- Email: [email protected]
High density lipoproteins down-regulate transcriptional expression of pro-inflammatory factors and oxidative burst in head kidney leukocytes from rainbow trout, Oncorhynchus mykiss
Fish & Shellfish Immunology 35 (2013) 180e183
Contents lists available at SciVerse ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Short communication
High density lipoproteins down-regulate transcriptional expression of pro-inflammatory factors and oxidative burst in head kidney leukocytes from rainbow trout, Oncorhynchus mykiss Franz Villarroel 1, Alin Casado 1, Rodolfo Amthauer, Margarita I. Concha* Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Campus Isla Teja, Universidad Austral de Chile, Valdivia, Chile
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 January 2013 Received in revised form 22 March 2013 Accepted 5 April 2013 Available online 15 April 2013
Teleosts are the first group of vertebrates possessing an acquired immune system; however, it is less developed than in mammals and is highly influenced by environmental changes. Therefore, innate immunity effectors play a more critical role in survival of pathogen-challenged fish. In a previous study we showed that trout high density lipoprotein (HDL), and its major apolipoprotein (ApoA-I) are widely expressed in primary defense barriers and other immune-relevant tissues, displaying important antibacterial activity in vitro. Here we show that trout HDL inhibits both basal and LPS-induced transcript expression of pro-inflammatory cytokines such as TNF-a and IL-1b, and the acute phase protein serum amyloid A (A-SAA), in head kidney leukocytes (HLK) from rainbow trout. In addition, trout HDL was able to block the respiratory burst of PMA-stimulated HKL, at physiological concentrations and in a dose dependent manner. Moreover, this effect was only partially mimicked by supra-physiologic concentrations of the HDL-transported carotenoid, astaxanthin. These results constitute the first data suggesting that in addition to its antimicrobial activity, HDL would have a relevant immunomodulatory role in salmonid fish. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Respiratory burst Head kidney leukocytes HDL LPS Cytokines
1. Introduction Although the main role of high density lipoprotein (HDL) has been long considered to be the reverse cholesterol transport and its anti-atherogenic and vascular-protective effects in mammals [1,2], evidences demonstrating a key role of HDL and its components in innate and adaptive immunity have accumulated during the last decade [3e6]. In fact, it has been demonstrated that HDL acts as a platform for the assembly of potent immunomodulatory complexes that regulate antimicrobial activity [3]. Among the immunomodulatory roles that have been described for mammalian HDL and its major apolipoprotein, are the ability of HDL and ApoA-I to interfere with the interaction between activated T cells and human monocytes and also to reduce neutrophil activation and the corresponding production of pro-inflammatory cytokines such as interleukin 1 beta (IL-1b) and tumour necrosis factor alpha (TNFa) [7e10]. Abbreviations: ROS, reactive oxygen species; HDL, high density lipoprotein; PMA, phorbol 12-myristate 13-acetate; A-SAA, acute serum amyloid A; Asx, astaxanthin. * Corresponding author. Tel.: þ56 63 221108; fax: þ56 63 221332. E-mail addresses: [email protected] (F. Villarroel), alin.casado@ gmail.com (A. Casado), [email protected] (R. Amthauer), [email protected], [email protected] (M.I. Concha). 1 These authors contributed equally to this work. 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.04.003
Our previous studies demonstrated that HDL and its major apolipoproteins (ApoA-I and ApoA-II) not only constitute one of the major plasma proteins in carp and rainbow trout but also display potent antimicrobial activity in vitro against Gram-positive and -negative bacteria [11e13]. In addition it was shown that apolipoprotein A-I transcript is abundantly expressed in the carp skin and ApoA-I protein is apparently secreted to the mucus constituting nascent HDL particles [11]. Similarly, high levels of ApoA-I transcript and protein have been detected in the main primary defense barriers in trout and its plasma concentration remains unaffected during the acute phase, in contrast with mammalian ApoA-I, which constitutes a negative acute phase reactant [13]. Taking in consideration these differences between mammalian and teleost HDL, the aim of the present study was to evaluate possible immunomodulatory functions of teleost HDL.
2. Material and methods 2.1. Experimental fish Healthy rainbow trout (Oncorhynchus mykiss) (150e200 g) were obtained from the fish farm (Cultivos Marinos Chiloe, Province of Valdivia, Chile) and maintained in 2500 l flow-through tanks with
F. Villarroel et al. / Fish & Shellfish Immunology 35 (2013) 180e183
aerated well water at 12 1 C under an 11-h D: 13-h L photoperiod. Fish were sacrificed by benzocaine overdose (250 mg l1) following the ethical guidelines for the use of animals in research from the University Committee, and dissected to obtain the head kidney for leukocyte isolation. Blood samples were extracted from the caudal vein of anaesthetised fish (50 mg l1 benzocaine) with a heparinised syringe and plasma samples from four fish were pooled after centrifugation at 2000 g for 3 min (twice) to assure the complete removal of blood cells, and after addition of protease inhibitors (1 mM PMSF and 2 mM benzamidine), kept in aliquots at 20 C. Plasma HDL fractions were isolated from whole plasma pools by affinity chromatography on Affi-GelÒ Blue-Gel (BIO-RAD), and its purity and integrity confirmed by SDS-PAGE and Western blot analyses, as previously described by [12]. 2.2. Isolation and stimulation of head kidney leukocytes Head kidney leukocytes (HLK) were isolated essentially as described previously [13]. Briefly, the head kidneys from at least three fish were resuspended in 0.97% (w/v) Hank’s salt solution, supplemented with 10 U ml1 heparin and 2% (v/v) foetal calf serum (FCS) and passed through a 100-mesh nylon cell strainer (Falcon). The resulting suspension was placed on a 31/46% discontinuous Percoll gradient and centrifuged at 400 g for 30 min. Finally, leukocytes were carefully collected from the interphase, suspended, washed twice in L-15 medium and viability tested by exclusion of trypan blue dye. Over 95% viable HKL preparations were suspended in L-15 medium supplemented with 10% FCS, 100 mg ml1 of streptomycin and 60 mg ml1 penicillin and then seeded on 6-well cell culture NuncÒ plates (2.5 106 cells per well). After 18 h of culture, non-adherent cells were removed and the remaining cells incubated in L-15 medium supplemented with 10% FCS. For the expression assays, HKL were incubated in L-15 medium supplemented with 5% FCS with or without 25 mg ml1 Escherichia coli 026:B6 LPS for 1 h at 16 C. Alternatively cells were incubated first during 18 h with 0.25 mg ml1 HDL, then the media containing trout HDL was completely removed and replaced by fresh medium containing 25 mg ml1 E. coli LPS. Afterwards, cells were lysed for RNA isolation and cytokine expression analyses. Expression experiments were performed in triplicates using three different HKL preparations. 2.3. Isolation of total RNA and cDNA synthesis For total RNA extraction from cultured HKL, the medium was removed and cells were immediately lysed by the addition of 1 ml of RNAwiz reagent (Ambion Inc.) for 4 106 cells according to the manufacturer’s instructions. RNA was quantified by absorbance at 260 nm and used immediately or stored precipitated in ethanol at 70 C until use. Total RNA integrity was evaluated as described previously [14]. After treating total RNAs with 5 units of amplification grade DNAse I RQ1 (Promega), semi-quantitative RT-PCR analyses were performed using the primers listed in Supplementary Table 1 and conditions described previously [14]. bactin was used as control gene since no changes in expression were observed for this gene under the conditions tested. To evaluate the relative expression of each gene, the ratio of the corresponding transcript to b-actin was determined after gel scanning, measuring the pixel intensity of the bands (same area) with a BIO-RAD Molecular Imager FX Pro System, using the Quantity One program for Windows. Total counts (arbitrary units) from each band were obtained after subtracting the background. An arbitrary value of one was assigned to the ratios obtained for the control cells and the mean fold change standard error was plotted for the treated cells.
181
2.4. Respiratory burst assays Head kidney leukocytes (1 106 cells per well) were placed in the wells of a flat-bottomed 96-well microtiter plate with 100 ml of L-15 and were preincubated at 18 C for 5 min with or without the addition of different concentrations of freshly prepared trout HDL (0.25 or 0.5 mg ml1) or 2 mM astaxanthin and then 20 mM of luminol (Sigma) and 1 mg ml1 phorbol-12-myristate 13-acetate (PMA, Sigma Chemical Co., St. Louis, MO, USA) were added. The plate was gently shaken and reactive oxygen species (ROS) production was immediately measured using a Luminoskan AscentÔ luminometer (Thermo Labsystems) for 5000 s. Luminescence backgrounds were determined using reagent solutions containing luminol but not PMA. The percentage of inhibition of the respiratory burst achieved by the different treatments was calculated measuring the area under the curve using the value obtained with PMA alone as the maximal response. 2.5. Statistical analyses All data are given as means standard error of the mean (SEM) of three independent experiments. Statistical analysis was carried out applying the Student’s t-test using the GraphPad Prism v5.0 software for Windows. Differences with p < 0.03 were considered statistically significant. 3. Results and discussion To evaluate if trout HDL was able to down-regulate the inflammatory response, we analysed by RT-PCR the relative levels of mRNAs for IL-1b, TNFa and the acute phase reactant, serum amyloid A (A-SAA) in unstimulated- and LPS-activated HKL. As expected, LPS promoted a significant increase in the relative transcript levels of all three cytokines (Fig. 1), while pre-incubation of the cells with 0.25 mg ml1 of purified trout HDL led to a reduction of more than 65% in the LPS-triggered response (Fig. 1B, C, D). Moreover, this inhibitory effect on tnf-a1 and il1-b1 expression was also observed in unstimulated HKL, since basal transcript levels became undetectable after pre-incubation with HDL. In contrast, basal levels of A-SAA transcript were negligible probably because different cytokines, including interferon-gamma, transforming growth factor beta, TNFa, and interleukins, either alone or in combination, have been shown to regulate the synthesis of this major acute-phase reactant at the transcriptional level [15,16]. Previously, we reported that A-SAA transcript was strongly upregulated during an in vivo- or in vitro-challenge with LPS in several trout tissues and HKL, respectively, suggesting that it could constitute a very sensitive marker for the detection of proinflammatory conditions [13,14]. Murphy et al. [17] described a potent attenuating effect of human HDL and its major apolipoprotein ApoA-I in monocyte activation and this effect could be reproduced by reconstituted HDL particles [18]. Furthermore, direct effects of HDL were found on myeloid cells including monocytes, macrophages and monocytes-derived dendritic cells, resulting in suppression of cytokine and chemokine production, downregulation of co-stimulatory molecules and inhibition of antigen presentation [4,19,20]. These data are consistent with the results presented herein suggesting that the anti-inflammatory role of HDL could be evolutionary conserved. We also studied the effect of trout HDL on the oxidative burst response in HKL stimulated with PMA, as overproduction of reactive oxygen species and other effective yet also toxic substances produced by leukocytes may participate in disturbed apoptosis and intensification of the inflammatory processes. As shown on Fig. 2, HDL showed a strong and significant reduction in the intracellular
182
F. Villarroel et al. / Fish & Shellfish Immunology 35 (2013) 180e183
Fig. 1. Trout HDL reduces pro-inflammatory cytokine expression in head-kidney leukocytes, under basal and LPS-stimulated conditions. Expression of il-1b1, tnfa1 and a-saa transcripts was evaluated by semi-quantitative RT-PCR, using b-actin as constitutive transcript. Database accession numbers of the genes used are detailed in Supplementary Table 1. (A) Representative RT-PCR results obtained from HKL without treatment (basal condition), stimulated with LPS (25 mg ml1) and pre-treated with 0.25 mg ml1 trout HDL with or without further LPS stimulation, respectively. Graphs B, C and D show the mean value standard error of the fold change obtained for a-saa, il1-b1 and tnf-a1, respectively from three independent experiments. Statistical significance was determined using the GraphPad Prism software for each treatment with respect to the control and also between LPStreated cells with or without pretreatment with HDL (xp < 0.03; *p < 0.01; #p < 0.002; **p < 0.001; ***p < 0.0001).
ROS production. Moreover, this inhibitory effect of trout HDL on PMA-stimulated superoxide generation was found to be dosedependent and detectable even at a concentration in the lower range of plasma HDL in rainbow trout [13]. In fact, a 5 min preincubation with 0.5 mg ml1 of HDL was enough to completely
abolish the respiratory burst in PMA-stimulated HKL (Fig. 2). A lower concentration of HDL (0.25 mg ml1) caused not only an 80% of inhibition but also delayed significantly the respiratory burst triggered by PMA, suggesting that trout HDL could have a very relevant role in controlling the timing and extension of ROS
Fig. 2. Trout HDL blocks PMA-induced respiratory burst in head-kidney leukocytes. (A) The respiratory burst was measured by a chemiluminescent assay based on luminal oxidation after stimulation of HKL with 1.0 mg ml1 PMA alone or previously incubated during 5 min with, physiological concentrations of trout HDL (0.25 and 0.5 mg ml1) or a supra-physiological concentration (2 mM) of astaxanthin (Asx). (B) The percentage of inhibition of the respiratory burst achieved by the different treatments was calculated measuring the area under the curve using the value obtained with PMA alone as the maximal response. Bars represent the mean value standard error (n ¼ 3).
F. Villarroel et al. / Fish & Shellfish Immunology 35 (2013) 180e183
production in fish leukocytes. HDL particles are very complex in composition and transport a large variety of molecules such as proteins, lipids, pigments and potent immunomodulatory complexes that can regulate its immunological activity and there is accumulating evidence that HDL composition determines its functional properties [21]. As HDL constitutes the main transporter protein for orally administered carotenoids, such as astaxanthin, which has been demonstrated to possess powerful antioxidant properties both in vitro and in vivo, especially as an inhibitor of ROSinduced production of NF-kappaB transcription factor and inflammatory cytokines production [22], we sought to evaluate the capacity of astaxanthin to reproduce at least in part the antioxidant effect of trout HDL. As depicted in Fig. 2B, a supraphysiological concentration of astaxanthin was able to inhibit ROS generation to a lesser extent than the lowest concentration of HDL evaluated suggesting that other components of trout HDL particles must be involved in its potent antioxidant effect. According to the studies performed with mammalian HDL, ApoA-I is one of the most important contributors to this effect since it inhibits fMLP- and PMA- activated neutrophil adhesion, oxidative burst, degranulation and L929 cell mortality [10]. Our results suggest that trout HDL and probably, its major apolipoprotein ApoA-I, could support resolution of inflammation in rainbow trout through the modulation of head kidney leukocytes activation, limiting the extent of the respiratory burst and pro-inflammatory gene expression. Taking in consideration that inflammation and tissue damage are tightly linked processes; the ability of HDL to regulate the amplitude of the inflammatory response might work in conjunction with its direct antimicrobial effects to influence the outcome of the infection in teleost fish. Acknowledgement This work was supported by grant 1050637 from FONDECYTChile. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2013.04.003. References [1] Feig JE, Rong JX, Shamir R, Sanson M, Vengrenyuk Y, Liu J, et al. HDL promotes rapid atherosclerosis regression in mice and alters inflammatory properties of plaque monocyte-derived cells. Proc Natl Acad Sci U S A 2011;108(17):7166e71.
183
[2] Mineo C, Shaul PW. Novel biological functions of high-density lipoprotein cholesterol. Circ Res 2012;111(8):1079e90. [3] Schifflet AM, Bishop JR, Pahwa A, Hajduk SL. Human high density lipoproteins are platforms for the assembly of multi-component innate immune complexes. J Biol Chem 2005;280(38):32578e85. [4] Norata GD, Pirillo A, Ammirati E, Catapano AL. Emerging role of high density lipoproteins as a player in the immune system. Atherosclerosis 2012;220(1): 11e21. [5] Murch O, Collin M, Hinds CJ, Thiemermann C. Lipoproteins in inflammation and sepsis. Intensive Care Med 2007;33:13e24. [6] Jahangiri A. High-density lipoprotein and the acute phase response. Curr Opin Endocrinol Diabetes Obes 2010;17:156e60. [7] Gruaz L, Delucinge-Vivier C, Descombes P, Dayer JM, Burger D. Blockade of T cell contact-activation of human monocytes by high density-lipoproteins reveals a new pattern of cytokine and inflammatory genes. PLoS One 2010;5(2): e9418. [8] Hyka N, Dayer JM, Modoux C, Kohno T, Edwards 3rd CK, Roux-Lombard P, et al. Apolipoprotein A-I inhibits the production of interleukin-1b and tumor necrosis factor-a by blocking contact-mediated activation of monocytes by T lymphocytes. Blood 2001;97:2381e9. [9] Carpintero R, Gruaz L, Brandt KJ, Scanu A, Faille D, Combes V, et al. HDL interfere with the binding of T cell microparticles to human monocytes to inhibit pro-inflammatory cytokine production. PLoS One 2010;5(7):e11869. [10] Liao X, Lou B, Ma J, Wu M. Neutrophils activation can be diminished by apolipoprotein A-I. Life Sci 2005;77:325e35. [11] Concha MI, Molina S, Oyarzún C, Villanueva J, Amthauer R. Local expression of apolipoprotein A-I gene and a possible role for HDL in primary defence in the carp skin. Fish Shellfish Immunol 2003;14:259e73. [12] Concha MI, Smith VJ, Castro K, Bastías A, Romero A, Amthauer R. Apolipoproteins A-I and A-II are potentially important effectors of innate immunity in the teleost fish Cyprinus carpio. Eur J Biochem 2004;271:2984e90. [13] Villarroel F, Bastías A, Casado A, Amthauer R, Concha MI. Apolipoprotein A-I, an antimicrobial protein in Oncorhynchus mykiss: evaluation of its expression in primary defence barriers and plasma levels in sick and healthy fish. Fish Shellfish Immunol 2007;23:197e209. [14] Villarroel F, Casado A, Vásquez J, Araneda B, Amthauer R, Matamala E, et al. Serum amyloid A: a typical acute-phase reactant in rainbow trout? Dev Comp Immunol 2008;32:1160e9. [15] Akira S, Hirano T, Taga T, Kishimoto T. Biology of multifunctional cytokines: IL 6 and related molecules (IL 1 and TNF). FASEB J 1990;4:2860e7. [16] Jensen LE, Whitehead AS. Regulation of serum amyloid-A protein expression during the acute-phase response. Biochem J 1998;334:489e503. [17] Murphy AJ, Woollard KJ, Hoang A, Mukhamedova N, Stirzaker RA, McCormick SP, et al. High-density lipoprotein reduces the human monocyte inflammatory response. Arterioscler Thromb Vasc Biol 2008;28:2071e7. [18] Murphy AJ, Hoang A, Aprico A, Sviridov D, Chin-Dusting J. Anti-inflammatory functions of apolipoprotein A-I and high-density lipoprotein are preserved in trimeric apolipoprotein A-I. J Pharmacol Exp Ther 2013;344:41e9. [19] Barter PJ, Nicholls S, Rye KA, Anantharamaiah GM, Navab M, Fogelman AM. Anti-inflammatory properties of HDL. Circ Res 2004;95:764e72. [20] Saemann MD, Poglitsch M, Kopecky C, Haidinger M, Horl WH, Weichhart T. The versatility of HDL: a crucial anti-inflammatory regulator. Eur J Clin Invest 2010;40:1131e43. [21] Marsche G, Saemann MD, Heinemann A, Holzer M. Inflammation alters HDL composition and function: implications for HDL-raising therapies. Pharmacol Ther 2013. http://dx.doi.org/10.1016/j.pharmthera.2012.12.001. [22] Speranza L, Pesce M, Patruno A, Franceschelli S, de Lutiis MA, Grilli A, et al. Astaxanthin treatment reduced oxidative induced pro-inflammatory cytokines secretion in U937: SHP-1 as a novel biological target. Mar Drugs 2012;10:890e9.
Contents lists available at SciVerse ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Short communication
High density lipoproteins down-regulate transcriptional expression of pro-inflammatory factors and oxidative burst in head kidney leukocytes from rainbow trout, Oncorhynchus mykiss Franz Villarroel 1, Alin Casado 1, Rodolfo Amthauer, Margarita I. Concha* Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Campus Isla Teja, Universidad Austral de Chile, Valdivia, Chile
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 January 2013 Received in revised form 22 March 2013 Accepted 5 April 2013 Available online 15 April 2013
Teleosts are the first group of vertebrates possessing an acquired immune system; however, it is less developed than in mammals and is highly influenced by environmental changes. Therefore, innate immunity effectors play a more critical role in survival of pathogen-challenged fish. In a previous study we showed that trout high density lipoprotein (HDL), and its major apolipoprotein (ApoA-I) are widely expressed in primary defense barriers and other immune-relevant tissues, displaying important antibacterial activity in vitro. Here we show that trout HDL inhibits both basal and LPS-induced transcript expression of pro-inflammatory cytokines such as TNF-a and IL-1b, and the acute phase protein serum amyloid A (A-SAA), in head kidney leukocytes (HLK) from rainbow trout. In addition, trout HDL was able to block the respiratory burst of PMA-stimulated HKL, at physiological concentrations and in a dose dependent manner. Moreover, this effect was only partially mimicked by supra-physiologic concentrations of the HDL-transported carotenoid, astaxanthin. These results constitute the first data suggesting that in addition to its antimicrobial activity, HDL would have a relevant immunomodulatory role in salmonid fish. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Respiratory burst Head kidney leukocytes HDL LPS Cytokines
1. Introduction Although the main role of high density lipoprotein (HDL) has been long considered to be the reverse cholesterol transport and its anti-atherogenic and vascular-protective effects in mammals [1,2], evidences demonstrating a key role of HDL and its components in innate and adaptive immunity have accumulated during the last decade [3e6]. In fact, it has been demonstrated that HDL acts as a platform for the assembly of potent immunomodulatory complexes that regulate antimicrobial activity [3]. Among the immunomodulatory roles that have been described for mammalian HDL and its major apolipoprotein, are the ability of HDL and ApoA-I to interfere with the interaction between activated T cells and human monocytes and also to reduce neutrophil activation and the corresponding production of pro-inflammatory cytokines such as interleukin 1 beta (IL-1b) and tumour necrosis factor alpha (TNFa) [7e10]. Abbreviations: ROS, reactive oxygen species; HDL, high density lipoprotein; PMA, phorbol 12-myristate 13-acetate; A-SAA, acute serum amyloid A; Asx, astaxanthin. * Corresponding author. Tel.: þ56 63 221108; fax: þ56 63 221332. E-mail addresses: [email protected] (F. Villarroel), alin.casado@ gmail.com (A. Casado), [email protected] (R. Amthauer), [email protected], [email protected] (M.I. Concha). 1 These authors contributed equally to this work. 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.04.003
Our previous studies demonstrated that HDL and its major apolipoproteins (ApoA-I and ApoA-II) not only constitute one of the major plasma proteins in carp and rainbow trout but also display potent antimicrobial activity in vitro against Gram-positive and -negative bacteria [11e13]. In addition it was shown that apolipoprotein A-I transcript is abundantly expressed in the carp skin and ApoA-I protein is apparently secreted to the mucus constituting nascent HDL particles [11]. Similarly, high levels of ApoA-I transcript and protein have been detected in the main primary defense barriers in trout and its plasma concentration remains unaffected during the acute phase, in contrast with mammalian ApoA-I, which constitutes a negative acute phase reactant [13]. Taking in consideration these differences between mammalian and teleost HDL, the aim of the present study was to evaluate possible immunomodulatory functions of teleost HDL.
2. Material and methods 2.1. Experimental fish Healthy rainbow trout (Oncorhynchus mykiss) (150e200 g) were obtained from the fish farm (Cultivos Marinos Chiloe, Province of Valdivia, Chile) and maintained in 2500 l flow-through tanks with
F. Villarroel et al. / Fish & Shellfish Immunology 35 (2013) 180e183
aerated well water at 12 1 C under an 11-h D: 13-h L photoperiod. Fish were sacrificed by benzocaine overdose (250 mg l1) following the ethical guidelines for the use of animals in research from the University Committee, and dissected to obtain the head kidney for leukocyte isolation. Blood samples were extracted from the caudal vein of anaesthetised fish (50 mg l1 benzocaine) with a heparinised syringe and plasma samples from four fish were pooled after centrifugation at 2000 g for 3 min (twice) to assure the complete removal of blood cells, and after addition of protease inhibitors (1 mM PMSF and 2 mM benzamidine), kept in aliquots at 20 C. Plasma HDL fractions were isolated from whole plasma pools by affinity chromatography on Affi-GelÒ Blue-Gel (BIO-RAD), and its purity and integrity confirmed by SDS-PAGE and Western blot analyses, as previously described by [12]. 2.2. Isolation and stimulation of head kidney leukocytes Head kidney leukocytes (HLK) were isolated essentially as described previously [13]. Briefly, the head kidneys from at least three fish were resuspended in 0.97% (w/v) Hank’s salt solution, supplemented with 10 U ml1 heparin and 2% (v/v) foetal calf serum (FCS) and passed through a 100-mesh nylon cell strainer (Falcon). The resulting suspension was placed on a 31/46% discontinuous Percoll gradient and centrifuged at 400 g for 30 min. Finally, leukocytes were carefully collected from the interphase, suspended, washed twice in L-15 medium and viability tested by exclusion of trypan blue dye. Over 95% viable HKL preparations were suspended in L-15 medium supplemented with 10% FCS, 100 mg ml1 of streptomycin and 60 mg ml1 penicillin and then seeded on 6-well cell culture NuncÒ plates (2.5 106 cells per well). After 18 h of culture, non-adherent cells were removed and the remaining cells incubated in L-15 medium supplemented with 10% FCS. For the expression assays, HKL were incubated in L-15 medium supplemented with 5% FCS with or without 25 mg ml1 Escherichia coli 026:B6 LPS for 1 h at 16 C. Alternatively cells were incubated first during 18 h with 0.25 mg ml1 HDL, then the media containing trout HDL was completely removed and replaced by fresh medium containing 25 mg ml1 E. coli LPS. Afterwards, cells were lysed for RNA isolation and cytokine expression analyses. Expression experiments were performed in triplicates using three different HKL preparations. 2.3. Isolation of total RNA and cDNA synthesis For total RNA extraction from cultured HKL, the medium was removed and cells were immediately lysed by the addition of 1 ml of RNAwiz reagent (Ambion Inc.) for 4 106 cells according to the manufacturer’s instructions. RNA was quantified by absorbance at 260 nm and used immediately or stored precipitated in ethanol at 70 C until use. Total RNA integrity was evaluated as described previously [14]. After treating total RNAs with 5 units of amplification grade DNAse I RQ1 (Promega), semi-quantitative RT-PCR analyses were performed using the primers listed in Supplementary Table 1 and conditions described previously [14]. bactin was used as control gene since no changes in expression were observed for this gene under the conditions tested. To evaluate the relative expression of each gene, the ratio of the corresponding transcript to b-actin was determined after gel scanning, measuring the pixel intensity of the bands (same area) with a BIO-RAD Molecular Imager FX Pro System, using the Quantity One program for Windows. Total counts (arbitrary units) from each band were obtained after subtracting the background. An arbitrary value of one was assigned to the ratios obtained for the control cells and the mean fold change standard error was plotted for the treated cells.
181
2.4. Respiratory burst assays Head kidney leukocytes (1 106 cells per well) were placed in the wells of a flat-bottomed 96-well microtiter plate with 100 ml of L-15 and were preincubated at 18 C for 5 min with or without the addition of different concentrations of freshly prepared trout HDL (0.25 or 0.5 mg ml1) or 2 mM astaxanthin and then 20 mM of luminol (Sigma) and 1 mg ml1 phorbol-12-myristate 13-acetate (PMA, Sigma Chemical Co., St. Louis, MO, USA) were added. The plate was gently shaken and reactive oxygen species (ROS) production was immediately measured using a Luminoskan AscentÔ luminometer (Thermo Labsystems) for 5000 s. Luminescence backgrounds were determined using reagent solutions containing luminol but not PMA. The percentage of inhibition of the respiratory burst achieved by the different treatments was calculated measuring the area under the curve using the value obtained with PMA alone as the maximal response. 2.5. Statistical analyses All data are given as means standard error of the mean (SEM) of three independent experiments. Statistical analysis was carried out applying the Student’s t-test using the GraphPad Prism v5.0 software for Windows. Differences with p < 0.03 were considered statistically significant. 3. Results and discussion To evaluate if trout HDL was able to down-regulate the inflammatory response, we analysed by RT-PCR the relative levels of mRNAs for IL-1b, TNFa and the acute phase reactant, serum amyloid A (A-SAA) in unstimulated- and LPS-activated HKL. As expected, LPS promoted a significant increase in the relative transcript levels of all three cytokines (Fig. 1), while pre-incubation of the cells with 0.25 mg ml1 of purified trout HDL led to a reduction of more than 65% in the LPS-triggered response (Fig. 1B, C, D). Moreover, this inhibitory effect on tnf-a1 and il1-b1 expression was also observed in unstimulated HKL, since basal transcript levels became undetectable after pre-incubation with HDL. In contrast, basal levels of A-SAA transcript were negligible probably because different cytokines, including interferon-gamma, transforming growth factor beta, TNFa, and interleukins, either alone or in combination, have been shown to regulate the synthesis of this major acute-phase reactant at the transcriptional level [15,16]. Previously, we reported that A-SAA transcript was strongly upregulated during an in vivo- or in vitro-challenge with LPS in several trout tissues and HKL, respectively, suggesting that it could constitute a very sensitive marker for the detection of proinflammatory conditions [13,14]. Murphy et al. [17] described a potent attenuating effect of human HDL and its major apolipoprotein ApoA-I in monocyte activation and this effect could be reproduced by reconstituted HDL particles [18]. Furthermore, direct effects of HDL were found on myeloid cells including monocytes, macrophages and monocytes-derived dendritic cells, resulting in suppression of cytokine and chemokine production, downregulation of co-stimulatory molecules and inhibition of antigen presentation [4,19,20]. These data are consistent with the results presented herein suggesting that the anti-inflammatory role of HDL could be evolutionary conserved. We also studied the effect of trout HDL on the oxidative burst response in HKL stimulated with PMA, as overproduction of reactive oxygen species and other effective yet also toxic substances produced by leukocytes may participate in disturbed apoptosis and intensification of the inflammatory processes. As shown on Fig. 2, HDL showed a strong and significant reduction in the intracellular
182
F. Villarroel et al. / Fish & Shellfish Immunology 35 (2013) 180e183
Fig. 1. Trout HDL reduces pro-inflammatory cytokine expression in head-kidney leukocytes, under basal and LPS-stimulated conditions. Expression of il-1b1, tnfa1 and a-saa transcripts was evaluated by semi-quantitative RT-PCR, using b-actin as constitutive transcript. Database accession numbers of the genes used are detailed in Supplementary Table 1. (A) Representative RT-PCR results obtained from HKL without treatment (basal condition), stimulated with LPS (25 mg ml1) and pre-treated with 0.25 mg ml1 trout HDL with or without further LPS stimulation, respectively. Graphs B, C and D show the mean value standard error of the fold change obtained for a-saa, il1-b1 and tnf-a1, respectively from three independent experiments. Statistical significance was determined using the GraphPad Prism software for each treatment with respect to the control and also between LPStreated cells with or without pretreatment with HDL (xp < 0.03; *p < 0.01; #p < 0.002; **p < 0.001; ***p < 0.0001).
ROS production. Moreover, this inhibitory effect of trout HDL on PMA-stimulated superoxide generation was found to be dosedependent and detectable even at a concentration in the lower range of plasma HDL in rainbow trout [13]. In fact, a 5 min preincubation with 0.5 mg ml1 of HDL was enough to completely
abolish the respiratory burst in PMA-stimulated HKL (Fig. 2). A lower concentration of HDL (0.25 mg ml1) caused not only an 80% of inhibition but also delayed significantly the respiratory burst triggered by PMA, suggesting that trout HDL could have a very relevant role in controlling the timing and extension of ROS
Fig. 2. Trout HDL blocks PMA-induced respiratory burst in head-kidney leukocytes. (A) The respiratory burst was measured by a chemiluminescent assay based on luminal oxidation after stimulation of HKL with 1.0 mg ml1 PMA alone or previously incubated during 5 min with, physiological concentrations of trout HDL (0.25 and 0.5 mg ml1) or a supra-physiological concentration (2 mM) of astaxanthin (Asx). (B) The percentage of inhibition of the respiratory burst achieved by the different treatments was calculated measuring the area under the curve using the value obtained with PMA alone as the maximal response. Bars represent the mean value standard error (n ¼ 3).
F. Villarroel et al. / Fish & Shellfish Immunology 35 (2013) 180e183
production in fish leukocytes. HDL particles are very complex in composition and transport a large variety of molecules such as proteins, lipids, pigments and potent immunomodulatory complexes that can regulate its immunological activity and there is accumulating evidence that HDL composition determines its functional properties [21]. As HDL constitutes the main transporter protein for orally administered carotenoids, such as astaxanthin, which has been demonstrated to possess powerful antioxidant properties both in vitro and in vivo, especially as an inhibitor of ROSinduced production of NF-kappaB transcription factor and inflammatory cytokines production [22], we sought to evaluate the capacity of astaxanthin to reproduce at least in part the antioxidant effect of trout HDL. As depicted in Fig. 2B, a supraphysiological concentration of astaxanthin was able to inhibit ROS generation to a lesser extent than the lowest concentration of HDL evaluated suggesting that other components of trout HDL particles must be involved in its potent antioxidant effect. According to the studies performed with mammalian HDL, ApoA-I is one of the most important contributors to this effect since it inhibits fMLP- and PMA- activated neutrophil adhesion, oxidative burst, degranulation and L929 cell mortality [10]. Our results suggest that trout HDL and probably, its major apolipoprotein ApoA-I, could support resolution of inflammation in rainbow trout through the modulation of head kidney leukocytes activation, limiting the extent of the respiratory burst and pro-inflammatory gene expression. Taking in consideration that inflammation and tissue damage are tightly linked processes; the ability of HDL to regulate the amplitude of the inflammatory response might work in conjunction with its direct antimicrobial effects to influence the outcome of the infection in teleost fish. Acknowledgement This work was supported by grant 1050637 from FONDECYTChile. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2013.04.003. References [1] Feig JE, Rong JX, Shamir R, Sanson M, Vengrenyuk Y, Liu J, et al. HDL promotes rapid atherosclerosis regression in mice and alters inflammatory properties of plaque monocyte-derived cells. Proc Natl Acad Sci U S A 2011;108(17):7166e71.
183
[2] Mineo C, Shaul PW. Novel biological functions of high-density lipoprotein cholesterol. Circ Res 2012;111(8):1079e90. [3] Schifflet AM, Bishop JR, Pahwa A, Hajduk SL. Human high density lipoproteins are platforms for the assembly of multi-component innate immune complexes. J Biol Chem 2005;280(38):32578e85. [4] Norata GD, Pirillo A, Ammirati E, Catapano AL. Emerging role of high density lipoproteins as a player in the immune system. Atherosclerosis 2012;220(1): 11e21. [5] Murch O, Collin M, Hinds CJ, Thiemermann C. Lipoproteins in inflammation and sepsis. Intensive Care Med 2007;33:13e24. [6] Jahangiri A. High-density lipoprotein and the acute phase response. Curr Opin Endocrinol Diabetes Obes 2010;17:156e60. [7] Gruaz L, Delucinge-Vivier C, Descombes P, Dayer JM, Burger D. Blockade of T cell contact-activation of human monocytes by high density-lipoproteins reveals a new pattern of cytokine and inflammatory genes. PLoS One 2010;5(2): e9418. [8] Hyka N, Dayer JM, Modoux C, Kohno T, Edwards 3rd CK, Roux-Lombard P, et al. Apolipoprotein A-I inhibits the production of interleukin-1b and tumor necrosis factor-a by blocking contact-mediated activation of monocytes by T lymphocytes. Blood 2001;97:2381e9. [9] Carpintero R, Gruaz L, Brandt KJ, Scanu A, Faille D, Combes V, et al. HDL interfere with the binding of T cell microparticles to human monocytes to inhibit pro-inflammatory cytokine production. PLoS One 2010;5(7):e11869. [10] Liao X, Lou B, Ma J, Wu M. Neutrophils activation can be diminished by apolipoprotein A-I. Life Sci 2005;77:325e35. [11] Concha MI, Molina S, Oyarzún C, Villanueva J, Amthauer R. Local expression of apolipoprotein A-I gene and a possible role for HDL in primary defence in the carp skin. Fish Shellfish Immunol 2003;14:259e73. [12] Concha MI, Smith VJ, Castro K, Bastías A, Romero A, Amthauer R. Apolipoproteins A-I and A-II are potentially important effectors of innate immunity in the teleost fish Cyprinus carpio. Eur J Biochem 2004;271:2984e90. [13] Villarroel F, Bastías A, Casado A, Amthauer R, Concha MI. Apolipoprotein A-I, an antimicrobial protein in Oncorhynchus mykiss: evaluation of its expression in primary defence barriers and plasma levels in sick and healthy fish. Fish Shellfish Immunol 2007;23:197e209. [14] Villarroel F, Casado A, Vásquez J, Araneda B, Amthauer R, Matamala E, et al. Serum amyloid A: a typical acute-phase reactant in rainbow trout? Dev Comp Immunol 2008;32:1160e9. [15] Akira S, Hirano T, Taga T, Kishimoto T. Biology of multifunctional cytokines: IL 6 and related molecules (IL 1 and TNF). FASEB J 1990;4:2860e7. [16] Jensen LE, Whitehead AS. Regulation of serum amyloid-A protein expression during the acute-phase response. Biochem J 1998;334:489e503. [17] Murphy AJ, Woollard KJ, Hoang A, Mukhamedova N, Stirzaker RA, McCormick SP, et al. High-density lipoprotein reduces the human monocyte inflammatory response. Arterioscler Thromb Vasc Biol 2008;28:2071e7. [18] Murphy AJ, Hoang A, Aprico A, Sviridov D, Chin-Dusting J. Anti-inflammatory functions of apolipoprotein A-I and high-density lipoprotein are preserved in trimeric apolipoprotein A-I. J Pharmacol Exp Ther 2013;344:41e9. [19] Barter PJ, Nicholls S, Rye KA, Anantharamaiah GM, Navab M, Fogelman AM. Anti-inflammatory properties of HDL. Circ Res 2004;95:764e72. [20] Saemann MD, Poglitsch M, Kopecky C, Haidinger M, Horl WH, Weichhart T. The versatility of HDL: a crucial anti-inflammatory regulator. Eur J Clin Invest 2010;40:1131e43. [21] Marsche G, Saemann MD, Heinemann A, Holzer M. Inflammation alters HDL composition and function: implications for HDL-raising therapies. Pharmacol Ther 2013. http://dx.doi.org/10.1016/j.pharmthera.2012.12.001. [22] Speranza L, Pesce M, Patruno A, Franceschelli S, de Lutiis MA, Grilli A, et al. Astaxanthin treatment reduced oxidative induced pro-inflammatory cytokines secretion in U937: SHP-1 as a novel biological target. Mar Drugs 2012;10:890e9.