Enzymatic Activity of HPGD in Treg Cells Suppresses Tconv Cells to Maintain Adipose Tissue Homeostasis and Prevent Metabolic Dysfunction.

Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.

Progesterone receptor mediates ovulatory transcription through RUNX transcription factor interactions and chromatin remodelling.

Progesterone receptor (PGR) plays diverse roles in reproductive tissues and thus coordinates mammalian fertility. In the ovary, rapid acute induction of PGR is the key determinant of ovulation through transcriptional control of a unique set of genes that culminates in follicle rupture. However, the molecular mechanisms for this specialized PGR function in ovulation is poorly understood. We have assembled a detailed genomic profile of PGR action through combined ATAC-seq, RNA-seq and ChIP-seq analysis in wildtype and isoform-specific PGR null mice. We demonstrate that stimulating ovulation rapidly reprograms chromatin accessibility in two-thirds of sites, correlating with altered gene expression. An ovary-specific PGR action involving interaction with RUNX transcription factors was observed with 70% of PGR-bound regions also bound by RUNX1. These transcriptional complexes direct PGR binding to proximal promoter regions. Additionally, direct PGR binding to the canonical NR3C motif enable chromatin accessibility. Together these PGR actions mediate induction of essential ovulatory genes. Our findings highlight a novel PGR transcriptional mechanism specific to ovulation, providing new targets for infertility treatments or new contraceptives that block ovulation.

RELA governs a network of islet-specific metabolic genes necessary for beta cell function.

AIMS/HYPOTHESIS: NF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role that NF-κB plays in normal metabolism. In this study we investigated how RELA impacts the beta cell transcriptional landscape and provides network control over glucoregulation. METHODS: We generated novel mouse lines harbouring beta cell-specific deletion of either the Rela gene, encoding the canonical NF-κB transcription factor p65 (ßp65KO mice), or the Ikbkg gene, encoding the NF-κB essential modulator NEMO (ßNEMOKO mice), as well as ßA20Tg mice that carry beta cell-specific and forced transgenic expression of the NF-κB-negative regulator gene Tnfaip3, which encodes the A20 protein. Mouse studies were complemented by bioinformatics analysis of human islet chromatin accessibility (assay for transposase-accessible chromatin with sequencing [ATAC-seq]), promoter capture Hi-C (pcHi-C) and p65 binding (chromatin immunoprecipitation-sequencing [ChIP-seq]) data to investigate genome-wide control of the human beta cell metabolic programme. RESULTS: Rela deficiency resulted in complete loss of stimulus-dependent inflammatory gene upregulation, consistent with its known role in governing inflammation. However, Rela deletion also rendered mice glucose intolerant because of functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as ßp65KO islets failed to secrete insulin ex vivo in response to a glucose challenge and were unable to restore metabolic control when transplanted into secondary chemical-induced hyperglycaemic recipients. Maintenance of glucose tolerance required Rela but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knockout of Ikbkg (NEMO), or beta cell overexpression of Tnfaip3 (A20), did not cause severe glucose intolerance. Thus, basal p65 activity has an essential and islet-intrinsic role in maintaining normal glucose homeostasis. Genome-wide bioinformatic mapping revealed the presence of p65 binding sites in the promoter regions of specific metabolic genes and in the majority of islet enhancer hubs (~70% of ~1300 hubs), which are responsible for shaping beta cell type-specific gene expression programmes. Indeed, the islet-specific metabolic genes Slc2a2, Capn9 and Pfkm identified within the large network of islet enhancer hub genes showed dysregulated expression in ßp65KO islets. CONCLUSIONS/INTERPRETATION: These data demonstrate an unappreciated role for RELA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism. These findings have clinical implications for the use of anti-inflammatories, which influence NF-κB activation and are associated with diabetes.

PKCζ activation promotes maturation of cord blood T cells towards a Th1 IFN-γ propensity.

A significant number of babies present transiently with low protein kinase C zeta (PKCζ) levels in cord blood T cells (CBTC), associated with reduced ability to transition from a neonatal Th2 to a mature Th1 cytokine bias, leading to a higher risk of developing allergic sensitisation, compared to neonates whose T cells have 'normal' PKCζ levels. However, the importance of PKCζ signalling in regulating their differentiation from a Th2 to a Th1 cytokine phenotype propensity remains undefined. To define the role of PKCζ signalling in the regulation of CBTC differentiation from a Th2 to a Th1cytokine phenotype we have developed a neonatal T cell maturation model which enables the cells to develop to CD45RA- /CD45RO+ T cells while maintaining the Th2 immature cytokine bias, despite having normal levels of PKCζ. The immature cells were treated with phytohaemagglutinin, but in addition with phorbol 12-myristate 13-acetate (PMA), an agonist which does not activate PKCζ. This was compared to development in CBTC in which the cells were transfected to express constitutively active PKCζ. The lack of PKCζ activation by PMA was monitored by western blot for phospho-PKCζ and translocation from cell cytosol to the membrane by confocal microscopy. The findings demonstrate that PMA fails to activate PKCζ in CBTC. The data show that CBTC matured under the influence of the PKC stimulator, PMA, maintain a Th2 cytokine bias, characterised by robust IL-4 and minimal interferon gamma production (IFN-γ), and lack of expression of transcriptional factor, T-bet. This was also reflected in the production of a range of other Th2/Th1 cytokines. Interestingly, introduction of a constitutively active PKCζ mutant into CBTC promoted development towards a Th1 profile with high IFN-γ production. The findings demonstrate that PKCζ signalling is essential for the immature neonatal T cells to transition from a Th2 to a Th1 cytokine production bias.

Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation.

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.

Long-term perturbation of the peripheral immune system months after SARS-CoV-2 infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious respiratory virus which is responsible for the coronavirus disease 2019 (COVID-19) pandemic. It is increasingly clear that recovered individuals, even those who had mild COVID-19, can suffer from persistent symptoms for many months after infection, a condition referred to as "long COVID", post-acute sequelae of COVID-19 (PASC), post-acute COVID-19 syndrome, or post COVID-19 condition. However, despite the plethora of research on COVID-19, relatively little is known about the molecular underpinnings of these long-term effects. METHODS: We have undertaken an integrated analysis of immune responses in blood at a transcriptional, cellular, and serological level at 12, 16, and 24 weeks post-infection (wpi) in 69 patients recovering from mild, moderate, severe, or critical COVID-19 in comparison to healthy uninfected controls. Twenty-one of these patients were referred to a long COVID clinic and > 50% reported ongoing symptoms more than 6 months post-infection. RESULTS: Anti-Spike and anti-RBD IgG responses were largely stable up to 24 wpi and correlated with disease severity. Deep immunophenotyping revealed significant differences in multiple innate (NK cells, LD neutrophils, CXCR3+ monocytes) and adaptive immune populations (T helper, T follicular helper, and regulatory T cells) in convalescent individuals compared to healthy controls, which were most strongly evident at 12 and 16 wpi. RNA sequencing revealed significant perturbations to gene expression in COVID-19 convalescents until at least 6 months post-infection. We also uncovered significant differences in the transcriptome at 24 wpi of convalescents who were referred to a long COVID clinic compared to those who were not. CONCLUSIONS: Variation in the rate of recovery from infection at a cellular and transcriptional level may explain the persistence of symptoms associated with long COVID in some individuals.

Associations between diet, the gut microbiome and short chain fatty acids in youth with islet autoimmunity and type 1 diabetes.

AIM: We aimed to characterize associations between diet and the gut microbiome and short chain fatty acid (SCFA) products in youth with islet autoimmunity or type 1 diabetes (IA/T1D) in comparison with controls. RESEARCH DESIGN AND METHODS: Eighty participants (25 diagnosed with T1D, 17 with confirmed IA, 38 sibling or unrelated controls) from the Australian T1D Gut Study cohort were studied (median [IQR] age 11.7 [8.9, 14.0] years, 43% female). A Food Frequency Questionnaire characterized daily macronutrient intake over the preceding 6 months. Plasma and fecal SCFA were measured by gas chromatography; gut microbiome composition and diversity by 16S rRNA gene sequencing. RESULTS: A 10 g increase in daily carbohydrate intake associated with higher plasma acetate in IA/T1D (adjusted estimate +5.2 (95% CI 1.1, 9.2) µmol/L p = 0.01) and controls (adjusted estimate +4.1 [95% CI 1.7, 8.5] µmol/L p = 0.04). A 5 g increase in total fat intake associated with lower plasma acetate in IA/T1D and controls. A 5% increase in noncore (junk) food intake associated with reduced richness (adjusted estimate -4.09 [95%CI -7.83, -0.35] p = .03) and evenness (-1.25 [95% CI -2.00, -0.49] p < 0.01) of the gut microbiome in IA/T1D. Fiber intake associated with community structure of the microbiome in IA/T1D. CONCLUSIONS: Modest increments in carbohydrate and fat intake associated with plasma acetate in all youth. Increased junk food intake associated with reduced diversity of the gut microbiome in IA/T1D alone. These associations with the gut microbiome in IA/T1D support future efforts to promote SCFA by using dietary interventions.

Thymus-Derived Regulatory T Cells Exhibit Foxp3 Epigenetic Modification and Phenotype Attenuation after Mating in Mice.

Regulatory T cells (Tregs) are essential for maternal tolerance in allogeneic pregnancy. In preeclampsia, Tregs are fewer and display aberrant phenotypes, particularly in the thymic Treg (tTreg) compartment, potentially because of insufficient priming to male partner alloantigens before conception. To investigate how tTregs as well as peripheral Tregs (pTregs) respond to male partner seminal fluid, Foxp3+CD4+ Tregs were examined in the uterus and uterus-draining lymph nodes in virgin estrus mice and 3.5 d postcoitum. Mating elicited 5-fold increases in uterine Tregs accompanied by extensive Treg proliferation in the uterus-draining lymph nodes, comprising 70% neuropilin 1+ tTregs and 30% neuropilin 1- pTregs. Proliferation marker Ki67 and suppressive competence markers Foxp3 and CTLA4 were induced after mating in both subsets, and Ki67, CTLA4, CD25, and GITR were higher in tTregs than in pTregs. Analysis by t-stochastic neighbor embedding confirmed phenotypically distinct tTreg and pTreg clusters, with the proportion of tTregs but not pTregs among CD4+ T cells expanding in response to seminal fluid. Bisulphite sequencing revealed increased demethylation of the Treg-specific demethylation region in the Foxp3 locus in tTregs but not pTregs after mating. These data show that tTregs and pTregs with distinct phenotypes both respond to seminal fluid priming, but the Foxp3 epigenetic signature is uniquely increased in tTregs. We conclude that reproductive tract tTregs as well as pTregs are sensitive to local regulation by seminal fluid, providing a candidate mechanism warranting evaluation for the potential to influence preeclampsia susceptibility in women.

Cord Blood T Cells Expressing High and Low PKCζ Levels Develop into Cells with a Propensity to Display Th1 and Th9 Cytokine Profiles, Respectively.

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA+ to CD45RO+ cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-ß) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases.

Characterization of the Transient Deficiency of PKC Isozyme Levels in Immature Cord Blood T Cells and Its Connection to Anti-Allergic Cytokine Profiles of the Matured Cells.

Cord blood T cells (CBTC) from a proportion of newborns express low/deficient levels of some protein kinase C (PKC) isozymes, with low levels of PKCζ correlating with increased risk of developing allergy and associated decrease in interferon-gamma (IFN-γ) producing T cells. Interestingly, these lower levels of PKCζ were increased/normalized by supplementing women during pregnancy with n-3 polyunsaturated fatty acids. However, at present, we have little understanding of the transient nature of the deficiency in the neonate and how PKCζ relates to other PKC isozymes and whether their levels influence maturation into IFN-γ producing T cells. There is also no information on PKCζ isozyme levels in the T cell subpopulations, CD4+ and CD8+ cells. These issues were addressed in the present study using a classical culture model of neonatal T cell maturation, initiated with phytohaemagglutinin (PHA) and recombinant human interleukin-2 (rhIL-2). Of the isozymes evaluated, PKCζ, ß2, δ, µ, ε, θ and λ/ι were low in CBTCs. The PKC isozyme deficiencies were also found in the CD4+ and CD8+ T cell subset levels of the PKC isozymes correlated between the two subpopulations. Examination of changes in the PKC isozymes in these deficient cells following addition of maturation signals showed a significant increase in expression within the first few hours for PKCζ, ß2 and µ, and 1-2 days for PKCδ, ε, θ and λ/ι. Only CBTC PKCζ isozyme levels correlated with cytokine production, with a positive correlation with IFN-γ, interleukin (IL)-2 and tumour necrosis factor-alpha (TNF), and a negative association with IL-9 and IL-10. The findings reinforce the specificity in using CBTC PKCζ levels as a biomarker for risk of allergy development and identify a period in which this can be potentially 'corrected' after birth.

Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples.

BACKGROUND: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. METHODS: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 106 cells/mL to 1.67 × 106 cells/mL. RESULTS: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 106 cells/mL. CONCLUSION: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.

Peptidase inhibitor 16 identifies a human regulatory T-cell subset with reduced FOXP3 expression over the first year of recent onset type 1 diabetes.

CD4+ T-cell subsets play a major role in the host response to infection, and a healthy immune system requires a fine balance between reactivity and tolerance. This balance is in part maintained by regulatory T cells (Treg), which promote tolerance, and loss of immune tolerance contributes to autoimmunity. As the T cells which drive immunity are diverse, identifying and understanding how these subsets function requires specific biomarkers. From a human CD4 Tconv/Treg cell genome wide analysis we identified peptidase inhibitor 16 (PI16) as a CD4 subset biomarker and we now show detailed analysis of its distribution, phenotype and links to Treg function in type 1 diabetes. To determine the clinical relevance of Pi16 Treg, we analysed PI16+ Treg cells from type 1 diabetes patient samples. We observed that FOXP3 expression levels declined with disease progression, suggesting loss of functional fitness in these Treg cells in Type 1 diabetes, and in particular the rate of loss of FOXP3 expression was greatest in the PI16+ve Treg. We propose that PI16 has utility as a biomarker of functional human Treg subsets and may be useful for tracking loss of immune function in vivo. The ability to stratify at risk patients so that tailored interventions can be applied would open the door to personalised medicine for Type 1 diabetes.

Changes in pancreatic exocrine function in young at-risk children followed to islet autoimmunity and type 1 diabetes in the ENDIA study.

BACKGROUNDS: We aimed to monitor pancreatic exocrine function longitudinally in relation to the development of islet autoimmunity (IA) and type 1 diabetes (T1D) in at-risk children with a first-degree relative with T1D, who were followed prospectively in the Environmental Determinants of Islet Autoimmunity (ENDIA) study. METHODS: Fecal elastase-1 (FE-1) concentration was measured longitudinally in 85 ENDIA children from median age 1.0 (IQR 0.7,1.3) year. Twenty-eight of 85 children (progressors) developed persistent islet autoantibodies at median age of 1.5 (IQR 1.1,2.5) years, of whom 11 went on to develop clinical diabetes. The other 57 islet autoantibody-negative children (non-progressors) followed similarly were age and gender-matched with the progressors. An adjusted linear mixed model compared FE-1 concentrations in progressors and non-progressors. RESULTS: Baseline FE-1 did not differ between progressors and non-progressors, or by HLA DR type or proband status. FE-1 decreased over time in progressors in comparison to non-progressors (Wald statistic 5.46, P = .02); in some progressors the fall in FE-1 preceded the onset of IA. CONCLUSIONS: Pancreatic exocrine function decreases in the majority of young at-risk children who progress to IA and T1D.

Higher frequency of vertebrate-infecting viruses in the gut of infants born to mothers with type 1 diabetes.

BACKGROUND: Microbial exposures in utero and early life shape the infant microbiome, which can profoundly impact on health. Compared to the bacterial microbiome, very little is known about the virome. We set out to characterize longitudinal changes in the gut virome of healthy infants born to mothers with or without type 1 diabetes using comprehensive virome capture sequencing. METHODS: Healthy infants were selected from Environmental Determinants of Islet Autoimmunity (ENDIA), a prospective cohort of Australian children with a first-degree relative with type 1 diabetes, followed from pregnancy. Fecal specimens were collected three-monthly in the first year of life. RESULTS: Among 25 infants (44% born to mothers with type 1 diabetes) at least one virus was detected in 65% (65/100) of samples and 96% (24/25) of infants during the first year of life. In total, 26 genera of viruses were identified and >150 viruses were differentially abundant between the gut of infants with a mother with type 1 diabetes vs without. Positivity for any virus was associated with maternal type 1 diabetes and older infant age. Enterovirus was associated with older infant age and maternal smoking. CONCLUSIONS: We demonstrate a distinct gut virome profile in infants of mothers with type 1 diabetes, which may influence health outcomes later in life. Higher prevalence and greater number of viruses observed compared to previous studies suggests significant underrepresentation in existing virome datasets, arising most likely from less sensitive techniques used in data acquisition.

Foxp3 heterozygosity does not overtly affect mammary gland development during puberty or the oestrous cycle in mice.

Female mice heterozygous for a genetic mutation in transcription factor forkhead box p3 (Foxp3) spontaneously develop mammary cancers; however, the underlying mechanism is not well understood. We hypothesised that increased cancer susceptibility is associated with an underlying perturbation in mammary gland development. The role of Foxp3 in mammary ductal morphogenesis was investigated in heterozygous Foxp3Sf/+ and wildtype Foxp3+/+ mice during puberty and at specific stages of the oestrous cycle. No differences in mammary ductal branching morphogenesis, terminal end bud formation or ductal elongation were observed in pubertal Foxp3Sf/+ mice compared with Foxp3+/+ mice. During adulthood, all mice underwent normal regular oestrous cycles. No differences in epithelial branching morphology were detected in mammary glands from mice at the oestrus, metoestrus, dioestrus and pro-oestrus stages of the cycle. Furthermore, abundance of Foxp3 mRNA and protein in the mammary gland and lymph nodes was not altered in Foxp3Sf/+ mice compared with Foxp3+/+ mice. These studies suggest that Foxp3 heterozygosity does not overtly affect mammary gland development during puberty or the oestrous cycle. Further studies are required to dissect the underlying mechanisms of increased mammary cancer susceptibility in Foxp3Sf/+ heterozygous mice and the function of this transcription factor in normal mammary gland development.

Gut microbiome dysbiosis and increased intestinal permeability in children with islet autoimmunity and type 1 diabetes: A prospective cohort study.

AIMS/HYPOTHESIS: To investigate the longitudinal relationship between the gut microbiome, circulating short chain fatty acids (SCFAs) and intestinal permeability in children with islet autoimmunity or type 1 diabetes and controls. METHODS: We analyzed the gut bacterial microbiome, plasma SCFAs, small intestinal permeability and dietary intake in 47 children with islet autoimmunity or recent-onset type 1 diabetes and in 41 unrelated or sibling controls over a median (range) of 13 (2-34) months follow-up. RESULTS: Children with multiple islet autoantibodies (≥2 IA) or type 1 diabetes had gut microbiome dysbiosis. Anti-inflammatory Prevotella and Butyricimonas genera were less abundant and these changes were not explained by differences in diet. Small intestinal permeability measured by blood lactulose:rhamnose ratio was higher in type 1 diabetes. Children with ≥2 IA who progressed to type 1 diabetes (progressors), compared to those who did not progress, had higher intestinal permeability (mean [SE] difference +5.14 [2.0], 95% confidence interval [CI] 1.21, 9.07, P = .006), lower within-sample (alpha) microbial diversity (31.3 [11.2], 95% CI 9.3, 53.3, P = .005), and lower abundance of SCFA-producing bacteria. Alpha diversity (observed richness) correlated with plasma acetate levels in all groups combined (regression coefficient [SE] 0.57 [0.21], 95% CI 0.15, 0.99 P = .008). CONCLUSIONS/INTERPRETATION: Children with ≥2 IA who progress to diabetes, like those with recent-onset diabetes, have gut microbiome dysbiosis associated with increased intestinal permeability. Interventions that expand gut microbial diversity, in particular SCFA-producing bacteria, may have a role to decrease progression to diabetes in children at-risk.

Ectodermal-Neural Cortex 1 Isoforms Have Contrasting Effects on MC3T3-E1 Osteoblast Mineralization and Gene Expression.

The importance of Wnt pathway signaling in development of bone has been well established. Here we investigated the role of a known Wnt target, ENC1 (ectodermal-neural cortex 1; NRP/B), in osteoblast differentiation. Enc1 expression was detected in mouse osteoblasts, chondrocytes, and osteocytes by in situ hybridization, and osteoblastic expression was verified in differentiating primary cultures and MC3T3-E1 pre-osteoblast cells, with 57 kDa and 67 kDa ENC1 protein isoforms detected throughout differentiation. Induced knockdown of both ENC1 isoforms reduced alkaline phosphatase staining and virtually abolished MC3T3-E1 mineralization. At culture confluence, Alpl (alkaline phosphatase liver/bone/kidney) expression was markedly reduced compared with control cells, and there was significant and coordinated alteration of other genes involved in cellular phosphate biochemistry. In contrast, with 67 kDa-selective knockdown mineralized nodule formation was enhanced and there was a two-fold increase in Alpl expression at confluence. There was enhanced expression of Wnt/ß-catenin target genes with knockdown of both isoforms at this time-point and a five-fold increase in Frzb (Frizzled related protein) with 67 kDa-selective knockdown at mineralization, indicating possible ENC1 interactions with Wnt signaling in osteoblasts. These results are the first to demonstrate a role for ENC1 in the control of osteoblast differentiation. Additionally, the contrasting mineralization phenotypes and transcriptional patterns seen with coordinate knockdown of both ENC1 isoforms vs selective knockdown of 67 kDa ENC1 suggest opposing roles for the isoforms in regulation of osteoblastic differentiation, through effects on Alpl expression and phosphate cellular biochemistry. This study is the first to report differential roles for the ENC1 isoforms in any cell lineage. J. Cell. Biochem. 118: 2141-2150, 2017. © 2016 Wiley Periodicals, Inc.

Single Cell Level Quantification of Nanoparticle-Cell Interactions Using Mass Cytometry.

Quantification of cell-associated nanoparticles (NPs) is a paramount question in both nanomedicine and nanotoxicology. Inductively coupled plasma mass spectrometry is a well-established method to resolve cell-associated (metal) NPs in bulk cell populations, however, such analysis at single cell level remains a challenge. Here we used mass cytometry, a technique that combines single cell analysis and time-of-flight mass spectrometry, to quantitatively analyze extra- and intracellular silver (Ag) in individual Ag NP exposed human T-lymphocytes. The results revealed significant population heterogeneity: for example, in lymphocytes exposed to 3 µg of 30 nm branched polyethylene imine coated Ag NPs/mL the extracellularly bound Ag varied from 79 to 560 fg and cellular uptake from 17 to 121 fg. Similar amplitude of heterogeneity was observed in cells exposed to various doses of Ag NPs with other sizes and surface coatings, demonstrating the importance of single cell analysis when studying NP-cell interactions. Although mass cytometry has some shortcomings such as inability to analyze potential transformation or dissolution of NPs in cells, we consider this method as the most promising for quantitative assessment of cell-NP interaction at single cell level.

MicroRNA regulation of immune events at conception.

The reproductive tract environment at conception programs the developmental trajectory of the embryo, sets the course of pregnancy, and impacts offspring phenotype and health. Despite the fundamental importance of this stage of reproduction, the rate-limiting regulatory mechanisms operating locally to control fertility and fecundity are incompletely understood. Emerging studies highlight roles for microRNAs (miRNAs) in regulating reproductive and developmental processes and in modulating the quality and strength of the female immune response. Since endometrial receptivity and robust placentation require specific adaptation of the immune response, we hypothesize that miRNAs participate in establishing pregnancy through effects on key gene networks in immune cells. Our recent studies investigated miRNAs that are induced in the peri-conception environment, focusing on miRNAs that have immune-regulatory roles-particularly miR-223, miR-155, and miR-146a. Genetic mouse models deficient in individual miRNAs are proving informative in defining roles for these miRNAs in the generation and stabilization of regulatory T cells (Treg cells) that confer adaptive immune tolerance. Overlapping and redundant functions between miRNAs that target multiple genes, combined with multiple miRNAs targeting individual genes, indicate complex and sensitive regulatory networks. Although to date most data on miRNA regulation of reproductive events are from mice, conserved functions of miRNAs across species imply similar biological pathways operate in all mammals. Understanding the regulation and roles of miRNAs in the peri-conception immune response will advance our knowledge of how environmental determinants act at conception, and could have practical applications for animal breeding as well as human fertility.