# Exploring the Mechanisms of Inflammatory Caspase Activation in Sickle Cell Disease

> **NIH NIH F32** · BAYLOR COLLEGE OF MEDICINE · 2022 · $42,175

## Abstract

PROJECT SUMMARY
Excessive hemolysis has been implicated in the inflammatory activation of monocytes, macrophages and endo-
thelial cells and often results in uncontrolled sterile inflammation that can augment susceptibility to infections and
vaso-occlusion. The hemolysis product, heme, activates caspase-1, an enzyme required to ensure correct reg-
ulation of inflammatory signaling through the maturation of the proinflammatory cytokines, interleukin (IL)-1β and
IL-18. Our preliminary data shows that heme promotes the release of IL-1β in primary human macrophages and
that SCD macrophages are more responsive to heme stimulation. Our data also show that caspase-5 is activated
by heme and that deletion of caspase-5 in a monocytic cell line results in a striking increase of IL-1β release
upon heme treatment. These results suggest that caspase-5 regulates caspase-1 activity. Furthermore, we have
identified five variants of the inflammatory caspases genes in SCD patients. A caspase-1 variant was associated
with a decreased risk stroke, while a variant of caspase-4 was associated with increased risk. Whether or not
these variants affect the response to pro-inflammatory stimuli remains elusive. My objective is to characterize
the activation mechanism of the inflammatory caspases in response to heme and to identify their role in clinical
outcomes in SCD patients. My central hypothesis is that heme activates the inflammatory caspases, which both
positively and negatively regulate inflammation in SCD. The specific aims are to: 1) determine the consequences
of heme-induced inflammatory caspases activation; 2) identify the molecular requirements for heme-induced
activation of the inflammatory caspases; and 3) determine how natural genetic variation in inflammatory caspa-
ses impacts inflammation in SCD. I will use Bimolecular Fluorescence Complementation (BiFC), an imaging-
based technique developed by our laboratory, to reveal the upstream requirements for inflammatory caspases
activation. To complete Aim 1, I will contrast levels of IL-1β, inflammatory caspase and substrate cleavage, and
inflammatory cell death in response to heme in a) immune cells from healthy donors and from SCD patients; and
b) immune cells wild-type or deficient in caspase-1, -4, or -5. For Aim 2, I will use BiFC to characterize the heme-
induced caspase activation and to determine the kinetics and localization of the BiFC onset in single cells. Com-
ponents of the heme-induced activation of caspase-1, -4, and -5 will be identified by BiFC experiments using
oligomerization-disrupting mutants and inflammasome genes silencing. Inflammasome interactions will be con-
firmed by immunoprecipitation or direct binding of heme will be explored using a cell free assay. Lastly, to com-
plete Aim 3, the impact of each variant on caspase activation, protein structure, and heme-binding will be deter-
mined using biochemical and cellular assays as well as in silico studies. Data from SCD patients will ...

## Key facts

- **NIH application ID:** 10318116
- **Project number:** 5F32DK121479-03
- **Recipient organization:** BAYLOR COLLEGE OF MEDICINE
- **Principal Investigator:** Beatriz Elena Bolivar-Vega
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $42,175
- **Award type:** 5
- **Project period:** 2019-12-01 → 2022-06-02

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10318116

## Citation

> US National Institutes of Health, RePORTER application 10318116, Exploring the Mechanisms of Inflammatory Caspase Activation in Sickle Cell Disease (5F32DK121479-03). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10318116. Licensed CC0.

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