Neutrophils drive type I interferon production and autoantibodies in patients with Wiskott-Aldrich syndrome.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficiency caused by mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in hematopoietic cells. A high proportion of patients experience autoimmunity caused by a breakdown in T- and B-cell tolerance. Moreover, excessive production of type I interferon (IFN-I) by plasmacytoid dendritic cells (pDCs) contributes to autoimmune signs; however, the factors that trigger excessive innate activation have not been defined. OBJECTIVE: Neutrophil extracellular traps (NETs) emerged as major initiating factors in patients with diseases such as systemic lupus erythematosus and rheumatoid arthritis. In this study we explored the possible involvement of aberrant neutrophil functions in patients with WAS. METHODS: We evaluated the expression of a set of granulocyte genes associated with NETs in a cohort of patients with WAS and the presence of NET inducers in sera. Using a mouse model of WAS, we analyzed NET release by WASp-null neutrophils and evaluated the composition and homeostasis of neutrophils in vivo. By using depletion experiments, we assessed the effect of neutrophils in promoting inflammation and reactivity against autoantigens. RESULTS: Transcripts of genes encoding neutrophil enzymes and antimicrobial peptides were increased in granulocytes of patients with WAS, and serum-soluble factors triggered NET release. WASp-null neutrophils showed increased spontaneous NETosis, induced IFN-I production by pDCs, and activated B cells through B-cell activating factor. Consistently, their depletion abolished constitutive pDC activation, normalized circulating IFN-I levels, and, importantly, abolished production of autoantibodies directed against double-stranded DNA, nucleosomes, and myeloperoxidase. CONCLUSIONS: These findings reveal that neutrophils are involved in the pathogenic loop that causes excessive activation of innate cells and autoreactive B cells, thus identifying novel mechanisms that contribute to the autoimmunity of WAS.

ALCAM-mediated cDC1 CD8 T cells interactions are suppressed in advanced lung tumors.

Conventional type 1 dendritic cells (cDC1) are critical regulators of anti-tumoral T-cell responses. The structure and abundance of intercellular contacts between cDC1 and CD8 T cells in cancer tissues is important to determine the outcome of the T-cell response. However, the molecular determinants controlling the stability of cDC1-CD8 interactions during cancer progression remain poorly investigated. Here, we generated a genetic model of non-small cell lung cancer crossed to a fluorescent cDC1 reporter (KP-XCR1venus) to allow the detection of cDC1-CD8T cell clusters in tumor tissues across tumor stages. We found that cDC1-CD8 clusters are abundant and productive at the early stages of tumor development but progressively diminish in advanced tumors. Transcriptional profiling and flow cytometry identified the adhesion molecule ALCAM/CD166 (Activated Leukocyte Cell Adhesion Molecule, ligand of CD6) as highly expressed by lung cDC1 and significantly downregulated in advanced tumors. Analysis of human datasets indicated that ALCAM is downregulated in non-small cell lung cancer and its expression correlates to better prognosis. Mechanistically, triggering ALCAM on lung cDC1 induces cytoskeletal remodeling and contact formation whereas its blockade prevents T-cell activation. Together, our results indicate that ALCAM is important to stabilize cDC1-CD8 interactions at early tumor stages, while its loss in advanced tumors contributes to immune evasion.

A mitochondrial quality control mechanism reverses the phagosome maturation arrest caused by Mycobacterium tuberculosis.

Phagosome maturation arrest (PMA) imposed by Mycobacterium tuberculosis ( Mtb ) is a classic tool that helps Mtb evade macrophage anti-bacterial responses. The exclusion of RAB7, a small GTPase, from Mtb -phagosomes underscores PMA. Here we report an unexpected mechanism that triggers crosstalk between the mitochondrial quality control (MQC) and the phagosome maturation pathways that reverses the PMA. CRISPR-mediated p62/SQSTM1 depletion ( p62 KD ) blocks mitophagy flux without impacting mitochondrial quality. In p62 KD cells, Mtb growth and survival are diminished, mainly through witnessing an increasingly oxidative environment and increased lysosomal targeting. The lysosomal targeting of Mtb is facilitated by enhanced TOM20 + mitochondria-derived vesicles (MDVs) biogenesis, a key MQC mechanism. In p62 KD cells, TOM20 + -MDVs biogenesis is MIRO1/MIRO2-dependent and delivered to lysosomes for degradation in a RAB7-dependent manner. Upon infection in p62 KD cells, TOM20 + -MDVs get extensively targeted to Mtb -phagosomes, inadvertently facilitating RAB7 recruitment, PMA reversal and lysosomal targeting of Mtb . Triggering MQC collapse in p62 KD cells further diminishes Mtb survival signifying cooperation between redox- and lysosome-mediated mechanisms. The MQC-anti-bacterial pathway crosstalk could be exploited for host-directed anti-tuberculosis therapies.

The SNARE VAMP7 Regulates Exocytic Trafficking of Interleukin-12 in Dendritic Cells.

Interleukin-12 (IL-12), produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesicles marked by the SNARE VAMP7. Dendritic cells (DCs) from VAMP7-deficient mice are partially impaired in the multidirectional release of IL-12. Upon encounter with antigen-specific T cells, IL-12-containing vesicles rapidly redistribute at the immune synapse and release IL-12 in a process entirely dependent on VAMP7 expression. Consistently, acquisition of effector functions is reduced in T cells stimulated by VAMP7-null DCs. These results provide insights into IL-12 intracellular trafficking pathways and show that VAMP7-mediated release of IL-12 at the immune synapse is a mechanism to transmit innate signals to T cells.