The multi-target regulation of cytokine storm by transcription factor Blimp-1

doi:10.21203/rs.3.rs-1867845/v1

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The multi-target regulation of cytokine storm by transcription factor Blimp-1

https://doi.org/10.21203/rs.3.rs-1867845/v1

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Objective

Cytokine storm syndrome is a fatal condition related to infectious and autoimmune diseases. Here we aim to investigate the regulatory mechanisms of Blimp-1 on cytokine storm.

Methods

The Blimp1 shRNA was transfected into RAW264.7 macrophages, followed by Toll-like receptor (TLR) ligand stimulation. The mRNA and protein levels of cytokines were detected by real-time PCR and flow cytometric bead array. The genomic binding sites of Blimp-1 were analyzed with chromatin immunoprecipitation-sequencing (ChIP-seq). The nuclear translocation of AP-1 and NF-κB p65 were measured by immunofluorescence staining.

Results

Blimp-1 significantly inhibited the expression and secretion of IL-1β, IL-6, and IL-18 in macrophages during stimulation with a variety of TLR ligands. ChIP-seq data showed that Blimp-1 stably combined with the promoters of Il1b and Tnf and the enhancers of Il6 and Il18, indicating a direct transcriptional repression of Blimp-1 on the pro-inflammatory cytokines. In addition, Blimp-1 strictly controlled nuclear translocation of NF-κB p65 in LPS-challenged macrophages, suggesting the regulatory mechanism of Blimp-1 on NF-κB activation.

Conclusion

Blimp-1 represses the production of multiple pro-inflammatory cytokines by directly binding the genomic region and restricting NF-κB p65 nuclear translocation. This finding may provide potential therapeutic strategies for the cytokine storm-related diseases.

Blimp-1

cytokine storm

macrophage

Toll-like receptor

NF-κB

nuclear translocation

Cytokine storm syndrome is a fatal condition that occurs when the immune system responds aggressively in infectious diseases, autoimmune diseases, and CAR T-cell therapy. Cytokine storm plays a key regulatory role in sepsis, unfortunately, so far, most clinical trials simultaneously targeting multiple cytokine production or effect failed [1]. Drugs blocking TNF-α and IL-6 have been approved for the treatment of rheumatoid arthritis, but the blockade of a single cytokine has a limited therapeutic effect on the disease [2]. Therefore, therapies targeting multiple pro-inflammatory cytokines may provide new ideas for the treatment of cytokine storm-related diseases.

Transcription factor B lymphocyte-induced maturation protein (Blimp-1) is a key regulator of plasma cell differentiation in B cells and effector/memory function in T cells [3, 4]. Blimp-1 is also required to maintain immune tolerance in myeloid cells and embryonic intestine [5, 6]. For example, conditional deletion of Blimp-1 in dendritic cells (DCs) showed an increased production of IL-6 and resulted in aberrant activation of the adaptive immune system in systemic lupus erythematous [7]. As mentioned above, Blimp-1 acts as a “Brake pad” in regulating immune functions, however, the regulatory mechanism of Blimp-1 in myeloid cells remains unclear. Blimp-1 is a SET domain and zinc finger–containing transcriptional repressor. Our previous study has demonstrated that it directly binds to the promoters of PD-1 and TIGIT in T cells [8]. In the present study, we aim to reveal the potential roles and mechanisms of Blimp-1 in regulating multiple cytokine production in macrophages. The results may provide new insight into the mechanisms of cytokine storm formation and potential therapeutic targets.

See supplementary material.

To elucidate the regulation of Blimp-1 on the pro-inflammatory cytokine production, we stably transfected RAW264.7 macrophages with Blimp1 shRNA followed by stimulation with different TLR ligands to mimic invasive pathogens. By stimulation with Pam3CSK4 (TLR2 ligand), PolyI:C (TLR3 ligand), Lipopolysaccharide (LPS; TLR4 ligand), and CpG-ODN (TLR9 ligand) for 3, 6, 12 and 24 hours, we detected the mRNA levels of Blimp1, Il1b, Il6, Il18, and Tnf with real-time PCR assay. The expression of Blimp-1 was significantly increased by TLR ligands and effectively inhibited by shRNA (Fig. 1a). With the down-regulation of Blimp-1, the mRNA levels of Il1β, Il6 and Il18 were remarkably elevated during TLR ligand stimulations (Fig. 1a). Exceptionally, the expression of Tnf in Blimp1 shRNA transfection was comparable to that in the non-specific control shRNA (Fig. 1a). The secretion of cytokine IL-1β, IL-6, IL-18 and TNF-α was similar to the mRNA expressions during TLR ligand stimulations (Fig. 1b). By analyzing the Blimp-1 ChIP-seq data from a previous study [9], we observed that Blimp-1 had stable binding at both the proximal promoters of Il1b and Tnf, and the distal enhancers of Il6 and Il18 (Fig. 1c). These results indicate that Blimp-1 has a strong inhibitory effect on a variety of pro-inflammatory cytokines at the transcription level during different infections.

TLRs activate the transcription factor NF-κB and activator protein-1 (AP-1), which consequently induces inflammatory responses. A hallmark activation of NF-κB and AP-1 is nuclear translocation, which is necessary for modulating gene transcription. By stimulating RAW264.7 cells with LPS, we found that Blimp-1 knockdown significantly up-regulated the expressions of p65 and AP-1 (Fig. 1d). Especially after Blimp-1 was knocked out, it was remarkably increased in the cytoplasmic to nuclear translocation of p65 (Fig. 1d). These data suggest that Blimp-1 inhibits the transcription of multiple pro-inflammatory cytokines by restricting the entry of NF-κB p65 into the nucleus.

Here we demonstrated the inhibitory effect of Blimp-1 in pro-inflammatory cytokine production in macrophages. Blimp-1 has been reported to regulate myeloid cell terminal differentiation. Blimp-1 acts as a regulation of the macrophages pyroptosis in response to L. donovani infection [10]. Our findings further revealed the function and mechanism of Blimp-1 in maintaining appropriate inflammation in macrophages against infections. Since macrophages possess immunomodulatory and inflammatory abilities and actively participate in the development of many diseases, Blimp-1 may play a broad role in inflammatory diseases in which macrophages are involved.

We found that Blimp-1 directly binds to promoters or enhancers of IL-1β, IL-6, and IL-18 coding genes and inhibits nuclear translocation of NF-κB p65, thereby reducing the cytokine secretion. Notably, although Blimp-1 binds to the Tnf promoter, it does not inhibit the expression and secretion of TNF-α. There are two possible explanations. First, transcriptional repression by Blimp-1 involves recruitment of histone deacetylase, and the inability to regulate Tnf transcription may be due to dysregulation of histone deacetylation [11]. Second, there may be other transcription factors that act antagonistically to regulate Tnf transcription. Therefore, the transcriptional regulation mechanism of TNF-α still needs to be further elucidated.

In conclusion, Blimp-1 tightly controls the expression and secretion of multiple pro-inflammatory cytokines, which may be the key hub for regulating the inflammatory response. Although it is still necessary to further demonstrate the pathophysiological role of Blimp-1, this study provides new ideas and potential therapeutic strategies for the cytokine storm-related diseases.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81871586 and 82172128) and Beijing Municipal Excellent Talents Foundation (2018000021223ZK27).

Conflict of interest

The authors declare that they have no conflict of interest.

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No competing interests reported.

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You are reading this latest preprint version

The multi-target regulation of cytokine storm by transcription factor Blimp-1

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Introduction

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