AP-1γ2 is an adaptor protein 1 variant required for endosome-to-Golgi trafficking of the mannose-6-P receptor (CI-MPR) and ATP7B copper transporter.

Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.

Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production.

Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites.IMPORTANCE The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process.

SARS-CoV-2-triggered neutrophil extracellular traps mediate COVID-19 pathology.

Severe COVID-19 patients develop acute respiratory distress syndrome that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that neutrophil extracellular traps (NETs) have been described as important mediators of tissue damage in inflammatory diseases, we investigated whether NETs would be involved in COVID-19 pathophysiology. A cohort of 32 hospitalized patients with a confirmed diagnosis of COVID-19 and healthy controls were enrolled. The concentration of NETs was augmented in plasma, tracheal aspirate, and lung autopsies tissues from COVID-19 patients, and their neutrophils released higher levels of NETs. Notably, we found that viable SARS-CoV-2 can directly induce the release of NETs by healthy neutrophils. Mechanistically, NETs triggered by SARS-CoV-2 depend on angiotensin-converting enzyme 2, serine protease, virus replication, and PAD-4. Finally, NETs released by SARS-CoV-2-activated neutrophils promote lung epithelial cell death in vitro. These results unravel a possible detrimental role of NETs in the pathophysiology of COVID-19. Therefore, the inhibition of NETs represents a potential therapeutic target for COVID-19.

Caspase-11-dependent IL-1α release boosts Th17 immunity against Paracoccidioides brasiliensis.

The granulomatous lesion resulting from infection with the fungus Paracoccidioides brasiliensis is characterized by a compact aggregate of mature cells, surrounded by a fibroblast- and collagen-rich content. Granuloma formation requires signaling elicited by inflammatory molecules such as members of the interleukin-1 family. Two members of this family have been thoroughly studied, namely IL-1α and IL-1ß. In this study, we addressed the mechanisms underlying IL-1α secretion and its functional role on the host resistance to fungal infection. We found that, the expression of caspase-11 triggered by P. brasiliensis infection of macrophages depends on IFN-ß production, because its inhibition reduced procaspase-11 levels. Curiously, caspase-11 deficiency did not impair IL-1ß production, however caspase-11 was required for a rapid pore-mediated cell lysis. The plasma membrane rupture facilitated the release of IL-1α, which was necessary to induce NO production and restrict fungal replication. Furthermore, P. brasiliensis-infected macrophages required IL-1α to produce optimal levels of IL-6, a major component of Th17 lymphocyte differentiation. Indeed, IL-1α deficiency accounted for a significant reduction of Th17 lymphocytes in lungs of infected mice, correlating with diminished neutrophil infiltration in the lungs. Strikingly, we identified that IL-1α directly reprograms the transcriptional profile of Th17-committed lymphocytes, increasing cellular proliferation, as for boosting IL-17 production by these cells. Beyond neutrophil chemotaxis in vivo, IL-17 also amplified IL-1α production by infected macrophages in vitro, endorsing a critical amplification loop of the inflammatory response. Therefore, our data suggest that the IFN-ß/caspase-11/IL-1α pathway shapes a protective antifungal Th17 immunity, revealing a molecular mechanism underlying the cross-talk between innate and adaptive immunity.

B lymphocyte-induced maturation protein 1 controls TH9 cell development, IL-9 production, and allergic inflammation.

BACKGROUND: The transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp-1) has a key role in terminal differentiation in various T-cell subtypes. However, whether Blimp-1 regulates TH9 differentiation and its role in allergic inflammation are unknown. OBJECTIVE: We aimed to investigate the role of Blimp-1 in TH9 differentiation and in the pathogenesis of allergic airway inflammation. METHODS: In vitro TH9 differentiation, flow cytometry, ELISA, and real-time PCR were used to investigate the effects of Blimp-1 on TH9 polarization. T cell-specific Blimp-1-deficient mice, a model of allergic airway inflammation, and T-cell adoptive transfer to recombination-activating gene 1 (Rag-1)-/- mice were used to address the role of Blimp-1 in the pathogenesis of allergic inflammation. RESULTS: We found that Blimp-1 regulates TH9 differentiation because deleting Blimp-1 increased IL-9 production in CD4+ T cells in vitro. In addition, we showed that in T cell-specific Blimp-1-deficient mice, deletion of Blimp-1 in T cells worsened airway disease, and this worsening was inhibited by IL-9 neutralization. In asthmatic patients CD4+ T cells in response to TGF-ß plus IL-4 increased IL-9 expression and downregulated Blimp-1 expression compared with expression in healthy control subjects. Blimp-1 overexpression in human TH9 cells inhibited IL-9 expression. CONCLUSION: Blimp-1 is a pivotal negative regulator of TH9 differentiation and controls allergic inflammation.