Journal of Innate Immunity最新文献

Cystic fibrosis (CF) is a hereditary disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The major causes of morbidity and mortality in CF are related to lung disease, involving neutrophil-dominated lung inflammation. Whether the altered inflammatory response of neutrophils in patients with CF is an intrinsic defect due to a lack of CFTR expression, or alternatively, exacerbated by chronic exposure to infection and inflammation, is extensively debated. Fuelling this dispute are conflicting studies on CFTR protein expression by neutrophils, with opposing results described at both the gene and protein level. This is pertinent in the era of CFTR modulator therapies, with clinicians and scientists exploring the impact of different CFTR mutation classes and CFTR modulators on neutrophil function. To address this, the focus of this article is to uncover the cause for the described disparity of data on neutrophil CFTR expression, by investigating methods utilised for CFTR detection, and to draw consensus on the most optimal protocol for identifying CFTR protein in neutrophils.

Introduction: Neutrophils are the most abundant innate immune cells in the peripheral blood and eliminate bacteria through phagocytosis and antimicrobial mechanisms. Early during infection, they often encounter high bacterial loads before full recruitment. Individual neutrophils can ingest many bacteria, but it remains unclear how high bacterial loads per neutrophil affect intracellular killing.

Methods: Neutrophils were isolated from healthy donor blood by fluorescence-activated cell sorting (FACS). Intracellular bacterial load was quantified using imaging flow cytometry to measure spot counts and green fluorescent protein (GFP) intensity after exposure to GFP-expressing Staphylococcus aureus. A single-cell killing assay assessed intracellular killing across bacterial-load categories by sorting individual GFP+ neutrophils into 384-well plates and counting wells with outgrowth after 100 hours. Phagolysosomal acidification was measured using dual-labeled (pH-sensitive pHrodo and pH-insensitive PromoFluor 520 LSS NHS ester [PF520]) S. aureus bioparticles.

Results: Bacterial uptake by neutrophils was highly heterogeneous in vivo and in vitro. GFP spot counts strongly correlated with GFP intensity (R² = 0.66), allowing stratification into GFP fluorescence intensity categories. In the single-cell killing assay, higher bacterial loads per neutrophil were associated with reduced intracellular killing (χ²(4) = 11.72, p = 0.0003). Higher bacterial loads per neutrophil corresponded with diminished phagolysosomal acidification capacity (χ²(4) = 24.00, p < 0.0001).

Conclusion: Neutrophils ingesting higher bacterial loads exhibit reduced intracellular killing, likely due to decreased phagolysosomal acidification. These findings highlight how bacterial load per neutrophil shapes antimicrobial capacity and early infection control.

Bothrops jararacussu venom contains snake venom metalloproteinases (SVMPs) that contribute to inflammation and tissue damage. BjussuMP-II, a PI-class SVMP, lacks hemorrhagic activity but retains proteolytic and immunomodulatory properties. Here, we uncover a previously unrecognized function of BjussuMP-II in triggering NLRP3 inflammasome activation in human neutrophils, leading to IL-1β release and pyroptosis. This discovery reveals a direct molecular link between SVMP activity and inflammasome-mediated inflammation, a fundamental mechanism with implications beyond snakebite pathology, and highlights inflammasomes as potential therapeutic targets to mitigate severe inflammatory responses in envenomed patients. Mechanistically, BjussuMP-II increased expression of NLRP3, ASC, CASPASE-1, NEK7, and HIF-1α, as well as IL-1β and Gasdermin D (GSDMD) cleavage, confirmed by immunoblotting and immunofluorescence. It promoted the release of IL-1β, LTB4, LDH, and dsDNA, consistent with pyroptosis, which was reduced by MCC950 or disulfiram. Studies in Gsdmd⁻/⁻ and HIF-1α-deficient neutrophils further demonstrated the requirement of these pathways for cytokine release. Collectively, these results indicate that BjussuMP-II modulates inflammasome-associated signaling pathways in neutrophils, contributing to the inflammatory responses triggered by snake venom metalloproteinases.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and older adults. Currently approved vaccines target only the viral fusion protein, but live-attenuated vaccines - e.g. through inactivation of nonstructural protein 1 (NS1) - likely induce a broader immune response. NS1 inhibits the immune response by repressing interferon production, but this has mostly been shown in immortalized cell lines, which do not necessarily represent the in vivo situation. Here, we assessed the effect of NS1 mutations on replication and host responses in physiologically relevant differentiated primary human nasal epithelial cells (HNEC) cultured at air-liquid interface (ALI). Using yeast-based reverse genetics, NS1-inactivating mutations were introduced. In differentiated HNECs, NS1 mutants showed delayed replication compared to wild-type virus. Bulk RNA sequencing early after infection revealed stronger antiviral signatures in HNEC infected with NS1 mutants compared to wild-type, characterized by upregulation of interferons and chemokines. Cytokine analysis confirmed these results. Finally, an indirect immune cell migration assay revealed that both WT and NS1-mutant viruses induce migration of mainly neutrophils. In conclusion, this study shows that RSV NS1 supports viral replication not only via inhibition of the production of interferons, but also by reducing early chemokine production and secretion by epithelial cells. Together, our data highlight the suitability of the ALI transwell model for preclinical assessment of live-attenuated vaccine candidates.

Background: Cardiovascular diseases, including myocardial infarction, are the leading cause of death worldwide. Neutrophils have emerged as actors in non-infectious pathologies and play major roles in cardiovascular diseases: after myocardial infarction, neutrophils are the first cell recruited to the ischemic heart and display multifaceted roles in orchestrating post myocardial infarction tissue healing and repair. Interestingly, recent studies described a high heterogeneity in neutrophils during this process.

Summary: At steady state, neutrophils present diversity during their development in hematopoietic organs, and in the circulation after reaching maturity. Inflammatory environments elicit neutrophil reprogramming and further complexify neutrophil heterogeneity, especially leading to the emergence of tissue-specific populations including SiglecF+ neutrophils. Neutrophils play beneficial and deleterious roles after myocardial infarction: they originate from diverse sources including the bone marrow, the spleen and from the marginated neutrophil pool, and display a time-dependent appearance of heterogeneous profile within the cardiac tissue.

Key messages: Neutrophil heterogeneity in the cardiac tissue could explain the contrasting roles ascribed to neutrophils after myocardial infarction. Increased knowledge in neutrophil diversity will enable the identification of specific targets to improve cardiac healing processes.

Objective: The cGAS-STING pathway is a critical sensor in the innate immune response to intracellular pathogens, yet its therapeutic potential for augmenting macrophage-mediated control of Mycobacterium tuberculosis (Mtb) remains incompletely understood. This study investigated whether pharmacological activation of the STING pathway could enhance autophagy to promote Mtb clearance in human macrophages.

Methods: Human THP-1 monocytes were differentiated into macrophages and infected with Mtb. The effects of the STING agonist MIW815 (ADU-S100) on Mtb phagocytosis, intracellular bacterial survival, and autophagic flux were assessed using a combination of molecular and cellular techniques, including qRT-PCR, western blotting, colony-forming unit (CFU) assays, and confocal immunofluorescence microscopy. The dependency on the cGAS-STING pathway was confirmed using siRNA-mediated gene silencing.

Results: Pharmacological activation of STING with ADU-S100 significantly enhanced Mtb phagocytosis and subsequent intracellular clearance. This enhanced bactericidal activity was mechanistically linked to an increase in autophagic flux, as evidenced by elevated LC3-II protein levels and significantly increased colocalization of Mtb with lysosomal compartments. Importantly, treatment with the autophagy inhibitor hydroxychloroquine or silencing of cGAS significantly reversed these phenotypes, confirming the pivotal role of the STING-autophagy axis.

Conclusion: Activating the STING pathway with ADU-S100 is a potent host-directed strategy to bolster macrophage autophagy and enhance the elimination of intracellular Mtb. This provides a strong rationale for exploring STING agonists as a novel therapeutic intervention for tuberculosis, addressing a significant and clinically relevant challenge in infectious disease.

Introduction: Chloroquine (CQ), a well-known antimalarial agent, has been proposed as a potential antiviral compound due to its ability to interfere with multiple cellular pathways critical for viral replication. Although CQ exhibits broad-spectrum antiviral activity, its effect on host innate immune responses remains incompletely understood. The timing of CQ administration, whether before or after infection, may lead to different immunological outcomes. Therefore, the immunomodulatory effects of CQ should be carefully evaluated before antiviral therapy.

Methods: To investigate the immunomodulatory role of CQ (50 μm), we used immunofluorescence staining, Western blotting, and reporter assays to evaluate innate immune activation in A549 cells. We established a doxycycline-inducible system to activate mitochondrial antiviral signaling (MAVS)-mediated signaling without viral infection. Plaque assays and antiviral tests were performed to measure viral replication, while cytokine array and RT-qPCR were used to quantify cytokine production. Mitochondrial morphology was assessed using immunofluorescence microscopy.

Results: CQ enhanced innate immune responses triggered by dengue virus infection and poly(I:C) stimulation. This enhancement was associated with the activation of the MAVS protein and its upstream receptors, including retinoic acid-inducible gene I and melanoma differentiation-associated protein 5. CQ strengthened MAVS-dependent antiviral signaling and increased IL-6 induction more than 13-fold. Alterations in mitochondrial morphology may contribute to this immunostimulatory effect.

Conclusion: CQ promotes MAVS-mediated antiviral and inflammatory cytokine responses, potentially through its effect on mitochondrial dynamics. These findings indicate that while CQ may enhance antiviral defense, its immune-stimulating properties should be carefully evaluated prior to its use as an antiviral agent in treating RNA virus infections.

Introduction: Interleukin (IL)-6 has an important role in limiting urinary tract infection (UTI). Mice lacking IL-6 are more susceptible to uropathogenic Escherichia coli (UPEC), including increased formation of intracellular bacterial communities (IBCs). How IL-6 promotes UPEC clearance is unknown. We hypothesize IL-6 reduces UTI susceptibility by limiting IBC formation through an early mechanism of infection.

Methods: Female mice were treated with vehicle or neutralizing antibodies to inhibit IL-6 or the IL-6 receptor (IL-6R) prior to transurethral UPEC infection. In rescue experiments, murine recombinant (r)IL-6 was administered to IL-6 knockout (KO) mice. Bladder IBCs, urinary and bladder bacterial burden, and UPEC expulsion were quantified. For clinical translation, human urothelial cells were pretreated with human rIL-6 and infected with UPEC. Bacterial attachment, invasion, and expulsion were quantified.

Results: Neutralization of IL-6 or IL-6R increased bladder IBC counts compared to isotype controls. Similarly, while IL-6 KO mice exhibited higher IBC counts than wild-type controls, this phenotype was reversed by rIL-6 administration. Gentamicin protection assays confirmed increased intracellular UPEC burden and reduced bacterial expulsion in IL-6 KO bladders. rIL-6 treatment enhanced UPEC expulsion in human urothelial cells without impacting bacterial attachment or invasion.

Conclusion: IL-6 facilitates UPEC expulsion, limiting intracellular UPEC early in infection and thus the initial formation of IBCs. Since IBC formation is a bottleneck in UPEC survival during UTI, these findings identify a mechanism whereby IL-6 reduces early UPEC urothelial infectivity.