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

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-1ß, 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.

Cofilin-1, peroxiredoxin-1, and galectin-3: Major proteins released by macrophages infected with Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis, a broad host-spectrum zoonotic pathogen, causes caseous lymphadenitis (CLA) in small ruminants and is responsible for considerable economic losses in the livestock industry worldwide. Macrophages play a pivotal role in the immunopathogenesis of CLA. However, the immunoregulatory mechanisms of macrophages against C. pseudotuberculosis remains poorly understood. In the present study, for the first time, the partial exoproteome of murine peritoneal macrophages infected with C. pseudotuberculosis was profiled and the differential expression of the identified proteins was analyzed. In macrophages, infection with C. pseudotuberculosis, rather than with heat-killed bacteria, induced release of diverse proteins. Three unconventional proteins: cofilin-1, peroxiredoxin-1, and galectin-3 were significantly expressed and released by infected macrophages into the culture supernatant. These proteins are involved in the host inflammatory response and may be responsible for the excessive inflammation of CLA. In C. pseudotuberculosis-infected macrophages, the release of cofilin-1 and peroxiredoxin-1 was predominant at later stages of infection, while the release of galectin-3 was independent of time. Taken together, the present work contributes to our understanding of the functional role of macrophage response to C. pseudotuberculosis infection.

Critical roles of NLRP3 inflammasome in IL-1ß secretion induced by Corynebacterium pseudotuberculosis in vitro.

Corynebacterium pseudotuberculosis is a prominent human and animal pathogen causing chronic inflammatory diseases. Interleukin-1ß (IL-1ß) is involved in the response to such pathogenic infections. However, the mechanism by which IL-1ß is secreted during C. pseudotuberculosis infection remains unclear. This study aimed to investigate the mechanism underlying IL-1ß secretion by macrophages infected with C. pseudotuberculosis. Herein, we firstly revealed that nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 (Casp1) play critical roles in IL-1ß secretion rather than IL-1ß precursor (pro-IL-1ß) expression in C. pseudotuberculosis-infected macrophages. Toll like receptor 4 (TLR4) is partially involved in IL-1ß secretion, while absent in melanoma 2 (AIM2) is not involved in IL-1ß secretion by C. pseudotuberculosis-infected macrophages. In addition, nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinases (p38 MAPK) inhibitors almost attenuated IL-1ß secretion, implying that NF-κB and p38MAPK pathway are involved in IL-1ß secretion in C. pseudotuberculosis-infected macrophages. Furthermore, C. pseudotuberculosis were significantly more numerous in Nlrp3-/-, Asc-/-, and Casp-1-/- macrophages than in WT macrophages at 24 h after infection (P < 0.05), indicating that NLRP3 inflammasome components limit C. pseudotuberculosis replication in macrophages. Together, these data provide novel insights into the mechanisms underlying IL-1ß secretion in C. pseudotuberculosis-infected macrophages and further the current understanding of the host pro-inflammatory immune response against this pathogen.

Sodium butyrate ameliorates Corynebacterium pseudotuberculosis infection in RAW264.7 macrophages and C57BL/6 mice.

Corynebacterium pseudotuberculosis (CP) infection in livestock has become highly difficult to control. To decrease the incidence of CP infection, the supplementation of feed with non-antibiotic antibacterial substances is a potential approach. The aim of this study was to assess the effects of sodium butyrate (NaB), a potential alternative to antibiotics, on CP infection in RAW264.7 macrophages and C57BL/6 mice. Our data showed that NaB (2 mM) significantly ameliorated CPinfection in RAW264.7 macrophages and decreased the bacterial load in the spleens of infected mice. By real-time PCR, we found that NaB induced significant decreases in zinc-dependent superoxide dismutase (sodC) and tip protein C (spaC) expression in CP from infected-RAW264.7 cells and in phospholipase D (pld) and spaC expression in CP from the spleens of infected mice. NaB treatment significantly up-regulated cathelicidin-related antimicrobial peptide (cramp) expression in spleens of mice infected with CP. Furthermore, NaB alleviated histopathological changes in spleens of CP-infected mice. In conclusion, NaB ameliorated CP infection in RAW264.7 macrophages and C57BL/6 mice, and these effects may be related to the modulation of sodC, spaC, pld, and cramp expression.