RECON-Dependent Inflammation in Hepatocytes Enhances Listeria monocytogenes Cell-to-Cell Spread.

The oxidoreductase RECON is a high-affinity cytosolic sensor of bacterium-derived cyclic dinucleotides (CDNs). CDN binding inhibits RECON's enzymatic activity and subsequently promotes inflammation. In this study, we sought to characterize the effects of RECON on the infection cycle of the intracellular bacterium Listeria monocytogenes, which secretes cyclic di-AMP (c-di-AMP) into the cytosol of infected host cells. Here, we report that during infection of RECON-deficient hepatocytes, which exhibit hyperinflammatory responses, L. monocytogenes exhibits significantly enhanced cell-to-cell spread. Enhanced bacterial spread could not be attributed to alterations in PrfA or ActA, two virulence factors critical for intracellular motility and intercellular spread. Detailed microscopic analyses revealed that in the absence of RECON, L. monocytogenes actin tail lengths were significantly longer and there was a larger number of faster-moving bacteria. Complementation experiments demonstrated that the effects of RECON on L. monocytogenes spread and actin tail lengths were linked to its enzymatic activity. RECON enzyme activity suppresses NF-κB activation and is inhibited by c-di-AMP. Consistent with these previous findings, we found that augmented NF-κB activation in the absence of RECON caused enhanced L. monocytogenes cell-to-cell spread and that L. monocytogenes spread correlated with c-di-AMP secretion. Finally, we discovered that, remarkably, increased NF-κB-dependent inducible nitric oxide synthase expression and nitric oxide production were responsible for promoting L. monocytogenes cell-to-cell spread. The work presented here supports a model whereby L. monocytogenes secretion of c-di-AMP inhibits RECON's enzymatic activity, drives augmented NF-κB activation and nitric oxide production, and ultimately enhances intercellular spread.IMPORTANCE To date, bacterial CDNs in eukaryotes are solely appreciated for their capacity to activate cytosolic sensing pathways in innate immunity. However, it remains unclear whether pathogens that actively secrete CDNs benefit from this process. Here, we provide evidence that secretion of CDNs leads to enhancement of L. monocytogenes cell-to-cell spread. This is a heretofore-unknown role of these molecules and suggests L. monocytogenes may benefit from their secretion in certain contexts. Molecular characterization revealed that, surprisingly, nitric oxide was responsible for the enhanced spread. Pathogens act to prevent nitric oxide production or, like L. monocytogenes, they have evolved to resist its direct antimicrobial effects. This study provides evidence that intracellular bacterial pathogens not only tolerate nitric oxide, which is inevitably encountered during infection, but can also capitalize on the changes this pleiotropic molecule enacts on the host cell.

Sensing of Bacterial Cyclic Dinucleotides by the Oxidoreductase RECON Promotes NF-κB Activation and Shapes a Proinflammatory Antibacterial State.

Bacterial and host cyclic dinucleotides (cdNs) mediate cytosolic immune responses through the STING signaling pathway, although evidence suggests that alternative pathways exist. We used cdN-conjugated beads to biochemically isolate host receptors for bacterial cdNs, and we identified the oxidoreductase RECON. High-affinity cdN binding inhibited RECON enzyme activity by simultaneously blocking the substrate and cosubstrate sites, as revealed by structural analyses. During bacterial infection of macrophages, RECON antagonized STING activation by acting as a molecular sink for cdNs. Bacterial infection of hepatocytes, which do not express STING, revealed that RECON negatively regulates NF-κB activation. Loss of RECON activity, via genetic ablation or inhibition by cdNs, increased NF-κB activation and reduced bacterial survival, suggesting that cdN inhibition of RECON promotes a proinflammatory, antibacterial state that is distinct from the antiviral state associated with STING activation. Thus, RECON functions as a cytosolic sensor for bacterial cdNs, shaping inflammatory gene activation via its effects on STING and NF-κB.

Modulation of steroidogenic enzymes in murine lymphoid organs after immune activation.

To study the effects of immune cell activation by a protein antigen or lymphoid tissue derived cytokines on peripheral steroidogenesis activities of 3beta HSD and 17beta HSD was measured in lymphoid organs of control and BSA immunized mice after 3 weeks treatment. We demonstrated the presence of 3betaHSD and 17betaHSD in the lymphoid organs after active immunization. We found elevated serum corticosterone after 3 weeks of antigen administration in presence of CFA and a higher serum IL-6 level that also alter lymphoid tissue cytokine responses like TNF-alpha, IL-12p70, and IL-6, among which IL-12p70 and TNF-alpha down-regulate the activity of steroidogenic enzymes in the thymus during an immune response.

Immunohistochemical localization of estrogen-specific 17 beta-hydroxysteroid oxidoreductase in the human and mouse prostate.

The estrogen-specific 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) enzyme protein was stained immunohistochemically in the newborn and adult human prostate as well as in the mouse prostate. In the newborn human prostate, ductal and urethral epithelia were faintly stained, whereas in the adult human prostate, intense staining for 17 beta-HSOR enzyme antigen could be detected in the epithelium of the collecting ducts and urethral epithelium as well as in the epithelium of the intraprostatic vas deferens and seminal vesicle epithelium. Immunostaining was weak in the prostatic tissues of both newborn and adult prostate. No positive cells were found in stroma. The activity of NADPH-dependent 3H-estrone reductase was detectable in cell-free homogenates prepared from human prostatic tissues. The activities showed a good correlation with immunocytochemical findings. In the mouse, neonatal estrogenization resulted in intensively stained epithelium of the collecting ducts at the age of 14 days. Moreover, when adult control and neonatally estrogenized mice were implanted with 17 beta-estradiol, the metaplastic epithelium of the periurethral collecting ducts of neonatally estrogenized mice was intensively stained with 17 beta-HSOR. These findings suggest that metaplastic epithelium rises from 17 beta-HSOR-positive cells. The similar distributions of 17 beta-HSOR-positive cells confirm the concept of homology in the posterior estrogen-responsive periurethral region (containing the periurethral ducts and periurethral glands) of the mouse and humans. Our findings further suggest that the 17 beta-HSOR-positive cells may have the same origin and hormonal control in both species.

Purification and properties of oestradiol 17 beta-dehydrogenase extracted from cytoplasmic vesicles of porcine endometrial cells.

Porcine endometrial oestradiol-17 beta dehydrogenase was solubilized from the particulate fraction of homogenates sedimenting between 1200 g and 10,000 g by treatment with 0.4% Brij 35 in neutral buffers. The extracts were processed by successive passage through DEAE-Sepharose, Amberlyte XAD-2 and Blue-Sepharose, and the enzyme was collected from the washed affinity matrix at 0.8 M of a 0-2 M-KCl gradient. A genuine oestrone reductase was eluted at 1.9 M-KCl. The dehydrogenase pool was resolved by butyl-Sepharose chromatography into a major (80%) peak (EDHM) eluted at 0.8 M-(NH4)2SO4 and a very hydrophobic fraction (VHF) recovered at 0.1 M. EDHM was further purified by filtration through Sephadex G-200 and cation-exchange chromatography on Mono S. Sephacryl 300 was used for VHF followed by Mono S. Enrichments from the homogenate amounted to 1074-fold for EDHM and 632-fold for VHF. A single silver-stained band at 32 kDa is seen on SDS/PAGE of EDHM, and VHF contains additional bands at 45 and 80 kDa. Polyclonal antibodies (G436) raised against EDHM and the monoclonal antibody F1 raised against VHF recognize the single 32 kDa band in EDHM and both the 32 kDa and 80 kDa bands in composite VHF. The 45 kDa band of VHF reacts with neither. Monoclonal antibody W1 raised against EDHM only recognizes the 32 kDa peptide of EDHM and VHF. The specific activity for oestradiol oxidation amounts to 4081 mu-units/mg for EDHM and to 2402 mu-units/mg for VHF. Both possess a minimal (1/260) endogenous reductase activity and are devoid of 3 beta, 3 alpha- and 20 alpha-dehydrogenases. We consider EDHM to be authentic oestradiol-17 beta dehydrogenase of porcine endometrium. The composite VHF could reflect the situation of the enzyme in vivo or result from aggregations occurring during processing.

Apparent immunologic nonidentity of human placental and endometrial 17beta-estradiol dehydrogenase.

Immunohistochemical techniques were used to search for the presence of 17beta-estradiol dehydrogenase activity in human endometrial and placental tissues, with the use of antibodies raised against highly purified human placental 17beta-estradiol dehydrogenase. Sensitivity and specificity of the antibodies were documented by radioimmunoassay and immunodiffusion on cellulose acetate. Although staining was consistently demonstrated in the syncytiotrophoblast layer of term placentas, in both cytoplasm and nuclei, no immunohistochemical reaction was observed in endometrial samples. These results support the contention that placental 17beta-estradiol dehydrogenase is immunologically dissimilar from the endometrial enzyme.