# Regulation of TLR signaling, Inflammation and Antigen Presentation by VPS33B

> **NIH NIH R01** · CINCINNATI CHILDRENS HOSP MED CTR · 2024 · $678,969

## Abstract

Project Summary
Initiation of innate immune responses depends on cognate interaction between germline-encoded pattern
recognition receptors and their ligands (expressed by microbes). Following recognition, the receptors initiate
activation of downstream signal transduction pathways that often involve recruitment of downstream adapters
and kinases. The Toll-like receptor family of PRRs, which are the subject of current investigation, are expressed
both on the plasma membrane and in the endosomes. Several recent studies have demonstrated that
endocytosis of the plasma membrane TLRs (especially TLR4) plays a critical role in regulating both quality and
magnitude of inflammatory responses in a responding macrophage. Other studies have demonstrated that TLR
signaling enhances phagocytosis of microbial cargo but not of apoptotic cell cargo suggesting a degree of
specificity that is not understood. In addition, although endocytosis of TLR4 and the events following endocytosis
of TLR4 that influence signal transduction are very well studied, it is not entirely clear if and how endocytosis
influences signaling downstream of other plasma membrane and endosomal TLRs. In our studies, we find that
a protein called Vps33B regulates handling of the phagocytic and endocytic cargo following pattern recognition
receptor activation. More importantly, Vps33B directly influences the outcome of signaling downstream of TLRs
in mice and Toll- and IMD pathways in Drosophila. Mutations in the genes VPS33B and VPS16B are linked to a
rare human disease called ARC (Arthrogryposis-renal dysfunction-cholestasis) syndrome. Both of these ARC
genes encode paralogs of HOPS complex subunits suggesting a role in membrane fusions but how perturbation
of function of these proteins results in a diverse spectrum of disease symptoms in ARC patients is not entirely
clear. It has however been documented that ARC patients suffer from sepsis and recurrent bacterial infections
and we were therefore investigated the role of these proteins in influencing immune responses. We find that in
the absence of VPS33B, Drosophila respond vigorously to microbial insult. Exaggerated immune responses are
generated in response to live or dead bacteria and purified ligands of the Toll and IMD pathway results in death
of Vps33B mutant, but not wild-type flies. This function of Vps33B is conserved in vertebrates and we find that
mouse macrophages lacking Vps33B secrete very high quantities of inflammatory cytokines, when stimulated
by either plasma membrane or endosomal TLR ligands. We therefore hypothesize that activation of pattern
recognition receptors and specifically TLRs leads to formation of specialized endosomes that depend on Vps33B
for lysosomal fusion. Lack of Vps33B is likely to affect several aspects of innate and adaptive immunity and to
test this hypothesis, we propose to 1. Define the molecular events that regulate Vps33B function in endosomal
maturation, 2. Define the role of Vps33B-regulated ...

## Key facts

- **NIH application ID:** 10864059
- **Project number:** 5R01AI155426-04
- **Recipient organization:** CINCINNATI CHILDRENS HOSP MED CTR
- **Principal Investigator:** Helmut J Kramer
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $678,969
- **Award type:** 5
- **Project period:** 2021-07-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10864059

## Citation

> US National Institutes of Health, RePORTER application 10864059, Regulation of TLR signaling, Inflammation and Antigen Presentation by VPS33B (5R01AI155426-04). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10864059. Licensed CC0.

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