Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

IL-32 is a newly described cytokine in the human found to be an in vitro inducer of tumor necrosis factor alpha (TNFα). We examined the in vivo relationship between IL-32 and TNFα, and the pathologic role of IL-32 in the TNFα-related diseases – arthritis and colitis. We demonstrated by quantitative PCR assay that IL-32 mRNA was expressed in the lymphoid tissues, and in stimulated peripheral T cells, monocytes, and B cells. Activated T cells were important for IL-32 mRNA expression in monocytes and B cells. Interestingly, TNFα reciprocally induced IL-32 mRNA expression in T cells, monocyte-derived dendritic cells, and synovial fibroblasts. Moreover, IL-32 mRNA expression was prominent in the synovial tissues of rheumatoid arthritis patients, especially in synovial-infiltrated lymphocytes by in situ hybridization. To examine the in vivo relationship of IL-32 and TNFα, we prepared an overexpression model mouse of human IL-32β (BM-hIL-32) by bone marrow transplantation. Splenocytes of BM-hIL-32 mice showed increased expression and secretion of TNFα, IL-1β, and IL-6 especially in response to lipopolysaccharide stimulation. Moreover, serum TNFα concentration showed a clear increase in BM-hIL-32 mice. Cell-sorting analysis of splenocytes showed that the expression of TNFα was increased in resting F4/80⁺ macrophages, and the expression of TNFα, IL-1β and IL-6 was increased in lipopolysaccharide-stimulated F4/80⁺ macrophages and CD11c⁺ dendritic cells. In fact, BM-hIL-32 mice showed exacerbation of collagen-antibody-induced arthritis and trinitrobenzen sulfonic acid-induced colitis. In addition, the transfer of hIL-32β-producing CD4⁺ T cells significantly exacerbated collagen-induced arthritis, and a TNFα blockade cancelled the exacerbating effects of hIL-32β. We therefore conclude that IL-32 is closely associated with TNFα, and contributes to the exacerbation of TNFα-related inflammatory arthritis and colitis.     

Gastric protective effect of peripheral PYY through PYY preferring receptors in anesthetized rats

The influence of intravenous peptide YY (PYY) on the gastric injury induced by 45% ethanol was investigated in urethane-anesthetized rats. PYY (25, 75, 125, and 250 pmol x kg(-1) x h(-1)) significantly reduced gastric lesions by 36, 59, 40, and 38%, respectively. Antibody against ratPYY (2 mg/rat) injected intravenously completely prevented the gastroprotective effect of intravenous PYY (75 pmol x kg(-1) x h(-1)), whereas injected intracisternally (460 microg/20 microl), it significantly prevented intracisternal PYY (24 pmol/rat)-induced 58% reduction of ethanol lesions but not that induced by intravenous PYY. Vagotomy did not influence the gastroprotective effect of intravenous PYY. The Y(1)/"PYY-preferring" receptor agonist [Pro(34)]PYY (75 pmol x kg(-1) x h(-1) iv) significantly decreased ethanol-induced gastric lesions by 82%, whereas [Leu(31), Pro(34)]NPY, a Y(1)/Y(3) agonist, and PYY-(3-36), a Y(2) agonist, had no effect. These data indicate that PYY-infused intravenously at doses reported to mimic postprandial peak blood levels prevents ethanol-induced gastric injury through vagal independent pathways and PYY-preferring receptors.     

Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis

The signaling molecule Wnt regulates bone homeostasis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Impairment of canonical Wnt signaling causes bone loss in arthritis and osteoporosis; however, it is unclear how noncanonical Wnt signaling regulates bone resorption. Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor (Ror) proteins. We showed that Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhanced osteoclastogenesis. Osteoblast-lineage cells expressed Wnt5a, whereas osteoclast precursors expressed Ror2. Mice deficient in either Wnt5a or Ror2, and those with either osteoclast precursor-specific Ror2 deficiency or osteoblast-lineage cell-specific Wnt5a deficiency showed impaired osteoclastogenesis. Wnt5a-Ror2 signals enhanced receptor activator of nuclear factor-κB (RANK) expression in osteoclast precursors by activating JNK and recruiting c-Jun on the promoter of the gene encoding RANK, thereby enhancing RANK ligand (RANKL)-induced osteoclastogenesis. A soluble form of Ror2 acted as a decoy receptor of Wnt5a and abrogated bone destruction in mouse arthritis models. Our results suggest that the Wnt5a-Ror2 pathway is crucial for osteoclastogenesis in physiological and pathological environments and represents a therapeutic target for bone diseases, including arthritis.     

Microbial Populations Responsive to Denitrification-Inducing Conditions in Rice Paddy Soil,as Revealed by Comparative 16S rRNA Gene Analysis

Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for nitrate respiration and denitrification have not been well characterized. In this study, we performed a clone library analysis of >1,000 clones of the nearly full-length 16S rRNA gene to characterize bacterial community structure in rice paddy soil. We also identified potential key players in nitrate respiration and denitrification by comparing the community structures of soils with strong denitrifying activity to those of soils without denitrifying activity. Clone library analysis showed that bacteria belonging to the phylum Firmicutes, including a unique Symbiobacterium clade, dominated the clones obtained in this study. Using the template match method, several operational taxonomic units (OTUs), most belonging to the orders Burkholderiales and Rhodocyclales, were identified as OTUs that were specifically enriched in the sample with strong denitrifying activity. Almost one-half of these OTUs were classified in the genus Herbaspirillum and appeared >10-fold more frequently in the soils with strong denitrifying activity than in the soils without denitrifying activity. Therefore, OTUs related to Herbaspirillum are potential key players in nitrate respiration and denitrification under the conditions used.Rice is one of the most important agronomic plants in the world (20). More than 135 million ha are used for rice cultivation worldwide, 88% of which consists of paddy fields (i.e., flooded fields) (16). Since rice paddy soil has limited available oxygen, various anaerobic biochemical processes can occur, including methane production, Mn⁴⁺ and Fe³⁺ reduction, nitrate respiration, and denitrification.Denitrification is a microbial respiratory process during which soluble nitrogen oxides (NO₃⁻ and NO₂⁻) are reduced to gaseous products (NO, N₂O, and N₂) (14, 43). Reduction of nitrate (NO₃⁻) to nitrite (NO₂⁻) is part of the denitrification process; however, this reaction can also be performed by nondenitrifiers. Reduction of nitrate to nitrite as an end product is called nitrate respiration (43). The emission of N₂O from rice paddy soils is less than that from upland crop fields (2), which is probably due to complete nitrate-nitrite reduction to N₂, since rice paddy soil is known to have strong denitrifying activity (28). However, the microbes responsible for denitrification in rice paddy soil are not well known.Denitrifying ability is sporadically distributed among taxonomically diverse groups of bacteria, as well as some archaea and fungi (14, 33, 43). Therefore, it is difficult to identify denitrifying organisms based only on their 16S rRNA gene sequences (33). However, culture-independent 16S rRNA gene analysis can be used to identify microbial populations responsive to denitrification-inducing conditions if they are properly differentiated from background populations. The 16S rRNA gene can provide taxonomic information about organisms which cannot be obtained from analyses targeting nitrite reductase genes (nirS and nirK) alone (34).One approach to differentiate functionally active populations from background populations is to use stable-isotope probing (SIP) (35). SIP was previously used to identify succinate-assimilating bacterial populations under denitrifying conditions in rice paddy soil, using nitrate and succinate as the electron acceptor and donor, respectively (37). Although SIP analysis can provide solid evidence that links function with taxonomy, it requires assimilation of isotopically labeled substrates. This may limit the application of SIP in studies of dissimilatory processes, such as nitrate respiration and denitrification. For example, previous SIP studies targeted bacteria assimilating ¹³C-labeled acetate, methanol, or succinate under denitrifying conditions (13, 30, 37).Another approach is to detect specifically enriched microbial populations under certain conditions by comparative analysis of 16S rRNA gene sequences (9). This approach does not necessarily require addition of isotopically labeled substrates and therefore has the potential to identify microbes performing dissimilatory processes. Furthermore, the community structure of the total population can also be elucidated in this manner (10, 25, 36). However, the usefulness of comparative analysis of 16S rRNA gene sequences has not been thoroughly tested. In addition, this approach has not been used to study nitrate respirators and denitrifiers.Consequently, the objectives of this study were (i) to characterize the soil bacterial population in rice paddy soil by clone library analysis of >1,000 clones of the nearly full-length 16S rRNA gene and (ii) to identify active bacterial populations under denitrification-inducing conditions by comparing clone libraries.     

Rhizonin, the first mycotoxin isolated from the zygomycota, is not a fungal metabolite but is produced by bacterial endosymbionts

Rhizonin is a hepatotoxic cyclopeptide isolated from cultures of a fungal Rhizopus microsporus strain that grew on moldy ground nuts in Mozambique. Reinvestigation of this fungal strain by a series of experiments unequivocally revealed that this "first mycotoxin from lower fungi" is actually not produced by the fungus. PCR experiments and phylogenetic studies based on 16S rRNA gene sequences revealed that the fungus is associated with bacteria belonging to the genus Burkholderia. By transmission electron microscopy, the bacteria were localized within the fungal cytosol. Toxin production and the presence of the endosymbionts were correlated by curing the fungus with an antibiotic, yielding a nonproducing, symbiont-free phenotype. The final evidence for a bacterial biogenesis of the toxin was obtained by the successful fermentation of the endosymbiotic bacteria in pure culture and isolation of rhizonin A from the broth. This finding is of particular interest since Rhizopus microsporus and related Rhizopus species are frequently used in food preparations such as tempeh and sufu.     

Estrogen regulation of nitric oxide synthesis in the procine oocyte

The endothelial type (NOS-3) of three isoforms of nitric oxide (NO) synthase occurs in porcine oocytes and granulosa cells, but the regulation of NO synthesis in oocytes remains unknown. The present study was designed to evaluate steroid control in the process of oocyte NO synthesis. Cumulus-oocyte complexes (COCs), obtained from small-sized antral follicles of immature porcine ovaries, were cultured in estrogen-deprived medium, and the effect of steroids or steroid-free porcine follicular fluids on the NO release from oocytes was investigated. Oocytes that were isolated from cultured COCs were incubated with 1 M ionomycin. The NO metabolites were identified using a NO detector-high-pressure liquid chromatography system. Oocytes from COCs cultured with 10 nM 17-estradiol (E₂) released NO in response to ionomycin, whereas progesterone and testosterone had little effect on the synthesis of NO. An inhibitor of NOS suppressed the synthesis of NO. The maximal synthesis was observed after a 15 h-culture with E₂. However, oocytes freshly obtained from antral follicles did not response to ionomycin, and the E₂ action was suppressed by the addition of steroid-free follicular fluids. Analyses of RT-PCR and Western blotting showed that E₂ did not increase NOS-3 expression. In addition, estrogen receptor was detected in oocytes and cumulus cells, and estrogen receptor was detected only in cumulus cells. These findings suggest that oocyte NOS-3 is promoted for the synthesis of NO by E₂ without increases in NOS-3 expression, but the synthesis of NO is suppressed, at least in the oocytes of early antral follicles.     

Multiple Primary Cilia Modulate the Fluid Transcytosis in Choroid Plexus Epithelium

Functional defects in cilia are associated with various human diseases including congenital hydrocephalus. Previous studies suggested that defects in cilia not only disrupt the flow of cerebrospinal fluid (CSF) generated by motile cilia in ependyma lining the brain ventricles, but also cause increased CSF production at the choroid plexus. However, the molecular mechanisms of CSF overproduction by ciliary dysfunction remain elusive. To dissect the molecular mechanisms, choroid plexus epithelial cells (CPECs) were isolated from porcine brain. These cells expressed clusters of primary cilia on the apical surface. Deciliation of CPECs elevated the intracellular cyclic AMP (cAMP) levels and stimulated basolateral‐to‐apical fluid transcytosis, without detrimental effects on other morphological and physiological features. The primary cilia possessed neuropeptide FF (NPFF) receptor 2. In deciliated cells, the responsiveness to NPFF was reduced at nanomolar concentrations. Furthermore, CPECs expressed NPFF precursor along with NPFFR2. An NPFFR antagonist, BIBP3226, increased the fluid transcytosis, suggesting the presence of autocrine NPFF signaling in CPECs for a tonic inhibition of fluid transcytosis. These results suggest that the clusters of primary cilia in CPECs act as a sensitive chemosensor to regulate CSF production.     

Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice

Listeria monocytogenes (LM), a facultative intracellular Gram-positive bacterium, often causes lethal infection of the host. In this study we investigated the molecular mechanism underlying LM eradication in the early phase of infection. Upon infection with LM, both IL-12 and IL-18 were produced, and then they synergistically induced IFN-gamma production, leading to normal LM clearance in the host. IFN-gamma knockout (KO) mice were highly susceptible to LM infection. IL-12/IL-18 double knockout mice were also highly susceptible. Their susceptibility was less than that of IFN-gamma KO mice, but more than that of single IL-12 or IL-18 KO mice. Mice deficient in myeloid differentiation factor 88 (MyD88), an essential adaptor molecule used by signal transduction pathways of all members of the Toll-like receptor (TLR) family, showed an inability to produce IL-12 and IFN-gamma following LM infection and were most susceptible to LM. Furthermore, MyD88-deficient, but not IFN-gamma-deficient, Kupffer cells could not produce TNF-alpha in response to LM in vitro, indicating the importance of MyD88-dependent TNF-alpha production for host defense. As TLR2 KO, but not TLR4 KO, mice showed partial impairment in their capacity to produce IL-12, IFN-gamma, and TNF-alpha, TLR2 activation partly contributed to the induction of IL-12-mediated IFN-gamma production. These results indicated a critical role for TLRs/MyD88-dependent IL-12/TNF-alpha production and for IL-12- and IL-18-mediated IFN-gamma production in early phase clearance of LM.