iNKT cells are a subset of innate T cells that recognize glycolipids presented on CD1d molecules and protect against bacterial infections, including S. pneumoniae. Using lung intravital imaging, we examined the behavior and mechanism of pulmonary iNKT cell activation in response to the specific iNKT cell ligand α-galactosylceramide or S. pneumoniae infection. In untreated mice, the major fraction of iNKT cells resided in the vasculature, but a small critical population resided in the extravascular space in proximity to monocyte-derived DCs. Administration of either α-GalCer or S. pneumoniae induced CD1d-dependent rapid recruitment of neutrophils out of the vasculature. The neutrophils guided iNKT cells from the lung vasculature via CCL17. Depletion of monocyte-derived DCs abrogated both the neutrophil and subsequent iNKT cell extravasation. Moreover, impairing iNKT cell recruitment by blocking CCL17 increased susceptibility to S. pneumoniae infection, suggesting a critical role for the influx of iNKT cells in host defense.

Periostin (POSTN) is a multifunctional extracellular component that regulates cell-matrix interactions and cell-cell crosstalk. POSTN deletion significantly decreases leukemia burden in mice; however, the underlying mechanisms by which POSTN promotes B cell acute lymphoblastic leukemia (B-ALL) progression remain largely unknown. Here, we demonstrate that bone marrow (BM)-derived mesenchymal stromal cells (BM-MSCs) express higher levels of POSTN when co-cultured with B-ALL cells in vitro and in vivo. POSTN deficiency in BM-MSCs significantly decreases CCL2 expression in co-cultured B-ALL cells in vitro and in vivo. Moreover, POSTN treatment increases expression of CCL2 in B-ALL cells by activating the integrin-ILK-NF-κB pathway. Conversely, CCL2 treatment upregulates expression of POSTN in BM-MSCs via STAT3 activation. Furthermore, there is a positive correlation between POSTN expression and CCL2 level in the BM of mice and patients with B-ALL. These findings suggest that B-ALL cell-derived CCL2 contributes to the increased leukemia burden promoted by BM-MSC-derived POSTN.

Mutations in proline-rich transmembrane protein 2 (PRRT2) are associated with a range of paroxysmal neurological disorders. PRRT2 predominantly localizes to the pre-synaptic terminals and is believed to regulate neurotransmitter release. However, the mechanism of action is unclear. Here, we use reconstituted single vesicle and bulk fusion assays, combined with live cell imaging of single exocytotic events in PC12 cells and biophysical analysis, to delineate the physiological role of PRRT2. We report that PRRT2 selectively blocks the trans SNARE complex assembly and thus negatively regulates synaptic vesicle priming. This inhibition is actualized via weak interactions of the N-terminal proline-rich domain with the synaptic SNARE proteins. Furthermore, we demonstrate that paroxysmal dyskinesia-associated mutations in PRRT2 disrupt this SNARE-modulatory function and with efficiencies corresponding to the severity of the disease phenotype. Our findings provide insights into the molecular mechanisms through which loss-of-function mutations in PRRT2 result in paroxysmal neurological disorders.

Long noncoding RNAs (lncRNAs) participate in host antiviral defense by modulating immune responses. However, it remains largely unexplored how viruses exploit interferon (IFN)-independent host lncRNAs to facilitate viral replication. Here, we have identified a group of human lncRNAs that modulate influenza A virus (IAV) replication in a loss-of-function screen and found that an IFN-independent lncRNA, called IPAN, is hijacked by IAV to assist IAV replication. IPAN is specifically induced by IAV infection independently of IFN and associates with and stabilizes viral RNA-dependent RNA polymerase PB1, enabling efficient viral RNA synthesis. Silencing IPAN results in PB1 degradation and severely impairs viral infection. Therefore, our data unveil an important role of host lncRNAs in promoting viral replication by modulating viral protein stability. Our findings may open avenues to the development of antiviral therapeutics.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages have escaped most receptor-binding domain (RBD)-targeting therapeutic neutralizing antibodies (NAbs), which proves that previous NAb drug screening strategies are deficient against the fast-evolving SARS-CoV-2. Better broad NAb drug candidate selection methods are needed. Here, we describe a rational approach for identifying RBD-targeting broad SARS-CoV-2 NAb cocktails. Based on high-throughput epitope determination, we propose that broad NAb drugs should target non-immunodominant RBD epitopes to avoid herd-immunity-directed escape mutations. Also, their interacting antigen residues should focus on sarbecovirus conserved sites and associate with critical viral functions, making the antibody-escaping mutations less likely to appear. Following these criteria, a featured non-competing antibody cocktail, SA55+SA58, is identified from a large collection of broad sarbecovirus NAbs isolated from SARS-CoV-2-vaccinated SARS convalescents. SA55+SA58 potently neutralizes ACE2-utilizing sarbecoviruses, including circulating Omicron variants, and could serve as broad SARS-CoV-2 prophylactics to offer long-term protection, especially for individuals who are immunocompromised or with high-risk comorbidities.

Retinoic acid-inducible-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and cyclic GMP-AMP synthase (cGAS) genes encode essential cytosolic receptors mediating antiviral immunity against viruses. Here, we show that OTUD3 has opposing role in response to RNA and DNA virus infection by removing distinct types of RIG-I/MDA5 and cGAS polyubiquitination. OTUD3 binds to RIG-I and MDA5 and removes K63-linked ubiquitination. This serves to reduce the binding of RIG-I and MDA5 to viral RNA and the downstream adaptor MAVS, leading to the suppression of the RNA virus-triggered innate antiviral responses. Meanwhile, OTUD3 associates with cGAS and targets at Lys279 to deubiquitinate K48-linked ubiquitination, resulting in the enhancement of cGAS protein stability and DNA-binding ability. As a result, Otud3-deficient mice and zebrafish are more resistant to RNA virus infection but are more susceptible to DNA virus infection. These findings demonstrate that OTUD3 limits RNA virus-triggered innate immunity but promotes DNA virus-triggered innate immunity.

Systemic metabolites serving as danger-associated molecular patterns play crucial roles in modulating the development, differentiation, and activity of innate immune cells. Yet, it is unclear how innate immune cells detect systemic metabolites for signal transmission. Here, we show that bile acids function as endogenous mitofusin 2 (MFN2) ligands and differentially modulate innate immune response to bacterial infection under cholestatic and physiological conditions. Bile acids at high concentrations promote mitochondrial tethering to the endoplasmic reticulum (ER), leading to calcium overload in the mitochondrion, which activates NLRP3 inflammasome and pyroptosis. By contrast, at physiologically relevant low concentrations, bile acids promote mitochondrial fusion, leading to enhanced oxidative phosphorylation and thereby strengthening infiltrated macrophages mediated phagocytotic clearance of bacteria. These findings support that bile acids, as endogenous activators of MFN2, are vital for tuning innate immune responses against infections, representing a causal link that connects systemic metabolism with mitochondrial dynamics in shaping innate immunity.

Bearing a relatively large single-stranded RNA genome in nature, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes sophisticated replication/transcription complexes (RTCs), mainly composed of a network of nonstructural proteins and nucleocapsid protein, to establish efficient infection. In this study, we develop an innovative interaction screening strategy based on phase separation in cellulo, namely compartmentalization of protein-protein interactions in cells (CoPIC). Utilizing CoPIC screening, we map the interaction network among RTC-related viral proteins. We identify a total of 47 binary interactions among 14 proteins governing replication, discontinuous transcription, and translation of coronaviruses. Further exploration via CoPIC leads to the discovery of extensive ternary complexes composed of these components, which infer potential higher-order complexes. Taken together, our results present an efficient and robust interaction screening strategy, and they indicate the existence of a complex interaction network among RTC-related factors, thus opening up opportunities to understand SARS-CoV-2 biology and develop therapeutic interventions for COVID-19.

The chromatin-associated protein WDR5 is a promising pharmacological target in cancer, with most drug discovery efforts directed against an arginine-binding cavity in WDR5 called the WIN site. Despite a clear expectation that WIN site inhibitors will alter the repertoire of WDR5 interaction partners, their impact on the WDR5 interactome remains unknown. Here, we use quantitative proteomics to delineate how the WDR5 interactome is changed by WIN site inhibition. We show that the WIN site inhibitor alters the interaction of WDR5 with dozens of proteins, including those linked to phosphatidylinositol 3-kinase (PI3K) signaling. As proof of concept, we demonstrate that the master kinase PDPK1 is a bona fide high-affinity WIN site binding protein that engages WDR5 to modulate transcription of genes expressed in the G2 phase of the cell cycle. This dataset expands our understanding of WDR5 and serves as a resource for deciphering the action of WIN site inhibitors.

Lung cancer is a highly aggressive and metastatic disease responsible for approximately 25% of all cancer-related deaths in the United States. Using high-throughput in vitro and in vivo screens, we have previously established Impad1 as a driver of lung cancer invasion and metastasis. Here we elucidate that Impad1 is a direct target of the epithelial microRNAs (miRNAs) miR-200 and miR∼96 and is de-repressed during epithelial-to-mesenchymal transition (EMT); thus, we establish a mode of regulation of the protein. Impad1 modulates Golgi apparatus morphology and vesicular trafficking through its interaction with a trafficking protein, Syt11. These changes in Golgi apparatus dynamics alter the extracellular matrix and the tumor microenvironment (TME) to promote invasion and metastasis. Inhibiting Impad1 or Syt11 disrupts the cancer cell secretome, regulates the TME, and reverses the invasive or metastatic phenotype. This work identifies Impad1 as a regulator of EMT and secretome-mediated changes during lung cancer progression.

The regulation of antiviral immunity is crucial in maintaining host immune homeostasis, a process that involves dynamic modulations of host organelles. The Golgi apparatus is increasingly perceived as a host organelle functioning as a critical platform for innate immunity, but the detailed mechanism by which it regulates antiviral immunity remains elusive. Here, we identify the Golgi-localized G protein-coupled receptor 108 (GPR108) as a regulator of type Ι interferon responses by targeting interferon regulatory factor 3 (IRF3). Mechanistically, GPR108 enhances the ubiquitin ligase Smad ubiquitylation regulatory factor 1 (Smurf1)-mediated K63-linked polyubiquitination of phosphorylated IRF3 for nuclear dot 10 protein 52 (NDP52)-dependent autophagic degradation, leading to suppression of antiviral immune responses against DNA or RNA viruses. Taken together, our study provides insight into the crosstalk between the Golgi apparatus and antiviral immunity via a dynamic and spatiotemporal regulation of GPR108-Smurf1 axis, thereby indicating a potential target for treating viral infection.

Leukemic-stem-cell-specific targeting may improve the survival of patients with acute myeloid leukemia (AML) by avoiding the ablative effects of standard regimens on normal hematopoiesis. Herein, we perform an unbiased screening of compounds targeting cell surface proteins and identify clinically used DPP4 inhibitors as strong suppressors of AML development in both murine AML models and primary human AML cells xenograft model. We find in retrovirus-induced AML mouse models that DPP4-deficient AML cell-transplanted mice exhibit delay and reversal of AML development, whereas deletion of DPP4 has no significant effect on normal hematopoiesis. DPP4 activates and sustains survival of AML stem cells that are critical for AML development in both human and animal models via binding with Src kinase and activation of nuclear factor κB (NF-κB) signaling. Thus, inhibition of DPP4 is a potential therapeutic strategy against AML development through suppression of survival and stemness of AML cells.

Cancer progresses through distinct stages, and mouse models recapitulating traits of this progression are frequently used to explore genetic, morphological, and pharmacological aspects of tumor development. To complement genomic investigations of this process, we here quantify phosphoproteomic changes in skin cancer development using the SILAC mouse technology coupled to high-resolution mass spectrometry. We distill protein expression signatures from our data that distinguish between skin cancer stages. A distinct phosphoproteome of the two stages of cancer progression is identified that correlates with perturbed cell growth and implicates cell adhesion as a major driver of malignancy. Importantly, integrated analysis of phosphoproteomic data and prediction of kinase activity revealed PAK4-PKC/SRC network to be highly deregulated in SCC but not in papilloma. This detailed molecular picture, both at the proteome and phosphoproteome level, will prove useful for the study of mechanisms of tumor progression.

Projections from sensory neurons of olfactory systems coalesce into glomeruli in the brain. The Kirrel receptors are believed to homodimerize via their ectodomains and help separate sensory neuron axons into Kirrel2- or Kirrel3-expressing glomeruli. Here, we present the crystal structures of homodimeric Kirrel receptors and show that the closely related Kirrel2 and Kirrel3 have evolved specific sets of polar and hydrophobic interactions, respectively, disallowing heterodimerization while preserving homodimerization, likely resulting in proper segregation and coalescence of Kirrel-expressing axons into glomeruli. We show that the dimerization interface at the N-terminal immunoglobulin (IG) domains is necessary and sufficient to create homodimers and fail to find evidence for a secondary interaction site in Kirrel ectodomains. Furthermore, we show that abolishing dimerization of Kirrel3 in vivo leads to improper formation of glomeruli in the mouse accessory olfactory bulb as observed in Kirrel3-/- animals. Our results provide evidence for Kirrel3 homodimerization controlling axonal coalescence.

Various culture conditions by small molecules have been explored to extend pluripotency of stem cells, but their impacts on cell fate in vivo remain elusive. We systematically compared the effects of various culture conditions on the pluripotency and cell fate in vivo of mouse embryonic stem cells (ESCs) by tetraploid embryo complementation assay. Conventional ESC cultures in serum/LIF-based condition produced complete ESC mice and also the survival to adulthood at the highest rates of all other chemical-based cultures. Moreover, long-term examination of the survived ESC mice demonstrated that conventional ESC cultures did not lead to visible abnormality for up to 1.5-2 years, whereas the prolonged chemical-based cultures developed retroperitoneal atypical teratomas or leiomyomas. The chemical-based cultures exhibited transcriptomes and epigenomes that typically differed from those of conventional ESC cultures. Our results warrant further refinement of culture conditions in promoting the pluripotency and safety of ESCs in future applications.

Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smoking and/or human papillomavirus (HPV). SCCs harbor 3q, 5p, and other recurrent chromosomal copy-number alterations (CNAs), DNA mutations, and/or aberrant methylation of genes and microRNAs, which are correlated with the expression of multi-gene programs linked to squamous cell stemness, epithelial-to-mesenchymal differentiation, growth, genomic integrity, oxidative damage, death, and inflammation. Low-CNA SCCs tended to be HPV(+) and display hypermethylation with repression of TET1 demethylase and FANCF, previously linked to predisposition to SCC, or harbor mutations affecting CASP8, RAS-MAPK pathways, chromatin modifiers, and immunoregulatory molecules. We uncovered hypomethylation of the alternative promoter that drives expression of the ΔNp63 oncogene and embedded miR944. Co-expression of immune checkpoint, T-regulatory, and Myeloid suppressor cells signatures may explain reduced efficacy of immune therapy. These findings support possibilities for molecular classification and therapeutic approaches.

N6-methyladenosine (m6A) modification has been implicated in many cell processes and diseases. YTHDF1, a translation-facilitating m6A reader, has not been previously shown to be related to allergic airway inflammation. Here, we report that YTHDF1 is highly expressed in allergic airway epithelial cells and asthmatic patients and that it influences proinflammatory responses. CLOCK, a subunit of the circadian clock pathway, is the direct target of YTHDF1. YTHDF1 augments CLOCK translation in an m6A-dependent manner. Allergens enhance the liquid-liquid phase separation (LLPS) of YTHDF1 and drive the formation of a complex comprising dimeric YTHDF1 and CLOCK mRNA, which is distributed to stress granules. Moreover, YTHDF1 strongly activates NLRP3 inflammasome production and interleukin-1β secretion leading to airway inflammatory responses, but these phenotypes are abolished by deleting CLOCK. These findings demonstrate that YTHDF1 is an important regulator of asthmatic airway inflammation, suggesting a potential therapeutic target for allergic airway inflammation.

NAD+ metabolism is involved in many biological processes. However, the underlying mechanism of how NAD+ metabolism is regulated remains elusive. Here, we find that PTIP governs NAD+ metabolism in macrophages by regulating CD38 expression and is required for macrophage inflammation. Through integrating histone modifications with NAD+ metabolic gene expression profiling, we identify PTIP as a key factor in regulating CD38 expression, the primary NAD+-consuming enzyme in macrophages. Interestingly, we find that PTIP deletion impairs the proinflammatory response of primary murine and human macrophages, promotes their metabolic switch from glycolysis to oxidative phosphorylation, and alters NAD+ metabolism via downregulating CD38 expression. Mechanistically, an intronic enhancer of CD38 is identified. PTIP regulates CD38 expression by cooperating with acetyltransferase p300 in establishing the CD38 active enhancer with enriched H3K27ac. Overall, our findings reveal a critical role for PTIP in fine-tuning the inflammatory responses of macrophages via regulating NAD+ metabolism.

Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.