TcpC Inhibits M1 but Promotes M2 Macrophage Polarization via Regulation of the MAPK/NF-κB and Akt/STAT6 Pathways in Urinary Tract Infection

Abstract TcpC is a multifunctional virulence factor of Uropathogenic Escherichia coli (UPEC). Macrophages can differentiate into two different subsets M1 and M2 that play distinct roles in anti-infection immunity. Here, we investigate the influence of TcpC on M1/M2 polarization and the potential mechanisms. Our data showed that M1 markers CD86 and iNOS were significantly inhibited, while the M2 markers CD163, CD206 and Arg-1 were enhanced in macrophages in kidneys from the TcpC-secreting wild-type CFT073 (CFT073wt)-infected pyelonephritis mouse model, compared with those in macrophages in kidneys from TcpC knockout CFT073 mutant (CFT073Δtcpc)-infected mice. CFT073wt or recombinant TcpC (rTcpC) treatment inhibits LPS + IFN-γ-induced CD80, CD86, TNF-α and iNOS expression, but promotes IL-4-induced CD163, CD206, Arg-1 and IL-10 expression in both human and mouse macrophage cell lines THP-1 and J774A.1. Moreover, rTcpC significantly attenuated LPS + IFN-γ-induced phosphorylation of p38, ERK, p50 and p65 but enhanced IL-4-induced phosphorylation of Akt and STAT6. These data suggest that TcpC inhibits M1 but promotes M2 macrophage polarization by down-regulation of p38, ERK/NF-κB and up-regulation of the Akt/STAT6 signaling pathway, respectively. Our findings not only illuminate the regulatory effects of TcpC on macrophage M1/M2 polarization and its related signaling pathways, but also provide a novel mechanism underlying TcpC-mediated immune evasion of macrophage-mediated innate immunity. Simple Summary We investigate the influence of TcpC, a multifunctional virulence factor of Uropathogenic Escherichia coli (UPEC), on M1/M2 macrophage polarization and the potential mechanisms. TcpC-secreting wild-type CFT073 (CFT073wt) or recombinant TcpC (rTcpC) treatment inhibits LPS + IFN-γ-induced CD80, CD86, TNF-α and iNOS expression, but promotes IL-4-induced CD163, CD206, Arg-1 and IL-10 expression in CFT073wt-infected pyelonephritis model mouse and both human and mouse macrophage cell lines THP-1 and J774A.1, respectively. Moreover, rTcpC significantly attenuated LPS + IFN-γ-induced phosphorylation of p38, ERK, p50 and p65 but en-hanced IL-4-induced phosphorylation of Akt and STAT6. These data suggest that TcpC inhibits M1 but promotes M2 macrophage polarization by down-regulation of p38, ERK/NF-κB and up-regulation of the Akt/STAT6 signaling pathway, respectively. Our findings not only illuminate the regulatory effects of TcpC on macrophage M1/M2 polarization and its related signaling pathways, but also provide a novel mechanism underlying TcpC-mediated immune evasion of macrophage-mediated innate immunity.


Introduction
Urinary tract infection (UTI) is urinary tract inflammation caused by various pathogens invading the urinary system, including urethritis, cystitis, pyelonephritis, etc. Some serious cases in patients can lead to renal failure or bacteremia [1,2]. It has been estimated that about 150 million people worldwide develop UTI each year, with high social costs in terms of hospitalizations and medical expenses [3,4]. Uropathogenic Escherichia coli (UPEC) is the most common pathogen of UTI; approximately 80% of UTIs are caused by UPEC [5,6].
Toll/interleukin-1 receptor (TIR) domain-containing protein (TcpC) encoded by the tcpc gene is a crucial virulence factor in most strains of UPEC [7]. The TIR domain of TcpC (TcpC-TIR) impedes the Toll-like receptor (TLR) signaling pathway by direct association with myeloid differentiation factor 88 (MyD88) and TLR4, promoting bacterial survival and increasing the severity of UTIs in human and mice [7]. Moreover, TcpC inhibits the activation of the NLRP3 inflammasome by binding to NLRP3 and caspase-1, leading to inhibition of IL-1β production in macrophages [8]. Finally, TcpC subverts the TLR-mediated nuclear factor-κB (NF-κB) signaling pathway and suppresses NETosis by serving as an MyD88-or peptidylarginine deiminase 4 (PAD4)-targeted E3 ubiquitin ligase that promotes degradation of MyD88 and PAD4 in macrophages and neutrophils, respectively [9,10]. Therefore, TcpC is a multifunctional virulence factor that inhibits innate immunity by dampening the inflammatory response.
Although TcpC was demonstrated to play a crucial role by blocking the NF-κB pathway in the immune evasion of macrophage-mediated innate immunity [7,9], its influence on M1/M2 macrophage polarization remains unknown. Here, we show that TcpC inhibits M1 but promotes M2 macrophage polarization via the MAPK/NF-κB/STAT pathway in urinary tract infection. Our findings not only illuminate the molecular mechanisms by which TcpC regulates M1/M2 macrophage polarization, but also provide novel clues to clarify the pathogenicity of UPEC. streptomycin (Sigma, Saint Louis, MO, USA), in a humidified 5% CO 2 incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37 • C.
TcpC-expressing wild-type uropathogenic E. coli CFT073 strain (CFT073 wt ) was kindly provided by Professor Jian-Guo Xu (State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, China). TcpC knockout CFT073 mutant strain (CFT073 ∆tcpc ) was constructed in our laboratory as described previously [25]. The strains were cultured in LB medium at 37 • C.

Mouse Pyelonephritis Model and In Situ M1/M2 Polarization Examination
Female C57BL/6 mice, 6-8 weeks of age, were purchased from SLC Laboratory Animal Co., Ltd. (Shanghai, China) and were housed in specific pathogen-free conditions. Mouse pyelonephritis (PN) models were prepared as per our previous report [9]. The model mice were sacrificed 3 days later, and kidneys were obtained for pathological examination. After the sections were fixed and tissue antigen repaired, the fixed tissues were incubated with FITC-F4/80, PE-CD86 and PerCP-CD163 (1:100 dilution) overnight at 4 • C in the dark, then we added ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) and DAPI (Sigma, Saint Louis, MO, USA) to each coverslip, then the coverslips were placed on the slides and confocal microscope images were taken.

Quantitative RT-PCR
The total RNA of treated macrophages was extracted by RNAiso Plus (TaKaRa, Shiga, Japan), and cDNA was synthesized by reverse transcription using a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Shiga, Japan). The mRNA levels of TNF-α, iNOS, IL-10 and Arg-1 in macrophages were detected by qRT-PCR, and GAPDH was set as an internal reference [9]. The Cq value was calculated by the 2 −∆∆CT method. The primers are listed in Supplementary Table S1.

ELISA and Griess Method
Expression of IL-10, TNF-α and Arg-1 in supernatants of macrophages with different treatments was measured by ELISA according to the instructions of the manufacturer (Novus, Saint Charles, MO, USA and eBioscience, San Diego, CA, USA). The production of NO was detected by the Griess method according to the instructions of the kit [28] (Promega, Madison, WI, USA).

Flow Cytometry
Macrophages were washed with PBS and resuspended to adjust the cell concentration to 1 × 10 6 /mL. A total of 1 × 10 5 cells in each group were stained using isotype control antibodies or M1 marker antibodies (CD80-PE, CD86-PE) and M2 marker antibodies (CD163-PerCP, CD206-FITC) at 37 • C for 30 min. After staining, cells were washed twice with PBS, and then analyzed on a FACS Calibur (BD, Franklin Lakes, NJ, USA). Data analysis was performed on FlowJo v10.0.7 (BD, Franklin Lakes, NJ, USA) software.

Statistical Analysis
Data shown are Mean ± SD of three independent experiments. Images were analyzed by ImageJ software. Differences between the groups were assessed by two-way analysis of variance (ANOVA). p < 0.05 is considered to be statistically significant and p < 0.01 is extremely significant. All source data are provided in source data file.

CFT073 wt Infection Inhibits In Vivo M1, but Enhances M2 Macrophage Polarization In Kidneys from Mouse PN Models
A mouse PN model was constructed by urethral instillation of CFT073 wt or CFT073 ∆tcpc as described previously [9]. Abscesses in kidneys could be observed in CFT073 wt -infected mice; no abscesses were present in kidneys from CFT073 ∆tcpc -infected mice (Supplementary Figure S1A). In addition, significantly increased infiltrates of neutrophil were observed in kidneys from the CFT073 wt group compared with those in kidneys from the CFT073 ∆tcpc group (Supplementary Figure S1B). In situ M1 macrophage marker CD86 in kidneys from the PN mouse model induced by CFT073 wt was significantly inhibited, while the M2 macrophage marker CD163 was promoted profoundly, when compared with those in kidneys from the CFT073 ∆tcpc -infected group ( Figure 1). These data suggest that TcpC may inhibit M1 but promote M2 macrophage polarization.
suppressed but CD206 (M2 marker) was significantly promoted in K-macrophages of the CFT073 wt group measured by flow cytometry when compared with those in K-macrophages from the CFT073 ∆tcpc -infected group (Figure 2A-C). The mRNA and protein levels of the M1 and M2 markers iNOS and Arg-1 were detected in K-macrophages. The results showed that iNOS was restrained, whereas Arg-1 was promoted in the CFT073 wt group compared with those in the CFT073 ∆tcpc group ( Figure 2D-F), demonstrating that CFT073 wt infection inhibits in vivo M1 but promotes M2 polarization in kidneys of mice with PN.  In order to further confirm the effect of CFT073 wt on the polarization of macrophages, kidney macrophages (K-macrophages) were sorted by flow cytometry as described in our previously published paper [9]. The cell purities were >95% when identified by the macrophage markers CD11b and F4/80 (Supplementary Figure S2). CD86 (M1 marker) was suppressed but CD206 (M2 marker) was significantly promoted in K-macrophages of the CFT073 wt group measured by flow cytometry when compared with those in Kmacrophages from the CFT073 ∆tcpc -infected group (Figure 2A-C). The mRNA and protein levels of the M1 and M2 markers iNOS and Arg-1 were detected in K-macrophages. The results showed that iNOS was restrained, whereas Arg-1 was promoted in the CFT073 wt group compared with those in the CFT073 ∆tcpc group ( Figure 2D-F), demonstrating that CFT073 wt infection inhibits in vivo M1 but promotes M2 polarization in kidneys of mice with PN.

CFT073 wt Suppresses M1 While Promoting M2 Macrophage Polarization In Vitro
Macrophage activation and polarization are crucial for inflammation and immune response. Proinflammatory M1 macrophages highly express CD80/CD86 on their surfaces, producing proinflammatory cytokines and NO. However, M2 macrophages up-regulate CD163/CD206, Arg-1 and anti-inflammatory cytokines [29,30]. Macrophage cell lines (THP-1 and J774A.1) were separately co-cultured, in the presence of LPS (100 ng/mL)/IFNγ (20 ng/mL) or IL-4 (20 ng/mL), in transwell with CFT073 wt or CFT073 ∆tcpc at MOI of 100 for 24 h. The expression of CD80/CD86 and CD163/CD206, as well as the production of iNOS and Arg-1, were analyzed. Our data showed that expression levels of the M1 markers CD80, CD86, NO and iNOS in CFT073 wt -treated macrophages were significantly inhibited, while the M2 markers CD163, CD206 and Arg-1 were enhanced, when compared with those in CFT073 Δtcpc -treated macrophages ( Figure 3). These results indicate that CFT073 wt suppresses M1 but promotes M2 macrophage polarization in vitro.

rTcpC Inhibits M1 but Promotes M2 Polarization in Both Human and Murine Macrophage Cell Lines
To further confirm if the aforementioned influence of CFT073 wt on polarization of macrophages was caused by TcpC, rTcpC was prepared and its effects on macrophage polarization were also examined. In accordance with previous reports [27,31,32], LPS + IFN-γ significantly induced the expression of M1 markers CD80 and CD86, and IL-4 treatment promoted the expression of M2 markers CD163 and CD206 in both THP-1 and
Cells 2022, 11, x FOR PEER REVIEW 10 of ( Figure 7A). Furthermore, when the mouse macrophage cell line J774A.1 was used, resul with the same trend were also obtained ( Figures 6B,D and 7B). These data demonstra that rTcpC inhibits M1 polarization via blocking the ERK/p38/NF-B pathway and pr motes M2 polarization via activating the Akt/STAT6 pathway.    Figure 7A). Furthermore, when the mouse macrophage cell line J774A.1 was used, resu with the same trend were also obtained ( Figures 6B,D and 7B). These data demonstr that rTcpC inhibits M1 polarization via blocking the ERK/p38/NF-B pathway and p motes M2 polarization via activating the Akt/STAT6 pathway.

Discussion
Pathogens are aggressors, and host immune cells such as macrophages and neutrophils are responsible for defense. UTIs are common infections, induced predominantly by UPEC, which cause severe pyelonephritis with significantly increased infiltrates of neutrophils [37,38]. We and others reported that UPEC CFT073 strains cause kidney abscesses TcpC-dependently in mice that contain an accumulation of neutrophils and bacteria, but inhibit the activation of NF-κB to attenuate inflammatory reaction in macrophages [7,9,10,39]. During the process of pathogen infection, the tissue cells could secrete chemokines to recruit macrophage infiltration to eliminate the pathogen [40]. Then, the infiltrated macrophages could also secrete chemokines to recruit other innate immune cells including neutrophils, which secrete inflammatory factors to kill pathogens together. As an important virulence factor, TcpC plays a crucial role in the pathogenecity of UPEC. TcpC-expressing UPEC cause death and tissue damage in normal hosts by creating a dysfunctional innate immune response [41]. In addition, TcpC promotes kidney cells to produce CXCL2 chemokine, which attracts neutrophil infiltration in kidneys [25]. Therefore, the inhibition of proinflammatory factors and enhanced neutrophil infiltration in CFT073 wt -infected mice kidneys are consistent with the findings which have been reported.
TcpC is a multifunctional virulence factor of UPEC that subverts the host innate immunity. TcpC inhibits phagocytic and bactericidal activity of macrophages [7,9]; it also attenuates the anti-infection activity of neutrophils by suppressing NETosis [10]. However, the regulatory effects of TcpC on macrophage M1/M2 polarization have been remained elusive. In the present study, we showed that TcpC-secreting UPEC CFT073 wt infection inhibits in situ M1 but promotes M2 polarization in a murine PN model (Figures 1 and 2). We also showed that CFT073 wt and rTcpC treatment inhibits in vitro M1 and promotes M2 polarization in both human and murine macrophage cell lines (Figures 3-5). These data demonstrate that TcpC-secreting CFT073 wt subverts macrophage polarization, which might contribute to the pathogenicity of UPEC.
The MAPK signaling pathway regulates many important cellular physiological and pathological processes, such as cell growth, differentiation and inflammatory response [58]. NF-κB, one of the major inflammatory regulators, is another key transcription factor that promotes transcription of genes encoding proinflammatory cytokines [59]. The STAT pathway is an evolutionarily conserved signal transduction paradigm, providing mechanisms for rapid receptor-to-nucleus communication and transcription control [60].
It was reported that complete polarization of macrophages to the M1 type requires activation of the MAPK and NF-κB signaling pathways [19,27,31,61], and M2 macrophage polarization requires activation of the STAT signaling pathway [62,63]. To explore the signal transduction pathways underlying the regulatory effects of TcpC on M1/M2 polarization, the influence of TcpC on MAPK/NF-κB and Akt/STAT pathways was examined. Our data clearly showed that TcpC significantly attenuated the LPS + IFN-γ-induced phosphorylation of ERK, p38, p50 and p65 ( Figure 6) and enhanced the IL-4-induced phosphorylation of Akt and STAT6 in both human and murine macrophage cell lines (Figure 7), demonstrating that TcpC inhibits M1 but promotes M2 polarization by regulating MAPK/NF-κB and Akt/STAT6 pathways. In our experiments, however, phosphorylation of JNK, one of the three members in MAPK family, remained unchanged after TcpC treatment ( Figure 6). The exact mechanism by which TcpC regulates the activation of different members of the MAPK family deserves further examination. Some studies have found that Lys63-linked polyubiquitination involves feedback mechanisms in control of JNK activity [64], and this modification does not cause its proteasomal degradation [65]. We have previously demonstrated that TcpC can also serve as an E3 ubiquitin ligase that promotes ubiquitination and degradation of MyD88 in macrophages, hereby suppressing the TLR-mediated innate immunity [9]. Therefore, whether TcpC promotes Lys63-linked ubiquitination of JNK and the role of JNK in TcpC regulation of M1/M2 polarization deserve further examination.
In summary, we demonstrated for the first time that TcpC suppresses M1 but promotes M2 macrophage polarization by down-regulation of ERK and p38/NF-κB and up-regulation of the Akt/STAT6 signaling pathway, respectively, hereby favoring UPEC to escape from the macrophage-mediated inflammatory anti-infection response (Figure 8). On the one hand, our findings not only illuminate the regulatory effects of TcpC on macrophage M1/M2 polarization and its related signaling pathways, but also provide a novel mechanism underlying TcpC-mediated immune evasion of macrophage-mediated innate immunity. On the other hand, our findings provide further evidence suggesting that TcpC is a potential target for clinical therapy of UTIs caused by TcpC + UPEC. On stimulation by TLR4 ligands, receptor tyrosine kinase or MyD88 recruit downstream signal molecules, leading to the activation of the MAPK/NF-κB pathway, while activation of the Akt/STAT pathway was stimulated by IL-4. Under the circumstance of TcpC-secreting UPEC infection, TcpC down-regulates ERK and p38/NF-κB and up-regulates the Akt/STAT6 signaling pathway, respectively, to inhibit M1 but promote M2 mac- On stimulation by TLR4 ligands, receptor tyrosine kinase or MyD88 recruit downstream signal molecules, leading to the activation of the MAPK/NF-κB pathway, while activation of the Akt/STAT pathway was stimulated by IL-4. Under the circumstance of TcpC-secreting UPEC infection, TcpC down-regulates ERK and p38/NF-κB and upregulates the Akt/STAT6 signaling pathway, respectively, to inhibit M1 but promote M2 macrophage polarization, hereby favoring UPEC to escape from the macrophage-mediated inflammatory anti-infection response.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11172674/s1, Figure S1: Abscesses and neutrophil infiltration could be observed in kidneys from CFT073 wt -induced mouse PN model; Figure S2: Purity identification of murine kidney macrophages; Figure S3: rTcpC inhibits the transcription of iNOS and TNF-α but promots the mRNA levels of IL-10 and Arg-1 in macrophages. Table S1: Information of primers used in this study.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data supporting the findings of this manuscript are available from the corresponding author upon request. Full-scan images of the Gels, Blots and numbers are provided with this paper.