Realveolarization with inhalable mucus-penetrating lipid nanoparticles for the treatment of pulmonary fibrosis in mice.

The stemness loss-associated dysregeneration of impaired alveolar type 2 epithelial (AT2) cells abolishes the reversible therapy of idiopathic pulmonary fibrosis (IPF). We here report an inhalable mucus-penetrating lipid nanoparticle (LNP) for codelivering dual mRNAs, promoting realveolarization via restoring AT2 stemness for IPF treatment. Inhalable LNPs were first formulated with dipalmitoylphosphatidylcholine and our in-house-made ionizable lipids for high-efficiency pulmonary mucus penetration and codelivery of dual messenger RNAs (mRNAs), encoding cytochrome b5 reductase 3 and bone morphogenetic protein 4, respectively. After being inhaled in a bleomycin model, LNPs reverses the mitochondrial dysfunction through ameliorating nicotinamide adenine dinucleotide biosynthesis, which inhibits the accelerated senescence of AT2 cells. Concurrently, pathological epithelial remodeling and fibroblast activation induced by impaired AT2 cells are terminated, ultimately prompting alveolar regeneration. Our data demonstrated that the mRNA-LNP system exhibited high protein expression in lung epithelial cells, which markedly extricated the alveolar collapse and prolonged the survival of fibrosis mice, providing a clinically viable strategy against IPF.

Stimulation of tumoricidal immunity via bacteriotherapy inhibits glioblastoma relapse.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor characterized by invasive behavior and a compromised immune response, presenting treatment challenges. Surgical debulking of GBM fails to address its highly infiltrative nature, leaving neoplastic satellites in an environment characterized by impaired immune surveillance, ultimately paving the way for tumor recurrence. Tracking and eradicating residual GBM cells by boosting antitumor immunity is critical for preventing postoperative relapse, but effective immunotherapeutic strategies remain elusive. Here, we report a cavity-injectable bacterium-hydrogel superstructure that targets GBM satellites around the cavity, triggers GBM pyroptosis, and initiates innate and adaptive immune responses, which prevent postoperative GBM relapse in male mice. The immunostimulatory Salmonella delivery vehicles (SDVs) engineered from attenuated Salmonella typhimurium (VNP20009) seek and attack GBM cells. Salmonella lysis-inducing nanocapsules (SLINs), designed to trigger autolysis, are tethered to the SDVs, eliciting antitumor immune response through the intracellular release of bacterial components. Furthermore, SDVs and SLINs administration via intracavitary injection of the ATP-responsive hydrogel can recruit phagocytes and promote antigen presentation, initiating an adaptive immune response. Therefore, our work offers a local bacteriotherapy for stimulating anti-GBM immunity, with potential applicability for patients facing malignancies at a high risk of recurrence.

Intracavitary Spraying of Nanoregulator-Encased Hydrogel Modulates Cholesterol Metabolism of Glioma-Supportive Macrophage for Postoperative Glioblastoma Immunotherapy.

Glioblastoma multiforme (GBM) is notoriously resistant to immunotherapy due to its intricate immunosuppressive tumor microenvironment (TME). Dysregulated cholesterol metabolism is implicated in the TME and promotes tumor progression. Here, it is found that cholesterol levels in GBM tissues are abnormally high, and glioma-supportive macrophages (GSMs), an essential "cholesterol factory", demonstrate aberrantly hyperactive cholesterol metabolism and efflux, providing cholesterol to fuel GBM growth and induce CD8+ T cells exhaustion. Bioinformatics analysis confirms that high 7-dehydrocholesterol reductase (DHCR7) level in GBM tissues associates with increased cholesterol biosynthesis, suppressed tumoricidal immune response, and poor patient survival, and DHCR7 expression level is significantly elevated in GSMs. Therefore, an intracavitary sprayable nanoregulator (NR)-encased hydrogel system to modulate cholesterol metabolism of GSMs is reported. The degradable NR-mediated ablation of DHCR7 in GSMs effectively suppresses cholesterol supply and activates T-cell immunity. Moreover, the combination of Toll-like receptor 7/8 (TLR7/8) agonists significantly promotes GSM polarization to antitumor phenotypes and ameliorates the TME. Treatment with the hybrid system exhibits superior antitumor effects in the orthotopic GBM model and postsurgical recurrence model. Altogether, the findings unravel the role of GSMs DHCR7/cholesterol signaling in the regulation of TME, presenting a potential treatment strategy that warrants further clinical trials.

Reversing immune evasion using a DNA nano-orchestrator for pancreatic cancer immunotherapy.

Immune evasion caused by the paucity of MHCI is a prominent characteristic of pancreatic adenocarcinoma (PAAD), which is thought to underlie dysfunctional even absent adaptive T cell immunity and is responsible for ineffective immunotherapy. Here, we report a ROS-responsive DNA nano-orchestrator to cascade reverse MHC I-associated immune evasion and boost anti-tumor T cell stimulation, stimulating the activation of tumoricidal immunity against PAAD. Chloroquine phosphate (CQP) as an autophagy inhibitor was first encapsulated with ferritin, and via DNA modular self-assembly technology, the generated ferritin nanocores (FNC) were then caged into ROS-responsive CpG-DNA nanoframe. After systemic injection, the FNC-laden DNA nanoframe (FNC@NF) was passively enriched in tumor tissues in which the DNA nanoframe was cleaved upon the ROS stimulation. Oligodeoxynucleotide (ODN) with CpG motifs was detached and functioned as a TLR9 agonist. The liberated FNC was then endocytosed in an actively targeted manner by binding to transferrin receptor 1. In the lysosome, CQP was burst released from FNC due to acid-triggering. Through CQP-mediated autophagy abrogation, MHC-I molecules were preserved. We demonstrated that cascade inhibiting autophagy and boosting TLR9 stimulation via our proposed DNA-based hybrid nanosystem restored MHC I on the tumor cell surface and reshaped the antigen presentation of DCs, and ultimately reversed immune evasion and synergistically reinforced the activation of cytotoxic T cells against PAAD cells. In sum, our work provides an alternative strategy for cascade reversing immune evasion and boosting anti-tumor T cell stimulation and holds great potential for pancreatic cancer immunotherapy. STATEMENT OF SIGNIFICANCE: A DNA nano-orchestrator was created by sequentially assembling chloroquine phosphate-laden ferritin nanocores with ROS-responsive CpG-DNA nanoframe. Through cascade inhibiting autophagy and boosting TLR9 stimulation, the nano-orchestrator efficiently reversed MHC I-associated immune evasion and augmented anti-tumor T cell stimulation, which ultimately activated tumoricidal immunity against pancreatic adenocarcinoma.

Convection-enhanced delivery of nanoencapsulated gene locoregionally yielding ErbB2/Her2-specific CAR-macrophages for brainstem glioma immunotherapy.

Locoregional delivery of chimeric antigen receptor (CAR)-modified T (CAR-T) cells has emerged as a promising strategy for brain tumors. However, the complicated ex vivo cell manufacturing procedures and the rapid progression of the disease have limited its broader applications. Macrophages (MΦs) exhibit unique effector functions and a high degree of infiltration within the solid tumor microenvironment (TME), especially in the brain, where MΦs function as structural support, and the main immune effector cells of the CNS represent 5-12% of brain cells. Here, we report a synthetic universal DNA nanocarrier for in situ genetic editing of intratumoral MΦs with an ErbB2-specific CAR to direct their phagocytic activity towards tumors and subsequently initiate a locoregional antitumor immune response. Specifically, we demonstrated that when delivered locoregionally, the RP-182 peptide, located in the shell of a nanoparticle, targeted MΦs and reprogrammed M2-like tumor-associated macrophages (TAMs) to an antitumor M1-like phenotype. Subsequently, the CAR gene-laden DNA nanocomplex can be used to introduce ErbB2-targeted CAR, and the generated CAR-MΦs then act as "living" cures, thereby serially clearing the invasive tumor cells. Our work demonstrates a practical antitumor immunotherapy for brainstem gliomas (BSGs) that may be broadly applicable for patients suffering from other ErbB2-positive solid malignancies.

Enterovirus 71 Antagonizes Antiviral Effects of Type III Interferon and Evades the Clearance of Intestinal Intraepithelial Lymphocytes.

Enterovirus 71 (EV71) is the major pathogen causing severe neurological complications and hand, foot, and mouth disease. The intestinal mucosal immune system has a complete immune response and immune regulation mechanism, consisting of densely arranged monolayer intestinal epithelial cells (IECs) and intestinal intraepithelial lymphocytes (iIELs) distributed among the IECs, which constitute the first line of intestinal mucosa against infection of foreign pathogens. As an enterovirus, EV71 is transmitted by the intestinal tract; however, the mechanisms it uses to evade the immunosurveillance of the intestinal mucosal immune system are still incompletely clarified. The present study investigated how EV71 evades from recognizing and eliminating IECs, iIELs, and iNK cells. We found that EV71 infection induced a higher level of type III interferons (IFN-λ) than type I interferons (IFN-ß) in IECs, and the addition of IFN-λ markedly restricted EV71 replication in IECs. These results indicate that IFN-λ plays a more important role in anti-EV71 intestinal infection. However, EV71 infection could markedly attenuate the antiviral responses of IFN-λ. Mechanistically, 2A protease (2Apro) and 3C protease (3Cpro) of EV71 inhibited the IFN-λ production and IFN-λ receptor expression and further decreased the response of IECs to IFN-λ. In addition, we found that EV71-infected IECs were less susceptible to the lysis of intestinal NK (iNK) cells and CD3+iIELs. We revealed that the viral 2Apro and 3Cpro could significantly reduce the expression of the ligands of natural killer group 2D (NKG2D) and promote the expression of PD-L1 on IECs, rendering them to evade the recognition and killing of iNK and CD3+iIELs. These results provide novel evasion mechanisms of EV71 from intestinal mucosal innate immunity and may give new insights into antiviral therapy.