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Reactive oxygen species (ROS) play a critical role in modulating a range of proinflammatory functions in neutrophils, as well as regulating neutrophil apoptosis and facilitating the resolution of an inflammatory response. Selenoproteins with the 21st amino acid, selenocysteine (Sec), regulate immune mechanisms through the modulation of redox homeostasis aiding in the efficient resolution of inflammation, while their role in neutrophil functions during diseases remains unclear. To study the role of selenoproteins in neutrophils during infection, we challenged the granulocyte-specific tRNA Sec (Trsp) knockout mice (Trsp N ) with Citrobacter rodentium (C. rodentium), a murine pathogenic bacterium. Reduced bacterial shedding during the disease-clearing phase and increased tissue damage and neutrophil accumulation in the colon of the Trsp N mice were observed following infection. Trsp N neutrophils showed increased intracellular ROS accumulation during ex vivo C. rodentium stimulation and upregulated fMLP or Cx3cl1-induced chemotaxis. We also observed delayed neutrophil apoptosis, reduced efferocytosis of Trsp N neutrophils, and increased abundance of apoptotic cells in the colon of Trsp N mice. Together, these studies indicate that selenoprotein depletion results in increased neutrophil migration to the gut accompanied by ROS accumulation, while downregulating neutrophil apoptosis and subsequent efferocytosis by macrophages. Such an increase in inflammation followed by impaired resolution culminates in decreased bacterial load but with exacerbated host tissue damage., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: K. Sandeep Prabhu reports financial support was provided by National Institutes of Health. K. Sandeep Prabhu reports financial support was provided by National Institute of Food and Agriculture. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Exposure to persistent organic pollutants (POPs) and disruptions in the gastrointestinal microbiota have been positively correlated with a predisposition to factors such as obesity, metabolic syndrome, and type 2 diabetes; however, it is unclear how the microbiome contributes to this relationship., Objective: This study aimed to explore the association between early life exposure to a potent aryl hydrocarbon receptor (AHR) agonist and persistent disruptions in the microbiota, leading to impaired metabolic homeostasis later in life., Methods: This study used metagenomics, nuclear magnetic resonance (NMR)- and mass spectrometry (MS)-based metabolomics, and biochemical assays to analyze the gut microbiome composition and function, as well as the physiological and metabolic effects of early life exposure to 2,3,7,8-tetrachlorodibenzofuran (TCDF) in conventional, germ-free (GF), and Ahr -null mice. The impact of TCDF on Akkermansia muciniphila ( A. muciniphila ) in vitro was assessed using optical density (OD 600), flow cytometry, transcriptomics, and MS-based metabolomics., Results: TCDF-exposed mice exhibited lower abundances of A. muciniphila , lower levels of cecal short-chain fatty acids (SCFAs) and indole-3-lactic acid (ILA), as well as lower levels of the gut hormones glucagon-like peptide 1 (GLP-1) and peptide YY (PYY), findings suggestive of disruption in the gut microbiome community structure and function. Importantly, microbial and metabolic phenotypes associated with early life POP exposure were transferable to GF recipients in the absence of POP carry-over. In addition, AHR-independent interactions between POPs and the microbiota were observed, and they were significantly associated with growth, physiology, gene expression, and metabolic activity outcomes of A. muciniphila , supporting suppressed activity along the ILA pathway., Conclusions: These data obtained in a mouse model point to the complex effects of POPs on the host and microbiota, providing strong evidence that early life, short-term, and self-limiting POP exposure can adversely impact the microbiome, with effects persisting into later life with associated health implications. https://doi.org/10.1289/EHP13356.

Background & Aims: The intestinal epithelium interfaces with a diverse milieu of luminal contents while maintaining robust digestive and barrier functions. Facultative intestinal stem cells are cells that survive tissue injury and divide to re-establish the epithelium. Prior studies have shown autophagic state as functional marker of facultative intestinal stem cells, but regulatory mechanisms are not known. The current study evaluated a post-transcriptional regulation of autophagy as an important factor for facultative stem cell state and tissue regeneration., Methods: We evaluated stem cell composition, autophagic vesicle content, organoid formation, and in vivo regeneration in mice with intestinal epithelial deletion of the RNA binding protein IGF2 messenger RNA binding protein 1 (IMP1). The contribution of autophagy to resulting in vitro and in vivo phenotypes was evaluated via genetic inactivation of Atg7. Molecular analyses of IMP1 modulation of autophagy at the protein and transcript localization levels were performed using IMP1 mutant studies and single-molecule fluorescent in situ hybridization., Results: Epithelial Imp1 deletion reduced leucine rich repeat containing G protein coupled receptor 5 cell frequency but enhanced both organoid formation efficiency and in vivo regeneration after irradiation. We confirmed prior studies showing increased autophagy with IMP1 deletion. Deletion of Atg7 reversed the enhanced regeneration observed with Imp1 deletion. IMP1 deletion or mutation of IMP1 phosphorylation sites enhanced expression of essential autophagy protein microtubule-associated protein 1 light chain 3β. Furthermore, immunofluorescence imaging coupled with single-molecule fluorescent in situ hybridization showed IMP1 colocalization with MAP1LC3B transcripts at homeostasis. Stress induction led to decreased colocalization., Conclusions: Depletion of IMP1 enhances autophagy, which promotes intestinal regeneration via expansion of facultative intestinal stem cells., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Intestinal epithelial transit-amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite these cells' critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit-amplifying cell function. We report that RNA methyltransferase-like 3 (METTL3) is required for survival of transit-amplifying cells in the murine small intestine. Transit-amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Sequencing of polysome-bound and methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation verified a relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit-amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine with important implications for both homeostatic tissue renewal and epithelial regeneration.

Relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia-initiating stem cells (LICs) that are typically not targeted by most existing therapies. Using a murine AML model, human AML cell lines, and patient samples, we show that AML LICs are sensitive to endogenous and exogenous cyclopentenone prostaglandin-J (CyPG), Δ 12 -PGJ 2 , and 15d-PGJ 2 , which are increased upon dietary selenium supplementation via the cyclooxygenase-hematopoietic PGD synthase pathway. CyPGs are endogenous ligands for peroxisome proliferator-activated receptor gamma and GPR44 (CRTH2; PTGDR2). Deletion of GPR44 in a mouse model of AML exacerbated the disease suggesting that GPR44 activation mediates selenium-mediated apoptosis of LICs. Transcriptomic analysis of GPR44 -/- LICs indicated that GPR44 activation by CyPGs suppressed KRAS-mediated MAPK and PI3K/AKT/mTOR signaling pathways, to enhance apoptosis. Our studies show the role of GPR44, providing mechanistic underpinnings of the chemopreventive and chemotherapeutic properties of selenium and CyPGs in AML., Competing Interests: Declaration of interests K.S.P. and R.F.P. have ownership interest (including patents) in OncOmega Inc. and Nemean Pharma Corporation., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Ribosome profiling and sequencing of methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras . Further investigation confirmed a novel relationship between METTL3 and Kras methylation and protein levels in vivo . Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine, with important implications for both homeostatic tissue renewal and epithelial regeneration., Competing Interests: Conflict-of-interest statement Premal Shah is a member of the Scientific Advisory Board of Trestle Biosciences and is Director at Ananke Therapeutics. All other authors declare they have no competing interests.

Selenoprotein W (Selenow) is a ~9 kDa selenoprotein suggested to play a beneficial role in resolving inflammation. However, the underlying mechanisms are poorly understood. SELENOW expression in the human GI tract using ScRNAseq Gut Cell Atlas and Gene Expression Omnibus (GEO) databases revealed its expression in the small intestine and colonic epithelial, endothelial, mesenchymal, and stem cells and correlated with a protective effect in ulcerative colitis patients. Selenow KO mice treated with 4% dextran sodium sulfate (DSS) showed exacerbated acute colitis, with greater weight loss, shorter colons, and increased fecal occult blood compared to the WT counterparts. Selenow KO mice expressed higher colonic Tnfα, increased Tnfα + macrophages in the colonic lamina propria, and exhibited loss in epithelial barrier integrity and decreased zonula occludens 1 (Zo-1) expression following DSS treatment. Expression of epithelial cellular adhesion marker (EpCam), yes-associated protein 1 (Yap1), and epidermal growth factor receptor (Egfr) were decreased along with CD24lo cycling epithelial cells in Selenow KO mice. Colonic lysates and organoids confirmed a crosstalk between Egfr and Yap1 that was regulated by Selenow. Overall, our findings suggest Selenow expression is key for efficient resolution of inflammation in experimental colitis that is mediated through the regulation of Egfr and Yap1.

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells (LSCs). An experimental AML rodent model to test therapies based on successfully transplanting these cells via retro-orbital injections in recipient mice is fraught with challenges. The aim of this study was to develop an easy, reliable, and consistent method to generate a robust murine model of AML using an intra-peritoneal route. In the present protocol, bone marrow cells were transduced with a retrovirus expressing human MLL-AF9 fusion oncoprotein. The efficiency of lineage negative (Lin - ) and Lin - Sca-1 + c-Kit + (LSK) populations as donor LSCs in the development of primary AML was tested, and intra-peritoneal injection was adopted as a new method to generate AML. Comparison between intra-peritoneal and retro-orbital injections was done in serial transplantations to compare and contrast the two methods. Both Lin - and LSK cells transduced with human MLL-AF9 virus engrafted well in the bone marrow and spleen of recipients, leading to a full-blown AML. The intra-peritoneal injection of donor cells established AML in recipients upon serial transplantation, and the infiltration of AML cells was detected in the blood, bone marrow, spleen, and liver of recipients by flow cytometry, qPCR, and histological analyses. Thus, intra-peritoneal injection is an efficient method of AML induction using serial transplantation of donor leukemic cells.

Polymorphonuclear neutrophils (PMNs)-derived ROS are involved in the regulation of multiple functions of PMNs critical in both inflammation and its timely resolution. Selenium is an essential trace element that functions as a gatekeeper of cellular redox homeostasis in the form of selenoproteins. Despite their well-studied involvement in regulating functions of various immune cells, limited studies have focused on the regulation of selenoproteins in PMN and their associated functions. Ex-vivo treatment of murine primary bone marrow derived PMNs with bacterial endotoxin lipopolysaccharide (LPS) indicated temporal regulation of several selenoprotein genes at the mRNA level. However, only glutathione peroxidase 4 (Gpx4) was significantly upregulated, while Selenof, Selenow, and Gpx1 were significantly downregulated in a temporal manner at the protein level. Exposure of PMNs isolated from tRNA Sec (Trsp) fl/fl S100A8 Cre (Trsp N ) PMN-specific selenoprotein knockout mice, to the Gram-negative bacterium, Citrobacter rodentium, showed decreased bacterial growth, reduced phagocytosis, as well as impaired neutrophil extracellular trap (NET) formation ability, when compared to the wild-type PMNs. Increased extracellular ROS production upon LPS stimulation was also observed in Trsp N PMNs that was associated with upregulation of Alox12, Cox2, and iNOS, as well as proinflammatory cytokines such as TNFα and IL-1β. Our data indicate that the inhibition of selenoproteome expression results in alteration of PMN proinflammatory functions, suggesting a potential role of selenoproteins in the continuum of inflammation and resolution., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The essential micronutrient Selenium (Se) is co-translationally incorporated as selenocysteine into proteins. Selenoproteins contain one or more selenocysteines and are vital for optimum immunity. Interestingly, many pathogenic bacteria utilize Se for various biological processes suggesting that Se may play a role in bacterial pathogenesis. A previous study had speculated that Francisella tularensis , a facultative intracellular bacterium and the causative agent of tularemia, sequesters Se by upregulating Se-metabolism genes in type II alveolar epithelial cells. Therefore, we investigated the contribution of host vs. pathogen-associated selenoproteins in bacterial disease using F. tularensis as a model organism. We found that F. tularensis was devoid of any Se utilization traits, neither incorporated elemental Se, nor exhibited Se-dependent growth. However, 100% of Se-deficient mice (0.01 ppm Se), which express low levels of selenoproteins, succumbed to F. tularensis -live vaccine strain pulmonary challenge, whereas 50% of mice on Se-supplemented (0.4 ppm Se) and 25% of mice on Se-adequate (0.1 ppm Se) diet succumbed to infection. Median survival time for Se-deficient mice was 8 days post-infection while Se-supplemented and -adequate mice was 11.5 and >14 days post-infection, respectively. Se-deficient macrophages permitted significantly higher intracellular bacterial replication than Se-supplemented macrophages ex vivo , corroborating in vivo observations. Since Francisella replicates in alveolar macrophages during the acute phase of pneumonic infection, we hypothesized that macrophage-specific host selenoproteins may restrict replication and systemic spread of bacteria. F. tularensis infection led to an increased expression of several macrophage selenoproteins, suggesting their key role in limiting bacterial replication. Upon challenge with F. tularensis , mice lacking selenoproteins in macrophages (TrspM) displayed lower survival and increased bacterial burden in the lung and systemic tissues in comparison to WT littermate controls. Furthermore, macrophages from TrspM mice were unable to restrict bacterial replication ex vivo in comparison to macrophages from littermate controls. We herein describe a novel function of host macrophage-specific selenoproteins in restriction of intracellular bacterial replication. These data suggest that host selenoproteins may be considered as novel targets for modulating immune response to control a bacterial infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Markley, Restori, Katkere, Sumner, Nicol, Tyryshkina, Nettleford, Williamson, Place, Dewan, Shay, Carlson, Girirajan, Prabhu and Kirimanjeswara.)

Enteropathogenic Escherichia coli (EPEC) leads to adverse colonic inflammation associated with poor resolution of inflammation and loss of epithelial integrity. Micronutrient trace element selenium (Se) is incorporated into selenoproteins as the 21st amino acid, selenocysteine (Sec). Previous studies have shown that such an incorporation of Sec into the selenoproteome is key for the anti-inflammatory functions of Se in macrophages and other immune cells. An intriguing mechanism underlying the anti-inflammatory and pro-resolving effects of Se stems from the ability of selenoproteins to skew arachidonic acid metabolism from pro-inflammatory mediators, prostaglandin E 2 (PGE 2 ) toward anti-inflammatory mediators derived from PGD 2 , such as 15-deoxy-Δ 12, 14 - prostaglandin J 2 (15d-PGJ 2 ), via eicosanoid class switching of bioactive lipids. The impact of Se and such an eicosanoid-class switching mechanism was tested in an enteric infection model of gut inflammation by C. rodentium , a murine equivalent of EPEC. C57BL/6 mice deficient in Se (Se-D) experienced higher mortality when compared to those on Se adequate (0.08 ppm Se) and Se supplemented (0.4 ppm Se) diets following infection. Decreased survival was associated with decreased group 3 innate lymphoid cells (ILC3s) and T helper 17 (Th17) cells in colonic lamina propria of Se-D mice along with deceased expression of epithelial barrier protein Zo-1. Inhibition of metabolic inactivation of PGE 2 by 15-prostaglandin dehydrogenase blocked the Se-dependent increase in ILC3 and Th17 cells in addition to reducing epithelial barrier integrity, as seen by increased systemic levels of FITC-dextran following oral administration; while 15d-PGJ 2 administration in Se-D mice alleviated the effects by increasing ILC3 and Th17 cells. Mice lacking selenoproteins in monocyte/macrophages via the conditional deletion of the tRNA [Sec] showed increased mortality post infection. Our studies indicate a crucial role for dietary Se in the protection against inflammation following enteric infection via immune mechanisms involving epithelial barrier integrity., (Copyright © 2020 Nettleford, Zhao, Qian, Herold, Arner, Desai, Amin, Xiong, Singh, Carlson and Prabhu.)

Inflammatory bowel disease (IBD), characterized by severe flares and remissions, is a debilitating condition. While the etiology is unknown, many immune cells, such as macrophages, T cells and innate lymphoid cells, are implicated in the pathogenesis of the disease. Previous studies have shown the ability of micronutrient selenium (Se) and selenoproteins to impact inflammatory signaling pathways implicated in the pathogenesis of the disease. In particular, two transcription factors, nuclear factor-κB (NF-κB), and peroxisome proliferator activated receptor (PPAR)γ, which are involved in the activation of immune cells, and are also implicated in various stages of inflammation and resolution, respectively, are impacted by Se status. Available therapies for IBD produce detrimental side effects, resulting in the need for alternative therapies. Here, we review the current understanding of the role of NF-κB and PPARγ in the activation of immune cells during IBD, and how Se and selenoproteins modulate effective resolution of inflammation to be considered as a promising alternative to treat IBD., Competing Interests: The authors declare no conflict of interest.