Engineering topics

Hum, Nicholas R.

Publications and source records attributed to Hum, Nicholas R..

At least 19 records

DOE OSTI2024

Spatiotemporal analysis of lung immune dynamics in lethal Coccidioides posadasii infection

Coccidioidomycosis, or Valley fever, is a lung disease caused by inhalation of Coccidioides fungi, prevalent in the Southwestern United States, Mexico, and parts of Central and South America. Annually, the United States reports 10,000–20,000 cases, although those numbers are expected to increase as climate change expands the fungal geographic range. While 60% of infections are asymptomatic, 40% symptomatic infections are often misdiagnosed due to similarities with bronchitis or pneumonia. A small subset of infection progress to severe illness, necessitating a better understanding of immune responses during lethal infection. Using single-cell RNA sequencing and spatial transcriptomics, we characterized lung responses during Coccidioides infection. We identified monocyte-derived Spp1-expressing macrophages as potential mediators of tissue remodeling and fibrosis, marked by high expression of profibrotic and proinflammatory transcripts. These macrophages showed elevated TGF-β and IL-6 signaling, pathways involved in fibrosis pathogenesis. Additionally, we observed significant neutrophil infiltration and defective lymphocyte responses, indicating severe adaptive immunity dysregulation in lethal, acute infection. These findings enhance our understanding of Coccidioides infection and suggest new therapeutic targets.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Osteocytic oxygen sensing: Distinct impacts of VHL and HIF-2alpha on bone integrity

Skeletal fracture resistance emerges from multiple components of bone structure like microarchitecture, matrix mineralization, and organization. These characteristics are engendered via mechanisms like the hypoxia-inducible factors (HIF) pathway, involving two paralogs, HIF-1α and HIF-2α. Under normoxia, HIF-α is targeted for degradation via von-Hippel Lindau (VHL); hypoxia enables HIF-α stabilization and induction of target genes. We previously showed that osteocytic Vhl deletion or expression of degradation-resistant HIF-2α cDR female mice each produced high bone mass, whereas degradation-resistant osteocytic HIF-1α produced no overt phenotype. We report within that Vhl cKO increased bone strength, while HIF-2α cDR displayed markedly reduced bone strength below Cre-negative controls. This suggests that VHL and HIF-2α drive distinct responses that promote disparate effects on bone strength. Both Vhl deletion or HIF-2α accumulation generated two discrete bone morphologies: an outer lamellar cortex and a woven, poorly mineralized endocortex that imparted dramatically different functional outcomes. Our studies reveal novel influence of osteocytic HIF-2α signaling on collagen matrix organization, mineralization, and bone strength.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Eucalyptus Wood Smoke Extract Elicits a Dose-Dependent Effect in Brain Endothelial Cells

The frequency, duration, and size of wildfires have been increasing, and the inhalation of wildfire smoke particles poses a significant risk to human health. Epidemiological studies have shown that wildfire smoke exposure is positively associated with cognitive and neurological dysfunctions. However, there is a significant gap in knowledge on how wildfire smoke exposure can affect the blood–brain barrier and cause molecular and cellular changes in the brain. Our study aims to determine the acute effect of smoldering eucalyptus wood smoke extract (WSE) on brain endothelial cells for potential neurotoxicity in vitro. Primary human brain microvascular endothelial cells (HBMEC) and immortalized human brain endothelial cell line (hCMEC/D3) were treated with different doses of WSE for 24 h. WSE treatment resulted in a dose-dependent increase in IL-8 in both HBMEC and hCMEC/D3. RNA-seq analyses showed a dose-dependent upregulation of genes involved in aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (NRF2) pathways and a decrease in tight junction markers in both HBMEC and hCMEC/D3. When comparing untreated controls, RNA-seq analyses showed that HBMEC have a higher expression of tight junction markers compared to hCMEC/D3. In summary, our study found that 24 h WSE treatment increases IL-8 production dose-dependently and decreases tight junction markers in both HBMEC and hCMEC/D3 that may be mediated through the AhR and NRF2 pathways, and HBMEC could be a better in vitro model for studying the effect of wood smoke extract or particles on brain endothelial cells.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Biphasic response of human iPSC-derived neural network activity following exposure to a sarin-surrogate nerve agent

Organophosphorus nerve agents (OPNA) are hazardous environmental exposures to the civilian population and have been historically weaponized as chemical warfare agents (CWA). OPNA exposure can lead to several neurological, sensory, and motor symptoms that can manifest into chronic neurological illnesses later in life. There is still a large need for technological advancement to better understand changes in brain function following OPNA exposure. The human-relevant in vitro multi-electrode array (MEA) system, which combines the MEA technology with human stem cell technology, has the potential to monitor the acute, sub-chronic, and chronic consequences of OPNA exposure on brain activity. However, the application of this system to assess OPNA hazards and risks to human brain function remains to be investigated. In a concentration-response study, we have employed a human-relevant MEA system to monitor and detect changes in the electrical activity of engineered neural networks to increasing concentrations of the sarin surrogate 4-nitrophenyl isopropyl methylphosphonate (NIMP). We report a biphasic response in the spiking (but not bursting) activity of neurons exposed to low (i.e., 0.4 and 4 μM) versus high concentrations (i.e., 40 and 100 μM) of NIMP, which was monitored during the exposure period and up to 6 days post-exposure. Regardless of the NIMP concentration, at a network level, communication or coordination of neuronal activity decreased as early as 60 min and persisted at 24 h of NIMP exposure. Once NIMP was removed, coordinated activity was no different than control (0 μM of NIMP). Interestingly, only in the high concentration of NIMP did coordination of activity at a network level begin to decrease again at 2 days post-exposure and persisted on day 6 post-exposure. Notably, cell viability was not affected during or after NIMP exposure. Also, while the catalytic activity of AChE decreased during NIMP exposure, its activity recovered once NIMP was removed. Gene expression analysis suggests that human iPSC-derived neurons and primary human astrocytes resulted in altered genes related to the cell’s interaction with the extracellular environment, its intracellular calcium signaling pathways, and inflammation, which could have contributed to how neurons communicated at a network level.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Host tracheal and intestinal microbiomes inhibit Coccidioides growth in vitro

Coccidioidomycosis, also known as Valley fever, is a disease caused by the fungal pathogen Coccidioides. Unfortunately, patients are often misdiagnosed with bacterial pneumonia, leading to inappropriate antibiotic treatment. The soil Bacillus subtilis-like species exhibits antagonistic properties against Coccidioides in vitro; however, the antagonistic capabilities of host microbiota against Coccidioides are unexplored. We sought to examine the potential of the tracheal and intestinal microbiomes to inhibit the growth of Coccidioides in vitro. We hypothesized that an uninterrupted lawn of microbiota obtained from antibiotic-free mice would inhibit the growth of Coccidioides, while partial in vitro depletion through antibiotic disk diffusion assays would allow a niche for fungal growth. We observed that the microbiota grown on 2×GYE (GYE) and Columbia colistin and nalidixic acid with 5% sheep’s blood agar inhibited the growth of Coccidioides, but microbiota grown on chocolate agar did not. Partial depletion of the microbiota through antibiotic disk diffusion revealed diminished inhibition and comparable growth of Coccidioides to controls. To characterize the bacteria grown and identify potential candidates contributing to the inhibition of Coccidioides, 16S rRNA sequencing was performed on tracheal and intestinal agar cultures and murine lung extracts. We found that the host bacteria likely responsible for this inhibition primarily included Lactobacillus and Staphylococcus. The results of this study demonstrate the potential of the host microbiota to inhibit the growth of Coccidioides in vitro and suggest that an altered microbiome through antibiotic treatment could negatively impact effective fungal clearance and allow a niche for fungal growth in vivo.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Mef2c regulates bone mass through Sost -dependent and -independent mechanisms

We report Mef2c is a transcription factor that mediates key cellular behaviors that promote endochondral ossification and bone formation. Previously, Mef2c has been shown to regulate Sost transcription via its osteocyte-specific enhancer, ECR5, and conditional deletions of Mef2 cfl/fl with either Col1-Cre or Dmp1-Cre produced generalized high bone mass (HBM) consistent with Van Buchem Disease phenotypes. However, Sost -/- ; Mef2 cfl/fl ; Dmp1-Cre mice produced a significantly higher bone mass phenotype that Sost-/- alone suggesting that Mef2c modulates bone mass through additional mechanisms, independent of Sost. To identify new Mef2c transcriptional targets important in bone metabolism, we profiled gene expression by single-cell RNA sequencing in subpopulations of cells isolated from Mef2 cfl/fl ; Dmp1-Cre and Mef2 cfl/fl ; Bglap-Cre femurs, both strains exhibiting similar high bone mass phenotypes. However, we found Mef2cfl/fl; Bglap-Cre to also display a growth plate defect characterized by an expansion of several osteoprogenitor subpopulations. Differential gene expression analysis identified a total of 96 up- and 2434 down- regulated genes in Mef2 cfl/fl ; Bglap-Cre and 176 up- and 1041 down- regulated genes in Mef2 cfl/fl ; Dmp1-Cre bone cell subpopulations compared to wildtype mice. Mef2c deletion affected the transcriptomes across several cell types including mesenchymal progenitors (MP), osteoprogenitors (OSP), osteoblast (OB), and osteocyte (OCY) subpopulations. Several energy metabolism genes such as Uqcrb, Ndufv2, Ndufs3, Ndufa13, Ndufb9, Ndufb5, Cox6a1, Cox5a, Atp5o, Atp5g2, Atp5b, Atp5 were significantly down regulated in Mef2c-deficient OBs and OCYs, in both strains. Binding motif analysis of promoter regions of differentially expressed genes identified Mef2c binding in Bone Sialoprotein (BSP/Ibsp), a gene known to cause increased trabecular BV/TV in the femurs of Ibsp -/- mice. Immunohistochemical analysis confirmed the absence of Ibsp protein in OBs and OCYs. These findings suggests that the HBM in Sost -/- ; Mef2 cfl/fl ; Dmp1-Cre is caused by a multitude of transcriptional changes in genes that regulate bone formation, two of which are Sost and Ibsp.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Loss of Cadherin-11 in pancreatic ductal adenocarcinoma alters tumor-immune microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is one of the top five deadliest forms of cancer with very few treatment options. The 5-year survival rate for PDAC is 10% following diagnosis. Cadherin 11 (Cdh11), a cell-to-cell adhesion molecule, has been suggested to promote tumor growth and immunosuppression in PDAC, and Cdh11 inhibition significantly extended survival in mice with PDAC. However, the mechanisms by which Cdh11 deficiency influences PDAC progression and anti-tumor immune responses have yet to be fully elucidated. To investigate Cdh11-deficiency induced changes in PDAC tumor microenvironment (TME), we crossed p48-Cre; LSL-Kras G12D/+ ; LSLTrp53 R172H/+ (KPC) mice with Cdh11 +/- mice and performed single-cell RNA sequencing (scRNA-seq) of the non-immune (CD45 - ) and immune (CD45 + ) compartment of KPC tumor-bearing Cdh11 proficient (KPC-Cdh11 +/+ ) and Cdh11 deficient (KPC-Cdh11 +/- ) mice. Our analysis showed that Cdh11 is expressed primarily in cancer-associated fibroblasts (CAFs) and at low levels in epithelial cells undergoing epithelial-to-mesenchymal transition (EMT). Cdh11 deficiency altered the molecular profile of CAFs, leading to a decrease in the expression of myofibroblast markers such as Acta2 and Tagln and cytokines such as Il6, Il33 and Midkine (Mdk). We also observed a significant decrease in the presence of monocytes/macrophages and neutrophils in KPC-Cdh11 +/- tumors while the proportion of T cells was increased. Additionally, myeloid lineage cells from Cdh11-deficient tumors had reduced expression of immunosuppressive cytokines that have previously been shown to play a role in immune suppression. In summary, our data suggests that Cdh11 deficiency significantly alters the fibroblast and immune microenvironments and contributes to the reduction of immunosuppressive cytokines, leading to an increase in anti-tumor immunity and enhanced survival.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Engineered bone marrow as a clinically relevant ex vivo model for primary bone cancer research and drug screening

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. While numerous other cancers now have promising therapeutic advances, treatment options for OS have remained unchanged since the advent of standard chemotherapeutics and offer less than a 25% 5-y survival rate for those with metastatic disease. This dearth of clinical progress underscores a lack of understanding of OS progression and necessitates the study of this disease in an innovative system. Here, we adapt a previously described engineered bone marrow (eBM) construct for use as a three-dimensional platform to study how microenvironmental and immune factors affect OS tumor progression. We form eBM by implanting acellular bone-forming materials in mice and explanting the cellularized constructs after 8 wk for study. We interrogate the influence of the anatomical implantation site on eBM tissue quality, test ex vivo stability under normoxic (5% O 2 ) and standard (21% O 2 ) culture conditions, culture OS cells within these constructs, and compare them to human OS samples. We show that eBM stably recapitulates the composition of native bone marrow. OS cells exhibit differential behavior dependent on metastatic potential when cultured in eBM, thus mimicking in vivo conditions. Furthermore, we highlight the clinical applicability of eBM as a drug-screening platform through doxorubicin treatment and show that eBM confers a protective effect on OS cells that parallel clinical responses. Combined, this work presents eBM as a cellular construct that mimics the complex bone marrow environment that is useful for mechanistic bone cancer research and drug screening.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Dose-dependent consequences of sub-chronic fentanyl exposure on neuron and glial co-cultures

Fentanyl is one of the most common opioid analgesics administered to patients undergoing surgery or for chronic pain management. While the side effects of chronic fentanyl abuse are recognized (e.g., addiction, tolerance, impairment of cognitive functions, and inhibit nociception, arousal, and respiration), it remains poorly understood what and how changes in brain activity from chronic fentanyl use influences the respective behavioral outcome. Here, we examined the functional and molecular changes to cortical neural network activity following sub-chronic exposure to two fentanyl concentrations, a low (0.01 μM) and high (10 μM) dose. Primary rat co-cultures, containing cortical neurons, astrocytes, and oligodendrocyte precursor cells, were seeded in wells on either a 6-well multi-electrode array (MEA, for electrophysiology) or a 96-well tissue culture plate (for serial endpoint bulk RNA sequencing analysis). Once networks matured (at 28 days in vitro ), co-cultures were treated with 0.01 or 10 μM of fentanyl for 4 days and monitored daily. Only high dose exposure to fentanyl resulted in a decline in features of spiking and bursting activity as early as 30 min post-exposure and sustained for 4 days in cultures. Transcriptomic analysis of the complex cultures after 4 days of fentanyl exposure revealed that both the low and high dose induced gene expression changes involved in synaptic transmission, inflammation, and organization of the extracellular matrix. Collectively, the findings of this in vitro study suggest that while neuroadaptive changes to neural network activity at a systems level was detected only at the high dose of fentanyl, transcriptomic changes were also detected at the low dose conditions, suggesting that fentanyl rapidly elicits changes in plasticity.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Single-cell RNA-Seq reveals changes in immune landscape in post-traumatic osteoarthritis

Osteoarthritis (OA) is the most common joint disease, affecting over 300 million people world-wide. Accumulating evidence attests to the important roles of the immune system in OA pathogenesis. Understanding the role of various immune cells in joint degeneration or joint repair after injury is vital for improving therapeutic strategies for treating OA. Post-traumatic osteoarthritis (PTOA) develops in ~50% of individuals who have experienced an articular trauma like an anterior cruciate ligament (ACL) rupture. Here, using the high resolution of single-cell RNA sequencing, we delineated the temporal dynamics of immune cell accumulation in the mouse knee joint after ACL rupture. Our study identified multiple immune cell types in the joint including neutrophils, monocytes, macrophages, B cells, T cells, NK cells and dendritic cells. Monocytes and macrophage populations showed the most dramatic changes after injury. Further characterization of monocytes and macrophages reveled 9 major subtypes with unique transcriptomics signatures, including a tissue resident Lyve1 hi Folr2 hi macrophage population and Trem2 hi Fcrls + recruited macrophages, both showing enrichment for phagocytic genes and growth factors such as Igf1 , Pdgfa and Pdgfc. We also identified several genes induced or repressed after ACL injury in a cell type-specific manner. This study provides new insight into PTOA-associated changes in the immune microenvironment and highlights macrophage subtypes that may play a role in joint repair after injury.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

IL-17A Increases Doxorubicin Efficacy in Triple Negative Breast Cancer

Due to lack of targetable receptors and intertumoral heterogeneity, triple negative breast cancer (TNBC) remains particularly difficult to treat. Doxorubicin (DOX) is typically used as nonselective neoadjuvant chemotherapy, but the diversity of treatment efficacy remains unclear. Comparable to variability in clinical response, an experimental model of TNBC using a 4T1 syngeneic mouse model was found to elicit a differential response to a seven-day treatment regimen of DOX. Single-cell RNA sequencing identified an increase in T cells in tumors that responded to DOX treatment compared to tumors that continued to grow uninhibited. Additionally, compared to resistant tumors, DOX sensitive tumors contained significantly more CD4 T helper cells (339%), γδ T cells (727%), Naïve T cells (278%), and activated CD8 T cells (130%). Furthermore, transcriptional profiles of tumor infiltrated T cells in DOX responsive tumors revealed decreased exhaustion, increased chemokine/cytokine expression, and increased activation and cytotoxic activity. γδ T cell derived IL-17A was identified to be highly abundant in the sensitive tumor microenvironment. IL-17A was also found to directly increase sensitivity of TNBC cells in combination with DOX treatment. In TNBC tumors sensitive to DOX, increased IL-17A levels lead to a direct effect on cancer cell responsiveness and chronic stimulation of tumor infiltrated T cells leading to improved chemotherapeutic efficacy. IL-17A’s role as a chemosensitive cytokine in TNBC may offer new opportunities for treating chemoresistant breast tumors and other cancer types.

4T1↗

DOE OSTI2022

MAVS mediates a protective immune response in the brain to Rift Valley fever virus

Rift Valley fever virus (RVFV) is a highly pathogenic mosquito-borne virus capable of causing hepatitis, encephalitis, blindness, hemorrhagic syndrome, and death in humans and livestock. Upon aerosol infection with RVFV, the brain is a major site of viral replication and tissue damage, yet pathogenesis in this organ has been understudied. Here, we investigated the immune response in the brain of RVFV infected mice. In response to infection, microglia initiated robust transcriptional upregulation of antiviral immune genes, as well as increased levels of activation markers and cytokine secretion that is dependent on mitochondrial antiviral-signaling protein (MAVS) and independent of toll-like receptors 3 and 7. In vivo , Mavs -/- mice displayed enhanced susceptibility to RVFV as determined by increased brain viral burden and higher mortality. Single-cell RNA sequence analysis identified defects in type I interferon and interferon responsive gene expression within microglia in Mavs -/- mice, as well as dysregulated lymphocyte infiltration. The results of this study provide a crucial step towards understanding the precise molecular mechanisms by which RVFV infection is controlled in the brain and will help inform the development of vaccines and antiviral therapies that are effective in preventing encephalitis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Preexisting Type 1 Diabetes Mellitus Blunts the Development of Posttraumatic Osteoarthritis

Type 1 diabetes mellitus (T1DM) affects 9.5% of the population. T1DM is characterized by severe insulin deficiency that causes hyperglycemia and leads to several systemic effects. T1DM has been suggested as a risk factor for articular cartilage damage and loss, which could expedite the development of osteoarthritis (OA). OA represents a major public health challenge by affecting 300 million people globally, yet very little is known about the correlation between T1DM and OA. In addition, current studies that have looked at the interaction between diabetes mellitus and OA have reported conflicting results with some suggesting a positive correlation whereas others did not. In this study, we aimed to evaluate whether T1DM exacerbates the development of spontaneous OA or accelerates the progression of posttraumatic osteoarthritis (PTOA) after joint injury. Histological evaluation of T1DM and control joints determined that T1DM mice displayed cartilage degeneration measurements consistent with mild OA phenotypes. RNA sequencing analyses identified significantly upregulated genes in T1DM corresponding to matrix-degrading enzymes known to promote cartilage matrix degradation, suggesting a role of these enzymes in OA development. Next, we assessed whether preexisting T1DM influences PTOA development subsequent to trauma. At 6 weeks post-injury, T1DM injured joints displayed significantly less cartilage damage and joint degeneration than injured non-diabetic joints, suggesting a significant delay in PTOA disease progression. At the single-cell resolution, we identified increased number of cells expressing the chondrocyte markers Col2a1, Acan, and Cytl1 in the T1DM injured group. Our findings demonstrate that T1DM can be a risk factor for OA but not for PTOA. This study provides the first account of single-cell resolution related to T1DM and the risk for OA and PTOA.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Interactions Between Diabetes Mellitus and Osteoarthritis: From Animal Studies to Clinical Data

Diabetes mellitus (DM) and osteoarthritis (OA) are commonly known metabolic diseases that affect a large segment of the world population. These two conditions share several risk factors such as obesity and aging; however, there is still no consensus regarding the direct role of DM on OA development and progression. Interestingly, both animal and human studies have yielded conflicting results, with some showing a significant role for DM in promoting OA, while others found no significant interactions between these conditions. In this review, we will discuss preclinical and clinical data that assessed the interaction between DM and OA. We will also discuss possible mechanisms associated with the effect of high glucose on the articular cartilage and chondrocytes. An emerging theme dominates the breath of published work in this area: most of the studies discussed in this review do not take into consideration the role of other factors such as the type of diabetes, age, biological sex, type of animal model, body mass index, and the use of pain medications when analyzing and interpreting data. Therefore, future studies should be more rigorous when designing experiments looking at DM and its effects on OA and should carefully account for these confounding factors, so that better approaches can be developed for monitoring and treating patients at risk of OA and DM.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Cadherin 11 Promotes Immunosuppression and Extracellular Matrix Deposition to Support Growth of Pancreatic Tumors and Resistance to Gemcitabine in Mice

Pancreatic ductal adenocarcinomas (PDACs) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). Here, we investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule CDH11, which has been associated with other fibrotic disorders and is expressed by activated fibroblasts. We compared levels of CDH11 messenger RNA in human pancreatitis and pancreatic cancer tissues and cells with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-Kras G12D/+ ;LSL-Trp53 R172H/+ (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11 +/+ and Cdh11 –/– mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11 +/+ (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored. Levels of CDH11 messenger RNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11 +/– and KPC/Cdh11 –/– mice survived significantly longer than KPC/Cdh11 +/+ mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11 +/+ mice and incompletely in KPC/Cdh11 +/– and KPC/Cdh11 –/– mice, whose lesions also contained fewer FOXP3 + cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11 +/+ mice, tumors of KPC/Cdh11 +/– mice had increased markers of antigen processing and presentation; more lymphocytes and associated cytokines; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival of KPC/Cdh11 +/– and KPC/Cdh11 –/– mice only or reduced subcutaneous tumor growth in mT3 engrafted Cdh11 +/+ mice when given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice. Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Sclerostin Depletion Induces Inflammation in the Bone Marrow of Mice

Romosozumab, a humanized monoclonal antibody specific for sclerostin (SOST), has been approved for treatment of postmenopausal women with osteoporosis at a high risk for fracture. Previous work in sclerostin global knockout (Sost-/- ) mice indicated alterations in immune cell development in the bone marrow (BM), which could be a possible side effect in romosozumab-treated patients. Here, we examined the effects of short-term sclerostin depletion in the BM on hematopoiesis in young mice receiving sclerostin antibody (Scl-Ab) treatment for 6 weeks, and the effects of long-term Sost deficiency on wild-type (WT) long-term hematopoietic stem cells transplanted into older cohorts of Sost-/- mice. Our analyses revealed an increased frequency of granulocytes in the BM of Scl-Ab-treated mice and WT→Sost -/- chimeras, indicating myeloid-biased differentiation in Sost-deficient BM microenvironments. This myeloid bias extended to extramedullary hematopoiesis in the spleen and was correlated with an increase in inflammatory cytokines TNFα, IL-1α, and MCP-1 in Sost-/- BM serum. Additionally, we observed alterations in erythrocyte differentiation in the BM and spleen of Sost-/- mice. Taken together, our current study indicates novel roles for Sost in the regulation of myelopoiesis and control of inflammation in the BM.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Functional and transcriptional characterization of complex neuronal co-cultures

Brain-on-a-chip systems are designed to simulate brain activity using traditional in vitro cell culture on an engineered platform. It is a noninvasive tool to screen new drugs, evaluate toxicants, and elucidate disease mechanisms. However, successful recapitulation of brain function on these systems is dependent on the complexity of the cell culture. In this study, we increased cellular complexity of traditional (simple) neuronal cultures by co-culturing with astrocytes and oligodendrocyte precursor cells (complex culture). We evaluated and compared neuronal activity (e.g., network formation and maturation), cellular composition in long-term culture, and the transcriptome of the two cultures. Compared to simple cultures, neurons from complex co-cultures exhibited earlier synapse and network development and maturation, which was supported by localized synaptophysin expression, up-regulation of genes involved in mature neuronal processes, and synchronized neural network activity. Also, mature oligodendrocytes and reactive astrocytes were only detected in complex cultures upon transcriptomic analysis of age-matched cultures. Functionally, the GABA antagonist bicuculline had a greater influence on bursting activity in complex versus simple cultures. Collectively, the cellular complexity of brain-on-a-chip systems intrinsically develops cell type-specific phenotypes relevant to the brain while accelerating the maturation of neuronal networks, important features underdeveloped in traditional cultures.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

LPS-Induced Inflammation Prior to Injury Exacerbates the Development of Post-Traumatic Osteoarthritis in Mice

ABSTRACT Osteoarthritis (OA) is a debilitating and painful disease characterized by the progressive loss of articular cartilage. Post-traumatic osteoarthritis (PTOA) is an injury-induced type of OA that persists in an asymptomatic phase for years before it becomes diagnosed in ~50% of injured individuals. Although PTOA is not classified as an inflammatory disease, it has been suggested that inflammation could be a major driver of PTOA development. Here we examined whether a state of systemic inflammation induced by lipopolysaccharide (LPS) administration 5-days before injury would modulate PTOA outcomes. RNA-seq analysis at 1-day post-injury followed by micro-computed tomography (μCT) and histology characterization at 6 weeks post-injury revealed that LPS administration causes more severe PTOA phenotypes. These phenotypes included significantly higher loss of cartilage and subchondral bone volume. Gene expression analysis showed that LPS alone induced a large cohort of inflammatory genes previously shown to be elevated in synovial M1 macrophages of rheumatoid arthritis (RA) patients, suggesting that systemic LPS produces synovitis. This synovitis was sufficient to promote PTOA in MRL/MpJ mice, a strain previously shown to be resistant to PTOA. The synovium of LPS-treated injured joints displayed an increase in cellularity, and immunohistological examination confirmed that this increase was in part attributable to an elevation in type 1 macrophages. LPS induced the expression of Tlr7 and Tlr8 in both injured and uninjured joints, genes known to be elevated in RA. We conclude that inflammation before injury is an important risk factor for the development of PTOA and that correlating patient serum endotoxin levels or their state of systemic inflammation with PTOA progression may help develop new, effective treatments to lower the rate of PTOA in injured individuals. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

59 BASIC BIOLOGICAL SCIENCES↗