- Email: [email protected]
Osteopontin exacerbates Pseudomonas aeruginosa-induced bacteremia in mice
Cellular Immunology xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Research paper
Osteopontin exacerbates Pseudomonas aeruginosa-induced bacteremia in mice ⁎
Zhenghao Piaoa,1, , Haiying Yuanb,1 a b
Department of Basic Medical Science, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang, China Department of Clinical Laboratory, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Osteopontin Bacteremia Pseudomonas aeruginosa
Osteopontin (OPN) is a multifunctional protein involved in various pathophysiological processes. However, the role of OPN in Pseudomonas aeruginosa-related sepsis is not yet clear. Here, we found that OPN expression was elevated in plasma and spleen samples from P. aeruginosa-infected mice. To determine the function of OPN in sepsis, we used wild-type (WT) and OPN-knockout (KO) mice with P. aeruginosa-induced bacteremia. We found that OPN-KO mice exhibited reduced mortality compared with WT mice and that OPN exacerbated spleen bleeding and functional impairment. OPN-KO mice exhibited reduced secretion of pro-inflammatory cytokines, such as interferon-γ, interleukin (IL)-1β, IL-12, and tumor necrosis factor-α, whereas levels of anti-inflammatory cytokine IL-10 and the leukocyte trafficking mediator macrophage inflammatory protein (MIP)-2 were not altered. Additionally, the percentages and absolute numbers of B cells were elevated in the spleens of OPN-KO mice. Thus, OPN promoted sepsis in P. aeruginosa-infected mice and potentially blocked B cell-dependent immunity.
1. Introduction Pseudomonas aeruginosa is a multidrug-resistant, gram-negative ubiquitous pathogen [1,2]. In surgical settings, organ transplantation and intravenous drug abuse can easily cause P. aeruginosa infections of the bloodstream [3–6]. Bacterial infections play a major role in the development of sepsis, and P. aeruginosa is frequently isolated from patients with sepsis [7,8]. Sepsis is a type of severe systemic inflammation; patients suffer from a “cytokine storm” as a result of excessive production of pro-inflammatory cytokines, chemokines, and other inflammatory mediators [9]. Additionally, due to this abnormal host response against invading pathogens, multi-organ failure and death often occur [10]. Unfortunately, early antibiotic therapy for bacterial infections is a challenge because of the increased number of infections caused by multidrug-resistant bacteria, including P. aeruginosa [11]. Osteopontin (OPN) is a phosphorylated glycoprotein expressed in many tissues and immune cells during a number of physiological and pathological processes [12,13]. OPN expression is elevated in the context of bacterial infection. For example, OPN is upregulated in Mycobacterium tuberculosis-infected mice and is responsible for
activating macrophages and increasing pathogen clearance [14]. During Klebsiella pneumoniae-induced pneumonia, OPN levels rapidly increase in the bronchoalveolar space, functioning to promote chemotaxis towards neutrophils and thereby facilitating an effective innate immune response [15]. Helicobacter pylori infection increases OPN expression in the stomach, but this is correlated with more severe gastric inflammation according to disease progression [16,17]. Moreover, Streptococcus pneumonia-induced pneumonia is associated with a rapid increase in pulmonary OPN concentrations in wild-type (WT) mice, whereas OPN-knockout (KO) mice show prolonged survival relative to WT mice [18]. These reports indicate that OPN is closely associated with bacterial infection; however, its specific functions are still unclear. Serum OPN levels are dramatically higher in patients with infection, suggesting that OPN may function to mediate the pathogenesis of systemic inflammatory response syndrome (SIRS) and sepsis [19]. Moreover, persistently elevated OPN serum concentrations are associated with unfavorable outcomes in patients with critical illnesses [20]. Therefore, OPN expression is correlated with experimental markers of the systemic inflammatory response and multi-organ failure. Despite intensive efforts to elucidate the role of OPN in bacterial infection-related inflammatory pathways, the function of OPN in the
Abbreviations: OPN, osteopontin; WT, wild-type; KO, knockout; SIRS, systemic inflammatory response syndrome ⁎ Corresponding author at: Department of Basic Medical Science, School of Medicine, Hangzhou Normal University, Xuelin Street 16#, Hangzhou 310036, China. E-mail address: [email protected] (Z. Piao). 1 Zhenghao Piao and Haiying Yuan contributed equally to this work. http://dx.doi.org/10.1016/j.cellimm.2017.05.004 Received 7 December 2016; Received in revised form 25 April 2017; Accepted 24 May 2017 0008-8749/ © 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Piao, Z., Cellular Immunology (2017), http://dx.doi.org/10.1016/j.cellimm.2017.05.004
Cellular Immunology xxx (xxxx) xxx–xxx
Z. Piao, H. Yuan
Fig. 1. OPN expression is promoted following P. aeruginosa infection in mice (A) WT mice were infected with the PAK strain of P. aeruginosa via the tail vein. At 3 h post-infection, RNA was extracted from the spleen for analysis of OPN expression by RT-PCR (n = 2 for each group). (B) Plasma samples were collected at 6 h post-infection, and OPN protein levels were quantified by ELISA (n = 3 for each group). All data are representative of at least three independent experiments and are expressed as means ± SDs. *p < 0.05.
mouse P. aeruginosa were homogenized on a cell strainer (cat. No. 352340; BD Falcon), serially diluted with 1 × PBS, and plated on tryptic soy agar plates. The plates were incubated at 37 °C overnight to allow colony formation, and viable counts were then determined.
pathogenesis of sepsis in patients with P. aeruginosa infection is unclear. Therefore, in this study, we investigated the functions of OPN in mice intravascularly infected with P. aeruginosa. P. aeruginosa infection increased mortality rates and spleen tissue damage and upregulated interferon (IFN)-γ, interleukin (IL)-1β, IL-12, and tumor necrosis factor (TNF)-α. In OPN-KO mice, the percentages and absolute numbers of B cells (B220+/CD19+) increased unexpectedly. These findings demonstrate that OPN, a pro-inflammatory inducer in P. aeruginosa-infected mice, promotes mortality in mice with bacteremia and possibly inhibits B cell-dependent immunity.
2.3. Enzyme-linked immunosorbent assay (ELISA) Plasma samples from PAK-infected WT and OPN-KO mice were subjected to ELISAs for the detection of inflammatory mediators. IFN-γ (cat. No. DY485), IL-10 (cat. No. DY417), and MIP-2 (cat. No. DY452) ELISA kits were purchased from R & D Systems. The OPN (rodent) ELISA kit (cat. No. ADI-900-090A) was purchased from Enzo Life Science.
2. Materials and methods 2.1. Mice and bacterial infections
2.4. Flow cytometric analysis of immune cells from mouse spleens Age- (7–10 weeks) and sex-matched C57BL/6 WT and OPN-KO mice were obtained from Hangzhou Normal University Experimental Animal Center (Hangzhou, China). All mice were maintained under specific pathogen-free standard conditions. The PAK strain of P. aeruginosa was cultured in Bacto Tryptic Soy Broth (cat. No. 6292241; BD Biosciences). Bacterial numbers were calculated by measuring the absorbance at a wavelength of 600 nm, and an absorbance of 0.5 was assumed to represent a bacterial concentration of 3 × 108 CFU/mL [21]. WT and OPN-KO mice were injected with the PAK strain of P. aeruginosa in 100 μL of 1 × PBS via the tail vein to construct a blood infection model. All animal experiments were approved by the Animal Ethics Committee of Hangzhou Normal University.
Spleens from infected WT and OPN-KO mice (5 × 107 CFU/mouse of PAK, intravenous injection) were homogenized, filtered through a cell strainer (cat. No. 352340; BD Falcon), harvested, and washed once with 1 × PBS prior to the lysis of red blood cells using RBC lysis buffer (cat. No. C3702; Beyotime). Isolated splenocytes were stained with antibodies for fluorescence-assisted cell sorting (FACS) analysis of the percentages of immune cells. The following antibodies (purchased from BD Biosciences) were used: NK1.1-PE (cat. No. 553165), CD3e-FITC (cat. No. 553062), CD11b-PE (cat. No. 557397), Ly-6G/Ly6C-FITC (cat. No. 553126), CD45R/B220-PE (cat. No. 553090), and CD19-FITC (cat. No. 553785).
2.2. Bacterial clearance assay
2.5. Statistical analysis
Spleens from WT and OPN-KO mice infected with 5 × 107 CFU/
All experiments were repeated at least three times. Results are 2
Cellular Immunology xxx (xxxx) xxx–xxx
Z. Piao, H. Yuan
mice, although no differences were observed at 24 h (Fig. 2C). Excessive inflammation owing to bacterial infections can lead to tissue damage and septic shock [10]. Bleeding (at 6 and 12 h post-infection) and white pulp damage (at 24 h post-infection) were more dramatic in WT mice than in OPN-KO mice, as shown in photographs of histological sections (Fig. 2D). These data suggest that OPN deficiency protects mice from P. aeruginosa-induced blood infection, as shown by increased mortality and spleen damage in OPN-expressing WT mice.
presented as means ± standard deviations. Student’s t-tests were used for statistical analyses. Differences with p values ≤ 0.05 were considered statistically significant. 3. Results 3.1. OPN is expressed at high levels in P. aeruginosa-infected mice OPN expression is elevated in the context of bacterial infection [14–16,18] and is closely related to sepsis [19]. To determine whether OPN was associated with P. aeruginosa infection, we detected OPN expression in infected mice. P. aeruginosa infection increased OPN expression in the spleen and plasma in a dose-dependent manner, as demonstrated by reverse transcription polymerase chain reaction (RTPCR) and ELISA, respectively (Fig. 1A–C).
3.3. OPN induces pro-inflammatory mediators in mice infected with P. aeruginosa Patients with sepsis suffer from a “cytokine storm” characterized by excessive production of pro-inflammatory cytokines, chemokines, and other inflammatory mediators [9]. Additionally, OPN functions as a pro-inflammatory inducer and can trigger cytokine and chemokine expression [17,22]. Analysis of circulating cytokine levels in the plasmas of WT and OPN-KO mice infected with P. aeruginosa revealed that INF-γ and IL-1β levels were lower in OPN-KO mice than in WT mice 6 h after inoculation (Fig. 3A and B). In contrast, IL-10 and MIP-2 expression were not dependent on OPN (Fig. 3C and D). RT-PCR analysis revealed that the expression levels of IL12 and TNFα were significantly lower in the spleens of OPN-KO mice than in those of WT mice (Fig. 3E–G). These data suggest that OPN functions as an inflammatory mediator to promote the expression of pro-inflammatory proteins in the context of P. aeruginosa-induced bacteremia.
3.2. OPN promotes P. aeruginosa-induced bacteremia in mice To determine the function of OPN in P. aeruginosa infection, we characterized WT and OPN-KO mice. At 10–12 weeks of age, WT mice exhibited gray or white hair, whereas sex- and age-matched OPN-KO mice still had black hair (Fig. 2A). At 7–8 weeks of age, mice were injected with 5 × 107 cells of the PAK strain. Interestingly, 100% of OPNKO mice survived, whereas only 30% of WT mice survived (Fig. 2B). Bacterial burden was closely related to mouse survival. During the early stages of infection, WT mice exhibited increased bacterial clearance; in particular, there was a threefold increase in bacterial clearance during the first 12 h post-infection in WT mice compared with that in OPN-KO
Fig. 2. OPN promotes bacteremia in P. aeruginosa-infected mice (A) At 10–12 weeks of age, non-infected WT and OPN-KO mice exhibited distinct hair colors. (B) At 7–8 weeks of age, WT and OPN-KO mice were injected with P. aeruginosa (PAK strain, 5 × 107 CFU/mouse) through the tail vein to construct a bacteremia model for monitoring mortality (n = 15 for each group). (C) Spleen samples were homogenized using a 40-μM cell strainer and serially diluted with sterile 1 × PBS. Cells were then plated on tryptic soy agar plates and cultured overnight for detection of colony formation (n = 5 for each time point). (D) At different time points after infection, spleen samples were collected and sectioned for H & E staining to monitor spleen bleeding (black arrow) and white pulp damage (yellow arrow). All data are representative of at least three independent experiments. *p < 0.05.
3
Cellular Immunology xxx (xxxx) xxx–xxx
Z. Piao, H. Yuan
Fig. 3. OPN promotes the expression of pro-inflammatory mediators in mice infected with P. aeruginosa WT and OPN-KO mice were injected with P. aeruginosa (PAK strain, 5 × 107 CFU/ mouse) through the tail vein. (A–D) At different time points after infection, plasma samples were collected for ELISA analysis of IFN-γ, IL-1β, IL-10, and MIP-2 (n = 4 for each time point). (E) RNA was extracted from the spleen at 0, 3, and 6 h post-infection for analysis of IL12, TNFα, and β-actin mRNAs by RT-PCR (n = 2 for each time point). The data are representative of at least three independent experiments. *p < 0.05.
the spleens of our experimental mice. We found that splenocyte numbers were twofold higher in OPN-KO mice than in WT mice (Table 1). Although the percentages of neutrophils (Gr1+) and macrophages (CD11b+) were high in WT mice (Fig. 4C and D), there were no significant differences in absolute numbers (Table 1). Unexpectedly, the percentages and absolute numbers of B cells (B220+/CD19+) were twofold higher in OPN-KO mice than in WT mice (Fig. 4B and Table 1).
3.4. OPN-KO mice exhibit higher numbers of splenic B cells after infection with P. aeruginosa Septic damage involves marked systemic lymphocyte apoptotic cell death in all lymphoid tissues [23,24]. To examine whether OPN-induced spleen damage affects the immune cell population in the context of P. aeruginosa infection, we evaluated the immune cell populations in
Table 1 Immune cell profile in mice spleen infected with P. aeruginosa. Absolute numbers of immune cells in total splenocytes. Comparative statistical analyses of immune cell populations in spleens 24 h after infection with P. aeruginosa (PAK strain, 5 × 107 CFU/mouse) are shown. The numbers of immune cells, including NK cells (NK1.1+), T cells (CD3e+), neutrophils (Gr1+), macrophages (CD11b+), and B cells (B220+/CD19+), were determined by calculating the absolute number of each cell type from the FACS profiles and total cell numbers in the spleen. Results are expressed as means ± SDs of three individual experiments (n = 3 for each group, *p < 0.05). ×105
NK1.1+
CD11b+
Gr1+
CD3e+
B220+/CD19+
Total
WT KO
5.1 ± 0.7 6.0 ± 1.4
21.2 ± 6.3 27.0 ± 3.7
68.4 ± 7.1 78.8 ± 12.9
113.7 ± 4.0 113.9 ± 24.2
77.9 ± 17.3 311.0 ± 101.0
220.0 ± 35.3 514.0 ± 65.0
4
Cellular Immunology xxx (xxxx) xxx–xxx
Z. Piao, H. Yuan
Fig. 4. OPN-KO mice infected with P. aeruginosa exhibited increased levels of B cells in the spleen Splenocytes were collected 24 h after tail vein injection with P. aeruginosa (PAK strain, 5 × 107 CFU/mouse) from WT and OPN-KO mice (n = 3 for each group). (A) FACS analysis was performed to calculate the percentages of NK cells (NK1.1+), T cells (CD3e+), B cells (B220+/CD19+), neutrophils (Gr1+), and macrophages (CD11b+). Results are representative of at least three independent experiments.
Authorship
These data suggest that OPN-KO mice may exhibit B cell-dependent protection from P. aeruginosa-induced bacteremia.
HaiYing Yuan wrote the manuscript. ZhengHao Piao designed and performed the experiments, analyzed data, and edited the manuscript. 4. Discussion Disclosures OPN functions as an inducer of pro-inflammatory cytokines and can trigger the expression of pro-inflammatory mediators [17,22]. Moreover, OPN expression is correlated with experimental markers of general inflammation and multi-organ failure [20]. We found that OPN promoted the expression of IFN-γ, IL-1β, IL-12, and TNF-α in the plasma and spleen but that IL-10 expression was not affected by OPN expression in mice infected with P. aeruginosa. Although the bacterial burden in WT mice was less than that in OPN-KO at 12 h, there were no differences in the numbers of surviving mice at 24 h. In mice infected with P. aeruginosa, levels of OPN increased markedly in the plasma and spleen, and OPN was detrimental to mice, promoting mortality and spleen damage during severe infections. These findings indicate that the presence of OPN may promote pro-inflammatory immune responses and render the host more susceptible to infection with P. aeruginosa, which may cause septic shock. OPN stimulation causes increased generation of B220+ B1 cells and increased serum IgG3 and IgM levels [25]. However, hind limb unloading increases OPN expression in circulating cells and suppresses bone marrow B-lymphogenesis [26]. Specific deletion of the innate response activator B (IRA-B, CD19+B220+MHCII+GM-CSF+) cell activity impairs bacterial clearance and elicits a cytokine storm, leading to septic shock [27]. In our study, we found that the percentages and absolute numbers of splenic B220+CD19+ cells were significantly elevated in P. aeruginosa-infected OPN-KO mice compared with those in WT mice. Further studies are required to determine how OPN regulates B220+CD19+ cells during sepsis induced by P. aeruginosa infection. In summary, we demonstrated that OPN depletion prevented the host from eliciting an excessive inflammatory response, as demonstrated by decreases in inflammatory mediators, inhibition of spleen damage, and reduced mortality in P. aeruginosa-infected mice. This process may involve the OPN-mediated regulation of the B-cell population. Thus, OPN deficiency had profound protective effects in mice infected with P. aeruginosa, which could have therapeutic implications for the management of bacteremia.
The authors have no conflicts of interest to disclose. Acknowledgments This work was supported by the Nature Science Fund sponsored by Zhejiang Provincial [grant number KZ13058] and the Research Fund from Hangzhou Normal University [grant number PD12002004121]. References [1] G.M. Rossolini, E. Mantengoli, Treatment and control of severe infections caused by multiresistant Pseudomonas aeruginosa, Clin. Microbiol. Infect. 11 (Suppl. 4) (2005) 17–32. [2] M.D. Obritsch, D.N. Fish, R. MacLaren, R. Jung, Nosocomial infections due to multidrug-resistant Pseudomonas aeruginosa: epidemiology and treatment options, Pharmacotherapy 25 (2005) 1353–1364. [3] C. van Delden, Pseudomonas aeruginosa bloodstream infections: how should we treat them? Int. J. Antimicrob. Agents 30 (Suppl 1) (2007) S71–75. [4] E. Tacconelli, M. Tumbarello, S. Bertagnolio, R. Citton, T. Spanu, et al., Multidrugresistant Pseudomonas aeruginosa bloodstream infections: analysis of trends in prevalence and epidemiology, Emerg. Infect. Dis. 8 (2002) 220–221. [5] M.D. Parkins, D.B. Gregson, J.D. Pitout, T. Ross, K.B. Laupland, Population-based study of the epidemiology and the risk factors for Pseudomonas aeruginosa bloodstream infection, Infection 38 (2010) 25–32. [6] Y. Siegman-Igra, R. Ravona, H. Primerman, M. Giladi, Pseudomonas aeruginosa bacteremia: an analysis of 123 episodes, with particular emphasis on the effect of antibiotic therapy, Int. J. Infect. Dis. 2 (1998) 211–215. [7] S.M. Opal, G.E. Garber, S.P. LaRosa, D.G. Maki, R.C. Freebairn, et al., Systemic host responses in severe sepsis analyzed by causative microorganism and treatment effects of drotrecogin alfa (activated), Clin. Infect. Dis. 37 (2003) 50–58. [8] M. Trautmann, P.M. Lepper, M. Haller, Ecology of Pseudomonas aeruginosa in the intensive care unit and the evolving role of water outlets as a reservoir of the organism, Am. J. Infect. Control 33 (2005) S41–49. [9] M. Aziz, A. Jacob, W.L. Yang, A. Matsuda, P. Wang, Current trends in inflammatory and immunomodulatory mediators in sepsis, J. Leukocyte Biol. 93 (2013) 329–342. [10] G. Ramachandran, Gram-positive and gram-negative bacterial toxins in sepsis: a brief review, Virulence 5 (2014) 213–218. [11] W. Duszynska, Strategies of empiric antibiotic therapy in severe sepsis, Anaesthesiol. Intensive Ther. 44 (2012) 96–103. [12] D.T. Denhardt, C.M. Giachelli, S.R. Rittling, Role of osteopontin in cellular signaling
5
Cellular Immunology xxx (xxxx) xxx–xxx
Z. Piao, H. Yuan
[20] C. Roderburg, F. Benz, D.V. Cardenas, M. Lutz, H.J. Hippe, et al., Persistently elevated osteopontin serum levels predict mortality in critically ill patients, Crit. Care 19 (2015) 271. [21] J.W. Chung, Z.H. Piao, S.R. Yoon, M.S. Kim, M. Jeong, et al., Pseudomonas aeruginosa eliminates natural killer cells via phagocytosis-induced apoptosis, PLoS Pathog. 5 (2009) e1000561. [22] Y. Zhu, Y. Wei, J. Chen, G. Cui, Y. Ding, et al., Osteopontin exacerbates pulmonary damage in influenza-induced lung injury, Jpn. J. Infect. Dis. 68 (2015) 467–473. [23] P.A. Efron, K. Tinsley, D.J. Minnich, V. Monterroso, J. Wagner, et al., Increased lymphoid tissue apoptosis in baboons with bacteremic shock, Shock 21 (2004) 566–571. [24] R.S. Hotchkiss, K.W. Tinsley, I.E. Karl, Role of apoptotic cell death in sepsis, Scand. J. Infect. Dis. 35 (2003) 585–592. [25] A. O'Regan, J.S. Berman, Osteopontin: a key cytokine in cell-mediated and granulomatous inflammation, Int. J. Exp. Pathol. 81 (2000) 373–390. [26] Y. Ezura, J. Nagata, M. Nagao, H. Hemmi, T. Hayata, et al., Hindlimb-unloading suppresses B cell population in the bone marrow and peripheral circulation associated with OPN expression in circulating blood cells, J. Bone Miner. Metab. 33 (2015) 48–54. [27] P.J. Rauch, A. Chudnovskiy, C.S. Robbins, G.F. Weber, M. Etzrodt, et al., Innate response activator B cells protect against microbial sepsis, Science 335 (2012) 597–601.
and toxicant injury, Annu. Rev. Pharmacol. Toxicol. 41 (2001) 723–749. [13] K.X. Wang, D.T. Denhardt, Osteopontin: role in immune regulation and stress responses, Cytokine Growth Factor Rev. 19 (2008) 333–345. [14] G.J. Nau, L. Liaw, G.L. Chupp, J.S. Berman, B.L. Hogan, et al., Attenuated host resistance against Mycobacterium bovis BCG infection in mice lacking osteopontin, Infect. Immun. 67 (1999) 4223–4230. [15] G.J. van der Windt, J.J. Hoogerwerf, A.F. de Vos, S. Florquin, T. van der Poll, Osteopontin promotes host defense during Klebsiella pneumoniae-induced pneumonia, Eur. Respir. J. 36 (2010) 1337–1345. [16] W.L. Chang, H.B. Yang, H.C. Cheng, C.H. Chuang, P.J. Lu, et al., Increased gastric osteopontin expression by Helicobacter pylori Infection can correlate with more severe gastric inflammation and intestinal metaplasia, Helicobacter 16 (2011) 217–224. [17] J.W. Park, S.H. Lee, M. Go du, H.K. Kim, H.J. Kwon, et al., Osteopontin depletion decreases inflammation and gastric epithelial proliferation during Helicobacter pylori infection in mice, Lab. Invest. 95 (2015) 660–671. [18] G.J. van der Windt, A.J. Hoogendijk, M. Schouten, T.J. Hommes, A.F. de Vos, et al., Osteopontin impairs host defense during pneumococcal pneumonia, J. Infect. Dis. 203 (2011) 1850–1858. [19] R. Vaschetto, S. Nicola, C. Olivieri, E. Boggio, F. Piccolella, et al., Serum levels of osteopontin are increased in SIRS and sepsis, Intensive Care Med. 34 (2008) 2176–2184.
6
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Research paper
Osteopontin exacerbates Pseudomonas aeruginosa-induced bacteremia in mice ⁎
Zhenghao Piaoa,1, , Haiying Yuanb,1 a b
Department of Basic Medical Science, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang, China Department of Clinical Laboratory, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Osteopontin Bacteremia Pseudomonas aeruginosa
Osteopontin (OPN) is a multifunctional protein involved in various pathophysiological processes. However, the role of OPN in Pseudomonas aeruginosa-related sepsis is not yet clear. Here, we found that OPN expression was elevated in plasma and spleen samples from P. aeruginosa-infected mice. To determine the function of OPN in sepsis, we used wild-type (WT) and OPN-knockout (KO) mice with P. aeruginosa-induced bacteremia. We found that OPN-KO mice exhibited reduced mortality compared with WT mice and that OPN exacerbated spleen bleeding and functional impairment. OPN-KO mice exhibited reduced secretion of pro-inflammatory cytokines, such as interferon-γ, interleukin (IL)-1β, IL-12, and tumor necrosis factor-α, whereas levels of anti-inflammatory cytokine IL-10 and the leukocyte trafficking mediator macrophage inflammatory protein (MIP)-2 were not altered. Additionally, the percentages and absolute numbers of B cells were elevated in the spleens of OPN-KO mice. Thus, OPN promoted sepsis in P. aeruginosa-infected mice and potentially blocked B cell-dependent immunity.
1. Introduction Pseudomonas aeruginosa is a multidrug-resistant, gram-negative ubiquitous pathogen [1,2]. In surgical settings, organ transplantation and intravenous drug abuse can easily cause P. aeruginosa infections of the bloodstream [3–6]. Bacterial infections play a major role in the development of sepsis, and P. aeruginosa is frequently isolated from patients with sepsis [7,8]. Sepsis is a type of severe systemic inflammation; patients suffer from a “cytokine storm” as a result of excessive production of pro-inflammatory cytokines, chemokines, and other inflammatory mediators [9]. Additionally, due to this abnormal host response against invading pathogens, multi-organ failure and death often occur [10]. Unfortunately, early antibiotic therapy for bacterial infections is a challenge because of the increased number of infections caused by multidrug-resistant bacteria, including P. aeruginosa [11]. Osteopontin (OPN) is a phosphorylated glycoprotein expressed in many tissues and immune cells during a number of physiological and pathological processes [12,13]. OPN expression is elevated in the context of bacterial infection. For example, OPN is upregulated in Mycobacterium tuberculosis-infected mice and is responsible for
activating macrophages and increasing pathogen clearance [14]. During Klebsiella pneumoniae-induced pneumonia, OPN levels rapidly increase in the bronchoalveolar space, functioning to promote chemotaxis towards neutrophils and thereby facilitating an effective innate immune response [15]. Helicobacter pylori infection increases OPN expression in the stomach, but this is correlated with more severe gastric inflammation according to disease progression [16,17]. Moreover, Streptococcus pneumonia-induced pneumonia is associated with a rapid increase in pulmonary OPN concentrations in wild-type (WT) mice, whereas OPN-knockout (KO) mice show prolonged survival relative to WT mice [18]. These reports indicate that OPN is closely associated with bacterial infection; however, its specific functions are still unclear. Serum OPN levels are dramatically higher in patients with infection, suggesting that OPN may function to mediate the pathogenesis of systemic inflammatory response syndrome (SIRS) and sepsis [19]. Moreover, persistently elevated OPN serum concentrations are associated with unfavorable outcomes in patients with critical illnesses [20]. Therefore, OPN expression is correlated with experimental markers of the systemic inflammatory response and multi-organ failure. Despite intensive efforts to elucidate the role of OPN in bacterial infection-related inflammatory pathways, the function of OPN in the
Abbreviations: OPN, osteopontin; WT, wild-type; KO, knockout; SIRS, systemic inflammatory response syndrome ⁎ Corresponding author at: Department of Basic Medical Science, School of Medicine, Hangzhou Normal University, Xuelin Street 16#, Hangzhou 310036, China. E-mail address: [email protected] (Z. Piao). 1 Zhenghao Piao and Haiying Yuan contributed equally to this work. http://dx.doi.org/10.1016/j.cellimm.2017.05.004 Received 7 December 2016; Received in revised form 25 April 2017; Accepted 24 May 2017 0008-8749/ © 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Piao, Z., Cellular Immunology (2017), http://dx.doi.org/10.1016/j.cellimm.2017.05.004
Cellular Immunology xxx (xxxx) xxx–xxx
Z. Piao, H. Yuan
Fig. 1. OPN expression is promoted following P. aeruginosa infection in mice (A) WT mice were infected with the PAK strain of P. aeruginosa via the tail vein. At 3 h post-infection, RNA was extracted from the spleen for analysis of OPN expression by RT-PCR (n = 2 for each group). (B) Plasma samples were collected at 6 h post-infection, and OPN protein levels were quantified by ELISA (n = 3 for each group). All data are representative of at least three independent experiments and are expressed as means ± SDs. *p < 0.05.
mouse P. aeruginosa were homogenized on a cell strainer (cat. No. 352340; BD Falcon), serially diluted with 1 × PBS, and plated on tryptic soy agar plates. The plates were incubated at 37 °C overnight to allow colony formation, and viable counts were then determined.
pathogenesis of sepsis in patients with P. aeruginosa infection is unclear. Therefore, in this study, we investigated the functions of OPN in mice intravascularly infected with P. aeruginosa. P. aeruginosa infection increased mortality rates and spleen tissue damage and upregulated interferon (IFN)-γ, interleukin (IL)-1β, IL-12, and tumor necrosis factor (TNF)-α. In OPN-KO mice, the percentages and absolute numbers of B cells (B220+/CD19+) increased unexpectedly. These findings demonstrate that OPN, a pro-inflammatory inducer in P. aeruginosa-infected mice, promotes mortality in mice with bacteremia and possibly inhibits B cell-dependent immunity.
2.3. Enzyme-linked immunosorbent assay (ELISA) Plasma samples from PAK-infected WT and OPN-KO mice were subjected to ELISAs for the detection of inflammatory mediators. IFN-γ (cat. No. DY485), IL-10 (cat. No. DY417), and MIP-2 (cat. No. DY452) ELISA kits were purchased from R & D Systems. The OPN (rodent) ELISA kit (cat. No. ADI-900-090A) was purchased from Enzo Life Science.
2. Materials and methods 2.1. Mice and bacterial infections
2.4. Flow cytometric analysis of immune cells from mouse spleens Age- (7–10 weeks) and sex-matched C57BL/6 WT and OPN-KO mice were obtained from Hangzhou Normal University Experimental Animal Center (Hangzhou, China). All mice were maintained under specific pathogen-free standard conditions. The PAK strain of P. aeruginosa was cultured in Bacto Tryptic Soy Broth (cat. No. 6292241; BD Biosciences). Bacterial numbers were calculated by measuring the absorbance at a wavelength of 600 nm, and an absorbance of 0.5 was assumed to represent a bacterial concentration of 3 × 108 CFU/mL [21]. WT and OPN-KO mice were injected with the PAK strain of P. aeruginosa in 100 μL of 1 × PBS via the tail vein to construct a blood infection model. All animal experiments were approved by the Animal Ethics Committee of Hangzhou Normal University.
Spleens from infected WT and OPN-KO mice (5 × 107 CFU/mouse of PAK, intravenous injection) were homogenized, filtered through a cell strainer (cat. No. 352340; BD Falcon), harvested, and washed once with 1 × PBS prior to the lysis of red blood cells using RBC lysis buffer (cat. No. C3702; Beyotime). Isolated splenocytes were stained with antibodies for fluorescence-assisted cell sorting (FACS) analysis of the percentages of immune cells. The following antibodies (purchased from BD Biosciences) were used: NK1.1-PE (cat. No. 553165), CD3e-FITC (cat. No. 553062), CD11b-PE (cat. No. 557397), Ly-6G/Ly6C-FITC (cat. No. 553126), CD45R/B220-PE (cat. No. 553090), and CD19-FITC (cat. No. 553785).
2.2. Bacterial clearance assay
2.5. Statistical analysis
Spleens from WT and OPN-KO mice infected with 5 × 107 CFU/
All experiments were repeated at least three times. Results are 2
Cellular Immunology xxx (xxxx) xxx–xxx
Z. Piao, H. Yuan
mice, although no differences were observed at 24 h (Fig. 2C). Excessive inflammation owing to bacterial infections can lead to tissue damage and septic shock [10]. Bleeding (at 6 and 12 h post-infection) and white pulp damage (at 24 h post-infection) were more dramatic in WT mice than in OPN-KO mice, as shown in photographs of histological sections (Fig. 2D). These data suggest that OPN deficiency protects mice from P. aeruginosa-induced blood infection, as shown by increased mortality and spleen damage in OPN-expressing WT mice.
presented as means ± standard deviations. Student’s t-tests were used for statistical analyses. Differences with p values ≤ 0.05 were considered statistically significant. 3. Results 3.1. OPN is expressed at high levels in P. aeruginosa-infected mice OPN expression is elevated in the context of bacterial infection [14–16,18] and is closely related to sepsis [19]. To determine whether OPN was associated with P. aeruginosa infection, we detected OPN expression in infected mice. P. aeruginosa infection increased OPN expression in the spleen and plasma in a dose-dependent manner, as demonstrated by reverse transcription polymerase chain reaction (RTPCR) and ELISA, respectively (Fig. 1A–C).
3.3. OPN induces pro-inflammatory mediators in mice infected with P. aeruginosa Patients with sepsis suffer from a “cytokine storm” characterized by excessive production of pro-inflammatory cytokines, chemokines, and other inflammatory mediators [9]. Additionally, OPN functions as a pro-inflammatory inducer and can trigger cytokine and chemokine expression [17,22]. Analysis of circulating cytokine levels in the plasmas of WT and OPN-KO mice infected with P. aeruginosa revealed that INF-γ and IL-1β levels were lower in OPN-KO mice than in WT mice 6 h after inoculation (Fig. 3A and B). In contrast, IL-10 and MIP-2 expression were not dependent on OPN (Fig. 3C and D). RT-PCR analysis revealed that the expression levels of IL12 and TNFα were significantly lower in the spleens of OPN-KO mice than in those of WT mice (Fig. 3E–G). These data suggest that OPN functions as an inflammatory mediator to promote the expression of pro-inflammatory proteins in the context of P. aeruginosa-induced bacteremia.
3.2. OPN promotes P. aeruginosa-induced bacteremia in mice To determine the function of OPN in P. aeruginosa infection, we characterized WT and OPN-KO mice. At 10–12 weeks of age, WT mice exhibited gray or white hair, whereas sex- and age-matched OPN-KO mice still had black hair (Fig. 2A). At 7–8 weeks of age, mice were injected with 5 × 107 cells of the PAK strain. Interestingly, 100% of OPNKO mice survived, whereas only 30% of WT mice survived (Fig. 2B). Bacterial burden was closely related to mouse survival. During the early stages of infection, WT mice exhibited increased bacterial clearance; in particular, there was a threefold increase in bacterial clearance during the first 12 h post-infection in WT mice compared with that in OPN-KO
Fig. 2. OPN promotes bacteremia in P. aeruginosa-infected mice (A) At 10–12 weeks of age, non-infected WT and OPN-KO mice exhibited distinct hair colors. (B) At 7–8 weeks of age, WT and OPN-KO mice were injected with P. aeruginosa (PAK strain, 5 × 107 CFU/mouse) through the tail vein to construct a bacteremia model for monitoring mortality (n = 15 for each group). (C) Spleen samples were homogenized using a 40-μM cell strainer and serially diluted with sterile 1 × PBS. Cells were then plated on tryptic soy agar plates and cultured overnight for detection of colony formation (n = 5 for each time point). (D) At different time points after infection, spleen samples were collected and sectioned for H & E staining to monitor spleen bleeding (black arrow) and white pulp damage (yellow arrow). All data are representative of at least three independent experiments. *p < 0.05.
3
Cellular Immunology xxx (xxxx) xxx–xxx
Z. Piao, H. Yuan
Fig. 3. OPN promotes the expression of pro-inflammatory mediators in mice infected with P. aeruginosa WT and OPN-KO mice were injected with P. aeruginosa (PAK strain, 5 × 107 CFU/ mouse) through the tail vein. (A–D) At different time points after infection, plasma samples were collected for ELISA analysis of IFN-γ, IL-1β, IL-10, and MIP-2 (n = 4 for each time point). (E) RNA was extracted from the spleen at 0, 3, and 6 h post-infection for analysis of IL12, TNFα, and β-actin mRNAs by RT-PCR (n = 2 for each time point). The data are representative of at least three independent experiments. *p < 0.05.
the spleens of our experimental mice. We found that splenocyte numbers were twofold higher in OPN-KO mice than in WT mice (Table 1). Although the percentages of neutrophils (Gr1+) and macrophages (CD11b+) were high in WT mice (Fig. 4C and D), there were no significant differences in absolute numbers (Table 1). Unexpectedly, the percentages and absolute numbers of B cells (B220+/CD19+) were twofold higher in OPN-KO mice than in WT mice (Fig. 4B and Table 1).
3.4. OPN-KO mice exhibit higher numbers of splenic B cells after infection with P. aeruginosa Septic damage involves marked systemic lymphocyte apoptotic cell death in all lymphoid tissues [23,24]. To examine whether OPN-induced spleen damage affects the immune cell population in the context of P. aeruginosa infection, we evaluated the immune cell populations in
Table 1 Immune cell profile in mice spleen infected with P. aeruginosa. Absolute numbers of immune cells in total splenocytes. Comparative statistical analyses of immune cell populations in spleens 24 h after infection with P. aeruginosa (PAK strain, 5 × 107 CFU/mouse) are shown. The numbers of immune cells, including NK cells (NK1.1+), T cells (CD3e+), neutrophils (Gr1+), macrophages (CD11b+), and B cells (B220+/CD19+), were determined by calculating the absolute number of each cell type from the FACS profiles and total cell numbers in the spleen. Results are expressed as means ± SDs of three individual experiments (n = 3 for each group, *p < 0.05). ×105
NK1.1+
CD11b+
Gr1+
CD3e+
B220+/CD19+
Total
WT KO
5.1 ± 0.7 6.0 ± 1.4
21.2 ± 6.3 27.0 ± 3.7
68.4 ± 7.1 78.8 ± 12.9
113.7 ± 4.0 113.9 ± 24.2
77.9 ± 17.3 311.0 ± 101.0
220.0 ± 35.3 514.0 ± 65.0
4
Cellular Immunology xxx (xxxx) xxx–xxx
Z. Piao, H. Yuan
Fig. 4. OPN-KO mice infected with P. aeruginosa exhibited increased levels of B cells in the spleen Splenocytes were collected 24 h after tail vein injection with P. aeruginosa (PAK strain, 5 × 107 CFU/mouse) from WT and OPN-KO mice (n = 3 for each group). (A) FACS analysis was performed to calculate the percentages of NK cells (NK1.1+), T cells (CD3e+), B cells (B220+/CD19+), neutrophils (Gr1+), and macrophages (CD11b+). Results are representative of at least three independent experiments.
Authorship
These data suggest that OPN-KO mice may exhibit B cell-dependent protection from P. aeruginosa-induced bacteremia.
HaiYing Yuan wrote the manuscript. ZhengHao Piao designed and performed the experiments, analyzed data, and edited the manuscript. 4. Discussion Disclosures OPN functions as an inducer of pro-inflammatory cytokines and can trigger the expression of pro-inflammatory mediators [17,22]. Moreover, OPN expression is correlated with experimental markers of general inflammation and multi-organ failure [20]. We found that OPN promoted the expression of IFN-γ, IL-1β, IL-12, and TNF-α in the plasma and spleen but that IL-10 expression was not affected by OPN expression in mice infected with P. aeruginosa. Although the bacterial burden in WT mice was less than that in OPN-KO at 12 h, there were no differences in the numbers of surviving mice at 24 h. In mice infected with P. aeruginosa, levels of OPN increased markedly in the plasma and spleen, and OPN was detrimental to mice, promoting mortality and spleen damage during severe infections. These findings indicate that the presence of OPN may promote pro-inflammatory immune responses and render the host more susceptible to infection with P. aeruginosa, which may cause septic shock. OPN stimulation causes increased generation of B220+ B1 cells and increased serum IgG3 and IgM levels [25]. However, hind limb unloading increases OPN expression in circulating cells and suppresses bone marrow B-lymphogenesis [26]. Specific deletion of the innate response activator B (IRA-B, CD19+B220+MHCII+GM-CSF+) cell activity impairs bacterial clearance and elicits a cytokine storm, leading to septic shock [27]. In our study, we found that the percentages and absolute numbers of splenic B220+CD19+ cells were significantly elevated in P. aeruginosa-infected OPN-KO mice compared with those in WT mice. Further studies are required to determine how OPN regulates B220+CD19+ cells during sepsis induced by P. aeruginosa infection. In summary, we demonstrated that OPN depletion prevented the host from eliciting an excessive inflammatory response, as demonstrated by decreases in inflammatory mediators, inhibition of spleen damage, and reduced mortality in P. aeruginosa-infected mice. This process may involve the OPN-mediated regulation of the B-cell population. Thus, OPN deficiency had profound protective effects in mice infected with P. aeruginosa, which could have therapeutic implications for the management of bacteremia.
The authors have no conflicts of interest to disclose. Acknowledgments This work was supported by the Nature Science Fund sponsored by Zhejiang Provincial [grant number KZ13058] and the Research Fund from Hangzhou Normal University [grant number PD12002004121]. References [1] G.M. Rossolini, E. Mantengoli, Treatment and control of severe infections caused by multiresistant Pseudomonas aeruginosa, Clin. Microbiol. Infect. 11 (Suppl. 4) (2005) 17–32. [2] M.D. Obritsch, D.N. Fish, R. MacLaren, R. Jung, Nosocomial infections due to multidrug-resistant Pseudomonas aeruginosa: epidemiology and treatment options, Pharmacotherapy 25 (2005) 1353–1364. [3] C. van Delden, Pseudomonas aeruginosa bloodstream infections: how should we treat them? Int. J. Antimicrob. Agents 30 (Suppl 1) (2007) S71–75. [4] E. Tacconelli, M. Tumbarello, S. Bertagnolio, R. Citton, T. Spanu, et al., Multidrugresistant Pseudomonas aeruginosa bloodstream infections: analysis of trends in prevalence and epidemiology, Emerg. Infect. Dis. 8 (2002) 220–221. [5] M.D. Parkins, D.B. Gregson, J.D. Pitout, T. Ross, K.B. Laupland, Population-based study of the epidemiology and the risk factors for Pseudomonas aeruginosa bloodstream infection, Infection 38 (2010) 25–32. [6] Y. Siegman-Igra, R. Ravona, H. Primerman, M. Giladi, Pseudomonas aeruginosa bacteremia: an analysis of 123 episodes, with particular emphasis on the effect of antibiotic therapy, Int. J. Infect. Dis. 2 (1998) 211–215. [7] S.M. Opal, G.E. Garber, S.P. LaRosa, D.G. Maki, R.C. Freebairn, et al., Systemic host responses in severe sepsis analyzed by causative microorganism and treatment effects of drotrecogin alfa (activated), Clin. Infect. Dis. 37 (2003) 50–58. [8] M. Trautmann, P.M. Lepper, M. Haller, Ecology of Pseudomonas aeruginosa in the intensive care unit and the evolving role of water outlets as a reservoir of the organism, Am. J. Infect. Control 33 (2005) S41–49. [9] M. Aziz, A. Jacob, W.L. Yang, A. Matsuda, P. Wang, Current trends in inflammatory and immunomodulatory mediators in sepsis, J. Leukocyte Biol. 93 (2013) 329–342. [10] G. Ramachandran, Gram-positive and gram-negative bacterial toxins in sepsis: a brief review, Virulence 5 (2014) 213–218. [11] W. Duszynska, Strategies of empiric antibiotic therapy in severe sepsis, Anaesthesiol. Intensive Ther. 44 (2012) 96–103. [12] D.T. Denhardt, C.M. Giachelli, S.R. Rittling, Role of osteopontin in cellular signaling
5
Cellular Immunology xxx (xxxx) xxx–xxx
Z. Piao, H. Yuan
[20] C. Roderburg, F. Benz, D.V. Cardenas, M. Lutz, H.J. Hippe, et al., Persistently elevated osteopontin serum levels predict mortality in critically ill patients, Crit. Care 19 (2015) 271. [21] J.W. Chung, Z.H. Piao, S.R. Yoon, M.S. Kim, M. Jeong, et al., Pseudomonas aeruginosa eliminates natural killer cells via phagocytosis-induced apoptosis, PLoS Pathog. 5 (2009) e1000561. [22] Y. Zhu, Y. Wei, J. Chen, G. Cui, Y. Ding, et al., Osteopontin exacerbates pulmonary damage in influenza-induced lung injury, Jpn. J. Infect. Dis. 68 (2015) 467–473. [23] P.A. Efron, K. Tinsley, D.J. Minnich, V. Monterroso, J. Wagner, et al., Increased lymphoid tissue apoptosis in baboons with bacteremic shock, Shock 21 (2004) 566–571. [24] R.S. Hotchkiss, K.W. Tinsley, I.E. Karl, Role of apoptotic cell death in sepsis, Scand. J. Infect. Dis. 35 (2003) 585–592. [25] A. O'Regan, J.S. Berman, Osteopontin: a key cytokine in cell-mediated and granulomatous inflammation, Int. J. Exp. Pathol. 81 (2000) 373–390. [26] Y. Ezura, J. Nagata, M. Nagao, H. Hemmi, T. Hayata, et al., Hindlimb-unloading suppresses B cell population in the bone marrow and peripheral circulation associated with OPN expression in circulating blood cells, J. Bone Miner. Metab. 33 (2015) 48–54. [27] P.J. Rauch, A. Chudnovskiy, C.S. Robbins, G.F. Weber, M. Etzrodt, et al., Innate response activator B cells protect against microbial sepsis, Science 335 (2012) 597–601.
and toxicant injury, Annu. Rev. Pharmacol. Toxicol. 41 (2001) 723–749. [13] K.X. Wang, D.T. Denhardt, Osteopontin: role in immune regulation and stress responses, Cytokine Growth Factor Rev. 19 (2008) 333–345. [14] G.J. Nau, L. Liaw, G.L. Chupp, J.S. Berman, B.L. Hogan, et al., Attenuated host resistance against Mycobacterium bovis BCG infection in mice lacking osteopontin, Infect. Immun. 67 (1999) 4223–4230. [15] G.J. van der Windt, J.J. Hoogerwerf, A.F. de Vos, S. Florquin, T. van der Poll, Osteopontin promotes host defense during Klebsiella pneumoniae-induced pneumonia, Eur. Respir. J. 36 (2010) 1337–1345. [16] W.L. Chang, H.B. Yang, H.C. Cheng, C.H. Chuang, P.J. Lu, et al., Increased gastric osteopontin expression by Helicobacter pylori Infection can correlate with more severe gastric inflammation and intestinal metaplasia, Helicobacter 16 (2011) 217–224. [17] J.W. Park, S.H. Lee, M. Go du, H.K. Kim, H.J. Kwon, et al., Osteopontin depletion decreases inflammation and gastric epithelial proliferation during Helicobacter pylori infection in mice, Lab. Invest. 95 (2015) 660–671. [18] G.J. van der Windt, A.J. Hoogendijk, M. Schouten, T.J. Hommes, A.F. de Vos, et al., Osteopontin impairs host defense during pneumococcal pneumonia, J. Infect. Dis. 203 (2011) 1850–1858. [19] R. Vaschetto, S. Nicola, C. Olivieri, E. Boggio, F. Piccolella, et al., Serum levels of osteopontin are increased in SIRS and sepsis, Intensive Care Med. 34 (2008) 2176–2184.
6