn injury. In this study, we aimed to improve antibacterial resistance of sub-acutely burned mice through macrophage polarization. Sepsis did not develop in mice at the sub-acute phase of 5% total body surface area (TBSA) burn after being infected with methicillin-resistant S. aureus (MRSA), and M1 macrophages (interleukin (IL)-10-IL-12+ inducible nitric oxide synthase+ Mφ) were isolated from these mice. In contrast, predominantly M2b macrophages (C-C motif chemokine ligand 1 (CCL1)+IL-10+IL-12- Mφ) were isolated from mice with >15% TBSA burn, and all of these mice died after the same MRSA infection. Comparing NOD scid gamma mice inoculated with Mφ with 25% TBSA burns, all mice treated with CCL1 antisense oligodeoxynucleotide (ODN) survived after MRSA infection, whereas all untreated mice given the same infection died within 4 days. CCL1 antisense ODN has been characterized as a specific polarizer of M2bMφ. M1Mφ were isolated from MRSA-infected mice with 25% TBSA burn after treatment with CCL1 antisense ODN, and these mice were shown to be resistant against a lethal dose of MRSA infection. M1Mφ were also isolated from 25% TBSA-burned mice infected with MRSA when the ODN was administered therapeutically, and subsequent sepsis was effectively controlled in these mice. These results indicate that the M2bMφ polarizer is beneficial for controlling MRSA infection in mice at the sub-acute phase of severe burn injury.

M2b macrophages (Mφ) play a major role in the increased susceptibility of subacutely burned patients, to sepsis stemming from enterococcal translocation. Certain opportunistic infections in severely burned mice have been controlled by murine CCL1 antisense oligodeoxynucleotide (ODN), a specific polarizer of mouse M2bMφ. In the present study, we have screened CCL1 antisense ODN, which is active against human M2bMφ. Among the 20 CCL1 antisense ODNs synthesized in our laboratory, HCA-11 was shown to be the most active polarizer for human CCL1+CD163+CD14+ cells. Burn patient CCL1+CD163+CD14+ cells (3 × 105 cells/mL) switched to quiescent CCL1−CD163−CD14+ cells within 48 h in cultures supplemented with 100 μg/mL of HCA-11. After treatment with a 25 μg/chimera dose of HCA-11, the bacterial growth was not observed in various organs of patient chimeras (γNSG mice inoculated with burn patient WBCs) infected with a lethal dose of Methicillin-resistant Staphylococcus aureus. The host antibacterial defenses against c...

Chattonella antiqua isolated in 2010 showed extremely more potent fish-killing activities against red sea bream, Japanese horse mackerel, and blue damselfish than those of Chattonella marina isolated in 1985. Chemiluminescence and electron spin resonance (ESR) analyses suggested greater reactive oxygen species (ROS)-producing activity of C. antiqua than that of C. marina. Sodium benzoate, a hydroxyl radical scavenger, significantly suppressed the fish-killing activity of C. antiqua on blue damselfish. The chlorophyll level in the gill tissue of blue damselfish exposed to flagellate cells increased along with the exposure time, and the cell count of gill-associated C. antiqua estimated with chlorophyll level was higher than that of C. marina. These results suggest that the ROS-producing activity and affinity of Chattonella cells to the gill surface may be important factors influencing the fish-killing activity of Chattonella species.

Chronic alcohol consumption markedly impairs host antibacterial defense against opportunistic infections. γ-irradiated NOD-SCID IL-2Rγnull mice inoculated with nonalcoholic PBMCs (control PBMC chimeras) resisted Klebsiella pneumonia and gut bacteria-associated sepsis, whereas the chimeras created with alcoholic PBMCs (alcoholic PBMC chimeras) were very susceptible to these infections. M1 monocytes (IL-12+IL-10−CD163−CD14+ cells), major effector cells in antibacterial innate immunity, were not induced by a bacterial Ag in alcoholic PBMC cultures, and M2b monocytes (CCL1+CD163+CD14+ cells), which predominated in alcoholic PBMCs, were shown to be inhibitor cells on the Ag-stimulated monocyte conversion from quiescent monocytes to M1 monocytes. CCL1, which functions to maintain M2b macrophage properties, was produced by M2b monocytes isolated from alcoholic PBMCs. These M2b monocytes reverted to quiescent monocytes (IL-12−IL-10−CCL1−CD163−CD14+ cells) in cultures supplemented with CCL1 antisense oligodeoxynucleotide, and the subsequent quiescent monocytes easily converted to M1 monocytes under bacterial Ag stimulation. Alcoholic PBMC chimeras treated with CCL1 antisense oligodeoxynucleotide were resistant against pulmonary infection by K. pneumoniae and sepsis stemming from enterococcal translocation. These results indicate that a majority of monocytes polarize to an M2b phenotype in association with alcohol abuse, and this polarization contributes to the increased susceptibility of alcoholics to gut and lung infections. Bacterial pneumonia and gut bacteria-associated sepsis, frequently seen in alcoholics, can be controlled through the polarization of macrophage phenotypes.

BACKGROUND Less than 10% of individuals infected with Mtb develop disease. Similarly, in the pre-antibiotic era, ~20% of Tuberculosis (TB) patients overcame the disease. This implies host immunity can control Mtb infection. We evaluated the role of cytotoxic immune cells (CD8, CD56 and CD3+CD56+) among individuals with chronic infections that increase risk of TB progression. METHODS DNA Methyl EPIC was evaluated in patients with helminths, HIV and TB (n = 32) and flow-based immune phenotyping was implemented on 108 individuals. Gene expression of 717 TB patients and 525 healthy controls was downloaded from publicly available databases. RESULTS All evaluated cytotoxic cells displayed DNA methylation perturbations in the IL-2-STAT5, PI3K and IFNγ signaling pathways. TB patients experienced DNA hyper-methylation of perforin in CD56 NK cells and in granzyme A and granzyme B of CD8+ cytotoxic lymphocytes (CTLs). By flow cytometry TB patients up-regulated mycobacterial-specific perforin in CTLs while CD3−CD56+ NK cells expressed decreased perforin. Early evidence suggests that the increased perforin expression from CTLs was heterogenous and only occurs in ~46% of TB patients. Further evidence for heterogenous TB sub-groups was demonstrated by gene expression as 333 TB patients had increased perforin gene expression, while 313 had decreased gene expression (log2 fold change with FDR < 1%). CONCLUSION To evaluate the DNA methylation-immune phenotype associations from this clinical cohort, mechanistic studies manipulating DNA methylation status are undergoing. Identifying how epigenetic modifications alter cytotoxic cells capacity to kill intracellular mycobacteria is critical to future host directed therapeutic options.

Epigenetic mechanisms, like DNA methylation, determine immune cell phenotype. To understand the epigenetic alterations induced by helminth co-infections, we evaluated the longitudinal effect of ascariasis and schistosomiasis infection on CD4+ T cell DNA methylation and the downstream tuberculosis (TB)-specific and BCG-induced immune phenotype. All experiments were performed on human primary immune cells from a longitudinal cohort of recently TB-exposed children. Compared to age-matched uninfected controls, children with active Schistosoma haematobium and Ascaris lumbricoides infection had 751 differentially DNA methylated genes with 72% hyper-methylated. Gene ontology pathway analysis identified inhibition of IFN-γ signaling, cellular proliferation, and the Th1 pathway. Targeted RT-PCR after methyl-specific endonuclease digestion confirmed DNA hyper-methylation of the transcription factors BATF3, ID2, STAT5A, IRF5, PPARg, RUNX2, IRF4 and NFATC1 and cytokines or cytokine receptors IFNGR1, TNFS11, RELT (TNF receptor), IL12RB2 and IL12B (p< 0.001; Sidak-Bonferroni). Functional blockage of the IFN-γ signaling pathway was confirmed with helminth-infected individuals having decreased up-regulation of IFN-γ-inducible genes (Mann-Whitney p < 0.05). Hypo-methylation of the IL-4 pathway and DNA hyper-methylation of the Th1 pathway was confirmed by antigen-specific multidimensional flow cytometry demonstrating decreased TB-specific IFN-γ and TNF and increased IL-4 production by CD4+ T cells (Wilcoxon signed rank P

Polysaccharides prepared from marine algae sometimes contain contaminants such as polyphenols and endotoxins that may mislead their bona fide biological activities. In this study, we examined bioactive contaminants in commercially available fucoindan from Fucus vesiculosus, along with ascophyllan and fucoidan from Ascophyllum nodosum. F. vesiculosus fucoidan inhibited the growth of Vibrio alginolyticus in a concentration-dependent manner (0-1,000 μg mL?1). However, the antibacterial activity of the fucoidan significantly reduced after methanol-extraction, and the methanol-extract showed a potent antibacterial activity. The extract also showed cytotoxicity to RAW264.7 and U937 cells, and induced apoptotic nuclear morphological changes in U937 cells. These results suggest that the antibacterial activity of the fucoidan is partly due to the methanol-extractable contaminants that can also contribute to the cytotoxicity on RAW264.7 and U937 cells. On the other hand, the activities to induce secretion of nitric oxide and tumor necrosis factor-α from RAW264.7 cells were observed in the fucoidan even after methanol extraction, and the extract had no such activities. Our observations suggest that commercially available fucoidan should be purified prior to biochemical use., ALGAE, 29(3), pp.237-247; 2014

In the pre-antibiotic era, ~20% of Tuberculosis (TB) patients overcame the disease without therapy, implying host immunity can sometimes control disease. The risk of disease progression is higher among HIV-infected individuals and children, therefore we evaluate how HIV and age alter the balance of cytotoxic versus immune regulatory NKT immune phenotype in individuals with TB and latent Mycobacterium tuberculosis infection (LTBi). We evaluated the NKT immune response from 49 individuals with TB (25 HIV-infected) and 48 with asymptomatic LTBi (14 HIV-infected). Using flow cytometry based multi-dimensional immune profiling, we analyzed the expression level of previously described determinants of NKT cytotoxicity (CD3dim) and immune regulatory (CD3bright) phenotype and function. CD3bright NKT cell population was correlated with age during children TB (p = 0.002). This cell type had increased in adults with TB (p = 0.034), but not in children. Also, high level of perforin producing CD3bright NKT cells had increased in adults with TB compare to adults with LTBi (p = 0.004). On the other hand, CD3dim NKT cell had no age correlation nor difference between TB and LTBi participants. While HIV-infected LTBi individuals had decreased perforin production in CD3dim NKT cells (p = 0.012). CD3bright and CD3dim NKT sub-populations previously reported influence regulatory versus cytotoxic function. We demonstrate that age and HIV status of participants have effected to regulatory and cytotoxic function, respectively. This suggested that these two factors have different mechanisms, decreasing perforin high producing CD3bright NKT cells and decreasing perforin production in CD3dim NKT, which promote TB disease progression.

BACKGROUND Chronic antigen stimulation from HIV, HPV, LCMV and schistosomiasis induce epigenetic-mediated immune exhaustion. Individuals with successful anti-TB therapy (ATT) have a thirteen-fold increased risk of developing recurrent TB. METHODS From a cohort of HIV infected and uninfected individuals with TB and their asymptomatic household contacts (n=32), genome-wide DNA methylation (DNA MethylEPIC) and epigenetic deconvolution (EDEC) identified cell-specific DNA methylation. Cell-specific results were validated with MSRE-PCR, gene expression and flow cytometry-based Mtb-specific multi-dimensional immune profiling. RESULTS Participants with TB had DNA hypermethylation in the IL-2, TCR and IFN-γ signalling pathways. The IFN-γ signalling pathway was affected in both innate (NK cells and monocytes) as well as adaptive (CD4 and CD8 T cells) lineages. Select DNA hyper-methylated differences were validated using MSRE-PCR. Pioneer and Transcription factors critical for cell-mediated immunity were also significantly differentially methylated with enrichment scores compatible with previous epigenetic-mediated immune exhaustion. After overnight stimulation with IFN-γ, TB patients have decreased IFN-γ-inducible gene expression. Six months after the completion of successful ATT, participants with TB have persistent DNA hypermethylation. DISCUSSION Persistent DNA hypermethylation may be a potential mechanism by which individuals with TB are at increased risk of recurrent disease. This may also assist in identifying potential new targets for host-directed therapy.

To investigate the role of sulfate groups on the macrophage-stimulating activities of ascophyllan, we prepared desulfated ascophyllan, and its effects on RAW264.7 cells were compared with native ascophyllan. The chemical structural analysis revealed that nearly 21% of sulfate groups of ascophyllan were removed by desulfation reaction, while no significant changes in the molecular mass and monosaccharide composition occurred after desulfation. NO- and cytokine- (TNF-α and G-CSF) inducing activities of the desulfated ascophyllan on RAW264.7 cells were significantly decreased as compared to native ascophyllan. Furthermore, the activity of desulfated ascophyllan to induce reactive oxygen species (ROS) generation from RAW264.7 cells decreased to almost negligible level. Our results suggest that the level of sulfate groups of ascophyllan is an important structural element responsible for the macrophage-stimulating activities. Probably, even the limited removal of sulfate residues sensitive to desulfation reaction may result in significant decrease in the bioactivities of ascophyllan.

M2b macrophages (Mφ) play a major role in the increased susceptibility of subacutely burned patients, to sepsis stemming from enterococcal translocation. Certain opportunistic infections in severely burned mice have been controlled by murine CCL1 antisense oligodeoxynucleotide (ODN), a specific polarizer of mouse M2bMφ. In the present study, we have screened CCL1 antisense ODN, which is active against human M2bMφ. Among the 20 CCL1 antisense ODNs synthesized in our laboratory, HCA-11 was shown to be the most active polarizer for human CCL1+CD163+CD14+ cells. Burn patient CCL1+CD163+CD14+ cells (3 × 105 cells/mL) switched to quiescent CCL1−CD163−CD14+ cells within 48 h in cultures supplemented with 100 μg/mL of HCA-11. After treatment with a 25 μg/chimera dose of HCA-11, the bacterial growth was not observed in various organs of patient chimeras (γNSG mice inoculated with burn patient WBCs) infected with a lethal dose of Methicillin-resistant Staphylococcus aureus. The host antibacterial defenses against certain opportunistic pathogens should be improved in severely burned patients treated with a human CCL1 antisense ODN, HCA-11.

A harmful dinoflagellate, Heterocapsa circularisquama, is highly toxic to shellfish and the zooplankton rotifer Brachionus plicatilis. A previous study found that H. circularisquama has both light-dependent and -independent haemolytic agents, which might be responsible for its toxicity. Detailed analysis of the haemolytic activity of H. circularisquama suggested that light-independent haemolytic activity was mediated mainly through intact cells, whereas light-dependent haemolytic activity was mediated by intracellular agents which can be discharged from ruptured cells. Because H. circularisquama showed similar toxicity to rotifers regardless of the light conditions, and because ultrasonic ruptured H. circularisquama cells showed no significant toxicity to rotifers, it was suggested that live cell-mediated light-independent haemolytic activity is a major factor responsible for the observed toxicity to rotifers. Interestingly, the ultrasonic-ruptured cells of H. circularisquama suppressed their own lethal effect on the rotifers. Analysis of samples of the cell contents (supernatant) and cell fragments (precipitate) prepared from the ruptured H. circularisquama cells indicated that the cell contents contain inhibitors for the light-independent cell-mediated haemolytic activity, toxins affecting H. circularisquama cells themselves, as well as light-dependent haemolytic agents. Ethanol extract prepared from H. circularisquama, which is supposed to contain a porphyrin derivative that displays photosensitising haemolytic activity, showed potent toxicity to Chattonella marina, Chattonella antiqua, and Karenia mikimotoi, as well as to H. circularisquama at the concentration range at which no significant toxicity to rotifers was observed. Analysis on a column of Sephadex LH-20 revealed that light-dependent haemolytic activity and inhibitory activity on cell-mediated light-independent haemolytic activity existed in two separate fractions (f-2 and f-3), suggesting that both activities might be derived from common compounds. Our results suggest that the photosensitising haemolytic toxin discharged from ruptured H. circularisquama cells has a relatively broad spectrum of phytoplankton toxicity, and that physical collapse of H. circularisquama cells can lead not only to the disappearance of its own toxicity, but also to mitigation of the effects of other HABs.

Luciferase, a bioluminescent protein, has been used as an analytical tool to visualize intracellular phenomena. Luciferase with red light emission is particularly useful for bioluminescence imaging because of its high transmittance in mammalian tissues. However, the luminescence intensity of existing luciferases with their emission over 600 nm is insufficient for imaging studies because of their weak intensities. We developed mutants of Emerald luciferase (Eluc) from Brazilian click beetle (Pyrearinus termitilluminans), which emits the strongest bioluminescence among beetle luciferases. We successively introduced four amino acid mutations into the luciferase based on a predicted structure of Eluc using homology modeling. Results showed that quadruple mutations R214K/H241K/S246H/H347A into the beetle luciferase emit luminescence with emission maximum at 626 nm, 88-nm red-shift from the wild-type luciferase. This mutant luciferase is anticipated for application in in vivo multicolor imaging in living samples.

In a murine model of γ-irradiation, bacterial translocation and subsequent sepsis have been effectively controlled by CCL1 antisense ODN, a M2b macrophage reverter. In the present study, we tried to obtain human CCL1 antisense ODN which is inhibitory on human M2b macrophages. Twenty human CCL1 antisense ODNs were synthesized based on the prediction of CCL1 mRNA secondary structure, calculation of GC content, and binding energy of ODNs. To evaluate the activity of these ODNs, an amount of CCL1 in culture fluids of M2b monocytes cultured with the ODNs was determined by ELISA. M2b monocytes were generated in vitro from healthy donor peripheral blood monocytes after stimulation with IL-1β and IgG in combination. In the results, 2 ODNs (HCA-15 ; CAACATCTGGAGAAGGGT : HCA-17 ; GGGCTGTGGTGATGATC), at doses of 20 and 100 µg/ml, suppressed 70-80% or more of CCL1 production by M2b monocytes. Also, decreased expression of IL-10 and CD163 was shown in M2b monocytes cultured with HCA-15 and HCA-17. The other 18 ODNs were not significantly active on the M2b macrophage reversion. These results suggest that HCA-15 and HCA-17 may be beneficial in controlling bacterial translocation and subsequent sepsis in patients with γ-irradiation.

In previous studies, sepsis stemming for E. faecalis translocation and MRSA local infection has been greatly controlled in SAB-mice prophylactically treated with CCL1 antisense ODN. Effector cells (M1Mφ) in host antibacterial immunities are isolated from the infected SAB-mice after treatment with the ODN. In this study, the antibacterial resistance of SAB-mice therapeutically treated with the ODN was examined against infections with E. faecalis and MRSA. Mice 2 weeks after flame burn injury (3rd degree 25% TBSA) were infected with MRSA (106 CFU/mouse, i.v.). Decontaminated mice 2 weeks after burn injury were infected with E. faecalis (8 x 106 CFU/mouse, p.o.). These mice were therapeutically treated with the ODN (s.c., 10 mg/mouse) at various schedules starting 12 h after the infection. The antibacterial efficacies of the ODN were evaluated by reducing bacterial growth in blood and organs as compared to those of control mice. In the results, bacteria grew in the blood (>105 CFU/ml), liver (>106 CFU/gram), spleen (>105 CFU/gram), and kidneys (>107 CFU/gram) of control mice 3 to 5 days after MRSA infection. However, significant growth of the pathogen in their organs was not demonstrated in SAB-mice treated with the ODN 12, 24, and 36 h after the infection. Similar results were obtained in SAB-mice orally infected with E. faecalis. These results indicate that infections with MRSA and E. faecalis in SAB-mice are controllable by the therapeutic treatment with CCL1 antisense ODN.

Gut bacteria-associated sepsis is a serious concern in persons who have radiation-induced injuries in the gastrointestinal system. Such infection does not normally occur in healthy hosts with M1Mϕ. However, M1Mϕ are not generated in γ-irradiated mice (WBI-mice), even though Mϕ are still present. WBI-mice are carriers of M2bMϕ, which inhibit Mϕ conversion from resident Mϕ to M1Mϕ. We have previously reported that sepsis stemming from Enterococcus faecalis oral infection is markedly mitigated in 5 Gy WBI-mice after M2bMϕ polarization using CCL1 antisense ODN. In this study, we examined the effect of CCL1 antisense ODN on spontaneous bacterial translocation and subsequent sepsis in 8 Gy WBI-mice. The ODN was administered (s.c., twice daily) to 8 Gy WBI-mice for 1 week starting 3 days after γ-irradiation. The efficacy of the treatment was evaluated by an increase in the percentage of survival in test groups, as compared with that of control groups. All control mice died within 22 days of γ-irradiation. However, 70% of the WBI-mice treated with 25 to 125 μg/mouse of the ODN survived more than 30 days after γ-irradiation. Severe bacteremia and bacterial growth in MLNs were not seen in WBI-mice treated with the ODN. M1Mϕ were isolated from MLNs of these mice, while M2bMϕ were not. These results indicate that the Mϕ polarization therapy using CCL1 antisense ODN is effective to mitigate spontaneous bacterial translocation and subsequent sepsis in mice exposed to 8 Gy of γ-irradiation.

Bacterial translocation from microbiota causes serious sepsis in mice with chronic alcohol consumption (CAC-mice). Since innate lymphoid cells (ILCs) are cells to be involved in the prosperity and decoy of host antibacterial innate immunities, in this study, we tried to isolate ILCs from intestinal mucosal tissues of CAC-mice with various durations of alcohol consumption. CAC-mice were prepared by an oral gavage of 0.6 ml/day of 20% EtOH for 4 to 14 weeks. Lineage negative (lin-) cells were isolated from the intestinal mucosal tissue of CAC-mice. These cells were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 6 hours. Obtained cells were analyzed for ILC2 and ILC3 by flow cytometry, and culture fluids of lin- cells were assayed for IL-22 and IL-17A by ELISA. In the results, the percentage of CD45+CD90+RORγt+ cells (ILC3) in the lin- cell population was gradually increased in mice with alcohol consumption for 10-12 weeks (7.84% in average), as compared to be percentage of ILC3 in normal mice (0.67%). On the other hand, CD127+ICOS+Sca-1+ST2+ cells (ILC2) were minimally isolated from the lin- cell population of 4-12 week CAC-mice. Lin- cells from 12 week CAC-mice produced 400 pg/ml of IL-22 and 1.7 ng/ml of IL-17A. These results indicate that 10-12 week CAC-mice are carriers of ILC3 which play a role in the increased susceptibility of CAC-mice to enterococcal translocation and subsequent sepsis.

Radiation victims who suffer from gastrointestinal injuries are extremely susceptible to gut-associated sepsis. In our previous studies, M1Mφ located in the lamina propria and mesenteric lymph nodes were shown to be major effector cells against bacterial translocation. However, M1Mφ were not generated in these organs of WBI-mice who are carriers of M2bMφ (inhibitor cells for Mφ conversion from resident Mφ to M1Mφ). CCL1 produced by M2bMφ is essential for the maintenance of M2bMφ properties. In this study, we examined the effect of CCL1 antisense ODN on gut-associated sepsis stemming from endogenous intestinal microflora in 9 Gy WBI-mice. CCL1 antisense ODN was administered s.c. to WBI-mice twice a day for 1 week starting 3 days post-exposure to γ-rays. This group of mice was designated as treated mice. The mortality rates and bacterial growth in the organs and blood of treated mice were compared to those of control mice (WBI-mice treated with scrambled ODN). In the results, control mice died within 14 days of γ-irradiation, while 30% of treated mice survived more than 30 days after the irradiation. Bacteria progressively grew in the mesenteric lymph nodes, liver and blood of control mice, while the pathogens were not isolated significantly from these organs of treated mice. These results indicate that sepsis stemming from spontaneous bacterial translocation is noticeably mitigated in WBI-mice treated with CCL1 antisense ODN.

Sub-acutely burned mice are shown to be carriers of M2bMφ, these cells are characterized as inhibitor cells for Mφ conversion from resident Mφ to M1Mφ. CCL1 released from M2bMφ has been identified as an essential chemokine for the maintenance of M2bMφ properties, and M2bMφ derived from sub-acutely burned mice have reverted to resident Mφ after cultivation with CCL1 antisense ODN. In this study, we tried to determine the effect of CCL1 antisense ODN on M1Mφ generation in sub-acutely burned mice infected with MRSA. Two weeks after burn injury, the mice were infected i.v. with 105 CFU/mouse of MRSA, and then, treated with the ODN (s.c., twice a day, 10 μg/mouse). Two days after the infection, F4/80+ cells were isolated from the peritoneal cavities of the mice and assayed for IL-12+ cells. Also, the killing activity of these cells against MRSA was tested in vitro. In the results, a majority of Mφ from normal mice infected with MRSA were shown to be IL-12+ cells (>82%), while 10% or less of Mφ from burn mice infected with the pathogen were IL-12+ cells. However, a majority of Mφ isolated from burn mice infected with MRSA and treated with the ODN were shown to be IL-12+ cells. A majority of Mφ from burn mice infected with MRSA were not bactericidal, while Mφ preparations from the same mice additionally treated with the ODN were bactericidal. These results indicate that M1Mφ are induced after treatment with CCL1 antisense ODN in sub-acutely burned mice infected with MRSA.

Staphylococcus aureus is frequently isolated from subacutely burned patients, and these infections easily develop into severe sepsis. M2bMφ have been characterized as the responsible cells on the increased susceptibility of SB-mice to staphylococcal infections, and SB-mice are carriers of M2bMφ. In this study, we tried to protect SB-mice (mice 10 to 30 days after burn injury) from a lethal dose infection of MRSA using CCL1 antisense ODN (an inhibitor of M2bMφ). Mice, 14 days after burn injury (20% total body surface area 3rd degree burns), were infected with 2 x 106 CFU/mouse of MRSA. Then, these mice were treated s.c. with 10 μg/mouse of CCL1 antisense ODN twice a day for 3 days (treated mice). As control mice, SB-mice were treated with scrambled ODN. The protective effect of the ODN against MRSA infection was evaluated by a reduction in the mortality rates of treated mice as compared with that of control mice. Also, bacterial growth in various organs of treated mice was compared with those of controls. In the results, all control mice died within 7 days of MRSA infection, while all treated mice were still alive after 7 days with the same infection. Bacteria grew in the liver, spleen and kidneys of control mice. However, the pathogen did not grow significantly in these organs of treated mice. These results indicate that sepsis caused by a lethal dose of MRSA infection in SB-mice is controlled by treatment with CCL1 antisense ODN.

An essential host defense against gut-associated sepsis is a local antibacterial innate immunity mainly expressed by M1Mφ (IL-10-IL-12+ Mφ with marked bactericidal activity). Although Mφ are radioresistant, M1Mφ are not generated in bacterial translocation sites (lamina propria and mesenteric lymph nodes) of WBI-mice. M2bMφ (IL-10+IL-12-CCL1+ Mφ) appearing in response to radiation injuries inhibit Mφ conversion from resident Mφ to M1Mφ. CCL1 has been characterized as an essential chemokine for the maintenance of M2bMφ properties, and M2bMφ are shown to be major CCL1 producer cells. In this study, we tried to eliminate M2bMφ and induce M1Mφ in the bacterial translocation sites of WBI-mice by treatment with CCL1 antisense ODN. The ODN was administered s.c. to WBI-mice twice a day for 1 week starting 3 days after γ-irradiation (8-11 Gy). One day after the final treatment, these mice were stimulated orally with 107 heat-killed E. faecalis. Then, F4/80+ Mφ were isolated from the bacterial translocation sties of these mice and tested for their M1Mφ and M2bMφ properties. In the results, M1Mφ were not detected in the translocation sites of WBI-mice stimulated with the antigen. However, M1Mφ were isolated from WBI-mice treated with the ODN and stimulated with the antigen. M2Mφ were not isolated significantly from these mice. These results indicate that M1Mφ are inducible by the bacterial antigen in 8-11 Gy WBI-mice after treatment with CCL1 antisense ODN.