Female WT Research Articles

T Cells Contribute to Lipid Deposition in Male and Female Dahl Rats Fed a High Saturated Fat Diet

BackgroundThe Dahl rat has been used as a model of high fat diet (HFD)‐induced hypertension and adiposity. Genetic deletion of T cells blunts HFD‐induced hypertension. However, it is not clear if deletion of T cells can also reduce the adiposity caused by the HFD. T cells have been shown to play a role in many inflammatory conditions which are characterized by dyslipidemia. Therefore, the goal of the current study was to test the hypothesis that deletion of T cells prevents lipid deposits in the liver and adipose tissue.Methods5‐week‐old male and female wildtype Dahl (WT; n=9‐19/group) or Dahl rats lacking T cells (CD247 knock out [KO] rats; n=3‐7/group) were randomized to either a control normal fat diet (NF; 7.2% calories from fat) or a high fat diet (HF; 36% calories from saturated fat) for 10 weeks. Following treatment, a subset of livers were processed for hematoxylin and eosin staining and liver steatosis was scored. Gonadal adipose tissue was extracted and weighed.ResultsTen weeks of HFD increased liver steatosis in WT males and females (PInteraction=0.84, PDiet=0.01, Psex=0.75, Table 1), but not in T cell deficient animals (PInteraction=0.35, PDiet=0.29, Psex=0.04). While HFD increased gonadal adipose tissue weight in both males and females, this weight was increased more in the females (PInteraction=0.006, PDiet<0.0001, Psex<0.0001). T cell deletion abolished the HFD‐induced weight gain in gonadal adipose tissue (PInteraction=0.98, PDiet=0.14, Psex=0.01).DiscussionThese data confirm the hypothesis that T cell KO prevents the HFD‐induced increases in adiposity in the liver and gonadal adipose tissue. Lipid deposits in these areas are detrimental for cardiovascular health. Lipid deposition in the liver is a strong predictor of insulin resistance. Furthermore, chronic ingestion of a HFD increases adipose tissue expansion, which promotes inflammation and oxidative stress. Since the majority of Americans are consuming large amounts of fat, investigating the mechanisms underlying HFD‐induced adiposity will help identify targets to improve cardiovascular health.

Abstract 285: Thrombospondin-5 Is Necessary For Males But Not Females In Hindlimb Ischemia Recovery

Introduction: Ameliorating critical limb ischemia (CLI), in part, relies on arteriogenesis and angiogenesis. We have shown thrombospondin-5 (TSP5) is proangiogenic in vitro. The role of TSP5 in CLI and the differences between males and females are unknown. Hypotheses: blood flow recovery, arteriogenesis and angiogenesis after limb ischemia is diminished (1) in TSP5 null mice compared to controls, and (2) in female compared to male TSP5 null mice. Methods: 14-16 week-old wild type (WT; C57Bl6) or TSP5 null male and female mice underwent left femoral artery ligation and transection (n=6-9/group). Laser Doppler data was collected preoperative, post ligation and at sacrifice (day 14). Blood flow recovery was expressed as a ratio of ischemic/non-ischemic limb. Immunohistochemistry was performed using anti-α-smooth-muscle-actin on the vastus lateralis to quantify arteriogenesis and anti-CD31 on the gastrocnemius to quantify angiogenesis. Arteriole and capillary density were determined by vessel counts/5HPF. Data was analyzed by ANOVA with post hoc testing, with p<0.05 being significant. Results: TSP5 null mice had decreased blood flow recovery in males (WT 0.85 ± 0.04 vs TSP5 0.48 ± 0.06, p<0.001) and females (WT 0.63 ± 0.07 vs TSP5 0.36 ± 0.04, p<0.01). No difference in flow recovery was seen between male and female TSP5 nulls (male 0.48 ± 0.06 vs female 0.36 ± 0.04 p<0.14). Arteriogenesis was impaired in male TSP5 null mice (WT 3.91 ± 0.24 vs TSP5 2.6 ± 0.42, p<0.02), but increased in females (WT 2.5 ± 0.26 vs TSP5 4.35 ± 0.73, p<0.01). Angiogenesis was decreased in TSP5 null males (WT 10.9 ± 0.93 vs TSP5 7.03 ± 0.84, p<0.01), but not females (WT 6.1 ± 0.50 vs TSP5 5.6 ± 0.80 p<0.9). In contrast, flow recovery, angiogenesis and arteriogenesis was decreased in WT females compared to WT males (p< 0.05). Conclusions: Our findings suggest TSP5 is relevant to vascular remodeling following hindlimb ischemia with a divergence in mechanism between males and females. TSP5 is necessary in males for arteriogenesis and angiogenesis. TSP5 in females may be supressive of arteriogenesis and has no effect on angiogenesis. Further investigation into these sexually dimorphic adaptive processes may lead to specific targeted CLI treatments.

The Integrated Stress Response and Eukaryotic Initiation Factor 2a Regulate Methylglyoxal-induced Pain Hypersensitivity

Diabetic neuropathic pain (DNP) is a debilitating condition. Current therapies are ineffective at treating DNP. We have previously demonstrated that methylglyoxal (MGO), a reactive metabolite of glycolysis that is elevated in the plasma of diabetic patients, pain hypersensitivity by engaging the integrated stress response (ISR) in mice. Induction of the ISR alters protein synthesis by inhibiting eukaryotic initiation factor 2α (eIF2α) and relying on eIF2A-mediated translation. We hypothesize that eIF2A regulates protein synthesis that contributes to pain hypersensitivity under conditions of ISR, especially after MGO application. We employed the recently characterized eIF2A-/- mouse to investigate how MGO treatment induces the ISR, facilitates eIF2A-mediated translation, and consequently causes pain hypersensitivity. We also subjected mice to tunicamycin (TUN), a well-known ISR inducer, and spared nerve injury (SNI). With donated human dorsal root ganglia (DRG) and spinal nerves (SpN), we aimed to translate our finding in rodents to humans. Intraplantar and intraperitoneal administrations of MGO induced pain hypersensitivity in a dose-dependent manner in male and female WT animals but not in eIF2A-/- mice. Following TUN and SNI, the eIF2A-/-¬¬ mice were protected against ISR-dependent pain (tunicamycin) but not against ISR-independent pain (SNI). MGO treatment of human DRG and SpN explants, and cultured human DRG neurons elevated p-eIF2α and eIF2A levels suggesting that MGO induces ISR not only in mice, but also in humans. Carboxyethyl lysine (CEL), a byproduct of reactive MGO and lysine residues, is increased in MGO treated cells and DRG and spinal nerve explants but not in tunicamycin treated explants, despite sharing elevated p-eIF2α and eIF2A levels. We demonstrate that eIF2A is necessary for the development of ISR-induced pain hypersensitivity. We further suggest that MGO and its byproduct, CEL, can be used as a biomarker to stratify DNP patients that would respond to ISR inhibitors like ISRIB. Diabetic neuropathic pain (DNP) is a debilitating condition. Current therapies are ineffective at treating DNP. We have previously demonstrated that methylglyoxal (MGO), a reactive metabolite of glycolysis that is elevated in the plasma of diabetic patients, pain hypersensitivity by engaging the integrated stress response (ISR) in mice. Induction of the ISR alters protein synthesis by inhibiting eukaryotic initiation factor 2α (eIF2α) and relying on eIF2A-mediated translation. We hypothesize that eIF2A regulates protein synthesis that contributes to pain hypersensitivity under conditions of ISR, especially after MGO application. We employed the recently characterized eIF2A-/- mouse to investigate how MGO treatment induces the ISR, facilitates eIF2A-mediated translation, and consequently causes pain hypersensitivity. We also subjected mice to tunicamycin (TUN), a well-known ISR inducer, and spared nerve injury (SNI). With donated human dorsal root ganglia (DRG) and spinal nerves (SpN), we aimed to translate our finding in rodents to humans. Intraplantar and intraperitoneal administrations of MGO induced pain hypersensitivity in a dose-dependent manner in male and female WT animals but not in eIF2A-/- mice. Following TUN and SNI, the eIF2A-/-¬¬ mice were protected against ISR-dependent pain (tunicamycin) but not against ISR-independent pain (SNI). MGO treatment of human DRG and SpN explants, and cultured human DRG neurons elevated p-eIF2α and eIF2A levels suggesting that MGO induces ISR not only in mice, but also in humans. Carboxyethyl lysine (CEL), a byproduct of reactive MGO and lysine residues, is increased in MGO treated cells and DRG and spinal nerve explants but not in tunicamycin treated explants, despite sharing elevated p-eIF2α and eIF2A levels. We demonstrate that eIF2A is necessary for the development of ISR-induced pain hypersensitivity. We further suggest that MGO and its byproduct, CEL, can be used as a biomarker to stratify DNP patients that would respond to ISR inhibitors like ISRIB.

Aging and G Protein‐Coupled Estrogen Receptor Exacerbates Carotid Artery Structural Remodeling

Aging is a nonmodifiable risk factor for cardiovascular disease associated with arterial stiffening and cardiac dysfunction. We previously showed an important role for G protein‐coupled estrogen receptor (GPER) in modulating female protection from arterial stiffening. In this study, we hypothesized that aging would decrease vascular compliance in GPER knockout (ko) mice to a greater extent than wildtype (wt) mice. Adult (25 weeks) and middle‐aged (57 weeks) mice were subjected to high frequency ultrasound for pulse wave velocity (PWV), and structural remodeling in the carotid artery was assessed with biaxial pressure myography followed by constitutive modeling. Data was analyzed by 3‐way ANOVA and Sidak’s post hoc tests. As we previously showed, PWV was significantly lower in adult female wt mice versus their male counterparts (1.4 ± 0.05 vs. 1.1 ± 0.02 m/s; P=0.0004) but was not different in adult ko mice (1.6 ± 0.07 vs 1.4 ± 0.08, P=0.20). Interestingly, PWV increased with aging in female but not male wt mice, so that the sex difference was lost (1.7 ± 0.03 vs 1.7 ± 0.06, P=0.99). Despite the lower PWV, circumferential linearized material stiffness in passive conditions was elevated in adult female wt versus male wt (1.9 ± 0.2 vs. 1.4 ± 0.2 MPa; P=0.02), but this sex difference was lost in middle‐aged wt mice (1.3 ± 0.1 vs. 1.2 ± 0.1 MPa; P=0.87). Aging significantly increased the ratio of thick to thin collagen only in male wt and female ko groups. We found that females but not males experience an increase in PWV at middle‐age along with a decrease in circumferential stiffness. In contrast, female GPER mice already display a higher PWV as adults, indicating that GPER deletion in females is a model of accelerated vascular aging.

TNF ALPHA AND SEX PREVALENCE OF ORAL MANIFESTATIONS IN CHRONIC INFLAMMATORY DISEASES: IT'S COMPLICATED

<h3>Objectives</h3> Several studies have linked chronic inflammatory conditions such as Crohn's disease (CD) or inflammatory arthritis to increased risk for periodontal disease (PD). It is attractive to attribute this to increased circulating pro-inflammatory cytokine levels such as tumor necrosis factor-alpha (TNF-α). Women show a higher incidence of CD, and women with CD have a higher risk of developing PD. How sex differences affect disease susceptibility and how this might be linked to pro-inflammatory cytokines is not well understood. The TNFΔARE (TNFΔ/WT) mouse is an established model for CD and inflammatory arthritis. The prolonged and increased TNF-α production led to spontaneous development of these 2 inflammatory diseases from a young age. Here, we used this model to test sex prevalence and the role of circulating TNF-α in the development of PD. <h3>Study Design</h3> Both male and female TNFΔARE (TNFΔ/WT) and control WT mice were analyzed at 2 and 4 months of age for changes to the temporomandibular joint (TMJ), buccal mucosa, gingival epithelium, and alveolar bone. MicroCT scans were acquired using a Milabs U-CT scanner and analyzed using Avizo 9.1.1. Oral manifestations were analyzed using histology (hematoxylin & eosin, Tartrate-resistant acid phosphatase (TRAP), Safranin O) and immunofluorescence to assess proliferation (Ki67), inflammation (CD45), and bone remodeling (Sclerostin (SOST), Receptor activator of nuclear factor kappa-B ligand (RANKL)). <h3>Results</h3> Four-month-old but not 2-month-old TNFΔ/WT showed various oral manifestations associated with arthritis (TMJ) or CD (cobblestone mucosa). No evidence of active periodontal disease was found. Four-month-old female mice showed gingival hyperplasia, severe TMJ arthritis, malocclusion, and alveolar bone loss in the absence of other signs of PD (epithelial inflammation, bleeding). Four-month-old TNFΔ/WT male mice presented TMJ arthritis, cobblestone mucosa, lack of hyperplasia but limited presence of leukocytes at the gingival epithelium, periodontal ligament, and buccal mucosa. Other signs of PD (epithelial inflammation, bleeding) were also missing. <h3>Conclusions</h3> We present strong evidence that circulating TNF-α is not sufficient to induce PD. The role of TNF-α in oral tissues is complex. TMJ arthritis and malocclusion might trigger alveolar bone loss in female TNFΔARE mice in the absence of PD. Sex-specific differences in oral manifestations (hyperplasia vs cobblestone mucosa) might be directly related to local rather than systemic differences in TNF-α.

Altered electrical properties in skeletal muscle of mice with glycogen storage disease type II

Electrical impedance methods, including electrical impedance myography, are increasingly being used as biomarkers of muscle health since they measure passive electrical properties of muscle that alter in disease. One disorder, Pompe Disease (Glycogen storage disease type II (GSDII)), remains relatively unstudied. This disease is marked by dramatic accumulation of intracellular myofiber glycogen. Here we assessed the electrical properties of skeletal muscle in a model of GSDII, the Pompe6neo/6neo (Pompe) mouse. Ex vivo impedance measurements of gastrocnemius (GA) were obtained using a dielectric measuring cell in 30-week-old female Pompe (N = 10) and WT (N = 10) mice. Longitudinal and transverse conductivity, σ, and the relative permittivity, εr, and Cole–Cole complex resistivity parameters at 0 Hz and infinite frequency, ρo and ρ∞, respectively, and the intracellular resistivity, ρintracellular were determined from the impedance data. Glycogen content (GC) was visualized histologically and quantified biochemically. At frequencies > 1 MHz, Pompe mice demonstrated significantly decreased longitudinal and transverse conductivity, increased Cole–Cole parameters, ρo and ρo-ρ∞, and decreased ρintracellular. Changes in longitudinal conductivity and ρintracellular correlated with increased GC in Pompe animals. Ex vivo high frequency impedance measures are sensitive to alterations in intracellular myofiber features considered characteristic of GSDII, making them potentially useful measures of disease status.

Involvement of GOBP2 in the perception of a sex pheromone component in both larval and adult Spodoptera litura revealed using CRISPR/Cas9 mutagenesis

General odorant-binding proteins (GOBPs) are long considered responsible for the perception of plant odorants. In this study with the important noctuid pest Spodoptera litura, we functionally characterized that GOBP2 is also involved in the perception of sex pheromone components using in vivo CRISPR/Cas9 technique. First, the GOBP2 sgRNA and Cas9 protein were injected into the newly laid insect eggs, resulting in a 35.6% target mutagenesis in G0 moths. Then, the homozygous GOBP2 knockout strain (GOBP2−/−) was obtained after the screening of three generations. The knockout male and female moths displayed a significant reduction in EAG responses to the sex pheromone components, and the knockout females also displayed a significant reduction to plant odorants. In the behavioral assay of food choice, GOBP2−/− larvae lost the preference to artificial diet added with the major sex pheromone component Z9, E11-tetradecadienyl acetate (Z9, E11-14:Ac), whereas the WT larvae highly preferred the pheromone diet. Y-tube olfactometer assay and direct pheromone stimulation assay showed that GOBP2−/− male adults reduced significantly than WT males in percentages of choice, hair pencil displaying and mating attempt to Z9, E11-14:Ac. In the oviposition test, GOBP2−/− females showed significantly reduced preference for the soybean plants compared to the WT females. Our study demonstrated that GOBP2 plays an important role in perceiving sex pheromones in adult and larval stages, providing new insight into sex pheromone perception and a potential target for sex pheromone-based behavioral regulation in the pest.

The amyloid-β1–42-oligomer interacting peptide D-AIP possesses favorable biostability, pharmacokinetics, and brain region distribution

We have previously developed a unique 8-amino acid Aβ42 oligomer-Interacting Peptide (AIP) as a novel anti-amyloid strategy for the treatment of Alzheimer’s disease. Our lead candidate has successfully progressed from test tubes (i.e., in vitro characterization of protease-resistant D-AIP) to transgenic flies (i.e., in vivo rescue of human Aβ42-mediated toxicity via D-AIP-supplemented food). In the present study, we examined D-AIP in terms of its stability in multiple biological matrices (i.e., ex-vivo mouse plasma, whole blood, and liver S9 fractions) using MALDI mass spectrometry, pharmacokinetics using a rapid and sensitive LC-MS method, and blood brain barrier (BBB) penetrance in WT C57LB/6 mice. D-AIP was found to be relatively stable over 3 h at 37 °C in all matrices tested. Finally, label-free MALDI imaging showed that orally administered D-AIP can readily penetrate the intact BBB in both male and female WT mice. Based upon the favorable stability, pharmacokinetics, and BBB penetration outcomes for orally administered D-AIP in WT mice, we then examined the effect of D-AIP on amyloid “seeding” in vitro (i.e., freshly monomerized versus preaggregated Aβ42). Complementary biophysical assays (ThT, TEM, and MALDI-TOF MS) showed that D-AIP can directly interact with synthetic Aβ42 aggregates to disrupt primary and/or secondary seeding events. Taken together, the unique mechanistic and desired therapeutic potential of our lead D-AIP candidate warrants further investigation, that is, testing of D-AIP efficacy on the altered amyloid/tau pathology in transgenic mouse models of Alzheimer’s disease.

Compound deletion of thrombospondin-1 and -2 results in a skeletal phenotype not predicted by the single gene knockouts.

The trimeric thrombospondin homologs, TSP1 and TSP2, are both components of bone tissue and contribute in redundant and distinct ways to skeletal physiology. TSP1-null mice display increased femoral cross-sectional area and thickness due to periosteal expansion, as well as diminished matrix quality and impaired osteoclast function. TSP2-null mice display increased femoral cross-sectional thickness and reduced marrow area due to increased endosteal osteoblast activity, with very little periosteal expansion. Osteoblast lineage cells are reduced in TSP2-null mice, but not in TSP1-null. The functional effects of combined TSP1 and TSP2 deficiency remain to be elucidated. Here, we examined the spectrum of detergent soluble proteins in diaphyseal cortical bone of growing (6-week old) male and female mice deficient in both thrombospondins (double knockout (DKO)). Of 3429 detected proteins, 195 were differentially abundant in both male and female DKO bones. Physiologically relevant annotation terms identified by Ingenuity Pathway Analysis included "ECM degradation" and "Quantity of Monocytes." Manual inspection revealed that a number of proteins with shared expression among osteoclasts and osteocytes were reduced in DKO bones. To associate changes in protein content with phenotype, we examined 12-week old male and female DKO and WT mice. DKO mice were smaller than WT and in male DKO, femoral cross section area was reduced. Some of the male DKO femora also had a flattened, less circular cross-section. Male DKO bones were less stiff in bending and they displayed reduced ultimate load. Displacements at yield load and at max load were both elevated in male DKO. However, the ratios of post-yield to pre-yield displacements significantly diminished in DKO suggesting proportionally reduced post-yield behavior. Male DKO mice also exhibited reductions in trabecular bone mass, which were surprisingly associated with equivalent osteoblast numbers and accordingly increased osteoblast surface. Marrow-derived colony forming unit-fibroblastic was reduced in male and female DKO mice. Together our data suggest that when both TSP1 and TSP2 are absent, a unique, sex-specific bone phenotype not predicted by the single knockouts, is manifested.

IL‐1 beta and TNF‐alpha play an essential role in modulating the risk of both periodontitis and Alzheimer’s disease

AbstractBackgroundPeriodontitis, a chronic systemic inflammatory disease, is a common health problem in the elderly. With increasing recognition that inflammation plays a key role in the pathophysiology of Alzheimer's disease (AD), our research team has been studying how systemic inflammation affects the brain immune responses. Aging is a major risk factor for both periodontitis and AD. As aging causes dysregulation of immune responses, we aimed to define the roles of cytokines in the pathogenesis of periodontitis and AD.MethodsTo induce experimental periodontitis, female WT mice were injected with heat‐killed P. gingivalis into their buccal mucosa three times per week every other week for a total of 5 weeks. Another group of mice were injected with IL‐1b and TNF‐a following the same timeline as above. Sickness Behavior and cognitive functions were assessed through open field and puzzle box test. Mandibular jaws were harvested for Micro‐CT scan. Different brain regions were harvested for biochemical analysis.ResultsMice injected with heat‐killed bacteria had worsened long‐term memory functions. It also significantly increased periodontal bone loss, which was accompanied by increased gene expression levels of IL‐1b and TNF‐a in the gums. Significant elevated levels of pro‐inflammatory cytokines in the liver as well as different brain regions were also observed. Immunostaining revealed increased levels of phospho‐tau in WT mice injected with heat‐killed bacteria, suggesting that elevated systemic cytokine levels caused by the injection of heat‐killed bacteria can lead to the appearance of AD pathology. Another group of mice injected with IL‐1b and TNF‐a displayed hyperactivity and worsened short‐term memory functions. Injections of cytokines also induced periodontal bone loss, which was accompanied by elevated levels of inflammatory cytokines in the liver as well as different brain regions.ConclusionTaken together, our study revealed that increased cytokine immune response in the gums led to periodontal bone loss, brain inflammation and modulated the cognitive functions of WT mice. This research demonstrated that cytokines play a crucial role in the pathogenesis of both periodontitis and AD.Acknowledgement: The study is supported by Health and Medical Research Fund (HMRF 04151216) to RCCC. RPHW is awarded by Hong Kong PhD Fellowship.

Abstract PO-110: Targeting cathepsin B in the pancreatic stellate cells stimulates CD8+ T cell dependent anti-tumor immune response

Abstract Background: Cathepsin B (CTSB) is a lysosomal cysteine protease which has been described to have a role in pancreatic cancer development. However, its significance in the tumor microenvironment has never been investigated. Pancreatic stellate cells (PSCs) are one of the main sources of Cancer Associated Fibroblasts (CAFs) in the pancreatic cancer microenvironment. Herein, we describe the effects of targeting CTSB in the pancreatic stellate cells (PSCs) in pancreatic cancer (PDAC). Methods: To specifically study the role of CTSB in PSCs, PSCs were isolated from C57BL/6J (WT) or CTSB -/- mice and were orthotopically injected with KPC cancer cells into the tail of pancreas of 6-week old female WT mice. Tumors were analyzed at endpoint using flow cytometry. To further study the dependence of effects of CTSB in PSCs on adaptive immune cells, WT or CTSB -/- PSCs were co-injected with KPC cells into Rag 1-/- mice or CD8+ T cell depleted mice (anti-CD8 mAb, 250ug/dose every 3 days). In vitro, WT or CTSB-/- PSCs were co-cultured with KPC across a transwell. CD8+ T cells, isolated from KPC tumor bearing mice, were exposed to conditioned media from this co-culture ex-vivo and their cytotoxicity was measured using calcein release assay. In another experiment, WT mice injected with KPC cancer cells were treated with vehicle control or the CTSB inhibitor, CA-074 methyl ester (10 mg/kg), through daily intraperitoneal injections. Results: Co-injections of WT PSC with KPC cells formed larger tumors as compared to KPC cancer cells alone, however, co-injection with CTSB-/- PSCs abrogated this tumor promoting effect. Flow cytometric analysis revealed increased infiltration of Ifn-γ secreting CD8+ T cells in the KPC+CB-/- PSC group as compared to KPC+WT PSC group. Tumor decreasing effect of CTSB-/- PSCs was lost when co-injections were performed in Rag 1-/- mice or CD8+ T cell depleted mice, indicating that this effect relies on a functional adaptive immune response, specifically CD8+ T cells. In vitro, co-culture of CTSB-/- PSCs with KPC led to reduced expression of inflammatory CAF (iCAF) markers like IL-6 and IL-11 when compared to co-culture of WT PSCs with KPC. CD8+ T cells exposed to conditioned media from CTSB-/- PSC co-culture showed increased cytotoxicity against KPC cells ex-vivo, as compared to those exposed to conditioned media from WT PSC co-culture. Finally, treatment with CA-074 was able to decrease orthotopic PDAC tumor growth with increased CD8+ T cell infiltration evident on flow cytometry. Conclusion: Targeting CTSB in the PSCs stimulates anti-tumor adaptive immune response against pancreatic cancer by inhibiting conversion of PSCs into the tumor-promoting iCAF phenotype. CTSB might emerge as a novel target for CAF-directed therapy in pancreatic cancer. Citation Format: Bharti Garg, Tejeshwar Jain, Utpreksha Vaish, Vikas Dudeja. Targeting cathepsin B in the pancreatic stellate cells stimulates CD8+ T cell dependent anti-tumor immune response [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-110.

Disruption of Glucocorticoid Action on CD11c+ Dendritic Cells Favors the Generation of CD4+ Regulatory T Cells and Improves Fetal Development in Mice.

A wealth of innate and adaptive immune cells and hormones are involved in mounting tolerance towards the fetus, a key aspect of successful reproduction. We could recently show that the specific cross talk between the pregnancy hormone progesterone and dendritic cells (DCs) is significantly engaged in the generation of CD4+ FoxP3+ regulatory T (Treg) cells while a disruption led to placental alterations and intra-uterine growth restriction. Apart from progesterone, also glucocorticoids affect immune cell functions. However, their functional relevance in the context of pregnancy still needs clarification. We developed a mouse line with a selective knockout of the glucocorticoid receptor (GR) on DCs, utilizing the cre/flox system. Reproductive outcome and maternal immune and endocrine adaptation of Balb/c-mated C57Bl/6 GRflox/floxCD11ccre/wt (mutant) females was assessed on gestation days (gd) 13.5 and 18.5. Balb/c-mated C57Bl/6 GRwt/wtCD11ccre/wt (wt) females served as controls. The number of implantation and fetal loss rate did not differ between groups. However, we identified a significant increase in fetal weight in fetuses from mutant dams. While the frequencies of CD11c+ cells remained largely similar, a decreased expression of co-stimulatory molecules was observed on DCs of mutant females on gd 13.5, along with higher frequencies of CD4+ and CD8+ Treg cells. Histomorphological and gene expression analysis revealed an increased placental volume and an improved functional placental capacity in mice lacking the GR on CD11c+ DCs. In summary, we here demonstrate that the disrupted communication between GCs and DCs favors a tolerant immune microenvironment and improves placental function and fetal development.

Amyloid-β Processing in Aged S100B Transgenic Mice Is Sex Dependent.

(1) Background: Calcium-binding protein S100B is involved in neuroregeneration but has also been associated with neurodegeneration. These contrasting effects may result from concentration or duration of exposure. We investigated the effect of long-term increased S100B levels on amyloid-β processing in one-year-old transgenic (tg) mice with 12 copies of the murine S100B gene with specific consideration of sex and specific brain regions. (2) Methods: S100B and amyloid-β 42 (Aβ42) were quantified in serum, cerebrospinal fluid (CSF), adipose tissue, and different brain regions by ELISA in wild-type (wt) and S100Btg mice (each n = 7 per group). Thioflavin T (ThT) and Aβ immunostaining were performed for visualization of Aβ deposition. (3) Results: S100B in serum, CSF, and brain was significantly increased in S100Btg mice of both sexes. Aβ42 was significantly increased in the hippocampus of male S100Btg mice (p = 0.0075), and the frontal cortex of female S100Btg mice (p = 0.0262). ThT and Aβ immunostaining demonstrated Aβ deposition in different brain regions in S100Btg mice of both sexes and female wt. (4) Conclusion: Our data validate this experimental model for studying the role of S100B in neurodegeneration and indicate that Aβ processing is sex-dependent and brain region-specific, which deserves further investigation of signaling pathways and behavioral responses.

Cigarette smoke and nicotine effects on behavior in HIV transgenic rats

HIV-related neurocognitive impairment can be worsened by cigarette smoking and be more severe in women. Therefore, we analyzed the effects of sex on behavioral function in HIV transgenic (Tg) rats that were exposed to either nicotine alone, to smoke from either nicotine-containing or nicotine-free cigarettes, or non-exposed. The animals were then assessed on the open field test for the total distance traveled and for the fraction of the total distance traveled and the total time spent in the center of the field, and the results then compared to WT rats subjected to the same exposures and testing. Higher total distances indicate greater locomotor activity and a higher center field measures imply a lower anxiety state. Total distances were overall higher for female and for Tg rats exposed to nicotine-free CS. Also, the total distance and both center field measures were overall higher for female rats in the control and nicotine-free CS-exposed groups. This was observed specifically for WT females as compared to WT males and, for the center field measures, for WT females as compared to Tg males. No genotype or sex-related differences were found for rats in the nicotine-free cigarette smoke (CS) and nicotine-containing CS exposed groups. Therefore, nicotine exposure did not impact genotype- and sex-related differences in motor responses and anxiety levels that were found in the control state. However, exposure to the non-nicotine components of CS resulted in locomotor activation in the presence of the HIV genes and was anxiogenic in WT and Tg male animals.

Abstract P389: Free Fatty Acid Receptor 4 Is Necessary For An Adaptive Response Following Heart Failure Induced By Metabolic Syndrome In Mice

Non-resolving inflammation is central to the pathogenesis of heart failure (HF). Heart failure preserved ejection fraction (HFpEF) is a type of HF that is particularly associated with inflammation provoked by metabolic syndrome (MetS). The G-protein coupled receptor, free fatty acid receptor 4 (Ffar4), is a receptor for medium and long chain fatty acids (FA) that regulates metabolism and attenuates inflammation. Ffar4 is expressed in the human heart, and downregulated in heart failure. Furthermore, polymorphisms in Ffar4 have been associated with eccentric remodeling in a patient cohort. Previously, Ffar4 was shown to protect the heart from pathologic stress by attenuating oxidative stress in a mouse model of pressure overload. Here, we tested the hypothesis that Ffar4 would attenuate the development of heart failure using a mouse model of MetS-induced HFpEF. Metabolic syndrome was induced in mice by feeding a high-fat, high-sucrose diet (42% fat, 30% sucrose) to produce obesity and delivering the nitric oxide synthase inhibitor, L-NAME, in the drinking water to induce hypertension. The combined intervention (referred to as HFpEF diet) resulted in mice developing excessive adiposity, glucose intolerance (in males only), and mild hypertension. After 20 weeks on the HFpEF diet, both male and female WT mice, developed diastolic dysfunction (increased E/A and E/e’) and preserved ejection fraction (EF), consistent with clinical HFpEF. In Ffar4KO male mice HFpEF diet induced a greater degree of diastolic dysfunction compared to WT mice, despite equivalent metabolic parameters. Female Ffar4KO mice fed the HFpEF diet had a greater increase in weight gain and adiposity compared to WT female mice. Surprisingly, diastolic function was equivalent between WT and FFAR4KO female mice, suggesting a sex-based difference in FFAR4 cardioprotection. Our data show that Ffar4 attenuates HFpEF secondary to MetS.

Abstract MP61: Sex Difference In The Response Of Human Renal Proximal Tubule Cells To A Nephrotoxic Agent.

Our previous work indicated that the renal dopamine D2 receptor (D2R) has a significant role in regulating renal inflammation and injury, as well as in blood pressure control. In mice, D2R has protective effects in the kidney by limiting the inflammatory and fibrotic reaction; impaired D2R function results in renal inflammation and damage. Some common single nucleotide polymorphisms (SNPs; rs 6276 and 6277) in the human DRD2 gene are associated with decreased D 2 R expression and function and high blood pressure. To determine the effects of the presence of SNPs in the response to the nephrotoxic aristolochic acid (AA, 5μg/ml, 24 h), we studied immortalized human renal proximal tubule cells isolated from normal tissue of nephrectomies and genotyped for DRD2 SNPs and DRD2 wild-type (WT). We also determined whether this response is sex dependent. D2R protein was higher in male than in female WT (135±5 vs 100±4%; n=3/group; P&lt;0.04) and lower in males with SNPs (43±2%, P&lt;0.05) and females with SNPs (23±2%,P&lt;0.05), compared with their respective WT counterparts. In both male groups (WT and SNPs), AA increased D2R protein by 80-100% but had no effect in WT females and increased ~50% in females with SNPs. The TNFα mRNA was higher in males with WT and SNPs which was increased by AA 9-10-fold in WT males and females but only 2-3-fold in those with SNPs. The TGFβ mRNA was similar in WT males and females and increased to the same extent in both groups with SNPs and was not affected by AA in all groups. Col1a1 mRNA was higher (30%) in WT males and females than those with SNPs; AA decreased Col1a1 mRNA in all groups. FN1 mRNA was higher (30-40%) in males and females with SNPs than WT; AA increased FN1 mRNA only in males and females with SNPs. The mRNA expression of the cell proliferation marker Ki-67 was higher in WT females than WT males (1.5-2-fold) and higher with SNPs than WT in both groups; AA increased Ki-67 mRNA in both groups and to a greater extent in males than in females. Taken together our data indicate that the presence of DRD2 SNPs affects the baseline expression of inflammatory and fibrotic factors and the response to AA is dependent on both sex and the presence of DRD2 SNPs. These data may have potential clinical translation since rs6276/6277 is commonly expressed (42%/23%) in the human population.

Accelerated developmental adipogenesis programs adipose tissue dysfunction and cardiometabolic risk in offspring born to dams with metabolic dysfunction.

This study determined if a perturbation in in utero adipogenesis leading to later life adipose tissue (AT) dysfunction underlies programming of cardiometabolic risk in offspring born to dams with metabolic dysfunction. Female mice heterozygous for the leptin receptor deficiency (Hetdb) had 2.4-fold higher prepregnancy fat mass and in late gestation had higher plasma insulin and triglycerides compared with wild-type (Wt) females (P < 0.05). To isolate the role of the intrauterine milieu, wild-type (Wt) offspring from each pregnancy were studied. Differentiation potential in isolated progenitors and cell size distribution analysis revealed accelerated adipogenesis in Wt pups born to Hetdb dams, accompanied by a higher accumulation of neonatal fat mass. In adulthood, whole body fat mass by NMR was higher in male (69%) and female (20%) Wt offspring born to Hetdb versus Wt pregnancies, along with adipocyte hypertrophy and hyperlipidemia (all P < 0.05). Lipidomic analyses by gas chromatography revealed an increased lipogenic index (16:0/18:2n6) after high-fat/fructose diet (HFFD). Postprandial insulin, ADIPO-IR, and ex vivo AT lipolytic responses to isoproterenol were all higher in Wt offspring born to Hetdb dams (P < 0.05). Intrauterine metabolic stimuli may direct a greater proportion of progenitors toward terminal differentiation, thereby predisposing to hypertrophy-induced adipocyte dysfunction.NEW & NOTEWORTHY This study reveals that accelerated adipogenesis during the perinatal window of adipose tissue development predisposes to later life hypertrophic adipocyte dysfunction, thereby compromising the buffering function of the subcutaneous depot.

Effects of D-serine treatment on outer retinal function

The role of the N-Methyl-D-Aspartate Receptor (NMDAR) in the outer retina is unclear despite expression of the NMDAR-complex and its subunits in the outer retina. The flash-electroretinogram (fERG) offers a non-invasive measurement of the retinal field potentials of the outer retina that can serve to clarify NMDAR contribution to early retinal processing. The role of the NMDAR in retinal function was assessed using a genetic mouse model for NMDAR hypofunction (SR-/-), where the absence of the enzyme serine racemase (SR) results in an 85% reduction of retinal D-serine. NMDAR hypo- and hyperfunction in the retina results in alterations in the components of the fERG. The fERG was examined after application of exogenous D-serine to the eye in order to determine whether pre- and post-topical delivery of D-serine would alter the fERG in SR-/- mice and their littermate WT controls. Amplitude and implicit time of the low-frequency components, the a- and b-wave, were conducted. Reduced NMDAR function resulted in a statistically significantly delayed a-wave and reduced b-wave in SR-/- animals. The effect of NMDAR deprivation was more prominent in male SR-/- mice. A hyperfunction of the NMDAR, through exogenous topical delivery of 5 mM D-serine, in WT mice caused a significantly delayed a-wave implicit time and reduced b-wave amplitude. These changes were not observed in female WT mice. There were temporal delays in the a-wave and amplitude and a decrease in the b-wave amplitude and implicit time in both hypo- and NMDAR hyperfunctional male mice. These results suggest that NMDAR and D-serine are involved in the retinal field potentials of the outer retina that interact based on the animal's sex. This implicates the involvement of gonadal hormones and D-serine in retinal functional integrity.

Ghrelin receptor signaling is not required for glucocorticoid-induced obesity in female mice.

Chronic exposure to high circulating glucocorticoid or ghrelin concentrations increases food intake, weight gain and adiposity, suggesting that ghrelin could contribute to the metabolic effects of chronic glucocorticoids. In male mice, however, blocking ghrelin receptor (GHSR) signaling increased the weight gain and adiposity induced by chronic corticosterone (CORT), rather than attenuating them. In the current study, we investigated the role of GHSR signaling in the metabolic effects of chronic exposure to high circulating CORT in female mice. To do this, female WT and GHSR KO mice were treated with either CORT in a 1% ethanol (EtOH) solution or 1% EtOH alone in their drinking water for 32 days (n = 5-8/group). Body weight, food, and water intake as well as vaginal cyclicity were assessed daily. As expected, CORT treatment-induced significant increases in body weight, food intake, adiposity and also impaired glucose tolerance. In contrast to results observed in male mice, WT and GHSR KO female mice did not differ on any of these parameters. Neither plasma levels of ghrelin, LEAP-2, the endogenous GHSR antagonist produced by the liver, nor their ratio were altered by chronic glucocorticoid exposure. In addition, CORT treatment disrupted vaginal cyclicity, produced a reduction in sucrose consumption and increased locomotor activity regardless of genotype. Chronic CORT also decreased exploration in WT but not GHSR KO mice. Collectively, these data suggest that most metabolic, endocrine, reproductive and behavioral effects of chronic CORT exposure are independent of GHSR signaling in female mice.

Fine-Tuning the PI3K/Akt Signaling Pathway Intensity by Sex and Genotype-Load: Sex-Dependent Homozygotic Threshold for Somatic Growth but Feminization of Anxious Phenotype in Middle-Aged PDK1 K465E Knock-In and Heterozygous Mice.

According to the Research Domain Criteria (RDoC), phenotypic differences among disorders may be explained by variations in the nature and degree of neural circuitry disruptions and/or dysfunctions modulated by several biological and environmental factors. We recently demonstrated the in vivo behavioral translation of tweaking the PI3K/Akt signaling, an essential pathway for regulating cellular processes and physiology, and its modulation through aging. Here we describe, for the first time, the in vivo behavioral impact of the sex and genetic-load tweaking this pathway. The anxiety-like phenotypes of 61 mature (11–14-month-old) male and female PDK1 K465E knock-in, heterozygous, and WT mice were studied. Forced (open-field) anxiogenic environmental conditions were sensitive to detect sex and genetic-load differences at middle age. Despite similar neophobia and horizontal activity among the six groups, females exhibited faster ethograms than males, with increased thigmotaxis, increased wall and bizarre rearing. Genotype-load unveiled increased anxiety in males, resembling female performances. The performance of mutants in naturalistic conditions (marble test) was normal. Homozygotic-load was needed for reduced somatic growth only in males. Factor interactions indicated the complex interplay in the elicitation of different negative valence system’s items and the fine-tuning of PI3K/Akt signaling pathway intensity by genotype-load and sex.