SAMHD1 Promotes the Antiretroviral Adaptive Immune Response in Mice Exposed to Lipopolysaccharide.

SAMHD1 is a potent HIV-1 restriction factor that blocks reverse transcription in monocytes, dendritic cells and resting CD4+ T cells by decreasing intracellular dNTP pools. However, SAMHD1 may diminish innate immune sensing and Ag presentation, resulting in a weaker adaptive immune response. To date, the role of SAMHD1 on antiretroviral immunity remains unclear, as mouse SAMHD1 had no impact on murine retrovirus replication in prior in vivo studies. Here, we show that SAMHD1 significantly inhibits acute Friend retrovirus infection in mice. Pretreatment with LPS, a significant driver of inflammation during HIV-1 infection, further unmasked a role for SAMHD1 in influencing immune responses. LPS treatment in vivo doubled the intracellular dNTP levels in immune compartments of SAMHD1 knockout but not wild-type mice. SAMHD1 knockout mice exhibited higher plasma infectious viremia and proviral DNA loads than wild-type mice at 7 d postinfection (dpi), and proviral loads inversely correlated with a stronger CD8+ T cell response. SAMHD1 deficiency was also associated with weaker NK, CD4+ T and CD8+ T cell responses by 14 dpi and weaker neutralizing Ab responses by 28 dpi. Intriguingly, SAMHD1 influenced these cell-mediated immune (14 dpi) and neutralizing Ab (28 dpi) responses in male but not female mice. Our findings formally demonstrate SAMHD1 as an antiretroviral factor in vivo that could promote adaptive immune responses in a sex-dependent manner. The requirement for LPS to unravel the SAMHD1 immunological phenotype suggests that comorbidities associated with a "leaky" gut barrier may influence the antiviral function of SAMHD1 in vivo.

Behavioral changes and hyperglycemia in NODEF mice following bisphenol S exposure are affected by diets.

Bisphenol S (BPS), an analogue of the controversial bisphenol A (BPA) that is found in epoxy resins and plastics, is a potential endocrine-disrupting chemical that can mimic endogenous hormone signaling. However, little is known about the behavioral or immunologic effects of BPS. The purpose of this study was to examine the impact of diets in BPS-treated mice in relation to hyperglycemia, development of type 1 diabetes, immunomodulation, and behavioral changes. Adult male and female nonobese diabetic excluded flora (NODEF) mice were exposed to environmentally relevant doses of BPS (VH, 30, or 300 µg/kg BW) and fed either a soy-based diet, a phytoestrogen-free diet, or a Western diet. NODEF male mice fed a soy-based diet exhibited a decreased curiosity/desire to explore, and possibly increased anxiety-like behavior and decreased short-term memory when exposed to BPS (300 µg/kg BW). In addition, these mice had significant increases in non-fasting blood glucose levels along with increased insulin sensitivity, impaired glucose tolerance, resistance to fasting and proinflammation. Although BPS had little effect on the glucose parameters in NODEF male mice fed a Western diet, there were decreases in %CD24+CD5+ and %B220+CD40L-cell populations and increases in distance traveled during the novel object test, suggesting hyperactivity. NODEF females fed a phytoestrogen-free diet exhibited slight decreases in time spent immobile during the tail suspension test in both the 30 and 300 µg/kg BW dose groups along with increases in %CD4+CD8+ and %Mac3+CD45R+ cell populations, signifying increased hyperactivity and anxiety-like behavior. In conclusion, BPS-exposed NODEF mice exhibited sex and diet-related changes in hyperglycemia, behaviors and immune endpoints.

Nucleic acid binding by SAMHD1 contributes to the antiretroviral activity and is enhanced by the GpsN modification.

SAMHD1 impedes infection of myeloid cells and resting T lymphocytes by retroviruses, and the enzymatic activity of the protein-dephosphorylation of deoxynucleotide triphosphates (dNTPs)-implicates enzymatic dNTP depletion in innate antiviral immunity. Here we show that the allosteric binding sites of the enzyme are plastic and can accommodate oligonucleotides in place of the allosteric activators, GTP and dNTP. SAMHD1 displays a preference for oligonucleotides containing phosphorothioate bonds in the Rp configuration located 3' to G nucleotides (GpsN), the modification pattern that occurs in a mechanism of antiviral defense in prokaryotes. In the presence of GTP and dNTPs, binding of GpsN-containing oligonucleotides promotes formation of a distinct tetramer with mixed occupancy of the allosteric sites. Mutations that impair formation of the mixed-occupancy complex abolish the antiretroviral activity of SAMHD1, but not its ability to deplete dNTPs. The findings link nucleic acid binding to the antiretroviral activity of SAMHD1, shed light on the immunomodulatory effects of synthetic phosphorothioated oligonucleotides and raise questions about the role of nucleic acid phosphorothioation in human innate immunity.

Effect of induced dNTP pool imbalance on HIV-1 reverse transcription in macrophages.

BACKGROUND: Terminally differentiated/nondividing macrophages, a key target cell type of HIV-1, harbor extremely low dNTP concentrations established by a host dNTP triphosphohydrolase, SAM domain and HD domain containing protein 1 (SAMHD1). We tested whether the induction of dNTP pool imbalance can affect HIV-1 replication in macrophages. For this test, we induced a large dNTP pool imbalance by treating human primary monocyte derived macrophages with either one or three of the four deoxynucleosides (dNs), which are phosphorylated to dNTPs in cells, to establish two different dNTP imbalance conditions in macrophages. RESULTS: The transduction efficiency and 2-LTR circle copy number of HIV-1 GFP vector were greatly diminished in human primary macrophages treated with the biased dN treatments, compared to the untreated macrophages. We also observed the induced dNTP bias blocked the production of infectious dual tropic HIV-1 89.6 in macrophages. Moreover, biochemical DNA synthesis by HIV-1 reverse transcriptase was significantly inhibited by the induced dNTP pool imbalance. Third, the induced dNTP bias increased the viral mutant rate by approximately 20-30% per a single cycle infection. Finally, unlike HIV-1, the single dN treatment did not significantly affect the transduction of SIVmac239-based GFP vector encoding Vpx in macrophages. This is likely due to Vpx, which can elevate all four dNTP levels even with the single dN treatment. CONCLUSION: Collectively, these data suggest that the elevated dNTP pool imbalance can induce kinetic block and mutation synthesis of HIV-1 in macrophages.

Bisphenol S Modulates Type 1 Diabetes Development in Non-Obese Diabetic (NOD) Mice with Diet- and Sex-Related Effects.

Bisphenol S (BPS) is a common replacement for bisphenol A (BPA) in plastics, which has resulted in widespread human exposure. Type 1 diabetes (T1D) is an autoimmune disease resulting from pancreatic ß-cell destruction and has been increasing in incidence globally. Because of the similarities (e.g., endocrine disrupting) between BPS and BPA, and the fact that BPA was previously shown to accelerate T1D development in female non-obese diabetic (NOD) mice, it was hypothesized that BPS could contribute to the increasing T1D incidence by altering immunity with sex-biased responses. Adult female non-obese diabetic (NOD) mice were orally administered BPS at environmentally relevant doses (3, 30, 150 and 300 µg/kg), and males were given 0 or 300 µg/kg BPS. Females following 30 µg/kg BPS treatment on a soy-based diet had significantly delayed T1D development at the end of the study and decreased non-fasting blood glucose levels (BGLs) during the study. In contrast, BPS-exposed males on a soy-based diet showed an increased insulin resistance and varied BGLs. This might be a mixture effect with phytoestrogens, since males on a phytoestrogen-free diet showed improved glucose tolerance and decreased insulin resistance and CD25+ T cells. Additionally, while BPS altered BGLs in soy-based diet mice, minimal effects were observed concerning their immunotoxicity. Thus, BPS had sex- and diet-dependent effects on T1D and glucose homeostasis, which were likely caused by other mechanisms in addition to immunomodulation.

Isoflavone daidzein regulates immune responses in the B6C3F1 and non-obese diabetic (NOD) mice.

Daidzein (DAZ), a dominant isoflavone in various natural products such as soybeans, has been gaining attention due to the beneficial health effects (e.g., protection against cancer and diabetes) of its metabolites. Our major hypothesis was that dietary exposure to the soy phytoestrogen DAZ could modulate the immune responses toward a protective effect and lead to improved metabolic functions (such as glucose metabolism). In this study, we applied complementary mouse models, the hybrid B6C3F1 and inbred type 1 diabetes prone non-obese diabetic (NOD) mice, to investigate if DAZ exposure modulated the immune responses. The animals were orally administered DAZ at various physiological doses (2-20 mg/kg body weight) during adulthood. DAZ significantly altered the relative organ weights in female B6C3F1 mice and decreased the B cell population (represented by CD3-IgM+), while the T cell populations (represented by CD3+IgM-, CD4+CD8- and CD4-CD8+) were increased. In addition, DAZ dosing produced a decrease in the percentage of late apoptotic thymocytes. However, the activities cytotoxic T cells and natural killer cells were not altered in the B6C3F1 mice. In NOD mice, the blood glucose level and glucose tolerance were not affected by DAZ exposure, but DAZ modulated the antibody production, as shown by increased levels of IgG2b in NOD females and IgG1 in NOD males. Further, DAZ increased CD8+CD25+ splenocytes in NOD females. Taken together, DAZ induced an immunomodulatory effect in both NOD and B6C3F1 mouse strains; however, minimal effects on glucose homeostasis were observed.

Bisphenol A alteration of type 1 diabetes in non-obese diabetic (NOD) female mice is dependent on window of exposure.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic ß-cell destruction can be mediated by dysbiosis, infiltration of pro-inflammatory immune cells, and cytokines/chemokines. Exposure to bisphenol A (BPA), an endocrine disruptor (ED), can lead to aberrant immunity and gut microbiota. We determined whether BPA had age-dependent effects on T1D by modulating immune homeostasis following various windows of exposure in non-obese diabetic (NOD) mice. Juvenile NOD females were orally exposed to 0 or 30 µg BPA/kg BW from postnatal day (PND) 28 to PND56. Adult NOD females were exposed to 0 or 300 µg BPA/kg BW. Female and male NOD offspring were exposed to 0 or 300 µg BPA/kg BW perinatally from gestation day 5 to PND28 by dosing the dams. It was found that BPA increased T1D risk in juvenile females with gut microbiota shifted towards pro-inflammation (e.g. increased Jeotgalicoccus). In agreement with our previous study, adult females had a trend of increased T1D and a general increase in immune responses. However, female offspring had a reduced T1D development. Consistently, female offspring had a shift towards anti-inflammation (e.g. decreased pro-inflammatory F4/80+Gr1+ cells). In contrast, BPA had minimal effects on immunity and T1D in male offspring. Thus, it was concluded that BPA had age- and sex-dependent effects on T1D with the alteration of gut microbiota and inflammation being the primary mechanisms for T1D exacerbation in juvenile exposure and decreases of inflammation being responsible for attenuated T1D in perinatally exposed females.

Sex-dependent effects of bisphenol A on type 1 diabetes development in non-obese diabetic (NOD) mice.

Type 1 diabetes (T1D) is an autoimmune disease caused by immune-mediated pancreatic ß-cell destruction. The endocrine disrupting chemical bisphenol A (BPA) has widespread human exposure and can modulate immune function and the gut microbiome (GMB), which may contribute to the increasing T1D incidence worldwide. It was hypothesized that BPA had sex-dependent effects on T1D by modulating immune homeostasis and GMB. Adult female and male non-obese diabetic (NOD) mice were orally administered BPA at environmentally relevant doses (30 or 300 µg/kg). Antibiotic-treated adult NOD females were exposed to 0 or 30 µg/kg BPA. BPA accelerated T1D development in females, but delayed males from T1D. Consistently, females had a shift towards pro-inflammation (e.g., increased macrophages and Bacteroidetes), while males had increases in anti-inflammatory immune factors and a decrease in both anti- and pro-inflammatory GMB. Although bacteria altered during sub-acute BPA exposure differed from bacteria altered from chronic BPA exposure in both sexes, the GMB profile was consistently pro-inflammatory in females, while males had a general decrease of both anti- and pro-inflammatory gut microbes. However, treatment of females with the antibiotic vancomycin failed to prevent BPA-induced glucose intolerance, suggesting changes in Gram-positive bacteria were not a primary mechanism. In conclusion, BPA exposure was found to have sex dimorphic effects on T1D with detrimental effects in females, and immunomodulation was identified as the primary mechanism.

Exacerbation of Type 1 Diabetes in Perinatally Genistein Exposed Female Non-Obese Diabetic (NOD) Mouse Is Associated With Alterations of Gut Microbiota and Immune Homeostasis.

Despite various hypothesized benefits of dietary isoflavone genistein (GEN) from soy-based products, many questions surrounding GEN's immunotoxic effects, especially during perinatal exposure, have yet to be answered. The objective of the study was to determine if there existed a sex-specific effect of GEN on type 1 diabetes (T1D) following perinatal exposure. We exposed offspring of non-obese diabetic (NOD) mice to GEN per oral at a physiological dose (20 mg/kg body weight) from embryonic day 7 to postnatal day (PND) 21. In female offspring, perinatal GEN dosing significantly increased the incidence of T1D at early time points, and the exacerbation was associated with decreased serum levels of interleukin (IL)-10, IgG2a, and IgM. In male offspring dosed with GEN, a decrease in serum IgG1 was also observed. Flow cytometric analysis in females suggested an increased pro-inflammatory splenic CD5+CD24- and CD4-CD8+ cell counts, while both %T cells and %CD4+ T cells were significantly decreased in males, suggesting an anti-inflammatory effect. Gut microbiota (GMB) analysis indicated that fecal microbiota from PND 90 female offspring exhibited an increased level of Enterobacteriales (suggesting a pro-inflammatory response), while the similar changes were not found in PND 30 females. Moreover, RNA sequencing showed that intestinal α-defensin expression was down-regulated in GEN-treated females, supporting a pro-inflammatory response. However, perinatal GEN administration perturbed GMB toward an anti-inflammatory response in PND 90 males. Taken together, a strong sex-specific effect was found in the perinatal GEN exposure window, and the T1D exacerbation in NOD females was associated with GMB-related immunomodulatory mechanisms.

Genistein prevention of hyperglycemia and improvement of glucose tolerance in adult non-obese diabetic mice are associated with alterations of gut microbiome and immune homeostasis.

Although studies have linked soy phytoestrogen 4,7,4-trihydroxyisoflavone genistein (GEN) to reduced type 1 diabetes (T1D) risk, the mechanism of dietary GEN on T1D remains unknown. In our studies, adult non-obese diabetic (NOD) mouse model was employed to investigate the effects of GEN exposure on blood glucose level (BGL), glucose tolerance, gut microbiome, and immune responses. Adult male and female NOD mice were fed with either soy-based or casein-based diet, and received GEN at 20mg/kg body weight by gavage daily. The BGL and immune responses (represented by serum antibodies, cytokines and chemokines, and histopathology) were monitored, while the fecal gut microbiome was sequenced for 16S ribosomal RNA to reveal any alterations in gut microbial communities. A significantly reduced BGL was found in NOD males fed with soy-based diet on day 98 after initial dosing, and an improved glucose tolerance was observed on both diets. In addition, an anti-inflammatory response (suggested by reduced IgG2b and cytokine/chemokine levels, and alterations in the microbial taxonomy) was accompanied by an altered ß-diversity in gut microbial species. Among the NOD females exposed to GEN, a later onset of T1D was observed. However, the profiles of gut microbiome, antibodies and cytokines/chemokines were all indicative of pro-inflammation. This study demonstrated an association among GEN exposure, gut microbiome alteration, and immune homeostasis in NOD males. Although the mechanisms underlying the protective effects of GEN in NOD mice need to be explored further, the current study suggested a GEN-induced sex-specific effect in inflammatory status and gut microbiome.

TCDD modulation of gut microbiome correlated with liver and immune toxicity in streptozotocin (STZ)-induced hyperglycemic mice.

An increasing body of evidence has shown the important role of the gut microbiome in mediating toxicity following environmental contaminant exposure. The goal of this study was to determine if the adverse metabolic effects of chronic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure would be sufficient to exacerbate hyperglycemia, and to further determine if these outcomes were attributable to the gut microbiota alteration. Adult male CD-1 mice were exposed to TCDD (6µg/kg body weight biweekly) by gavage and injected (i.p.) with STZ (4×50mg/kg body weight) to induced hyperglycemia. 16S rRNA sequencing was used to characterize the changes in the microbiome community composition. Glucose monitoring, flow cytometry, histopathology, and organ characterization were performed to determine the deleterious phenotypic changes of TCDD exposure. Chronic TCDD treatment did not appear to exacerbate STZ-induced hyperglycemia as blood glucose levels were slightly reduced in the TCDD treated mice; however, polydipsia and polyphagia were observed. Importantly, TCDD exposure caused a dramatic change in microbiota structure, as characterized at the phylum level by increasing Firmicutes and decreasing Bacteroidetes while at the family level most notably by increasing Lactobacillaceae and Desulfovibrionaceae, and decreasing Prevotellaceae and ACK M1. The changes in microbiota were further found to be broadly associated with phenotypic changes seen from chronic TCDD treatment. In particular, the phylum level Bacteroidetes to Firmicutes ratio negatively correlated with both liver weight and liver pathology, and positively associated with %CD3(+)NK(+) T cells, a key mediator of host-microbial interactions. Collectively, these findings suggest that the dysregulated gut microbiome may contribute to the deleterious effects (e.g., liver toxicity) seen with TCDD exposure.

Developmental Bisphenol A Exposure Modulates Immune-Related Diseases.

Bisphenol A (BPA), used in polycarbonate plastics and epoxy resins, has a widespread exposure to humans. BPA is of concern for developmental exposure resulting in immunomodulation and disease development due to its ability to cross the placental barrier and presence in breast milk. BPA can use various mechanisms to modulate the immune system and affect diseases, including agonistic and antagonistic effects on many receptors (e.g., estrogen receptors), epigenetic modifications, acting on cell signaling pathways and, likely, the gut microbiome. Immune cell populations and function from the innate and adaptive immune system are altered by developmental BPA exposure, including decreased T regulatory (Treg) cells and upregulated pro- and anti-inflammatory cytokines and chemokines. Developmental BPA exposure can also contribute to the development of type 2 diabetes mellitus, allergy, asthma and mammary cancer disease by altering immune function. Multiple sclerosis and type 1 diabetes mellitus may also be exacerbated by BPA, although more research is needed. Additionally, BPA analogs, such as bisphenol S (BPS), have been increasing in use, and currently, little is known about their immune effects. Therefore, more studies should be conducted to determine if developmental exposure BPA and its analogs modulate immune responses and lead to immune-related diseases.