Immunomics analysis of rheumatoid arthritis identified precursor dendritic cells as a key cell subset of treatment resistance.

OBJECTIVES: Little is known about the immunology underlying variable treatment response in rheumatoid arthritis (RA). We performed large-scale transcriptome analyses of peripheral blood immune cell subsets to identify immune cells that predict treatment resistance. METHODS: We isolated 18 peripheral blood immune cell subsets of 55 patients with RA requiring addition of new treatment and 39 healthy controls, and performed RNA sequencing. Transcriptome changes in RA and treatment effects were systematically characterised. Association between immune cell gene modules and treatment resistance was evaluated. We validated predictive value of identified parameters for treatment resistance using quantitative PCR (qPCR) and mass cytometric analysis cohorts. We also characterised the identified population by synovial single cell RNA-sequencing analysis. RESULTS: Immune cells of patients with RA were characterised by enhanced interferon and IL6-JAK-STAT3 signalling that demonstrate partial normalisation after treatment. A gene expression module of plasmacytoid dendritic cells (pDC) reflecting the expansion of dendritic cell precursors (pre-DC) exhibited strongest association with treatment resistance. Type I interferon signalling was negatively correlated to pre-DC gene expression. qPCR and mass cytometric analysis in independent cohorts validated that the pre-DC associated gene expression and the proportion of pre-DC were significantly higher before treatment in treatment-resistant patients. A cluster of synovial DCs showed both features of pre-DC and pro-inflammatory conventional DC2s. CONCLUSIONS: An increase in pre-DC in peripheral blood predicted RA treatment resistance. Pre-DC could have pathophysiological relevance to RA treatment response.

Identifying the most influential gene expression profile in distinguishing ANCA-associated vasculitis from healthy controls.

OBJECTIVE: Previous gene expression analyses seeking genes specific to antineutrophil cytoplasmic antibody-associated vasculitis (AAV) have been limited due to crude cell separation and the use of microarrays. This study aims to identify AAV-specific gene expression profiles in a way that overcomes those limitations. METHODS: Blood samples were collected from 26 AAV patients and 28 healthy controls (HCs). Neutrophils were isolated by negative selection, whereas 19 subsets of peripheral blood mononuclear cells were sorted by fluorescence assisted cell sorting. RNA-sequencing was then conducted for each sample, and iterative weighted gene correlation network analysis (iterativeWGCNA) and random forest were consecutively applied to identify the most influential gene module in distinguishing AAV from HCs. Correlations of the identified module with clinical parameters were evaluated, and the biological role was assessed with hub gene identification and pathway analysis. Particularly, the module's association with neutrophil extracellular trap formation, NETosis, was analyzed. Finally, the module's overlap with GWAS-identified autoimmune disease genes (GADGs) was assessed for validation. RESULTS: A neutrophil module (Neu_M20) was ranked top in the random forest analysis among 255 modules created by iterativeWGCNA. Neu_M20 correlated with disease activity and neutrophil counts but not with the presence of antineutrophil cytoplasmic antibody. The module comprised pro-inflammatory genes, including those related to NETosis, supported by experimental evidence. The genes in the module significantly overlapped GADGs. CONCLUSION: We identified the distinct group of pro-inflammatory genes in neutrophils, which characterize AAV. Further investigations are warranted to confirm our findings as they could serve as novel therapeutic targets.

A Physiological and Genomic Comparison of Nitrosomonas Cluster 6a and 7 Ammonia-Oxidizing Bacteria.

Ammonia-oxidizing bacteria (AOB) within the genus Nitrosomonas perform the first step in nitrification, ammonia oxidation, and are found in diverse aquatic and terrestrial environments. Nitrosomonas AOB were grouped into six defined clusters, which correlate with physiological characteristics that contribute to adaptations to a variety of abiotic environmental factors. A fundamental physiological trait differentiating Nitrosomonas AOB is the adaptation to either low (cluster 6a) or high (cluster 7) ammonium concentrations. Here, we present physiological growth studies and genome analysis of Nitrosomonas cluster 6a and 7 AOB. Cluster 6a AOB displayed maximum growth rates at ≤ 1 mM ammonium, while cluster 7 AOB had maximum growth rates at ≥ 5 mM ammonium. In addition, cluster 7 AOB were more tolerant of high initial ammonium and nitrite concentrations than cluster 6a AOB. Cluster 6a AOB were completely inhibited by an initial nitrite concentration of 5 mM. Genomic comparisons were used to link genomic traits to observed physiological adaptations. Cluster 7 AOB encode a suite of genes related to nitrogen oxide detoxification and multiple terminal oxidases, which are absent in cluster 6a AOB. Cluster 6a AOB possess two distinct forms of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and select species encode genes for hydrogen or urea utilization. Several, but not all, cluster 6a AOB can utilize urea as a source of ammonium. Hence, although Nitrosomonas cluster 6a and 7 AOB have the capacity to fulfill the same functional role in microbial communities, i.e., ammonia oxidation, differentiating species-specific and cluster-conserved adaptations is crucial in understanding how AOB community succession can affect overall ecosystem function.

Hybrid Nitrous Oxide Production from a Partial Nitrifying Bioreactor: Hydroxylamine Interactions with Nitrite.

The goal of this study was to elucidate the mechanisms of nitrous oxide (N2O) production from a bioreactor for partial nitrification (PN). Ammonia-oxidizing bacteria (AOB) enriched from a sequencing batch reactor (SBR) were subjected to N2O production pathway tests. The N2O pathway test was initiated by supplying an inorganic medium to ensure an initial NH4+-N concentration of 160 mg-N/L, followed by 15NO2- (20 mg-N/L) and dual 15NH2OH (each 17 mg-N/L) spikings to quantify isotopologs of gaseous N2O (44N2O, 45N2O, and 46N2O). N2O production was boosted by 15NH2OH spiking, causing exponential increases in mRNA transcription levels of AOB functional genes encoding hydroxylamine oxidoreductase (haoA), nitrite reductase (nirK), and nitric oxide reductase (norB) genes. Predominant production of 45N2O among N2O isotopologs (46% of total produced N2O) indicated that coupling of 15NH2OH with 14NO2- produced N2O via N-nitrosation hybrid reaction as a predominant pathway. Abiotic hybrid N2O production was also observed in the absence of the AOB-enriched biomass, indicating multiple pathways for N2O production in a PN bioreactor. The additional N2O pathway test, where 15NH4+ was spiked into 400 mg-N/L of NO2- concentration, confirmed that the hybrid N2O production was a dominant pathway, accounting for approximately 51% of the total N2O production.

Tepidicaulis marinus gen. nov., sp. nov., a marine bacterium that reduces nitrate to nitrous oxide under strictly microaerobic conditions.

A moderately thermophilic, aerobic, stalked bacterium (strain MA2T) was isolated from marine sediments in Kagoshima Bay, Japan. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain MA2T was most closely related to the genera Rhodobium,Parvibaculum, and Rhodoligotrophos (92-93 % similarity) within the class Alphaproteobacteria. Strain MA2T was a Gram-stain-negative and stalked dimorphic bacteria. The temperature range for growth was 16-48 °C (optimum growth at 42 °C). This strain required yeast extract and NaCl (>1 %, w/v) for growth, tolerated up to 11 % (w/v) NaCl, and was capable of utilizing various carbon sources. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The DNA G+C content was 60.7 mol%. Strain MA2T performed denitrification and produced N2O from nitrate under strictly microaerobic conditions. Strain MA2T possessed periplasmic nitrate reductase (Nap) genes but not membrane-bound nitrate reductase (Nar) genes. On the basis of this morphological, physiological, biochemical and genetic information a novel genus and species, Tepidicaulis marinus gen. nov., sp. nov., are proposed, with MA2T ( = NBRC 109643T = DSM 27167T) as the type strain of the species.

Methyloceanibacter caenitepidi gen. nov., sp. nov., a facultatively methylotrophic bacterium isolated from marine sediments near a hydrothermal vent.

A moderately thermophilic, methanol-oxidizing bacterium (strain Gela4(T)) was isolated from methane-utilizing mixed-culture originating from marine sediment near a hydrothermal vent. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain Gela4(T) was closely related to members of the genus 'Methyloligella' (94.7% similarity) within the class Alphaproteobacteria. Strain Gela4(T) was a Gram-staining-negative and aerobic organism. Cells were rod-shaped and non-motile. The temperature range for growth of strain Gela4(T) was 19-43 °C (optimal growth at 35 °C). Strain Gela4(T) tolerated up to 9% NaCl with an optimum at 1%. The organism was a facultative methylotroph that could utilize methanol, methylamine, trimethylamine and a variety of multi-carbon compounds. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The predominant phospholipids were phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 63.9 mol%. On the basis of the morphological, physiological, biochemical and genetic information, a novel genus and species, Methyloceanibacter caenitepidi is proposed, with Gela4(T) ( = NBRC 109540(T) = DSM 27242(T)) as the type strain.

The role of dendritic cells and their immunometabolism in rheumatoid arthritis.

Dendritic cells (DCs) play crucial roles in the pathogenesis of rheumatoid arthritis (RA), a prototypic autoimmune disease characterized by chronic synovitis and joint destruction. Conventional dendritic cells (cDCs) with professional antigen-presenting functions are enriched in the RA synovium. In the synovium, the cDCs are activated and show both enhanced migratory capacities and T cell activation in comparison with peripheral blood cDCs. Plasmacytoid dendritic cells, another subtype of DCs capable of type I interferon production, are likely to be tolerogenic in RA. Monocyte-derived dendritic cells (moDCs), once called "inflammatory DCs", are localized in the RA synovium, and they induce T-helper 17 cell expansion and enhanced proinflammatory cytokine production. Recent studies revealed that synovial proinflammatory hypoxic environments are linked to metabolic reprogramming. Activation of cDCs in the RA synovium is accompanied by enhanced glycolysis and anabolism. In sharp contrast, promoting catabolism can induce tolerogenic DCs from monocytes. Herein, we review recent studies that address the roles of DCs and their immunometabolic features in RA. Immunometabolism of DCs could be a potential therapeutic target in RA.

Pulmonary Hypertension Associated with Anti-synthetase Syndrome: A Case Report and Literature Review.

Pulmonary hypertension (PH) is a serious condition in which there is an abnormally high pressure in the pulmonary arteries that can occur as a complication of connective tissue diseases. Although the relationship between PH and systemic lupus erythematosus or systemic sclerosis has been well-characterized, PH rarely occurs in patients with anti-synthetase syndrome (ASS), and little is known about the pathophysiology and clinical outcome of patients with ASS-PH. We herein report a patient with anti-Jo-1-positive ASS complicated by PH and discuss the treatment strategy through a review of previously reported cases.

Genome sequence of Nitrosomonas sp. strain AL212, an ammonia-oxidizing bacterium sensitive to high levels of ammonia.

Nitrosomonas sp. strain AL212 is an obligate chemolithotrophic ammonia-oxidizing bacterium (AOB) that was originally isolated in 1997 by Yuichi Suwa and colleagues. This organism belongs to Nitrosomonas cluster 6A, which is characterized by sensitivity to high ammonia concentrations, higher substrate affinity (lower K(m)), and lower maximum growth rates than strains in Nitrosomonas cluster 7, which includes Nitrosomonas europaea and Nitrosomonas eutropha. Genome-informed studies of this ammonia-sensitive cohort of AOB are needed, as these bacteria are found in freshwater environments, drinking water supplies, wastewater treatment systems, and soils worldwide.

The physiological potential of anammox bacteria as revealed by their core genome structure.

We present here the second complete genome of anaerobic ammonium oxidation (anammox) bacterium, Candidatus (Ca.) Brocadia pituitae, along with those of a nitrite oxidizer and two incomplete denitrifiers from the anammox bacterial community (ABC) metagenome. Although NO2- reduction to NO is considered to be the first step in anammox, Ca. B. pituitae lacks nitrite reductase genes (nirK and nirS) responsible for this reaction. Comparative genomics of Ca. B. pituitae with Ca. Kuenenia stuttgartiensis and six other anammox bacteria with nearly complete genomes revealed that their core genome structure contains 1,152 syntenic orthologues. But nitrite reductase genes were absent from the core, whereas two other Brocadia species possess nirK and these genes were horizontally acquired from multiple lineages. In contrast, at least five paralogous hydroxylamine oxidoreductase genes containing candidate ones (hao2 and hao3) encoding another nitrite reductase were observed in the core. Indeed, these two genes were also significantly expressed in Ca. B. pituitae as in other anammox bacteria. Because many nirS and nirK genes have been detected in the ABC metagenome, Ca. B. pituitae presumably utilises not only NO supplied by the ABC members but also NO and/or NH2OH by self-production for anammox metabolism.

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4 +/15NH4 + Analyses via Sequential Conversion to N2O.

The importance of understanding the fate of nitrate (NO3-), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has been increasing because global nitrogen loads have dramatically increased following industrialization. Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are both microbial processes that use NO3- for respiration. Compared to denitrification, quantitative determinations of the DNRA activity have been carried out only to a limited extent. This has led to an insufficient understanding of the importance of DNRA in NO3- transformations and the regulating factors of this process. The objective of this paper is to provide a detailed procedure for the measurement of the potential DNRA rate in environmental samples. In brief, the potential DNRA rate can be calculated from the 15N-labeled ammonium (15NH4+) accumulation rate in 15NO3- added incubation. The determination of the 14NH4+ and 15NH4+ concentrations described in this paper is comprised of the following steps. First, the NH4+ in the sample is extracted and trapped on an acidified glass filter as ammonium salt. Second, the trapped ammonium is eluted and oxidized to NO3- via persulfate oxidation. Third, the NO3- is converted to N2O via an N2O reductase deficient denitrifier. Finally, the converted N2O is analyzed using a previously developed quadrupole gas chromatography-mass spectrometry system. We applied this method to salt marsh sediments and calculated their potential DNRA rates, demonstrating that the proposed procedures allow a simple and more rapid determination compared to previously described methods.

Characterization of diverse heterocyclic amine-degrading denitrifying bacteria from various environments.

Although, there have been many published bacterial strains aerobically degrading the heterocyclic amine compounds, only one strain to date has been reported to degrade pyrrolidine under denitrifying conditions. In this study, denitrifying bacteria degrading pyrrolidine and piperidine were isolated from diverse geological and ecological origins through selective enrichment procedures. Based on the comparative sequence results of 16S rRNA genes, 30 heterocyclic amine-degrading isolates were grouped into ten distinct phylotypes belonging to the genera Thauera, Castellaniella, Rhizobium, or Paracoccus of the phylum Proteobacteria. The representative isolates of individual phylotypes were characterized by phylogenetic, phenotypic and chemotaxonomical traits, and dissimilatory nitrite reductase gene (nirK and nirS). All isolates completely degraded pyrrolidine and piperidine under both aerobic and anaerobic conditions. The anaerobic degradations were coupled to nitrate reduction. A metabolic pathway for the anaerobic degradation of pyrrolidine was proposed on the basis of enzyme activities implicated in pyrrolidine metabolism from three isolates. The three key pyrrolidine-metabolizing enzymes pyrrolidine dehydrogenase, gamma-aminobutyrate/alpha-ketoglutarate aminotransferase, and succinic semialdehyde dehydrogenase, were induced by heterocyclic amines under denitrifying conditions. They were also induced in cells grown aerobically on heterocyclic amines, suggesting that the anaerobic degradation of pyrrolidine shares the pathway with aerobic degradation.

Nitrogen removal by co-occurring methane oxidation, denitrification, aerobic ammonium oxidation, and anammox.

The pathway for removing NO(3)(-) and NH(4)(+) from wastewater in the presence of both CH(4) and O(2) was clarified by studying microbial activity and community. Batch incubation tests were performed to characterize the microbial activity of the sludge, which was acclimatized in a bioreactor in which O(2) and CH(4) were supplied to treat wastewater containing NO(3)(-) and NH(4)(+) . The tests showed that the sludge removed significant amounts of NO(3)(-) and NH(4)(+) in the presence of CH(4) and O(2), and the presence of the activity of methane oxidation, denitrification, nitrification, and anammox in the sludge. It was estimated that the total inorganic nitrogen removal was attributed to denitrification associated with methane oxidation as 53.4%, microbial assimilation as 37.9%, and anammox as 8.7%. Nitrification also contributed to NH(4)(+) decrease as 34.5% and anammox as 6.4%. Anammox activity was unambiguously demonstrated by (29)N(2) production in anaerobic batch incubation with (15)N-labeled inorganic nitrogen compounds. The presence of methane-oxidizing bacteria and candidate denitrifiers in the sludge was shown by denaturing gradient gel electrophoresis of 16S rRNA gene fragments. Clone library analysis of the PCR-amplified 16S rRNA gene fragment using specific primers for aerobic ammonium oxidizer and anammox revealed the presence of these bacteria. The results reveal that complex nitrogen-removal processes occur in the presence of CH(4) and O(2) by methanotroph, denitrifier, aerobic ammonium oxidizer, and anammox.

Anaerobic ammonium oxidation (anammox) irreversibly inhibited by methanol.

Methanol inhibition of anaerobic ammonium oxidation (anammox) activity was characterized. An enrichment culture entrapped in a polyethylene glycol gel carrier was designed for practical uses of wastewater treatment. Batch experiments demonstrated that anammox activity decreased with increases in methanol concentration, and relative activity reached to 29% of the maximum when 5 mM methanol was added. Also, batch experiments were conducted using anammox sludge without immobilization. Anammox activity was evaluated by quantifying (14)N(15)N ((29)N) emission by combined gas chromatography-quadrupole mass spectrometry, and the anammox activity was found to be almost as sensitive to methanol as in the earlier trials in which gel carriers were used. These results indicated that methanol inhibition was less severe than previous studies. When methanol was added in the influent of continuous feeding system, relative activity was decreased to 46% after 80 h. Although the addition was halted, afterwards the anammox activity was not resumed in another 19 days of cultivation, suggesting that methanol inhibition to anammox activity was irreversible. It is notable that methanol inhibition was not observed if anammox activity was quiescent when substrate for anammox was not supplied. These results suggest that methanol itself is not inhibitory and may not directly inhibit the anammox activity.

Limitations of the use of group-specific primers in real-time PCR as appear from quantitative analyses of closely related ammonia-oxidising species.

To study the ecology of ammonia-oxidising bacteria (AOB), quantitative techniques are essential. Real-time PCR assays based on the 16S rRNA or on the structural amoA gene are routinely used. The CTO primer set rooted on the 16S rRNA gene has a number of mismatches with some of the cultures of AOB. To examine if these mismatches have an effect on the outcome of real-time PCR assays, the assay was tested with DNA from a number of closely related isolates of AOB. Standard curves of known amounts of initial DNA were similar among most of the tested cultures of AOB, except for the standard curves of Nitrosomonas strain AL212 and Nitrosospira strain NpAV. Nitrosomonas strain AL212 had 3 mismatches with the CTO primer set. Adaptation of the CTO primer set in order to perfectly match the Nitrosomonas strain AL212 gave a standard curve similar to the majority of the AOB tested. As Nitrosospira strain NpAV has no mismatches with the original CTO primer set, there must be another reason for the less efficient amplification than the sequence itself. Application of an existing sigmoidal mathematical model gave no other results with respect to the standard curves of Nitrosomonas europaea and Nitrosomonas strain AL212, but also demonstrated that primer mismatches can seriously underestimate the initial target concentration. It was concluded that in general correct interpretation of real-time PCR results requires knowledge of the target community composition, in particular of the target sequences of the dominant community members.

Quantitative evaluation of inhibitory effect of various substances on anaerobic ammonia oxidation (anammox).

The inhibitory effect of 20 substances of various chemical species on the anaerobic ammonia oxidation (anammox) activity of an enrichment culture, predominated by Candidatus Brocadia, was determined systematically by using a 15N tracer technique. The initial anammox rate was determined during first 25 min with a small-scale anaerobic batch incubation supplemented with possible inhibitors. Although Cu2+ and Mn2+ did not inhibit anammox, the remaining 18 substances [Ni2+, Zn2+, Co2+, [Formula: see text] , Fe2+, 4 amines, ethylenediaminetetraacetic acid (EDTA), ethylenediamine-N,N'-bis (2-hydroxyphenylacetic acid) (EDDHA), citric acid, nitrilotriacetic acid (NTA), N,N-dimethylacetamide (DMA), 1,4-dioxane, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and tetrahydrofuran (THF)] were inhibitory. Inhibitory effect of NTA, EDDHA, THF, DMF, DMA and amines on anammox was first determined in this study. Inhibitory effects of metals were re-evaluated because chelators, which may interfere inhibitory effect, have been used to dissolve metal salts into assay solution. The relative anammox activities as a function of concentration of each substance were described successfully (R2 > 0.91) either with a linear inhibition model or with a Michaelis-Menten-based inhibition model. IC50 values were estimated based on either model, and were compared. The IC50 values of the 4 chelators (0.06-2.7 mM) and 5 metal ions (0.02-1.09 mM) were significantly lower than those of the 4 amines (10.6-29.1 mM) and 5 organic solvents (3.5-82 mM). Although it did not show any inhibition within 25 min, 0.1 mM Cu2+ completely inhibited anammox activity in 240 min, suggesting that the inhibitory effect caused by Cu2+ is time-dependent.

Sporadic Inclusion Body Myositis Manifesting as Isolated Muscle Weakness of the Finger Flexors Three Years after Disease Onset.

Sporadic inclusion body myositis (sIBM) is a chronic progressive myopathy characterized by muscle weakness of both the quadriceps femoris and finger flexors. We herein present the case of a typical male patient with sIBM, which manifested as the isolated weakness of the finger flexors three years after the disease onset. We have identified several patients with sIBM in our cohort with muscle weakness of the flexors but not the quadriceps femoris. Examination of the flexor digitorum profundus muscle is important for the early and proper diagnosis of sIBM, even if a patient only presents with isolated finger flexor muscle weakness.

Complete genome of Nitrosospira briensis C-128, an ammonia-oxidizing bacterium from agricultural soil.

Nitrosospira briensis C-128 is an ammonia-oxidizing bacterium isolated from an acid agricultural soil. N. briensis C-128 was sequenced with PacBio RS technologies at the DOE-Joint Genome Institute through their Community Science Program (2010). The high-quality finished genome contains one chromosome of 3.21 Mb and no plasmids. We identified 3073 gene models, 3018 of which are protein coding. The two-way average nucleotide identity between the chromosomes of Nitrosospira multiformis ATCC 25196 and Nitrosospira briensis C-128 was found to be 77.2 %. Multiple copies of modules encoding chemolithotrophic metabolism were identified in their genomic context. The gene inventory supports chemolithotrophic metabolism with implications for function in soil environments.

An anaerobic continuous-flow fixed-bed reactor sustaining a 3-chlorobenzoate-degrading denitrifying population utilizing versatile electron donors and acceptors.

An anaerobic continuous-flow fixed-bed column reactor capable of degrading 3-chlorobenzoate (3-CBA) under denitrifying conditions was established, and its rate reached 2.26 mM d(-1). The denitrifying population completely degraded 3-CBA when supplied at 0.1-0.54 mM, but its activity was partly suppressed when 3-CBA was supplied at 0.89 mM. Nitrate was concomitantly consumed throughout the operation of the reactor, the amount of which was similar to or up to 35% higher than the theoretical stoichiometric value that was calculated by assuming that 3-CBA degradation is coupled with denitrification. Batch incubation experiments proved that nitrate is strictly required for 3-CBA degradation in the absence of molecular oxygen. The population also degraded 3-CBA aerobically. Benzoate and 4-CBA were degraded under denitrifying conditions as well as 3-CBA, but 2-CBA was not. Considering that the previously reported denitrifying 3-CBA-degrading cultures do not exhibit 4-CBA degradation under denitrifying conditions, nor aerobic 3-CBA degradation [FEMS Microbiol. Lett. 144 (1996) 213, Appl. Environ. Microbiol. 66 (2000) 3446], the microbial population developed in this experiment was physiologically versatile with respect to the utilization of both electron donors and electron acceptors.