The Antiviral Activity of Interferon-Induced Transmembrane Proteins and Virus Evasion Strategies.

Interferons (IFNs) are antiviral cytokines that defend against viral infections by inducing the expression of interferon-stimulated genes (ISGs). Interferon-inducible transmembrane proteins (IFITMs) 1, 2, and 3 are crucial ISG products and members of the CD225 protein family. Compelling evidence shows that IFITMs restrict the infection of many unrelated viruses by inhibiting the virus-cell membrane fusion at the virus entry step via the modulation of lipid composition and membrane properties. Meanwhile, viruses can evade IFITMs' restrictions by either directly interacting with IFITMs via viral glycoproteins or by altering the native entry pathway. At the same time, cumulative evidence suggests context-dependent and multifaceted roles of IFITMs in modulating virus infections and cell signaling. Here, we review the diverse antiviral mechanisms of IFITMs, the viral antagonizing strategies, and the regulation of IFITM activity in host cells. The mechanisms behind the antiviral activity of IFITMs could aid the development of broad-spectrum antivirals and enhance preparedness for future pandemics.

Interferon-induced transmembrane protein-1 competitively blocks Ephrin receptor A2-mediated Epstein-Barr virus entry into epithelial cells.

Epstein-Barr virus (EBV) can infect both B cells and epithelial cells (ECs), causing diseases such as mononucleosis and cancer. It enters ECs via Ephrin receptor A2 (EphA2). The function of interferon-induced transmembrane protein-1 (IFITM1) in EBV infection of ECs remains elusive. Here we report that IFITM1 inhibits EphA2-mediated EBV entry into ECs. RNA-sequencing and clinical sample analysis show reduced IFITM1 in EBV-positive ECs and a negative correlation between IFITM1 level and EBV copy number. IFITM1 depletion increases EBV infection and vice versa. Exogenous soluble IFITM1 effectively prevents EBV infection in vitro and in vivo. Furthermore, three-dimensional structure prediction and site-directed mutagenesis demonstrate that IFITM1 interacts with EphA2 via its two specific residues, competitively blocking EphA2 binding to EBV glycoproteins. Finally, YTHDF3, an m6A reader, suppresses IFITM1 via degradation-related DEAD-box protein 5 (DDX5). Thus, this study underscores IFITM1's crucial role in blocking EphA2-mediated EBV entry into ECs, indicating its potential in preventing EBV infection.

GADD45B in the ventral hippocampal CA1 modulates aversive memory acquisition and spatial cognition.

AIMS: This study was designed to investigate the role of growth arrest and DNA damage-inducible ß (GADD45B) in modulating fear memory acquisition and elucidate its underlying mechanisms. MAIN METHODS: Adeno-associated virus (AAV) that knockdown or overexpression GADD45B were injected into ventral hippocampal CA1 (vCA1) by stereotactic, and verified by fluorescence and Western blot. The contextual fear conditioning paradigm was employed to examine the involvement of GADD45B in modulating aversive memory acquisition. The Y-maze and novel location recognition (NLR) tests were used to examine non-aversive cognition. The synaptic plasticity and electrophysiological properties of neurons were measured by slice patch clamp. KEY FINDINGS: Knockdown of GADD45B in the vCA1 significantly enhanced fear memory acquisition, accompanied by an upregulation of long-term potentiation (LTP) expression and intrinsic excitability of vCA1 pyramidal neurons (PNs). Conversely, overexpression of GADD45B produced the opposite effects. Notably, silencing the activity of vCA1 neurons abolished the impact of GADD45B knockdown on fear memory development. Moreover, mice with vCA1 GADD45B overexpression exhibited impaired spatial cognition, whereas mice with GADD45B knockdown did not display such impairment. SIGNIFICANCE: These results provided compelling evidence for the crucial involvement of GADD45B in the formation of aversive memory and spatial cognition.

Gentulizumab, a novel anti-CD47 antibody with potent antitumor activity and demonstrates a favorable safety profile.

BACKGROUND: Targeting CD47/SIRPα axis has emerged as a promising strategy in cancer immunotherapy. Despite the encouraging clinical efficacy observed in hematologic malignancies through CD47-SIRPα blockade, there are safety concerns related to the binding of anti-CD47 antibodies to CD47 on the membrane of peripheral blood cells. METHODS: In order to enhance the selectivity and therapeutic efficacy of the antibody, we developed a humanized anti-CD47 monoclonal antibody called Gentulizumab (GenSci059). The binding capacity of GenSci059 to CD47 was evaluated using flow cytometry and surface plasmon resonance (SPR) methods, the inhibitory effect of GenSci059 on the CD47-SIRPα interaction was evaluated through competitive ELISA assays. The anti-tumor activity of GenSci059 was assessed using in vitro macrophage models and in vivo patient-derived xenograft (PDX) models. To evaluate the safety profile of GenSci059, binding assays were conducted using blood cells. Additionally, we investigated the underlying mechanisms contributing to the weaker binding of GenSci059 to erythrocytes. Finally, toxicity studies were performed in non-human primates to assess the potential risks associated with GenSci059. RESULTS: GenSci059 displayed strong binding to CD47 in both human and monkey, and effectively inhibited the CD47-SIRPα interaction. With doses ranging from 5 to 20 mg/kg, GenSci059 demonstrated potent inhibition of the growth of subcutaneous tumor with the inhibition rates ranged from 30.3% to complete regression. Combination of GenSci059 with 2.5 mg/kg Rituximab at a dose of 2.5 mg/kg showed enhanced tumor inhibition compared to monotherapy, exhibiting synergistic effects. GenSci059 exhibited minimal binding to hRBCs compared to Hu5F9-G4. The binding of GenSci059 to CD47 depended on the cyclization of N-terminal pyroglutamic acid and the spatial conformation of CD47, but was not affected by its glycosylation modifications. A maximum tolerated dose (MTD) of 450 mg/kg was observed for GenSci059, and no significant adverse effects were observed in repeated dosages up to 10 + 300 mg/kg, indicating a favorable safety profile. CONCLUSION: GenSci059 selectively binds to CD47, effectively blocks the CD47/SIRPα axis signaling pathway and enhances the phagocytosis effects of macrophages toward tumor cells. This monoclonal antibody demonstrates potent antitumor activity and exhibits a favorable safety profile, positioning it as a promising and effective therapeutic option for cancer.

Remazolam affects the phenotype and function of astrocytes to improve traumatic brain injury by regulating the Cx43.

PURPOSE: To explore the mechanism by which Remazolam affects the phenotype and function of astrocytes to improve traumatic brain injury (TBI). METHODS: The oxygen -glucose deprivation/recovery (OGD/R) cell model was constructed to simulate the pathological state of astrocytes in a TBI environment. The viability of astrocytes was measured by CCK-8, and the cytoskeleton changes were observed by Phalloidin- TRITC staining. The expressions of differentiation markers, Cx43 and phosphorylated Cx43 (P-Cx43) of A1/A2 astrocytes were detected by Western blot, and the complement C3 and S100A10 of A1/A2 astrocytes were detected by ELISA. The TBI rat model was established. The water content of brain tissue was measured by dry-wet specific gravity method, the pathological morphology of brain tissue in cortical injury area was observed by HE staining method, ROS was detected by fluorescence quantitative method, Cx43 expression was detected by immunohistochemistry method, and the differentiation markers of A1/A2 astrocytes were detected by immunofluorescence. RESULTS: In the TBI environment, astrocytes showed decreased cell viability, blurred skeleton, and increased expression of Cx43. In TBI rats, the water content of brain tissue increased, the brain tissue in the cortex injury area was seriously damaged, ROS and Cx43 expression were significantly increased, and mainly distributed in A2 astrocytes. Remazolam can reverse the above results after the intervention. CONCLUSION: Remazolam affects the phenotype and function of astrocytes to improve TBI via regulating Cx43, and plays a role in protecting the neurological function of TBI rats.

IFITM1 and IFITM2 inhibit the replication of senecavirus A by positive feedback with RIG-I signaling pathway.

The role of host factors in the replication of emerging senecavirus A (SVA) which induced porcine idiopathic vesicular disease (PIVD) distributed worldwide remains obscure. Here, interferon-induced transmembrane (IFITM) protein 1 and 2 inhibit SVA replication by positive feedback with RIG-I signaling pathway was reported. The expression levels of IFITM1 and IFITM2 increased significantly in SVA infected 3D4/21 cells. Infection experiments of cells with over and interference expression of IFITM1 and IFITM2 showed that these two proteins inhibit SVA replication by regulating the expression of interferon beta (IFN-ß), IFN-stimulated gene 15 (ISG-15), interleukin 6 (IL-6), IL-8, tumor necrosis factor alpha (TNF-α), IFN regulatory factor-3 (IRF3), and IRF7. Further results showed that antiviral responses of IFITM1 and IFITM2 were achieved by activating retinoic acid-inducible gene I (RIG-I) signaling pathway which in turn enhanced the expression of IFITM1 and IFITM2. It is noteworthy that conserved domains of these two proteins also paly the similar role. These findings provide new data on the role of host factors in infection and replication of SVA and help to develop new agents against the virus.

Cancer stem cells: The important role of CD markers, Signaling pathways, and MicroRNAs.

For the first time, a subset of small cancer cells identified in acute myeloid leukemia has been termed Cancer Stem Cells (CSCs). These cells are notorious for their robust proliferation, self-renewal abilities, significant tumor-forming potential, spread, and resistance to treatments. CSCs are a global concern, as it found in numerous types of cancer, posing a real-world challenge today. Our review encompasses research on key CSC markers, signaling pathways, and MicroRNA in three types of cancer: breast, colon, and liver. These factors play a critical role in either promoting or inhibiting cancer cell growth. The reviewed studies have shown that as cells undergo malignant transformation, there can be an increase or decrease in the expression of different Cluster of Differentiation (CD) markers on their surface. Furthermore, alterations in essential signaling pathways, such as Wnt and Notch1, may impact CSC proliferation, survival, and movement, while also providing potential targets for cancer therapies. Additionally, some research has focused on MicroRNAs due to their dual role as potential therapeutic biomarkers and their ability to enhance CSCs' response to anti-cancer drugs. MicroRNAs also regulate a wide array of cellular processes, including the self-renewal and pluripotency of CSCs, and influence gene transcription. Thus, these studies indicate that MicroRNAs play a significant role in the malignancy of various tumors. Although the gathered information suggests that specific CSC markers, signaling pathways, and MicroRNAs are influential in determining the destiny of cancer cells and could be advantageous for therapeutic strategies, their precise roles and impacts remain incompletely defined, necessitating further investigation.

The hybrid CL-SP-D molecule has the potential to regulate xenogeneic rejection by human neutrophils more efficiently than CD47.

OBJECTIVE: Innate immunity plays a vital role in xenotransplantation. A CD47 molecule, binding to the SIRPα expressed on monocyte/macrophage cells, can suppress cytotoxicity. Particularly, the SIRPα contains ITIM, which delivers a negative signal. Our previous study demonstrated that the binding between CL-P1 and surfactant protein-D hybrid (CL-SP-D) with SIRPα regulates macrophages' phagocytic activity. In this study, we examined the effects of human CD47 and CL-SP-D expression on the inhibition of xenograft rejection by neutrophils in swine endothelial cells (SECs). METHODS: We first examined SIRPα expression on HL-60 cells, a neutrophil-like cell line, and neutrophils isolated from peripheral blood. CD47-expressing SECs or CL-SP-D-expressing SECs were generated through plasmid transfection. Subsequently, these SECs were co-cultured with HL-60 cells or neutrophils. After co-culture, the degree of cytotoxicity was calculated using the WST-8 assay. The suppressive function of CL-SP-D on neutrophils was subsequently examined, and the results were compared with those of CD47 using naïve SECs as controls. Additionally, we assessed ROS production and neutrophil NETosis. RESULTS: In initial experiments, the expression of SIRPα on HL-60 and neutrophils was confirmed. Exposure to CL-SP-D significantly suppressed the cytotoxicity in HL-60 (p = 0.0038) and neutrophils (p = 0.00003). Furthermore, engagement with CD47 showed a suppressive effect on neutrophils obtained from peripheral blood (p = 0.0236) but not on HL-60 (p = 0.4244). The results of the ROS assays also indicated a significant downregulation of SEC by CD47 (p = 0.0077) or CL-SP-D (p = 0.0018). Additionally, the suppression of NETosis was confirmed (p = 0.0125) in neutrophils co-cultured with S/CL-SP-D. CONCLUSION: These results indicate that CL-SP-D is highly effective on neutrophils in xenogeneic rejection. Furthermore, CL-SP-D was more effective than CD47 at inhibiting neutrophil-mediated xenograft rejection.

Biochemical evidence for conformational variants in the anti-viral and pro-metastatic protein IFITM1.

Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.

Shikonin reactivates TSGs GADD45B and PPP3CC to block NSCLC cell proliferation and migration through JNK/P38/MAPK signaling pathways.

BACKGROUND: Shikonin, a natural naphthoquinone compound extracted from the Chinese traditional herbal medicine "Lithospermum erythrorhizon", possesses antitumor activity against various cancer types. Tumor-suppressor genes (TSGs) negatively regulate cell growth, proliferation, and differentiation, thereby inhibiting tumor formation. However, the molecular mechanism of action of shikonin on TSGs in non-small-cell lung cancer (NSCLC) remains unclear. METHODS: The inhibitory effect of shikonin on the proliferation and migration abilities of lung cancer cells were measured by Cell Counting Kit 8 (CCK8) and wound healing assays. The alteration of genes by shikonin treatment was detected by mRNA high-throughput sequencing and further confirmed by qPCR and western blotting experiments. The dominant functions of the upregulated genes were analyzed by GO and KEGG profiling. RESULTS: Shikonin inhibited the proliferation and migration of A549 and H1299 NSCLC cells in a dose-dependent manner. mRNA high-throughput sequencing revealed a total of 1794 upregulated genes in shikonin-treated NSCLC cells. Moreover, bioinformatic analysis of GO and KEGG profiling revealed that the up-regulated genes were mostly involved in the JNK/P38/MAPK signaling pathway, among which the expression of GADD45B and PPP3CC was significantly enhanced. Finally, we confirmed that GADD45B and PPP3CC were indeed upregulated in JNK/P38/MAPK pathway. CONCLUSIONS: Taken together, these results suggested that shikonin might affect the expression of GADD45B and PPP3CC through the JNK/P38/MAPK pathway, therefore exerting an inhibitory effect on the proliferation and migration of cancer cells. To our knowledge, this is the first study reporting the role of shikonin in upregulating TSGs to activate the JNK/P38/MAPK signaling pathways in NSCLC.

Extracellular Vesicles Carrying RUNX3 Promote Differentiation of Dental Pulp Stem Cells.

BACKGROUND: This study aims to clarify the mechanism underlying dental pulp cells-extracellular vesicles (DPC-EVs) carrying runt-related transcription factor 3 (RUNX3) in mediating odontogenic differentiation of dental pulp stem cells (DPSCs) with the involvement of miR-30a-5p-regulated NOTCH1. METHODS: Extracellular vesicles (EVs) were isolated from human DPSCs, and identified using transmission electron microscopy, and nanoparticle tracking analysis. PBS, EVs, or EV inhibitor GW4869 was added to DPSCs for co-culture, whilst odontogenic differentiation was assessed in terms of ratio of mineralized nodules and expression odontoblast differentiation markers. Dual luciferase reporter gene assay and chromatin immunoprecipitation for binding relation among RUNX3, miR-30a-5p and NOTCH1were employed to evaluate their roles in odontogenic differentiation was determined. Animal experiment was established to confirm the effect of DPC-EVs-loaded RUNX3 on dental pulp. RESULTS: In vitro finding demonstrated that EVs delivered RUNX3 to DPSCs, thereby activated miR-30a-5p expression and inhibited NOTCH1 expression, which was reversed by addition of GW4869. RUNX3 upregulation promoted miR-30a-5p while miR-30a-5p targeted and inhibited NOTCH1. Silencing of RUNX3 in EVs decreased expression of those differentiation markers, downregulated miR-30a-5p and upregulated NOTCH1. CONCLUSION: DPSC-EVs can carry RUNX3 to the DPSCs, promote the transcription of miR-30a-5p, and then inhibit the expression of NOTCH1, and finally promote the odontogenic differentiation of DPSCs.

Clinicopathological analysis of CD47 and signal regulatory protein alpha expression in myeloid sarcoma patients: CD47 expression is a favourable prognostic factor.

Myeloid sarcoma is a rare extramedullary haematopoietic malignancy. Interaction between CD47 and signal regulatory protein α (SIRPα) inhibits phagocytosis. CD47-positive tumours confer poor prognoses in various malignant tumours, including acute myeloid leukaemia. This study aimed to investigate the clinicopathological effects of CD47 and SIRPα expression in myeloid sarcoma. Immunohistochemistry (IHC) of CD47 and SIRPα was performed in 84 biopsy samples obtained from patients with myeloid sarcoma, some of which were CD47-positive. Patients were categorised into the following two groups based on IHC of SIRPα: those with SIRPα-positive neoplastic cells (nSIRPα) and, SIRPα expression on non-neoplastic stromal cells in tumour microenvironment (miSIRPα). In addition, patients with CD47 positivity had higher lymphocytic infiltration into the tumour microenvironment. Overall, these patients had significantly higher overall survival, however, no significant difference was observed in progression-free survival. No significant prognostic differences were observed between the nSIRPα and miSIRPα groups. This is the first study to demonstrate an association between CD47 expression and improved prognosis in myeloid sarcoma. Nonetheless, it will be necessary to conduct additional research on gene expression and genomic abnormalities to elucidate the corresponding pathogenesis of myeloid sarcoma.

Effect of DHCR7 on adipocyte differentiation in goats.

Cholesterol is regarded as a signaling molecule in regulating the metabolism and function of fat cells, in which 7-Dehydrocholesterol reductase (DHCR7) is a key enzyme that catalyzes the conversion of 7-dehydrocholesterol to cholesterol, however, the exact function of DHCR7 in goat adipocytes remains unknown. Here, the effect of DHCR7 on the formation of subcutaneous and intramuscular fat in goats was investigated in vitro, and the result indicated that the mRNA level of DHCR7 showed a gradual downward trend in subcutaneous adipogenesis, but an opposite trend in intramuscular adipogenesis. In the process of subcutaneous preadipocytes differentiation, overexpression of DHCR7 inhibited the expression of adipocytes differentiation marker genes (CEBP/α, CEBP/ß, SREBP1 and AP2), lipid metabolism-related genes (AGPAT6, FASN, SCD1 and LPL), and the lipid accumulation. However, in intramuscular preadipocyte differentiation, DHCR7 overexpression showed a promoting effect on adipocyte differentiation marker genes (CEBP/α, CEBP/ß, PPARγ and SREBP1) and lipid metabolism-related genes (GPAM, AGPAT6, DGAT1 and SCD1) expression, and on lipid accumulation. In summary, our work demonstrated that DHCR7 played an important role in regulating adipogenic differentiation and lipid metabolism in preadipocytes in goats, which is of great significance for uncovering the underlying molecular mechanism of adipocyte differentiation and improving goat meat quality.

Has-miR-300-GADD45B promotes melanoma growth via cell cycle.

Response to oncogenic factors like UV, GADD45 family in skin participates in scavenging ROS, DNA repair and cell cycle control. Because of this, the previous study of the chronic UVB injury model has found that hsa-miR-300 can conduct intercellular transport by exosomes and target regulation of GADD45B. Whether the hsa-miR-300-GADD45B still regulates tumor development by cell cycle pathway is unclear. Through transcriptomic analysis of primary (n=39) and metastatic (n=102) melanoma, it was confirmed that in metastatic samples, some of the 97 down-regulated genes participate in maintaining skin homeostasis while 42 up-regulated genes were enriched in cancer-related functions. Furthermore, CDKN1A, CDKN2A, CXCR4 and RAD51 in the melanoma pathway, were also differentially expressed between normal skin and melanoma. CDKN1A and CDKN2A were also found to be involved in TP53-dependent cell cycle regulation. In conclusion, it was speculated that CDKN1A, CDKN2A, TP53, GADD45B and hsa-miR-300 may have regulatory relationships. It was demonstrated that there is a bidirectional regulation between hsa-miR-300 and TP53. In addition, miR-300 can regulate CDKN1A by GADD45B/TP53 and promote melanoma growth by accelerating the cell cycle transition from G1/S to G2 phase.

Heavy-chain antibody targeting of CD38 NAD+ hydrolase ectoenzyme to prevent fibrosis in multiple organs.

The functionally pleiotropic ectoenzyme CD38 is a glycohydrolase widely expressed on immune and non-hematopoietic cells. By converting NAD+ to ADP-ribose and nicotinamide, CD38 governs organismal NAD+ homeostasis and the activity of NAD+-dependent cellular enzymes. CD38 has emerged as a major driver of age-related NAD+ decline underlying adverse metabolic states, frailty and reduced health span. CD38 is upregulated in systemic sclerosis (SSc), a chronic disease characterized by fibrosis in multiple organs. We sought to test the hypothesis that inhibition of the CD38 ecto-enzymatic activity using a heavy-chain monoclonal antibody Ab68 will, via augmenting organismal NAD+, prevent fibrosis in a mouse model of SSc characterized by NAD+ depletion. Here we show that treatment of mice with a non-cytotoxic heavy-chain antibody that selectively inhibits CD38 ectoenzyme resulted in NAD+ boosting that was associated with significant protection from fibrosis in multiple organs. These findings suggest that targeted inhibition of CD38 ecto-enzymatic activity could be a potential pharmacological approach for SSc fibrosis treatment.

Biomimetic Design of Peptide Inhibitor to Block CD47/SIRPα Interactions.

CD47 on the surface of tumor cells has become a research hot spot in immunotherapy and anticancer therapy, as it can bind to SIRPα protein on the surface of macrophages, which ultimately leads to immune escape of tumor cells. In the present study, molecular interactions between CD47 and human SIRPα proteins (including variant 1, V1 and variant 2, V2) were analyzed through molecular dynamics (MD) simulation and the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. Hydrophobic interactions were found as the main driving force for the binding of CD47 on SIRPα. The residues including pyroglutamate acid (Z)1, L2, E35, Y37, E97, L101, and T102 of CD47 were identified with a significant favorable contribution to the binding of CD47 on SIRPα (both V1 and V2). Based on this, a peptide inhibitor library with the sequence ZLXRTLXEXY was designed (X represents the arbitrary residue of 20 standard amino acids) and then screened using molecular docking, MD simulations, and experimental validation. Finally, a peptide ZLIRTLHEWY was determined with high affinity with SIRPα from 8000 candidates, containing 6/10 residues favorable for the binding on SIRPα V1 and 8/10 residues favorable for the binding on SIRPα V2, which was thus considered to have potential anticancer function.

IFITM1 and IFITM3 Proteins Inhibit the Infectivity of Progeny HIV-1 without Disrupting Envelope Glycoprotein Clusters.

Human interferon-induced transmembrane (IFITM) proteins inhibit the fusion of a broad spectrum of enveloped viruses, both when expressed in target cells and when present in infected cells. Upon expression in infected cells, IFITMs incorporate into progeny virions and reduce their infectivity by a poorly understood mechanism. Since only a few envelope glycoproteins (Envs) are present on HIV-1 particles, and Env clustering has been proposed to be essential for optimal infectivity, we asked if IFITM protein incorporation modulates HIV-1 Env clustering. The incorporation of two members of the IFITM family, IFITM1 and IFITM3, into HIV-1 pseudoviruses correlated with a marked reduction of infectivity. Super-resolution imaging of Env distribution on single HIV-1 pseudoviruses did not reveal significant effects of IFITMs on Env clustering. However, IFITM3 reduced the Env processing and incorporation into virions relative to the control and IFITM1-containing viruses. These results show that, in addition to interfering with the Env function, IFITM3 restricts HIV-1 Env cleavage and incorporation into virions. The lack of notable effect of IFITMs on Env clustering supports alternative restriction mechanisms, such as modification of the properties of the viral membrane.

CD47 masks pro-phagocytic ligands in cis on tumor cells to suppress antitumor immunity.

Cancer cells often overexpress CD47, which triggers the inhibitory receptor SIRPα expressed on macrophages, to elude phagocytosis and antitumor immunity. Pharmacological blockade of CD47 or SIRPα is showing promise as anticancer therapy, although CD47 blockade has been associated with hematological toxicities that may reflect its broad expression pattern on normal cells. Here we found that, in addition to triggering SIRPα, CD47 suppressed phagocytosis by a SIRPα-independent mechanism. This mechanism prevented phagocytosis initiated by the pro-phagocytic ligand, SLAMF7, on tumor cells, due to a cis interaction between CD47 and SLAMF7. The CD47-SLAMF7 interaction was disrupted by CD47 blockade and by a first-in-class agonist SLAMF7 antibody, but not by SIRPα blockade, thereby promoting antitumor immunity. Hence, CD47 suppresses phagocytosis not only by engaging SIRPα, but also by masking cell-intrinsic pro-phagocytic ligands on tumor cells and knowledge of this mechanism may influence the decision between CD47 blockade or SIRPα blockade for therapeutic purposes.

Inokosterone activates the BMP2 to promote the osteogenic differentiation of bone marrow mesenchymal stem cells and improve bone loss in ovariectomized rats.

Evidence suggests that enhancing the osteogenic ability of bone marrow-derived mesenchymal stem cells (BMSCs) may be beneficial in the fight against osteoporosis (OP) effects. Inokosterone (IS) is a major active constituent of Achyranthis bidentatae radix (ABR), which stimulates osteogenic differentiation of mouse embryonic osteoblasts. This study aims to investigate effect of IS on OP using osteogenic differentiated BMSCs and ovariectomy (OVX)-induced OP rats. The BMSCs were treated with 50, 100, or 200 mg/L IS and OP rats were given 2 or 4 mg/kg of IS by gavage. Cell viability, the osteogenic differentiation marker protein expression level, and mineralization were observed. This study proved that IS improved cell viability, osteogenic differentiation, and cellular mineralization in BMSCs and raised expression levels of bone morphogenetic protein-2 (BMP2), Smad1, runt-related transcription factor 2 (RUNX2), collagen I, ALP, and OCN. By BMP2 knockdown/overexpression, this study also proved the BMP2 signaling pathway activation is a potential biological mechanism of IS to improve osteogenic differentiation and mineralization in osteogenic differentiated BMSCs. In OVX-induced OP rats, IS was observed to antagonize bone loss, improve osteogenic differentiation marker protein expression levels, and activate BMP-2, smad1, and RUNX2. These findings provide scientific support for further investigation of the biological mechanisms of IS in ameliorating OP.

Galectin-3 Cooperates with CD47 to Suppress Phagocytosis and T-cell Immunity in Gastric Cancer Peritoneal Metastases.

The peritoneal cavity is a common site of gastric adenocarcinoma (GAC) metastasis. Peritoneal carcinomatosis (PC) is resistant to current therapies and confers poor prognosis, highlighting the need to identify new therapeutic targets. CD47 conveys a "don't eat me" signal to myeloid cells upon binding its receptor signal regulatory protein alpha (SIRPα), which helps tumor cells circumvent macrophage phagocytosis and evade innate immune responses. Previous studies demonstrated that the blockade of CD47 alone results in limited clinical benefits, suggesting that other target(s) might need to be inhibited simultaneously with CD47 to elicit a strong antitumor response. Here, we found that CD47 was highly expressed on malignant PC cells, and elevated CD47 was associated with poor prognosis. Galectin-3 (Gal3) expression correlated with CD47 expression, and coexpression of Gal3 and CD47 was significantly associated with diffuse type, poor differentiation, and tumor relapse. Depletion of Gal3 reduced expression of CD47 through inhibition of c-Myc binding to the CD47 promoter. Furthermore, injection of Gal3-deficient tumor cells into either wild-type and Lgals3-/- mice led to a reduction in M2 macrophages and increased T-cell responses compared with Gal3 wild-type tumor cells, indicating that tumor cell-derived Gal3 plays a more important role in GAC progression and phagocytosis than host-derived Gal3. Dual blockade of Gal3 and CD47 collaboratively suppressed tumor growth, increased phagocytosis, repolarized macrophages, and boosted T-cell immune responses. These data uncovered that Gal3 functions together with CD47 to suppress phagocytosis and orchestrate immunosuppression in GAC with PC, which supports exploring a novel combination therapy targeting Gal3 and CD47. SIGNIFICANCE: Dual inhibition of CD47 and Gal3 enhances tumor cell phagocytosis and reprograms macrophages to overcome the immunosuppressive microenvironment and suppress tumor growth in peritoneal metastasis of gastric adenocarcinoma.