Ligand Stimulation Research Articles

Pattern recognition receptor ligand-induced differentiation of human transitional B cells

B cells represent a critical component of the adaptive immune response whose development and differentiation are determined by antigen-dependent and antigen-independent interactions. In this study, we explored the effects of IL-4 and pattern-recognition receptor (PRR) ligands on B cell development and differentiation by investigating their capacity to drive the in vitro maturation of human transitional B cells. In the presence of IL-4, ligands for TLR7/8, TLR9, and NOD1 were effective in driving the in vitro maturation of cord blood transitional B cells into mature, naïve B cells as measured by CD23 expression, ABCB1 transporter activation and upregulation of sIgM and sIgD. In addition, several stimulation conditions, including TLR9 ligand alone, favored an expansion of CD27+ IgM memory B cells. Transitional B cells stimulated with TLR7/8 ligand + IL-4 or TLR9 ligand, with or without IL-4, induced a significant subpopulation of CD23+CD27+ B cells expressing high levels of sIgM and sIgD, a minor B cell subpopulation found in human peripheral blood. These studies illustrate the heterogeneity of the B cell populations induced by cytokine and PRR ligand stimulation. A comparison of transitional and mature, naïve B cells transcriptomes to identify novel genes involved in B cell maturation revealed that mature, naïve B cells were less transcriptionally active than transitional B cells. Nevertheless, a subset of differentially expressed genes in mature, naïve B cells was identified including genes associated with the IL-4 signaling pathway, PI3K signaling in B lymphocytes, the NF-κB signaling pathway, and the TNFR superfamily. When transitional B cells were stimulated in vitro with IL-4 and PRR ligands, gene expression was found to be dependent on the nature of the stimulants, suggesting that exposure to these stimulants may alter the developmental fate of transitional B cells. The influence of IL-4 and PRR signaling on transitional B cell maturation illustrates the potential synergy that may be achieved when certain PRR ligands are incorporated as adjuvants in vaccine formulations and presented to developing B cells in the context of an inflammatory cytokine environment. These studies demonstrate the potential of the PRR ligands to drive transitional B cell differentiation in the periphery during infection or vaccination independently of antigen mediated BCR signaling.

Cellular Electrical Impedance as a Method to Decipher CCR7 Signalling and Biased Agonism.

The human C-C chemokine receptor type 7 (CCR7) has two endogenous ligands, C-C chemokine ligand 19 (CCL19) and CCL21, displaying biased agonism reflected by a pronounced difference in the level of β-arrestin recruitment. Detecting this preferential activation generally requires the use of separate, pathway-specific label-based assays. In this study, we evaluated an alternative methodology to study CCR7 signalling. Cellular electrical impedance (CEI) is a label-free technology which yields a readout that reflects an integrated cellular response to ligand stimulation. CCR7-expressing HEK293 cells were stimulated with CCL19 or CCL21, which induced distinct impedance profiles with an apparent bias during the desensitisation phase of the response. This discrepancy was mainly modulated by differential β-arrestin recruitment, which shaped the impedance profile but did not seem to contribute to it directly. Pathway deconvolution revealed that Gαi-mediated signalling contributed most to the impedance profile, but Gαq- and Gα12/13-mediated pathways were also involved. To corroborate these results, label-based pathway-specific assays were performed. While CCL19 more potently induced β-arrestin2 recruitment and receptor internalisation than CCL21, both chemokines showed a similar level of Gαi protein activation. Altogether, these findings indicate that CEI is a powerful method to analyse receptor signalling and biased agonism.

Titania nanospikes activate macrophage phagocytosis by ligand-independent contact stimulation

Macrophage phagocytosis is an important research target to combat various inflammatory or autoimmune diseases; however, the phenomenon has never been controlled by artificial means. Titania nanospikes created by alkaline etching treatment can tune macrophage polarization toward a M1-like type and might regulate macrophage phagocytosis. This in vitro study aimed to determine whether the two-dimensional titania nanosurfaces created by alkaline etching treatment activated the macrophage phagocytosis by nanospike-mediated contact stimulation. On two-dimensional pure titanium sheets, alkaline etching treatments with different protocols created superhydrophilic nanosurfaces with hydroxyl function groups and moderate or dense nanospikes. Both types of titania nanosurfaces promoted the phagocytic activity of the mouse macrophage-like cell line, J774A.1, through upregulation of M1 polarization markers and phagocytosis-related receptors, such as toll-like receptors (TLR2 and 4). In contrast, the hydrophobic smooth or micro-roughened titanium surfaces did not activate macrophage phagocytosis or the expression of related receptors. These phenomena remained unchanged even under the antibody blockade of macrophage TLR2 but were either suppressed or augmented for each surface excited by ultraviolet irradiation. Titania nanospikes induced paxillin expression and provided physical stimuli to macrophages, the extent of which was positively correlated with TLR expression levels. Ligand stimulation with lipopolysaccharide did not upregulate macrophage TLR expression but further enhanced M1 marker expression by titania nanosurfaces. These results showed that the two-dimensional titania nanosurfaces activated macrophage phagocytosis by enhancing expression of phagocytosis-related receptors through nanospike-mediated contact stimulation, in assistance with physical surface properties, in a ligand-independent manner.

Mechanosensor Piezo1 mediates bimodal patterns of intracellular calcium and FAK signaling.

Piezo1 belongs to mechano-activatable cation channels serving as biological force sensors. However, the molecular events downstream of Piezo1 activation remain unclear. In this study, we used biosensors based on fluorescence resonance energy transfer (FRET) to investigate the dynamic modes of Piezo1-mediated signaling and revealed a bimodal pattern of Piezo1-induced intracellular calcium signaling. Laser-induced shockwaves (LIS) and its associated shear stress can mechanically activate Piezo1 to induce transient intracellular calcium (Ca[i] ) elevation, accompanied by an increase in FAK activity. Interestingly, multiple pulses of shockwave stimulation caused a more sustained calcium increase and a decrease in FAK activity. Similarly, tuning the degree of Piezo1 activation by titrating either the dosage of Piezo1 ligand Yoda1 or the expression level of Piezo1 produced a similar bimodal pattern of FAK responses. Further investigations revealed that SHP2 serves as an intermediate regulator mediating this bimodal pattern in Piezo1 sensing and signaling. These results suggest that the degrees of Piezo1 activation induced by both mechanical LIS and chemical ligand stimulation may determine downstream signaling characteristics.

Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer.

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer.

Human Beta Papillomavirus Type 8 E1 and E2 Proteins Suppress the Activation of the RIG-I-Like Receptor MDA5.

Persistent infections of the skin with the human papillomavirus of genus beta (β-HPV) in immunocompetent individuals are asymptomatic, but in immunosuppressed patients, β-HPV infections exhibit much higher viral loads on the skin and are associated with an increased risk of skin cancer. Unlike with HPV16, a high-risk α-HPV, the impact of β-HPV early genes on the innate immune sensing of viral nucleic acids has not been studied. Here, we used primary skin keratinocytes and U2OS cells expressing HPV8 or distinct HPV8 early genes and well-defined ligands of the nucleic-acid-sensing receptors RIG-I, MDA5, TLR3, and STING to analyze a potential functional interaction. We found that primary skin keratinocytes and U2OS cells expressed RIG-I, MDA5, TLR3, and STING, but not TLR7, TLR8, or TLR9. While HPV16-E6 downregulated the expression of RIG-I, MDA5, TLR3, and STING and, in conjunction with HPV16-E7, effectively suppressed type I IFN in response to MDA5 activation, the presence of HPV8 early genes showed little effect on the expression of these immune receptors, except for HPV8-E2, which was associated with an elevated expression of TLR3. Nevertheless, whole HPV8 genome expression, as well as the selective expression of HPV8-E1 or HPV8-E2, was found to suppress MDA5-induced type I IFN and the proinflammatory cytokine IL-6. Furthermore, RNA isolated from HPV8-E2 expressing primary human keratinocytes, but not control cells, stimulated a type I IFN response in peripheral blood mononuclear cells, indicating that the expression of HPV8-E2 in keratinocytes leads to the formation of stimulatory RNA ligands that require the active suppression of immune recognition. These results identify HPV8-E1 and HPV8-E2 as viral proteins that are responsible for the immune escape of β-HPV from the innate recognition of viral nucleic acids, a mechanism that may be necessary for establishing persistent β-HPV infections.

Abstract 5412: Role of her3 mutations on breast cancer oncogenesis

Abstract We sought to determine if naturally occurring mutations in HER3 could drive oncogenic growth of HER3KO HER2+ HCC1569 cells in which endogenous HER3 has been eliminated via CRISPR-Cas9. A series of HER3 mutations identified in breast cancer patients (F94L, V104L, G284R, D297Y, T355I, and E928G) were introduced using lentiviral transduction and stable cell lines were generated in HER3KOHCC1569 cells via puromycin selection. We identified HER3V104L mutation to have higher cell proliferation and higher p-HER3 expression compared to wild-type (wt) and empty-vector (EV) HER3. We observed that HCC1569HER3KO cells stably expressing WT and V104L were sensitive to increasing doses of neratinib (0.1-0.5 µM) concentration. Next we analyzed if this mutation rendered resistance to the recently FDA- approved irreversible HER2 tyrosine kinase inhibitor, tucatinib. Our data indicated that both that V104L cells were sensitive to higher concentration of tucatinib compared to neratinib. In parallel experiments, we utilized COS7 cells to examine the signaling properties of HER3V104L. Our data indicated that transient transfection of COS7 cells with HER3V104L mutant significantly induces p-HER3/p-AKT and p-HER2 expression compared to WT HER3 in a ligand dependent manner. In addition, we observed that the V104L mutation stabilizes HER3 protein expression independent of HER2 and ligand stimulation. Experiments are ongoing to determine whether V104L induced HER3 stabilization and downstream signaling activation is dependent on HER3 binding partners including EGFR, HER2 or HER4. We also aim to understand how the V104L mutation stabilizes HER3 expression. Structural modeling of V104L mutation will provide insight about the mechanism of stabilization of the HER3 protein. We will use MCF10A and HEK293 cells to determine the effect of the V104L mutation on HER3 expression and downstream signaling. We also aim to decipher the signaling mechanism that drives the oncogenic potential of V104L mutation. In addition, we are using various PDXs with different HER3 mutations to determine other driver HER3 mutations in breast cancer and how this can be targeted in the clinic using HER targeted therapy. Citation Format: Rosalin Mishra, Mary Kate Kilroy, Hima Patel, Samar Alanazi, Joan T. Garrett. Role of her3 mutations on breast cancer oncogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5412.

Abstract 663: Systemic targeting of therapeutic RNA to cancer via a novel, cell-penetrating and nucleic acid binding, monoclonal antibody

Abstract There is intense interest in the development of nucleic acid ligands for immune stimulation of the tumor microenvironment via pattern recognition receptors (PRRs), particularly for the treatment of “cold” tumors. However, delivery of these ligands in a tumor-specific manner to avoid systemic toxicity has been a challenge. Many current efforts rely on direct intra-tumoral injection of RNAs or other stimulatory immune ligands, which is therapeutically sub-optimal, especially for metastatic disease. Here we report studies evaluating the utility of a lupus-derived, cell-penetrating antibody, to deliver nucleic acids to tumors in vivo. This antibody, a modified version of 3E10-D31N, designated GMAB, forms non-covalent complexes with RNAs and can mediate highly specific delivery into tumors via intravenous injection by targeting the nucleoside transporter ENT2, which is highly expressed in tumor cells. Studies with labeled RNAs showed tumor specific delivery and functional expression by imaging, with minimal delivery to healthy tissues. Following these proof-of-concept studies, we investigated whether our antibody could be used to deliver RIG-I agonists to tumors. Using a known agonist of RIG-I, 3p-hpRNA, we demonstrated single agent activity of our GMAB/RNA complexes in multiple tumor models, including a mouse model of melanoma (B16) and an orthotopic model of pancreatic cancer (KPC). Measuring 3p-hpRNA by RT-PCR, there was a 1000X fold difference in RNA uptake in KPC tumor cells relative to CD45+ cells isolated from tumors. In addition, synergy between our GMAB/RNA complexes and anti-PD-1 was additionally observed in mouse models of breast (EMT6) and colon (MC38) cancer. Given the expression of ENT2 along the blood brain barrier (BBB), we demonstrate single agent activity of our GMAB/RNA complexes in an orthotopic mouse model of medulloblastoma, resulting in suppression of tumor growth and spinal metastases. Together, these studies demonstrate that the GMAB antibody can 1) localize to orthotopic and flank tumor models, 2) cross the BBB, and 3) deliver RNA payloads to tumors, providing a novel platform for delivery of immunogenic RNAs, as well as mRNAs and siRNAs, directly to tumors with high specificity following systemic administration. Citation Format: Elias Quijano, Diana Martinez Saucedo, Minsoo Khang, Yanfeng Liu, Dale Ludwig, Bruce C. Turner, Stephen Squinto, Ranjit Bindra, W. Mark Saltzman, Luisa Escobar-Hoyos, Peter M. Glazer. Systemic targeting of therapeutic RNA to cancer via a novel, cell-penetrating and nucleic acid binding, monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 663.

Abstract 1298: An allosteric SHP2 inhibitor RMC-4550 induces immunogenicity in pancreatic adenocarcinoma (PDA) and enhances the cytotoxicity of CD8+ effector T cells

Abstract Background: Immunotherapy is ineffective in MSS PDA. Src homology-2 domain-containing protein tyrosine phosphatase (SHP2) mediates multiple immunosuppressive mechanisms in tumor cells and tumor microenvironment (TME). SHP2 plays an important role in oncogenic RAS signaling, the hallmark of PDA, that reduces tumor MHC-I expression and promotes immunotherapy resistance by stabilizing PD-L1. It modulates multiple T cell inhibitory pathways including PD-1 signaling via dephosphorylation of ITIM and ITSM. We hypothesize that SHP2i will increase anti-PDA immunity and help overcome resistance to PD-1 blockade when combined with gemcitabine, an active cytotoxic agent against PDA with pleiotropic immunomodulatory effects on its TME. Methods: Mouse KPC-luc cells (derived from LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre GEMM) and human RAS mutant PDA cells were treated in vitro with various concentrations of RMC-4550, a potent and selective allosteric SHP2 inhibitor, to test its cytotoxicity using cell viability assay and measure tumor MHC-I and PD-L1 expression upon IFNy stimulation. Human PBMC were used for T cells in vitro experiments to examine the effect of RMC-4550 on T-cell proliferation inhibition by PD-1 ligand (PD-L1) stimulation. In vivo antitumor activity of RMC-4550 combined with αPD-1 or with αPD-1 and gemcitabine was investigated in a syngeneic KPC-luc subcutaneous model. After treatment, tumors and spleens were harvested, digested to single cell suspensions, and immune infiltrates quantified using flow cytometry. Results: In vitro, there was no direct cytotoxicity of RMC-4550 on mouse KPC or human PDAC cells and no additive effect to gemcitabine. SHP2i increases IFNy mediated upregulation of MHC-I, and downregulates PD-L1 in both KPC mouse and MiaPaca-2 human PDA cells. In vitro, RMC-4550 reverses the PD-L1-mediated inhibition of T cell proliferation. In vivo, SHP2i with αPD-1 has a combinatorial effect on tumor growth inhibition. Gemcitabine seems to augment this effect. Preliminary data revealed no significant changes in number of immune cells in treatment groups when compared to the control group while an increase in proportion of Granzyme B+ CD8+ T cells and Ki-67+ CD8+ T cells in spleen and a decrease in proportion of PD1+ CD8+ T cells in TME was observed in RMC-4550 plus αPD-1 plus gemcitabine group compared to the control group. Conclusion: SHP2i RMC-4550 and αPD-1 combination induces significant anti-tumor benefit in an in vivo PDA tumor model, an effect that is enhanced by adding gemcitabine. SHP2i may promote antitumor immunity in part by increasing MHC-I expression and reducing PD-L1 expression in RAS mutant PDA cells. PD-L1 mediated suppression of T cell proliferation was overcome by RMC-4550. In vivo, RMC-4550 may potentiate anti-PD-1 therapy by enhancing cytotoxicity of CD8+ effector T cells. Citation Format: Agnieszka Looney, Uma Giri, Yezi Zhu, Alex Somma, Nisha Holay, Milad Soleimani, Heta Gandhi, Anna Capasso, Jeanne Kowalski, William Matsui, Carla Van Berg, S Gail Eckhardt, Todd Triplett, Kyaw Lwin Aung. An allosteric SHP2 inhibitor RMC-4550 induces immunogenicity in pancreatic adenocarcinoma (PDA) and enhances the cytotoxicity of CD8+ effector T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1298.

Abstract 957: Cadherin-11 contributes to the heterogenous and dynamic Wnt-β-catenin pathway activation in Ewing sarcoma

Abstract Introduction: Metastatic Ewing sarcoma (ES) has a dismal prognosis, and clarifying the molecular and cellular mechanism of metastasis is the most pressing need to improving prognosis. Recently, the heterogenous expression of the driving fusion gene EWS-FLI1, as well as canonical Wnt/β-catenin signaling activity has been increasingly recognized as a potential mediator of metastatic progression. However, targeting the pathway has been difficult due to its complexity and cross-activity, and not much is known of the mechanism of how ES cells maintain Wnt signaling heterogeneity. Cadherin-11 (CDH11) is a member of the cadherin super-family, which modulates calcium ion-dependent cell-cell adhesion and signal transduction, and is highly expressed in ES. CDH11 has been found to regulate the expression level of β-catenin, and play a key role in enhancing the stem cell, migration and invasion in other carcinomas, however the relationship between CDH11 and β-catenin in ES is not clear. Methods: Wnt/β-catenin dependent transcriptional activity of ES cells in vitro and in vivo were assessed by flow cytometry, immune fluorescence, as well as single cell RNA-seq. β-catenin and CDH11 knock-down cell lines were generated, and affected on invasion/migration, and metastatic progression was assessed in vitro and in vivo. Furthermore, the interaction of CDH11 with β-catenin, and the contribution of the protein-protein interaction to the heterogenous activation of Wnt/β-catenin was assessed by immunofluorescence, flowcytometry, Western blot, and co-immunoprecipitation. Results: At baseline, only a small fraction of ES cells have activated Wnt/β-catenin dependent transcriptional activity both in vitro and in vivo, which can respond rapidly with Wnt ligand stimulation. β-catenin knockdown reduced clonogenicity in vitro, and metastatic tumor burden in the orthotopic implantation amputation mouse model of ES. We further identified that most of the tested ES cells had abundant β-catenin bound to CDH11 in the cell membrane, which would rapidly be released through the cytoplasm and into the nucleus within 2 hours of adding exogenous Wnt ligand. CDH11 knockdown reduced β-catenin expression, rapid activation of signaling by Wnt ligands, and metastasis progression. Conclusion: Our data suggests that the cell surface CDH11- β-catenin complex contributes to the heterogenous and dynamic activation of the Wnt/β-catenin pathway in ES that drives ES metastasis. Targeting the CDH11- β-catenin complex can present a novel strategy to interfere with Wnt driven metastasis in ES. Citation Format: Ryota Shirai, Tyler Biebighauser, Sarah Nelson-Taylor, Avery Bodlak, Timothy Porfilio, Deandra Walker, Naoki Oike, Masanori Hayashi. Cadherin-11 contributes to the heterogenous and dynamic Wnt-β-catenin pathway activation in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 957.

Serodolin, a β-arrestin–biased ligand of 5-HT7 receptor, attenuates pain-related behaviors

G protein–coupled receptors (GPCRs) are involved in regulation of manifold physiological processes through coupling to heterotrimeric G proteins upon ligand stimulation. Classical therapeutically active drugs simultaneously initiate several downstream signaling pathways, whereas biased ligands, which stabilize subsets of receptor conformations, elicit more selective signaling. This concept of functional selectivity of a ligand has emerged as an interesting property for the development of new therapeutic molecules. Biased ligands are expected to have superior efficacy and/or reduced side effects by regulating biological functions of GPCRs in a more precise way. In the last decade, 5-HT7 receptor (5-HT7R) has become a promising target for the treatment of neuropsychiatric disorders, sleep and circadian rhythm disorders, and pathological pain. In this study, we showed that Serodolin is unique among a number of agonists and antagonists tested: it behaves as an antagonist/inverse agonist on Gs signaling while inducing ERK activation through a β-arrestin–dependent signaling mechanism that requires c-SRC activation. Moreover, we showed that Serodolin clearly decreases hyperalgesia and pain sensation in response to inflammatory, thermal, and mechanical stimulation. This antinociceptive effect could not be observed in 5-HT7R knockout (KO) mice and was fully blocked by administration of SB269-970, a specific 5-HT7R antagonist, demonstrating the specificity of action of Serodolin. Physiological effects of 5-HT7R stimulation have been classically shown to result from Gs-dependent adenylyl cyclase activation. In this study, using a β-arrestin–biased agonist, we provided insight into the molecular mechanism triggered by 5-HT7R and revealed its therapeutic potential in the modulation of pain response.

Carnosol inhibits osteoclastogenesis invivo and invitro by blocking the RANKL‑induced NF‑κB signaling pathway.

Bone homeostasis is maintained by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Disruption of bone homeostasis due to excessive osteoclastogenesis or reduced osteogenesis results in various disorders, such as postmenopausal osteoporosis. Receptor activator of NF-κB ligand (RANKL) stimulation of the NF-κB signaling pathway is essential in osteoclastogenesis. The aim of the present study was to investigate the novel effects of carnosol, an active compound found in Rosmarinus officinalis, on RANKL-induced osteoclastogenesis both in vitro and in vivo. TRAP staining showed that carnosol significantly inhibited osteoclasts differentiation of bone marrow monocytes and RAW264.7 cells. Western blot results showed that the protein expression levels of osteoclastogenesis-associated genes, including cathepsin K, tartrate-resistant acid phosphatase and MMP-9, were markedly inhibited by carnosol, which may have suppressed osteoclast function. Furthermore, western blot and immunofluorescent staining results revealed that carnosol markedly suppressed the phosphorylation of p65 induced by RANKL and blocked its nuclear translocation, indicating the suppression of NF-κB signaling pathway. H&E staining and micro-CT results showed that in vivo treatment with carnosol significantly attenuated ovariectomy-induced bone loss in mice. In conclusion, the present study indicated that carnosol may suppress osteoclastogenesis both in vivo and in vitro by inhibiting the activation of the NF-κB signaling pathway. Carnosol may therefore be a potential novel therapeutic candidate for the clinical treatment of osteoclast-related disorders.

Interspecies Single-Cell RNA-Seq Analysis Reveals the Novel Trajectory of Osteoclast Differentiation and Therapeutic Targets.

ABSTRACTBone turnover is finely tuned by cells in the bone milieu, including osteoblasts, osteoclasts, and osteocytes. Osteoclasts are multinucleated giant cells with a bone‐resorbing function that play a critical role in regulating skeletal homeostasis. Osteoclast differentiation is characterized by dramatic changes in morphology and gene expression following receptor activator of nuclear factor‐kappa‐Β ligand (RANKL) stimulation. We performed single‐cell RNA‐sequencing analyses of human and murine osteoclast‐lineage cells (OLCs) and found that OLCs in the mitotic phase do not differentiate into mature osteoclasts. We also identified a guanosine triphosphatase (GTPase) family member, RAB38, as a highly expressed molecule in both human and murine osteoclast clusters; RAB38 gene expression is associated with dynamic changes in histone modification and transcriptional regulation. Silencing Rab38 expression by using short hairpin RNA (shRNA) inhibited osteoclast differentiation and maturation. In summary, we established an integrated fate map of human and murine osteoclastogenesis; this will help identify therapeutic targets in bone diseases. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Location Bias of G Protein‐Coupled Receptor Kinases Promotes Biased Signaling at CXCR3

G protein‐coupled receptors (GPCRs) are the largest family of cell‐surface receptors and are known to canonically signal through two main effector proteins: G proteins and β‐arrestins. Biased agonism at GPCRs refers to the functional selectivity of a ligand towards certain signaling pathways over others. In addition to biased agonists, there are various other sources of bias that influence GPCR signaling, such as receptor and location bias, which affect the downstream pathways of receptor activation, leading to disproportionate levels of signaling between the G protein and β‐arrestin pathway. One of the mechanisms underlying this signaling bias is the phosphorylation barcode hypothesis which states that ligand induced receptor phosphorylation promotes differential engagement with downstream transducers. Across the entire GPCR superfamily, GPCR kinases (GRKs) are known to be primarily responsible for receptor phosphorylation. Of the seven known isoforms in the GRK family, GRK2, GRK3, GRK5, and GRK6 are expressed ubiquitously, whereas GRK1 and GRK7 are mainly found in the eye while GRK4 is predominantly located in the testes. Although there is supportive evidence demonstrating that differential phosphorylation promotes biased signaling, there is relatively little known about the role of GRKs in directing GPCR functional selectivity. To study the role of GRKs in biased signaling, we studied the GPCR CXCR3, a chemokine receptor primarily involved in leukocyte migration, which has three endogenous biased ligands, CXCL9, CXCL10, and CXCL11, and two synthetic biased ligands, VUF10661 and VUF11418. We found that, following ligand stimulation, GRK2 and GRK3 are recruited to CXCR3, with VUF10661 and CXCL11 being the most efficacious ligands. However, we also observed that GRK3 and GRK5 are able to traffic to endosomes following ligand stimulation with VUF10661, even though all ligands are able to promote receptor internalization. Given biased engagement of the GRKs observed at both the receptor and in endosomes, we next studied the functional consequences of ligand and location bias by creating GRK 2, 3, 5 and 6 constructs that are specifically targeted to the plasma membrane or the endosome. Using GRK2/3/5/6 KO cells, we determined that localization of the GRKs impacts receptor internalization, ERK activity, and transcription in a ligand specific manner. Together, we observed that the endogenous and synthetic ligands of CXCR3 demonstrate biased engagement of the GRKs at different subcellular locations and highlight the functional significance of these findings on downstream receptor signaling. These data provide evidence that the functional selectivity of biased signaling extends beyond β‐arrestins and G‐proteins to other effector proteins that interact with GPCRs.

CD16 and NKG2D co-clustering facilitates quality of primary NK cell responses

Abstract Cooperation between CD16 and NKG2D receptors has been demonstrated both in animal and NK cell line models leading to an enhanced cytotoxicity and tumor elimination. However, the mechanisms of the receptor cooperation are not known, and whether the receptors form co-clusters has not been determined either. We exposed human primary polyclonal NK cells with CD16 158V/V-genotype to planar lipid bilayers (PLB) presenting Herceptin and anti-NKG2D Ab in the presence of ICAM-1. NK cells interacting with Herceptin-containing PLBs + ICAM-1 formed mostly unstable synapse, i.e., kinapses, while stimulation with anti-NKG2D alone or Herceptin+anti-NKG2D bilayers + ICAM-1 led to formation of mature synapses showing complete segregation of the receptors from ICAM-1, increased synapse stability, and efficient NK cell cytotoxicity. The cells exposed to PLBs with both anti-NKG2D and Herceptin ligands were mostly confined within the initial contact area while the cells responding to Herceptin alone were significantly more motile. Regardless of stimulatory ligands displayed on PLB, the frequency of adhered cells was very similar, but the degranulation was observed within 40% of kinapses and 80% of mature synapses, suggesting that degranulation efficiency is linked to synapse stability. Importantly, more efficient degranulation was associated with rapid and sustained Ca2+ signaling as opposed to oscillating Ca2+ signaling observed for NK cells showing less efficient degranulation. Our data demonstrated that Herceptin alone functions as a weak agonist, while co-activation of CD16 and NKG2D results in rapid and sustained Ca2+ signaling that mediates stable synaptic interface and efficient cytolytic activity by human NK cells. Supported by Grant from NIH U01AI148117

Chronic Alcohol Exposure Among People Living with HIV Is Associated with Innate Immune Activation and Alterations in Monocyte Phenotype and Plasma Cytokine Profile.

Despite advances in antiretroviral therapy, chronic immune activation continues to be observed among individuals with well-controlled HIV viral loads, and is associated with non-AIDS defining morbidities among people living with HIV. Alcohol use disorder impacts a significant proportion of individuals living with HIV, and alcohol exposure is known to damage the intestinal epithelium which may increase translocation of pathogens and their molecular products, driving systemic immune activation and dysregulation. The aim of this study was to determine if adults living with HIV with well-controlled viral loads, who also suffer from alcohol use disorder with and without hepatitis C virus co-infection (n=23), exhibit evidence of advanced systemic immune activation, intestinal damage, and microbial translocation, as compared to adults living with HIV who are not exposed to chronic alcohol or other substances of abuse (n=29). The impact of a 1-month intervention to treat alcohol-use disorder was also examined. Alcohol-use disorder was associated with evidence of advanced innate immune activation, alterations in monocyte phenotype including increased expression of Toll-like receptor 4, increased burden of stimulatory ligands for Toll-like receptor 4, and alterations in plasma cytokine signature, most notably elevations in soluble CD40 ligand and transforming growth factor beta. Alcohol-associated immune activation was more pronounced among individuals with hepatitis C virus co-infection. Although the 1-month intervention to treat alcohol use disorder did not result in significant reductions in the interrogated indicators of immune activation, our findings suggest that chronic alcohol exposure is a major modifiable risk factor for chronic immune activation and dysregulation among people-living with HIV.

Influence of exposure to microbial ligands, immunosuppressive drugs and chronic kidney disease on endogenous immunomodulatory gene expression in feline adipose-derived mesenchymal stem cells.

Feline autologous mesenchymal stem cells (MSCs) show promise for immunomodulatory activity, but the functional impact of chronic kidney disease (CKD), concurrent immunosuppressive drug administration or infection is unknown. The study objectives compare endogenous cytokine gene expression (interleukin [IL]-6, IL-10, IL-12p40, IL-18 and transforming growth factor beta [TGF-β]) in adipose-derived MSCs (aMSCs) from cats with and without CKD, following in vitro exposure to microbial ligands and treatment with common immunosuppressive drugs. Previously obtained aMSCs, phenotype CD44+, CD90+, CD105+ and MHCII-, from cats with (n = 6) and without (n = 6) CKD were compared via real-time PCR (RT-PCR) for immunomodulatory gene expression. aMSCs were exposed in vitro to lipopolysaccharide (LPS), peptidoglycan or polyinosinic:polycytidylic acid (Poly I:C), simulating bacterial or viral exposure, respectively. aMSCs were also exposed to ciclosporin, dexamethasone or methotrexate. Gene expression was measured using RT-PCR, and Cq was utilized after each run to calculate the delta cycle threshold. aMSCs isolated from healthy and CKD cats showed no significant differences in gene expression in the five measured cytokines. No significant changes in measured gene expression after drug treatment or microbial ligand stimulation were observed between normal or CKD affected cats. Proinflammatory genes (IL-6, IL-12p40 and IL-18) showed altered expression in aMSCs from both groups when compared with the same cells in standard culture after exposure to methotrexate. Poly I:C altered IL-6 and TGF-β gene expression in aMSCs from both healthy and CKD cats when compared with the same cells in standard culture. The five genes tested showed no statistical differences between aMSCs from healthy or CKD cats. There was altered cytokine gene expression between the control and treatment groups of both healthy and CKD cats suggesting feline aMSCs have altered function with immunosuppressive treatment or microbial ligand exposure. Although the current clinical relevance of this pilot study comparing brief exposure to select agents in vitro in aMSCs from a small number of cats is unknown, the study highlights a need for continued investigation into the effects of disease and concurrent therapies on use of cell-based therapies in feline patients.

The Effect of the Nanoparticle Shape on T Cell Activation.

The mechanism of extracellular ligand nano-geometry in ex vivo T cell activation for immunotherapy remains elusive. Herein, the authors demonstrate large aspect ratio (AR) of gold nanorods (AuNRs) conjugated on cell culture substrate enhancing both murine and human T cell activation through the nanoscale anisotropic presentation of stimulatory ligands (anti-CD3(αCD3) and anti-CD28(αCD28) antibodies). AuNRs with large AR bearing αCD3 and αCD28 antibodies significantly promote T cell expansion and key cytokine secretion including interleukin-2 (IL-2), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). High membrane tension observed in large AR AuNRs regulates actin filament and focal adhesion assembly and develops maturation-related morphological features in T cells such as membrane ruffle formation, cell spreading, and large T cell receptor (TCR) cluster formation. Anisotropic stimulatory ligand presentation promotes differentiation of naïve CD8+ T cells toward the effector phenotype inducing CD137 expression upon co-culture with human cervical carcinoma. The findings suggest the importance of manipulating extracellular ligand nano-geometry in optimizing T cell behaviors to enhance therapeutic outcomes.

Rosmarinic acid alleviates diabetic osteoporosis by suppressing the activation of NLRP3 inflammasome in rats.

Diabetic osteoporosis is a common metabolic bone disorder characterized by bone loss in diabetic patients, which causes an enormous social burden due to the unsatisfactory outcome of current therapeutic strategy. Based on the importance of inflammasome activation in diabetic osteoporosis, we evaluated the protective effect of an antioxidant, rosmarinic acid (RA) in diabetic osteoporosis. Bone marrow-derived monocytes isolated from rats were treated with receptor activator of nuclear factor kappa-Β ligand (RANKL) and macrophage colony stimulating factor to differentiate into mature osteoclasts (OCs). Next OCs were stimulated with RA under high glucose condition to evaluate bone resorption. Next, streptozotocin (STZ)-injected rats were orally treated with 50 mg kg-1 RA to analyze its effect on diabetic osteoporosis. RA inhibited high glucose-stimulated inflammation and inflammasome activation in OCs. Bone resorption was also reduced after RA treatment as shown by the resorption pits assay. Moreover, RA significantly reduced bone resorption, alleviated bone weight loss and increased bone mineral density by inhibiting the activation of NACHT-LRR-PYD domains-containing protein 3 (NLRP3) inflammasome in STZ-induced diabetic rats, leading to the improvement of diabetic osteoporosis. RA effectively ameliorates diabetic osteoporosis in STZ-induced rats by inhibiting the activation of NLRP3 inflammasome in OCs, which suggests that RA might serve as a potential candidate drug for treating diabetic osteoporosis.

Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib-induced KIT mutations in gastrointestinal stromal tumours.

Drug resistance remains a major challenge in the clinical treatment of gastrointestinal stromal tumours (GISTs). While acquired on‐target mutations of mast/stem cell growth factor receptor (KIT) kinase is the major resistance mechanism, activation of alternative signalling pathways may also play a role. Although several second‐ and third‐generation KIT kinase inhibitors have been developed that could overcome some of the KIT mutations conferring resistance, the low clinical responses and narrow safety window have limited their broad application. The present study revealed that nintedanib not only overcame resistance induced by a panel of KIT primary and secondary mutations, but also overcame ERK‐reactivation‐mediated resistance caused by the upregulation of fibroblast growth factor (FGF) activity. In preclinical models of GISTs, nintedanib significantly inhibited the proliferation of imatinib‐resistant cells, including GIST‐5R, GIST‐T1/T670I and GIST patient‐derived primary cells. In addition, it also exhibited dose‐dependent inhibition of ERK phosphorylation upon FGF ligand stimulation. In vivo antitumour activity was also observed in several xenograft GIST models. Considering the well‐documented safety and pharmacokinetic profiles of nintedanib, this finding provides evidence for the repurposing of nintedanib as a new therapy for the treatment of GIST patients with de novo or acquired resistance to imatinib.