Mycobacterium tuberculosis Induces Irg1 in Murine Macrophages by a Pathway Involving Both TLR-2 and STING/IFNAR Signaling and Requiring Bacterial Phagocytosis.

Irg1 is an enzyme that generates itaconate, a metabolite that plays a key role in the regulation of inflammatory responses. Previous studies have implicated Irg1 as an important mediator in preventing excessive inflammation and tissue damage in Mycobacterium tuberculosis (Mtb) infection. Here, we investigated the pattern recognition receptors and signaling pathways by which Mtb triggers Irg1 gene expression by comparing the responses of control and genetically deficient BMDMs. Using this approach, we demonstrated partial roles for TLR-2 (but not TLR-4 or -9), MyD88 and NFκB signaling in Irg1 induction by Mtb bacilli. In addition, drug inhibition studies revealed major requirements for phagocytosis and endosomal acidification in Irg1 expression triggered by Mtb but not LPS or PAM3CSK4. Importantly, the Mtb-induced Irg1 response was highly dependent on the presence of the bacterial ESX-1 secretion system, as well as host STING and Type I IFN receptor (IFNAR) signaling with Type II IFN (IFN-γ) signaling playing only a minimal role. Based on these findings we hypothesize that Mtb induces Irg1 expression in macrophages via the combination of two independent triggers both dependent on bacterial phagocytosis: 1) a major signal stimulated by phagocytized Mtb products released by an ESX-1-dependent mechanism into the cytosol where they activate the STING pathway leading to Type I-IFN production, and 2) a secondary TLR-2, MyD88 and NFκB dependent signal that enhances Irg1 production independently of Type I IFN induction.

IRG1 induced by heme oxygenase-1/carbon monoxide inhibits LPS-mediated sepsis and pro-inflammatory cytokine production.

The immunoresponsive gene 1 (IRG1) protein has crucial functions in embryonic implantation and neurodegeneration. IRG1 promotes endotoxin tolerance by increasing A20 expression in macrophages through reactive oxygen species (ROS). The cytoprotective protein heme oxygenase-1 (HO-1), which generates endogenous carbon monoxide (CO), is expressed in the lung during Lipopolysaccharide (LPS) tolerance and cross tolerance. However, the detailed molecular mechanisms and functional links between IRG1 and HO-1 in the innate immune system remain unknown. In the present study, we found that the CO releasing molecule-2 (CORM-2) and chemical inducers of HO-1 increased IRG1 expression in a time- and dose-dependent fashion in RAW264.7 cells. Furthermore, inhibition of HO-1 activity by zinc protoporphyrin IX (ZnPP) and HO-1 siRNA significantly reduced expression of IRG1 under these conditions. In addition, treatment with CO and HO-1 induction significantly increased A20 expression, which was reversed by ZnPP and HO-1 siRNA. LPS-stimulated TNF-α was significantly decreased, whereas IRG1 and A20 were increased by CORM-2 application and HO-1 induction, which in turn were abrogated by ZnPP. Interestingly, siRNA against IRG1 and A20 reversed the effects of CO and HO-1 on LPS-stimulated TNF-α production. Additionally, CO and HO-1 inducers significantly increased IRG1 and A20 expression and downregulated TNF-α production in a LPS-stimulated sepsis mice model. Furthermore, the effects of CO and HO-1 on TNF-α production were significantly reversed when ZnPP was administered. In conclusion, CO and HO-1 induction regulates IRG1 and A20 expression, leading to inhibition of inflammation in vitro and in an in vivo mice model.

A novel chimeric flagellum fused with the multi-epitope vaccine CTB-UE prevents Helicobacter pylori-induced gastric cancer in a BALB/c mouse model.

Helicobacter pylori (H. pylori) infection causes peptic ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. The eradication of H. pylori might be an effective means of preventing gastric cancer. A dual-antigen epitope and dual-adjuvant vaccine called CTB-UE-CF (CCF) was constructed by combining a multi-epitope vaccine CTB-UE with a novel chimeric flagellum (CF) to simultaneously activate Toll-like receptor (TLR) 5-agonist activity and preserve the immunogenicity of H. pylori flagellum FlaA. The evaluation of efficacy to reduce H. pylori colonization was performed using BALB/c mice by oral immunization with a triple dose of this vaccine strain. Two weeks after the last immunization, mice were sacrificed to determine specific antibody levels and proinflammatory cytokine production. To determine the presence of H. pylori, we detected the number of H. pylori by real-time quantitative PCR (qPCR) and measured the urease activity in the gastric tissue. The results showed that the immunogenicity and mucosal immune responses of CCF performed significantly better than those of CTB-UE. This dual-antigen epitope and dual-adjuvant system might greatly contribute to the development of a safe and efficient therapeutic vaccine for humans against H. pylori infection.

Immune responsive gene 1 (IRG1) promotes endotoxin tolerance by increasing A20 expression in macrophages through reactive oxygen species.

Sepsis-associated immunosuppression (SAIS) is regarded as one of main causes for the death of septic patients at the late stage because of the decreased innate immunity with a more opportunistic infection. LPS-tolerized macrophages, which are re-challenged by LPS after prior exposure to LPS, are regarded as the common model of hypo-responsiveness for SAIS. However, the molecular mechanisms of endotoxin tolerance and SAIS remain to be fully elucidated. In addition, negative regulation of the Toll-like receptor (TLR)-triggered innate inflammatory response needs further investigation. Here we show that expression of immune responsive gene 1 (IRG1) was highly up-regulated in the peripheral blood mononuclear cells of septic patients and in LPS-tolerized mouse macrophages. IRG1 significantly suppressed TLR-triggered production of proinflammatory cytokines TNF-α, IL-6, and IFN-ß in LPS-tolerized macrophages, with the elevated expression of reactive oxygen species (ROS) and A20. Moreover, ROS enhanced A20 expression by increasing the H3K4me3 modification of histone on the A20 promoter domain, and supplement of the ROS abrogated the IRG1 knockdown function in breaking endotoxin tolerance by increasing A20 expression. Our results demonstrate that inducible IRG1 promotes endotoxin tolerance by increasing A20 expression through ROS, indicating a new molecular mechanism regulating hypoinflammation of sepsis and endotoxin tolerance.

Double knockdown of alpha1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC.

BACKGROUND: Antibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans in vivo. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies. RESULTS: Firstly, loss-of-function analyses using small interfering RNAs (siRNAs) against the three key genes involved in oligosaccharide fucose modification, i.e. alpha1,6-fucosyltransferase (FUT8), GDP-mannose 4,6-dehydratase (GMD), and GDP-fucose transporter (GFT), revealed that single-gene knockdown of each target was insufficient to completely defucosylate the products in antibody-producing cells, even though the most effective siRNA (>90% depression of the target mRNA) was employed. Interestingly, beyond our expectations, synergistic effects of FUT8 and GMD siRNAs on the reduction in fucosylation were observed, but not when these were used in combination with GFT siRNA. Secondly, we successfully developed an effective short hairpin siRNA tandem expression vector that facilitated the double knockdown of FUT8 and GMD, and we converted antibody-producing Chinese hamster ovary (CHO) cells to fully non-fucosylated antibody producers within two months, and with high converting frequency. Finally, the stable manufacture of fully non-fucosylated antibodies with enhanced ADCC was confirmed using the converted cells in serum-free fed-batch culture. CONCLUSION: Our results suggest that FUT8 and GMD collaborate synergistically in the process of intracellular oligosaccharide fucosylation. We also demonstrated that double knockdown of FUT8 and GMD in antibody-producing cells could serve as a new strategy for producing next-generation therapeutic antibodies fully lacking core fucosylation and with enhanced ADCC. This approach offers tremendous cost- and time-sparing advantages for the development of next-generation therapeutic antibodies.

Characterization of 17beta-hydroxysteroid dehydrogenase type 4 in human ovarian surface epithelial cells.

The human ovarian surface epithelium (hOSE) is a single layer of mesothelial-type primitive epithelial cells that are potential estrogen targets. It has been reported that hOSE cells can produce estrogen. However, the mechanisms that regulate estrogen level(s) in hOSE cells are not yet known. To elucidate the enzymes involved in these reactions, we examined gene expression of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) in primary hOSE (POSE) and OSE2a cells using RT-PCR. We found that POSE cells and cells of the immortalized hOSE line, OSE2a, bidirectionally converted estrone (E1) and 17beta-estradiol (E2). Both cell types expressed mRNA for 17beta-HSD type 1 (17beta-HSD1), suggesting that the enzyme is involved in the E1 to E2 conversion. Interestingly, both cells expressed 17beta-HSD4 mRNA but not 17beta-HSD2 mRNA. We prepared an antibody against the carboxyl terminal of 17beta-HSD4 (anti-17beta-HSD4 antibody), which recognized the 80 and 48 kDa proteins in POSE and OSE2a cells based on immunoblot analysis. Furthermore, immunohistochemical study revealed the presence of 17beta-HSD4 in hOSE cells in the human ovary. These results suggest that 17beta-HSD4 is involved in estrogen inactivation and may protect against an excessive accumulation of E2 in hOSE cells.

Insertional inactivation of pac and rmlB genes reduces the release of tumor necrosis factor alpha, interleukin-6, and interleukin-8 induced by Streptococcus mutans in monocytic, dental pulp, and periodontal ligament cells.

Streptococcus mutans possesses different cell wall molecules, such as protein of the I/II family, the serotype f polysaccharide rhamnose glucose polymer (RGP), and lipoteichoic acid (LTA), which act as adhesins and modulins, allowing S. mutans to colonize teeth and cause dental caries and pulpitis. We tested several isogenic mutants of S. mutans defective in protein I/II and/or RGP, as well as purified modulins such as protein I/II, RGP, and LTA, for their binding and activation abilities on monocytic, dental pulp (DP), and periodontal ligament (PDL) cells. Our results demonstrate that both protein I/II and RGP play important roles in streptococcal adherence to human monocytic and fibroblastic cells, whereas LTA is only a minor adhesin. In the activation process, the cytokine response elicited is polarized toward a Th1 response which seems principally due to protein I/II and RGP. Even if protein I/II seems to be more efficient in its purified form in triggering cells to release interleukin-8 (IL-8), RGP is the most efficient cytokine-stimulating component in intact bacteria, while LTA plays only a minor role. In cell activation, we showed, by using either cytochalasin D or coated ligands, that internalization of either S. mutans, S. mutans isogenic mutants, or purified ligands is not necessary to trigger cells to release IL-8. We also showed that, besides the implication of monocytes in pulpal inflammation, fibroblast-like cells such as DP and PDL cells are also actively implicated in local inflammation and in the generation of a Th1 response after stimulation with S. mutans cells or antigens.

Dimerization co-factor of hepatocyte nuclear factor 1/pterin-4alpha-carbinolamine dehydratase is necessary for pigmentation in Xenopus and overexpressed in primary human melanoma lesions.

Dimerization co-factor of hepatocyte nuclear factor 1 (HNF1)/pterin-4alpha-carbinolamine dehydratase (DCoH/PCD) is both a positive co-factor of the HNF1 homeobox transcription factors and thus involved in gene regulation as well as an enzyme catalyzing the regeneration of tetrahydrobiopterin. Dysfunction of DCoH/PCD is associated with the human disorders hyperphenylalaninemia and vitiligo. In Xenopus, overexpression of the protein during development induces ectopic pigmentation. In this study loss of function experiments using DCoH/PCD-specific antibodies demonstrated that the protein is also absolutely necessary for pigment cell formation in Xenopus. In normal human skin DCoH/PCD protein is weakly expressed in the basal layer of the epidermis that consists of keratinocytes and melanocytes. Whereas only 4 of 25 benign nevi reacted with DCoH/PCD-specific antibodies, high protein levels were detectable in melanoma cell lines and 13 of 15 primary malignant melanoma lesions. The comparison with the commonly used melanoma markers S100 and HMB45 demonstrated that DCoH/PCD has an overlapping but distinct expression pattern in melanoma lesions. In addition to human colon cancer, this is the second report about the overexpression of DCoH/PCD in human tumor cells indicating that the protein might be involved in cancerogenesis.

Characterization of qa-2 mutants of Neurospora crassa by genetic, enzymatic, and immunological techniques.

Genetic and complementation mapping studies using 20 qa-2 mutants defective for catabolic dehydroquinase indicate that the qa-2 gene encodes a single polypeptide chain and is the structural gene for catabolic dehydroquinase, a 220,000-molecular-weight protein composed of identical 10,000-molecular-weight subunits. Many qa-2 mutants are capable of reversion, but no evidence has yet been obtained for nonsense mutations in this gene. The biochemical consequences of the mutations in two complementing qa-2 strains (M239 and M204) have been determined. Both mutants have extremely low levels of catalytic activity and form a heterocaryon with about 4% of the wild-type activity. As assayed by immunological cross-reactivity, mutant M239 and the heterocaryon have nearly wild-type levels of native-molecular-weight catabolic dehydroquinase protein, whereas M204 has no detectable amount of this protein. Thus it is concluded that M239 has a mutation at or near the catalytic site which reduces the activity 10,000-fold but has little or no influence on the formation of the native multimeric structure. In contrast, M204 apparently has a mutation that severely inhibits aggregation and may have only a minor effect on the inherent potential for catalytic conversion at the reactive site. The heterocaryon would appear to form a mixed multimer with the monomeric subunits from M239 providing the aggregated structure and those from M204, the catalytically active moiety.

Biodegradative L-threonine deaminase of Salmonella typhimurium.

The threonine deaminase formed under anaerobic conditions by Salmonella typhimurium is induced by l-serine and l-threonine, is catabolite repressible, requires cyclic adenosine 3',5'-monophosphate for its synthesis and adenylic acid for optimal activity, and is immunologically different from biosynthetic threonine deaminase.