Aeromonas sobria regulates proinflammatory immune response in mouse macrophages via activating the MAPK, AKT, and NF-κB pathways

Aeromonas sobria regulates proinflammatory immune response in mouse macrophages via activating the MAPK, AKT, and NF-κB pathways MAPK、AKT和NF-κB信号通路在温和气单胞菌诱导宿主细胞炎性应答中的作用及分子机制研究 1 equal first-author 2 equal 1 1 1 1 1 1 3 corresp Panpan ZHAO, zhaopp19@mails.jlu.edu.cn Panpan ZHAO, zhaopp19@mails.jlu.edu.cn zhaopp19@mails.jlu.edu.cn https://orcid.org/0000-0002-2494-3148 1 corresp Jingquan DONG, 2018000029@jou.edu.cn Jingquan DONG, 2018000029@jou.edu.cn 2018000029@jou.edu.cn https://orcid.org/0000-0001-9696-9681 Key Jiangsu Institute of Marine Resources Development, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang222005, China Key Jiangsu Institute of Marine Resources Development, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang222005, China Lianyungang Academy of Agricultural Sciences, Lianyungang222006, China Lianyungang Academy of Agricultural Sciences, Lianyungang222006, China Key Laboratory of Zoonosis of Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun130062, China Key Laboratory of Zoonosis of Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun130062, China 目的 2 以利用小鼠原代腹腔巨噬细胞建立了一种温和气单胞菌体外感染模型，探究MAPK、AKT和NF-κB信号通路在温和气单胞菌诱导宿主细胞炎性应答中的作用及相关的分子机制，为防治温和气单胞菌造成的感染提供新的治疗靶点。 创新点 2 首次探究了丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）、蛋白激酶B（protein kinase B，AKT）和核因子κB（nuclear factor kappa B，NF-κB）信号通路在温和气单胞菌诱导宿主细胞炎性应答中的作用及相关的分子机制，并分别从正反向进行了研究，围绕NF-κB、MAPK、磷酯酰肌醇-3-激酶（phosphatidylinositol 3 kinase，PI3K）/AKT等与炎症反应密切相关的信号通路，探索温和气单胞菌调控宿主细胞免疫应答的作用机制，为防治温和气单胞菌感染的治疗提供新的靶点。 方法 2 本研究利用小鼠原代腹腔巨噬细胞建立了一种温和气单胞菌体外感染模型，通过测定感染细胞培养上清中的炎性细胞因子分泌水平，初步确定其可引起小鼠原代腹腔巨噬细胞产生炎性应答。应用荧光定量PCR、蛋白质免疫印迹、阻断实验、酶联免疫吸附试验和免疫荧光等技术，围绕NF-κB、MAPK、PI3K/AKT等与炎症反应密切相关的信号通路，探索温和气单胞菌调控宿主细胞免疫应答的作用机制，为防治温和气单胞菌感染的治疗提供新的靶点。 结论 2 温和气单胞菌体外刺激小鼠腹腔巨噬细胞可以引起多种炎性细胞因子分泌水平的升高，如白细胞介素1β（interleukin-1β，IL-1β）、白细胞介素6（interleukin-6，IL-6）、白细胞介素12（interleukin-12，IL-12）和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）。同时温和气单胞菌可以通过p38MAPK、c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）/MAPK及NF-κB信号通路调节小鼠腹腔巨噬细胞促炎性因子的转录和表达，并通过AKT信号通路负调节促炎性因子的转录和表达等。因此，p38MAPK、JNK/MAPK和NF-κB信号通路均可作为潜在的药物干预靶点治疗温和气单胞菌引起的各类炎症感染。 温和气单胞菌 免疫应答 丝裂原活化蛋白激酶（MAPK） 蛋白激酶B（AKT） 核因子κB（NF-κB） Aeromonas sobria , a Gram-negative bacterium that can colonize both humans and animals, is found in a variety of environments, including water, seafood, meat, and vegetables ( Cahill, 1990 Cahill, 1990 ; Galindo et al., 2004 Galindo et al., 2004 ; Song et al., 2019 Song et al., 2019 ). Aeromonas spp. are conditionally pathogenic bacteria in aquaculture, which can rapidly proliferate, causing disease and even death in fish, especially when the environment is degraded ( Neamat-Allah et al., 2020 Neamat-Allah et al., 2020 , 2021a 2021a , 2021b 2021b ). In developing countries, Aeromonas spp. have been associated with a wide spectrum of infections in humans, including gastroenteritis, wound infections, septicemia, and lung infections ( San Joaquin and Pickett, 1988 San Joaquin and Pickett, 1988 ; Wang et al., 2009 Wang et al., 2009 ; Su et al., 2013 Su et al., 2013 ). Infections caused by Aeromonas spp. are usually more severe in immunocompromised individuals ( Miyamoto et al., 2017 Miyamoto et al., 2017 ). The presence of a plasmid encoding a β‍-lactamase in A . sobria that confers resistance to β-lactam antibiotics poses a huge challenge to the treatment of diseases caused by this microorganism ( Lim and Hong, 2020 Lim and Hong, 2020 ). Consequently, an in-depth understanding of the interaction between A . sobria and its hosts is urgently required to enable the development of effective strategies for the treatment of A . sobria infections. Innate immunity represents the first line of defense against pathogen invasion through the recognition of a vast array of pathogen-associated or danger-associated molecular patterns released by invading microbes. Macrophages as the most important phagocytic cells are widely present in mammals and play key roles in the elimination of pathogens ( Wang XC et al., 2019 Wang XC et al., 2019 ). Aeromonas hydrophila has been reported to trigger host proinflammatory responses through the activation of caspase-1 and the release of interleukin-1β (IL-1β) ( McCoy et al., 2010 McCoy et al., 2010 ). Other studies have shown that this bacterium can induce the activation of mitogen-activated protein kinase (MAPK) signaling and classical caspase-associated apoptosis in macrophages and intestinal epithelial cells ( Galindo et al., 2004 Galindo et al., 2004 ). Aeromonas veronii infection was shown to activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome in mouse macrophages, leading to apoptosis ( McCoy et al., 2010 McCoy et al., 2010 ). A . sobria bacteremia was associated with the highest mortality among members of the genus Aeromonas (56%) ( Martins et al., 2002 Martins et al., 2002 ). However, the mechanisms underlying innate immune responses against A . sobria infection in mammals remain elusive. The MAPK signaling pathway includes a variety of kinases, namely, p38 MAPK, extracellular signal-related kinase (ERK), and c-Jun-activated kinase (JNK), that play significant roles in cell development and differentiation, as well as in controlling cell responses to cytokines and stress ( Lewiset al., 1998 Lewis et al., 1998 ; Garrington and Johnson, 1999 Garrington and Johnson, 1999 ; Morrison, 2012 Morrison, 2012 ). Nuclear factor-κB (NF-‍κB), a member of the Rel family of nuclear transcription factors, regulates the expression of a set of important genes in a wide variety of biological processes, such as innate immunity, adaptive immunity, inflammation, and stress responses ( Perkins, 2006 Perkins, 2006 ). The phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway, which can be activated by insulin or a variety of growth and survival factors, has essential functions in controlling cell survival and apoptosis ( Frankeet al., 1995 Franke et al., 1995 , 1997 1997 ). Whether these signaling pathways are activated during A . sobria infection and involved in the interaction between A . sobria and macrophages needs to be further explored; such in-depth research on the mechanisms implicated in A . sobria -induced inflammation may provide new targets for the treatment of A . sobria -associated infections. Therefore, in the present study, we established an A . sobria infection model using primary mouse peritoneal macrophages (PMφ); measured proinflammatory cytokine expression levels using quantitative real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) methods; researched the roles of p38, ERK, JNK, AKT, and NF-κB signaling pathways through inhibition assays; explored the subcellular localization of NF-κB p65 by immunofluorescence assay; and found that A . sobria induced inflammatory responses through the activation of the MAPK and NF-κB signaling pathways. In order to evaluate the effects of A . sobria on PMφ, the levels of secreted proinflammatory cytokines in the cell supernatants were determined after 12 h of stimulation with A . sobria at a multiplicity of infection (MOI) of 10. As shown in Fig. 1 Fig. 1 , the secretion levels of IL-1β, IL-6, IL-12, and tumor necrosis factor-‍α (TNF-‍α) were significantly upregulated ( P < 0.001) compared with those of the control (unstimulated) group. These data revealed that A . sobria triggered an inflammatory response in PMφ. The MAPK signaling pathway is involved in immune responses triggered by a variety of pathogens. To explore the roles of this signaling pathway in A . sobria -induced proinflammatory cytokine production, mouse macrophages were inoculated with A . sobria , and cells were collected at 0, 0.5, 1.0, 2.0, and 4.0 h for the determination of total protein and phosphorylated protein (p38 MAPK, JNK, and MAPK (ERK1/2)) expression levels using western blotting ( Fig. 2 Fig. 2 a). As shown in Fig. 2 Fig. 2 b, the expression of phospho-p38 MAPK (P-p38) was significantly upregulated compared with uninoculated controls at the time points of 0.5, 1.0, and 2.0 h ( P < 0.05), and was highly upregulated in PMφ 4.0 h post A . sobria infection ( P < 0.001). The levels of phospho-JNK (P-JNK) were significantly higher at all assessed time points, but the change was the most notable at 0.5 h ( P < 0.001; Fig. 2 Fig. 2 d). The gray analysis showed that phospho-ERK (P-ERK) protein expression was highly upregulated ( P < 0.001) at 0.5 and 1.0 h after A . sobria inoculation, but gradually decreased thereafter; however, the differences remained significant in comparison with the levels in uninfected cells ( P < 0.01) ( Fig. 2 Fig. 2 c). In order to determine the roles of the activated MAPK signaling pathway in A . sobria -mediated inflammatory responses in PMφ, inhibition assays were conducted with pathway-specific inhibitors (SB203580, SCH772984, and SP600125). Cells were incubated with inhibitors prior to A . sobria infection, and the supernatants were collected for the determination of proinflammatory cytokine levels. As shown in Figs. 3 Figs. 3 a‍–‍3d, the treatment with either SB203580 or SP600125 led to a significant reduction in the levels of secreted IL-1β, IL-6, IL-12, and TNF-‍α ( P < 0.05, P < 0.01, or P < 0.001). Although SCH772984 treatment also decreased the levels of these cytokines, only IL-1β showed a significant reduction ( P < 0.01; Fig. 3 Fig. 3 a). These results revealed that the MAPK signaling pathway was activated in response to A . sobria infection, and the p38 MAPK and JNK pathways were involved in A . sobria -mediated inflammation in PMφ. Next, we examined whether the PI3K/AKT signaling pathway, an important regulator of cell survival and apoptosis, was involved in A . sobria -mediated inflammation in PMφ. The total AKT protein and phospho-AKT (P-AKT) protein levels were quantified using ImageJ software. The results showed that P-AKT levels were significantly increased ( P < 0.001) at the time points of 0.5, 1.0, and 2.0 h, and then gradually decreased after 4.0 h ( P < 0.05; Figs. 4a 4a and 4b 4b ). In order to explore whether a correlation existed between the activated AKT pathway and A . sobria -mediated inflammatory response in PMφ, the macrophages were pretreated with the inhibitor MK-2206 2HCl and then inoculated with A . sobria . The levels of all assessed proinflammatory cytokines (IL-1β, IL-6, IL-12, and TNF-α) were found to be significantly elevated to a varying extent ( P < 0.001, P < 0.05, P < 0.05, and P < 0.01, respectively) compared with those of uninoculated controls ( Figs. 5 Figs. 5 a‍–‍5d). These findings indicated that the AKT signaling pathway negatively regulated A . sobria -induced inflammation in PMφ. Since the NF-κB signaling pathway is known to regulate the expression of numerous genes following cytokine stimulation, we explored its role in A . sobria -triggered inflammation in PMφ. The subcellular locali‑zation analysis of NF-‍κB p65 by immunofluorescence assay showed that P-p65 was localized in the cytoplasm in control PMφ cells; however, following A . sobria stimulation, P-p65 fluorescence could be clearly observed in the nucleus ( Fig. 6 Fig. 6 a). The western blot analysis showed that the levels of P-p65 were significantly increased at the 0.5 h collection time point and peaked at 4.0 h compared with uninoculated control ( P < 0.001; Figs. 6b 6b and 6c 6c ). In contrast, the protein levels of inhibitor of κBα (IκBα) significantly decreased with the infection time, reaching their minimum at 4.0 h ( P < 0.001; Figs. 6b 6b and 6d 6d ). Moreover, similarly to P-p65, the levels of phosphorylated inhibitor of κB kinase α/β (P-IKKα/β) increased with prolonged infection time ( P < 0.05 and P < 0.001) and peaked at 4.0 h (Figs. 6b 6b and 6e 6e ). Then, the effects of blocking the NF-κB signaling pathway on proinflammatory cytokine levels in A . sobria -treated PMφ were investigated using the inhibitor BAY11-7082. Blocking the NF-κB pathway extremely significantly downregulated the expression of IL-1β, IL-6, and TNF-α ( P < 0.001; Figs. 7a 7a , 7b 7b , and 7d), and markedly reduced that of IL-12 ( P < 0.01; Fig. 7 Fig. 7 c). Overall, these results revealed that the NF-‍κB signaling pathway is actively involved in the A . sobria infection process in PMφ, and it enhances the secretion of proinflammatory cytokines. Aeromonas spp. are becoming more widely recognized as human pathogens, particularly in developing countries and in immunocompromised individuals suffering from diseases such as cancer, liver cirrhosis, and diabetes ( Parker and Shaw, 2011 Parker and Shaw, 2011 ). Moreover, Aeromonas infection has been associated with a high fatality rate ranging from 25% to 70% in bacteremia-affected immunocompromised patients ( Figueras et al., 2007 Figueras et al., 2007 ). Among members of the genus Aeromonas , A . sobria was reported as the most virulent species in cases of bacteremia. Nevertheless, the mechanisms underlying the associated pathogenesis have not been clarified ( Martins et al., 2002 Martins et al., 2002 ). In this study, we explored the effects of A . sobria on innate immunity using primary mouse PMφ and investigated the potential underlying mechanisms to provide a reference for future studies on the treatment of A . sobria infections in mammals. Research has indicated that the aerolysin-related cytotoxic enterotoxin of A . hydrophila can activate the NF-‍κB signaling pathway in murine macrophages. Furthermore, proinflammatory genes controlled by the NF-‍κB pathway were upregulated in the gills of rainbow trout infected by Aeromonas salmonicida , indicating that NF-‍κB pathway-mediated inflammation responses are involved in host defenses against this microorganism ( Wang WW et al., 2019 Wang WW et al., 2019 ). These observations demonstrate that proinflammatory cytokines are essential for organisms to eliminate and control Aeromonas -related infections in both humans and animals. In the current study, the levels of various proinflammatory cytokines were measured in PMφ infected with A . sobria . Similarly to those reported for other Aeromonas spp., the secretion levels of IL-1β, IL-6, IL-12, and TNF-‍α were significantly upregulated, which indicated that A . sobria triggered inflammatory responses in PMφ ( Cahill, 1990 Cahill, 1990 ; Galindo et al., 2004 Galindo et al., 2004 ; Song et al., 2019 Song et al., 2019 ). The MAPK signaling cascades are crucial regulators of immune responses to infection by a variety of pathogens; they influence the levels of proinflammatory cytokines through the phosphorylation of p38, ERK, and JNK, and promote chromatin remodeling ( Kirk et al., 2020 Kirk et al., 2020 ). The cytotoxic enterotoxin of A . hydrophila has been shown to induce the phosphorylation of p38, JNK, and ERK kinases as well as cell apoptosis in both RAW 264.7 cells/murine PMφ and T84/HT-29 human intestinal epithelial cells ( Galindoet al., 2004 Galindo et al., 2004 ). The NF-κB signaling pathway is known to be involved in cellular stress responses, immunity, and inflammation ( Perkins, 2006 Perkins, 2006 ). The nuclear activity of NF-‍κB is controlled by IκB proteins, which provide transient or dynamic regulation through their stimuli-responsive degradation and resynthesis ( Adelaja and Hoffmann, 2019 Adelaja and Hoffmann, 2019 ). Moreover, studies have suggested that the transition from p50–p65 heterodimers to p50 homodimers in NF-‍κB may be related to inflammation ( Lawrenceet al., 2001 Lawrence et al., 2001 ; Ashall et al., 2009 Ashall et al., 2009 ). We found that A . sobria activated the p38, ERK, and JNK pathways, while the inhibition assays indicated that only the p38 and JNK pathways played a promotive role in proinflammatory cytokine secretion. A . sobria infection decreased IκBα levels and promoted the phosphorylation of NF-‍κB p65, leading to its translocation into the nucleus and thus enhancing the proinflammatory response. In addition, A . sobria activated the AKT signaling pathway, and the inhibition of AKT phosphorylation increased proinflammatory cytokine levels. AKT is a serine/threonine-protein kinase with important roles in processes such as cell survival, proliferation, and metabolism ( Lee et al., 2011 Lee et al., 2011 ). The opposing functions between the AKT and MAPK/NF-‍κB signaling pathways were consistent with the reported negative feedback roles of AKT in IL-12 production in dendritic cells ( Fukao et al., 2002 Fukao et al., 2002 ). In summary, this study established an A . sobria infection model in vitro using primary mouse PMφ to explore the defense mechanisms involved in host cell response to A . sobria invasion. We found that A . sobria infection triggered proinflammatory cytokine production in PMφ through the activation of p38 MAPK, p44/42 MAPK (ERK1/2), and NF-‍κB signaling pathways, and this process was averted by the AKT signaling pathway. 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Mar Biotechnol , 21 ( 3 ): 335 - 347 . https://doi.org/10.1007/s10126-019-09883-0 https://doi.org/10.1007/s10126-019-09883-0 GWAS analysis indicated importance of NF-‍κB signaling pathway in host resistance against motile Aeromonas septicemia disease in catfish Wang XC , Gong PT , Zhang N , et al. , 2019 . Inflammasome activation restrains the intracellular Neospora caninum proliferation in bovine macrophages . Vet Parasitol , 268 : 16 - 20 . https://doi.org/10.1016/j.vetpar.2019.02.008 https://doi.org/10.1016/j.vetpar.2019.02.008 Inflammasome activation restrains the intracellular Neospora caninum proliferation in bovine macrophages Panpan ZHAO and Jingquan DONG conceptualized and designed the study. Wei ZHANG collected the data. Wei ZHANG, Bello Babatunde KAZEEM, Haitao YANG, Guanglu WANG, Zhixing LI, and Tao GUO performed the data processing and data analysis. Wei ZHANG, Bello Babatunde KAZEEM, and Panpan ZHAO wrote and edited the manuscript. Wei ZHANG and Bello Babatunde KAZEEM drew the figures. Jingquan DONG and Gang LIU checked the final version, and provided program finance support. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the Open Foundation of Key Jiangsu Institute of Marine Resources Development (No. JSIMR202016) and the Jiangsu Distinguished Professor Program (No. KK19515), China. We thank Charlesworth Author Services for the English language editing. 10.1631/jzus.B2100456 1673-1581（2021）09-0782-09 2021-05-27 2021-07-08 2021-09-15 2022-05-09 2021-08-20 © Zhejiang University Press 2021 Wei ZHANG, Bello Babatunde KAZEEM, Haitao YANG, Gang LIU, Guanglu WANG, Zhixing LI, Tao GUO, Panpan ZHAO, and Jingquan DONG declare that they have no conflict of interest. All animal experiments were strictly performed according to the guidelines for the Animal Welfare and Research Ethics Committee of Jiangsu Ocean University (Permit Number: 2017124242). All institutional and national guidelines for the care and use of laboratory animals were followed. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 9 22