ARC Syndrome-Linked Vps33B Protein Is Required for Inflammatory Endosomal Maturation and Signal Termination

Abstract

Summary Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) sense microbial ligands and initiate signaling to induce inflammatory responses. Although the quality of inflammatory responses is influenced by internalization of TLRs, the role of endosomal maturation in clearing receptors and terminating inflammatory responses is not well understood. Here, we report that Drosophila and mammalian Vps33B proteins play critical roles in the maturation of phagosomes and endosomes following microbial recognition. Vps33B was necessary for clearance of endosomes containing internalized PRRs, failure of which resulted in enhanced signaling and expression of inflammatory mediators. Lack of Vps33B had no effect on trafficking of endosomes containing non-microbial cargo. These findings indicate that Vps33B function is critical for determining the fate of signaling endosomes formed following PRR activation. Exaggerated inflammatory responses dictated by persistence of receptors in aberrant endosomal compartments could therefore contribute to symptoms of ARC syndrome, a disease linked to loss of Vps33B.

Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling

“…Thus, Imd amyloid fibrils must be regulated such that they trigger rapid NF-κB activation and robust AMP gene expression, but for only a limited time. Hyperactivation of Imd signaling is highly deleterious; overexpression of Imd leads to spontaneous immune signaling and excessive cell death (Akbar et al, 2016; Cao et al, 2013; Georgel et al, 2001). However, cell death is not observed during experimental immune challenge.…”

Escherichia coli Infection of Drosophila

“…The diagram shows the fraction of surviving wild-type flies (OreR) or Vps33B mutants after injection with heat-killed E. coli , similar to experiments previously published (Akbar et al , 2016). Statistical analysis was performed in Prism using the Log-rank (or Mantel-Cox) test to determine a P value smaller than 0.0001.…”

“…Unlike these protocols, our experimental procedure allows us to determine the site of infection and precisely control the dose of bacteria injected into each fly. This provides homogeneity and reproducibility, and allows us to adapt bacterial load for different experiments (Akbar et al , 2011 and 2016). …”

“…This protocol has been adapted and modified from our previously published work (Akbar et al , 2016; Akbar et al , 2011). …”

Isolation and Infection of Drosophila Primary Hemocytes

“…Such bacterial uptake is straightforward to measure using FACS analysis (Tirouvanziam et al ., 2004). For a detailed analysis of phagosomal maturation, however, we found it advantageous to examine individual hemocytes attached to a glass cover slip (Akbar et al , 2011; Rahman et al ., 2012; Akbar et al ., 2016) as this procedure offered us the best combination of temporal and spatial resolution for our studies of phagosome maturation.…”