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F′ does not trigger inflammation in the bovine mammary gland
Microbial Pathogenesis 51 (2011) 396e401
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Purified Staphylococcus aureus leukotoxin LukM/F0 does not trigger inflammation in the bovine mammary gland Angélina Fromageau, Patricia Cunha, Florence B. Gilbert, Pascal Rainard* INRA, UR 1282 Infectiologie Animale et Santé Publique, IASP Bat. 311, F-37380 Nouzilly, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 May 2011 Received in revised form 12 September 2011 Accepted 15 September 2011 Available online 22 September 2011
An early recruitment of neutrophils in mammary tissue and milk is considered an important component of the defense of the mammary gland against Staphylococcus aureus. We investigated whether the leukotoxin LukM/F0, which is produced by a proportion of mastitis-causing strains of S. aureus, would be able to trigger inflammation in the udder. Infusion of purified LukM/F0 toxin in lactating mammary glands did not cause neutrophil influx in milk, showing that the toxin was not able to cause mastitis on its own. Purified LukM/F0 did not kill or stimulate mammary epithelial cells in culture. As expected, LukM bound to mammary macrophages and the complete LukM/F0 toxin killed these cells, but subcytotoxic LukM/F0 concentrations did not induce secretion of IL-8, TNF-a, IL-1b or IL-6 by macrophages. On the contrary, the production of these pro-inflammatory mediators by adhesion-stimulated macrophages was reduced. Overall, these results indicate that purified leukotoxin LukM/F0 is not likely to contribute to the initiation of the inflammatory response and could even play an anti-inflammatory role in the mammary gland by inactivating macrophages. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Staphylococcus aureus Mastitis Cattle Epithelial cell Macrophage Leukotoxin
1. Introduction In ruminants, mastitis is by far the main pathology elicited by Staphylococcus aureus. Among mastitis-causing bacteria, S. aureus is prominent owing to its capacity to induce severe cases or long-lasting infections and because of its prevalence in certain countries [1e3]. Because of its contagious nature and its low cure rates, S. aureus mastitis is also difficult to control [4]. The influx of neutrophils in mammary tissue and milk is an important component of the defense of the mammary gland against S. aureus [5]. The mammary gland is likely to sense intruding S. aureus mainly through the detection of microbial-associated molecular patterns (MAMPs) such as peptidoglycan fragments, lipopeptides and lipoteichoic acid (LTA), as it was shown in other pathological settings in most host species [6]. Staphylococcal LTA or muramyl dipeptide induce inflammation and neutrophil influx when infused in the lumen of the mammary gland [7,8]. Besides MAMPs, exotoxins are likely to play a part in setting off inflammation [6]. For example, staphylococcal alpha toxin was shown to induce inflammation when infused in rabbit mammary glands [9]. Staphylococcal leukotoxins may have pro-inflammatory properties. Administration of the Panton-Valentine leucocidin (PVL) in the * Corresponding author. Tel.: þ33 2 47 42 76 33; fax: þ33 2 47 42 77 79. E-mail address: [email protected] (P. Rainard). 0882-4010/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2011.09.005
lungs of mice was sufficient to induce inflammation and tissue damage [10], and PVL contributed to muscle injury through induction of chemokines and recruitment of neutrophils [11]. Recently, several studies indicated that staphylococcal leukotoxins could be sensed by the innate immune system and trigger inflammatory responses [12e14]. Staphylococcal leukotoxins are bi-components toxins which assemble on their target cells to form trans-membrane pores [15]. The cells most susceptible to leukotoxins belong to the myeloid lineage, such as neutrophils, monocytes and macrophages. Several distinct staphylococcal leukotoxins have been described, but certain of these toxins are host-specific: the PVL targets mainly human and rabbit leucocytes but not bovine cells [15], whereas the LukM/LukF0 leukotoxin is active on bovine but not human cells [16]. The genes coding the leukotoxin LukM/LukF0 have not been found in S. aureus strains of human origin, and seem to be harbored only by strains isolated from mastitis of ruminants [17,18]. Among these bi-component poreforming toxins, LukM, associating with its partner component LukF0, composes the leukotoxin most active on bovine cells [19]. Very few data are available concerning the role of this toxin in the pathology of staphylococcal mastitis, because little is known of the local effects of this leucocidal product in vivo. Yet, the toxin is produced during mastitis and can be found in mammary secretions from severe cases of mastitis [20]. Pro-inflammatory effects of staphylococcal leukotoxins could result from a direct effect on target cells through induction of
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mediators such as leucotriene B4, IL-8, IL-6 and IL-12 as shown with human neutrophils stimulated with PVL or gamma hemolysin [21]. Another possibility is an indirect effect through the release of damaging components released by neutrophils attracted to the site of infection after leukotoxin-induced necrosis [22]. Lately it was reported that the Panton-Valentine leucocidin was able to interact with and activate the Toll-like receptor TLR2 [13], and that another leucocidin, hemolysin gamma (also named LukS/F) was able to activate TLR4 [12]. The mammary gland and mammary epithelial cells are known to express both TLR2 and TLR4 and to respond to the corresponding ligands such as LTA and LPS [7,8,23e25]. Thus the question arises of the reactivity of the ruminant mammary gland to the ruminant-associated leukotoxin (LukM/F0 ) and to the contribution of this exotoxin to the detection of S. aureus during infection. In this line of thought, a preparation of the leukotoxin LukM/LukF0 has been reported to induce inflammation in the bovine mammary gland [14]. The aim of our study was to investigate the pro-inflammatory activity of LukM/LukF0 in the bovine mammary gland. We used LukM/LukF0 purified from the culture supernatant of a mastitis isolate of S. aureus. A particular attention was paid to contamination of the preparation by bacterial agonists able to induce inflammation in the mammary gland, which is very susceptible to MAMPs such as lipopolysaccharide (LPS), lipoteichoic acid (LTA) or MDP, and even more so when these components are in association [8,26]. Our preparations of LukM/LukF0, which were fully active on bovine neutrophils, failed to induce inflammation in the lactating mammary gland. The absence of pro-inflammatory responses of mammary epithelial cells (MEC) or mammary macrophages to LukM/LukF0 was in line with this finding. 2. Materials and methods 2.1. Infusion of mammary glands with purified LukM/F0 Five healthy Holstein cows from the institutional experimental herd (PFIE, Nouzilly) that had no detectable bacterial growth and cell counts less than 100,000 cells/ml in milk samples of their four quarters in two consecutive weeks were used. These cows had no record of clinical mastitis in the period between calving and time of challenge. They were in their second or third lactation, and between three to seven months in lactation. Cows were milked twice daily, at 08:00 and 16:00 h. The use and care of the cows conformed to the practices in effect at the INRA Nouzilly Research Center. The two components of the leukotoxin LukM/F0 were purified as described [19]. Separately, LukM and LukF0 had no activity on bovine neutrophils, whereas after combination at equimolar concentrations, the mixture induced cytopathic effects (flattening of neutrophils) at 0.15 nM and higher concentrations, as previously reported [27]. The presence of endotoxin in the purified LukM and LukF0 was assessed with a Limulus amoebocyte lysate assay (E-toxate test, Sigma). Before infusion, the purified components LukM and LukF0 were diluted in HBSS which was supplemented with bovine serum albumin (BSA; cell culture tested, Sigma) at a final concentration of 0.5 mg/ml (HBSS-A), as a vehicle to minimize losses of toxin components during preparation of diluted solutions. Each cow received at time zero HBSS-A in one quarter, 15 mg of LukM in a second quarter, 15 mg in a third quarter, and 15 mg of LukM plus 15 mg of LukF’ in the fourth quarter. The infused quarters were aseptically sampled just before infusion after the morning milking, then 4, 8, 12, 24, and 48 h post-infusion. Milk samples were used for bacteriological examination by plating over a blood-esculin-agar plate and somatic cell count (SCC) by use of a cell counter (Fossomatic model 90; Foss Food Technology, Hillerod, Denmark) as previously described [8]. Milk samples were then centrifuged at
397
1000 g for 30 min at 4 C. Skimmed milk was stored in portions at 18 C. 2.2. Interaction of LukM/F0 with mammary epithelial cells Mammary secretory tissue from five different cows of the institutional dairy herd at Nouzilly was obtained at the slaughterhouse from lactating quarters free from infection and inflammation. Bovine mammary epithelial cells (bMEC) were isolated and cryopreserved in liquid nitrogen [28]. When required, bMEC were thawed and cultured without serum and antibiotics in Dulbecco’s modified Eagle’s medium (D-MEM)eF12 Advanced medium (Gibco), which contains insulin (10 mg/ml), albumin (0.4 mg/ml), and transferrin (7.5 mg/ml), supplemented with 2 mM L-glutamine, 10 ng/ml insulin-like growth factor I (IGF-I) (Peprotech), 5 ng/ml fibroblast growth factor (FGF) (Peprotech), 5 ng/ml human recombinant epidermal growth factor (EGF) (Sigma), 1 mg/ml hydrocortisone (Sigma), and 20 mM HEPES (Cambrex Biowhittaker). Cells were used at their 3rd passage after harvest from mammary glands. Cells were seeded in 24-well tissue culture plates at a density of 1 105 cells/well and cultured until confluence, then the growth medium was replaced with stimulation medium made up of D-MEM/F12 Advanced medium with 2 mM Lglutamine, 20 mM HEPES and 4 ng/ml of hydrocortisone as additives. Stimulations were carried out 16e24 h later. MEC were incubated with 1, 5 or 20 nM of LukM, LukF’ or LukM/F0 for 24 h at 37 C and 5% CO2. Staphylococcal LTA (Cayla/InvivoGen, Toulouse, France) was used at 250 ng/ml as a positive control of stimulation [8]. The effect of LukM/F0 on the viability of bMEC was assessed with an alamarBlueÒ (Biosource International Camarillo, CA, USA) assay, which is an oxidation-reduction indicator. AlamarBlue stock solution was prepared in the assay medium and distributed in the wells to yield a final concentration of 10% alamarBlue. The plates were incubated for 4 h at 37 C and 5% CO2. The fluorescence was then measured with an excitation wavelength of 530 nm and an emission wavelength at 590 nm (Cytofluor 2300 System, Applied Biosystems). Results were expressed as arbitrary units (AU) of fluorescence. The positive control of toxicity was obtained by exposing the cells to 0.1% Triton 100 for 5 min before washing and addition of alamarBlue. After this treatment, fluorescence was hardly detectable. Results were expressed as % metabolic activity: (control fluorescence/ residual fluorescence) 100. 2.3. Interaction of LukM/F0 with mammary macrophages Macrophages were obtained from secretions of involuted mammary glands as described [29]. After daily washing of a driedoff udder quarter with 50 ml of non-pyrogenic saline for 7 days, cells were harvested from the lavage fluid by centrifugation at 400 g for 15 min, washed once in Dulbecco’s PBS, resuspended and adjusted to 5 105 cells/ml in RPMI containing HEPES 10 mM, FCS 10%, penicillin 50 U, streptomycin 200 mg/ml. To determine the cytotoxic effect of LukM/F0 on macrophages, the cells were distributed in a 96-well flat-bottomed microtiter plate (80 ml of suspension per well) with increasing twofold dilutions (20 ml per well) of LukM, LukF’ alone or in association (equimolar amounts, from 0.03 nM to 4 nM) in RPMI-A containing 10% alamarBlue. Plates were incubated for 2 h at 37 C, 5% CO2, and the fluorescence was measured as described above. To determine the stimulating effect of LukM/F0, macrophages in 12-well plates (7.5 105 cells/well) were incubated for 2 h at 37 C, 5% CO2. Thereafter, 0.1, 0.2 and 0.4 nM of LukM, LukF0 or LukM/F0 were added and incubation carried out for 6 h. LPS (0.1 mg/ml) was added as positive control of cytokines secretion. Supernatants were conserved to determine cytokine concentrations.
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2.4. Interaction of LukM/F0 with neutrophils in milk
2.5. ELISAs for TNF-a, IL-1b, IL-6 and IL-8 Enzyme-linked immunosorbent assays (ELISAs) were used to measure cytokine concentrations in culture supernatants of macrophage incubated with LukM/F0. The ELISAs for TNF-a and IL-8 were performed as previously described [31,32]. Commercially available kits (Thermo Scientific, Rockford, IL, USA) were used according to the manufacturer’s instructions to measure bovine IL1b and IL-6. 2.6. Statistical analysis Statistical analyses of the milk cell concentrations or the concentrations of analytes in cell culture supernatants were performed with the nonparametric Friedman test (GraphPad Prism). A probability level of a P value < 0.05 was considered significant. 3. Results 3.1. Intramammary infusion of purified LukM/F0 The capacity of LukM/F0 to induce inflammation in the mammary gland was investigated by infusing the purified components in the lumen of uninflamed glands of 5 cows through the teat canal. To check whether the active toxin, comprised of the two components LukM and LukF’ assembling on the target cells, was necessary to trigger inflammation, each cow received either LukM (15 mg), LukF0 (15 mg), or LukM/F0 (15mg þ 15 mg) in different udder quarters. The fourth quarter was infused with the vehicle only and served as a negative control. None of the cows developed inflammation, which was monitored through the influx of leucocytes in milk (Fig. 1), apart from the physiological slight increase in milk cell concentrations between milkings, which remained in the range of cell concentrations in healthy glands (less than 200,000 cells per ml milk). For comparison, intramammary infusion of PAMPs elicits influx of leucocytes in milk of several million cells per ml [7,8]. Incidentally, the absence of response suggested that the preparation of purified LukM/F0 was not contaminated with PAMPs, as the mammary gland is very susceptible to these bacterial agonists of the innate immune system. Because mammary epithelial cells (MEC) are the first and most numerous cells in contact with the infused toxin, we tested their susceptibility to LukM/F0. 3.2. Effect of LukM/F0 on bovine MEC MEC are able to secrete several chemokines in response to various bacterial agonists [24,28,33], and the secretion of IL-8 can be used as a sensitive and convenient stimulation read-out [8]. Consequently, we monitored the reactivity of MEC by measuring IL-
Cells / ml (/1000)
HBSS A HBSS-A LukM LukF' LukM/F'
1000 100 10 1 0
12 24 36 Hours post-infusion
48
Fig. 1. The leukotoxin LukM/F0 did not induce inflammation in the mammary gland. Healthy glands of 5 cows were infused with either HBSS-A (vehicle), 15 mg LukM, 15 mg LukF0 or the LukM þ LukF0 (15 mg each). At 4, 8, 12, 24 and 48 h post-infusion, milk samples were taken and the concentration of cells (recruited leucocytes) was determined as correlate of inflammation. Results are expressed as numbers (median values/ 1000) of cells per ml of milk and interquartiles (Q1 and Q3).
8 concentrations in culture supernatant following incubation with LukM/F0. After 24 h of incubation with 5 or 20 nM LukM/F0, the concentrations of IL-8 in cell culture supernatants were not significantly different from concentration in the supernatant of control unstimulated cells, whereas a positive control (S. aureus LTA at 250 ng/ml) induced a strong response (4680 pg/ml) (Fig. 2). As the absence of IL-8 secretion could have resulted from a cytotoxic effect of LukM/F0, the viability of MEC was checked with an alamarBlue test. The alamarBlue fluorescence was not reduced even in the presence of 20 nM LukM/F0, showing that the metabolic activity of MEC was not affected by the toxin (results not shown). These results suggest that LukM/F0 is not likely to induce mammary inflammation by stimulating MEC, an inference that is in keeping with the results of the in vivo experiment. Another cell type which is likely to be in contact with intramammarily infused LukM/F0 is mammary macrophages. Consequently, we tested the
5000 4000 IL- 8 (pg/mL)
Milk was obtained from one cow. Bacteriological status (sterility) of milk was checked before storage at 20 C in portions. Bovine blood neutrophils were prepared from the blood of dairy cows (Holstein breed) selected for their low concentration of circulating eosinophils, as described [30] with some modifications [16]. Cells were resuspended in RPMI-A (2 106 cells/ml). Twenty ml of neutrophil suspension was distributed in the wells of a 96-well flat-bottomed microtiter plate with 80 ml of increasing twofold dilutions of LukM/F0 (equimolar amounts of the two components) in RPMI-A in one plate and in milk in another one, in the presence of 10% alamarBlue. After 1.5 h of incubation at 37 C, the fluorescence was measured with an excitation wavelength of 530 nm and an emission wavelength at 590 nm (Cytofluor 2300 System).
10000
3000 2000 1000 0 ed M
iu
m Lu
'1 /F kM
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'2 /F M uk
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A
Fig. 2. The leukotoxin did not elicit IL-8 production by mammary epithelial cells. Primary bovine MEC were incubated with either the separate components or the two components LukM and LukF’ at the indicated concentrations (5 nM or 20 nM) for 24 h. Concentrations of IL-8 were determined in the culture supernatants as a read-out of MEC stimulation. Staphylococcal lipoteichoic acid (LTA, 250 ng/ml) was used as a positive control, yielding an IL-8 concentration of 4577 pg/ml. Concentrations of IL-8 after incubation with LukM/F0 did not differ from the concentration in supernatant of unstimulated cells (Friedman test, P ¼ 0.095).
A. Fromageau et al. / Microbial Pathogenesis 51 (2011) 396e401
pro-inflammatory cytokines and tended to reduce the secretion of the chemokine IL-8.
% metabolic activity
120,0 100,0 80,0
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3.4. Effect of milk on the cytotoxic activity of LukM/F0
M/F' 1
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Fig. 3. LukM/F0 is cytotoxic for mammary macrophages. Macrophages from 3 cows were incubated with various concentrations of LukM/F0 for 6 h (M/F’ 1, 2, 3) or with only one of the two components of the toxin at 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2 and 4 nM (M or F0 ). Then the metabolic activity (reduction of alamarBlue) was determined.
Milk components, such as antibodies, could have dampened or neutralized the activity of LukM/F0, thereby inhibiting its capacity to induce an inflammatory response after infusion in lactating mammary glands. The cytotoxic activity of LukM/F0 was determined in milk from a healthy gland and compared to the activity in RPMI-A with the alamarBlue assay. Milk did not reduce the activity of LukM/F0, on the contrary it exacerbated the cytotoxic activity at LukM/F0 concentrations of 0.5 nM and higher (Fig. 5). This suggests that the lack of pro-inflammatory activity of the leukotoxin in the mammary gland did not result from interference by milk. 4. Discussion
capacity of LukM/F0 to induce the secretion of pro-inflammatory cytokines by these cells. 3.3. Effect of LukM/F0 on bovine mammary macrophages Mammary macrophages bind LukM and are susceptible to LukM/F0 [16]. We speculated that subcytotoxic concentrations of LukM/F0 could induce the secretion of pro-inflammatory cytokines by macrophages. We first determined the minimal concentrations of LukM/F0 which induced the killing of mammary macrophages, with the alamarBlue assay. Cell metabolic activity was reduced by concentrations as low as 0.5 nM LukM/F0, as measured after 6 h of incubation with LukM/F0 (Fig. 3). Subsequently, mammary macrophages were incubated with concentrations of 0.1, 0.2 and 0.4 nM LukM/F0 for 6 h and IL-8, TNF-a, IL-1b and IL-6 concentrations were measured in the culture supernatant. LPS from Escherichia coli (0.1 mg/ml), used as positive control, induced the secretion of IL-8, TNF-a and IL-1b but not of IL-6. LukM/F0 did not induce a detectable secretion of TNF-a or IL-6 (results not shown). There was a constitutive secretion of IL-8 by macrophages under our culture conditions. This secretion was reduced by LukM/F0 with a doseeresponse effect, although the decrease was not significant (Fig. 4A). There was some constitutive secretion of IL-1b, but there was no significant dose-effect in response to LukM/F0 (Fig. 4B). These results indicate that LukM/F0 did not induce the secretion of
This study aimed at defining the capacity of the staphylococcal leukotoxin LukM/F0 to induce an inflammatory response in the mammary gland. Our results demonstrate that LukM/F0 did not trigger an inflammation in the mammary gland, even when a sizeable amount was infused in its lumen. The amount of LukM/F0 infused in mammary quarters (15 mg) would yield a concentration of about 150 ng/ml, assuming a volume of 100 ml of residual milk in the mammary cistern after milking. This is 15 fold the minimal concentration inducing cytotoxity to milk macrophages (this study), and a high concentration compared to the leucotoxic activity towards bovine neutrophils that begins at 4 ng/ml [27]. Also, we checked that milk from healthy glands did not dampen the toxic activity of LukM/ F0. Thus we can conclude that LukM/F0 is not pro-inflammatory for the mammary gland on its own. This incapacity correlated with the absence of pro-inflammatory response of the two main cell types accessible from the lumen, epithelial cells and macrophages, when exposed to subcytotoxic or high concentrations of the toxin. Mammary epithelial cells proved to be insensitive to LukM/LukF0 : they did not secrete IL-8, and where not killed even at high concentrations of the complete toxin. It should be noted that MEC are efficient detectors of MAMPs, in particular when they are in association because MAMPs can act in synergy [8]. Thus, the absence of response of infused udder quarters or MEC to the purified LukM/LukF0 preparations used in this study is a strong indication that they were not contaminated with bio-active concentrations of MAMPs. Mammary gland macrophages bind
1400
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S LP
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0 m
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/F '0 .4
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Fig. 4. LukM/F0 did not elicit IL-8 or IL-1b production by mammary macrophages. Macrophages were exposed to subcytotoxic (0.1 and 0.2 nM) or low cytotoxic (0.4 nM) concentrations of LukM/F0 for 6 h, then IL-8 (A) and IL-1b (B) concentrations in culture supernatant were determined by ELISA. Concentrations of IL-8 (p ¼ 0.078) and IL-1b (p ¼ 0.087) in LukM/F0 -exposed macrophage culture supernatants did not differ significantly from control (Friedman test).
400
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100 % metabolic activity
RPMI 90
Milk
80 70 60 50 0
0,5
1
1,5
2
LukM/F' (nM) Fig. 5. Milk did not inhibit the cytotoxic activity of LukM/F0 on neutrophils. Bovine neutrophils were exposed to various concentrations of LukM/F0 diluted either in normal bovine milk or in RPMI supplemented with serumalbumin for 1.5 h. Then the metabolic activity (reduction of alamarBlue) was determined. Difference between values in milk versus RPMI was statistically significant (Friedman test, p ¼ 0.025).
proliferation of lymphocytes, a property of superantigenic enterotoxins. The proliferative activity of the supernatant fraction may have been due to trace contamination with an enterotoxin. Moreover, human cells do not bind LukM/F0 efficiently and have a low sensitivity if any to this toxin [16]. Controls demonstrating absence of PAMPs were lacking in the study showing pro-inflammatory activity of the LukM/F0 fraction [14]. In conclusion, our study failed to support a unique role for LukM/F0 in inflammation triggering in the course of S. aureus mastitis. Instead, our data suggest that LukM/F0 could exert some anti-inflammatory activity by inactivating mammary macrophages. Further studies are needed to define the capacity of LukM/F0 to interfere with the inflammatory response in the mammary gland via activities on target cells. Acknowledgements We are grateful for the excellent services provided by the Experimental Infectiology Platform (PFIE) Unit at Nouzilly. References
LukM/F0
LukM [16] and were susceptible to low concentrations of (this study). We speculated that concentrations just below the minimal concentration inducing cellular alterations (0.5 nM) would induce production of pro-inflammatory cytokines or chemokines by macrophages. It has been shown that at low, sublytic concentrations, human neutrophils and monocytes are activated and secrete inflammatory mediators such as leucotriene B4 and interleukin-8 when exposed to Panton-Valentine leucocidin [21,34]. Mammary gland macrophages did not secrete TNF-a, IL-6 or IL-1b in response to subcytotoxic concentrations of LukM/F0, and the basal secretion of IL8 was reduced (Fig. 4). It can be put forward that the progressive decrease in IL-8 secretion as the concentration of LukM/F0 augmented resulted from a parallel decrease in cell viability as indicated by the dose-dependent decrease in metabolic activity illustrated in Fig. 3. This is not contradictory to the sustained or even slightly increased (although not significantly) production of IL-1b as the concentration of LukM/F0 increased (Fig. 4), because it has been shown that S. aureus pore-forming toxins activate the maturation of pro-IL-1b by caspase1 through activation of the inflammasome NLRP3 [35]. Overall, these results suggest that LukM/F0 could have an anti-inflammatory rather than pro-inflammatory effect on mammary macrophages. Nevertheless, our results do not rule out completely a pro-inflammatory role for LukM/F0 in certain settings, because we have not monitored other pro-inflammatory macrophages responses such as degranulation and production of eicosanoids (leucotrienes, prostaglandins). It also cannot be totally excluded that in the context of infection by a living S. aureus the LukM/F0 toxin contributes to the inflammatory response of the mammary gland. Yet, the in vivo absence of inflammatory response of the udder to intraluminal infusion of the toxin is a strong case against its responsibility in the initiating of inflammation by mastitis-causing S. aureus. Our results are conflicting with those reported by another group [14]. In this latter report, a S. aureus culture supernatant chromatographic fraction containing both LukM and LukF0 components was able to induce inflammation after infusion in the mammary gland, proliferation of bovine blood mononuclear cells, and secretion of TNF-a, IL-1b, IL-6 and IL-8 in a human whole blood assay. We infused in the mammary gland four times as much leukotoxin as in the supernatant fraction used in [14], and there was no inflammatory response. The simplest explanation for the discrepancy is that the supernatant fraction used contained bio-active compounds other than the leukotoxin LukM/F0. Several lines of reasoning back up this hypothesis. Leukotoxins have not been associated with
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A. Fromageau et al. / Microbial Pathogenesis 51 (2011) 396e401 [19] Barrio MB, Rainard P, Prévost G. LukM/LukF’-PV is the most active Staphylococcus aureus leukotoxin on bovine neutrophils. Microbes Infect 2006;8:2068e74. [20] Rainard P. Staphylococcus aureus leucotoxin LukM/F’ is secreted and stimulates neutralising antibody response in the course of intramammary infection. Vet Res 2007;38:685e96. [21] König B, Prévost G, Piémont Y, König W. Effects of Staphylococcus aureus leukocidins on inflammatory mediator release from human granulocytes. J Infect Dis 1995;171:607e13. [22] Boyle-Vavra S, Daum RS. Community-acquired methicillin-resistant Staphylococcus aureus: the role of Panton-Valentine leukocidin. Lab Invest 2007;87: 3e9. [23] Goldammer T, Zerbe H, Molenaar A, Schuberth HJ, Brunner RM, Kata SR, et al. Mastitis increases mammary mRNA abundance of beta-defensin 5, toll-likereceptor 2 (TLR2), and TLR4 but not TLR9 in cattle. Clin Diagn Lab Immunol 2004;11:174e85. [24] Gunther J, Koczan D, Yang W, Nurnberg G, Repsilber D, Schuberth HJ, et al. Assessment of the immune capacity of mammary epithelial cells: comparison with mammary tissue after challenge with Escherichia coli. Vet Res 2009;40:31. [25] Strandberg Y, Gray C, Vuocolo T, Donaldson L, Broadway M, Tellam R. Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells. Cytokine 2005;31:72e86. [26] Shuster DE, Kehrli Jr ME, Stevens MG. Cytokine production during endotoxininduced mastitis in lactating dairy cows. Am J Vet Res 1993;54:80e5. [27] Rainard P, Corrales JC, Barrio MB, Cochard T, Poutrel B. Leucotoxic activities of Staphylococcus aureus strains isolated from cows, ewes, and goats with
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Purified Staphylococcus aureus leukotoxin LukM/F0 does not trigger inflammation in the bovine mammary gland Angélina Fromageau, Patricia Cunha, Florence B. Gilbert, Pascal Rainard* INRA, UR 1282 Infectiologie Animale et Santé Publique, IASP Bat. 311, F-37380 Nouzilly, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 May 2011 Received in revised form 12 September 2011 Accepted 15 September 2011 Available online 22 September 2011
An early recruitment of neutrophils in mammary tissue and milk is considered an important component of the defense of the mammary gland against Staphylococcus aureus. We investigated whether the leukotoxin LukM/F0, which is produced by a proportion of mastitis-causing strains of S. aureus, would be able to trigger inflammation in the udder. Infusion of purified LukM/F0 toxin in lactating mammary glands did not cause neutrophil influx in milk, showing that the toxin was not able to cause mastitis on its own. Purified LukM/F0 did not kill or stimulate mammary epithelial cells in culture. As expected, LukM bound to mammary macrophages and the complete LukM/F0 toxin killed these cells, but subcytotoxic LukM/F0 concentrations did not induce secretion of IL-8, TNF-a, IL-1b or IL-6 by macrophages. On the contrary, the production of these pro-inflammatory mediators by adhesion-stimulated macrophages was reduced. Overall, these results indicate that purified leukotoxin LukM/F0 is not likely to contribute to the initiation of the inflammatory response and could even play an anti-inflammatory role in the mammary gland by inactivating macrophages. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Staphylococcus aureus Mastitis Cattle Epithelial cell Macrophage Leukotoxin
1. Introduction In ruminants, mastitis is by far the main pathology elicited by Staphylococcus aureus. Among mastitis-causing bacteria, S. aureus is prominent owing to its capacity to induce severe cases or long-lasting infections and because of its prevalence in certain countries [1e3]. Because of its contagious nature and its low cure rates, S. aureus mastitis is also difficult to control [4]. The influx of neutrophils in mammary tissue and milk is an important component of the defense of the mammary gland against S. aureus [5]. The mammary gland is likely to sense intruding S. aureus mainly through the detection of microbial-associated molecular patterns (MAMPs) such as peptidoglycan fragments, lipopeptides and lipoteichoic acid (LTA), as it was shown in other pathological settings in most host species [6]. Staphylococcal LTA or muramyl dipeptide induce inflammation and neutrophil influx when infused in the lumen of the mammary gland [7,8]. Besides MAMPs, exotoxins are likely to play a part in setting off inflammation [6]. For example, staphylococcal alpha toxin was shown to induce inflammation when infused in rabbit mammary glands [9]. Staphylococcal leukotoxins may have pro-inflammatory properties. Administration of the Panton-Valentine leucocidin (PVL) in the * Corresponding author. Tel.: þ33 2 47 42 76 33; fax: þ33 2 47 42 77 79. E-mail address: [email protected] (P. Rainard). 0882-4010/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2011.09.005
lungs of mice was sufficient to induce inflammation and tissue damage [10], and PVL contributed to muscle injury through induction of chemokines and recruitment of neutrophils [11]. Recently, several studies indicated that staphylococcal leukotoxins could be sensed by the innate immune system and trigger inflammatory responses [12e14]. Staphylococcal leukotoxins are bi-components toxins which assemble on their target cells to form trans-membrane pores [15]. The cells most susceptible to leukotoxins belong to the myeloid lineage, such as neutrophils, monocytes and macrophages. Several distinct staphylococcal leukotoxins have been described, but certain of these toxins are host-specific: the PVL targets mainly human and rabbit leucocytes but not bovine cells [15], whereas the LukM/LukF0 leukotoxin is active on bovine but not human cells [16]. The genes coding the leukotoxin LukM/LukF0 have not been found in S. aureus strains of human origin, and seem to be harbored only by strains isolated from mastitis of ruminants [17,18]. Among these bi-component poreforming toxins, LukM, associating with its partner component LukF0, composes the leukotoxin most active on bovine cells [19]. Very few data are available concerning the role of this toxin in the pathology of staphylococcal mastitis, because little is known of the local effects of this leucocidal product in vivo. Yet, the toxin is produced during mastitis and can be found in mammary secretions from severe cases of mastitis [20]. Pro-inflammatory effects of staphylococcal leukotoxins could result from a direct effect on target cells through induction of
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mediators such as leucotriene B4, IL-8, IL-6 and IL-12 as shown with human neutrophils stimulated with PVL or gamma hemolysin [21]. Another possibility is an indirect effect through the release of damaging components released by neutrophils attracted to the site of infection after leukotoxin-induced necrosis [22]. Lately it was reported that the Panton-Valentine leucocidin was able to interact with and activate the Toll-like receptor TLR2 [13], and that another leucocidin, hemolysin gamma (also named LukS/F) was able to activate TLR4 [12]. The mammary gland and mammary epithelial cells are known to express both TLR2 and TLR4 and to respond to the corresponding ligands such as LTA and LPS [7,8,23e25]. Thus the question arises of the reactivity of the ruminant mammary gland to the ruminant-associated leukotoxin (LukM/F0 ) and to the contribution of this exotoxin to the detection of S. aureus during infection. In this line of thought, a preparation of the leukotoxin LukM/LukF0 has been reported to induce inflammation in the bovine mammary gland [14]. The aim of our study was to investigate the pro-inflammatory activity of LukM/LukF0 in the bovine mammary gland. We used LukM/LukF0 purified from the culture supernatant of a mastitis isolate of S. aureus. A particular attention was paid to contamination of the preparation by bacterial agonists able to induce inflammation in the mammary gland, which is very susceptible to MAMPs such as lipopolysaccharide (LPS), lipoteichoic acid (LTA) or MDP, and even more so when these components are in association [8,26]. Our preparations of LukM/LukF0, which were fully active on bovine neutrophils, failed to induce inflammation in the lactating mammary gland. The absence of pro-inflammatory responses of mammary epithelial cells (MEC) or mammary macrophages to LukM/LukF0 was in line with this finding. 2. Materials and methods 2.1. Infusion of mammary glands with purified LukM/F0 Five healthy Holstein cows from the institutional experimental herd (PFIE, Nouzilly) that had no detectable bacterial growth and cell counts less than 100,000 cells/ml in milk samples of their four quarters in two consecutive weeks were used. These cows had no record of clinical mastitis in the period between calving and time of challenge. They were in their second or third lactation, and between three to seven months in lactation. Cows were milked twice daily, at 08:00 and 16:00 h. The use and care of the cows conformed to the practices in effect at the INRA Nouzilly Research Center. The two components of the leukotoxin LukM/F0 were purified as described [19]. Separately, LukM and LukF0 had no activity on bovine neutrophils, whereas after combination at equimolar concentrations, the mixture induced cytopathic effects (flattening of neutrophils) at 0.15 nM and higher concentrations, as previously reported [27]. The presence of endotoxin in the purified LukM and LukF0 was assessed with a Limulus amoebocyte lysate assay (E-toxate test, Sigma). Before infusion, the purified components LukM and LukF0 were diluted in HBSS which was supplemented with bovine serum albumin (BSA; cell culture tested, Sigma) at a final concentration of 0.5 mg/ml (HBSS-A), as a vehicle to minimize losses of toxin components during preparation of diluted solutions. Each cow received at time zero HBSS-A in one quarter, 15 mg of LukM in a second quarter, 15 mg in a third quarter, and 15 mg of LukM plus 15 mg of LukF’ in the fourth quarter. The infused quarters were aseptically sampled just before infusion after the morning milking, then 4, 8, 12, 24, and 48 h post-infusion. Milk samples were used for bacteriological examination by plating over a blood-esculin-agar plate and somatic cell count (SCC) by use of a cell counter (Fossomatic model 90; Foss Food Technology, Hillerod, Denmark) as previously described [8]. Milk samples were then centrifuged at
397
1000 g for 30 min at 4 C. Skimmed milk was stored in portions at 18 C. 2.2. Interaction of LukM/F0 with mammary epithelial cells Mammary secretory tissue from five different cows of the institutional dairy herd at Nouzilly was obtained at the slaughterhouse from lactating quarters free from infection and inflammation. Bovine mammary epithelial cells (bMEC) were isolated and cryopreserved in liquid nitrogen [28]. When required, bMEC were thawed and cultured without serum and antibiotics in Dulbecco’s modified Eagle’s medium (D-MEM)eF12 Advanced medium (Gibco), which contains insulin (10 mg/ml), albumin (0.4 mg/ml), and transferrin (7.5 mg/ml), supplemented with 2 mM L-glutamine, 10 ng/ml insulin-like growth factor I (IGF-I) (Peprotech), 5 ng/ml fibroblast growth factor (FGF) (Peprotech), 5 ng/ml human recombinant epidermal growth factor (EGF) (Sigma), 1 mg/ml hydrocortisone (Sigma), and 20 mM HEPES (Cambrex Biowhittaker). Cells were used at their 3rd passage after harvest from mammary glands. Cells were seeded in 24-well tissue culture plates at a density of 1 105 cells/well and cultured until confluence, then the growth medium was replaced with stimulation medium made up of D-MEM/F12 Advanced medium with 2 mM Lglutamine, 20 mM HEPES and 4 ng/ml of hydrocortisone as additives. Stimulations were carried out 16e24 h later. MEC were incubated with 1, 5 or 20 nM of LukM, LukF’ or LukM/F0 for 24 h at 37 C and 5% CO2. Staphylococcal LTA (Cayla/InvivoGen, Toulouse, France) was used at 250 ng/ml as a positive control of stimulation [8]. The effect of LukM/F0 on the viability of bMEC was assessed with an alamarBlueÒ (Biosource International Camarillo, CA, USA) assay, which is an oxidation-reduction indicator. AlamarBlue stock solution was prepared in the assay medium and distributed in the wells to yield a final concentration of 10% alamarBlue. The plates were incubated for 4 h at 37 C and 5% CO2. The fluorescence was then measured with an excitation wavelength of 530 nm and an emission wavelength at 590 nm (Cytofluor 2300 System, Applied Biosystems). Results were expressed as arbitrary units (AU) of fluorescence. The positive control of toxicity was obtained by exposing the cells to 0.1% Triton 100 for 5 min before washing and addition of alamarBlue. After this treatment, fluorescence was hardly detectable. Results were expressed as % metabolic activity: (control fluorescence/ residual fluorescence) 100. 2.3. Interaction of LukM/F0 with mammary macrophages Macrophages were obtained from secretions of involuted mammary glands as described [29]. After daily washing of a driedoff udder quarter with 50 ml of non-pyrogenic saline for 7 days, cells were harvested from the lavage fluid by centrifugation at 400 g for 15 min, washed once in Dulbecco’s PBS, resuspended and adjusted to 5 105 cells/ml in RPMI containing HEPES 10 mM, FCS 10%, penicillin 50 U, streptomycin 200 mg/ml. To determine the cytotoxic effect of LukM/F0 on macrophages, the cells were distributed in a 96-well flat-bottomed microtiter plate (80 ml of suspension per well) with increasing twofold dilutions (20 ml per well) of LukM, LukF’ alone or in association (equimolar amounts, from 0.03 nM to 4 nM) in RPMI-A containing 10% alamarBlue. Plates were incubated for 2 h at 37 C, 5% CO2, and the fluorescence was measured as described above. To determine the stimulating effect of LukM/F0, macrophages in 12-well plates (7.5 105 cells/well) were incubated for 2 h at 37 C, 5% CO2. Thereafter, 0.1, 0.2 and 0.4 nM of LukM, LukF0 or LukM/F0 were added and incubation carried out for 6 h. LPS (0.1 mg/ml) was added as positive control of cytokines secretion. Supernatants were conserved to determine cytokine concentrations.
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2.4. Interaction of LukM/F0 with neutrophils in milk
2.5. ELISAs for TNF-a, IL-1b, IL-6 and IL-8 Enzyme-linked immunosorbent assays (ELISAs) were used to measure cytokine concentrations in culture supernatants of macrophage incubated with LukM/F0. The ELISAs for TNF-a and IL-8 were performed as previously described [31,32]. Commercially available kits (Thermo Scientific, Rockford, IL, USA) were used according to the manufacturer’s instructions to measure bovine IL1b and IL-6. 2.6. Statistical analysis Statistical analyses of the milk cell concentrations or the concentrations of analytes in cell culture supernatants were performed with the nonparametric Friedman test (GraphPad Prism). A probability level of a P value < 0.05 was considered significant. 3. Results 3.1. Intramammary infusion of purified LukM/F0 The capacity of LukM/F0 to induce inflammation in the mammary gland was investigated by infusing the purified components in the lumen of uninflamed glands of 5 cows through the teat canal. To check whether the active toxin, comprised of the two components LukM and LukF’ assembling on the target cells, was necessary to trigger inflammation, each cow received either LukM (15 mg), LukF0 (15 mg), or LukM/F0 (15mg þ 15 mg) in different udder quarters. The fourth quarter was infused with the vehicle only and served as a negative control. None of the cows developed inflammation, which was monitored through the influx of leucocytes in milk (Fig. 1), apart from the physiological slight increase in milk cell concentrations between milkings, which remained in the range of cell concentrations in healthy glands (less than 200,000 cells per ml milk). For comparison, intramammary infusion of PAMPs elicits influx of leucocytes in milk of several million cells per ml [7,8]. Incidentally, the absence of response suggested that the preparation of purified LukM/F0 was not contaminated with PAMPs, as the mammary gland is very susceptible to these bacterial agonists of the innate immune system. Because mammary epithelial cells (MEC) are the first and most numerous cells in contact with the infused toxin, we tested their susceptibility to LukM/F0. 3.2. Effect of LukM/F0 on bovine MEC MEC are able to secrete several chemokines in response to various bacterial agonists [24,28,33], and the secretion of IL-8 can be used as a sensitive and convenient stimulation read-out [8]. Consequently, we monitored the reactivity of MEC by measuring IL-
Cells / ml (/1000)
HBSS A HBSS-A LukM LukF' LukM/F'
1000 100 10 1 0
12 24 36 Hours post-infusion
48
Fig. 1. The leukotoxin LukM/F0 did not induce inflammation in the mammary gland. Healthy glands of 5 cows were infused with either HBSS-A (vehicle), 15 mg LukM, 15 mg LukF0 or the LukM þ LukF0 (15 mg each). At 4, 8, 12, 24 and 48 h post-infusion, milk samples were taken and the concentration of cells (recruited leucocytes) was determined as correlate of inflammation. Results are expressed as numbers (median values/ 1000) of cells per ml of milk and interquartiles (Q1 and Q3).
8 concentrations in culture supernatant following incubation with LukM/F0. After 24 h of incubation with 5 or 20 nM LukM/F0, the concentrations of IL-8 in cell culture supernatants were not significantly different from concentration in the supernatant of control unstimulated cells, whereas a positive control (S. aureus LTA at 250 ng/ml) induced a strong response (4680 pg/ml) (Fig. 2). As the absence of IL-8 secretion could have resulted from a cytotoxic effect of LukM/F0, the viability of MEC was checked with an alamarBlue test. The alamarBlue fluorescence was not reduced even in the presence of 20 nM LukM/F0, showing that the metabolic activity of MEC was not affected by the toxin (results not shown). These results suggest that LukM/F0 is not likely to induce mammary inflammation by stimulating MEC, an inference that is in keeping with the results of the in vivo experiment. Another cell type which is likely to be in contact with intramammarily infused LukM/F0 is mammary macrophages. Consequently, we tested the
5000 4000 IL- 8 (pg/mL)
Milk was obtained from one cow. Bacteriological status (sterility) of milk was checked before storage at 20 C in portions. Bovine blood neutrophils were prepared from the blood of dairy cows (Holstein breed) selected for their low concentration of circulating eosinophils, as described [30] with some modifications [16]. Cells were resuspended in RPMI-A (2 106 cells/ml). Twenty ml of neutrophil suspension was distributed in the wells of a 96-well flat-bottomed microtiter plate with 80 ml of increasing twofold dilutions of LukM/F0 (equimolar amounts of the two components) in RPMI-A in one plate and in milk in another one, in the presence of 10% alamarBlue. After 1.5 h of incubation at 37 C, the fluorescence was measured with an excitation wavelength of 530 nm and an emission wavelength at 590 nm (Cytofluor 2300 System).
10000
3000 2000 1000 0 ed M
iu
m Lu
'1 /F kM
LukM/F0
nM Lu
'5 /F kM
nM L
'2 /F M uk
0
nM
LT
A
Fig. 2. The leukotoxin did not elicit IL-8 production by mammary epithelial cells. Primary bovine MEC were incubated with either the separate components or the two components LukM and LukF’ at the indicated concentrations (5 nM or 20 nM) for 24 h. Concentrations of IL-8 were determined in the culture supernatants as a read-out of MEC stimulation. Staphylococcal lipoteichoic acid (LTA, 250 ng/ml) was used as a positive control, yielding an IL-8 concentration of 4577 pg/ml. Concentrations of IL-8 after incubation with LukM/F0 did not differ from the concentration in supernatant of unstimulated cells (Friedman test, P ¼ 0.095).
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pro-inflammatory cytokines and tended to reduce the secretion of the chemokine IL-8.
% metabolic activity
120,0 100,0 80,0
399
3.4. Effect of milk on the cytotoxic activity of LukM/F0
M/F' 1
60,0 60 0
M/F' / 2
40,0
M/F' 3
20,0
F'
M
0,0 0,01
0,1 1 Concentration (nM)
10
Fig. 3. LukM/F0 is cytotoxic for mammary macrophages. Macrophages from 3 cows were incubated with various concentrations of LukM/F0 for 6 h (M/F’ 1, 2, 3) or with only one of the two components of the toxin at 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2 and 4 nM (M or F0 ). Then the metabolic activity (reduction of alamarBlue) was determined.
Milk components, such as antibodies, could have dampened or neutralized the activity of LukM/F0, thereby inhibiting its capacity to induce an inflammatory response after infusion in lactating mammary glands. The cytotoxic activity of LukM/F0 was determined in milk from a healthy gland and compared to the activity in RPMI-A with the alamarBlue assay. Milk did not reduce the activity of LukM/F0, on the contrary it exacerbated the cytotoxic activity at LukM/F0 concentrations of 0.5 nM and higher (Fig. 5). This suggests that the lack of pro-inflammatory activity of the leukotoxin in the mammary gland did not result from interference by milk. 4. Discussion
capacity of LukM/F0 to induce the secretion of pro-inflammatory cytokines by these cells. 3.3. Effect of LukM/F0 on bovine mammary macrophages Mammary macrophages bind LukM and are susceptible to LukM/F0 [16]. We speculated that subcytotoxic concentrations of LukM/F0 could induce the secretion of pro-inflammatory cytokines by macrophages. We first determined the minimal concentrations of LukM/F0 which induced the killing of mammary macrophages, with the alamarBlue assay. Cell metabolic activity was reduced by concentrations as low as 0.5 nM LukM/F0, as measured after 6 h of incubation with LukM/F0 (Fig. 3). Subsequently, mammary macrophages were incubated with concentrations of 0.1, 0.2 and 0.4 nM LukM/F0 for 6 h and IL-8, TNF-a, IL-1b and IL-6 concentrations were measured in the culture supernatant. LPS from Escherichia coli (0.1 mg/ml), used as positive control, induced the secretion of IL-8, TNF-a and IL-1b but not of IL-6. LukM/F0 did not induce a detectable secretion of TNF-a or IL-6 (results not shown). There was a constitutive secretion of IL-8 by macrophages under our culture conditions. This secretion was reduced by LukM/F0 with a doseeresponse effect, although the decrease was not significant (Fig. 4A). There was some constitutive secretion of IL-1b, but there was no significant dose-effect in response to LukM/F0 (Fig. 4B). These results indicate that LukM/F0 did not induce the secretion of
This study aimed at defining the capacity of the staphylococcal leukotoxin LukM/F0 to induce an inflammatory response in the mammary gland. Our results demonstrate that LukM/F0 did not trigger an inflammation in the mammary gland, even when a sizeable amount was infused in its lumen. The amount of LukM/F0 infused in mammary quarters (15 mg) would yield a concentration of about 150 ng/ml, assuming a volume of 100 ml of residual milk in the mammary cistern after milking. This is 15 fold the minimal concentration inducing cytotoxity to milk macrophages (this study), and a high concentration compared to the leucotoxic activity towards bovine neutrophils that begins at 4 ng/ml [27]. Also, we checked that milk from healthy glands did not dampen the toxic activity of LukM/ F0. Thus we can conclude that LukM/F0 is not pro-inflammatory for the mammary gland on its own. This incapacity correlated with the absence of pro-inflammatory response of the two main cell types accessible from the lumen, epithelial cells and macrophages, when exposed to subcytotoxic or high concentrations of the toxin. Mammary epithelial cells proved to be insensitive to LukM/LukF0 : they did not secrete IL-8, and where not killed even at high concentrations of the complete toxin. It should be noted that MEC are efficient detectors of MAMPs, in particular when they are in association because MAMPs can act in synergy [8]. Thus, the absence of response of infused udder quarters or MEC to the purified LukM/LukF0 preparations used in this study is a strong indication that they were not contaminated with bio-active concentrations of MAMPs. Mammary gland macrophages bind
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Fig. 4. LukM/F0 did not elicit IL-8 or IL-1b production by mammary macrophages. Macrophages were exposed to subcytotoxic (0.1 and 0.2 nM) or low cytotoxic (0.4 nM) concentrations of LukM/F0 for 6 h, then IL-8 (A) and IL-1b (B) concentrations in culture supernatant were determined by ELISA. Concentrations of IL-8 (p ¼ 0.078) and IL-1b (p ¼ 0.087) in LukM/F0 -exposed macrophage culture supernatants did not differ significantly from control (Friedman test).
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A. Fromageau et al. / Microbial Pathogenesis 51 (2011) 396e401
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LukM/F' (nM) Fig. 5. Milk did not inhibit the cytotoxic activity of LukM/F0 on neutrophils. Bovine neutrophils were exposed to various concentrations of LukM/F0 diluted either in normal bovine milk or in RPMI supplemented with serumalbumin for 1.5 h. Then the metabolic activity (reduction of alamarBlue) was determined. Difference between values in milk versus RPMI was statistically significant (Friedman test, p ¼ 0.025).
proliferation of lymphocytes, a property of superantigenic enterotoxins. The proliferative activity of the supernatant fraction may have been due to trace contamination with an enterotoxin. Moreover, human cells do not bind LukM/F0 efficiently and have a low sensitivity if any to this toxin [16]. Controls demonstrating absence of PAMPs were lacking in the study showing pro-inflammatory activity of the LukM/F0 fraction [14]. In conclusion, our study failed to support a unique role for LukM/F0 in inflammation triggering in the course of S. aureus mastitis. Instead, our data suggest that LukM/F0 could exert some anti-inflammatory activity by inactivating mammary macrophages. Further studies are needed to define the capacity of LukM/F0 to interfere with the inflammatory response in the mammary gland via activities on target cells. Acknowledgements We are grateful for the excellent services provided by the Experimental Infectiology Platform (PFIE) Unit at Nouzilly. References
LukM/F0
LukM [16] and were susceptible to low concentrations of (this study). We speculated that concentrations just below the minimal concentration inducing cellular alterations (0.5 nM) would induce production of pro-inflammatory cytokines or chemokines by macrophages. It has been shown that at low, sublytic concentrations, human neutrophils and monocytes are activated and secrete inflammatory mediators such as leucotriene B4 and interleukin-8 when exposed to Panton-Valentine leucocidin [21,34]. Mammary gland macrophages did not secrete TNF-a, IL-6 or IL-1b in response to subcytotoxic concentrations of LukM/F0, and the basal secretion of IL8 was reduced (Fig. 4). It can be put forward that the progressive decrease in IL-8 secretion as the concentration of LukM/F0 augmented resulted from a parallel decrease in cell viability as indicated by the dose-dependent decrease in metabolic activity illustrated in Fig. 3. This is not contradictory to the sustained or even slightly increased (although not significantly) production of IL-1b as the concentration of LukM/F0 increased (Fig. 4), because it has been shown that S. aureus pore-forming toxins activate the maturation of pro-IL-1b by caspase1 through activation of the inflammasome NLRP3 [35]. Overall, these results suggest that LukM/F0 could have an anti-inflammatory rather than pro-inflammatory effect on mammary macrophages. Nevertheless, our results do not rule out completely a pro-inflammatory role for LukM/F0 in certain settings, because we have not monitored other pro-inflammatory macrophages responses such as degranulation and production of eicosanoids (leucotrienes, prostaglandins). It also cannot be totally excluded that in the context of infection by a living S. aureus the LukM/F0 toxin contributes to the inflammatory response of the mammary gland. Yet, the in vivo absence of inflammatory response of the udder to intraluminal infusion of the toxin is a strong case against its responsibility in the initiating of inflammation by mastitis-causing S. aureus. Our results are conflicting with those reported by another group [14]. In this latter report, a S. aureus culture supernatant chromatographic fraction containing both LukM and LukF0 components was able to induce inflammation after infusion in the mammary gland, proliferation of bovine blood mononuclear cells, and secretion of TNF-a, IL-1b, IL-6 and IL-8 in a human whole blood assay. We infused in the mammary gland four times as much leukotoxin as in the supernatant fraction used in [14], and there was no inflammatory response. The simplest explanation for the discrepancy is that the supernatant fraction used contained bio-active compounds other than the leukotoxin LukM/F0. Several lines of reasoning back up this hypothesis. Leukotoxins have not been associated with
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