TRIM21 negatively regulates Corynebacterium pseudotuberculosis-induced inflammation and is critical for the survival of C. pseudotuberculosis infected C57BL6 mice

Highlights

• C. pseudotuberculosis infection activates TRIM21 expression in macrophages.

• TRIM21 limits C. pseudotuberculosis replication in macrophages.

• TRIM21 protects C. pseudotuberculosis infected macrophages from death.

• TRIM21 protects the host from C. pseudotuberculosis infection in vivo.

• TRIM21 negatively regulates C. pseudotuberculosis-induced inflammation.

Abstract

Corynebacterium pseudotuberculosis, a facultative intracellular bacterium, is an important zoonotic pathogen responsible for chronic inflammatory diseases. TRIM21, an E3 ubiquitin-protein ligase, plays pivotal roles in inflammation regulation. However, its role during C. pseudotuberculosis infection is unclear. Here, we found that TRIM21 expression was significantly increased in C. pseudotuberculosis-infected macrophages. Following infection by C. pseudotuberculosis, we observed a significantly higher number of bacteria and a higher degree of LDH release from Trim21^-/- macrophages compared to wild-type (WT) macrophages, suggesting that TRIM21 limits C. pseudotuberculosis replication in macrophages and protects the infected cells from death. Further in vivo experiments showed a significantly higher mortality, higher bacterial load, much more severe abscess formation, and lesions in the organs of C. pseudotuberculosis-infected Trim21^-/- mice compared to those of the infected WT mice, suggesting that TRIM21 plays critical roles in protecting against C. pseudotuberculosis infection. Moreover, the secretory levels of IL-1α, IL-1β, IL-6, and TNF-α were significantly higher in C. pseudotuberculosis-infected Trim21^-/- macrophages compared to infected WT macrophages; the levels of these cytokines were also higher in the sera, organs, and ascites of C. pseudotuberculosis-infected Trim21^-/- mice compared to infected WT mice. These findings suggest that TRIM21 negatively regulates the secretion of pro-inflammatory cytokines in macrophages, sera, organs, and ascites of mice following C. pseudotuberculosis infection. Collectively, the present study demonstrates that TRIM21 plays a vital role in preventing C. pseudotuberculosis infection, which may be related to the negative regulation of pro-inflammatory cytokines production by TRIM21 during this pathogen infection.

Introduction

Corynebacterium pseudotuberculosis - a gram positive, polymorphic, facultative intracellular bacterium - is one of the most important animal/human pathogens responsible for chronic diseases characterized by abscesses and pyogranuloma formation (Lopes Bastos, 2012). It causes caseous lymphadenitis (CLA) in goat and sheep (Windsor and Bush, 2016), ulcerative lymphangitis in horses (Haas et al., 2017), mastitis in cows (Silva et al., 2010), and necrotizing lymphadenitis in humans (Trost et al., 2011), Among these, CLA has been widely reported in small ruminants in most countries worldwide, and it has often resulted in significant economic losses (Lopes Bastos, 2012). Since C. pseudotuberculosis possess strong resistance to the external environment, and long course of infection, it is extremely difficult to completely eradicate C. pseudotuberculosis infection once introduced in a herd (Dorella et al., 2009). In order to develop ideal vaccines and control measures for CLA, a deeper understanding of the pathogenic mechanisms and the host immune response to C. pseudotuberculosis infection are necessary (Dorella et al., 2009).

The tripartite motif-containing (TRIM) protein family, which contains up to 100 members in mammals, represents the largest E3 ubiquitin ligase family (Bottermann and James, 2018; Hos et al., 2020; Hu and Shu, 2017), and plays important roles in many biological processes, including proliferation, apoptosis, and autophagy (Wan et al., 2021). In addition, it is also involved in the regulation of innate immune responses, thereby enabling the direct detection and restriction of pathogens (Wang and Hur, 2020). Most TRIMs have been reported to enhance the innate immune response at both the pre- and post-transcriptional stages of signaling pathways (Versteeg et al., 2013). Furthermore, some TRIMs have been found to play key roles as pathogen sensors or restriction factors (McEwan, 2016; Zhao et al., 2011), rather than just being involved in the up/downregulation of the activities of immune signaling molecules (Wang and Hur, 2020). TRIM21, also known as Ro52/SS-A, has long been known as an autoantigen. Autoantibodies against Ro52 have been found in many autoimmune diseases, including systemic lupus erythematosus (SLE), Sjögren’s syndrome, and systemic sclerosis (SSc) (Lee, 2017). TRIM21 is also the only known cytosolic IgG receptor in mammals; it detects antibody-coated viruses or bacteria that have entered the cytosol (Bottermann and James, 2018) and plays critical roles in the detection and neutralization of human adenovirus type 5 (AdV5), foot and mouth virus (FMDV), and Salmonella enterica, through a process called antibody-dependent intracellular neutralization (ADIN) (Fan et al., 2016; Foss et al., 2016; Rakebrandt et al., 2014). In addition, TRIM21 catalyzes Lys63 (K63)-linked ubiquitin chain formation and stimulates the signaling pathways of NF-κB, AP-1, IRF3, IRF5, and IRF7 transcription factors, resulting in the production of pro-inflammatory cytokines (McEwan et al., 2013).

To this point, research on TRIM21 response to infections has primarily focused on its intracellular response to viral infections. However, the importance and regulation of TRIM21 during bacterial infections, especially gram positive bacteria, remains poorly understood. Infection by C. pseudotuberculosis, a gram positive and facultative intracellular bacterium, causes a significant increase in the levels of inflammatory cytokines (Pepin et al., 1997; Zhou et al., 2019a). Therefore, the aim of this study was to evaluate the response of TRIM21 to C. pseudotuberculosis infection through in vivo and in vitro experiments.