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

Ou, Qian; Fang, Jia-qi; Zhang, Zhe-sheng; Chi, Zhe; Fang, Jie; Xu, Di-yan; Lu, Kai-zhong; Qian, Meng-qing; Zhang, Da-yong; Guo, Jun-ping; Gao, Wei; Zhang, Na-ru; Pan, Jian-ping

doi:10.1038/s41467-021-23881-8

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Published: 09 June 2021

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

Qian Ou^1,2^na1,
Jia-qi Fang^1,2^na1,
Zhe-sheng Zhang^1,3^na1,
Zhe Chi^1,2,
Jie Fang^1,3,
Di-yan Xu^1,3,
Kai-zhong Lu^1,3,
Meng-qing Qian^1,3,
Da-yong Zhang^1,3,
Jun-ping Guo^1,3,
Wei Gao^1,3,
Na-ru Zhang^1,3 &
…
Jian-ping Pan ORCID: orcid.org/0000-0001-9636-1561^1,3

Nature Communications volume 12, Article number: 3481 (2021) Cite this article

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Abstract

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 (CFT073^wt) and LPS-induced in vitro NETosis with CFT073^wt 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.

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Introduction

Urinary tract infection (UTI) is one of the most common bacterial infections affecting 150 million people worldwide each year^1,2. It is estimated that the medical burden of UTI exceeds $6 billion annually³. These significant infections can be a serious health problem to reduce the quality of life. E. coli is the most common pathogen of UTIs, and ~80% of UTI are caused by E. coli³. Urinary pathogenic E. coli (UPEC) can specifically adhere to and implant into urothelial mucosal epithelial cells, leading to UTIs, including asymptomatic bacteriuria, urethritis, urocystitis, and pyelonephritis (PN)⁴.

It was found that most strains of UPEC secrete a protein containing the Toll/interleukin 1 receptor (TIR) domain or TcpC⁵. TcpC is an important virulence factor of UPEC that subverts the toll like receptor (TLR) signaling pathway by direct association of myeloid differentiation factor 88 (MyD88)^5,6. TcpC inhibits the bactericidal activity of macrophages and promotes the survival of UPEC in the host, which favors the pathogenicity of the pathogen^5,7.

The characteristic pathological changes of acute pyelonephritis are neutrophil infiltration⁵. It is well-known that neutrophils are important effector arms of the innate immune system. Neutrophils play crucial roles in clearance of extracellular bacterial infection by phagocytosis. Over the past years, increasing evidence has demonstrated that neutrophils have, in addition to phagocytosis, a special anti-infection mechanism that produces neutrophil extracellular traps (NETs), the process of NETs formation is referred to as NETosis⁸. NETs are three dimensional meshwork consisting of chromatin, antimicrobial components such as myeloperoxidase (MPO) and neutrophil elastase (NE)⁹. These fibrous networks can catch and eliminate bacteria, fungi, and viral particles as well^9,10,11. NETosis can be induced by a wide range of substances, including bacteria, lipopolyssacharide (LPS), phorbol-12-myristate-13-acetate (PMA), and viruses, etc¹². The specific requirements for NETs formation depend on the stimulus, but histone citrullination mediated by the peptidylarginine deiminase 4 (PAD4) has been demonstrated to be an essential step in NETosis^9,13,14,15.

PAD4 is primarily expressed in neutrophils, it catalyzes the conversion of histone arginine to citrullination¹⁶. Citrullination is a post-translational modification that plays an important role in many physiological processes such as cell differentiation and renewing, but abnormal citrullination, however, can lead to the development of diseases, including autoimmune diseases, cancers, and cardiovascular diseases¹³. Numerous stimuli have been confirmed to activate PAD4 and NETosis^17,18.

There are reactive oxygen species (ROS)-dependent and ROS-independent NETosis. ROS-dependent NETs release requires the production of ROS by NADPH oxidase¹⁹. ROS stimulate MPO to trigger the activation and translocation of NE from the azurophilic granules to the nucleus where NEs digest nucleosomal histones and promote chromatin relaxation, which favors the citrullination of histone (CitH3) by PAD4²⁰. Citrullination of histone leads to chromatin decondensation, which causes entropic chromatin swelling⁹. Subsequently, MPO associate with chromatin and, synergistically with NE and PAD4, promote massive chromatin decondensation¹⁵. In association with diverse granular and cytoplasmic proteins, decondensed chromatin is eventually released into the extracellular space¹⁷.

Although TcpC was demonstrated to play a crucial role in the immune evasion of macrophage-mediated innate immunity^5,6,21, its influence on NETosis remains elusive. In the present study, we show that TcpC suppresses NETosis by promoting degradation of PAD4 through ubiquitin proteasome pathway. Our findings provide not only a novel mechanism underlying TcpC-mediated innate immune evasion, but also new insight into the pathogenicity of pathological microbes.

Results

NETosis is inhibited in PN mouse model induced by TcpC-secreting wild-type CFT073 (CFT073^wt) and in CFT073^wt-treated neutrophils

The PN mouse models were induced by urethral instillation of CFT073^wt or tcpc-knock out CFT073 mutant (CFT073^Δtcpc) as described previously^5,21,22. Abscesses in kidneys could be observed in CFT073^wt-induced PN mouse model, no abscess was present in kidneys from CFT073^Δtcpc-infected mice (Fig. 1a, b). Accordingly, significantly increased infiltrates of neutrophil were observed in kidneys from the CFT073^wt group compared with those in kidneys from the CFT073^Δtcpc group (Fig. 1c). These data demonstrate again that TcpC is a crucial virulence factor of UPEC. In situ NETosis in kidneys from PN mouse model induced by CFT073^wt was significantly inhibited when compared with that in kidneys from the CFT073^Δtcpc infected group (Fig. 1d, e). To further confirm that this inhibition of in situ NETosis was caused by CFT073^wt, human neutrophils were separately co-cultured in transwell with CFT073^wt or CFT073^Δtcpc at a multiplicity of infection (MOI) = 100 for 12 h. 4 h before the end of the co-culture, 1 μg/ml LPS was added into the corresponding wells and NETosis was examined. NETosis in the CFT073^wt + LPS group was significantly inhibited compared with the LPS or CFT073^Δtcpc + LPS groups. No significant inhibition of NETosis was observed in the group of CFT073^Δtcpc + LPS compared with the LPS treatment group (Fig. 1f, g). These data demonstrate that CFT073^wt inhibits NETosis.

**Fig. 1: NETosis is inhibited in Kidneys of CFT073^wt induced pyelonephritis murine model and in CFT073^wt-treated neutrophils.**

Recombinant TcpC protein inhibits NETosis

Since CFT073^wt treatment caused inhibition of NETosis, recombinant TcpC protein (rTcpC) was prepared and its influence on NETosis was examined. The entry of rTcpC into neutrophils was verified by confocal and western blot analyses. rTcpC could enter the cells gradually with the treatment time (Supplementary Fig. 1a–c). Endocytosis is the process by which cells actively internalize molecules and surface proteins via an endocytic vesicle²³. In order to confirm which endocytic vesicle mediated the entry of rTcpC, the cells were treated by clathrin-mediated endocytosis inhibitor dynasore^24,25 and caveolin-dependent endocytosis inhibitor Methyl-β-cyclodextrin (MCD)^24,26 for 30 min before treatment with rTcpC respectively. MCD but not dynasore blocked the entry of rTcpC into neutrophils, demonstrating that rTcpC could get into neutrophils through caveolin-mediated endocytosis (Supplementary Fig. 1d–f). LPS and PMA-induced ROS-dependent NETs formation, which is in line with previous report²⁷. Although TAK-242, a TLR4 inhibitor²⁸, did not influence PMA-induced NETosis, it completely blocked the LPS-induced NETosis. In the presence of rTcpC, both the LPS- and PMA-induced NETosis were significantly suppressed (Fig. 2a–c). In ROS-dependent NETosis, oxidative stress occurs in neutrophils. NADPH oxidase catalyzes the intracellular O₂ to generate ROS that destroy pathogenic bacteria and promote translocation of MPO and NE to the nucleus, where NE proteolytically processes histones to disrupt chromatin packaging and MPO binds decondensing chromatin²⁹, meanwhile expression of IL-1β, IL-6, TNF-α, and other proinflammatory factors is enhanced to achieve antibacterial effect^30,31. The influence of rTcpC on ROS and proinflammatory cytokine production was examined. In the presence of rTcpC, LPS-induced production of ROS was profoundly inhibited while rTcpC itself did not affect the generation of ROS (Fig. 2d–f). Upon stimulation of LPS, enhanced expression of IL-1β, IL-6, and TNF-α occurred in neutrophils. However, rTcpC substantially inhibited this LPS-induced production of proinflammatory cytokines at both protein and mRNA levels (Fig. 2g, h). Moreover, when neutrophils were treated by CFT073^wt, results with same trend were also obtained (Supplementary Fig. 2a, b). These data suggest that TcpC inhibits ROS-dependent NETosis.

rTcpc not only inhibits the citrullination of chromatin histone, but also affects the transcription of related genes in nucleus

During the ROS-dependent NETosis, NE digested histones are citrullinated by PAD4, resulting in chromatin decondensation^17,32. Dynamic analyses of the influence of rTcpC on chromatin decondensation were carried out as described in previous reports^33,34. Decondensation of chromatin increased along with the time in the LPS group. In the presence of rTcpC, however, the LPS-induced chromatin decondensation was significantly inhibited (Fig. 3a–c). Since CitH3 is also one of the indicators of chromatin decondensation³⁵, CitH3 levels were dynamically analyzed. As shown in Fig. 3d, e, CitH3 levels also increased along with the time in LPS group, but this LPS-induced increase of CitH3 was suppressed significantly in the LPS + rTcpC group. Furthermore, rTcpC itself dynamically suppressed the levels of CitH3. Taken together, these data further indicate that rTcpC can inhibit chromatin depolymerization to form NETs. To determine the relevance of transcriptional activity during NETosis, a genome-wide transcriptomics analysis of neutrophils treated with LPS and/or rTcpC was conducted. rTcpC promoted gene transcripts down-regulation more than up-regulation (Fig. 4a). A large number of gene transcripts related to superoxidation and cytokines of ROS-dependent NETosis were inhibited (Fig. 4b), but ubiquitination-related gene transcripts were significantly up-regulated in rTcpC-treated neutrophils (Fig. 4c). Therefore, TcpC not only significantly inhibits the chromatin depolymerization, but also affects gene transcription during NETosis.

**Fig. 3: rTcpc inhibits chromatin decondensation in NETosis.**

**Fig. 4: rTcpC affects the transcription of genes in neutrophils.**

rTcpC dose-dependently and dynamically decreases protein but not mRNA levels of PAD4 in neutrophils

Because PAD4-mediated histone citrullination is the essential step in NETs formation^9,13,36, dose-dependent and dynamic analyses of the influence of rTcpC on PAD4 expression were examined. In accordance with the results of transcriptomics analyses (Fig. 4b), upon LPS stimulation, both mRNA and protein levels of PAD4 increased significantly (Fig. 5a–f). Although rTcpC treatment did not affect LPS-induced mRNA expression of PAD4 (Figs. 4b and 5a, d), rTcpC itself could dose-dependently inhibit protein levels of PAD4 compared with the control group (Fig. 5b, c). Importantly, dose-dependent and dynamical inhibition of PAD4 protein levels were observed in the LPS + rTcpC group when compared with the LPS group (Fig. 5b, c and e, f). Furthermore, when neutrophils were treated with CFT073^wt and CFT073^Δtcpc, similar results were obtained (Supplementary Fig. 3a, b) These data show that TcpC decreases protein but not mRNA levels of PAD4 in neutrophils.

**Fig. 5: rTcpC inhibits protein but not mRNA levels of PAD4 in LPS-treated neutrophils.**

rTcpC is an E3 ubiquitin ligase that promotes ubiquitination of PAD4

Ubiquitination is an important protein post-translational modification pathway in eukaryotic cells³⁷. Ubiquitination is mediated by ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzyme cascade reactions³⁸. In the process of ubiquitination, E3 binds not only to E2, but also specifically to substrate proteins³⁹. In human genome, >600 annotated E3 ligases have been found, they are divided into three types of domains: RING, HETC, and RBR⁴⁰. In the process of ubiquitination by HETC E3 ligase, the conserved cysteine (Cys) residue at N-terminus of the enzyme is necessary^37,41. HECT E3 ligase contains tryptophan-tryptophan (WW) domains that bind to proline-tyrosine (P-Y) motif containing substrates^42,43,44. In our previous studies, we have demonstrated for the first time that TcpC is a HECT family E3 ubiquitin ligase that can promote ubiquitination of MyD88⁴⁵. To examine whether TcpC can also serve as the E3 that promote PAD4 ubiquitination, we did bioinformatics analyses at first and found that human and mouse PAD4 contain P-Y motifs (Fig. 6a, b), suggesting that PAD4 is probably one of the TcpC E3 enzyme substrates. Then, the influence of rTcpC on ubiquitination of PAD4 was examined by co-immunoprecipitation and immunoblotting assays. Although rTcpC treatment decreased the PAD4 protein level in lysates of neutrophils, the ubiquitination of PAD4 was significantly enhanced compared with the LPS-treated group. When the proteasome activity was blocked by MG-132, the ubiquitination of PAD4 was enhanced further in the rTcpC-treated groups (Fig. 6c, d). These data demonstrate that rTcpC promotes ubiquitination of PAD4. At last, the PAD4-targeted E3 activity of rTcpC was further examined by in vitro ubiquitination kit analyses which mimic the ubiquitination process as previously described^46,47. In these assays, the GST-MuRF (E3) and its substrate S5a were served as the positive control. When the E3 in the kit was replaced by rTcpC and lysates of neutrophils and commercially available human recombinant PAD4 (Hu-rPAD4) used as the substrates, substantially enhanced ubiquitination of PAD4 could be observed. Furthermore, this rTcpC mediated enhanced ubiquitination of PAD4 could be abrogated when the E3 inhibitor Nutlin-3 was employed (Fig. 6e). These data firmly demonstrate that TcpC is an E3 ubiquitin ligase which promotes ubiquitination of PAD4.

**Fig. 6: TcpC is an E3 ligase that promotes ubiquitination of PAD4.**

TcpC promotes accumulation of PAD4 in proteasomes

In the process of ubiquitination, E3 ubiquitin ligase transports substrate protein to proteasome for degradation^48,49. In order to verify if rTcpC treatment leads to accumulation of PAD4 in the proteasomes, confocal microscopy was employed to examine co-localization of PAD4 with the proteasome marker, non-ATPase 2 (PSMD2)^50,51. Dynamic analyses of PAD4 accumulation in proteasomes showed that, in the absence of MG-132, co-localization of PAD4 and PSMD2 increased 60 min later, and reached a plateau 120 min after rTcpC treatment (Fig. 7a–c). In the presence of MG-132, however, co-localization of PAD4 with PSMD2 increased steadily after 60 min treatment with rTcpC (Fig. 7d–f). These data suggest that rTcpC promotes PAD4 accumulation in proteasomes in LPS-stimulated neutrophils.

**Fig. 7: rTcpC treatment leads to accumulation of PAD4 in proteasomes.**

C12S point mutant rTcpC-C12S loses the ability to inhibit NETosis

In our previous studies, we have demonstrated that C12 is the key amino acid to retain the E3 activity of TcpC⁴⁵, the influence of C12S point mutant rTcpC-C12S on degradation of PAD4 and LPS-induced NETosis was examined. Compared with the rTcpC prototype, rTcpC-C12S showed significantly attenuated activity to promote degradation of PAD4 (Fig. 8a, b) and to inhibit LPS-induced NETosis (Fig. 8c–e), suggesting further that the inhibitory effect of TcpC on NETosis is mediated by promoting degradation of PAD4.

**Fig. 8: rTcpC-C12S loses ability to promote degradation of PAD4 and inhibit LPS-induced NETosis.**

Discussion

Although the pathophysiological roles of NETosis have not been fully understood, NETosis is an important anti-infection mechanism of neutrophils. In ROS-dependent NETosis, NADPH oxidase complex catalyzes O₂ to produce ROS which activate transcription and activation of downstream NETosis essential enzyme genes⁵². Among them, PAD4 is of the most important. The conversion of histone arginine into citrulline catalyzed by PAD4, which reduces the positive charges of histone and causes chromatin entropic swelling, is the most important event that drives the release of NETs⁹. After chromatin entropic swelling the process of NET formation cannot be reversed. Therefore, histone citrullination can be regarded as the “turning-point” in NETosis. Although the exact signaling pathway that regulates PAD4 expression has not been clarified, manipulating PAD4 to inhibit NETosis might be a vital strategy for pathogens to evade effector function of neutrophils. On the one hand, it was found that TcpC impedes TLR signaling pathway, hereby inhibiting macrophage-mediated innate immunity^5,21. On the other hand, TcpC promotes kidney cells to produce CXCL2 chemokine which attracts neutrophil infiltration in kidney, the characteristic pathological changes of acute PN²². But the influence of TcpC on NETosis has been remained elusive.

In the present study, we show that in situ NETosis in kidneys of PN murine model induced by CFT073^wt was significantly inhibited compared with that in CFT073^Δtcpc infected mouse (Fig. 1d, e). In line with the results of in situ NETosis, LPS-induced in vitro NETosis was also suppressed significantly in CFT073^wt- (Fig. 1f, g) or rTcpC-treated (Fig. 2a–c) human neutrophils. These data demonstrate that TcpC suppresses NETosis.

During ROS-dependent NETosis, ROS is produced to kill pathogens and stimulate NE to digest histone for citrullination by PAD4 in nucleus^20,53. Furthermore, proinflammatory cytokines such as IL-1β, IL-6, TNF-α are up-regulated when neutrophils undergo NETosis⁵⁴. In order to explore the mechanisms underlying TcpC-mediated inhibition of NETosis, we examined the influence of TcpC on expression of ROS and proinflammatory cytokines as well as PAD4. We found that rTcpC profoundly inhibited LPS-induced production of ROS (Fig. 2d–f) and expression of proinflammatory cytokines at both protein and mRNA levels (Fig. 2g, h) in neutrophils. But unlike the influence on proinflammatory cytokines, rTcpC suppressed, in a dose- and time-dependent manner, protein (Fig. 5b, c, e, f) but not mRNA (Fig. 5a, d) levels of PAD4, suggesting that rTcpC decreases PAD4 protein by post-transcriptional modification. Because level of CitH3 is determined by PAD4 and rTcpC decreases concentration of PAD4 in neutrophils, it is no wonder that rTcpC itself can dynamically suppress levels of CitH3 (Fig. 3d).

It has been demonstrated that ubiquitination plays an important role in protein post-translational modification in eukaryotic cells that regulates many biological processes^{39,48,55,56,57}, and in many aspects of the immune system, including innate and adaptive immunity and antimicrobial autophagy. In addition, increasing evidence indicates that microbial pathogens exploit the ubiquitin pathway to evade the host immune system^58,59. Furthermore, our previous studies have demonstrated that TcpC is an E3 ubiquitin ligase of HECT family⁴⁵, the influence of rTcpC on ubiquitination of PAD4 was examined. Our data clearly show that rTcpC treatment caused significantly enhanced ubiquitination of PAD4 in neutrophils (Fig. 6c, d). Bioinformatics analyses showed that in the amino acid sequence of human (Fig. 6a) and mouse (Fig. 6b) PAD4 there are P-Y motifs of the E3 ligase substrates, suggesting that TcpC might be a PAD4-targetd E3 ubiquitin ligase. This speculation was further verified by in vitro ubiquitination kit assays. rTcpC could serve as the E3 to promote ubiquitination of both proteins in the lysate of neutrophils and Hu-rPAD4, and this E3 activity of rTcpC could be abrogated by the E3 inhibitor Nutlin-3 (Fig. 6e). These data confirm that TcpC is a PAD4-targeted E3 ubiquitin ligase.

To examine whether rTcpC treatment promotes accumulation of PAD4 in proteasome. Co-localization of PAD4 with the proteasome maker PSMD2 in neutrophils was observed by confocal microscopy. rTcpC treatment led to increased accumulation of PAD4 in proteasomes in LPS-treated neutrophils (Fig. 7a–c). Particularly, when the proteasome activity was blocked by MG-132, accumulation of PAD4 in proteasome increased steadily during the whole treatment period (Fig. 7d–f). These data confirm that TcpC promotes degradation of PAD4 through ubiquitin-proteasome pathway.

At last, we examined the influence of rTcpC-C12S on degradation of PAD4 and LPS-induced NETosis. In line with the changes of activity in degrading PAD4 (Fig. 8a, b), rTcpC-C12S showed significantly decreased activity to inhibit the LPS-induced NETosis compared with the rTcpC prototype (Fig. 8c–e). These data suggest that the inhibitory effect of TcpC on NETosis is primarily mediated by promoting ubiquitinated degradation of PAD4.

In summary, this study demonstrated that TcpC inhibits NETosis by serving as a PAD4-targeted E3 ubiquitin ligase that promote degradation of PAD4 via ubiquitin-proteasome pathway (Fig. 9). Our findings provide not only a novel mechanism by which TcpC-secreting UPECs evade host innate immune response, but also new clues to clarify the pathogenicity of other microbial pathogens.

**Fig. 9: Schematic diagram of the mechanisms underlying TcpC-mediated inhibition of NETosis.**

Methods

Ethics statement

Animal experiments were performed in accordance with the National Regulations for the Administration of Experimental Animals of China (1988–002) and the National Guidelines for Experimental Animal Welfare of China (2006–398). All the animal experimental protocols were approved by the Ethics Committee for Animal Experiment of Zhejiang University City College School of Medicine and fully informed consent of all healthy blood donors was obtained after clearing the possible consequences of the study.

Reagents

Fetal bovine serum (FBS, Gibco, USA, 10100147), RPMI 1640 medium (Gibco, USA, 22400105), LPS (Sigma, USA, L2630), Sytox Green (Invitrogen, USA, S7020), IL-1β, IL-6, TNF-α ELISA Kit (eBioscience, USA, KAC1211, EH2IL6, KHC3011), DCFH-DA probe dye (Thermo, USA, I36007), BCA Protein Concentration Kit (Beyotime, China, P0012S), rabbit anti-PAD4-IgG (Abcam, UK, ab96758), rabbit anti-MPO-IgG (Abcam, UK, ab208670), rabbit anti-ubiquitin-IgG (Abcam, UK, ab134953), anti-GAPDH-IgG (Abcam, UK, ab181602), Anti-PSMD2-IgG (Abcam, UK, ab125914), Alexa fluor 647 nm goat anti-rabbit IgG (Invitrogen, USA, A-21244) and Alexa fluor 488 nm goat anti-mouse IgG fluorescent secondary antibody (Invitrogen, USA, A-11001), DAPI (Invitrogen, USA, D1306), Ly-6G/Ly-6C (PE, Thermo, USA, 14-5931-81), CD11b (FITC, Thermo, USA, 11-0112-41), Nutlin-3 (Selleckchem, USA, S1061), MG-132 (Selleckchem, USA, S2619), primer (Invitrogen, USA), RNAiso Plus (TaKaRa, Japan, 9109), PrimeScript^TM RT reagent Kit with gDNA Eraser (TaKaRa, Japan, RR047A), TB Green TM Premix Ex Taq^TM (TaKaRa, Japan, RR071A), Pierce^TM Protein A/G magnetic beads (Thermo, USA, 88802), MuRF1 Ubiquitin Ligase Kit-S5a Substrate (Boston Biochem, USA, K-102), rabbit anti-Ly-6G-IgG (Abcam, UK, ab238132), Alexa fluor 546 nm goat anti-rabbit IgG (Thermo, USA, A-11035), rabbit anti-CitH3-IgG (Abcam, UK, ab5103), Alexa fluor 488 nm goat anti-rabbit IgG fluorescent secondary antibody (Invitrogen, USA, A-11008), ProLong Diamond Antifade Mountant (Thermo, USA, P36990), Dynasore (MCE, USA, HY-15304), TAK-242 (MCE, USA, HY-11109), Methyl-β-cyclodextrin (MCE, USA, HY-101461), and recombinant TcpC protein and rabbit anti-TcpC-IgG (made in our laboratory).

Mouse PN models and in situ NETosis examination

Female C57BL/6 mice, 6–8 weeks of age, were provided by Shanghai Slack Laboratory Animals Co., Ltd (Shanghai, China). PN murine models were induced by transurethral instillation of CFT073^wt or CFT073^Δtcpc as described in our previous report²². Normal saline-treated mice served as the control group. In all, 72 h later, the model mice were sacrificed and kidneys were removed for pathological and in situ NETosis examinations as described previously⁶⁰. After the sections were fixed and tissue antigen repairing, the fixed tissues were incubated with rabbit anti-Ly-6G-IgG, rabbit anti-CitH3-IgG, and rabbit anti-MPO-IgG (Abcam, UK, 1:200 dilution) overnight at 4 °C and stained with different fluorescent secondary antibody (Alexa fluor 546 nm goat anti-rabbit IgG, Alexa fluor 488 nm goat anti-rabbit IgG and Alexa fluor 647 nm goat anti-rabbit IgG, 1:500 dilution) in dark respectively, then added ProLong Diamond Antifade Mountant (Thermo, USA) and DAPI to each coverslip, the coverslips were placed on the slides and confocal microscope images were taken.

Histological examination

Kidneys were fixed in freshly prepared 4% paraformaldehyde soon after dissection and incubated overnight at 4 °C. The fixed tissues were incubated in 15% sucrose at 4 °C and washed in 25% ice cold sucrose at 4 °C. Tissues were then frozen in isopentane at −80 °C. Cryostat sections were made, mounted onto poly-d-lysine-coated glass slides and stained with htx-eosin²¹.

Isolation of human neutrophils

EDTA-anticoagulated peripheral blood from healthy donors was used to isolate neutrophils using EasySep Direct Human Neutrophil Isolation Kit (StemCell Technologies, USA) following the manufacturer’s instructions. Erythrocytes were lysed with red blood cell lysis buffer and washed with PBS. Subsequently, purity of the human neutrophils was evaluated by FACS using antibodies against Ly-6G/Ly-6C (1:500 dilution, Ly-6G/Ly-6C monoclonal antibody, PE, Thermo, USA) and CD11b (1:500 dilution, CD11b monoclonal antibody, FITC, Thermo, USA). Neutrophil purity was >93% (Supplementary Fig. 4).

Transwell co‑culture

Neutrophils (1 × 10⁶) were separately co-cultured for 12 h, in transwell (Corning, USA) at a multiplicity of infection (MOI) = 100, with CFT073^wt or CFT073^Δtcpc. Neutrophil cultures without E. coli treatment served as the control group. In all, 4 h before the end of the culture, 1 μg/ml LPS was added into corresponding wells. Then the cells were harvested and subjected to confocal microscopy to examine NETosis.

rTcpC treatment of neutrophils

In all, 1 × 10⁶ neutrophils were treated, in the presence or absence of 1 μg/ml LPS, 100 nM PMA, with or without 20 μM TAK-242, 4 μg/ml rTcpC for 210 min. Neutrophils cultured in medium alone was set as control group. Culture supernatants were collected for measurement of proinflammatory cytokines and cells were subjected to detection of NETosis, qRT-PCR analyses of cytokine and pad4 mRNA and western blot analyses of PAD4, respectively.

Confocal microscopy to examine NETosis

The above treated neutrophils were fixed by 4% paraformaldehyde in PBS overnight at 4 °C, then permeabilized with 0.1% TritonX-100 in PBS for 20 min, and washed three times with PBS. Cells were blocked with 1% BSA, 0.1% Tween 20 in PBS for 2 h at room temperature, and then incubated with rabbit anti-MPO antibodies (Abcam, UK, 1:200 diluted in 0.1% Tween 20 in PBS) overnight at 4 °C. Next, neutrophils were gently washed three times with PBS and incubated in the dark with goat anti-rabbit IgG antibody (Alex Flour 647 nm, 1:500 diluted in the PBS) for 2 h at room temperature. Before staining with 100 ng/ml DAPI, neutrophils were incubated with rabbit anti-CitH3 antibodies (Abcam, UK, 1:200 diluted in 0.1% Tween 20 in PBS) overnight at 4 °C, then washed three times and incubated with Alexa fluor 488 nm goat anti-rabbit IgG fluorescent secondary antibody, finally washed with PBS and ProLong Diamond Antifade Mountant (Thermo, USA) was added to each coverslip, the coverslips were placed on the slides, and then observed with a confocal fluoresce microscope (FV3000 Olympus, Japan). Images were analyzed with the ImageJ software from National Institutes of Health^33,61.

Quantification of NETs

NETs formation was quantified by detecting DNA release spectrophotometrically with the DNA-binding dye Sytox Green as previously described³³. Briefly, neutrophils were treated for 210 min with or without 4 μg/ml rTcpC in the presence or absence of 1 μg/ml LPS, at the beginning of the incubation 500 nM Sytox Green were added into each well. Fluorescence values were measured every 30 min with the 488 nm excitation. Scale was set and threshold adjusted to define the area of fluorescent DNA. Total area of all fluorescent particles indicated the amount of NETs formation. Unstimulated neutrophils had an area of 62 ± 6.1 µm². Thus only particles >70 µm² were considered NETs.

Confocal microscopy to detect ROS production

In all, 1 × 10⁶/ml neutrophils were treated with or without 4 μg/ml TcpC and 1 μg/ml LPS for 210 min. Cells were harvested and resuspended in 1 ml serum-free culture medium. Cells were stained with 1 μl DCFH-DA probe dye (Invitrogen, USA) and 100 ng DAPI (Invitrogen, USA) in dark for 20 min at 37 °C. After three times wash with PBS, cells in each group were adjusted to a density of 1 × 10⁶ cells/ml. In total, 10 μl cells were put on a Poly-d-Lys (Sigma, USA) pre-coated coverslip. In all, 1 min later the coverslip was put on a slide and observed with a confocal microscope (FV3000 Olympus, Japan). Images were analyzed with the ImageJ software.

ELISA

Concentrations of IL-1β, IL-6, and TNF-α in supernatants of neutrophils were measured by ELISA according to the instructions of the manufacturer (eBioscience, USA).

qRT-PCR

Total RNA of treated neutrophils was extracted by RNAiso Plus (TaKaRa, Japan), and cDNA was synthesized by reverse transcription using PrimeScript^TM RT reagent kit with gDNA Eraser (TaKaRa, Japan). The mRNA levels of IL-1β, IL-6, TNF-α, and PAD4 in neutrophils were detected by qRT-PCR, and β-actin was set as an internal reference. The primers are listed in Table 1. The Cq value was calculated by 2^−ΔΔCT method.

Table 1 Information of primers used in this study.

Full size table

Flow cytometry analyses of ROS

The ROS generation during NETosis was detected by FACS using DCFH-DA (Invitrogen, USA). Before stimulation, neutrophils were preloaded with DCFH-DA for 20 min, cells were washed three times with PBS. Then, neutrophils were treated with LPS or rTcpC for 210 min. Finally, the fluorescence signals of DCFH-DA were read at FITC channel on a BD FACSCalibur.

Western blot analyses of PAD4

Proteins in supernatants of lysates from treated neutrophils were prepared. After 12% SDS-PAGE, proteins were electro-transferred onto PVDF membranes. Proteins were probed with rabbit anti-PAD4 IgG (1:10000 dilution, Abcam, UK) as primary antibody and IRDye® 680RD goat anti-rabbit-IgG (H + L) (1:5000 dilution, LI-COR, USA) as secondary antibody. Images were developed using ODYSSEY® CLx Infrared Imaging System (LI-COR). Gray scale values of bands were analyzed by ImageJ software.

Dynamic analyses of CitH3 levels

Neutrophils (1 × 10⁶) were treated with or without 4 μg/ml rTcpC for the indicated time in the absence or presence of 1 μg/ml LPS. Proteins in supernatants of lysates from treated neutrophils were prepared. After 12% SDS-PAGE, proteins were electro-transferred onto PVDF membranes. Proteins were probed with rabbit anti-CitH3 IgG (1:5000 dilution, Abcam, UK) as primary antibody and IRDye® 680RD goat anti-rabbit-IgG (H + L) (1:5000 dilution, LI-COR, USA) as secondary antibody. Images were developed using ODYSSEY® CLx Infrared Imaging System (LI-COR). Gray scale values of bands were analyzed by ImageJ software.

Co-immunoprecipitation and immunoblotting to detect ubiquitination of PAD4

The treated neutrophils were washed twice with PBS and were lysed with RIPA. After centrifugation at 12,000×g for 30 min at 4 °C, the supernatants were collected and protein concentrations were determined by a BCA Protein Assay Kit (Beyotime, China). Proteins in cell lysates were then immunoprecipitated with the rabbit anti-PAD4 IgG (1:1000 dilution, Abcam, UK) using Pierce™ Protein A/G Magnetic Beads (Thermo Scientific) and the released proteins (rTcpC, PAD4 and ubiquitin) were detected by immunoblotting. In detection of ubiquitin and rTcpC, rabbit anti-ubiquitin IgG (1:1000 dilution, Abcam, UK) and the self-made rabbit anti-rTcpC IgG were used as the primary antibody respectively, and IRDye® 680RD goat anti-rabbit-IgG (H + L) (1:5000 dilution, LI-COR, USA) as secondary antibody.

In vitro ubiquitination kit assays

MuRF1 Ubiquitin Ligase Kit-S5a Substrate (Boston Biochem, USA) was used to detect ubiquitination of PAD4 and determine the E3 ligase function of rTcpC. When the E3 was replaced by rTcpC, proteins in the neutrophil lysates and rPAD4 (Abcam, UK) were used as the substrates. All the experiments were performed according to instructions of the manufacturer. Ubiquitination of PAD4 was detected by western blotting as described above.

Confocal microscopy to examine co-localization of PAD4 with PSMD2

Neutrophils were treated with or without 4 μg/ml rTcpC and 1 μg/ml LPS for the indicated time, cells were collected and fixed with 4% paraformaldehyde overnight at 4 °C. The fixed cells were permeabilized with 0.1% TritonX-100 in PBS for 20 min, and washed three times with PBS. After blocked with 1% BSA, 0.1% Tween 20 in PBS for 2 h at room temperature, cells were stained with rabbit anti-PAD4 IgG (1:100 dilution, Abcam, UK) overnight at 4 °C, and then probed with the secondary antibody Alex Flour 647 goat anti-rabbit IgG fluorescence antibody (1:500 dilution, Invitrogen, USA) for 2 h at room temperature in dark. After three times wash with PBS, the cells were also probed with rabbit anti-PSMD2 (1:100 dilution, Abcam, UK) and Alexa Fluor 488 goat anti-rabbit IgG fluorescent antibody (1:200 dilution, Invitrogen, USA) as primary and secondary antibodies respectively. Neutrophils were finally washed with PBS and stained with 100 ng/ml DAPI for 30 min. Co-localization of PAD4 with PSMD2 was observed by confocal microscopy (Olympus) (495/519, 652/668 or 485 nm excitation/emission wavelengths for Alexa Fluor 488, Alexa Fluor 647 or DAPI detection). Percentages and yellow FI reflecting the co-localization were analyzed by ImageJ software⁶². To examine the influence of MG-132 on rTcpC induced accumulation of PAD4 in proteasomes, neutrophils were treated with 1 μM MG-132 for 30 min before treatment with 4 μg/ml rTcpC for the indicated time, and then the treated cells were subjected to confocal microscopy as described above.

Transcriptome sequencing

Neutrophils (1 × 10⁶ cells) were treated for 120 min with 1 μg/ml LPS, 4 μg/ml rTcpC, 4 μg/ml rTcpC + 1 μg/ml LPS, respectively, and untreated neutrophils were served as the control group. Cells were collected (1500 rpm, 10 min) and frozen at −80 °C with RNAiso Plus (TaKaRa, Japan). Total RNA was extracted and a genome-wide transcriptomics analysis was conducted (LC-BIOTECHNOLOGIS (HANGZHOU) CO., LTD). The differentially expressed mRNAs were selected with fold change >2 or fold change <0.5 and p-value < 0.05 by R package edgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) or DESeq2 (http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html).

Bioinformatics analyses

The structure and functional domains in PAD4 of human and mouse were analyzed using NCBI-Batch CD-Search software⁶³.

Statistics and reproducibility

Data shown are mean ± SD of three independent experiments. Images were analyzed by ImageJ software. Dunnett and Mann–Whitney tests of SPSS 22.0 were used for variance analysis. Data from quantification of NETs formation were analyzed by Mann–Whitney test. p < 0.05 is considered to be statistically significant and p < 0.01 is extremely significant. NS is considered to be not significant. All source data are provided in separate Raw Data file of Source Data file.

Reporting summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper.

Data availability

Data supporting the findings of this manuscript are available from the corresponding author upon request. Full scan images of the Gels and Blots and Source data for figures and numbers are provided with this paper. We deposited our data including the transcriptomic data in GEO DataSets, the accession number is GSE173807. Source data are provided with this paper.

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Acknowledgements

We thank Professor Jianguo Xu (National Institute for Communicable Disease Control and Prevention, China) for providing the CFT073 strain of UPEC, and all blood donors. This work was supported by grants from the National Natural Science Foundation of China (81671613 to J.P.P.), the Postdoctoral Science Foundation of China (2020M671747 to J.Q.F), the Natural Science Foundation of Zhejiang Province (LQ20H100001 to J.Q.F.), and the Science and Technology Bureau of Zhejiang Province (LGD20H100001 to J.F.).

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These authors contributed equally: Qian Ou, Jia-qi Fang, Zhe-sheng Zhang.

Authors and Affiliations

Institute of Translational Medicine, Zhejiang University City College, Hangzhou, P. R. China
Qian Ou, Jia-qi Fang, Zhe-sheng Zhang, Zhe Chi, Jie Fang, Di-yan Xu, Kai-zhong Lu, Meng-qing Qian, Da-yong Zhang, Jun-ping Guo, Wei Gao, Na-ru Zhang & Jian-ping Pan
Department of Basic Medical Sciences, Zhejiang University School of Medicine, Hangzhou, P. R. China
Qian Ou, Jia-qi Fang & Zhe Chi
Department of Clinical Medicine, Zhejiang University City College School of Medicine, Hangzhou, P. R. China
Zhe-sheng Zhang, Jie Fang, Di-yan Xu, Kai-zhong Lu, Meng-qing Qian, Da-yong Zhang, Jun-ping Guo, Wei Gao, Na-ru Zhang & Jian-ping Pan

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Qian Ou
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Q.O., J.Q.F., and J.P.P. contributed to the design of this study. Q.O., J.Q.F., Z.S.Z., Z.C., J.F., D.Y.X., K.Z.L., M.Q.Q, D.Y.Z, J.P.G., W.G., and N.R.Z. performed the experiments. Q.O., J.Q.F., Z.S.Z., and J.P.P analyzed the experimental data. J.P.P., Q.O., and J.Q.F. wrote the manuscript. Q.O. and J.Q.F. created Fig. 9. All authors reviewed and approved the final version of the manuscript.

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Ou, Q., Fang, Jq., Zhang, Zs. et al. TcpC inhibits neutrophil extracellular trap formation by enhancing ubiquitination mediated degradation of peptidylarginine deiminase 4. Nat Commun 12, 3481 (2021). https://doi.org/10.1038/s41467-021-23881-8

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Received: 01 August 2020
Accepted: 24 May 2021
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DOI: https://doi.org/10.1038/s41467-021-23881-8

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Abstract

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Introduction

Results

NETosis is inhibited in PN mouse model induced by TcpC-secreting wild-type CFT073 (CFT073wt) and in CFT073wt-treated neutrophils

Recombinant TcpC protein inhibits NETosis

rTcpc not only inhibits the citrullination of chromatin histone, but also affects the transcription of related genes in nucleus

rTcpC dose-dependently and dynamically decreases protein but not mRNA levels of PAD4 in neutrophils

rTcpC is an E3 ubiquitin ligase that promotes ubiquitination of PAD4

TcpC promotes accumulation of PAD4 in proteasomes

C12S point mutant rTcpC-C12S loses the ability to inhibit NETosis

Discussion

Methods

Ethics statement

Reagents

Mouse PN models and in situ NETosis examination

Histological examination

Isolation of human neutrophils

Transwell co‑culture

rTcpC treatment of neutrophils

Confocal microscopy to examine NETosis

Quantification of NETs

Confocal microscopy to detect ROS production

ELISA

qRT-PCR

Flow cytometry analyses of ROS

Western blot analyses of PAD4

Dynamic analyses of CitH3 levels

Co-immunoprecipitation and immunoblotting to detect ubiquitination of PAD4

In vitro ubiquitination kit assays

Confocal microscopy to examine co-localization of PAD4 with PSMD2

Transcriptome sequencing

Bioinformatics analyses

Statistics and reproducibility

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

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Competing interests

Additional information

Supplementary information

Source data

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NETosis is inhibited in PN mouse model induced by TcpC-secreting wild-type CFT073 (CFT073^wt) and in CFT073^wt-treated neutrophils