Biology subjects

Hsu, A. Y.

Publications and source records attributed to Hsu, A. Y..

7 recordsLinked to original sources

bioRxiv2026

S1PR1 signaling biases neutrophils toward long-lived low-inflammatory functional states

Sphingosine 1-phosphate (S1P), a lipid mediator that signals through five G protein-coupled S1P receptors (S1PRs), regulates T cell trafficking, tissue residency, and inflammatory processes. In contrast to the established roles of S1P signaling in T cell trafficking, its role in neutrophil biology remains poorly understood. Here, we demonstrate that S1PR1, one of the two S1PRs expressed in neutrophils, promotes mitochondrial fitness, enhances survival, and reduces inflammatory output. Using myeloid- and neutrophil-selective S1PR1 overexpression (S1PR1hi) mouse models, we show that elevated S1PR1 signaling promotes redistribution of neutrophils from the bone marrow to peripheral tissues under steady-state conditions, without inducing overt inflammation or tissue injury. S1PR1hi neutrophils display altered surface marker profiles consistent with a less mature state. These cells also exhibit reduced in vivo turnover, increased mitochondrial membrane potential and oxidative phosphorylation, and transcriptional programs linked to survival and dampened inflammatory signaling. Functionally, S1PR1hi neutrophils exhibit a reduced oxidative burst while preserving phagocytic capacity. However, in vivo bacterial challenge revealed impaired bacterial clearance in the lung. In contrast, in a model of influenza A virus infection of the lung, enhanced neutrophil-intrinsic S1PR1 signaling conferred reduced lung injury, decreased inflammatory output, and improved survival. Together, these findings support a model in which S1PR1 reprograms neutrophils, enabling their survival and dampening inflammatory potential in a context-dependent manner, thereby differentially shaping host defense and tissue protection during microbial infections.

immunology↗

bioRxiv2023

Palmitoylation of gasdermin D directs its membrane translocation and pore formation in pyroptosis

Gasdermin D (GSDMD)-mediated macrophage pyroptosis plays a critical role in inflammation and host defense. Plasma membrane perforation elicited by caspase-cleaved GSDMD N-terminal domain (GSDMD-NT) triggers membrane rupture and subsequent pyroptotic cell death, resulting in release of pro-inflammatory IL-1{beta} and IL-18. However, the biological processes leading to its membrane translocation and pore formation are not fully understood. Here, using a proteomics approach, we identified fatty acid synthase (FASN) as a GSDMD-binding partner and demonstrated that post-translational palmitoylation of GSDMD at Cys191/Cys192 (human/mouse) led to membrane translocation of GSDMD-NT but not full-length GSDMD. GSDMD lipidation, mediated by palmitoyl acyltransferases ZDHHC5/9 and facilitated by LPS-induced reactive oxygen species (ROS), was essential for GSDMD pore-forming activity and pyroptosis. Inhibition of GSDMD palmitoylation with palmitate analog 2-bromopalmitate or a cell permeable GSDMD-specific competing peptide suppressed pyroptosis and IL-1{beta} release in macrophages, mitigated organ damage, and extended the survival of septic mice. Collectively, we establish GSDMD-NT palmitoylation as a key regulatory mechanism controlling GSDMD membrane localization and activation, providing a novel target for modulating immune activity in infectious and inflammatory diseases. One Sentence SummaryLPS-induced palmitoylation at Cys191/Cys192 is required for GSDMD membrane translocation and its pore-forming activity in macrophages.

immunology↗

bioRxiv2021

RORA regulates neutrophil migration and activation in zebrafish

Neutrophil migration and activation are essential for defense against pathogens. However, this process may also lead to collateral tissue injury. We used microRNA overexpression as a platform and discovered protein-coding genes that regulate neutrophil migration. Here we show that miR-99 decreased the chemotaxis of zebrafish neutrophils and human neutrophil-like cells. In zebrafish neutrophils, miR-99 directly targets the transcriptional factor RAR-related orphan receptor alpha (roraa). Inhibiting ROR, but not the closely related ROR{gamma}, reduced chemotaxis of zebrafish and primary human neutrophils without causing cell death, and increased susceptibility of zebrafish to bacterial infection. Expressing a dominant-negative form of Ror or disrupting the roraa locus specifically in zebrafish neutrophils reduced cell migration. At the transcriptional level, ROR regulates transmembrane signaling receptor activity and protein phosphorylation pathways. Our results, therefore, reveal previously unknown functions of miR- 99 and ROR in regulating neutrophil migration and anti-microbial defense.

cell biology↗

bioRxiv2021

Autophagy Induced by Palmitic Acid: a Brake in NAFLD Neutrophils

Innate immune suppression and high blood fatty acid levels are the pathological basis of multiple metabolic diseases. Neutrophil vacuolation is an indicator of the immune status of patients, which is associated with autophagy-dependent granule degradation. Vacuolated neutrophils are observed in ethanol toxicity and septicemia patients due to the changes in their blood constituents, but how about the neutrophils in nonalcoholic fatty liver disease (NAFLD) patient is unknown. Here, we confirmed that an adhesion deficiency and an increased autophagy level existed in NAFLD neutrophils, and the three neutrophil granule subunits, namely, the azurophil granules, specific granules and gelatinase granules, could be engulfed by autophagosomes for degradation, and these autophagy-triggered granule degradation events were associated with vacuolation in palmitic acid (PA)-treated and NAFLD neutrophils. Concordantly, the adhesion-associated molecules CD11a, CD11b, CD18 and Rap1 on the three granule subunits were degraded during PA induced autophagy. Moreover, the cytosolic CD11a, CD11b, CD18 and Rap1 were targeted by Hsc70 and then delivered to lysosomal-like granules for degradation. Notably, in vitro and ex vivo, PA induced autophagy by inhibiting the p-PKC/PKD2 pathway. Overall, we showed that high blood PA level inhibited the p-PKC/PKD2 pathway to induce NAFLD neutrophil autophagy, which promoted the degradation of CD11a, CD11b, CD18 and Rap1 and further decreased the adhesion of neutrophils, thereby impairing the neutrophil function of NAFLD patients. This theory provides a new therapeutic strategy to improve the immune deficiency in NAFLD patients. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/438261v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@454a53org.highwire.dtl.DTLVardef@112a7e7org.highwire.dtl.DTLVardef@1cd397forg.highwire.dtl.DTLVardef@7a0058_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIVacuolation and adhesion deficiency of NAFLD neutrophils are associated with autophagy-dependent granule degradation C_LIO_LIPA inhibits p-PKC/PKD2 to induce autophagy, which induces the degradation of CD11a, CD11b, CD18 and Rap1 and decreases neutrophil adhesion C_LI

cell biology↗

bioRxiv2020

A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish

Tissue-specific knockout techniques are widely applied in biological studies to probe the tissue-specific roles of specific genes in physiology, development, and disease. CRISPR/Cas9 is a widely used technology to perform fast and efficient genome editing in vitro and in vivo. Here, we report a robust CRISPR-based gateway system for tissue-specific gene inactivation in zebrafish. A transgenic fish line expressing Cas9 under the control of a neutrophil-restricted promoter was constructed. As proof of principle, we transiently disrupted rac2 or cdk2 in neutrophils using plasmids driving the expression of sgRNAs from U6 promoters. Loss of the rac2 or cdk2 gene in neutrophils resulted in significantly decreased cell motility, which could be restored by re-expressing Rac2 or Cdk2 in neutrophils in the corresponding knockout background. The subcellular location of Rac activation and actin structure and stress in the context of neutrophil migration was determined in both the wild-type and rac2 knockout neutrophils in vivo. In addition, we evaluated an alternative approach where the Cas9 protein is ubiquitously expressed while the sgRNA is processed by ribozymes and expressed in a neutrophil-restricted manner. Cell motility was also reduced upon rac2 sgRNA expression. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identification and characterization of gene functions in neutrophils.

genetics↗

bioRxiv2019

Mitofusin 2 regulates neutrophil adhesive migration via suppressing Rac activation

Neutrophils rely on glycolysis for energy production. How mitochondria regulate neutrophil function is not fully understood. Here, we report that mitochondrial outer membrane protein Mitofusin 2 (Mfn2) regulates neutrophil homeostasis in vivo. Mfn2-deficient neutrophils are released from the hematopoietic tissue and trapped in the vasculature in zebrafish embryos. Human neutrophil-like cells deficient with MFN2 fail to arrest on activated endothelium under sheer stress or perform chemotaxis. Deletion of Mfn2 results in a significant reduction of neutrophil infiltration to the inflamed peritoneal cavity in mice. Mfn2, but not Mfn1, -null mouse embryonic fibroblast cells have altered actin structure and are impaired in wound closure. MFN2-deficient neutrophil-like cells display heightened intracellular calcium levels and Rac activation after chemokine stimulation. Mechanistically, MFN2 maintains mitochondria-ER interaction. Restoring mitochondria-ER tether rescues the chemotaxis defect and Rac activation resulted from MFN2 depletion. Finally, inhibition of Rac restores chemotaxis in MFN2-deficient neutrophils. Altogether, we identified that MFN2 regulates neutrophil migration via suppressing Rac activation and uncovered a previously unrecognized role of MFN2 in regulating the actin cytoskeleton.

cell biology↗

bioRxiv2019

MicroRNA Overexpression screen reveals a noncanonical role of CDK2 in regulating neutrophil migration

Neutrophil migration is essential for inflammatory responses to kill pathogens, however it also causes tissue injury. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA overexpression screen in zebrafish, and identified eight microRNAs as potent suppressors of neutrophil migration. Among those, miR-199 decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (cdk2), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting CDK2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils. Chemotaxis of primary human neutrophils are also reduced by CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. Together, our results reveal previously unknown functions of miR-199 and CDK2 in regulating neutrophil migration and provide new directions in alleviating systemic inflammation.\n\nOne Sentence SummarymiR-199 directly suppresses cdk2 expression, neutrophil chemotaxis and systemic inflammation.

immunology↗