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Notoginsenoside R1 (NGR1) Attenuates Chronic Atrophic Gastritis in Rats

Background This study investigated the effect and mechanism of notoginsenoside R1 (NGR1) on chronic atrophic gastritis (CAG) in a rat model. Material/Methods To perform our investigation, a rat model of CAG was established, and then rats were treated with various doses of NGR1. After treatment, hematoxylin-eosin (HE) staining was used for histopathological observation and further scoring. Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of gastrointestinal hormones, inflammatory factors, gastric mucosal destruction factors, and gastric mucosal-protective factors. Gene and protein expressions were measured using quantitative real-time PCR and Western blot assay, respectively. Results Results indicated that NGR1 relieved rat CAG. NGR1 treatment significantly increased the levels of gastrin (GAS) and somatostatin (SS) and reduced motilin (MTL) in the serum of CAG rats. The serum levels of interleukin (IL)-1β and IL-6 were significantly reduced by NGR1 treatment in CAG rats in a dose-dependent manner. Additionally, the increased levels of prostaglandin (PG)E2, nitric oxide synthase (NOS), and endothelin (ET) in CAG rats were significantly decreased by NGR1 administration. Moreover, the decreased level of secretory IgA (sIgA) and glutathione (GSH) in rats caused by MNNG was notably increased by NGR1 treatment. No significant changes were found in glutathione disulfide (GSSG) secretion. Finally, we found that the increased Bcl-2 expression and reduced Bax expression in the stomach tissues of rats caused by MNNG were eliminated by NGR1 treatment. Conclusions NGR1 exerts a protective effect on CAG, and it is a multi-target, multi-linked, comprehensive process.

Background
Chronic atrophic gastritis (CAG) is one of the most common digestive diseases in the world, and it is characterized by atrophy of gastric mucosal epithelium and glands, reduced number of glands, thinning of gastric mucosa, and thickening of mucosal basal layer, or is accompanied by pyloric adenosis and intestinal metaplasia, as well as dysplasia [1]. CAG is often clinically manifested as fullness, belching, abdominal pain, loss of appetite, and weight loss. CAG is a well-known sign of precancerous lesions of gastric cancer, which is ranked fourth in cancer incidence and is currently the second most common cause of cancer-related deaths worldwide [2,3). In recent years, the incidence of CAG has been rising year by year and the average age of patients is tending to be younger [4]. Therefore, active treatment of CAG is an effective means of reducing cancer and preventing gastric cancer. The pathogenesis of CAG is not clear, but it is generally believed that it is caused by repeated damage to the gastric mucosa caused by a variety of endogenous and exogenous factors (such as emotional stress, physicochemical factors, and microbial infection) [5]. In-depth studies on the pathogenesis of CAG have focused increased attention on the treatment of CAG. However, there is no effective treatment for CAG other than drug treatments such as H. pylori eradication, acid suppression, and non-steroidal antiinflammatory drugs [2,[6][7][8]. Therefore, it is of great importance to develop more effective CAG treatments.
Notoginsenoside R1 (NGR1) is the major phytoestrogen extracted from the plant P. notoginseng; it exhibits antiapoptotic, anti-oxidant, and anti-inflammatory properties [9]. Recent studies have shown that NGR1 plays an important role in the treatment of cardiac dysfunction [10,11], acute liver failure [12], and diabetic nephropathy [13]. However, to the best of our knowledge, the effect of NGR1 on CAG is still unclear. Therefore, we performed the present study. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a carcinogen that has been widely used in recent years, and it can directly act on epithelial cells of the stomach without affecting enzymes [14]. MNNG simulates improper intake of nitrate, which can transform into carcinogenic substances such as nitrite in the stomach, which lead to chronic atrophic gastritis and precancerous lesions or even gastric cancer [14][15][16][17]. MNNG has a strong mutagenic ability, and can cause mutations in bases in the original DNA chain, leading to cancer [18]. At present, MNNG is widely used in the establishment of animal models of CAG [15][16][17]. Therefore, in the present study, a CAG rat model was established by administration of MNNG.
The purpose of our study was to investigate the effect of NGR1 on CAG in a rat model and to explore the underlying mechanism. We hope to provide a stronger theoretical basis for the treatment of CAG.

Animals
We obtained 60 Sprague-Dawley (SD) rats (age: 8 weeks; body weight: 200±20 g) from the Vital River Company (Beijing, China). All rats were kept under specific pathogen-free (SPF) conditions with a 12 h light/dark cycle and free access to water and food. Our study was performed according to the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals and was approved by the Ethics Committee of the Second Affiliated Hospital of Nanjing University of Chinese Medicine (Second Hospital of Jiangsu Province).

CAG model establishment and TNR1 treatment
After 1 week of adaptation, the rats were randomly divided into 6 groups (n=10 per group): 1) control group; 2) CAG model group; 3) CAG+Vehicle group; 4) CAG+TGR1-5 group; 5) CAG+TGR1-10 group; and 6) CAG+TGR1-20 group. The CAG rat model was established by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) combined with irregular diet for 12 weeks according to previous studies (15,16). Rats in the CAG+Vehicle and CAG+NGR1 groups were administrated vehicle (sterile distilled water, by gavage) and various doses of NGR1 (5, 10, and 20 mg/kg/day, by gavage) [19] for 60 consecutive days. From week 12, all rats in each group were fed normally and sterile water was given ad libitum. Rats were weighed weekly and their weight changes were recorded. After NGR1 treatment was finished, all rats were anesthetized and killed and their stomachs were quickly removed, then abdominal aortic blood was collected for experiments. NGR1 (purity >98%) was from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO).

Histopathological observation
Gastric tissues were collected from rats of each group, then the tissue samples were fixed with 10% formalin (Invitrogen) and embedded in paraffin, followed by sectioning (5-μm thickness) and hematoxylin and eosin (HE) staining. Finally, we observed and analyzed the sections by using light microscopy. The histological score was assessed according to the diagnostic criteria of gastritis in Houston in 1996 [20]. We calculated the inflammatory score in 10 microscopic fields of each section and scored them on a 4-point scale [16,21] (0=normal; 1=mild, small amount of inflammatory cells infiltrating into the depression or basal area of gastric glands; 2=moderate, moderate amount of inflammatory cell infiltration, which is located in two-thirds of the gastric gland; and 3=significant, massive infiltration of inflammatory cells throughout the gastric gland). The atrophy score of gastric glands was also calculated in 10 microscopic fields of each section and scored on a 4-point scale (2) (0=normal; 1=mild, atrophy less than onethird of the stomach gland; 2=moderate, atrophy is limited to two-thirds of the stomach glands; and 3=significant, atrophy more than two-thirds of the stomach glands). The histological score in this study included the inflammation score and the atrophy score.

Enzyme-linked immunosorbent assay (ELISA)
All rats in each group were anesthetized and abdominal aortic blood were collected. Blood was centrifuged (3000 rpm, 10 min) to collect the serum. Then, the level of GAS (Cat no.

Western blot assay
Total proteins from stomach tissues of rats from each group were extracted by using RIPA buffer (Beyotime Institute of Biotechnology) with a protease/phosphatase inhibitor cocktail (Cell Signaling Technology). We used the bicinchoninic acid assay kit (BCA; Pierce Biotechnology, Rockford, IL, USA) to detect the protein concentrations according to the manufacturer's instructions. Equal amounts of protein samples (25 μg/lane) were    The relative mRNA expression level was calculated using the 2 -DDCT method [22].

Statistical analysis
Data are displayed as mean ±SD. SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. We used t tests or one-way ANOVA followed by NSK tests to analyze differences between groups. A value of P<0.05 was considered statistically significant.

Results
Alleviating effects of NGR1 on rat CAG As shown in Figure 1A, compared with the control group, the body weights in the CAG model group were significantly lower at week 8, week 12, and 60 days after treatment. However, after 60 days of NGR1 treatment, the body weights in the NGR1 treatment groups were significantly higher than that in the CAG model group. As shown in Figure 1B-1D, compared with the control group, the inflammatory score, atrophy score, and histological score in the CAG model group were significantly increased, and these increases were eliminated by NGR1 treatment. These results indicate that NGR1 treatment had a significant effect in protecting against CAG in rats.

Effects of NGR1 on gastrointestinal hormones (GAS, SS, MTL) in MNNG-induced CAG Rats
We assessed the levels of gastrointestinal hormones (GAS, SS, MTL) in serum of rats by using ELISA. As shown in Figure 2, compared with the control group, the levels of GAS and SS were significantly reduced and MTL was significantly increased in rats in the MNNG treatment alone group. However, NGR1 treatment significantly increased the levels of GAS and SS and reduced MTL level in the serum of CAG rats in a dose-dependent way.

Effects of NGR1 on inflammatory factors (IL-1b, IL-6) in MNNG-induced CAG Rats
CAG is a process of chronic inflammation of the stomach mucosa; thus, inflammation plays critical roles in the development of CAG. Therefore, we assessed the effect of NGR1 on the expression of inflammatory factors. As shown in Figure 3, the serum levels of IL-1b and IL-6 in rats were significantly enhanced by MNNG treatment, and these enhancements were notably reduced by NGR1 treatment in a dose-dependent manner.

Effects of NGR1 on gastric mucosal destruction factors (PGE2, NOS, ET) in MNNG-induced CAG Rats
The expression levels of gastric mucosal destruction factors including PGE2, NOS, and ET were analyzed in the present study. The findings suggested that compared with the control group, the serum levels of PGE2, NOS, and ET were significantly increased in the CAG model group. As expected, NGR1 treatment dose-dependently reduced the serum levels of PGE2, NOS, and ET in CAG rats (Figure 4).

Effects of NGR1 on gastric mucosa protective factors (sIgA, GSSG, and GSH) in MNNG-induced CAG Rats
As shown in Figure 5, our results showed that MNNG administration significantly decreased the levels of sIgA and GSH in the serum of rats, and these changes were notably inhibited by NGR1 treatment in a dose-dependent manner. No significant changes were found in the level of GSSG in different groups.

Effects of NGR1 on the expression of Bcl-2 and Bax in MNNG-induced CAG Rats
Finally, the anti-apoptotic gene Bcl-2 and pro-apoptotic gene Bax were analyzed. We found that the increased Bcl-2 expression and reduced Bax expression in the stomach tissues of rats caused by MNNG were dose-dependently eliminated by NGR1 treatment (Figure 6).

Discussion
We found that NGR1 administration relieved MNNG-induced pathological changes and symptoms of CAG in rats. Our findings suggest that NGR1 treatment affected the expression of gastrointestinal hormones (GAS, SS, MTL), inflammatory factors (IL-1b, IL-6), gastric mucosal destruction factors (PGE2, NOS, ET), and gastric mucosa-protective factors (sIgA and GSH) in the serum of CAG rats. Moreover, we found that Bcl-2 and Bax were involved in the influences of NGR1 on CAG. Our data show that NGR1 treatment mitigated the CAG induced by MNNG administration.
CAG is one of the most common digestive diseases worldwide [1]. In recent years, the incidence of CAG has been rising year by year and the average of patients is becoming younger [4]. CAG severely affects the health and quality of life , and ET (C) expression in serum of rats with or without CAG were detected using ELISA. Control: rats without any treatment; CAG Model: rats were treated with MNNG; CAG+Vehicle: rats were treated with MNNG and administered sterile distilled water; CAG+TGR1-5: rats were treated with MNNG and administered 5 mg/kg/day NGR1; CAG+TGR1-10: rats were treated with MNNG and administered 10 mg/kg/day NGR1; CAG+TGR1-20: rats were treated with MNNG and administered 20 mg/kg/day NGR1. Data are displayed as mean ±SD. ** p<0.01 vs. of patients [23,24]. However, there is still no effective treatment method for CAG. Finding new effective CAG treatments is clinically significant. NGR1 is the major phytoestrogen extracted from P. notoginseng, and increasing evidence indicates that NGR1 has a variety of pharmacological effects, including antiapoptotic, anti-oxidant, and anti-inflammatory properties [9]. More and more studies indicate that NGR1 has an important influence on the development of many diseases. Fang et al. reported that NGR1 prevented vascular smooth muscle cell growth, metastasis, and neointimal hyperplasia via regulating the PI3K/Akt pathway [25]. Tu et al. suggested that NGR1 plays a protective role in neonatal cerebral hypoxic-ischemic brain injury [26]. Zhai et al. indicated that NGR1 can relieve diabetic encephalopathy [27]. Zhao et al. reported that NGR1 prevented wear particle-induced periprosthetic osteolysis [28]. NGR1 was also found to ameliorate podocyte injury in diabetic nephropathy rats [19]. Another study indicated that NGR1 plays a protective role in myocardial inflammation through inhibiting cardiomyocyte inflammatory response and apoptosis [29]. However, the effect of NGR1 on CAG is still unclear. Therefore, we conducted the present study.
Firstly, to perform our investigation, a rat model of CAG was established by administration of MNNG. After treatment with various doses of NGR1, we found that NGR1 significantly increased the body weights of rats, which were reduced by MNNG treatment. NGR1 dose-dependently reduced the inflammation score, atrophy score, and histological score in the stomach tissues of CAG rats. These data show the protective role of NGR1 in CAG.    Figure 6. Effect of NGR1 on Bcl-2 and Bax expression the stomach tissues of rats with or without CAG. After specific treatment, the protein (A) and mRNA (B, C) level of Bcl-2 and Bax expression the stomach tissues of rats with or without CAG was detected using Western blot and qRT-PCR assay, respectively. Control: rats without any treatment; CAG Model: rats were treated with MNNG; CAG+Vehicle: rats were treated with MNNG and administered sterile distilled water; CAG+TGR1-5: rats were treated with MNNG and administered 5 mg/kg/day NGR1; CAG+TGR1-10: rats were treated with MNNG and administered 10 mg/kg/day NGR1; CAG+TGR1-20: rats were treated with MNNG and administered 20 mg/kg/day NGR1. Data are displayed as mean ±SD. ** p<0.01 vs. Control group; # , ## p<0.05, 0.01 vs. CAG model group.

GAS is produced by gastric antral G cells and is
secreted by endocrine cells (D cells) and has a wide inhibitory effect on the gastrointestinal system. With the development of CAG, gastric antrum tissue atrophy is aggravated, G cells gradually decrease, GAS content decreases, and SS also decreases. The dysfunction of GAS-SS-gastric acid secretion axis can accelerate atrophic lesions of gastric mucosal glands. MTL has a drastic effect on gastrointestinal motility and gastrointestinal electrical activity, aiding gastric emptying and food digestion. Our present study showed that the levels of GAS and SS were significantly reduced, and MTL level was significantly increased in rats in the MNNG treatment alone group. However, NGR1 treatment significantly increased the levels of GAS and SS, and reduced MTL level, indicating that NGR1 has a good regulatory effect on rat gastrointestinal hormones and is an important mechanism for NGR1 in the treatment of CAG.
Studies have shown that the secretion of pro-inflammatory cytokines (IL-1b and IL-6) plays an important role in the development of CAG [30]. The expression of IL-1b and IL-6 in the serum of CAG rats was analyzed in our study, and we found that the increased serum levels of IL-1b and IL-6 in CAG rats were significantly reduced by NGR1 treatment. These data suggest that NGR1 improves the degree of inflammation of the gastric mucosa by reducing IL-1b and IL-6 expression.
Next, we studied whether NGR1 has inhibitory effects on the factors causing or aggravating CAG, and the serum levels of PEG2, NOS, and ET were determined after NGR1 treatment. The results indicated that the serum levels of PEG2, NOS, and ET were significantly increased in the CAG rats, and NGR1 treatment dose-dependently reduced the serum levels of PEG2, NOS, and ET. This experiment confirmed that NGR1 inhibits the destruction of gastric mucosal destruction factor in CAG rats, reduces the degree of inflammation, ensures the blood supply to the gastric mucosa, maintains the stability of the internal environment of the body, and may be effective in treating CAG. Then, we explored whether NGR1 also plays a role in gastric mucosal protective factors (sIgA, GSH, and GSSG). Our findings show that MNNG administration significantly decreased the levels of sIgA and GSH in the serum of rats, and these changes were notably inhibited by NGR1 treatment in a dose-dependent manner. However, no significant changes were found in the level of GSSG in rats from different groups. NGR1 enhanced GSH expression, increased the ratio of GSH/ GSSG, scavenged oxygen free radicals, regulated immunity, and treated CAG.
Finally, as abnormalities of apoptosis and proliferation of gastric epithelial cells are involved in the progression of CAG, we analyzed whether the anti-apoptotic gene Bcl-2 and the proapoptotic gene Bax are involved in the effect of NGR1 on CAG. We found that the increased Bcl-2 expression and reduced Bax expression in the stomach tissues of rats caused by MNNG treatment were dose-dependently eliminated by NGR1 treatment. These data indicate that NGR1 promotes the normal apoptosis of gastric epithelial cells to accelerate its normal renewal and repair and prevent cancer damage.
Taken together, our results show that NGR1 relieved rat CAG through regulating secretion of gastrointestinal hormones, inhibiting production of inflammatory factors, reducing expression of gastric mucosal destruction factors, increasing expression of gastric mucosa protective factors, and regulating Bcl-2 and Bax expression. However, this study is only a preliminary study of the effect of NGR1 on atrophic gastritis. The effects and precise mechanisms of NGR1 in chronic atrophic gastritis need to be further explored, and we will conduct in-depth research on these issues in the future.

Conclusions
NGR1 can treat CAG, and the process is a multi-target and multi-linked process. Therefore, NGR1 is a promising agent for the treatment of CAG.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

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TET Enzymes and 5hmC in Adaptive and Innate Immune Systems

DNA methylation is an abundant and stable epigenetic modification that allows inheritance of information from parental to daughter cells. At active genomic regions, DNA methylation can be reversed by TET (Ten-eleven translocation) enzymes, which are responsible for fine-tuning methylation patterns. TET enzymes oxidize the methyl group of 5-methylcytosine (5mC) to yield 5-hydroxymethylcytosine (5hmC) and other oxidized methylcytosines, facilitating both passive and active demethylation. Increasing evidence has demonstrated the essential functions of TET enzymes in regulating gene expression, promoting cell differentiation, and suppressing tumor formation. In this review, we will focus on recent discoveries of the functions of TET enzymes in the development and function of lymphoid and myeloid cells. How TET activity can be modulated by metabolites, including vitamin C and 2-hydroxyglutarate, and its potential application in shaping the course of immune response will be discussed.

INTRODUCTION
Cells rely on the proper propagation and preservation of epigenetic information in order to regulate gene expression appropriately. 5-methylcytosine (5mC), described as the 5th base of DNA, is a chemically stable modification that is one of the most reliable ways of transmitting epigenetic information. In most cells, 5mC is present primarily at symmetrically-methylated CG dinucleotides in DNA, although methylation of cytosines in other contexts (CH=CA, CT, CC) has been reported in stem cells and in neurons (1). During DNA replication, methylated CGs are replaced by unmodified cytosines in the newly synthesized DNA strand, and the resulting hemimethylated CGs are recognized by a complex of UHRF1 and the maintenance methyl-transferase DNMT1 (2)(3)(4). The remethylation of hemi-methylated CpGs in newly replicated DNA is complete within 20 min, accounting for the stable inheritance of DNA methylation (5). In contrast to DNMT1, which depends on 5mC deposition at CpG motifs for maintenance DNA methylation, the de novo methyltransferases DNMT3A and DNMT3B can methylate unmodified cytosines in both CG and CH sequence contexts. While the writers for DNA methylation (DNMTs) have been known for decades, how DNA methylation is removed remained unclear until the discovery of TET (Ten-Eleven Translocation) enzymes and their ability to oxidize 5mC to 5-hydroxymethyl-cytosine (5hmC) [(6); reviewed in (3,4)].
5hmC, the so-called 6th base, is a stable epigenetic modification that accounts for 1-10% of 5mC depending on the cell type: ∼10% in embryonic stem cells (6) and as high as 40% in Purkinje neurons (7). While 5hmC or related modifications have been known to exist in simpler organisms including T-even phages for more than half a century (8), it was not until 2009 that 5hmC was rediscovered in mammalian cells (6,7). The mammalian enzymes responsible for generating this modification are the three TET dioxygenases (TET1, TET2, and TET3) that utilize the co-factors α-ketoglutarate (αKG), reduced iron (Fe 2+ ), and molecular oxygen to oxidize the methyl group at the 5 position of 5mC (6). TET proteins can be found in every metazoan organism that contains DNMTs, even simple organisms such as comb jellies (9)(10)(11).
Besides being a potential epigenetic mark, 5hmC is the key intermediate for TET-mediated active (replication-independent) and passive (replication-dependent) DNA demethylation FIGURE 1 | TET-mediated DNA modifications and demethylation. (A) Unmodified cytosine (C) is methylated by DNA methyltransferases (DNMTs) at the 5 position to become 5-methylcytosine (5mC). TET proteins oxidize 5mC into 5-hydroxymethylcytosine (5hmC), a stable epigenetic mark, and subsequently to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TET can demethylate DNA via replication-dependent (passive) or replication-independent (active) mechanisms. (B) Left, passive DNA demethylation. DNMT1/UHRF1 complex recognizes 5mC at the hemi-methylated CpG motif during DNA replication and methylates the unmodified cytosine on the newly synthesized DNA strand (left; pink strand). However, the oxidized methylcytosines 5hmC, 5fC, and 5caC (together, oxi-mC) are not recognized by DNMT1/UHRF1, resulting in unmodified cytosine on the new DNA strand. Further DNA replication in the presence of continuing TET activity will result in progressive dilution of 5mC in the daughter cells. Right panel, active DNA demethylation. While 5hmC is stable and persists in the genome, 5fC and 5caC can be recognized and excised by thymine DNA glycosylase (TDG), and the resulting abasic sites are repaired as unmodified C by base excision repair (BER). Other mechanisms (e.g., decarboxylation of 5caC) have been suggested but have not yet been proven to exist. (C) The approximate abundance of unmodified and modified cytosines in the haploid human/mouse genome. About 5% of cytosine is methylated (5mC); in most cells, the vast majority of 5mC is present at CG dinucleotides although it is low at CpG islands. 5hmC amounts to about 1-10% of 5mC (estimated at 10% here as in embryonic stem cells), while the levels of 5fC and 5caC are each about an order of magnitude lower than the previous oxidative modification. It is not known whether the low levels of 5fC and 5caC are due to features of TET enzymes that cause them to arrest at 5hmC, or to their continuing removal by TDG or other mechanisms. (Figure 1). TET enzymes iteratively oxidize 5mC and 5hmC into other oxidized cytosines (oxi-mCs) including 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (12); in active DNA demethylation, 5fC and 5caC are recognized and excised by thymine DNA glycosylase (TDG), repaired by the base-excision repair system, and replaced by unmodified C, thus resulting in DNA demethylation (13). In replication-dependent passive DNA demethylation, the DNMT1/UHRF1 complex does not recognize hemi-modified CGs with 5hmC, 5fC, or 5caC and thus the cytosine on the newly synthesized DNA strand is not methylated (5,14,15). Thus, the interplay between DNMT and TET proteins sculpts the DNA methylation landscape and TET proteins, with the co-factors Fe 2+ and α-ketoglutarate (αKG), use oxygen to oxidize 5mC into 5hmC, generating CO 2 and succinate as by-products. The enzymatic activity of TET can be modulated by additional factors. For instance, vitamin C (ascorbate) can enhance TET activity, potentially via reduction of the iron ion. On the other hand, the "oncometabolite" 2-hydroxyglutarate (2-HG), generated in acute myeloid leukemia and glioblastoma by recurrent dominant-active mutants of isocitrate dehydrogenase 1 or 2 (IDH1/2), inhibits TET activity. Furthermore, lack of oxygen in hypoxia also inhibits TET function. (B) Model of TET-mediated enhancer regulation. Prior to the commissioning of an enhancer, pioneer transcription factor (indicated as TF 1 ) binds to nucleosomal DNA and recruits TET which oxidizes the surrounding 5mC into 5hmC (and/or other oxi-mCs), facilitating DNA demethylation. TET proteins and, TF 1 promote enhancer accessibility by recruiting nucleosome remodeling complexes, thus allowing binding of secondary transcription factors (indicated here as TF 2 ) that are otherwise inhibited by DNA methylation or the presence of nucleosomes.
enables the flow of epigenetic information across cell generations.
DNA modification by TET proteins is essential for gene regulation (Figure 2). TET3 is expressed in the oocyte and the zygote; all three TET proteins are expressed in blastocysts; TET1 and TET2 are expressed in embryonic stem (ES) cells; and TET2 and TET3 are expressed ubiquitously in differentiated cells (3,4). The three TET enzymes appear to have overlapping but distinct targets in the genome. For instance, in mouse ES cells, TET2 rather than TET1 is responsible for the vast majority of 5hmC generation, and TET1 preferentially facilitates promoter demethylation while TET2 and TET3 act on enhancers (16,17). The longstanding association of high-level gene transcription with low levels of promoter methylation may be explained by TET-mediated conversion of 5mC to 5hmC at promoters, and subsequent DNA demethylation.
The genome-wide distribution of 5hmC reflects the strong association of TET enzymes with gene transcription. 5hmC is enriched at the most active enhancers and the gene bodies of the most highly transcribed genes (18). Moreover, multiple transcription factors important in cell differentiation and lineage specification, including NANOG, SALL4A, WT1, EBF1, PU.1, and E2A, have been shown to recruit TET proteins to specific genomic loci (primarily enhancers) for 5hmC modification, in most cases marking them for subsequent demethylation (19)(20)(21)(22)(23)(24). As a result, TET function is particularly essential for gene transcription during cell activation and lineage specification, and deficiencies of TET protein expression or activity result in skewed or arrested cell differentiation in multiple lineages, including those in neural and hematopoietic systems (25)(26)(27)(28)(29)(30).
TET loss-of-function is strongly connected to oncogenesis (31,32). Especially in the hematopoietic system, arrested or skewed cell differentiation is often associated with cell transformation (22,26). In humans, TET2 is one of the most frequently mutated genes in hematopoietic cancers of both myeloid and lymphoid origin (26). Using mouse models, we and other groups have shown that deletion of Tet2 alone, or deletion of both Tet2 and Tet3 (the two TET enzymes with the greatest overlap in expression and function), leads to myeloid or lymphoid expansion and the development of aggressive cancers with 100% penetrance (22,25,33). For instance, a striking example is the inducible deletion of both Tet2 and Tet3 in adult mice, which leads to acute myeloid leukemia with the mice succumbing as early as 3 weeks post-deletion (25). Since the role of TET proteins in malignancies has been reviewed extensively (26,(34)(35)(36), we will focus here on their roles in immune cell development and function. In the sections below, we outline our current understanding of the roles of TET proteins in regulating the adaptive and innate immune systems. The major findings are summarized in Figures 3, 4.

TET PROTEINS IN T AND B CELL DIFFERENTIATION AND FUNCTION
During development and immune responses, T and B cells continuously receive signals from antigen and cytokine receptors. These external signals converge and are interpreted by combinations of ubiquitously expressed and cell typespecific transcription factors, which function together with chromatin regulators to remodel the epigenome. The epigenetic changes associated with immune cell activation and differentiation include DNA and histone modifications, which allow information to be stored and/or inherited by daughter cells. As noted above, analyses of genome-wide 5hmC distribution reveal a close relationship between 5hmC and gene transcription. In thymic and peripheral T cell subsets, the level of 5hmC at gene bodies shows a striking positive correlation with the level of gene expression, as well as occupancy by RNA polymerase II and the level of H3K36me3 histone modification, an epigenetic mark reflective of RNA transcription into the gene body (17,18,50,51). Similarly, in lymphoid cells, 5hmC showed a strong positive correlation with enhancer activity, denoted by the level of H3K27 acetylation (H3K27ac), suggesting that TET is important for regulating enhancer function (16,18,52). Indeed, recent studies of T and B cells from our lab (see below) and others demonstrated that one of the functions of TET proteins is to facilitate chromatin accessibility at enhancers (22,28,33,37). TET-mediated conversion of 5mC to 5hmC potentially disrupts the binding of 5mC-binding proteins including MeCP2 and MBD (Methyl-CpG-binding domain) proteins, facilitating nucleosome remodeling and the binding of transcription factors (53,54). These changes in the epigenetic status of enhancers  (22,37). (B) Acute deletion of Tet2/3 using Cre ERT2 in B cells resulted in decreased Aicda expression and thus class switch recombination (28). (C) Deletion of Tet2 using Vav-Cre and Cd19-Cre resulted in hyperplasia of germinal center B cells. Vav-Cre-driven Tet2 deletion resulted in decreased plasma cell differentiation (38). (D) Cd4-cre Tet2/3-deficient mice exhibited skewed differentiation toward iNKT17 cells, partly due to decreased expression of Tbx21 and Zbtb7b expression, and a massive T-cell-receptor-dependent expansion of affected T cells (33). (E) Tet proteins facilitate the in vitro differentiation of naïve CD4 T cells to iTreg cells by demethylating Foxp3 enhancer CNS2, a process enhanced by the presence of vitamin C. All three TET proteins have a role in stabilizing the expression of Foxp3 in Treg cells in vivo (39,40). (F) CD4 T cells from Cd2-cre Tet2-deficient mice showed impaired Th1, Th2, and Th17 differentiation and cytokine production (41). (G) Increased differentiation of CD8 memory cells from Cd4-cre Tet2-deficient mice in response to lymphocytic choriomeningitis virus infection (42).
are likely transmitted to daughter cells, thus facilitating the establishment of lineage identity.

TET Proteins in B Cell Development and Function
From bone marrow progenitors to peripheral memory and plasma cells, the B cell genome undergoes progressive demethylation following differentiation (55). Furthermore, TET2 is one of the most frequently mutated genes (6-12%) in diffuse large B cell lymphoma, a malignancy originating from germinal center B cells (56)(57)(58). These observations suggest that TET proteins play an important role in B cell biology. Indeed, when Tet2 and Tet3 were deleted in early B cells during bone marrow development using Mb1-Cre, B cell differentiation was arrested at the transition from the pro-B to the pre-B stage (22,37) (Figure 3A). One function of these TET proteins during early B cell development is to regulate the arrangement  (43,44). (B) TET2 regulates the function of myeloid cells. In mouse and human macrophages, TET2 repressed expression of the pro-inflammatory cytokines IL-1β and IL-6 (45-48). TET2 was shown to associate with Iκbζ, bind to the Il6 promoter, recruit HDAC2, and repress Il6 expression (46). Tet2-deficient macrophages also expressed a high level of Socs3 mRNA, which in normal mice was suggested to be demethylated by TET2 and subsequently degraded by ADAR1 (44). In plasmacytoid dendritic cells, CXXC5 recruited TET2 to an intragenic CpG island in Irf7, facilitating the demethylation and maintaining basal expression. As a result, loss of Cxxc5, and to a lesser extent Tet2, resulted in decreased levels of IRF7, decreased type I interferon (IFN-I) production, and decreased anti-viral responses (49).
of the Ig kappa (Igκ) light chain genes, that pairs with the rearranged Ig heavy chain to form the complete B cell receptor. TET proteins regulate Igκ rearrangement by oxidizing 5mC at Igκ enhancers and facilitating their DNA demethylation and chromatin accessibility.
Mechanistically, TET proteins appear to be recruited to enhancers by "pioneer" transcription factors, defined by their ability to associate with their binding motifs on nucleosomebound DNA. Our data indicate that in pro-B cells, the pioneer transcription factor PU.1 binds to the Igκ enhancers prior to light chain rearrangement as a placeholder and recruits Tet proteins for DNA demethylation, facilitating the binding of additional B cell transcription factors including E2A ( Figure 3A). Tet proteins also regulate the expression of IRF4 and IRF8, both of which are important for Igκ rearrangement (22). Similar to the expansion phenotype observed in T cells (described in more detail below), Mb1-Cre Tet2/3-deficient mice developed massive expansion of immature B cells resembling acute lymphoblastic lymphoma (22). Therefore, Tet proteins are essential for B cell development by controlling the expression of multiple key genes.
In mature B cells, TET proteins are important for the antibody response. Recently we showed that B cell activation induced Tet protein expression and changes in the genome-wide distribution of 5hmC (the hydroxymethylome): lipopolysaccharide (LPS) and IL-4 stimulation induced a progressive TET-dependent hydroxymethylation at ∼8,000 regions in the span of 3 days (28). Functionally, the two major members in naïve B cells TET2 and TET3 are crucial for antibody class switch recombination (CSR) (Figure 3B). Acute deletion of Tet2 and Tet3 by Cre ERT2 resulted in an ∼50% decrease the expression of AID (Activation Induced Deaminase; encoded by Aicda), the critical enzyme for CSR; reconstitution of catalytically active AID in Tet2/3deficient B cells restored CSR. Interestingly, the CSR phenotype is reminiscent of that resulting from Aicda haploinsufficiency (59,60), suggesting that TET proteins are required for optimal expression of Aicda. Mechanistically, we showed that the transcription factor BATF recruits Tet proteins to the Aicda superenhancer, facilitating hydroxymethylation and chromatin accessibility of two Tet-responsive elements, TetE1 and TetE2, within the superenhancer and augmenting the expression of Aicda (28) (Figure 3B).
Recently, Vav-Cre and Cd19-Cre, which are expressed in the entire hematopoietic system and during B cell development, respectively, were used to show that disruption of Tet2 resulted in germinal center hyperplasia (38) (Figure 3C). However, germinal center B cells appeared to be normal in Tet2 deletion driven by Cγ 1-Cre, which is expressed in germinal center B cells. Consistent with our findings, Tet2 was shown to be required for CSR and affinity maturation of antibody ( Figure 3C). More importantly, TET2 positively regulated the expression of the transcription factor Prdm1 (encoding BLIMP1), and plasma cell differentiation was impaired in Tet2-deficient mice. Interestingly, the gene signature of TET2-deficient DLBCL resembles that of cells with mutations in the histone acetyltransferase CREBBP, suggesting that TET2 and CREBBP may cooperate to regulate enhancer H3K27 acetylation. Taken together, these observations demonstrate that TET proteins regulate multiple processes in B cells by preferentially strengthening the activity of enhancers, including individual enhancer elements located within superenhancers (Igκ and Aicda) (61).

TET Proteins in T Cell Development
Tet2 and Tet3 are expressed at higher levels than Tet1 in thymocytes and peripheral T cells, and are responsible for the majority of 5hmC modification in these cells. Deletion of Tet2 alone in the germline, in the hematopoietic system using Cd2cre, or in T cells (Cd4-cre) did not lead to any obvious defect in T cell development (41,42,62), suggesting that Tet3 was able to compensate for the loss of Tet2. Indeed, data from our lab showed that the deletion of both Tet2 and Tet3 in T cells using Cd4-cre caused a massive lymphoproliferative phenotype with enlarged spleen and lymph nodes, and the mice succumbed by 8 weeks of age (33). At 3-4 weeks of age, young Tet2/3 Cd4-cre DKO mice showed decreased thymic cellularity, a lower percentage of CD4 + CD8 + double positive cells, and an increased percentage of CD4 + and CD8 + single positive cells, phenotypes reminiscent of thymic atrophy induced by stress or inflammation. Further examination showed that the expanded cells in the periphery were invariant natural killer T (iNKT or NKT) cells that expressed the transcription factor Rorγt and produced IL-17 ( Figure 3D). These cells thus resemble the NKT17 subset, one of the three subsets of NKT cells besides NKT1 (T-bet-expressing) and NKT2 (Gata3-expressing). In contrast, NKT cells from wildtype mice are primarily of the NKT1 and NKT2 subsets (63).
Genome-wide analyses provided mechanistic explanations for the lineage skewing observed in Tet2/3 Cd4-cre DKO mice. Briefly, the profiles of transcriptome, whole-genome methylome, and chromatin accessibility showed that Tet2/3 deficiency resulted in decreased expression of Tbx21 (encoding T-bet) and Zbtb7b (encoding Th-POK), likely because of hypermethylation at the corresponding regulatory elements (Figure 3D). Both Tbet and Th-POK repress Rorc (encoding Rorγt) expression, thus the decreased levels of T-bet and Th-POK transcription factors in Tet2/3-deficient cells permitted increased Rorγt expression and skewed the cells to the NKT17 lineage (33). Interestingly, the Tet2/3-deficient iNKT cells were able to expand upon transfer to fully immunocompetent, wild-type (WT) but not Cd1d-deficient recipient mice (33), suggesting (i) that the expansion was secondary to recognition of "self " antigens presented by CD1d and (ii) that expansion was not suppressible by WT regulatory T (Treg) cells (see below). Together, these observations indicate that TET enzymes are important to maintain the proper expression of lineage-specifying transcription factors, and to limit the differentiation and proliferation of overly self-reactive cells including iNKT cells.

Maintenance of Foxp3 + Treg Cells Requires TET Proteins
TET enzymes are important for the homeostasis of T regulatory (Treg) cells, which are distinguished from other T cell lineages by their expression of the transcription factor FOXP3. In Treg cells, TET2 and TET3 are required for stable Foxp3 expression through their ability to demethylate two intronic enhancers, termed conserved non-coding sequence (CNS) 1 and CNS2 (39, 64) ( Figure 3E). Bisulfite sequencing showed that the Foxp3 CNS1 and CNS2 enhancers were hypermethylated in Treg cells from Tet2/3 Cd4-cre DKO mice (39). Moreover, overexpression of the TET1 catalytic domain in CD4 cells induced to differentiate into Foxp3-expressing induced Treg cells (iTreg) in vitro and resulted in partial demethylation of CNS2 (65), suggesting that TET enzymes may be in constant balance with the methylation machinery. Hypermethylation at Foxp3 CNS2 was also observed in Tet1/2-deficient mice, suggesting all three Tet proteins may function redundantly in regulating Foxp3 (40).
Several proteins have been identified to partner with TET proteins in regulating Foxp3 CNS2. For instance, loss of the DNA methyl-binding protein MBD2 also resulted in hypermethylation of CNS2 (also termed TSDR), potentially because of decreased TET2 binding (66). How MBD2 cooperates with TET to demethylate CNS2 remained to be determined. Besides MBD2, the transcription factors SMAD3 and STAT5, induced by TGFβ and IL-2 respectively, recruit TET proteins to Foxp3 CNS2 and facilitate DNA demethylation (40). In addition, the level of TCR and cytokine stimulation has been linked to the degree of DNA demethylation at Foxp3 CNS2 (67). Since there is only one functional allele of Foxp3 per Treg cell, this observation implies that stronger a TCR stimulation might increase the probability of TET-mediated DNA demethylation at Foxp3 CNS2 and concomitantly, the stability of Foxp3 expression.

TET Proteins Link Metabolism to Foxp3 Expression
The enzymatic activity of TET can be influenced by various factors, including the level of co-factors αKG, oxygen, and vitamin C (Figure 2). In a chemical screen using mouse embryonic stem cells, vitamin C was found to enhance the expression of gene expression in germ cells and ES cells by facilitating TET-mediated DNA demethylation at their promoters (68). Vitamin C treatment also facilitated TET-mediated demethylation of Foxp3 CNS2 and stabilized Foxp3 expression in differentiating induced Treg (iTreg) cells ( Figure 3E). Inhibition of the vitamin C transporter by sulfinpyrazone confirmed the role of vitamin C and TET proteins in CNS2 demethylation and the generation of peripheral Treg cells in vivo (69). In addition, vitamin C facilitated the conversion of mouse and human naïve CD4 T cells into iTreg cells induced by TGFβ and retinoic acid with improved stability and suppressive function (39). Besides vitamin C, another metabolite hydrogen sulfide (H 2 S) was shown to be required for Treg cell differentiation, at least in part by increasing Tet1 and Tet2 expression (40).
TET activity can be inhibited by the "oncometabolite" 2hydroxyglutarate (2-HG), a competitive inhibitor of αKGdependent dioxygenases including TET (70, 71) (Figure 2). 2-HG is a normal metabolite that exists as two stereoisomers, R-2-HG and S-2-HG; the latter is considerably more potent at inhibiting TET activity (72). In the past few years, it has become clear that 2-HG can be generated via multiple pathways; for instance, recurrent mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2) give rise to dominant-active enzymes with the novel property of converting isocitrate to the R enantiomer of 2-HG (R-2-HG) (70,71). A recent study identified a compound, (aminooxy)acetic acid (AOA), that is able to reprogram differentiating Th17 cells into Foxp3-expressing iTreg cells (73). Metabolic profiling identified the target of AOA in Th17 cells as GOT1 (glutamate oxaloacetate transaminase 1), an enzyme that catalyzes the conversion of glutamate to αKG. Th17 cells express a high level of GOT1 compared to iTreg cells, consistent with their elevated level of αKG. However, instead of facilitating the function of TET enzymes and other dioxygenases, the αKG is converted by wild-type IDH1/2 into R-2-HG, which inhibits TET activity, promotes increased methylation at Foxp3 CNS2, and represses Foxp3 expression. By targeting GOT1, the small molecule AOA effectively decreased the intracellular level of R-2-HG and allowed TET proteins to demethylate CNS2, favoring differentiation to iTreg cells at the expense of the Th17 lineage (73). Therefore, these observations suggest that, besides conveying signals from cell surface receptors, TET proteins also integrate environmental cues into the epigenome.

TET Proteins Regulate Peripheral T Cell Differentiation and Function
After stimulation and depending on the extracellular signal received, naïve CD4 T cells can differentiate into multiple lineages, including Th1, Th2, Th17, follicular T helper cells (Tfh), and Treg. Analysis of 5hmC distribution in peripheral T cells showed a positive correlation between gene expression level and 5hmC modification at gene bodies, including those of the lineage-specific transcription factor Tbx21 and Gata3 for Th1 and Th2 cells, respectively. This observation suggests that TET proteins may regulate the differentiation of peripheral T cells (18,41). Similar lineage-specific 5hmC modifications during Th1 and Th2 polarization were also reported in human CD4 T cells (74). Indeed, Tet2-deficient murine CD4 T cells produced less IFNγ and IL-17 when polarized in vitro to Th1 and Th17, respectively (41) (Figure 3F). Compared to WT cells, adoptively transferred Tet2-deficient CD4 T cells were more pathogenic in an experimental autoimmune encephalomyelitis (EAE) model, and immunization with myelin oligodendrocyte glycoprotein (MOG) peptide induced significantly less IFNγ and IL-10 but a similar level of IL-17 (41). These observations reinforce the idea that Tet proteins are important for proper lineage differentiation and gene expression.
Analysis of Tet2-deficient (Tet2 fl/fl Cd4-cre) CD8 T cells responding to infection with lymphocytic choriomeningitis virus (LCMV) showed increased LCMV gp33-specific memory precursor cells (KLRG1 − CD127 + ) and decreased short-lived effector cells (KLRG1 + CD127 − ) on day 8 post-infection (42) (Figure 3G). These memory-like cells expressed CD27, CD62L, and CXCR3, a phenotype similar to central memory cells, and persisted for at least 45 days post-infection with a higher level of Eomes compared to WT. Transfer of Tet2-deficient memory cells conferred better protection against gp33-expressing Listeria monocytogenes compared to WT memory cells, strongly suggesting that TET2 represses memory cell differentiation (42). In addition to TCR-induced TET protein expression (42), TET activity can also be modulated by physiologically produced 2-HG. CD8 T cells generate substantial levels of the potent 2-HG enantiomer "oncometabolite" S-2-HG as early as day two after TCR stimulation, coinciding with the decrease in 5hmC (75). Similar to genetic ablation of Tet2, S-2-HG treatment of CD8 T cells induced higher expression of Eomes and CD62L, markers for central memory cells. Surprisingly, OT-I CD8 T cells cultured in the presence of S-2-HG in vitro displayed enhanced survival and tumor clearance upon adoptive transfer in vivo (75), suggesting the effect of S-2-HG is long lasting by stably remodeling the epigenome.
In humans, TET loss-of-function was shown to have a major potentiating role in a case of cancer immunotherapy against B cell malignancy using T cells bearing the anti-CD19 chimeric antigen receptor (CAR). The patient bore a hypomorphic mutation in one allele of TET2, and the CAR lentivirus serendipitously became integrated into the other TET2 allele. The resulting profound loss of function of TET2 resulted in an almost monoclonal expansion of this particular CAR-T cell, and the patient went into complete remission (76). Thus the loss of TET2 activity resulting from insertional mutation of one TET2 allele due to lentiviral integration, combined with the preexisting hypomorphic mutation in the other TET2 allele, led to superior anti-tumor function and again conferred a central memory phenotype on the expanded CAR-T cells. Together, these observations show that TET proteins are important in regulating peripheral T cell differentiation.

TET2 Regulates Myeloid Differentiation
TET2 mutation has been closely linked to myeloid malignancies including myelodysplastic syndrome and acute myeloid leukemia in human (26). In mice, germline disruption of the Tet2 gene decreased the global level of 5hmC in hematopoietic stem cells (HSCs), enhanced HSC survival and proliferation, inhibited T, B, and erythroid differentiation, and biased differentiation toward the myeloid lineage (62). Similarly, knockdown of TET2 in human cord blood CD34 + progenitor cells decreased total 5hmC in the cells and skewed their differentiation toward the granulomonocytic lineage, specifically monocytes, at the expense of both lymphoid and erythroid lineages (77). These and other studies suggested that, compared to other lineages, the myeloid lineage requires less reconfiguration of the DNA methylome during differentiation and therefore is relatively unaffected in the absence of TET2.
Beyond HSC, TET2 also regulates the differentiation of mast cells (Figure 4A). In a model of in vitro mast cell differentiation in which bone marrow progenitors were cultured with IL-3, loss of Tet2 inhibited mast cell differentiation, decreased cytokine production, and induced aberrant hyperproliferation (43). Two major transcription factors involved in myeloid development, C/EBPα and C/EBPε, were up-regulated in Tet2deficient mast cells, and both contributed to the observed defect in differentiation. Similar to another observation in macrophages (see below), both catalytically active and inactive TET2 could partially rescue these phenotypic defects, suggesting that part of the function of TET2 is to maintain the structure of a repressive protein complex (43). In vivo, Tet2 is important for the expansion of mast cells induced by parasites (44).
Human monocytes can differentiate into macrophages (MACs), dendritic cells (DCs), and osteoclasts (OCs) in vitro depending on cytokine signals, and the epigenetic regulation of this process has been studied extensively (78). During postmitotic differentiation of DCs from monocytes, stimulation with cytokines GM-CSF and IL-4 induced DNA demethylation. Since these cells do not proliferate prior to differentiation, the mechanism of demethylation is assumed to involve an active replication-independent process. Similar observations were made during MAC and OC differentiation (20,(79)(80)(81). TET2-mediated oxidation of 5mC into 5hmC preceded and was required for DNA demethylation, which was accompanied by the presence of active histone modifications (H3K4me1, H3K4me3, H3/H4 acetylation) ( Figure 4A). In general, the degree of DNA demethylation at distal elements or promoters showed a loose positive correlation with gene expression with numerous exceptions, suggesting that additional mechanisms contributed to gene regulation, such as H3K27 methylation by the polycomb complex (82). In monocyte to DC differentiation, IL-4-activated STAT6 induced TET2-dependent demethylation, and this was important for acquiring the proper cell identity and priming the expression of inducible genes (e.g., IL1B, CCL20) (81). During monocyte to OC or to MAC differentiation, the transcription factor PU.1 was found to associate with both hypoand hypermethylated regions and to directly bind to TET2 as well as to the DNA methyltransferase DNMT3B (20). TET2 functioned together with thymine-DNA glycosylase (TDG), and to a lesser extent with activation-induced deaminase (AID), to hydroxymethylate and demethylate DNA, facilitating the establishment of cell-type-specific gene expression programs (83). The same study also showed that TET2 was responsible for recruiting the histone H3K4 methyltransferase SETD1A, and for increasing H3K4me3 modification at cell-type specific genes examined (83).
Together, these in vitro human studies showed that postmitotic myeloid cells utilize TET2 and TDG for replicationindependent, active DNA demethylation to establish cell-specific gene expression patterns or to prime gene for subsequent induction. Besides regulating lymphoid development, Tet proteins are required for the differentiation of multiple lineages of myeloid cells.

TET Proteins Regulate Immune Responses by Myeloid Cells
One function of TET proteins in normal myeloid cells appears to be the repression of inflammatory gene expression (Figure 4B). For instance, Tet2-deficient macrophages and dendritic cells expressed a higher level of IL-6 in response to stimulation (45,46). Mechanistically, TET2 was shown to associate with Iκbζ and bind to the Il6 promoter, recruiting the histone deacetylase HDAC2 and repressing Il6 expression. As discussed for mast cells above, the repression appeared to be independent of TET2 catalytic activity, suggesting that TET2 provided a structural scaffold for the formation of a repressive complex (46). Compared to WT controls, Tet2-deficient mice were more susceptible to endotoxin-induced septic shock and dextran sulfate sodium (DSS)-induced colitis, coincident with an increased IL-6 level (46).
TET proteins also repressed another inflammatory cytokine, IL-1β (47,48). Moreover, loss of Tet2 accelerated atherosclerosis development in a mouse model of low-density lipoprotein receptor (Ldrl) deficiency (47). Tet2-deficient macrophages increased IL-1β secretion via the NLRP3 inflammasome, the inhibition of which protects mice from atherosclerosis (47). Interestingly, IL-1R/MyD88 signaling was shown to induce Tet2 mRNA and protein expression in bone marrow-derived macrophages (84), suggesting a potential negative feedback loop controlling IL-1β expression by TET proteins. Lastly, TET2 facilitated immunosuppression by tumor-infiltrating myeloid cells in a melanoma model and loss of TET2 in myeloid cells inhibited melanoma growth in vivo (84), consistent with the role of TET proteins in suppressing inflammation in myeloid cells. TET proteins contribute to osteoclast differentiation and suppress inflammation, and osteoclast activation has been linked to rheumatoid arthritis (RA) (85), warranting further detailed investigation of the role of TET proteins in autoimmune and auto-inflammatory diseases.
TET proteins appear to have different functions in myeloid cells depending on the circumstances. For instance, TET proteins have been reported to promote myeloid immune responses and production of inflammatory cytokines rather than suppressing inflammation. Plasmacytoid dendritic cells (pDCs) are fast responders to infection and are able to produce a large quantity of type I interferon. This ability has previously been attributed to their high basal level of the transcription factor IRF7, the expression of which is regulated by an intronic CpG island (CGI) (49). TET2 is recruited to this locus by the zinc-finger protein CXXC5, and is required to maintain the demethylated status of the CGI (Figure 4B). As a result, mice deficient in Cxxc5, or to a lesser extent Tet2, were more vulnerable to infection by herpes simplex virus and vesicular stomatitis virus due to an impaired interferon response (49). Similarly, in a model of abdominal sepsis, Tet2 deficiency was shown to reduce infection-induced myelopoiesis with a decreased level of TNFα and chemokines (44). The authors suggested that instead of oxidizing DNA, TET2 repressed Socs3 expression by oxidizing methylcytosine in the 3 ′ untranslated region of Socs3 RNA, thereby facilitating ADAR1-mediated destabilization of the mRNA in a manner independent of the normal RNAediting function of ADAR1 (44) (Figure 4B). Although TET proteins are capable of oxidizing methylcytosine on RNA (86,87), whether TETs can demethylate RNA (i.e., replace 5mC with unmodified C) is still an open question as neither passive nor active mechanisms for DNA demethylation would apply in RNA (Figure 1).
Finally, it is worth noting that the phenotypes in Tet2deficient mice may be complicated due to environmental influences. Whole-body Tet2 deficiency was shown to result in a compromised intestinal barrier, allowing bacteria to translocate from the intestinal lumen to internal organs and induce IL-6 production and inflammation; in turn, the proinflammatory signal facilitated pre-leukemic myeloproliferation (88). Therefore, depending on the microbiota at a given facility, Tet2-deficient mice may display differing basal levels of inflammation, a feature that may account for the variable reported phenotypes of different strains of Tet2-deficient mice (26). Since most TET2 mutations in human are acquired somatically rather than through the germline, the extent to which inflammation plays a role in human myeloid neoplasms remains to be determined. Taken together, these studies provide clear evidence that TET proteins regulate innate immune responses in myeloid cells.

OUR CURRENT UNDERSTANDING OF TET-MEDIATED GENE REGULATION TET Regulation of Transcription Factor Expression in Immune System
Transcription factors have emerged as one of the major targets of TET-mediated regulation. For instance, TET2 is important for inducing Blimp1 expression in peripheral B cells by demethylating intronic CpGs (38). On the other hand, TET proteins may be required for repressing BCL6 expression. In the human BCL6 locus, DNA methylation of intragenic CpG islands at the first intron prevents CTCF binding and promotes BCL6 expression. DNA demethylation at these CpG islands allowed CTCF binding, resulting in repressed BCL6 expression (89). However, whether TET proteins regulate BCL6 expression remains to be demonstrated.
Many loci encoding transcription factors are heavily hydroxymethylated, including Tbx21, Zbtb7b, and Gata3 in iNKT and T cells (18,33,41). Loss of Tet2 alone, however, has no significant effect on Tbx21 expression in CD4 and CD8 T cells (41,42). It is likely that other TET proteins such as TET3 can compensate, since Tbx21 expression is decreased in iNKT cells that are deficient in both Tet2 and Tet3 (33). In contrast to Tbx21 which is decreased in TET-deficient iNKT cells, loss of TET activity, either by gene targeting or inhibition by 2-HG, facilitates Eomes expression in iNKT and CD8 T cells (33,42,75). Whether TET proteins directly regulate Tbx21 and Eomes expression by binding to regulatory elements in the Tbx21 and Eomes loci remains to be determined.

TET-Mediated Regulation of Enhancers
Consistent with the functions of TET proteins in gene regulation, enhancers are usually enriched in 5hmC. TET proteins can be recruited to specific regulatory elements through interaction with multiple transcription factors including NANOG, SALL4A, WT-1, PU.1, E2A, and EBF1 (19)(20)(21)(22)(23)(24). The pleiotropic interaction between TET proteins and transcription factors is reminiscent of histone acetyltransferase p300, which interacts with hundreds of transcription factors (90). Once recruited to enhancers, TET proteins can oxidize 5mC into 5hmC, marking enhancers for DNA demethylation.
TET-dependent DNA modifications potentially affect gene expression via at least two non-mutually exclusive mechanisms. First, 5hmC, other oxi-mCs, and the ensuing DNA demethylation increase chromatin accessibility (22,28,33). In this scenario, unmodified C and oxi-mC potentially relieve the nucleosome rigidity caused by DNA methylation (91,92); additionally, TET proteins may recruit nucleosome remodeling complexes to displace nucleosomes from enhancers. Second, TET-generated oxi-mC modifications may exert immediate effects on gene expression by modulating transcription factor binding, and TET proteins may also exert more long-term effects. Specifically, 5mC and oxi-mCs are known to modify the binding of several transcription factors with CG or TG dinucleotides in their recognition sequences (54). The methyl group of thymine is located at the 5th position, corresponding to the methyl group of 5mC. Thus, transcription factors with TG dinucleotides in their preferred binding sequences often also bind the same sequences with methylated CGs (93), and their DNA binding is likely to be modified by the presence of oxi-mCs. Other transcription factors, including WT1, can bind sequences containing 5caC in a CG context with higher affinity than the corresponding sequence with unmodified CG (94). The exact mechanisms of enhancer regulation by TET enzymes and oxi-mCs remain to be delineated.

TET-Mediated DNA Oxidation and Demethylation
TET proteins can oxidize 5mC into oxi-mCs and mediate DNA demethylation. Depending on the conditions, TET2 can iteratively oxidize 5mC to 5hmC and then to all other oxidized cytosines in a single encounter (95). However, in the genome, most 5mC oxidation appears to pause at 5hmC and to a lesser extent 5fC (Figure 1C), a notion supported by mass spectrometric analyses showing that both 5hmC and 5fC are rather stable in cells (96,97). It remains to be determined why 5hmC is the most abundant of the oxi-mCs. Two mechanisms (not mutually exclusive) may be involved: (i) TET-mediated oxidation preferentially arrests at 5hmC or 5fC; (ii) 5fC and 5caC, but not 5hmC, are continuously removed by TDG/ BER or by other mechanisms (Figure 1B). Regardless of the mechanism, the modified cytosines can facilitate active or passive demethylation and affect gene regulation. In addition, 5hmC may act as a bookmark to label CpG sites in cis-elements such as promoters, enhancers and insulators marked by CTCF binding (5,98,99) for subsequent demethylation upon cell division, thus affecting gene expression patterns in the daughter cells (a latent effect).
Potential Co-transcriptional 5hmC Modification 5hmC distribution at gene bodies is positively correlated with gene expression levels, suggesting that TET activity is coupled to transcription by RNA polymerase II (RNA pol II) (18). One of the possible links between TET and RNA pol II is via their mutual association with the histone H3K4 methyltransferase Set1/COMPASS complex (100). Another possible link between 5hmC and RNA transcription is via the gene body histone mark H3K36me3: the levels of 5hmC and H3K36me3 in gene bodies are positively correlated with one another and with gene expression. During transcription, the methyltransferase SETD2 associates with the phosphorylated C-terminal domain of RNA pol II and co-transcriptionally methylates H3K36 to yield H3K36me3 (94). H3K36me3 is subsequently recognized by the de novo DNA methyltransferases DNMT3B, and to a lesser extent DNMT3A, via the PWWP domain (101)(102)(103), mediating gene body DNA methylation. Since all three TET proteins have been shown to co-immunoprecipitate with the maintenance methyltransferase DNMT1, and all three DNMT proteins coimmunoprecipitate with TET2 (104), the extensive interaction between TET and DNMT may provide a possible mechanism for transcription-coupled 5hmC modification. The biological significance of gene body 5hmC modification remains to be determined.

Potential Model for TET-Mediated Asymmetric Cell-Fate Decision
Hypothetically, it may also be possible to facilitate asymmetric gene regulation by engineering an asymmetric distribution of DNA methylation between two daughter cells via strand-biased 5hmC modifications. In one potential scenario, 5mC bases at CpG motifs on one strand at a given locus are preferentially oxidized by TET into 5hmC, while the complementary strand remains as 5mC (e.g., the template strand during transcription). As a result, after cell division, the CpG motifs at the locus in one of the daughter cell will remain methylated because the DNMT1/UHRF1 complex restores symmetrical methylation; the CpG motifs in the other daughter cell will contain 5hmC and unmodified C. This is an attractive putative mechanism by which TET enzymes could regulate cell fate decisions.

HARNESSING THE POWER OF THE DARK SIDE FOR THE LIGHT SIDE
TET loss-of-function, either through genetic mutations or catalytic inhibition, has shown a strong causal relationship with multiple malignancies (31,32). TET deficiency appears to enhance cell survival and increase "stemness, " as in the case of TET-deficient HSCs which could be passaged for a much longer period of time in vitro and out-competed WT HSCs after transplantation in vivo. Interestingly, at least some of the phenotypes are reversible by re-introducing TET or enhancing the remaining TET activity by vitamin C (105), raising the possibility of temporarily inhibiting TET activity to enhance immune responses. In fact, two recent studies of human and mouse CD8 T cells provided supporting evidence for this approach. In both cases, TET2-deficiency facilitated the differentiation and expansion of CD8 T cells with central memory phenotype that could provide long-lasting protection against tumor and virus (discussed above). Using non-specific inhibitors such as the oncometabolite 2-HG or other TET-specific inhibitors that remain to be developed, it should be possible to inhibit TET activity and boost antigen-specific responses and immune cell expansion during vaccination or infusion of cancer-specific T cells. It would be of great interest to borrow the trick of losing TET function from cancer cells to arm immune cells with the superpower to fight against the cancer cells themselves and other pathogens.

CONCLUDING REMARKS
TET proteins and 5hmC were identified/rediscovered almost 10 years ago. Numerous studies have shown their importance in gene regulation, tumor suppression, and cell differentiation. Yet, much remains to be learned about TET and 5hmC. For instance, how do TET enzymes suppress cancer progression? How does TET-mediated DNA modification affect cell identity? What is the relative contribution of enzymatic activitydependent and -independent (structural) mechanisms to the functions of TET? Besides being intermediates for DNA demethylation, what is the function of 5hmC and other oxidized methylcytosines as potential epigenetic marks? Who are the "readers" of these epigenetic marks? Also, given their seemingly opposite functions, why do mutations of Tet and Dnmt3a/b result in similar phenotypes in hematopoiesis? Besides all these fundamental questions, modulating the activity of epigenetic regulating enzymes including TET proteins may provide a promising way to alter and to achieve the desired magnitude and direction of immune responses.


extfieldsofstudy: Medicine Biology

text:
Miracula Moenusica , a New Member of the Holocarpic Parasitoid Genus from the Invasive Freshwater Diatom Pleurosira Laevis

Holocarpic oomycetes are poorly known but widespread parasites in freshwater and marine ecosystems. Most of the holocarpic species seem to belong to clades that diverge before the two crown lineages of the oomycetes, the Saprolegniomycetes and the Peronosporomycetes. Recently, the genus Miracula was described to accommodate Miracula helgolandica, a holocarpic parasitoid of Pseudo-nitzschia diatoms, which received varying support for its placement as the earliest-diverging oomycete lineage. In the same phylogenetic reconstruction, Miracula helgolandica was grouped with some somewhat divergent sequences derived from environmental sequencing, indicating that Miracula would not remain monotypic. Here, a second species of Miracula is reported, which was found as a parasitoid in the limnic centric diatom Pleurosira leavis. Its life-cycle stages are described and depicted in this study and its phylogenetic placement in the genus Miracula revealed. As a consequence, the newly discovered species is introduced as Miracula moenusica.

INTRODUCTION
Despite their global distribution in various habitats, including streams, lakes, and oceans, holocarpic oomycetes are still poorly known (Scholz et al. 2016). However, these organisms play a pivotal role in the breakdown of plankton blooms, as parasitoids of multicellular and unicellular algae (Scholz et al. 2016, Raghukumar 2017, Buaya et al. 2017. Most work on diatom parasitoids has been published in the late 19 th and early 20 th century, with the monographic treatments of Karling (1942) and Sparrow (1960) pretty much reflecting the current knowledge of this group. Only recently, research interest in oomycete parasitoids of diatoms has increased again, leading to the phylogenetic characterisation of Lagenisma coscinodisci, a pathogen of centric diatoms of the genus Coscinodiscus (Thines et al. 2015a), and the description of two new diatom parasitoids, Olpidiopsis drebesii in Rhizosolenia spp. and Miracula helgolandica in species of the genus Pseudo-nitzschia (Buaya et al. 2017). While L. coscinodisci was found to belong to the early-diverging members of one of the two crown oomycete lineages, the Saprolegniomycetes, the other two parasitoids were branching below the Peronosporomycetes/ Saprolegniomycetes split. Olpidiopsis drebesii grouped loosely with other Olpidiopsis species on red algae, while Miracula helgolandica was inferred to likely be the most early-divergent oomycete lineage (Buaya et al. 2017). Both new species were grouped with several somewhat divergent environmental sequences, suggesting a widespread nature and the presence of additional, still undiscovered species. While screening for diatom-infecting oomycetes in water and sediment samples from the river Main, a tributary to the central to western European stream Rhine, an unusual parasitoid was found in Pleurosira leavis, an invasive species (Litchman 2010) which had not been reported as host for holocarpic oomycetes before. It was the aim of this study to characterise this pathogen in terms of phylogenetic relationships and life cycle and to clarify its taxonomic assignment.

Diatom sampling
In September 2018, sediment surface samples were taken from the banks of the river Main in Frankfurt am Main Germany, by scraping biofilms into 1 L plastic bottles, which were subsequently filled half with water from the river. Samples were brought to the laboratory and screened by pouring sediment suspension into 9-cm-diam Petri dishes and observing them at 50-100 × magnification on an inverted microscope (AE31, Motic, China). Infected diatom cells were transferred to droplets of tap water and observed at 400 × using a Zeiss Imager equipped with DIC and an AxioCam (Zeiss, Oberkochen, Germany). For phylogenetic investigations, around 20 infected filaments were collected in a 2 mL vial containing 1 mL of Ambion RNA Later TM solution (Sigma-Aldrich, Munich, Germany).

DNA extraction, PCR, and sequencing
For DNA extraction, the tube was centrifuged in a table centrifuge at 19 000 g for 2 min and the RNA Later was removed by pipetting. Subsequently, samples were disrupted, and DNA was extracted using the innuprep plant DNA extraction kit (analyticjena, Jena, Germany), as described earlier (Buaya et al. 2017). PCR for the amplification of partial small ribosomal subunit (18S nrDNA) and sequencing were performed as described in Buaya et al. (2017). Sequencing was done by the Laboratory Centre of the Senckenberg Biodiversity and Climate Research Centre, with the primers Euk573 and Euk1422 (Wang et al. 2014), which were also used in PCR. The consensus sequence of the parasite of Pleurosira laevis was deposited in GenBank under the accession number MK239934.

Phylogenetic inference
Sequences were added to the dataset of Buaya et al. 2017 and aligned using MUSCLE with standard settings in MEGA v. 5 (Tamura et al. 2011), except for using a gap opening penalty of -200 and a gap extension penalty of -4. Phylogenetic inference was done using RAxML v. 8 (Stamatakis 2014) with the GTRGAMMA model and running 1 000 bootstrap replicates for Maximum Likelihood analysis, and using MEGA v. 5 (Tamura et al. 2011) with the Tamura-Nei model and running 1 000 bootstrap replicates for Minimum Evolution analysis.

Life-cycle observation
Filaments infected with oomycete parasitoids were observed from September 2018 to November 2018, usually at low abundance (less than 5 % of filaments infested). The parasitoid becomes first visible near the central nucleus, rod-shaped, elongating towards the periphery. Subsequently the central part enlarges, giving the thalli a lemon-shaped appearance. Parasitoids remain at this shape for some time, steadily increasing in volume. Towards the end of this stage, chloroplasts degrade into irregular shapes and assume a reddish-brown colouration. Subsequently, a large, central vacuole is forming, the thallus again increasing in size, until almost filling the diatom cells. Within the cytoplasm, the formation of refractive structures can be observed, and zoospores start to mature. When compartmentation is almost concluded, tubular exit tubes with a slightly thickened base develop at or close to the girdle region and push between the valves. Zoospores begin moving within the mature thallus, and then the discharge tube ruptures at the apex, releasing roundish, biflagellate zoospores into the surrounding medium, which swim away from the host cell. After a few minutes, zoospores come to rest. If they assume movement again has not been seen. Frequently, a few zoospores come to rest within the empty thallus and take a globose shape. If they develop further into meioszoospores or if they start moving again has not been observed. The different stages of the life-cycle are illustrated in Fig. 1.

Phylogenetic inference
In the phylogenetic trees based on partial small ribosomal subunit sequences of the parasitoid of Pleurosira leavis grouped together with Miracula helgolandica and two sequences derived from environmental sequencing with maximum support in all analyses (Fig. 2). Collectively, they formed the earliest-diverging oomycete lineage, but without strong support. The parasitoid from the river Main was sister to all remaining lineages in Miracula, which were grouped together with maximum support in all analyses. Apart from Lagenisma coscinodisci, which grouped with other early diverging members of the Saprolegniomycetes, all other holocarpic parasitoids of algae and diatoms branched before the split of Peronosporomycetes and Saprolegniomycetes. These crown oomycete classes were grouped together with moderate to strong support. While the Peronosporomycetes and the crown Saprolegniomycetes were each grouped together with strong support, the sister-group relationship of the crown Saprolegniomycetes and the early-diverging lineages was only weakly supported. The branching order of the early-diverging subclades was not well resolved, but some of the groups received moderate (Haptoglossa and Eurychasma; Haliphthoros, Halocrusticia, and Halodaphnea), to strong support (phaeophyte parasitoids -Anisolpidium ectocarpi, Olpidiopsis drebesii, and three sequences derived from environmental sequencing). The parasitoids of red algae did not form a monophyletic assemblage, however, without support.

Taxonomy
Due to its unique development, diatom host and phylogenetic placement, a new species of Miracula is introduced here. Diagnosis: Differs from Miracula helgolandica by its lemon-shaped maturing thallus, its more elongated discharge tube, its host in Coscinodiscophyceae, and its occurrence in a freshwater habitat.

Miracula moenusica
Description: Thallus hyaline, normally one, rarely two to three, endobiotic in Pleurosira leavis, rod shaped when young, lemonshaped during maturation, expanding until large parts of the host cell are filled, up to 100 µm long; wall thin, smooth, colourless; zoospore cleavage from a large central vacuole; zoospores roundish to grape-seed-shaped, 2-3 µm in diameter, beginning movement within the thallus; exit tube single, with a somewhat thickened base, 4-6 µm wide, 4-8 µm long.
Typus: Germany, Hessen, Frankfurt, northern bank, in Coscinodiscophyceae in freshwater, leg. A. Buaya, Sep. 2018 (holotype specimen in the Herbarium Senckenbergianum under the accession number FR0046007). Ex-type sequence deposited in Gen-Bank under the accession number MK239934.
Known distribution: Germany, river Main.

DISCUSSION
While the past two decades have seen huge advances towards a natural system of the crown oomycetes, in particular for both obligate biotrophic plant pathogens (Constantinescu 1998, Constantinescu & Fatehi 2002, Göker et al. 2003, Voglmayr et al. 2004, Constantinescu et al. 2005, Thines & Spring 2005, Thines et al. 2006, 2007, 2015b, Voglmayr & Constantinescu 2008, Telle & Thines 2011) and cultivable Peronosporomycetes (Bala et al. 2010, Hulvey et al. 2010, Uzuhashi et al. 2010, Li et al. 2016, Bennett et al. 2017, Jung et al. 2017, many genera of the Saprolegniomycetes and even more that were assumed to belong to the early-diverging oomycetes have not been revised, so far (Beakes & Sekimoto 2009, Beakes et al. 2014, Beakes & Thines 2017. However, the finding that some oomycete lineages diverged before the two major classes (Hudspeth et al. 2003), has spurred some interest in holocarpic oomycetes and has revealed the genera Haptoglossa and Eurychasma as the earliest-diverging lineages (e.g. Sekimoto et al. 2008, Gachon et al. 2017, a placement that only recently has been contested by the holocarpic diatom parasitoid Miracula helgolandica (Buaya et al. 2017). Miracula helgolandica parasitises the filamentous diatoms of the genus Pseudo-nitzschia (Hanic et al. 2009, Buaya et al. 2017) and could not be assigned to any of the five holocarpic genera known to parasitize diatoms (Aphanomycopsis, Ectrogella, Lagenidium, Lagenisma, and Olpidiopsis), which was the reason the new genus Miracula had been introduced. Overall, the evolutionary diversity of oomycetes parasitising diatoms seems to be very high, as witnessed by the relatively many descriptions of such organisms in the second half of the 19 th and the first half of the 20 th century (Zopf 1884, Karling 1942, Sparrow 1960, Drebes 1968, and references therein). So far, sequence data are available only from Lagenisma, Miracula, and Olpidiopsis diatom parasitoids. For all these genera, sequences from environmental sequencing exist, suggesting the presence of additional species that await their discovery. The finding of a second member of the genus Miracula in this study supports this notion. That Miracula moenusica was found in a freshwater environment is another example of the wide ecological amplitude of water-borne oomycetes, in which the border between marine and freshwater environments has been crossed several times, e.g. in Haptoglossa (Beakes & Sekimoto 2009), Phytopythium (Thines 2014), and Halophytophthora (Yang & Hong 2014). Miracula moenusica bears some similarity to Ectrogella monostoma (Scherffel 1925, Sparrow 1960, in the central swellings of the thallus. However, the behaviour or the zoospores is rather olpidioid in the former species, as they swim away from the host after emergence, rather than encysting directly at the orifice for the formation of secondary zoospores in the latter species. As in addition to these differences, the host species, Pleurosira leavis, has not been reported as a host of oomycete parasitoids, it seems that the species has not been observed previously. The unexpected observation of a second species of Miracula in a freshwater diatom, as well as the recent finding of a marine Olpidiopsis species in Rhizosolenia diatoms highlights that the diversity of holocarpic oomycetes is largely uncharted and promises to hold additional surprises for the future.


s2fieldsofstudy: Biology Medicine

text:
Successful rescue treatment of sepsis due to a pandrug-resistant, NDM-producing Klebsiella pneumoniae using aztreonam powder for nebulizer solution as intravenous therapy in combination with ceftazidime/avibactam

70% would seem appropriate. This is longer than the Gramnegative fT.MIC targets for cephalosporins of 30%–40% 7 and for carbapenems of 20%–35% and more similar to that for penicillins of 30%–60%. All the above data show the robustness of dilutional in vitro models in defining pharmacodynamic target sizes and their close alignment with animal models. Previous concerns about washout have also been shown to be baseless and indeed dilutional models may be superior to hollow-fibre systems in terms of cost-effectiveness, biofilm formation and modelled drug concentrations.

Sir, Pandrug-resistant Klebsiella pneumoniae that produces New Delhi MBL (NDM) is increasingly reported worldwide. 1 These strains contain multiple b-lactamase genes but also may have acquired resistance to last-resort options such as colistin and tigecycline. Combining aztreonam and avibactam is potentially effective in MDR, NDM-producing Enterobacterales. 2 Avibactam inhibits class A, C and D ESBLs, cephalosporinases and carbapenemases, while aztreonam is stable to hydrolysis by class B MBLs such as NDM. Until this drug combination becomes available, one could combine This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com aztreonam and ceftazidime/avibactam to treat serious infections with such strains. A small number of studies have reported on 13 patients with serious infections with NDM-producing Enterobacterales who were successfully treated with aztreonam and ceftazidime/avibactam. [3][4][5][6][7] Evidence of clinical efficacy and safety is therefore limited at present. Also, aztreonam for IV use is not registered and readily available in many countries, including the Netherlands. We describe successful rescue treatment of a patient with sepsis due to a pandrug-resistant, NDM-producing K. pneumoniae using aztreonam powder for nebulizer solution as IV therapy in combination with ceftazidime/avibactam.
A woman in her sixties who had received a kidney transplant 2 months earlier underwent routine urinary culture screening. The culture grew K. pneumoniae resistant to all antimicrobial classes ( Table 1). An in-house carbapenemase PCR showed that the strain carried an NDM carbapenemase gene. As she was asymptomatic, no treatment was started, but 9 days later she presented signs and symptoms of a kidney transplant pyelonephritis. We regarded aztreonam combined with ceftazidime/avibactam as the only promising treatment option for the pandrug-resistant K. pneumoniae. However, it was not clear whether we could import aztreonam for IV use to the Netherlands within a reasonable time. As the patient's condition deteriorated, we decided to administer aztreonam powder for nebulizer solution intravenously (1000 mg three-times daily, prepared from 14 vials of 75 mg, in extended infusion) in combination with ceftazidime/avibactam (2000 ! 500 mg three-times daily by continuous infusion). The subsequent day the blood culture became positive with the pandrug-resistant K. pneumoniae. Immunosuppressive therapy was reduced and the patient received supportive care for sepsis. Her condition improved within 1 day after the start of combination therapy and she recovered completely with 14 days of therapy without signs of adverse events. Aztreonam for IV solution was imported from France 11 days after our urgent request to the Dutch Government. The patient had one mild pyelonephritis recurrence with the same strain and unchanged susceptibility pattern 1 month later and recovered with the same treatment. She consented to the publication of this report. Table 1 shows phenotypic characteristics of the isolate. We assessed in vitro synergy by using gradient test superposition as previously described in this journal. 5 We compared MICs of the gradient test superposition with MICs of single gradient tests. We only found clinically relevant synergy (i.e. inhibition of the strain at drug concentrations below the breakpoints of both antimicrobials with gradient test superposition compared with single gradient tests) when combining aztreonam and ceftazidime/avibactam (Table 1).
Next-generation sequencing (HiSeq 2500 sequencer, BaseClear, Leiden, the Netherlands) reads were uploaded to the European nucleotide archive (accession number PRJEB33296) and used to perform resistome and replicome composition analysis (ResFinder, version 2.1, PlasmidFinder, version 1.3). These analyses showed that the isolate (MLST ST15) carried the bla NDM-1 carbapenemase gene, as well as the bla CMY-6 , bla CTX-M-15 , bla OXA-1 , bla OXA-10 , bla SHV-28 b-lactamase genes and aac(3)-IIa, aac(6 0 )Ib-3 resistance genes, among others, conferring resistance to all b-lactams, aminoglycosides and fluoroquinolones. Similar to a previously described comparable strain, we did not identify resistance genes conferring resistance to colistin and tigecycline. 1 We considered carefully before using aztreonam powder for nebulizer solution as off-label and unlicensed IV therapy. Aztreonam powder for nebulizer solution is a sterile product, without any additives that are known to be harmful. Also, it has a similar composition to the IV product. We expected that the benefits of the product, i.e. potential survival and no other treatment alternatives, weighed against potential risks of the product, i.e. unexpected side effects. Before providing aztreonam powder for nebulizer solution intravenously, we asked for consent from the patient and the medical director of our hospital.
In conclusion, we report successful rescue treatment of a patient with sepsis due to a pandrug-resistant, NDM-producing K. pneumoniae using aztreonam powder for nebulizer solution as IV therapy in combination with ceftazidime/avibactam and reducing immunosuppressive therapy. As such strains have been reported worldwide, we request the pharmaceutical industry to make aztreonam for IV use and ceftazidime/avibactam readily available in all countries. When aztreonam for IV use is not registered in a country, our case demonstrates that rescue treatment with aztreonam powder for nebulizer solution as IV therapy may be considered after careful assessment of the potential benefits and harms. Future studies are awaited to define the efficacy and safety of the promising treatment combination of aztreonam and avibactam in patients with serious infections due to pandrug-resistant, NDM-producing K. pneumoniae and other Enterobacterales.
Sir, Human cytomegalovirus (CMV) remains a serious complication of HSCT. In 2017, letermovir was approved for prophylaxis of CMV infection for high-risk patients following allogeneic HSCT. 1,2 Letermovir is an inhibitor of CYP3A4 and inducer of CYP2C19/2C9, which are common enzymatic pathways for many medications used in HSCT, including voriconazole. [2][3][4] Voriconazole is metabolized by CYP2C9 and CYP2C19, and co-administration with letermovir may lead to reduced voriconazole exposure through induction of these pathways. 3,4 In a study of healthy subjects who received letermovir 480 mg daily with voriconazole, voriconazole AUC and maximum serum concentration were reduced by 44% and 39%, respectively. 4 In addition, interpatient variability can be significant, with plasma concentrations of voriconazole varying up to 100-fold between patients. 5 Although letermovir is known to reduce voriconazole exposure, there are limited published data describing the implications of this interaction in clinical practice. Here, we report two cases of a clinically significant drug interaction between voriconazole and letermovir.