TcpC inhibits neutrophil extracellular trap formation by enhancing ubiquitination mediated degradation of peptidylarginine deiminase 4

TcpC is a multifunctional virulence factor of uropathogenic E. coli (UPEC). Neutrophil extracellular trap formation (NETosis) is a crucial anti-infection mechanism of neutrophils. Here we show the influence of TcpC on NETosis and related mechanisms. We show NETosis in the context of a pyelonephritis mouse model induced by TcpC-secreting wild-type E. coli CFT073 (CFT073wt) and LPS-induced in vitro NETosis with CFT073wt or recombinant TcpC (rTcpC)-treated neutrophils are inhibited. rTcpC enters neutrophils through caveolin-mediated endocytosis and inhibits LPS-induced production of ROS, proinflammatory cytokines and protein but not mRNA levels of peptidylarginine deiminase 4 (PAD4). rTcpC treatment enhances PAD4 ubiquitination and accumulation in proteasomes. Moreover, in vitro ubiquitination kit analyses show that TcpC is a PAD4-targetd E3 ubiquitin-ligase. These data suggest that TcpC inhibits NETosis primarily by serving as an E3 ligase that promotes degradation of PAD4. Our findings provide a novel mechanism underlying TcpC-mediated innate immune evasion.

Anti-peptidylarginine deiminase-4 antibodies at mucosal sites can activate peptidylarginine deiminase-4 enzyme activity in rheumatoid arthritis

Background Mucosal sites are hypothesized to play a role in the development of rheumatoid arthritis (RA). Since serum anti-peptidylarginine deiminase (PAD)4 antibodies, including a subset that cross-react with PAD3 (PAD3/4), are specific for RA and associate with severe disease, we sought to examine whether anti-PAD4 and anti-PAD3/4 antibodies were present in the lung and oral mucosa of subjects with RA and “at-risk” for RA. Methods We included 37 RA, 25 healthy control, and 46 subjects “at-risk” for RA based on familial RA and/or serum anti-citrullinated protein antibody (ACPA) positivity. Paired serum, sputum, and saliva were evaluated for anti-PAD4 and anti-PAD3/4 using immunoprecipitation and ACPA using ELISA. Immunoglobulins (Ig) were purified from representative samples, and their effect on citrullination of histone H3 by recombinant human PAD4 was measured by anti-citH3 immunoblot. Results Anti-PAD4 antibodies were detected in the serum of 6/37 (16.2%), sputum of 3/37 (8.1%), and saliva of 3/33 (9.1%) RA subjects and in the serum and sputum of 1/46 (2.2%) at-risk subjects. None of the healthy controls had anti-PAD4 antibodies at any site. Serum, sputum, and salivary anti-PAD4 antibodies were more prevalent in RA subjects with RA duration >2 years. Purified antibodies from representative anti-PAD4-positive and anti-PAD3/4-positive sputum were primarily of the IgA isotype and able to increase PAD4 enzymatic activity. Conclusions Anti-PAD4 antibodies are present in the sputum and saliva of a portion of RA patients and are infrequent in at-risk subjects. Importantly, the ability of anti-PAD4, and particularly anti-PAD3/4, antibodies in the sputum to enhance PAD4 enzymatic activity suggests that anti-PAD4 may play an active role in the RA lung.

Factors Associated with Promoted Proliferation of Osteosarcoma by Peptidylarginine Deiminase 4

Osteosarcoma is the most common type of bone malignancy, and the pathogenesis has not been entirely elucidated yet. An important deimination modification enzyme PADI4 (peptidylarginine deiminase 4) has attracted much attention in recent years for its important function in several kinds of human tumors. However, the role of PADI4 on osteosarcoma tumorigenesis remains largely unrevealed. Here, we first assessed the effect of PADI4 on osteosarcoma proliferation by the CCK8 method and colony formation assay. Ectopically expressing PADI4 positively regulates the colony formation capacity of both U2OS and Saos-2 cells. Furthermore, we explored the related mechanism and showed that PADI4 could stimulate Wnt/β-catenin and MEK/ERK signaling in both U2OS and Saos-2 cells. Then, we detected expression of PADI4 in human tissues of osteosarcoma and revealed that differential expression of PADI4 was associated with tumorigenesis of osteosarcoma. Last, we performed the in vivo experiment in nude mice and results also showed PADI4 could affect the tumor growth. In conclusion, this work revealed that PADI4 could upregulate the proliferation of osteosarcoma, mainly via the Wnt/β-catenin and MEK/ERK signaling pathway. This study gives us new insight into the regulation mechanism of osteosarcoma proliferation and highlights PADI4 as a promising target for osteosarcoma diagnosis and treatment.

Evaluation of peptidylarginine deiminase 4 and PADI4 polymorphisms in sepsis-induced acute kidney injury

SUMMARY BACKGROUND: The aim of this study is to evaluate the peptidylarginine deiminase 4 (PAD 4) concentration and PADI4 polymorphisms as predictors of acute kidney injury (AKI) development, the need for renal replacement therapy (RRT), and mortality in patients with septic shock. METHODS: We included all individuals aged ≥ 18 years, with a diagnosis of septic shock at ICU admission. Blood samples were taken within the first 24 hours of the patient's admission to determine serum PAD4 concentration and its PADI4 polymorphism (rs11203367) and (rs874881). Patients were monitored during their ICU stay and the development of SAKI was evaluated. Among the patients in whom SAKI developed, mortality and the need for RRT were also evaluated. RESULTS: There were 99 patients, 51.5% of whom developed SAKI and of these, 21.5% needed RRT and 80% died in the ICU. There was no difference between PAD4 concentration (p = 0.116) and its polymorphisms rs11203367 (p = 0.910) and rs874881 (p = 0.769) in patients in whom SAKI did or did not develop. However, PAD4 had a positive correlation with plasma urea concentration (r = 0.269 and p = 0.007) and creatinine (r = 0.284 and p = 0.004). The PAD4 concentration and PADI4 polymorphisms were also not associated with RRT and with mortality in patients with SAKI. CONCLUSION: PAD4 concentration and its polymorphisms were not associated with SAKI development, the need for RRT, or mortality in patients with septic shock. However, PAD4 concentrations were associated with creatinine and urea levels in these patients.