Multiple Experimental Models Research Articles

Molecular and Cellular Effects of Microplastics and Nanoplastics: Focus on Inflammation and Senescence.

Microplastics and nanoplastics (MNPs) are ubiquitous environmental contaminants. Their prevalence, persistence, and increasing industrial production have led to questions about their long-term impact on human and animal health. This narrative review describes the effects of MNPs on oxidative stress, inflammation, and aging. Exposure to MNPs leads to increased production of reactive oxygen species (ROS) across multiple experimental models, including cell lines, organoids, and animal systems. ROS can cause damage to cellular macromolecules such as DNA, proteins, and lipids. Direct interaction between MNPs and immune cells or an indirect result of oxidative stress-mediated cellular damage may lead to increased production of pro-inflammatory cytokines throughout different MNP-exposure conditions. This inflammatory response is a common feature in the pathogenesis of neurodegenerative, cardiovascular, and other age-related diseases. MNPs also act as cell senescence inducers by promoting mitochondrial dysfunction, impairing autophagy, and activating DNA damage responses, exacerbating cellular aging altogether. Increased senescence of reproductive cells and transfer of MNPs/induced damages from parents to offspring in animals further corroborates the transgenerational health risks of the tiny particles. This review aims to provoke a deeper investigation into the notorious effects these pervasive particles may have on human well-being and longevity.

CD11b suppresses TLR7-driven inflammatory signaling to protect against lupus nephritis.

Lupus Nephritis (LN) is a severe complication of systemic lupus erythematosus (SLE) that affects kidney function. Here, we investigated the role of CD11b, a protein encoded by the ITGAM gene, in the development of LN and its functional activation as a therapeutic strategy. Genetic coding variants of ITGAM significantly increase the risk for SLE and LN by producing a less active CD11b and leading to elevated levels of type I interferon (IFN I). However, a molecular mechanism for how these variants increase LN risk has been unclear. Here, we determined that these variants also significantly associate with elevations in soluble urokinase plasminogen activator receptor (suPAR), a known biomarker linked to kidney disease, suggesting a novel molecular connection. Pharmacologic activation of CD11b with a novel, clinical-stage agonist ONT01 significantly suppressed suPAR production in myeloid cells and reduced systemic inflammation and kidney damage in multiple experimental models of LN. Importantly, delaying treatment with ONT01 until after disease onset also significantly reduced serum suPAR and inflammatory cytokines, and decreased immune complex deposition in the glomerulus, glomerulonephritis and albuminuria, suggesting that CD11b activation is therapeutic for LN. Genetic activation of CD11b via a gain-of-function CD11b mutation also showed complete protection from LN, whereas genetic deletion of CD11b worsened the disease in mice, providing further evidence of the role of CD11b activation in regulating LN. Finally, transfer of human LN PBMCs generated human LN like disease in mice that was significantly reduced by ONT01. Together, these data provide strong evidence that ONT01 mediated CD11b activation can therapeutically modulate TLR7-driven inflammation and protect against LN. These findings support clinical development of CD11b agonists as novel therapeutics for treating lupus nephritis in human patients.

O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis. We characterized the O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT)/O-GlcNAc axis in IPF using single-cell RNA-sequencing (scRNA-seq) data and human lung sections and isolated fibroblasts from IPF and non-IPF donors. The underlying mechanism(s) of IPF were further investigated using multiple experimental models to modulate collagen expression and accumulation by genetically and pharmacologically targeting OGT. Furthermore, we hone in on the transforming growth factor-beta (TGF-β) effector molecule, Smad3, by co-expressing it with OGT to determine if it is modified and its subsequent effect on Smad3 activation. We found that OGT and O-GlcNAc levels are upregulated in patients with IPF compared to non-IPF. We report that the OGT regulates collagen deposition and fibrosis resolution, which is an evolutionarily conserved process demonstrated across multiple species. Co-expression of OGT and Smad3 showed that Smad3 is O-GlcNAc modified. Blocking OGT activity resulted in decreased phosphorylation at Ser-423/425 of Smad3 attenuating the effects of TGF-β1 induced collagen expression/deposition. OGT inhibition or knockdown successfully blocked and reversed collagen expression and accumulation, respectively. Smad3 is discovered to be a substrate of OGT and its O-GlcNAc modification(s) directly affects its phosphorylation state. These data identify OGT as a potential target in pulmonary fibrosis resolution, as well as other diseases that might have aberrant ECM/collagen accumulation.

Ciliogenesis-associated Kinase 1 Promotes Breast Cancer Cell Proliferation and Chemoresistance via Phosphorylating ERK1.

Ciliogenesis-associated kinase 1 (CILK1) plays a key role in the ciliogenesis and ciliopathies. It remains totally unclear whether CILK1 is involved in tumor progression and therapy resistance. Here, we report that the aberrant high-expression of CILK1 in breast cancer is required for tumor cell proliferation and chemoresistance. Two compounds, CILK1-C30 and CILK1-C28, were identified with selective inhibitory effects towards the Tyr-159/Thr-157 dual-phosphorylation of CILK1, pharmacological inhibition of CILK1 significantly suppressed tumor cell proliferation and overcame chemoresistance in multiple experimental models. Large-scale screen of CILK1 substrates confirmed that the kinase directly phosphorylates ERK1, which is responsible for CILK1-mediated oncogenic function. CILK1 is also indicated to be responsible for the chemoresistance of small-cell lung cancer cells. Our data highlight the importance of CILK1 in cancer, implicating that targeting CILK1/ERK1 might offer therapeutic benefit to cancer patients.

Long-term administration of royal jelly regulates age-related disorders and improves gut function in naturally aging mice.

A natural aging mouse model can exhibit physiological characteristics that closely resemble those of human aging. Through long-term observation, it reflects the occurrence and development of the aging process more accurately. Although numerous beneficial effects of royal jelly (RJ) have been extensively demonstrated in multiple experimental models, the effects of RJ on naturally aging mice have not yet been investigated. In this study, middle-aged male C57BL/6J mice were given RJ for 9 consecutive months to investigate its impact on the intestinal barrier function, gut microbiota, short-chain fatty acids (SCFAs) content and possible mechanisms. The results confirmed that RJ modulated serum lipids by reducing the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C). Additionally, it protected the liver by increasing antioxidant enzyme levels while decreasing inflammatory cytokines TNF-α (by 51.97%), IL-6 (by 29.73%), and IL-1β (by 43.89%). Furthermore, RJ inhibited the expression of cell cycle-dependent kinase inhibitors including p16, p21, and p53. Importantly, RJ ameliorated gut dysfunctions by inhibiting reduction of tight junction proteins and reducing inflammatory cytokines content in the colon. We also observed an alteration in gut microbiota characterized by an elevated ratio of Firmicutes to Bacteroides (F/B) along with increased abundance of beneficial bacteria, i.e., Lachnospiraceae NK4A136 and Akkermansia. Correlation analysis revealed positive associations between most bacterial genera and SCFAs production. Functional profiling of gut microbiota composition indicated that RJ intervention regulated amino acid metabolism, glycan biosynthesis, and cofactor/vitamin metabolism. Overall, our findings provide an effective dietary intervention strategy for modulating age-associated frailty through the modulation of the gut microbiota.

LTX-315 triggers anticancer immunity by inducing MyD88-dependent maturation of dendritic cells.

LTX-315 is a synthetic cationic oncolytic peptide with potent anticancer activity but limited toxicity for non-malignant cells. LTX-315 induces both immunogenic tumor cell death and generation of tumor-specific immune responses in multiple experimental tumor models. Given the central role of dendritic cell (DC) maturation in the induction of antigen-specific immunity, we investigated the effect of LTX-315 treatment on the maturation of tumor-infiltrating DCs (TiDCs) and the generation of anti-melanoma immunity. We found that LTX-315 treatment induces the maturation of DCs, both indirectly through the release of cancer cell-derived damage-associated molecular patterns (DAMPs)/alarmins and nucleic acids (DNA and RNA) capable of triggering distinct Toll-like receptor (TLR) signaling, and, directly by activating TLR7. The latter results in the ignition of multiple intracellular signaling pathways that promotes DC maturation, including NF-κB, mitogen activated protein kinases (MAPKs), and inflammasome signaling, as well as increased type 1 interferon production. Critically, the effects of LTX-315 on DCs the consequent promotion of anti-melanoma immunity depend on the cytosolic signal transducer myeloid differentiation response gene 88 (MyD88). These results cast light on the mechanisms by which LTX-315 induces DC maturation and hence elicits anticancer immunity, with important implications for the use of LTX-315 as an anticancer immunotherapeutic.

Astrogliosis in multiple sclerosis and neuro-inflammation: what role for the notch pathway?

Multiple sclerosis is an autoimmune inflammatory disease of the central nervous system leading to neurodegeneration. It affects 2.3 million people worldwide, generally younger than 50. There is no known cure for the disease, and current treatment options - mainly immunotherapies to limit disease progression - are few and associated with serious side effects. In multiple sclerosis, disruption of the blood-brain barrier is an early event in the pathogenesis of lesions, predisposing to edema, excito-toxicity and inflammatory infiltration into the central nervous system. Recently, the vision of the blood brain barrier structure and integrity has changed and include contributions from all components of the neurovascular unit, among which astrocytes. During neuro-inflammation, astrocytes become reactive. They undergo morphological and molecular changes named "astrogliosis" driving the conversion from acute inflammatory injury to a chronic neurodegenerative state. Astrogliosis mechanisms are minimally explored despite their significance in regulating the autoimmune response during multiple sclerosis. Therefore, in this review, we take stock of the state of knowledge regarding astrogliosis in neuro-inflammation and highlight the central role of NOTCH signaling in the process of astrocyte reactivity. Indeed, a very detailed nomenclature published in nature neurosciences in 2021, listing all the reactive astrocyte markers fully identified in the literature, doesn't cover the NOTCH signaling. Hence, we discuss evidence supporting NOTCH1 receptor as a central regulator of astrogliosis in the pathophysiology of neuro-inflammation, notably multiple sclerosis, in human and experimental models.

Honokiol decreases alpha-synuclein mRNA levels and reveals novel targets for modulating alpha-synuclein expression.

Intracytoplasmic inclusions comprised of aggregated alpha-synuclein (αsyn) represent a key histopathological feature of neurological disorders collectively termed "synucleinopathies," which includes Parkinson's disease (PD). Mutations and multiplications in the SNCA gene encoding αsyn cause familial forms of PD and a large body of evidence indicate a correlation between αsyn accumulation and disease. Decreasing αsyn expression is recognized as a valid target for PD therapeutics, with down-regulation of SNCA expression potentially attenuating downstream cascades of pathologic events. Here, we evaluated if Honokiol (HKL), a polyphenolic compound derived from magnolia tree bark with demonstrated neuroprotective properties, can modulate αsyn levels in multiple experimental models. Human neuroglioma cells stably overexpressing αsyn, mouse primary neurons, and human iPSC-derived neurons were exposed to HKL and αsyn protein and SNCA messenger RNA levels were assessed. The effect of HKL on rotenone-induced overexpression of αsyn levels was further assessed and transcriptional profiling of mouse cortical neurons treated with HKL was performed to identify potential targets of HKL. We demonstrate that HKL can successfully reduce αsyn protein levels and SNCA expression in multiple in vitro models of PD with our data supporting a mechanism whereby HKL acts by post-transcriptional modulation of SNCA rather than modulating αsyn protein degradation. Transcriptional profiling of mouse cortical neurons treated with HKL identifies several differentially expressed genes (DEG) as potential targets to modulate SNCA expression. This study supports a HKL-mediated downregulation of SNCA as a viable strategy to modify disease progression in PD and other synucleinopathies. HKL has potential as a powerful tool for investigating SNCA gene modulation and its downstream effects.

Deletion of the Transient Receptor Potential Vanilloid 1 Channel Attenuates Sympathoexcitation and Hypertension and Improves Glomerular Filtration Rate in 2-Kidney-1-Clip Rats.

Renal denervation lowers arterial blood pressure in both clinical populations and multiple experimental models of hypertension. This therapeutic effect is partly attributed to the removal of overactive renal sensory nerves. The TRPV1 (transient receptor potential vanilloid 1) channel is highly expressed in renal sensory nerves and detects changes in noxious and mechanosensitive stimuli, pH, and chemokines. However, the extent to which TRPV1 channels contribute to 2-kidney-1-clip (2K1C) renovascular hypertension has not been tested. We generated a novel Trpv1-/- (TRPV1 knockout) rat using CRISPR/Cas9 and 26-bp deletion in exon 3 and induced 2K1C hypertension. The majority (85%) of rat renal sensory neurons retrogradely labeled from the kidney were TRPV1-positive. Trpv1-/- rats lacked TRPV1 immunofluorescence in the dorsal root ganglia, had a delayed tail-flick response to hot but not cold water, and lacked an afferent renal nerve activity response to intrarenal infusion of the TRPV1 agonist capsaicin. Interestingly, 2K1C hypertension was significantly attenuated in male Trpv1-/- versus wild-type rats. 2K1C hypertension significantly increased the depressor response to ganglionic blockade, total renal nerve activity (efferent and afferent), and afferent renal nerve activity in wild-type rats, but these responses were attenuated in male Trpv1-/- rats. 2K1C hypertension was attenuated in female rats with no differences between female strains. Finally, glomerular filtration rate was reduced by 2K1C in wild-type rats but improved in Trpv1-/- rats. These findings suggest that renovascular hypertension requires activation of the TRPV1 channel to elevate renal afferent and sympathetic nerve activity, reduce glomerular filtration rate, and increase arterial blood pressure.

Dual Targeting of the PDZ1 and PDZ2 Domains of MDA-9/Syntenin Inhibits Melanoma Metastasis.

Genome-wide gene expression analysis and animal modeling indicate that melanoma differentiation associated gene-9 (mda-9, Syntenin, Syndecan binding protein, referred to as MDA-9/Syntenin) positively regulates melanoma metastasis. The MDA-9/Syntenin protein contains two tandem PDZ domains serving as a nexus for interactions with multiple proteins that initiate transcription of metastasis-associated genes. Although targeting either PDZ domain abrogates signaling and prometastatic phenotypes, the integrity of both domains is critical for full biological function. Fragment-based drug discovery and NMR identified PDZ1i, an inhibitor of the PDZ1 domain that effectively blocks cancer invasion in vitro and in vivo in multiple experimental animal models. To maximize disruption of MDA-9/Syntenin signaling, an inhibitor has now been developed that simultaneously binds and blocks activity of both PDZ domains. PDZ1i was joined to the second PDZ binding peptide (TNYYFV) with a PEG linker, resulting in PDZ1i/2i (IVMT-Rx-3) that engages both PDZ domains of MDA-9/Syntenin. IVMT-Rx-3 blocks MDA-9/Syntenin interaction with Src, reduces NF-κB activation, and inhibits MMP-2/MMP-9 expression, culminating in repression of melanoma metastasis. The in vivo antimetastatic properties of IVMT-Rx-3 are enhanced when combined with an immune-checkpoint inhibitor. Collectively, our results support the feasibility of engineering MDA-9 dual-PDZ inhibitors with enhanced antimetastatic activities and applications of IVMT-Rx-3 for developing novel therapeutic strategies effectively targeting melanoma and in principle, a broad spectrum of human cancers that also overexpress MDA-9/Syntenin.

Cerebrospinal fluid, brain, and spinal cord levels of L-aspartate signal excitatory neurotransmission abnormalities in multiple sclerosis patients and experimental autoimmune encephalomyelitis mouse model.

The neuroinflammatory process characterizing multiple sclerosis (MS) is associated with changes in excitatory synaptic transmission and altered central concentrations of the primary excitatory amino acid, L-glutamate (L-Glu). Recent findings report that cerebrospinal fluid (CSF) levels of L-Glu positively correlate with pro-inflammatory cytokines in MS patients. However, to date, there is no evidence about the relationship between the other primary excitatory amino acid, L-aspartate (L-Asp), its derivative D-enantiomer, D-aspartate, and the levels of pro-inflammatory and anti-inflammatory cytokines in the CSF of MS. In the present study, we measured by HPLC the levels of these amino acids in the cortex, hippocampus, cerebellum, and spinal cord of mice affected by experimental autoimmune encephalomyelitis (EAE). Interestingly, in support of glutamatergic neurotransmission abnormalities in neuroinflammatory conditions, we showed reduced L-Asp levels in the cortex and spinal cord of EAE mice and increased D-aspartate/total aspartate ratio within the cerebellum and spinal cord of these animals. Additionally, we found significantly decreased CSF levels of L-Asp in both relapsing-remitting (n = 157) MS (RR-MS) and secondary progressive/primary progressive (n = 22) (SP/PP-MS) patients, compared to control subjects with other neurological diseases (n = 40). Importantly, in RR-MS patients, L-Asp levels were correlated with the CSF concentrations of the inflammatory biomarkers G-CSF, IL-1ra, MIP-1β, and Eotaxin, indicating that the central content of this excitatory amino acid, as previously reported for L-Glu, reflects a neuroinflammatory environment in MS. In keeping with this, we revealed that CSF L-Asp levels were positively correlated with those of L-Glu, highlighting the convergent variation of these two excitatory amino acids under inflammatory synaptopathy occurring in MS.

Abstract LB344: Complement-mediated signaling during cross-presentation of tumor antigen augments T cell responses

Abstract Production of complement C3a and C5a locally at the immune synapse by activated dendritic cells (DCs) enhances T cell responses in multiple experimental models. Complement signaling through cognate C3a receptor (C3aR) and C5a receptor (C5aR) modulate effector T cell stability, proliferation and differentiation. We have previously shown that complement C3 plays a mechanistic role in driving synergistic anti-tumor effects of blocking indoleamine 2,3-dioxygenase (IDO) during radiation and chemotherapy in a murine brain tumor model that expresses the gp10025-33 peptide antigen. We hypothesize that, when anti-tumor responses are allowed to happen, complement C3 production by local DCs acts as a costimulation factor, operating through C3aR and C5aR on the T cell surface to allow optimal T cell activation and proliferation. To study the role of complement C3 as a costimulation factor during T cell responses against a nominal tumor antigen (gp10025-33 peptide, expressed by B16F10 melanoma), we used T cells from syngeneic pmel-1 mice (TCR transgenic with CD8 T cells that recognize the cross-presented gp10025-33 peptide) co-cultured with gp10025-33 peptide and dendritic cells from either wild-type (WT) or C3-defiecient (C3-KO) mice. In addition, we used in vitro culture conditions where gp10025-33 tumor peptide was in stringent limited supply to model homeostatic conditions of the tumor microenvironment. We found that, when stimulated by DCs from C3-KO mice, pmel-1 CD8 T cells had decreased activation marker expression, proliferation, and cytokine effector function relative to stimulation by DCs from WT mice. Next, we bred pmel-1 mice onto a syngeneic background deficient in receptors for both C3a and C5a complement factors (C3aR-KO/C5aR-KO double knock-out mice). We used CD8 T cells from the resulting mouse strain (pmel-1/C3aR-KO/C5aR-KO) co-cultured with WT DCs to study the role of complement signaling in T cells during responses to tumor antigen. Using the same stringent antigen conditions, CD8 T cells from the pmel-1/C3aR-KO/C5aR-KO mice had decreased activation marker expression and proliferation, and differences in cytokine function compared to CD8 T cells from the pmel-1 mice. These findings suggest that lack of local complement production by the DCs in the tumor microenvironment may inhibit anti-tumor immune responses, and that complement signaling through C3aR and C5aR on T cells may play an important role during such anti-tumor responses. Citation Format: Caryn L. Bird, Gabriela A. Pacholczyk, Theodore S. Johnson. Complement-mediated signaling during cross-presentation of tumor antigen augments T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB344.

Abstract 3394: Simultaneous targeting of the PDZ1 and PDZ2 domains of MDA-9 inhibits melanoma metastasis

Abstract Metastasis poses significant obstacles to effective therapy and drug development. Melanoma differentiation-associated gene-9/Syntenin-1/Syndecan Binding Protein (MDA-9) is a pro-metastatic gene robustly and differentially expressed in cancer cells in comparison with corresponding adjacent normal/primary cells and represents an attractive therapeutic target to inhibit cancer cell dissemination and tumor growth. MDA-9 contains two tandem PDZ domains that provide a nexus for interactions with multiple proteins that can initiate the transcription of metastasis-associated genes. The goal of this project was to evaluate the efficiency of a unique first-in-class, second-generation MDA-9 dual-PDZ targeted pharmacological inhibitor generated using a combination of a PDZ1 small molecule and a PDZ2 selective peptide. Fragment-based drug discovery and NMR identified PDZ1i, an inhibitor of the PDZ1 domain, that effectively blocks cancer invasion in vitro and in vivo in multiple experimental animal models. To maximize the disruption of MDA-9 signaling an inhibitor has now been developed that simultaneously binds and blocks the activity of both PDZ domains. PDZ1i was joined to the second PDZ binding peptide (TNYYFV) with a PEG linker, resulting in IVMT-Rx-3 that simultaneously engages both PDZ domains of MDA-9. Mechanistic studies involved a combination of molecular and cell biology approaches and included both established and early passaged patient-derived melanoma cell lines. To test the efficacy of potential combinatorial effects in vivo we used a murine syngeneic model and B16F10, an immunotherapy-resistant murine cell line. After confirming synthesis using multiple analytical methods, e.g., HPLC, LC/MS, and NMR, the dissociation constant (determined by microscale thermophoresis (MST) analysis, of the IVMT-Rx-3 recombinant MDA-9 complex was 63 +/- 11 μM. This compound is not toxic to normal melanocytes or melanoma cells, but significantly inhibits invasion in vitro in a wide range of melanomas. IVMT-Rx-3 interrupts c-Src binding to MDA-9 and downregulates NF-kB activation. Inactivation of this transcription factor causes suppression of MMP2/MMP9 and inhibits the secretion of cytokines/growth factors. These molecular changes block cellular invasiveness and facilitate immune cytotoxicity by converting an unfavorable into a favorable tumor microenvironment culminating in the repression of melanoma metastasis. In addition, the in vivo anti-metastatic properties of IVMT-Rx-3 are enhanced when combined with an immune-checkpoint inhibitor. IVMT-Rx-3 is a unique MDA-9 antagonist in terms of composition that effectively inhibits melanoma metastasis as does our small molecule PDZ1i and holds potential for developing novel therapeutic strategies effectively targeting melanoma and in principle, a broad spectrum of human cancers that also overexpress MDA-9. Citation Format: Anjan K. Pradhan, Jinkal Modi, Santanu Maji, Amit Kumar, Praveen Bhoopathi, Padmanabhan Mannangatti, Chunqing Guo, Daniel K. Afosah, Mark Mochel, Nitai D. Mukhopadhyay, John M. Kirkwood, Xiang-Yang Wang, Umesh R. Desai, Devanand Sarkar, Luni Emdad, Swadesh K. Das, Paul B. Fisher. Simultaneous targeting of the PDZ1 and PDZ2 domains of MDA-9 inhibits melanoma metastasis [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 3394.

Abstract 3285: Targeting TBK1 to overcome resistance to cancer immunotherapy

Abstract Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking. We identified the innate immune kinase TANK-binding kinase 1 (TBK1) as a candidate immune evasion gene in a pooled genetic screen. Using a suite of genetic and pharmacologic tools across multiple experimental model systems, we confirm a role for TBK1 as an immune evasion gene. Targeting TBK1 enhances response to PD-1 blockade by lowering the cytotoxicity threshold to effector cytokines (TNFα/IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumor models. Cancer cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNFα/IFNγ in a JAK/STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is a novel and effective strategy to overcome resistance to cancer immunotherapy. Citation Format: Yi Sun, Or-Yam Revach, Seth Anderson, Anne Jenney, Caitlin E. Mills, Payal Tiwari, Robert T. Manguso, Russell William Jenkins. Targeting TBK1 to overcome resistance to cancer immunotherapy [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 3285.

Age-dependent loss of hepatic SIRT1 enhances NLRP3 inflammasome signaling and impairs capacity for liver fibrosis resolution.

Our studies indicate that the longevity factor SIRT1 is implicated in metabolic disease; however, whether and how hepatocyte-specific SIRT1 signaling is involved in liver fibrosis remains undefined. We characterized a functional link of age-mediated defects in SIRT1 to the NLRP3 inflammasome during age-related liver fibrosis. In multiple experimental murine models of liver fibrosis, we compared the development of liver fibrosis in young and old mice, as well as in liver-specific SIRT1 knockout (SIRT1 LKO) mice and wild-type (WT) mice. Liver injury, fibrosis, and inflammation were assessed histologically and quantified by real-time PCR analysis. In a model of hepatotoxin-induced liver fibrosis, old mice displayed more severe and persistent liver fibrosis than young mice during liver injury and after injury cessation, as characterized by inhibition of SIRT1, induction of NLRP3, infiltration of macrophages and neutrophils, activation of hepatic stellate cells (HSCs), and excessive deposition and remodeling of the extracellular matrix. Mechanistically, deletion of SIRT1 in hepatocytes resulted in NLRP3 and IL-1β induction, pro-inflammatory response, and severe liver fibrosis in young mice, mimicking the ability of aging to impair the resolution of established fibrosis. In an aging mouse model, chronic-plus-binge alcohol feeding-induced liver fibrosis was attenuated by treatment with MCC950, a selective NLRP3 inhibitor. NLRP3 inhibition ameliorated alcoholic liver fibrosis in old mice by repressing inflammation and reducing hepatocyte-derived danger signaling-ASK1 and HMGB1.In conclusion, age-dependent SIRT1 defects lead to NLRP3 activation and inflammation, which in turn impairs the capacity to resolve fibrosis during aging.

Microglia play beneficial roles in multiple experimental seizure models.

Seizure disorders are common, affecting both the young and the old. Currently available antiseizure drugs are ineffective in a third of patients and have been developed with a focus on known neurocentric mechanisms, raising the need for investigations into alternative and complementary mechanisms that contribute to seizure generation or its containment. Neuroinflammation, broadly defined as the activation of immune cells and molecules in the central nervous system (CNS), has been proposed to facilitate seizure generation, although the specific cells involved in these processes remain inadequately understood. The role of microglia, the primary inflammation-competent cells of the brain, is debated since previous studies were conducted using approaches that were less specific to microglia or had inherent confounds. Using a selective approach to target microglia without such side effects, we show a broadly beneficial role for microglia in limiting chemoconvulsive, electrical, and hyperthermic seizures and argue for a further understanding of microglial contributions to contain seizures.

Harnessing the therapeutic potential of the stem cell secretome in neonatal diseases

Preterm birth and intrapartum related complications account for a substantial amount of mortality and morbidity in the neonatal period despite significant advancements in neonatal-perinatal care. Currently, there is a noticeable lack of curative or preventative therapies available for any of the most common complications of prematurity including bronchopulmonary dysplasia, necrotizing enterocolitis, intraventricular hemorrhage, periventricular leukomalacia and retinopathy of prematurity or hypoxic-ischemic encephalopathy, the main cause of perinatal brain injury in term infants. Mesenchymal stem/stromal cell-derived therapy has been an active area of investigation for the past decade and has demonstrated encouraging results in multiple experimental models of neonatal disease. It is now widely acknowledged that mesenchymal stem/stromal cells exert their therapeutic effects via their secretome, with the principal vector identified as extracellular vesicles. This review will focus on summarizing the current literature and investigations on mesenchymal stem/stromal cell-derived extracellular vesicles as a treatment for neonatal diseases and examine the considerations to their application in the clinical setting.

Targeting TBK1 to overcome resistance to cancer immunotherapy.

Despite thesuccess of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.

The role of calcium, Akt and ERK signaling in cadmium-induced hair cell death

Exposure to heavy metals has been shown to cause damage to a variety of different tissues and cell types including hair cells, the sensory cells of our inner ears responsible for hearing and balance. Elevated levels of one such metal, cadmium, have been associated with hearing loss and shown to cause hair cell death in multiple experimental models. While the mechanisms of cadmium-induced cell death have been extensively studied in other cell types they remain relatively unknown in hair cells. We have found that calcium signaling, which is known to play a role in cadmium-induced cell death in other cell types through calmodulin and CaMKII activation as well as IP3 receptor and mitochondrial calcium uniporter mediated calcium flow, does not appear to play a significant role in cadmium-induced hair cell death. While calmodulin inhibition can partially protect hair cells this may be due to impacts on mechanotransduction activity. Removal of extracellular calcium, and inhibiting CaMKII, the IP3 receptor and the mitochondrial calcium uniporter all failed to protect against cadmium-induced hair cell death. We also found cadmium treatment increased pAkt levels in hair cells and pERK levels in supporting cells. This activation may be protective as inhibiting these pathways enhances cadmium-induced hair cell death rather than protecting cells. Thus cadmium-induced hair cell death appears distinct from cadmium-induced cell death in other cell types where calcium, Akt and ERK signaling all promote cell death.

Probiotics and Intestinal Microbiome: A Review of Literature

Probiotics, prebiotics, and synbiotics modify various aspects of local and systemic immune function in multiple experimental models. However, their impact and mechanisms of action are not known across all products or noticed in every population studied, and impacts on in vitro, ex vivo, or other measures of immune function do not necessarily result in an impact on infection and illness in vivo. Studies have discussed that intestinal microbiota has an essential role in enhancing the immune system against viruses. The regulatory impact of the intestinal microbiota on viral infection is connected with local and systemic immune responses and plays a part in congenital and adaptive immune responses. The microbiota composition critically modulates the production of virus-specific CD4 and CD8 T cells and antibody responses following influenza virus infection. The intestinal microbiota has an important role in the stabilizing of immune homeostasis by augmenting the integrity of the barrier functions of the gut mucosa, which is a crucial aspect of systemic immunity. In conclusion, the intestinal microbiota can influence organismal immunity locally and systemically, proximally, and distally. Studying the possible mechanism by which the intestinal microbiota maintains host immunity can provide a clearer understanding of the occurrence and development of diseases.