Research Background

Sepsis is a life-threatening organ dysfunction syndrome caused by the body’s abnormal response to microbial infection. It has high morbidity and mortality, mainly due to excessive inflammatory response and metabolic disorders. Studies have shown that myeloid cells (such as monocytes and macrophages) play a key role in the pathogenesis of sepsis. In the early stage of sepsis, the accumulation of lipid metabolites in the body significantly affects disease progression, but the specific mechanism is not yet clear. The relationship between lipid metabolism abnormalities and the NLRP3 inflammasome is particularly worthy of attention.

The NLRP3 inflammasome is a multi-protein complex consisting of the sensor NLRP3, the adaptor ASC, and the effector caspase-1. NLRP3 can recognize various pathogen- or damage-associated molecular patterns (PAMPs and DAMPs), triggering the assembly and activation of the inflammasome, ultimately leading to the enzymatic activation of caspase-1, as well as the maturation of downstream IL-1β and Gasdermin D-dependent cell pyroptosis.

However, abnormal activation of the NLRP3 inflammasome often leads to excessive inflammatory response in early sepsis, so its activity must be strictly regulated. Post-translational modifications (PTMs) of proteins are considered one of the important ways to control NLRP3 inflammasome activation.

The research team in this paper demonstrates how glucocorticoid-induced tumor necrosis factor-related protein (GITR) mediates systemic inflammatory injury related to lipid metabolism abnormalities by regulating NLRP3’s PTMs, revealing GITR as a potential target for treating inflammatory diseases.

Paper Source

This paper was written by Siping Liang et al., with research conducted at Zhongshan School of Medicine, Sun Yat-sen University and its affiliated hospitals. The paper was published in “Cellular & Molecular Immunology” in May 2024, detailing GITR’s new role in sepsis.

Research Workflow

1. Relationship between GITR and LPC: The study first observed GITR expression on monocytes of sepsis patients and found a positive correlation with serum levels of LysoPC (lysophosphatidylcholine) and sepsis severity.

2. GITR promotes macrophage pyroptosis: In vitro experiments demonstrated that LPC treatment could induce GITR expression in macrophages, and GITR could enhance LPC uptake and NLRP3 inflammasome-mediated macrophage pyroptosis.

3. Rat model validation: Using cecal ligation and puncture (CLP) and LPS-induced sepsis mouse models, it was further confirmed that LPC exacerbated sepsis severity and mortality, while specific knockout of GITR in myeloid cells or NLRP3/caspase-1/IL-1β deficient mouse models significantly reduced sepsis severity and mortality.

4. Mechanism study: Mechanistically, GITR specifically enhances inflammasome activation by regulating NLRP3’s post-translational modifications (reducing its ubiquitination but increasing its acetylation). GITR competes with E3 ligase MARCH7 for binding, reducing NLRP3 ubiquitination and promoting its acetylation, thereby increasing inflammasome activity.

Detailed Interpretation of Research Results

GITR expression in monocytes correlates with sepsis LPC levels

The study found that LPC can significantly induce GITR expression in monocytes of sepsis patients, and GITR expression positively correlates with serum LPC levels. This suggests that LPC may mediate macrophage inflammatory response through GITR.

Specific data show: - In sepsis patients with high SOFA scores, low-density lipoprotein cholesterol (LDL-C) and LPC levels were significantly elevated. - Through flow cytometry, it was found that the proportion of GITR+ cells in CD11b+ monocytes of sepsis patients increased and positively correlated with serum LDL-C and LPC levels. - In CLP-induced septic mice, GITR expression significantly increased in PBMCs and spleen and lung, while serum LPC levels also significantly increased.

GITR promotes pyroptosis by increasing macrophage uptake of LPC

In in vitro experiments, LPC induced time-dependent expression of GITR in BMDMs (bone marrow-derived macrophages). In GITR-deficient BMDMs, both LPC uptake and LPC-induced cell death were significantly reduced, indicating that GITR is involved in LPC-induced macrophage inflammatory injury.

Specific experiments showed: - LPC induced GITR expression in BMDMs, while LPS challenge failed to induce GITR expression. - Treatment with JAK1/2 inhibitor ruxolitinib significantly attenuated LPC-induced GITR expression. - GITR deficiency significantly inhibited LPC-induced pyroptosis by reducing IL-1β secretion and GSMD-N, caspase-1, and mature IL-1β protein levels.

GITR promotes inflammasome activation by regulating NLRP3’s PTMs

Experiments found: - GITR deficiency significantly increased NLRP3 ubiquitination levels, indicating that GITR inhibits NLRP3 ubiquitination. - Through co-immunoprecipitation experiments, it was found that GITR competitively binds to MARCH7, reducing MARCH7-mediated NLRP3 ubiquitination. - The study further showed that GITR deficiency upregulated Sirt2 protein levels in BMDMs (Sirt2 is an NLRP3 deacetylase), reducing NLRP3 acetylation levels. - Animal experiments showed that GITR deficiency or NLRP3/caspase-1/IL-1β deficiency in mice could significantly reduce sepsis mortality and inflammatory injury.

Through these experiments, it was proven that GITR plays an important role in LPC-induced various inflammatory injuries in the body by regulating NLRP3’s PTMs.

Research Significance and Value

This study reveals the important role of GITR in regulating the NLRP3 inflammasome and early inflammatory injury in sepsis. Specific conclusions are as follows: - LPC enhances NLRP3 inflammasome-mediated macrophage pyroptosis by upregulating GITR expression, thereby exacerbating sepsis severity. - GITR, as a potential therapeutic target, has important significance in regulating inflammatory diseases. - The study provides new insights into LPC and GITR in sepsis, advancing the understanding of sepsis pathogenesis and potential therapeutic strategies.

By revealing the relationship between GITR and the NLRP3 inflammasome, this study provides new directions and possibilities for the treatment of sepsis and other inflammatory diseases.