# Intracellular Membrane Attack Complexes and COVID-19 Immune Hyperactivation

> **NIH NIH R21** · YALE UNIVERSITY · 2024 · $209,375

## Abstract

PROJECT SUMMARY
 During severe COVID-19, immune hyperactivation contributes to tissue injury and worsened patient
outcomes. Complement (C’) proteins are believed to centrally mediate COVID-19-related immune
hyperactivation. C’ are a conserved set of immune proteins involved in host defense that become activated on
various cell types in the lung during severe COVID-19. Upon terminal activation, C’ proteins assemble to form
pore-like membrane attack complexes (MACs) that insert into target cell surfaces as transmembrane
structures. The presence of MACs in pulmonary tissues strongly correlates with immune hyperactivation, but
underlying mechanisms are unknown.
 The immune effects of MACs have been widely attributed to their cytolytic properties. However,
widespread MAC deposition occurs on alveolar endothelial cells (ECs) during severe COVID-19 in the absence
of significant EC death or vessel rarefaction. Instead, MAC-bound ECs show signs of EC dysfunction with
dysregulated NF-B activation and elaboration of pro-inflammatory cytokines. These patient-level observations
suggest immune effects of MACs that are separable from their cytolytic properties.
 In this application we examine a novel role for intracellular MAC proteins as alarmins. We used a
SARS-CoV-2-derived antigen, S protein, to induce non-cytolytic MAC assembly on human ECs. Following
assembly on ECs, surface-bound MACs became rapidly internalized and transferred to Rab5+ endosomes.
The pool of intracellular, but not extracellular or surface-bound MACs, activated NF-B to induce elaboration of
inflammatory cytokines. The intraluminal milieu of Rab5+ endosomes caused a MAC protein, C9, to form
insoluble aggregates that stimulated aggrephagy, a specialized form of selective macroautophagy, to activate
NF-B. Via proteomic profiling of solubilized MAC complexes (Sc5b-9) in COVID-19 sera, we identified
guanylate binding protein 4 (GBP4) as a C9-binding protein regulating aggrephagy and NF-B activity.
 Based on these exciting preliminary data we propose 2 Specific Aims to explore the hypothesis that
intracellular MAC proteins act as alarmins to initiate inflammatory signaling. We will consolidate role(s) for
GBP4 as an immune sensor by examining binding interactions with C9 using COVID-19 patient sera.
Secondly, we will identify mouse orthologs for human GBP4 that mediate C9 sensing and MAC-induced
immune hyperactivation in vivo in response to S protein-induced MAC and following infection by replication
competent SARS-CoV-2. Our studies introduce a new paradigm for understanding MAC-related immunity and
contribute to our long-term aim of understanding C’-induced inflammation. By doing this, druggable targets
ameliorating MAC-induced immune complications of severe COVID-19 may emerge.

## Key facts

- **NIH application ID:** 10844646
- **Project number:** 5R21AI178582-02
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Dan Jane-Wit
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $209,375
- **Award type:** 5
- **Project period:** 2023-05-19 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10844646

## Citation

> US National Institutes of Health, RePORTER application 10844646, Intracellular Membrane Attack Complexes and COVID-19 Immune Hyperactivation (5R21AI178582-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10844646. Licensed CC0.

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