WT Control Mice Research Articles

Development of a New Positron Emission Tomography Imaging Radioligand Targeting RIPK1 in the Brain and Characterization in Alzheimer's Disease.

Targeting receptor-interacting protein kinase 1 (RIPK1) has emerged as a promising therapeutic stratagem for neurodegenerative disorders, particularly Alzheimer's disease (AD). A positron emission tomography (PET) probe enabling brain RIPK1 imaging can provide a powerful tool to unveil the neuropathology associated with RIPK1. Herein, the development of a new PET radioligand, [11C]CNY-10 is reported, which may enable brain RIPK1 imaging. [11C]CNY-10 is radiosynthesized with a high radiochemical yield (41.8%) and molar activity (305 GBq/µmol). [11C]CNY-10 is characterized by PET imaging in rodents and a non-human primate, demonstrating good brain penetration, binding specificity, and a suitable clearance kinetic profile. It is performed autoradiography of[11C]CNY-10 in human AD and healthy control postmortem brain tissues, which shows strong radiosignal in AD brains higher than healthy controls. Subsequently, it is conducted further characterization of RIPK1 in AD using [11C]CNY-10-based PET studies in combination with immunohistochemistry leveraging the 5xFAD mouse model. It is found that AD mice revealed RIPK1 brain signal significantly higher than WT control mice and that RIPK1 is closely related to amyloid plaques in the brain. The studies enable further translational studies of [11C]CNY-10 for AD and potentially other RIPK1-related human studies.

Parkinson's LRRK2-G2019S risk gene mutation drives sex-specific behavioral and cellular adaptations to chronic variable stress.

Anxiety is a psychiatric non-motor symptom of Parkinson's that can appear in the prodromal period, prior to significant loss of brainstem dopamine neurons and motor symptoms. Parkinson's-related anxiety affects females more than males, despite the greater prevalence of Parkinson's in males. How stress, anxiety and Parkinson's are related and the basis for a sex-specific impact of stress in Parkinson's are not clear. We addressed this using young adult male and female mice carrying a G2019S knockin mutation of leucine-rich repeat kinase 2 ( Lrrk2 G2019S ) and Lrrk2 WT control mice. In humans, LRRK2 G2019S significantly elevates the risk of late-onset Parkinson's. To assess within-sex differences between Lrrk2 G2019S and control mice in stress-induced anxiety-like behaviors in young adulthood, we used a within-subject design whereby Lrrk2 G2019S and Lrrk2 WT control mice underwent tests of anxiety-like behaviors before (baseline) and following a 28 day (d) variable stress paradigm. There were no differences in behavioral measures between genotypes in males or females at baseline, indicating that the mutation alone does not produce anxiety-like responses. Following chronic stress, male Lrrk2 G2019S mice were affected similarly to male wildtypes except for novelty-suppressed feeding, where stress had no impact on Lrrk2 G2019S mice while significantly increasing latency to feed in Lrrk2 WT control mice. Female Lrrk2 G2019S mice were impacted by chronic stress similarly to wildtype females across all behavioral measures. Subsequent post-stress analyses compared cFos immunolabeling-based cellular activity patterns across several stress-relevant brain regions. The density of cFos-activated neurons across brain regions in both male and female Lrrk2 G2019S mice was generally lower compared to stressed Lrrk2 WT mice, except for the nucleus accumbens of male Lrrk2 G2019S mice, where cFos-labeled cell density was significantly higher than all other groups. Together, these data suggest that the Lrrk2 G2019S mutation differentially impacts anxiety-like behavioral responses to chronic stress in males and females that may reflect sex-specific adaptations observed in circuit activation patterns in stress-related brain regions.

Abstract 5344: Myeloid populations upregulated in prostate cancer are associated with colitis induced colorectal cancer

Abstract Chronic systemic inflammation caused by diseases like ulcerative colitis (UC) and Crohn’s disease (CD) increases the risk of developing colorectal cancer (CRC). Research indicates that patients with UC are more susceptible to prostate cancer (PCa) and individuals with PCa may also be at a higher risk for developing CRC. However, these relationships are not well defined. Here, we explored these relationships using preclinical mouse models of dextran sulfate sodium (DSS)-induced colitis (DSS-UC) and in young and aged healthy wild-type mice and conditional transgenic mice harboring Pten-deficient (KO) or Pten/Trp53-deficient (DKO) PCa. After a 25-week follow-up, DSS-induced UC caused more severe colitis in prostate tumor-bearing DKO compared to healthy WT mice. None of the DSS WT mice developed tumors, whereas 75% (3/4 mice) of DSS DKO mice did, including two out of four mice that developed multiple tumors. Overall, DKO mice with prostate tumors had increased levels of CD11b+/Gr1+ in peripheral blood, prostate, and colon compared with WT mice. Quantitative immunohistochemical analyses revealed that DKO mice with prostate tumors had a significantly higher 2.8-fold increase in the levels of Gr1+ cells in the colon compared to heathy WT mice. Overall, DSS-induced colitis further increased Gr1+ cells in the colon of WT and DKO mice by 2.5- and 2-fold, respectively. Gr1+ cells comprised 3.0%, 4.7%, and 10.1% of cells in normal colon from control WT mice and inactive and active regions of the colon in DSS-exposed WT mice, respectively. In DKO mice, Gr1+ cells comprised 7.0%, 19.2%, and 23.6% of cells in normal colon from control mice and inactive and active regions of the colon in DSS-exposed mice, respectively. CD68+, which corresponds to M1 polarized macrophages comprised 0.9%, 1.8%, and 20.6% of cells in normal colon from control WT mice and inactive and active regions of the colon in DSS-exposed WT mice, respectively, However, in DKO mice CD68+ cells comprised 3.0%, 2.8% and 1.6% of cells in normal colon from control mice and inactive and active regions of the colon in DSS-exposed mice, respectively. Our study suggests that Gr1 cells that were abundant in PCa-bearing mice were also present in colons and could have contributed to CRC. Moreover, CD68+ macrophages in the colons of healthy mice may have decreased tumor promoting effects of DSS. This study provides preclinical evidence showing that systemic inflammation in the presence of an existing malignancy increases the risk of a second malignancy, in this case linking PCa to colitis-induced-CRC. Citation Format: Yurie Kura, Marco A. De Velasco, Kazuko Sakai, Mamoru Hashimoto, Syogo Adomi, Takafumi Minami, Kazuhiro Yoshimura, Kazutoshi Fujita, Kazuto Nishio, Hirotsugu Uemura. Myeloid populations upregulated in prostate cancer are associated with colitis induced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5344.

Parkinson's LRRK2-G2019S risk gene mutation drives sex-specific behavioral and cellular adaptations to chronic variable stress.

Anxiety is a psychiatric non-motor symptom of Parkinson's that can appear in the prodromal period, prior to significant loss of midbrain dopamine neurons and motor symptoms. Parkinson's-related anxiety affects females more than males, despite the greater prevalence of Parkinson's in males. How stress, anxiety and Parkinson's are related and the basis for a sex-specific impact of stress in Parkinson's are not clear. We addressed this using young adult male and female mice carrying a G2019S knockin mutation of leucine-rich repeat kinase 2 (Lrrk2 G2019S) and Lrrk2 WT control mice. In humans, LRRK2 G2019S significantly elevates the risk of late-onset Parkinson's. To assess within-sex differences between Lrrk2 G2019S and control mice in stress-induced anxiety-like behaviors in young adulthood, we used a within-subject design whereby Lrrk2 G2019S and Lrrk2 WT control mice underwent tests of anxiety-like behaviors before (baseline) and following a 28 day (d) variable stress paradigm. There were no differences in behavioral measures between genotypes in males or females at baseline, indicating that the mutation alone does not produce anxiety-like responses. Following chronic stress, male Lrrk2 G2019S mice were affected similarly to male wildtypes except for novelty-suppressed feeding, where stress had no impact on Lrrk2 G2019S mice while significantly increasing latency to feed in Lrrk2 WT control mice. Female Lrrk2 G2019S mice were impacted by chronic stress similarly to wildtype females across all behavioral measures. Subsequent post-stress analyses compared cFos immunolabeling-based cellular activity patterns across several stress-relevant brain regions. The density of cFos-activated neurons across brain regions in both male and female Lrrk2 G2019S mice was generally lower compared to stressed Lrrk2 WT mice, except for the nucleus accumbens of male Lrrk2 G2019S mice, where cFos-labeled cell density was significantly higher than all other groups. Together, these data suggest that the Lrrk2 G2019S mutation differentially impacts anxiety-like behavioral responses to chronic stress in males and females that may reflect sex-specific adaptations observed in circuit activation patterns in some, but not all stress-related brain regions.

Tubular mitochondrial pyruvate carrier disruption elicits redox adaptations that protect from acute kidney injury

ObjectiveEnergy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Although tubular metabolism changes markedly following acute kidney injury (AKI), it remains unclear which metabolic alterations are beneficial or detrimental. By analyzing large-scale, publicly available datasets, we observed that AKI consistently leads to downregulation of the mitochondrial pyruvate carrier (MPC). This investigation aimed to understand the contribution of the tubular MPC to kidney function, metabolism, and acute injury severity. MethodsWe generated tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice and employed renal function tests, in vivo renal 13C-glucose tracing, mechanistic enzyme activity assays, and tests of injury and survival in an established rhabdomyolysis model of AKI. ResultsMPC TubKO mice retained normal kidney function, displayed unchanged markers of kidney injury, but exhibited coordinately increased enzyme activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, compared to WT control mice, MPC TubKO mice showed increased glycolysis, decreased kidney injury and oxidative stress markers, and strikingly increased survival. ConclusionsOur findings suggest that decreased renal tubular mitochondrial pyruvate uptake hormetically upregulates oxidant defense systems before AKI and is a beneficial adaptive response after rhabdomyolysis-induced AKI. This raises the possibility of therapeutically modulating the MPC to attenuate AKI severity.

Abstract 16602: The PlGF-Nrp1 Interaction Promotes the Splenic Immune Response in Hypertension After Neuroimmune Activation

Introduction: We previously discovered that Placental Growth Factor (PlGF) is released in the spleen by a sympathetic discharge to elicit hypertension. Neuropilin1 (Nrp1) is one of PlGF receptors and, interestingly, is a transmembrane protein expressed on precursors of Dendritic cells (DCs). Hypothesis: Here we investigated the involvement of Nrp1 as the PlGF receptor mediating the neuroimmune activation of immune response during hypertension. Methods: We used flow cytometry and tail cuff to evaluate the involvement of PlGF-Nrp1 pathway in neuroimmune activation of splenic immune response in hypertension. Results: To define Nrp1 role in transducing PlGF effect during hypertension, we evaluate its expression by flow cytometry finding that monocyte-derived DCs (MoDCs) increased Nrp1 expression in response to AngII (12.7±1.3 vs 28.8±3 % Nrp1 Veh vs AngII; p<0.01). This effect was blocked by splenic sympathetic denervation obtained by celiac ganglionectomy (CGX) (28.8±3 vs 14±1.5 % Nrp1 Sham AngII vs CGX AngII; p<0.01). To test the relevance of PlGF-Nrp1 pathway in hypertension, we generated a transgenic mouse model with a selective deletion of Nrp1 in myeloid lineage utilizing the LysMcre (Nrp1 myeKO mice). By using an in vitro standard differentiation protocol of maturation of monocytes in DCs, we observed that CD86 and MHCII expression was up-regulated in MoDCs from the spleen of WT mice cultured in the standard medium plus rmPlGF (0.014±0.001 vs 0.035±0.005 CD86+ DCs/total cells count respectively, p<0.05; 0.010±0.003 vs 0.030±0.003 MHCII+ DCs/total cells count respectively, p<0.01). On the contrary, CD86 and MHCII were similarly expressed on MoDCs from Nrp1 myeKO mice cultured with and without rmPlGF indicating that PlGF is involved in the maturation of MoDCs through its interaction with Nrp1. To confirm the involvement of Nrp1 in blood pressure (BP) increase, we infused AngII to Nrp1 myeKO mice, finding that they were protected from hypertension as compared to WT control mice (Systolic BP 106±1 vs 126±2 mmHg respectively, p<0.001). Conclusions: These data demonstrate that PlGF-Nrp1 pathway is a key mechanism that promote the immune response involved in hypertension and blocking the splenic innervation, hampered this response and BP increase.

Rker-050, a Modified Activin Receptor Type Iia Ligand Trap, Promoted Erythropoiesis in a Murine Model of Myelofibrosis

Myelofibrosis (MF) is one of several myeloproliferative neoplasms most often associated with anemia due to ineffective hematopoiesis, inflammation, aberrant bone metabolism and progressive fibrosis in the bone marrow (BM). As a compensatory mechanism, extramedullary hematopoiesis is initiated in the spleen, resulting in splenomegaly. Megakaryocyte hyperplasia/dysplasia are thought to be one of the main drivers of MF disease progression. Evidence also suggests that dysregulated TGF-β superfamily activity contributes to ineffective hematopoiesis. KER-050 and its research form, RKER-050, are investigational modified activin receptor type IIA ligand traps designed to inhibit specific TGF-β superfamily ligands, including activin A, activin B, growth and differentiation factor (GDF) 8 and GDF11, to promote erythropoiesis and thrombopoiesis. Additionally, the KER-050 target ligands promoted fibrosis and bone resorption in preclinical studies. Therefore, by inhibiting these ligands, KER-050 could potentially rebalance the bone marrow microenvironment, including reducing fibrosis, allowing restoration of hematopoiesis and alleviating extramedullary hematopoiesis and associated splenomegaly. To investigate the potential of RKER-050 to reverse ineffective hematopoiesis in MF, a hypomorphic Gata1 MF mouse model (Gata1Low; MF mice) was utilized which presents similar characteristics to human MF, including ineffective hematopoiesis, cytopenias, extramedullary hematopoiesis, defective bone deposition and BM fibrosis. this study, we also observed aberrant bone growth in the cortex of the hind limb long bones which inappropriately extended into the bone marrow, resulting in limited BM compartment space and demonstrating a severe disease state in these mice. Anemia status of MF mice was confirmed at the start of the treatment. MF mice showed a 16% reduction in hemoglobin (Hgb) compared to WT control mice. After 12 weeks of treatment, MF+VEH mice continued to exhibit a significant or trending decrease in red blood cells (RBCs; -19%), Hgb (-20%), and hematocrit (Hct; -10%) compared to WT mice. In contrast, MF+050 mice had a significant recovery of RBCs (+31%), Hgb (+24%) and Hct (+20%), compared to MF+VEH mice, demonstrating that RKER-050 reversed anemia in this advanced disease MF model. To assess the mechanism of action of RKER-050, we evaluated erythroid precursor populations in the BM. While MF+VEH mice showed a significant reduction in erythroid progenitors compared to WT mice, These data support increased BM erythropoiesis with RKER-050 treatment. In addition, we evaluated the effect of RKER-050 on thrombopoiesis. MF+VEH mice were severely thrombocytopenic and had significantly increased megakaryocyte progenitors (MkP; +591%) compared to WT mice. Although RKER-050 significantly reduced MkP (-33%; MF+050), suggesting RKER-050 may positively influence the megakaryocyte lineage, this did not translate to improvements in thrombocytopenia, and platelet counts remained similar to MF+VEH. Additional data will be presented that investigates the potential of RKER-050 treatment in (6-month vs 10-month-old mice, where disease is less severe) to further evaluate the potential to impact cytopenias, extramedullary hematopoiesis and BM fibrosis. Overall, in the context of severe disease, our results suggest RKER-050 can promote erythropoiesis and reverse anemia in the BM in by rebalancing hematopoiesis in the BM niche. Reestablishing BM hematopoiesis could obviate the need for compensatory extramedullary hematopoiesis in the spleen, the major driver of splenomegaly in MF patients. In conclusion, KER-050 represents a potentially promising approach for patients with MF and other hematological diseases where ineffective hematopoiesis occurs.

Abstract 059: (Pro)renin Receptor Ablation In Tyrosine Hydroxylase Neurons In The Paraventricular Nucleus Of The Hypothalamus Improves Insulin Sensitivity In Mice Fed A High-fat Diet

We recently reported that neuron-specific (pro)renin receptor (PRR) deletion reduces blood pressure (BP), fasting blood glucose (BG) and improves glucose tolerance in high-fat diet (HFD)-treated mice. To investigate underlying mechanisms, we focused on tyrosine hydroxylase (TH) neurons in the hypothalamic paraventricular nucleus (TH PVN neurons), a largely GABAergic population that expresses PRR and projects to autonomic centers important for BP and BG regulation. The PRR was ablated specifically in the TH-positive neurons in the PVN using Cre-LoxP approach. Radio telemetric BP recording, fasting blood glucose (FBG) and frequently sampled intravenous glucose tolerance test (FSIVGTT) were performed following 6-week HFD (60% calories from fat). HFD induced elevation of BP within the first week ( p < 0.05) and maintained elevated during 6-week protocol; however, there was no difference in BP between TH PVN PRRKO and WT control mice in both sexes following HFD. In male mice, the controls had higher FBG when compared with the TH PVN PRRKO mice (144.1 ± 5.74 vs. 121.9 ± 4.34 mg/dl, p < 0.05) at the end of the 6-week HFD treatment. Interestingly, PRR ablation in the TH PVN neurons did not reduce FBG in female mice. The TH PVN PRRKO mice showed improved glucose handling during FSIVGTT (AUC: 3481 ± 247.5 vs. 4263 ± 153.9, p < 0.05), while there was no difference in insulin secretion (AUC: 1782 ± 339.2 vs. 2324 ± 495.6, p > 0.05) between the two groups suggesting that TH PVN PRR ablation attenuates HFD-induced glucose intolerance through either an increase in insulin sensitivity or an insulin-independent pathway. Detected by western blot, the glucose transporter 4, an insulin-dependent glucose transporter, was higher on the plasma membrane of skeletal muscle in the TH PVN PRRKO mice (1.9 ± 0.2 vs. 1.0 ± 0.1, p < 0.05) compared with the controls. The hepatic mRNA expression of glucose-6-phosphatase, a gluconeogenic gene transcriptional negative-regulated by insulin, was lower in the TH PVN PRRKO (0.60 ± 0.07 vs. 0.95 ± 0.10, p < 0.05) compared with the control mice, suggesting improvement of insulin sensitivity in the TH PVN PRRKO mice. In conclusion, PRR ablation in TH PVN neurons attenuates FBG and glucose tolerance through improvement of insulin sensitivity without impacting BP.

#4369 MYELOID CELL-DERIVED HMGB1 AGGRAVATES THE PROGRESSION OF CHRONIC KIDNEY DISEASE INDUCED BY CHRONIC OXALATE EXPOSURE

Abstract Background and Aims HMGB1, long form High Mobility Group Box 1, a highly conserved DNA chaperone stabilizes chromatin in the nucleus, regulates gene transcription, and supports DNA repair. In the cytosol, it regulates mitochondrial function, autophagy, and apoptosis. In addition, when secreted or passively released into the extracellular space during cell necrosis, it acts as danger signal (DAMP). As such HMGB1 amplifies necroinflammation in ischemic and toxic acute kidney injury. In contrast, little is known about the contribution of HMGB1 release from infiltrating immune cells in this context. We hypothesized that HMGB1 from resident and infiltrating myeloid cells would contribute to the progression of CKD by driving tubular atrophy and tissue remodeling. Method We generated myeloid deletion of HMGB1 by crossing Hmgb1 floxed mice (Hmgb1f/f) with endogenous Lyz2 promoter (Lyz2Cre/+) to investigate the role of myeloid cell HMGB1 in a model of chronic kidney disease induced by sodium oxalate-rich diet (50 μmol/g), i.e., calcium oxalate nephropathy. The Lyz2Cre/+ mice were on a C57BL/6J background and crossed with mice homozygous for the floxed Hmgb1 gene to generate mice with a constitutive deletion of the Hmgb1 gene in all myeloid cells. We used littermates of 6- or 8-week-old males for all experiments. Mice with no Lyz2Cre/+ but the homozygous of floxed Hmgb1 gene served as wildtype controls (Lyz2Cre/−Hmgb1f/f WT) and mice with Lyz2Cre/+ and the homozygous of floxed Hmgb1 gene were used as Lyz2Cre/+Hmgb1f/f KO. The primary endpoint for the comparison between Lyz2Cre/+Hmgb1f/f KO and WT mice was glomerular filtration rate at 21 days of oxalate feeding, assessed by FITC-sinistrin clearance. Plasma samples were collected only on day 21 before sacrifice by cervical dislocation. Urine samples were collected as well as GFR was measured from all experimental groups on day 0, day7, day14 and before sacrifice by cervical dislocation on day 21. Kidneys were harvested after sacrifice. The kidney was divided into two equal parts. One part was kept in RNA later solution at −80°C for RNA isolation and the second part was kept in 4% formalin to be embedded in paraffin for histology analysis. Results KO animals did not reveal any abnormalities within an observation phase of 6 months. Healthy mice showed normal kidney function parameters in urine and blood. Observation is continued and will be supplemented by histopathological analyzes at the age of 12 months. Primary endpoint: Deletion of HMGB1 from myeloid cells attenuated the decline of GFR as compared to WT control mice. Secondary endpoints: The difference in GFR was consistent with the respective levels of serum creatinine at day 21 upon oxalate-rich diet compared with controls. The ablation of HMGB1 in myeloid cells was associated with less tubulointerstitial fibrosis at day 21 after oxalate-rich diet Conclusion Our data suggest that HMGB1 release from myeloid cells promotes crystal-induced chronic kidney injury, which is consistent with the role of HMGB1 as an extracellular DAMP and persistent inflammation in CKD. However, this effect may also relate to the intracellular role of HMGB1 as a transcriptional regulator.

Complement Turning Violent in Ultra-Rare Genetic Disorder

Abstract Sandhoff disease (SD) is an ultra-rare lysosomal storage disorder (LSD), which affects ~ 1/1000, 000 live birth. SD is caused by genetic deficiency of beta (β) Hexosaminidase and resulting excess central nervous system (CNS) synthesis of GM2 ganglioside (GM2) and its impact on neuron death. The exact mechanisms underlying such GM2-driven neuron death are unknown in SD. Glucosylceramide (GC) induced complement 5a (C5a) and its C5aR1 activation causes tissue inflammation in experimental and clinical Gaucher disease. Additionally, C5a-C5aR1 axis was found to sparks CNS inflammation and neurodegeneration in several brain diseases including, intracerebral hemorrhage, traumatic brain injury, myasthenia gravis, amyotrophic lateral sclerosis, neuromyelitis optica spectrum, Alzheimer, and Huntington’s diseases. Here, we determined the increased brain levels of C5a and C5aR1 in a beta (β) hexosaminidase inhibitor (HABI)-induced experimental mouse model of SD, when compared to vehicle (PBS) injected WT control mice. Also, HABI-induced experimental mouse model of SD showed increased microglial cells activation, massive brain generation of pro-inflammatory cytokines and loss of neurons, when compared to vehicle injected WT mice. To assess the relevance of C5a-C5aR1 axis activation for brain inflammation in SD, we targeted β hexosaminidase with HAB-I in WT and C5aR1 −/−mice. Strikingly, as compared to HABI-injected WT mice, HABI-injected C5aR1 −/−mice caused marked reduction in microglial cell activation, brain production of pro-inflammatory cytokines, and the loss of neurons. These data suggest that the C5a-C5aR1 axis is a critical driver of neuroinflammation and neurodegeneration in SD. No Funding

Assessment of the Response of Young and Aged Sarcospan-Deficient Mice to Nutrient Excess

This study follows up on human genome-wide association studies that have identified variants in the SSPN locus. This locus encodes the protein sarcospan that segregates with several anthropometric traits. One of these traits includes an increase in waist circumference. In this study, we compared the response of young and old SSPN-deficient (SSPN-/-) and WT control mice to high fat diet (HFD) (60% fat). The HFD was initiated in 2 month and 1 year old male and female for 4 months and 5 months respectively. This study showed that SSPN-deficiency provided partial protection from weight gain in young male mice when compared to age-matched WT mice and a significantly smaller increase in body weight (BW) compared to controls. Also, young female SSPN-/- mice gained 50% less BW than WT age matched mice compared to controls and visceral WAT fat pad weights were significantly lower. To examine whether these differences are lost in aging (e.g. due to hormonal decline), we subjected 12-month old WT and SSPN-/- mice to 5 months of HFD and assessed anthropometric traits, blood lipids, glucose tolerance, body composition, and alterations of cytokine/adipokine/chemokine levels in blood serum and adipose tissue. There were signs of inflammation/fibrosis in young WT male WAT. This was largely absent in SSPN-/- mice. Interestingly, a specific subset of upregulated inflammatory mediators was found in WT WAT including leptin. However, another distinct subset of adipokines/chemokines were upregulated including adiponectin in SSPN-/- WAT. Therefore, we are investigating whether SSPN affects obesity-associated inflammatory signaling in adipocytes that influences adipose tissue expansion and ectopic triglyceride accumulation in the liver. FDOH JEK 21K12 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Enhanced B Cell Receptor Signaling Partially Compensates for Impaired Toll-like Receptor 4 Responses in LPS-Stimulated IκBNS-Deficient B Cells.

Lipopolysaccharide (LPS) stimulates dual receptor signaling by bridging the B cell receptor and Toll-like receptor 4 (BCR/TLR4). B cells from IκBNS-deficient bumble mice treated with LPS display reduced proliferative capacity and impaired plasma cell differentiation. To improve our understanding of the regulatory role of IκBNS in B cell activation and differentiation, we investigated the BCR and TLR4 signaling pathways separately by using dimeric anti-IgM Fab (F(ab')2) or lipid A, respectively. IκBNS-deficient B cells exhibited reduced survival and defective proliferative capacity in response to lipid A compared to B cells from wildtype (wt) control mice. In contrast, anti-IgM stimulation of bumble B cells resulted in enhanced viability and increased differentiation into CD138+ cells compared to control B cells. Anti-IgM-stimulated IκBNS-deficient B cells also showed enhanced cycle progression with increased levels of c-Myc and cyclin D2, and augmented levels of pCD79a, pSyk, and pERK compared to control B cells. These results suggest that IκBNS acts as a negative regulator of BCR signaling and a positive regulator of TLR4 signaling in mouse B cells.

Tff3-/- Knock-Out Mice with Altered Lipid Metabolism Exhibit a Lower Level of Inflammation following the Dietary Intake of Sodium Chloride for One Week.

A high salt intake causes hemodynamic changes and promotes immune response through cell activation and cytokine production, leading to pro-inflammatory conditions. Transgenic Tff3-/- knock-out mice (TFF3ko) (n = 20) and wild-type mice (WT) (n = 20) were each divided into the (1) low-salt (LS) group and (2) high-salt (HS) group. Ten-week-old animals were fed with standard rodent chow (0.4% NaCl) (LS) or food containing 4% NaCl (HS) for one week (7 days). Inflammatory parameters from the sera were measured by Luminex assay. The integrin expression and rates of T cell subsets of interest from the peripheral blood leukocytes (PBLs) and mesenteric lymph nodes (MLNs) were measured using flow cytometry. There was a significant increase in high-sensitivity C reactive protein (hsCRP) only in the WT mice following the HS diet, while there were no significant changes in the serum levels of IFN-γ, TNF-α, IL-2, IL-4, or IL-6 as a response to treatment in either study groups. The rates of CD4+CD25+ T cells from MLNs decreased, while CD3+γδTCR+ from peripheral blood increased following the HS diet only in TFF3ko. γδTCR expressing T cell rates decreased in WT following the HS diet. The CD49d/VLA-4 expression decreased in the peripheral blood leukocytes in both groups following the HS diet. CD11a/LFA-1 expression significantly increased only in the peripheral blood Ly6C-CD11ahigh monocytes in WT mice following salt loading. In conclusion, salt-loading in knock-out mice caused a lower level of inflammatory response compared with their control WT mice due to gene depletion.

Abstract 5919: The significant role of HMGB1 in mesothelial cells in contributing to the mesothelioma development induced by asbestos

Abstract Diffuse Malignant mesothelioma is a very aggressive cancer that is related to asbestos exposure. We found previously that following asbestos deposition in the pleura and in the peritoneum, mesothelial cells release HMGB1 that attract granulocytes and macrophages that, in turn, secrete HMGB1. It was unknown whether the primary source of HMGB1 that drives the inflammatory process caused by asbestos, which may ensue in mesothelioma, derives from the mesothelial cells exposed to asbestos or from the granulocytes and macrophages. To study separately the contribution of mesothelial cells and of the inflammatory cells to HMGB1 secretion that drives mesothelioma development, we used two tissue-specific HMGB1 knockout mouse models: (1) An inducible mesothelial conditional HMGB1 knockout (HMGB1ΔpMeso) mouse model, and (2) a constitutive myelomonocytic-lineage knock out (HMGB1ΔMylc) mouse model. To investigate the role of HMGB1 in asbestos induced carcinogenesis in vivo, we conducted both short-term and long-term experiments in HMGB1ΔpMeso mice and in HMGB1ΔMylc mice. For the Short-term experiment, we observed that one week following the completion of the 10-weekly crocidolite i.p. injections, all the mice in experimental and control groups showed inflammation with multiple granulomas around asbestos deposits in the various tissues and organs present in the abdomen, and diffuse mesothelial hyperplasia. However, the granulomas in HMGB1ΔpMeso mice were significantly smaller compared to those found in the control WT mice. TNFα IHC stain was minimal to non-detectable in the granulomas and areas of mesothelial hyperplasia of HMGB1ΔpMeso mice, compared to a strong TNFα staining in both HMGB1ΔMylc and WT mice. For the Long-term experiment, we observed that the incidence of mesothelioma was significantly lower in HMGB1ΔpMeso mice compared with the 3 control groups including WT mice, HMGB1F/F mice and Wt1ERT2Cre/+ control mice. Moreover, HMGB1ΔpMeso mice had a significantly longer mesothelioma-specific survival compared to all three controls. Histologically, the HMGB1ΔpMeso mesotheliomas were smaller and grew largely over the surface rather than infiltrating the abdominal organs, compared to the mesotheliomas that developed in the three control groups. In summary, our findings underscore the role of HMGB1 in asbestos pathogenesis, including asbestos induced chronic inflammation and mesothelioma. Moreover, our findings elucidated the mechanisms responsible for asbestos induced inflammation and asbestos carcinogenesis, and the causal link between HMGB1, TNFα secretion and mesothelioma. Our results point to the HMGB1 secreted by mesothelial cells as the culprit and thus the ideal target to prevent the growth of mesothelioma upon asbestos exposure and during the early phases of mesothelioma growth. Citation Format: Joelle S. Suarez, Giovanni Gaudino, Tak W. Mak, Michele Carbone, Haining Yang. The significant role of HMGB1 in mesothelial cells in contributing to the mesothelioma development induced by asbestos [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5919.

Integrin-linked kinase expression in myeloid cells promotes colon tumorigenesis.

Colorectal cancer (CRC) is one of the most common forms of cancer worldwide and treatment options for advanced CRC, which has a low 5-year survival rate, remain limited. Integrin-linked kinase (ILK), a multifunctional, scaffolding, pseudo-kinase regulating many integrin-mediated cellular processes, is highly expressed in many cancers. However, the role of ILK in cancer progression is yet to be fully understood. We have previously uncovered a pro-inflammatory role for myeloid-specific ILK in dextran sodium sulfate (DSS)-induced colitis. To establish a correlation between chronic intestinal inflammation and colorectal cancer (CRC), we investigated the role of myeloid-ILK in mouse models of CRC. When myeloid-ILK deficient mice along with the WT control mice were subjected to colitis-associated and APCmin/+-driven CRC, tumour burden was reduced by myeloid-ILK deficiency in both models. The tumour-promoting phenotype of macrophages, M2 polarization, in vitro was impaired by the ILK deficiency and the number of M2-specific marker CD206-expressing tumour-associated macrophages (TAMs) in vivo were significantly diminished in myeloid-ILK deficient mice. Myeloid-ILK deficient mice showed enhanced tumour infiltration of CD8+ T cells and reduced tumour infiltration of FOXP3+ T cells in colitis-associated and APCmin/+-driven CRC, respectively, with an overall elevated CD8+/FOXP3+ ratio suggesting an anti-tumour immune phenotypes. In patient CRC tissue microarrays we observed elevated ILK+ myeloid (ILK+ CD11b+) cells in tumour sections compared to adjacent normal tissues, suggesting a conserved role for myeloid-ILK in CRC development in both human and animal models. This study identifies myeloid-specific ILK expression as novel driver of CRC, which could be targeted as a potential therapeutic option for advanced disease.

The receptor for advanced glycation end products (RAGE) is involved in mitochondrial function and cigarette smoke-induced oxidative stress

The mechanisms underlying muscle dysfunction with Chronic Obstructive Pulmonary Disease (COPD) are poorly understood. Indirect evidence has recently suggested a role of Advanced Glycation End Products (AGEs) and their receptor (RAGE) in the pathophysiology of COPD. Accordingly, this study aimed to examine the redox balance and mitochondrial alterations in the skeletal muscle of a mouse model deficient in the receptor for AGE (RAGE-KO) and wild-type C57BL/6 exposed to cigarette smoke for 8-months using immunoblotting, spectrophotometry, and high-resolution respirometry. Cigarette smoke exposure increased by two-fold 4-HNE levels (P < 0.001), a marker of oxidative stress, and markedly downregulated contractile proteins, mitochondrial respiratory complexes, and uncoupling proteins levels (P < 0.001). Functional alterations with cigarette smoke exposure included a greater reliance on complex-I supported respiration (P < 0.01) and lower relative respiratory capacity for fatty acid (P < 0.05). RAGE knockout resulted in 47% lower 4-HNE protein levels than the corresponding WT control mice exposed to cigarette smoke (P < 0.05), which was partly attributed to increased Complex III protein levels. Independent of cigarette smoke exposure, RAGE KO decreased mitochondrial specific maximal respiration (P < 0.05), resulting in a compensatory increase in mitochondrial content measured by citrate synthase activity (P < 0.001) such that muscle respiratory capacity remained unaltered. Together, these findings suggest that knockout of RAGE protected the skeletal muscle against oxidative damage induced by 8 months of cigarette smoke exposure. In addition, this study supports a role for RAGE in regulating mitochondrial content and function and can thus serve as a potential therapeutic target.

Psychosocial Stress‐Accelerates Alzheimer’s Disease Progression via the Xanthine Oxidase Pathway

AbstractBackgroundVascular contributions to cognitive impairment and dementia are a key pathology associated with Alzheimer’s disease (AD). We have shown that chronic stress induces cerebrovascular dysfunction due to a pro‐oxidative environment, a phenotype similar to that noted with AD. Epidemiological studies also suggest a link between chronic stress and dementia. We tested the hypothesis that chronic stress accelerates the AD pathology via the xanthine oxidase (XO) pathway.MethodsWild type (WT) and Triple transgenic (AD) mice were separated into 1) WT control, 2) WT+UCMS, 3) AD control, 4) AD control+Feb, 5) AD+UCMS, 6) AD+UCMS+Feb. At 4 months‐of‐age, dependent upon group, the unpredicted chronic mild stress (UCMS) model with or without Febuxostat (Feb) treated water (50mg/L) began for 8 weeks. Mice were either euthanized at 6 or 9 months‐of‐age. The middle cerebral artery (MCA) was removed for pressure myography. Brains were homogenized to extract soluble and insoluble fractions of tau proteins using Western blot normalized to GAPDH or B‐actin.ResultsAt 6 months‐of‐age, AD mice displayed a 72% impaired MCA dilation to acetylcholine compared to WT control mice (p&lt;0.05). WT and AD mice that underwent UCMS displayed similar MCA dysfunction compared to AD control mice. UCMS accelerated total and phosphorylated tau protein expression by 83% (p&lt;0.05) and 350% (p&lt;0.001), respectively, compared to AD controls. Treatment with Feb prevented MCA dysfunction in AD control mice, WT UCMS mice, and AD UCMS mice. Feb prevented the increased expression of phosphorylated tau and lowered the total tau protein expression in the AD‐UCMS group. However, total tau protein expression remained higher compared to AD control levels. At 9 months‐of‐age, the majority of these trends persisted with the one exception being a significant impairment in the AD UCMS group compared to both AD control or WT UCMS mice. Tau levels have yet to be processed for 9‐months.ConclusionOverall, these data suggest that chronic stress accelerates cerebrovascular pathology with AD and via a pro‐oxidative state driven by activation of the XO pathway. We will further explore Aβ1‐42 and XO in 3xTg brain homogenates at 6 and 9 months‐of‐age, to better understand how stress accelerates AD pathology.

Platelet αIIbβ3 Receptor Antagonist Impedes the Hemostatic Efficacy of Platelet-Specific FVIII Gene Therapy in Hemophilia a Mice

Hemophilia A is a prime model for gene therapy. Data from the current human clinical trial using liver-specific AAV-mediated FVIII gene therapy are encouraging, but FVIII levels steadily decline, raising serious concerns about durability and safety. Furthermore, pediatric and adult patients who have severe liver disease, neutralizing antibodies to AAV, which are present in 30-50% of the population, are excluded from AAV liver gene therapy. Our previous studies have demonstrated that platelet-targeted FVIII expression under the control of the platelet-specific αIIb promoter (2bF8) is effective in rescuing the bleeding phenotype of hemophilia (FVIII-/-) mice even in the presence of anti-FVIII inhibitory antibodies (inhibitors). Currently, our gene therapy approach is in Phase I clinical trial. Since antiplatelet drugs are commonly used in the clinic, it is important to investigate how platelet antagonists affect the clinical efficacy of platelet-derived FVIII. The αIIbβ3 complex is a receptor for fibrinogen and VWF, with a crucial role in platelet activation and platelet plug formation at the site of injury. The αIIbβ3 complex is the target of several drugs used in cardiovascular medicine. Thus, we propose to use an αIIbβ3 antibody to block the integrin αIIbβ3 receptor to investigate how platelet aggregation impacts the clinical efficacy of platelet-derived FVIII in FVIII-/- mice. We used anti-mouse integrin αIIbβ3 antibody (Leo.H4) to block the αIIbβ3 complex, inhibiting its binding to fibrinogen and therefore inhibiting platelet aggregation. Hemostatic properties of platelet-FVIII were assessed by ex vivo ROTEM and native whole blood TGA (nWB-TGA) analysis of whole blood and in vivo tail FeCl3-induced carotid artery injury model and lateral tail vein transection (TVT) injury model.In 2bF8/FVIII-/- mice, platelet-FVIII levels were 6.89±0.65 mU/108 platelets, with all parameters from ROTEM analysis comparable to those in WT control mice. However, all parameters, including clotting time, clot formation time, maximal clot firmness, α-angle, and A10 were significantly impaired in the presence of Leo.H4 compared to native samples from 2bF8 mice (8.5±1.96 min, 3.1±0.41min, 67.25±2.63mm, 55.75±3.59°, 44.25±4.03mm in native samples versus 10.68±1.77min, 13.5±3.11min, 28.5±0.58mm, 33±2.83°, 18.25±1.26mm in those with Leo.H4, respectively). In contrast, in WT mice, only α-angle and A10 were significantly tempered in the presence of Leo.H4. Interestingly, in 2bF8/FVIII-/- mice, peak thrombin and endogenous thrombin potential determined by TGA were significantly enhanced in the presence of Leo.H4 compared to native samples (58.4±23.72nM and 600.8±185.83nM vs. 97.93±13.89nM and 921.03±72.95nM, respectively), but there were no significant differences in other parameters. Leo.H4 did not impact the hemostatic parameters determined by nWB-TGA in WT mice. Complete occlusion occurred in the FeCl3-induced carotid injury in all 2bF8 and WT mice, but none occluded when Leo.H4 was infused. In the TVT injury model, when Leo.H4 was infused, none of 2bF8/FVIII-/- mice stopped bleeding in the 10-minute bleeding test, and animals lost a significant amount of blood (525.98 ± 92.06 μL). In contrast, when animals were infused with IgG isotype control, the bleeding time was 0.99 ± 0.05 min, and blood loss in the IgG control group was 17.05 ± 9.37 μL, which was 30-fold lower than in the Leo.H4 group. There were no significant differences in the bleeding time and blood loss between the 2bF8 infused with IgG and WT groups in the TVT test. In conclusion, our results demonstrated that αIIbβ3-mediated platelet aggregation is critical in maintaining the hemostatic functions and clinical efficacy of platelet-derived FVIII in hemophilia A. Anti-αIIbβ3 antibody, Leo.H4, impedes the hemostatic efficacy of platelet-FVIII in FVIII-/- mice.

Novel Evidence That Intracellular Complement Network (complosome) Is Expressed in Hematopoietic Stem Cells (HSPCs) and Regulates Their Trafficking, Metabolism, and Proliferation in an Intracrine-Dependent Manner By Engaging Pattern Recognition Receptor Nlrp3 Inflammasome

Background. Complement cascade (ComC) proteins, including C3 and C5 components, are synthesized in the liver; however, recent reports indicate their expression in lymphocytes as well (Immunity 2020;52(3):513-527). Accordingly, intracellular ComC proteins have been reported to be cleaved to active C3a and C5a mediators that interact with corresponding receptors in an intracrine-depended manner. This novel regulatory loop operating in lymphocytes has been named ''complosome''. Aim of the study.Because hematopoiesis and lymphopoiesis have a common stem cell origin and similar mechanisms are involved in regulating the biology of both lineages, we asked if complosome is also expressed and functional in normal murine hematopoietic stem progenitor cells (HSPCs). We also address a challenging question if intracellular ComC mediators could activate pattern recognition receptor Nlrp3 inflammasome that, as we reported, regulates trafficking, proliferation and metabolism of HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). Materials and Methods. First, we phenotyped purified murine and human HSPCs for expression of major complement elements, including C3, C5, C3aR, C5aR, and cathepsin L that cleaves intracellular C3 to C3a. Next, we focused on a crucial distal executive part of ComC, protein C5, and evaluated in C5-KO and C5aR-KO mice the number of HSPCs and their clonogeneic potential. We also studied hematopoietic recovery of these animals after sublethal irradiation (650 cGy). Activation of Nlrp3 inflammasome in C5a-KO and C5aR-KO in response to hematopoietic growth factors and cytokines was evaluated by immunofluorescence Glow assay. We also studied homing and engraftment of C5-KO and C5aR-KO mice-derived HSPCs transplanted into normal WT recipients. Finally, we performed a metabolomic analysis of C5-KO, C5aR-KO, and control WT mice in steady-state conditions and after stimulation with selected hematopoietic growth factors and cytokines. Results. We show for the first time that purified murine and human HSPCs express complosome elements, including C3, C5, C3aR, C5aR, and cathepsin L. Next, C5-KO and C5aR-KO mice also have a lower number of HSPCs and clonogenic progenitors in bone marrow (BM), and display defect of hematopoietic recovery from sublethal irradiation. Furthermore, BM cells isolated from these animals had impaired homing and engraftment in normal WT recipients, supported by the observation that HSPCs from complosome deficient mice had a defect in migration to stromal-derived factor-1 (SDF-1), which correlated with decreased activation of Nlrp3 inflammasome. This defect could be alleviated by direct activation of Nlrp3 inflammasome by nigericin. Finally, our metabolic studies revealed a defect in glucose, amino acids, and lipid metabolism in C5-KO and C5aR-KO mice which correlates with complosome deficiency and Nlrp3 inflammasome activation. Conclusions. We provide for the first time evidence that complosome is expressed and functional in normal HSPCs and by engaging Nlrp3 inflammasome regulates metabolism, proliferation, and migration of HSPCs. Thus, our data sheds new intriguing light on innate immunity as a regulator of hematopoiesis that involves intracellular activation of Nlrp3 inflammasome. We currently focus on Nlrp3 inflammasome-released alarmins released from the cells in response to complosome-Nlrp3 inflammasome activation that accounts for observed phenomena. These studies are essential to develop more efficient ex vivo expansion strategies of HSPCs and to understand better intrinsic mechanisms that regulate metabolism of these cells.

β2-AR Signaling Enhances MDSC Survival through Metabolic Reprograming and Impairs Therapeutic Efficacy in Hematologic Malignancies

Nerve-driven chronic adrenergic stress can suppress anti-tumor immunity. However, the mechanistic details of how this occurs are still poorly understood. Catecholamines (epinephrine, norepinephrine) released by the autonomic nervous system (ANS) can activate adrenergic receptors (AR) present on nearly every cell type, including myeloid derived suppressor cells (MDSCs). MDSCs are important regulatory cells that hinder anti-tumor immune responses in hematological malignancies by inhibiting numerous cell types, including effector NK cells, CD8+ T cells, CD4+ T cells, and processes, such as blocking cytokine secretion. Because first line treatment for most hematologic malignancies includes chemotherapy, and MDSCs are known to be detrimental to its efficacy, understanding their role, including through chronic stress pathways, is relevant. Recently, using preclinical models of solid tumors, we have shown β2 adrenergic receptor (β2-AR) signaling, activated by chronic stress, increases the accumulation of MDSCs in the tumor microenvironment (TME). However, the role of β2-AR in MDSC survival and accumulation in hematological malignancies after chemotherapy, and the precise mechanisms by which β2-AR increases MDSC accumulation, remain unresolved. We have discovered β2-AR signaling drives MDSC survival in tumor bearing mice treated with chemotherapy. We have also shown the combination of propranolol, a non-selective β-AR blocker, with doxorubicin prolongs survival in mice bearing EL4 and C1498 tumors. Examination of immune cell compartments demonstrated a significant reduction of both M-MDSC and PMN-MDSC abundance in mice receiving the combination therapy, indicating the key effect of stress induced β2-AR signaling on MDSC accumulation. Further, we implanted EL4 tumors into β2-AR-/- and β2ARfl/fl LysmCre mice and confirmed doxorubicin increases survival in both β2-AR-/- and β2ARfl/fl LysmCre mice compared to WT control mice, suggesting β2-AR signaling in MDSCs is the main receptor driving the effects of propranolol in combination therapy. Using both in vitro and in vivo systems, we showed β2-AR signaling increased MDSC survival by the induction of BCL-2 expression and the suppression of caspase-3 and caspase-8. Seahorse metabolic studies further showed β2-AR signaling induced a metabolic shift to increased mitochondrial respiration (OCR) and ATP production in MDSCs. We then applied single cell RNA sequencing (scRNAseq) pathway analysis and multiplexed D7-Glc plus 13C5-Gln tracing in Stable Isotope-Resolved Metabolomics (SIRM). We found β2-AR signaling induces a metabolic shift toward higher ATP generation and decreased mitochondrial reactive oxygen species (mROS) that serves as a survival signal for MDSCs. We also found β2-AR signaling suppressed the expression of ATPase Inhibitory Factor 1 (ATPIF1) in MDSCs, which is responsible for increased ATP generation. These results show β2-AR signaling induces MDSC survival through metabolic reprogramming in mitochondria by enhancing ATP generation and modulating key survival signals to increase the ratio of ATP/mROS. Altogether, these studies reveal β2-AR signaling plays an important role in mitochondrial metabolic fitness in MDSCs, priming them to survive chemotherapy-induced cytotoxicity. The combination of the stress blocker propranolol, with chemotherapy regimens, could potentially improve the current therapeutic regimens used for hematological malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal