Bosutinib Stimulates Macrophage Survival, Phagocytosis, and Intracellular Killing of Bacteria.

Host-acting compounds are emerging as potential alternatives to combating antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduce the bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodeling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-κB activation, which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, cotreatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infections through multiple mechanisms and could be used to boost the host innate immunity to combat drug-resistant bacterial infections.

Nonpathogenic E. coli engineered to surface display cytokines as a new platform for immunotherapy.

Given the safety, tumor tropism, and ease of genetic manipulation in non-pathogenic Escherichia coli (E. coli), we designed a novel approach to deliver biologics to overcome poor trafficking and exhaustion of immune cells in the tumor microenvironment, via the surface display of key immune-activating cytokines on the outer membrane of E. coli K-12 DH5α. Bacteria expressing murine decoy-resistant IL18 mutein (DR18) induced robust CD8+ T and NK cell-dependent immune responses leading to dramatic tumor control, extending survival, and curing a significant proportion of immune-competent mice with colorectal carcinoma and melanoma. The engineered bacteria demonstrated tumor tropism, while the abscopal and recall responses suggested epitope spreading and induction of immunologic memory. E. coli K-12 DH5α engineered to display human DR18 potently activated mesothelin-targeting CAR NK cells and safely enhanced their trafficking into the tumors, leading to improved control and survival in xenograft mice bearing mesothelioma tumor cells, otherwise resistant to NK cells. Gene expression analysis of the bacteria-primed CAR NK cells showed enhanced TNFα signaling via NFkB and upregulation of multiple activation markers. Our novel live bacteria-based immunotherapeutic platform safely and effectively induces potent anti-tumor responses in otherwise hard-to-treat solid tumors, motivating further evaluation of this approach in the clinic.

Si-enriched biochars improved soil properties, reduced Cd bioavailability while enhanced Cd translocation to grains of rice.

Biochar and silicon (Si) have been widely considered to play an important role in mitigating cadmium (Cd) toxicity. In this study, wild-type rice (WT, high-Si) and Si-deficient mutant rice (lsi1, low-Si) were used as raw materials to prepare biochar at 500℃; the Si concentrations of high- and low-Si biochar were 15.9% and 5.3%, respectively. The impacts of different application rates (0%, 2%, 4%) of high- and low-Si biochars on soil chemical properties, Si and Cd fractions and availability, Cd absorption, and translocation were investigated. The results showed that both types of biochars increased soil pH, soil available nitrogen, and available phosphorus and potassium; and promoted Si uptake and plant growth of rice. Soil available Si, CaCl2-Si, acetic-Si, H2O2-Si, oxalate-Si, and Na2CO3-Si were also increased by biochar supply, especially for high-Si biochar treatments. In addition, both types of biochars had no effects on soil total Cd, but reduced soil available Cd by 2-17% in early season 2022, and reduced oxidizable Cd and residual Cd. Biochar application did not influence Cd concentrations in roots, stems, and leaves, but significantly increased Cd uptake and transport from stems and leaves to grains. The results suggested that Si-rich biochar could improve soil nutrients, change soil Si/Cd fractions and availability, promote rice growth but increase the risk of Cd toxicity in grains, indicating the complex of straw biochar in remediating Cd-contaminated paddy soil.

Activation of GPR3-ß-arrestin2-PKM2 pathway in Kupffer cells stimulates glycolysis and inhibits obesity and liver pathogenesis.

Kupffer cells are liver resident macrophages and play critical role in fatty liver disease, yet the underlying mechanisms remain unclear. Here, we show that activation of G-protein coupled receptor 3 (GPR3) in Kupffer cells stimulates glycolysis and protects mice from obesity and fatty liver disease. GPR3 activation induces a rapid increase in glycolysis via formation of complexes between ß-arrestin2 and key glycolytic enzymes as well as sustained increase in glycolysis through transcription of glycolytic genes. In mice, GPR3 activation in Kupffer cells results in enhanced glycolysis, reduced inflammation and inhibition of high-fat diet induced obesity and liver pathogenesis. In human fatty liver biopsies, GPR3 activation increases expression of glycolytic genes and reduces expression of inflammatory genes in a population of disease-associated macrophages. These findings identify GPR3 activation as a pivotal mechanism for metabolic reprogramming of Kupffer cells and as a potential approach for treating fatty liver disease.

Monovalent SARS-COV-2 mRNA vaccine using optimal UTRs and LNPs is highly immunogenic and broadly protective against Omicron variants.

The emergence of highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that are resistant to the current COVID-19 vaccines highlights the need for continued development of broadly protective vaccines for the future. Here, we developed two messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccines, TU88mCSA and ALCmCSA, using the ancestral SARS-CoV-2 spike sequence, optimized 5' and 3' untranslated regions (UTRs), and LNP combinations. Our data showed that these nanocomplexes effectively activate CD4+ and CD8+ T cell responses and humoral immune response and provide complete protection against WA1/2020, Omicron BA.1 and BQ.1 infection in hamsters. Critically, in Omicron BQ.1 challenge hamster models, TU88mCSA and ALCmCSA not only induced robust control of virus load in the lungs but also enhanced protective efficacy in the upper respiratory airways. Antigen-specific immune analysis in mice revealed that the observed cross-protection is associated with superior UTRs [Carboxylesterase 1d (Ces1d)/adaptor protein-3ß (AP3B1)] and LNP formulations that elicit robust lung tissue-resident memory T cells. Strong protective effects of TU88mCSA or ALCmCSA against both WA1/2020 and VOCs suggest that this mRNA-LNP combination can be a broadly protective vaccine platform in which mRNA cargo uses the ancestral antigen sequence regardless of the antigenic drift. This approach could be rapidly adapted for clinical use and timely deployment of vaccines against emerging and reemerging VOCs.

IGFBPL1 is a master driver of microglia homeostasis and resolution of neuroinflammation in glaucoma and brain tauopathy.

Microglia shift toward an inflammatory phenotype during aging that is thought to exacerbate age-related neurodegeneration. The molecular and cellular signals that resolve neuroinflammation post-injury are largely undefined. Here, we exploit systems genetics methods based on the extended BXD murine reference family and identify IGFBPL1 as an upstream cis-regulator of microglia-specific genes to switch off inflammation. IGFBPL1 is expressed by mouse and human microglia, and higher levels of its expression resolve lipopolysaccharide-induced neuroinflammation by resetting the transcriptome signature back to a homeostatic state via IGF1R signaling. Conversely, IGFBPL1 deficiency or selective deletion of IGF1R in microglia shifts these cells to an inflammatory landscape and induces early manifestation of brain tauopathy and retinal neurodegeneration. Therapeutic administration of IGFBPL1 drives pro-homeostatic microglia and prevents glaucomatous neurodegeneration and vision loss in mice. These results identify IGFBPL1 as a master driver of the counter-inflammatory microglial modulator that presents an endogenous resolution of neuroinflammation to prevent neurodegeneration in eye and brain.

Mitoxantrone targets both host and bacteria to overcome vancomycin resistance in Enterococcus faecalis.

Antibiotic resistance critically limits treatment options for infection caused by opportunistic pathogens such as enterococci. Here, we investigate the antibiotic and immunological activity of the anticancer agent mitoxantrone (MTX) in vitro and in vivo against vancomycin-resistant Enterococcus faecalis (VRE). We show that, in vitro, MTX is a potent antibiotic against Gram-positive bacteria through induction of reactive oxygen species and DNA damage. MTX also synergizes with vancomycin against VRE, rendering the resistant strains more permeable to MTX. In a murine wound infection model, single-dose MTX treatment effectively reduces VRE numbers, with further reduction when combined with vancomycin. Multiple MTX treatments accelerate wound closure. MTX also promotes macrophage recruitment and proinflammatory cytokine induction at the wound site and augments intracellular bacterial killing in macrophages by up-regulating the expression of lysosomal enzymes. These results show that MTX represents a promising bacterium- and host-targeted therapeutic for overcoming vancomycin resistance.

Pharmacological functions of salidroside in renal diseases: facts and perspectives.

Rhodiola rosea is a valuable functional medicinal plant widely utilized in China and other Asian countries for its anti-fatigue, anti-aging, and altitude sickness prevention properties. Salidroside, a most active constituent derived from Rhodiola rosea, exhibits potent antioxidative, hypoxia-resistant, anti-inflammatory, anticancer, and anti-aging effects that have garnered significant attention. The appreciation of the pharmacological role of salidroside has burgeoned over the last decade, making it a beneficial option for the prevention and treatment of multiple diseases, including atherosclerosis, Alzheimer's disease, Parkinson's disease, cardiovascular disease, and more. With its anti-aging and renoprotective effects, in parallel with the inhibition of oxidative stress and inflammation, salidroside holds promise as a potential therapeutic agent for kidney damage. This article provides an overview of the microinflammatory state in kidney disease and discuss the current therapeutic strategies, with a particular focus on highlighting the recent advancements in utilizing salidroside for renal disease. The potential mechanisms of action of salidroside are primarily associated with the regulation of gene and protein expression in glomerular endothelial cells, podocytes, renal tubule cells, renal mesangial cells and renal cell carcinoma cell, including TNF-α, TGF-ß, IL-1ß, IL-17A, IL-6, MCP-1, Bcl-2, VEGF, ECM protein, caspase-3, HIF-1α, BIM, as well as the modulation of AMPK/SIRT1, Nrf2/HO-1, Sirt1/PGC-1α, ROS/Src/Cav-1, Akt/GSK-3ß, TXNIP-NLRP3, ERK1/2, TGF-ß1/Smad2/3, PI3K/Akt, Wnt1/Wnt3a ß-catenin, TLR4/NF-κB, MAPK, JAK2/STAT3, SIRT1/Nrf2 pathways. To the best of our knowledge, this review is the first to comprehensively cover the protective effects of salidroside on diverse renal diseases, and suggests that salidroside has great potential to be developed as a drug for the prevention and treatment of metabolic syndrome, cardiovascular and cerebrovascular diseases and renal complications.

Selective expression of variant surface antigens enables Plasmodium falciparum to evade immune clearance in vivo.

Plasmodium falciparum has developed extensive mechanisms to evade host immune clearance. Currently, most of our understanding is based on in vitro studies of individual parasite variant surface antigens and how this relates to the processes in vivo is not well-understood. Here, we have used a humanized mouse model to identify parasite factors important for in vivo growth. We show that upregulation of the specific PfEMP1, VAR2CSA, provides the parasite with protection from macrophage phagocytosis and clearance in the humanized mice. Furthermore, parasites adapted to thrive in the humanized mice show reduced NK cell-mediated killing through interaction with the immune inhibitory receptor, LILRB1. Taken together, these findings reveal new insights into the molecular and cellular mechanisms that the parasite utilizes to coordinate immune escape in vivo. Identification and targeting of these specific parasite variant surface antigens crucial for immune evasion provides a unique approach for therapy.

Memory-like NK cells armed with a neoepitope-specific CAR exhibit potent activity against NPM1 mutated acute myeloid leukemia.

Acute myeloid leukemia (AML) remains a therapeutic challenge, and a paucity of tumor-specific targets has significantly hampered the development of effective immune-based therapies. Recent paradigm-changing studies have shown that natural killer (NK) cells exhibit innate memory upon brief activation with IL-12 and IL-18, leading to cytokine-induced memory-like (CIML) NK cell differentiation. CIML NK cells have enhanced antitumor activity and have shown promising results in early phase clinical trials in patients with relapsed/refractory AML. Here, we show that arming CIML NK cells with a neoepitope-specific chimeric antigen receptor (CAR) significantly enhances their antitumor responses to nucleophosphmin-1 (NPM1)-mutated AML while avoiding off-target toxicity. CIML NK cells differentiated from peripheral blood NK cells were efficiently transduced to express a TCR-like CAR that specifically recognizes a neoepitope derived from the cytosolic oncogenic NPM1-mutated protein presented by HLA-A2. These CAR CIML NK cells displayed enhanced activity against NPM1-mutated AML cell lines and patient-derived leukemic blast cells. CAR CIML NK cells persisted in vivo and significantly improved AML outcomes in xenograft models. Single-cell RNA sequencing and mass cytometry analyses identified up-regulation of cell proliferation, protein folding, immune responses, and major metabolic pathways in CAR-transduced CIML NK cells, resulting in tumor-specific, CAR-dependent activation and function in response to AML target cells. Thus, efficient arming of CIML NK cells with an NPM1-mutation-specific TCR-like CAR substantially improves their innate antitumor responses against an otherwise intracellular mutant protein. These preclinical findings justify evaluating this approach in clinical trials in HLA-A2+ AML patients with NPM1c mutations.

Ovarian Cancer Ascites Inhibits Transcriptional Activation of NK Cells Partly through CA125.

Malignant ascites is a common clinical problem in ovarian cancer. NK cells are present in the ascites, but their antitumor activity is inhibited. The underlying mechanisms of the inhibition have yet to be fully elucidated. Using an Fcγ receptor-mediated NK cell activation assay, we show that ascites from ovarian cancer patients potently inhibits NK cell activation. Part of the inhibitory activity is mediated by CA125, a mucin 16 fragment shed from ovarian cancer tumors. Moreover, transcriptional analyses by RNA sequencing reveal upregulation of genes involved in multiple metabolic pathways but downregulation of genes involved in cytotoxicity and signaling pathways in NK cells purified from ovarian cancer patient ascites. Transcription of genes involved in cytotoxicity pathways are also downregulated in NK cells from healthy donors after in vitro treatment with ascites or with a CA125-enriched protein fraction. These results show that ascites and CA125 inhibit antitumor activity of NK cells at transcriptional levels by suppressing expression of genes involved in NK cell activation and cytotoxicity. Our findings shed light on the molecular mechanisms by which ascites inhibits the activity of NK cells and suggest possible approaches to reactivate NK cells for ovarian cancer immunotherapy.

Current Development and Challenges of Tetravalent Live-Attenuated Dengue Vaccines.

Dengue is the most common arboviral disease caused by one of four distinct but closely related dengue viruses (DENV) and places significant economic and public health burdens in the endemic areas. A dengue vaccine will be important in advancing disease control. However, the effort has been challenged by the requirement to induce effective protection against all four DENV serotypes and the potential adverse effect due to the phenomenon that partial immunity to DENV may worsen the symptoms upon subsequent heterotypic infection. Currently, the most advanced dengue vaccines are all tetravalent and based on recombinant live attenuated viruses. CYD-TDV, developed by Sanofi Pasteur, has been approved but is limited for use in individuals with prior dengue infection. Two other tetravalent live attenuated vaccine candidates: TAK-003 by Takeda and TV003 by National Institute of Allergy and Infectious Diseases, have completed phase 3 and phase 2 clinical trials, respectively. This review focuses on the designs and evaluation of TAK-003 and TV003 vaccine candidates in humans in comparison to the licensed CYD-TDV vaccine. We highlight specific lessons from existing studies and challenges that must be overcome in order to develop a dengue vaccine that confers effective and balanced protection against all four DENV serotypes but with minimal adverse effects.

Expansion, persistence, and efficacy of donor memory-like NK cells infused for posttransplant relapse.

BackgroundResponses to conventional donor lymphocyte infusion for postallogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell-based therapy is a promising modality to treat post-HCT relapse.MethodsWe initiated this ongoing phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5 to 10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High-resolution profiling with mass cytometry and single-cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion.ResultsIn the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires.ConclusionGiven their rapid expansion and long-term persistence in an immune-compatible environment, CIML NK cells serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies.Trial RegistrationClinicalTrials.gov NCT04024761.FundingDunkin' Donuts, NIH/National Cancer Institute, and the Leukemia and Lymphoma Society.

Current Developments and Challenges of mRNA Vaccines.

mRNA vaccines have brought about a great revolution in the vaccine fields owing to their simplicity and adaptability in antigen design, potential to induce both humoral and cell-mediated immune responses and demonstrated high efficacy, and rapid and low-cost production by using the same manufacturing platform for different mRNA vaccines. Multiple mRNA vaccines have been investigated for both infectious diseases and cancers, showing significant superiority to other types of vaccines. Although great success of mRNA vaccines has been achieved in the control of the coronavirus disease 2019 pandemic, there are still multiple challenges for the future development of mRNA vaccines. In this review, the most recent developments of mRNA vaccines against both infectious diseases and cancers are summarized for an overview of this field. Moreover, the challenges are also discussed on the basis of these developments.

In Vitro Engineering Chimeric Antigen Receptor Macrophages and T Cells by Lipid Nanoparticle-Mediated mRNA Delivery.

Chimeric antigen receptor (CAR)-engineered adoptive cell therapy marks a revolution in cancer treatment based on the highly successful responses to CAR T cell therapy in the treatment of blood cancers. Due to the versatile structure of CARs, this technology can be easily adapted to other immune cell types, including macrophages and NKs, and applied in the treatment of many other cancers. However, high costs and fatal adverse effects represent significant concerns for future development. In vitro transcribed (IVT) mRNA therapeutics, which possess a high safety profile and straightforward production methods, could provide a useful alternative for CAR cell construction. However, the low stability and transfection efficiency of IVT-mRNA in immune cells limit further applications. In this work, we successfully engineered CAR macrophages (CAR-Ms) and CAR T cells with CAR mRNA using lipid nanoparticles (LNPs). Both the LNP formulations and mRNA modifications were optimized for in vitro mRNA transfection. More importantly, the CAR macrophages and CAR T cells both demonstrated significant cytotoxic effects on B lymphoma in vitro, underscoring the great potential of mRNA-engineered adoptive cell therapy.

In situ cancer vaccination using lipidoid nanoparticles.

In situ vaccination is a promising strategy for cancer immunotherapy owing to its convenience and the ability to induce numerous tumor antigens. However, the advancement of in situ vaccination techniques has been hindered by low cross-presentation of tumor antigens and the immunosuppressive tumor microenvironment. To balance the safety and efficacy of in situ vaccination, we designed a lipidoid nanoparticle (LNP) to achieve simultaneously enhancing cross-presentation and STING activation. From combinatorial library screening, we identified 93-O17S-F, which promotes both the cross-presentation of tumor antigens and the intracellular delivery of cGAMP (STING agonist). Intratumor injection of 93-O17S-F/cGAMP in combination with pretreatment with doxorubicin exhibited excellent antitumor efficacy, with 35% of mice exhibiting total recovery from a primary B16F10 tumor and 71% of mice with a complete recovery from a subsequent challenge, indicating the induction of an immune memory against the tumor. This study provides a promising strategy for in situ cancer vaccination.

High-throughput phenotypic screen and transcriptional analysis identify new compounds and targets for macrophage reprogramming.

Macrophages are plastic and, in response to different local stimuli, can polarize toward multi-dimensional spectrum of phenotypes, including the pro-inflammatory M1-like and the anti-inflammatory M2-like states. Using a high-throughput phenotypic screen in a library of ~4000 FDA-approved drugs, bioactive compounds and natural products, we find ~300 compounds that potently activate primary human macrophages toward either M1-like or M2-like state, of which ~30 are capable of reprogramming M1-like macrophages toward M2-like state and another ~20 for the reverse repolarization. Transcriptional analyses of macrophages treated with 34 non-redundant compounds identify both shared and unique targets and pathways through which the tested compounds modulate macrophage activation. One M1-activating compound, thiostrepton, is able to reprogram tumor-associated macrophages toward M1-like state in mice, and exhibit potent anti-tumor activity. Our compound-screening results thus help to provide a valuable resource not only for studying the macrophage biology but also for developing therapeutics through modulating macrophage activation.

CAR-T cells targeting a nucleophosmin neoepitope exhibit potent specific activity in mouse models of acute myeloid leukaemia.

Therapies employing chimeric antigen receptor T cells (CAR-T cells) targeting tumour-associated antigens (TAAs) can lead to on-target-off-tumour toxicity and to resistance, owing to TAA expression in normal tissues and to TAA expression loss in tumour cells. These drawbacks can be circumvented by CAR-T cells targeting tumour-specific driver gene mutations, such as the four-nucleotide duplication in the oncogene nucleophosmin (NPM1c), which creates a neoepitope presented by the human leukocyte antigen with the A2 serotype (HLA-A2) that has been observed in about 35% of patients with acute myeloid leukaemia (AML). Here, we report a human single-chain variable fragment (scFv), identified via yeast surface display, that specifically binds to the NPM1c epitope-HLA-A2 complex but not to HLA-A2 or to HLA-A2 loaded with control peptides. In vitro and in mice, CAR-T cells with the scFv exhibit potent cytotoxicity against NPM1c+HLA-A2+ leukaemia cells and primary AML blasts, but not NPM1c-HLA-A2+ leukaemia cells or HLA-A2- tumour cells. Therapies using NPM1c CAR-T cells for the treatment of NPM1c+HLA-A2+ AML may limit on-target-off-tumour toxicity and tumour resistance.