- Email: [email protected]
14 does not contribute to dissemination or inflammation in a murine model of Lyme borreliosis
Immunobiology xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Immunobiology journal homepage: www.elsevier.com/locate/imbio
MRP8/14 does not contribute to dissemination or inflammation in a murine model of Lyme borreliosis ⁎
Lauren M.K. Masona, , Jeroen Coumoua, Jasmin I. Ersöza, Anneke Oeib, Joris J.T.H Roelofsc, Thomas Vogld, Tom van der Polla,e, Joppe W.R. Hoviusa,e a
Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands Department of Medical Microbiology, Academic Medical Center, Amsterdam, The Netherlands c Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands d Institute of Immunology, University of Muenster, Muenster, Germany e Division of Infectious Diseases, Academic Medical Center, Amsterdam, The Netherlands b
A R T I C LE I N FO
A B S T R A C T
Keywords: Borrelia Lyme MRP8/14 Calprotectin S100A8 S100A9
Myeloid-related protein (MRP)8 and MRP14 form a complex (MRP8/14) that is released by activated neutrophils and monocytes during infection. MRP8/14 has been shown to have bacteriostatic activity in vitro against Borrelia burgdorferi, the spirochete that causes Lyme borreliosis. Furthermore, levels of MRP8/14 have been shown to be elevated in the joints of patients with Lyme arthritis. We hypothesized that MRP8/14 has a protective effect during B. burgdorferi infection. To determine the role of MRP8/14 in the immune response to B. burgdorferi, we studied the course of B. burgdorferi infection in wildtype (wt) and mrp14−/− mice. In addition, we studied the response of leukocytes from mice lacking MRP8/14 to B. burgdorferi ex vivo. We demonstrated similar levels of B. burgdorferi dissemination, cytokine and immunoglobulin production in infected wt and mrp14−/− mice after 21 days. Neutrophils and monocytes lacking MRP8/14 were undiminished in their ability to become activated or phagocytose B. burgdorferi. In conclusion, we did not find a central role of MRP8/14 in the immune response against B. burgdorferi. As the levels of MRP8/14 in the serum of infected mice were low, we speculate that MRP8/ 14 is not released in levels great enough to influence the course of B. burgdorferi infection.
1. Introduction Lyme borreliosis is caused by Borrelia burgdorferi sensu lato and transmitted by Ixodes ticks. In disseminated disease, the bacteria can affect the joints, heart, central nervous system and the skin (Steere, 2001). It has been demonstrated that both innate and adaptive immune mechanisms are deployed to combat the bacteria, however the complex immunopathogenesis of the disease and the host factors that affect it are not entirely understood (Mason et al., 2014). In the early stages of infection, B. burgdorferi is recognised by skin immune cells, which instigates an immune response against the invading pathogen. Monocytes and neutrophils are recruited to the tickbite site where they phagocytose B. burgdorferi and produce cytokines, which modulate the immune response (Mason et al., 2015) together with other local inflammatory mediators such as damage-associated molecular patterns (DAMPs). The DAMPs myeloid-related protein (MRP)8 and MRP14 form a heterodimer (calprotectin), secreted by activated neutrophils and
⁎
monocytes during infection which amplifies immune responses to pathogens, is involved in cytoskeleton modulation during phagocytosis and has antimicrobial properties (Ehrchen et al., 2009). Indeed, MRP8/ 14 from human neutrophils was found to have bacteriostatic activity in vitro by chelating zinc essential for growth of B. burgdorferi (Lusitani et al., 2003). Furthermore, elevated levels of MRP8/14 have been measured in the joints of Lyme arthritis patients, suggesting that it may contribute to or prolong anti-Borrelia immunity locally (Montgomery et al., 2006). We hypothesised that the bacteriostatic effect of MRP8/14 in vitro may confer protection against dissemination during infection and that MRP8/14 may augment the response of immune cells against B. burgdorferi. To investigate this, we infected wildtype (wt) and mrp14−/− mice and studied the dissemination of B. burgdorferi and the immune response. In addition, we studied the ability of phagocytes ex vivo from mrp14−/− mouse whole blood to phagocytose and produce cytokines in response to B. burgdorferi.
Corresponding author at: Academic Medical Center, Postbus 22660, 1100 DD, Amsterdam, The Netherlands. E-mail address: [email protected] (L.M.K. Mason).
https://doi.org/10.1016/j.imbio.2018.07.017 Received 5 March 2018; Received in revised form 6 July 2018; Accepted 14 July 2018 0171-2985/ © 2018 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
Please cite this article as: Mason, L.M.K., Immunobiology (2018), https://doi.org/10.1016/j.imbio.2018.07.017
Immunobiology xxx (xxxx) xxx–xxx
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Fig. 1. MRP8/14 deficiency does not affect B. burgdorferi loads or immune responses in mice. Wildtype and mrp14−/− mice were syringe inoculated with 106 spirochetes and sacrificed after 21 days. a) Dot plots show MRP8/14 levels in serum of wt infected and uninfected mice ± SEM. b) Dot plots show B. burgdorferi loads measured in the skin, bladders, tibiotarsi and hearts of infected mrp14-/- and wt mice ± SEM. c) Dot plots show IgG and IgM levels measured in the serum of infected wt and mrp14−/− mice ± SEM.
2. Materials and methods
sacrificed by bleeding from the inferior vena cava. Paraffin-embedded sections of skin, ankle and heart were processed and H&E stained by routine histological techniques. Inflammation was scored on a scale from 0 to 3 by a pathologist who was blinded to the experimental design, as previously described (Hovius et al., 2009).
2.1. B. burgdorferi B. burgdorferi sensu stricto strain N40, previously recovered from an experimentally infected mouse was cultured in modified KellyPettenkofer medium (MKP; AMC, Amsterdam, Netherlands). Low passage (< 5) spirochetes were washed and resuspended in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Paisley, UK) supplemented with 10% foetal calf serum (FCS, Lonza, Verviers, Belgium) for ex vivo experiments, or phosphate buffered saline (PBS, Fresenius Kabi, Graz, Austria) for mouse inoculation. Organs of infected mice were cultured in MKP with rifampicin (50 μg/ml) and phosphomycin (100 μg/ml) at 33 °C and checked weekly for presence of spirochetes.
2.3. Cytokine and immunoglobulin measurements Serum MRP8/14 was measured by ELISA as previously described (Achouiti et al., 2012). N40 strain-specific IgG and IgM in serum was measured by ELISA as previously described (Hovius et al., 2009). Cytokines were measured in whole blood stimulation supernatant and the serum of infected mice using a mouse inflammation CBA kit (BD, Franklin Lakes, NJ), according to manufacturer’s instructions.
2.2. Mice 2.4. qPCR Female wt C57Bl/6 mice were purchased from Charles River Laboratories (Maastricht, the Netherlands). Female mrp14−/−mice, backcrossed > 10 times to a C57BL/6 background were generated as described (Manitz et al., 2003) and were bred in the animal facility of the Academic Medical Center (Amsterdam, the Netherlands). Six to eight-week-old mice were infected by subcutaneous inoculation in the midline of the back with 1 × 106spirochetes in 100 μl PBS, or PBS control, as described previously (Hovius et al., 2009). Mice were
DNA from murine tissues was obtained by blood and tissue kit (Qiagen, Venlo, The Netherlands) according to manufacturer’s instructions. Quantitative (q)PCR detecting Borrelia flaB and mouse βactin was performed using the lightcycler480 (Roche, Nutley, NJ) and SYBR green dye (Roche) in triplicate as described previously (Hovius et al., 2009). Results were analyzed using LinregPCR software (Amsterdam, the Netherlands). 2
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Fig. 2. MRP8/14-deficient phagocytes have undiminished capacity to respond to B. burgdorferi. Whole blood of eight mrp14−/− and eight wt mice was stimulated for 16 h with B. burgdorferi and analysed by flow cytometry. a) FACS plots show how neutrophils were identified and gated as Ly6G + cells and monocytes were identified and gated as F4-80+ cells (left panel). The forward scatter (FSC-A) and side scatter (SSC-A) of Ly6C + cells and F4-80+ cells are shown (right panel). b) Histograms show the mean fluorescence intensity (MFI) of CD11b of wt and mrp14-/- neutrophils and monocytes after stimulation with B. burgdorferi (Bb), compared to unstimulated and LPS-stimulated cells, ± SEM. c) Levels of IL-6, TNF, MCP-1 and IL-10 measured in stimulated, unstimulated and LPS-stimulated wt and mrp14-/- whole blood are shown, ± SEM. d) The whole blood of eight mrp14-/- and eight wt mice was incubated with CFSE-stained B. burgdorferi at 37 °C for various time points and flow cytometry was performed to measure extent of phagocytosis. FACS plots show how neutrophils were stained and gated as Gr-1+ cells (left panel) and the FSC-A and SSC-A of Gr-1+ cells is shown in black (right panel). e) The CFSE MFI is shown in histograms for neutrophils of one representative wt and one representative mrp14-/-mouse. f) The mean phagocytosis index (CFSE MFI x % CFSE + cells at 37 °C CFSE MFI x % CFSE + cells at 4 °C) of all eight wt (circles) and all eight mrp14-/- (triangles) cells is displayed over time ± SEM. Figures show the results of one experiment, representative of three independent experiments.
2.5. Whole blood stimulation
2.6. Phagocytosis
Heparinised whole blood from eight naïve mrp14−/− and eight wt mice was stimulated in 96-well plates with viable B. burgdorferi, 10 ng/ ml Escherichia coli lipopolysaccharide (LPS, Invitrogen, Paisley, UK) or medium control for 16 h at 37 °C, 5% CO2 as previously described (Mason et al., 2015; Hovius et al., 2009). Supernatant was collected and cells were washed and stained with αLy6G-FITC, αF4/80-APC and αCD11b-PE (BD) and analysed by flow cytometry using FACS Canto II (BD) and Flow-Jo (Treestar, Ashland, OR).
Phagocytosis assays were performed in essence as described before (Mason et al., 2015; Hovius et al., 2009). Viable B. burgdorferi were labeled with CFSE (Invitrogen, Breda, the Netherlands) and incubated at a multiplicity of infection (MOI) of 10 with 50 μl heparinised whole blood from eight mrp14−/− and eight wt mice at 37 °C for various time points. Phagocytosis was stopped by transferring samples to 4 °C. Erythrocytes were lysed using isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, 100 mM EDTA) and cells were washed and stained with 3
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αGr-1-PE and αF4/80-APC and analysed by flow cytometry.
respectively (Fig. 2a). CD11b expression of both cell types was assessed to measure activation. B. burgdorferi induced significant CD11b upregulation of both wt and mrp14-/- monocytes and a trend was seen towards CD11b upregulation on neutrophils at an MOI of 1. No differences in activation were seen between wt and mrp14−/− cells (Fig. 2b). Similar results were seen at an MOI of 0.2 and 5 (data not shown). Levels of IL-6 and TNFα, MCP-1 and IL-10 were increased in the supernatant of stimulated cells however no differences were seen between wt and mrp14−/− mice (Fig. 2c). Additionally, we investigated the phagocytic capacity of mrp14-/- neutrophils. Whole blood of mrp14−/− or wt mice was incubated at 37 °C for various time points with CFSEstained B. burgdorferi at an MOI of 5 and transferred to 4 °C to prevent further phagocytosis. Flow cytometry was performed and neutrophils were identified by Gr-1 expression (Fig. 2d). The percentage of CFSEpositive cells (Fig. 2e) and phagocytosis index (Fig. 2f) increased over time in both mrp14−/− and wt mice at a comparable rate. Thus, MRP8/ 14 did not influence the uptake of B. burgdorferi. In previous studies whether MRP8/14 enhanced the phagocytic capacity of immune cells varied per pathogen (Achouiti et al., 2012; De Jong et al., 2015). Similarly, activation and cytokine production of neutrophils and monocytes from mrp14-/- whole blood stimulated with B. burgdorferi was unchanged. This is in line with other studies, which demonstrated unaffected or modestly affected cytokine levels produced by stimulated whole blood (Achouiti et al., 2012, 2014). In conclusion, we did not find evidence for a role of MRP8/14 in the course of B. burgdorferi infection in this murine model. We speculate that B. burgdorferi does not induce MRP8/14 release in levels great enough to influence the course of infection. Although we did not see evidence for an effect on phagocytosis and immune response of monocytes and neutrophils we cannot rule out possible local effects of MRP8/14 on the immune response of B. burgdorferi in organs and joints.
2.7. Statistics Data was analysed using a Kruskal-Wallis test for overall significance. Differences between groups were analysed using Dunn’s post test (Graphpad Prism, San Diego, CA). A p value of < 0.05 was considered significant, where * indicated p < 0.05, ** p < 0.01 and *** p < 0.001. 2.8. Ethics Experiments were carried out in accordance with the Dutch Experiment on Animals Act and approved by the Animal Care and Use Committee of the University of Amsterdam (Permit number: DIX103041). 3. Results and discussion 3.1. Mice lacking MRP8/14 have unaltered course of B. burgdorferi infection In order to study the role of MRP8/14 in the course of B. burgdorferi infection, we syringe-inoculated eight wt and eight mrp14−/− mice with strain N40 and sacrificed after 21 days. MRP8 is unstable in the absence of MRP14, therefore mrp14−/− mice are functional double knockouts (Manitz et al., 2003). Serum levels of MRP8/14 were not significantly higher in infected than uninfected wt mice (Fig. 1a). B. burgdorferi was detected in inoculation site and bladder cultures of all infected mice, seven and fourteen days respectively after sacrifice (data not shown). Wt and mrp14−/− mice developed comparable Borrelia loads in the skin, bladder, ankle and heart (Fig. 1b) and displayed a similar IgG and IgM response to B. burgdorferi (Fig. 1c), comparable skin, heart and ankle histopathology scores (data not shown) and similar serum levels of interleukin (IL)-12, monocyte chemoattractant protein (MCP)-1 and IL-10 (data not shown). Systemic levels of MRP8/14 in B. burgdorferi-infected wt mice were much lower than observed previously in sepsis models, which give rise to stronger systemic infection and inflammation (Achouiti et al., 2012, 2014; Achouiti et al., 2015; De Jong et al., 2015; van Zoelen et al., 2009). It is likely that systemic MRP8/14 levels are too low to affect B. burgdorferi growth as knock-out mice were just as prone to dissemination and developed comparable loads at distant organs. Similarly, although MRP8/14 had a bacteriostatic effect on Staphylococcus aureus, Steptococcus pneumoniae, Salmonella typhimurium and Burkholderia pseudomallei in vitro (Achouiti et al., 2014, 2015; De Jong et al., 2015), it did not prevent bacterial outgrowth in wt mice in in vivo infection models of these pathogens compared to mrp14−/− mice. C57Bl6 mice are fairly resistant to developing joint and heart inflammation during the course of Lyme borreliosis. As MRP8/14 is elevated in Lyme arthritic joints (Montgomery et al., 2006), it could be useful to study its role in C3H/HeN mice, which develop more severe arthritis (Radolf et al., 2012), or in an intra-articular inoculation model. Although levels of MRP8/14 are systemically low, locally released MRP8/14 may contribute to local inflammatory processes in such an experimental setup.
Conflict of interest The authors declare that no conflict of interest exists. Acknowledgements This work was supported by a “Veni” grant (91611065) from JWH received from The Netherlands Organisation for health research and development (ZonMw). References Achouiti, A., Vogl, T., Urban, C.F., Rohm, M., Hommes, T.J., van Zoelen, M.A., Florquin, S., Roth, J., van’ t Veer, C., de Vos, A.F., van der Poll, T., 2012. Myeloid-related protein-14 contributes to protective immunity in gram-negative pneumonia derived sepsis. PLoS Pathog. 8 (10), e1002987. Achouiti, A., Vogl, T., Endeman, H., Mortensen, B.L., Laterre, P.F., Wittebole, X., van Zoelen, M.A., Zhang, Y., Hoogerwerf, J.J., Florquin, S., Schultz, M.J., Grutters, J.C., Biesma, D.H., Roth, J., Skaar, E.P., van’ t Veer, C., de Vos, A.F., van der Poll, T., 2014. Myeloid-related protein-8/14 facilitates bacterial growth during pneumococcal pneumonia. Thorax 69 (11), 1034–1042. Achouiti, A., Vogl, T., Van der Meer, A.J., Stroo, I., Florquin, S., de Boer, O.J., Roth, J., Zeerleder, S., van’ t Veer, C., de Vos, A.F., van der Poll, T., 2015. Myeloid-related protein-14 deficiency promotes inflammation in staphylococcal pneumonia. Eur. Respir. J. 46 (2), 464–473. De Jong, H.K., Achouiti, A., Koh, G.C., Parry, C.M., Baker, S., Faiz, M.A., van Dissel, J.T., Vollaard, A.M., van Leeuwen, E.M., Roelofs, J.J., de Vos, A.F., Roth, J., van der Poll, T., Vogl, T., Wiersinga, W.J., 2015. Expression and function of S100A8/A9 (calprotectin) in human typhoid fever and the murine Salmonella model. PLoS Negl. Trop. Dis. 9 (4), e0003663. Ehrchen, J.M., Sunderkotter, C., Foell, D., Vogl, T., Roth, J., 2009. The endogenous Tolllike receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer. J. Leukoc. Biol. 86 (3), 557–566. Hovius, J.W., Bijlsma, M.F., van der Windt, G.J., Wiersinga, W.J., Boukens, B.J., Coumou, J., Oei, A., de Beer, R., de Vos, A.F., van’ t Veer, C., van Dam, A.P., Wang, P., Fikrig, E., Levi, M.M., Roelofs, J.J., van der Poll, T., 2009. The urokinase receptor (uPAR) facilitates clearance of Borrelia burgdorferi. PLoS Pathog. 5 (5), e1000447. Lusitani, D., Malawista, S.E., Montgomery, R.R., 2003. Calprotectin, an abundant cytosolic protein from human polymorphonuclear leukocytes, inhibits the growth of Borrelia burgdorferi. Infect. Immun. 71 (8), 4711–4716.
3.2. MRP8/14-deficient leukocytes are undiminished in their ability to respond to B. burgdorferi To investigate whether MRP8/14 is implicated in local inflammation in Lyme borreliosis, we examined the contribution of MRP8/14 to the response of monocytes and neutrophils to B. burgdorferi ex vivo. Whole blood of mrp14−/− or wt mice was incubated with B. burgdorferi for 16 h. Subsequently, flow cytometry was performed and neutrophils and monocytes were identified as Ly6G and F4-80 expressing cells 4
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Montgomery, R.R., Schreck, K., Wang, X., Malawista, S.E., 2006. Human neutrophil calprotectin reduces the susceptibility of Borrelia burgdorferi to penicillin. Infect. Immun. 74 (4), 2468–2472. Radolf, J.D., Caimano, M.J., Stevenson, B., Hu, L.T., 2012. Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat. Rev. Microbiol. 10 (2), 87–99. Steere, A.C., 2001. Lyme disease. N. Engl. J. Med. 345 (2), 115–125. van Zoelen, M.A., Vogl, T., Foell, D., Van Veen, S.Q., van Till, J.W., Florquin, S., Tanck, M.W., Wittebole, X., Laterre, P.F., Boermeester, M.A., Roth, J., van der Poll, T., 2009. Expression and role of myeloid-related protein-14 in clinical and experimental sepsis. Am. J. Respir. Crit. Care Med. 180 (11), 1098–1106.
Manitz, M.P., Horst, B., Seeliger, S., Strey, A., Skryabin, B.V., Gunzer, M., Frings, W., Schonlau, F., Roth, J., Sorg, C., Nacken, W., 2003. Loss of S100A9 (MRP14) results in reduced interleukin-8-induced CD11b surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants in vitro. Mol. Cell. Biol. 23 (3), 1034–1043. Mason, L.M., Veerman, C.C., Geijtenbeek, T.B., Hovius, J.W., 2014. Menage a trois: Borrelia, dendritic cells, and tick saliva interactions. Trends Parasitol. 30 (2), 95–103. Mason, L.M., Herkes, E.A., Krupna-Gaylord, M.A., Oei, A., van der Poll, T., Wormser, G.P., Schwartz, I., Petzke, M.M., Hovius, J.W., 2015. Borrelia burgdorferi clinical isolates induce human innate immune responses that are not dependent on genotype. Immunobiology 220 (10), 1141–1150.
5
Contents lists available at ScienceDirect
Immunobiology journal homepage: www.elsevier.com/locate/imbio
MRP8/14 does not contribute to dissemination or inflammation in a murine model of Lyme borreliosis ⁎
Lauren M.K. Masona, , Jeroen Coumoua, Jasmin I. Ersöza, Anneke Oeib, Joris J.T.H Roelofsc, Thomas Vogld, Tom van der Polla,e, Joppe W.R. Hoviusa,e a
Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands Department of Medical Microbiology, Academic Medical Center, Amsterdam, The Netherlands c Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands d Institute of Immunology, University of Muenster, Muenster, Germany e Division of Infectious Diseases, Academic Medical Center, Amsterdam, The Netherlands b
A R T I C LE I N FO
A B S T R A C T
Keywords: Borrelia Lyme MRP8/14 Calprotectin S100A8 S100A9
Myeloid-related protein (MRP)8 and MRP14 form a complex (MRP8/14) that is released by activated neutrophils and monocytes during infection. MRP8/14 has been shown to have bacteriostatic activity in vitro against Borrelia burgdorferi, the spirochete that causes Lyme borreliosis. Furthermore, levels of MRP8/14 have been shown to be elevated in the joints of patients with Lyme arthritis. We hypothesized that MRP8/14 has a protective effect during B. burgdorferi infection. To determine the role of MRP8/14 in the immune response to B. burgdorferi, we studied the course of B. burgdorferi infection in wildtype (wt) and mrp14−/− mice. In addition, we studied the response of leukocytes from mice lacking MRP8/14 to B. burgdorferi ex vivo. We demonstrated similar levels of B. burgdorferi dissemination, cytokine and immunoglobulin production in infected wt and mrp14−/− mice after 21 days. Neutrophils and monocytes lacking MRP8/14 were undiminished in their ability to become activated or phagocytose B. burgdorferi. In conclusion, we did not find a central role of MRP8/14 in the immune response against B. burgdorferi. As the levels of MRP8/14 in the serum of infected mice were low, we speculate that MRP8/ 14 is not released in levels great enough to influence the course of B. burgdorferi infection.
1. Introduction Lyme borreliosis is caused by Borrelia burgdorferi sensu lato and transmitted by Ixodes ticks. In disseminated disease, the bacteria can affect the joints, heart, central nervous system and the skin (Steere, 2001). It has been demonstrated that both innate and adaptive immune mechanisms are deployed to combat the bacteria, however the complex immunopathogenesis of the disease and the host factors that affect it are not entirely understood (Mason et al., 2014). In the early stages of infection, B. burgdorferi is recognised by skin immune cells, which instigates an immune response against the invading pathogen. Monocytes and neutrophils are recruited to the tickbite site where they phagocytose B. burgdorferi and produce cytokines, which modulate the immune response (Mason et al., 2015) together with other local inflammatory mediators such as damage-associated molecular patterns (DAMPs). The DAMPs myeloid-related protein (MRP)8 and MRP14 form a heterodimer (calprotectin), secreted by activated neutrophils and
⁎
monocytes during infection which amplifies immune responses to pathogens, is involved in cytoskeleton modulation during phagocytosis and has antimicrobial properties (Ehrchen et al., 2009). Indeed, MRP8/ 14 from human neutrophils was found to have bacteriostatic activity in vitro by chelating zinc essential for growth of B. burgdorferi (Lusitani et al., 2003). Furthermore, elevated levels of MRP8/14 have been measured in the joints of Lyme arthritis patients, suggesting that it may contribute to or prolong anti-Borrelia immunity locally (Montgomery et al., 2006). We hypothesised that the bacteriostatic effect of MRP8/14 in vitro may confer protection against dissemination during infection and that MRP8/14 may augment the response of immune cells against B. burgdorferi. To investigate this, we infected wildtype (wt) and mrp14−/− mice and studied the dissemination of B. burgdorferi and the immune response. In addition, we studied the ability of phagocytes ex vivo from mrp14−/− mouse whole blood to phagocytose and produce cytokines in response to B. burgdorferi.
Corresponding author at: Academic Medical Center, Postbus 22660, 1100 DD, Amsterdam, The Netherlands. E-mail address: [email protected] (L.M.K. Mason).
https://doi.org/10.1016/j.imbio.2018.07.017 Received 5 March 2018; Received in revised form 6 July 2018; Accepted 14 July 2018 0171-2985/ © 2018 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
Please cite this article as: Mason, L.M.K., Immunobiology (2018), https://doi.org/10.1016/j.imbio.2018.07.017
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Fig. 1. MRP8/14 deficiency does not affect B. burgdorferi loads or immune responses in mice. Wildtype and mrp14−/− mice were syringe inoculated with 106 spirochetes and sacrificed after 21 days. a) Dot plots show MRP8/14 levels in serum of wt infected and uninfected mice ± SEM. b) Dot plots show B. burgdorferi loads measured in the skin, bladders, tibiotarsi and hearts of infected mrp14-/- and wt mice ± SEM. c) Dot plots show IgG and IgM levels measured in the serum of infected wt and mrp14−/− mice ± SEM.
2. Materials and methods
sacrificed by bleeding from the inferior vena cava. Paraffin-embedded sections of skin, ankle and heart were processed and H&E stained by routine histological techniques. Inflammation was scored on a scale from 0 to 3 by a pathologist who was blinded to the experimental design, as previously described (Hovius et al., 2009).
2.1. B. burgdorferi B. burgdorferi sensu stricto strain N40, previously recovered from an experimentally infected mouse was cultured in modified KellyPettenkofer medium (MKP; AMC, Amsterdam, Netherlands). Low passage (< 5) spirochetes were washed and resuspended in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Paisley, UK) supplemented with 10% foetal calf serum (FCS, Lonza, Verviers, Belgium) for ex vivo experiments, or phosphate buffered saline (PBS, Fresenius Kabi, Graz, Austria) for mouse inoculation. Organs of infected mice were cultured in MKP with rifampicin (50 μg/ml) and phosphomycin (100 μg/ml) at 33 °C and checked weekly for presence of spirochetes.
2.3. Cytokine and immunoglobulin measurements Serum MRP8/14 was measured by ELISA as previously described (Achouiti et al., 2012). N40 strain-specific IgG and IgM in serum was measured by ELISA as previously described (Hovius et al., 2009). Cytokines were measured in whole blood stimulation supernatant and the serum of infected mice using a mouse inflammation CBA kit (BD, Franklin Lakes, NJ), according to manufacturer’s instructions.
2.2. Mice 2.4. qPCR Female wt C57Bl/6 mice were purchased from Charles River Laboratories (Maastricht, the Netherlands). Female mrp14−/−mice, backcrossed > 10 times to a C57BL/6 background were generated as described (Manitz et al., 2003) and were bred in the animal facility of the Academic Medical Center (Amsterdam, the Netherlands). Six to eight-week-old mice were infected by subcutaneous inoculation in the midline of the back with 1 × 106spirochetes in 100 μl PBS, or PBS control, as described previously (Hovius et al., 2009). Mice were
DNA from murine tissues was obtained by blood and tissue kit (Qiagen, Venlo, The Netherlands) according to manufacturer’s instructions. Quantitative (q)PCR detecting Borrelia flaB and mouse βactin was performed using the lightcycler480 (Roche, Nutley, NJ) and SYBR green dye (Roche) in triplicate as described previously (Hovius et al., 2009). Results were analyzed using LinregPCR software (Amsterdam, the Netherlands). 2
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Fig. 2. MRP8/14-deficient phagocytes have undiminished capacity to respond to B. burgdorferi. Whole blood of eight mrp14−/− and eight wt mice was stimulated for 16 h with B. burgdorferi and analysed by flow cytometry. a) FACS plots show how neutrophils were identified and gated as Ly6G + cells and monocytes were identified and gated as F4-80+ cells (left panel). The forward scatter (FSC-A) and side scatter (SSC-A) of Ly6C + cells and F4-80+ cells are shown (right panel). b) Histograms show the mean fluorescence intensity (MFI) of CD11b of wt and mrp14-/- neutrophils and monocytes after stimulation with B. burgdorferi (Bb), compared to unstimulated and LPS-stimulated cells, ± SEM. c) Levels of IL-6, TNF, MCP-1 and IL-10 measured in stimulated, unstimulated and LPS-stimulated wt and mrp14-/- whole blood are shown, ± SEM. d) The whole blood of eight mrp14-/- and eight wt mice was incubated with CFSE-stained B. burgdorferi at 37 °C for various time points and flow cytometry was performed to measure extent of phagocytosis. FACS plots show how neutrophils were stained and gated as Gr-1+ cells (left panel) and the FSC-A and SSC-A of Gr-1+ cells is shown in black (right panel). e) The CFSE MFI is shown in histograms for neutrophils of one representative wt and one representative mrp14-/-mouse. f) The mean phagocytosis index (CFSE MFI x % CFSE + cells at 37 °C CFSE MFI x % CFSE + cells at 4 °C) of all eight wt (circles) and all eight mrp14-/- (triangles) cells is displayed over time ± SEM. Figures show the results of one experiment, representative of three independent experiments.
2.5. Whole blood stimulation
2.6. Phagocytosis
Heparinised whole blood from eight naïve mrp14−/− and eight wt mice was stimulated in 96-well plates with viable B. burgdorferi, 10 ng/ ml Escherichia coli lipopolysaccharide (LPS, Invitrogen, Paisley, UK) or medium control for 16 h at 37 °C, 5% CO2 as previously described (Mason et al., 2015; Hovius et al., 2009). Supernatant was collected and cells were washed and stained with αLy6G-FITC, αF4/80-APC and αCD11b-PE (BD) and analysed by flow cytometry using FACS Canto II (BD) and Flow-Jo (Treestar, Ashland, OR).
Phagocytosis assays were performed in essence as described before (Mason et al., 2015; Hovius et al., 2009). Viable B. burgdorferi were labeled with CFSE (Invitrogen, Breda, the Netherlands) and incubated at a multiplicity of infection (MOI) of 10 with 50 μl heparinised whole blood from eight mrp14−/− and eight wt mice at 37 °C for various time points. Phagocytosis was stopped by transferring samples to 4 °C. Erythrocytes were lysed using isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, 100 mM EDTA) and cells were washed and stained with 3
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αGr-1-PE and αF4/80-APC and analysed by flow cytometry.
respectively (Fig. 2a). CD11b expression of both cell types was assessed to measure activation. B. burgdorferi induced significant CD11b upregulation of both wt and mrp14-/- monocytes and a trend was seen towards CD11b upregulation on neutrophils at an MOI of 1. No differences in activation were seen between wt and mrp14−/− cells (Fig. 2b). Similar results were seen at an MOI of 0.2 and 5 (data not shown). Levels of IL-6 and TNFα, MCP-1 and IL-10 were increased in the supernatant of stimulated cells however no differences were seen between wt and mrp14−/− mice (Fig. 2c). Additionally, we investigated the phagocytic capacity of mrp14-/- neutrophils. Whole blood of mrp14−/− or wt mice was incubated at 37 °C for various time points with CFSEstained B. burgdorferi at an MOI of 5 and transferred to 4 °C to prevent further phagocytosis. Flow cytometry was performed and neutrophils were identified by Gr-1 expression (Fig. 2d). The percentage of CFSEpositive cells (Fig. 2e) and phagocytosis index (Fig. 2f) increased over time in both mrp14−/− and wt mice at a comparable rate. Thus, MRP8/ 14 did not influence the uptake of B. burgdorferi. In previous studies whether MRP8/14 enhanced the phagocytic capacity of immune cells varied per pathogen (Achouiti et al., 2012; De Jong et al., 2015). Similarly, activation and cytokine production of neutrophils and monocytes from mrp14-/- whole blood stimulated with B. burgdorferi was unchanged. This is in line with other studies, which demonstrated unaffected or modestly affected cytokine levels produced by stimulated whole blood (Achouiti et al., 2012, 2014). In conclusion, we did not find evidence for a role of MRP8/14 in the course of B. burgdorferi infection in this murine model. We speculate that B. burgdorferi does not induce MRP8/14 release in levels great enough to influence the course of infection. Although we did not see evidence for an effect on phagocytosis and immune response of monocytes and neutrophils we cannot rule out possible local effects of MRP8/14 on the immune response of B. burgdorferi in organs and joints.
2.7. Statistics Data was analysed using a Kruskal-Wallis test for overall significance. Differences between groups were analysed using Dunn’s post test (Graphpad Prism, San Diego, CA). A p value of < 0.05 was considered significant, where * indicated p < 0.05, ** p < 0.01 and *** p < 0.001. 2.8. Ethics Experiments were carried out in accordance with the Dutch Experiment on Animals Act and approved by the Animal Care and Use Committee of the University of Amsterdam (Permit number: DIX103041). 3. Results and discussion 3.1. Mice lacking MRP8/14 have unaltered course of B. burgdorferi infection In order to study the role of MRP8/14 in the course of B. burgdorferi infection, we syringe-inoculated eight wt and eight mrp14−/− mice with strain N40 and sacrificed after 21 days. MRP8 is unstable in the absence of MRP14, therefore mrp14−/− mice are functional double knockouts (Manitz et al., 2003). Serum levels of MRP8/14 were not significantly higher in infected than uninfected wt mice (Fig. 1a). B. burgdorferi was detected in inoculation site and bladder cultures of all infected mice, seven and fourteen days respectively after sacrifice (data not shown). Wt and mrp14−/− mice developed comparable Borrelia loads in the skin, bladder, ankle and heart (Fig. 1b) and displayed a similar IgG and IgM response to B. burgdorferi (Fig. 1c), comparable skin, heart and ankle histopathology scores (data not shown) and similar serum levels of interleukin (IL)-12, monocyte chemoattractant protein (MCP)-1 and IL-10 (data not shown). Systemic levels of MRP8/14 in B. burgdorferi-infected wt mice were much lower than observed previously in sepsis models, which give rise to stronger systemic infection and inflammation (Achouiti et al., 2012, 2014; Achouiti et al., 2015; De Jong et al., 2015; van Zoelen et al., 2009). It is likely that systemic MRP8/14 levels are too low to affect B. burgdorferi growth as knock-out mice were just as prone to dissemination and developed comparable loads at distant organs. Similarly, although MRP8/14 had a bacteriostatic effect on Staphylococcus aureus, Steptococcus pneumoniae, Salmonella typhimurium and Burkholderia pseudomallei in vitro (Achouiti et al., 2014, 2015; De Jong et al., 2015), it did not prevent bacterial outgrowth in wt mice in in vivo infection models of these pathogens compared to mrp14−/− mice. C57Bl6 mice are fairly resistant to developing joint and heart inflammation during the course of Lyme borreliosis. As MRP8/14 is elevated in Lyme arthritic joints (Montgomery et al., 2006), it could be useful to study its role in C3H/HeN mice, which develop more severe arthritis (Radolf et al., 2012), or in an intra-articular inoculation model. Although levels of MRP8/14 are systemically low, locally released MRP8/14 may contribute to local inflammatory processes in such an experimental setup.
Conflict of interest The authors declare that no conflict of interest exists. Acknowledgements This work was supported by a “Veni” grant (91611065) from JWH received from The Netherlands Organisation for health research and development (ZonMw). References Achouiti, A., Vogl, T., Urban, C.F., Rohm, M., Hommes, T.J., van Zoelen, M.A., Florquin, S., Roth, J., van’ t Veer, C., de Vos, A.F., van der Poll, T., 2012. Myeloid-related protein-14 contributes to protective immunity in gram-negative pneumonia derived sepsis. PLoS Pathog. 8 (10), e1002987. Achouiti, A., Vogl, T., Endeman, H., Mortensen, B.L., Laterre, P.F., Wittebole, X., van Zoelen, M.A., Zhang, Y., Hoogerwerf, J.J., Florquin, S., Schultz, M.J., Grutters, J.C., Biesma, D.H., Roth, J., Skaar, E.P., van’ t Veer, C., de Vos, A.F., van der Poll, T., 2014. Myeloid-related protein-8/14 facilitates bacterial growth during pneumococcal pneumonia. Thorax 69 (11), 1034–1042. Achouiti, A., Vogl, T., Van der Meer, A.J., Stroo, I., Florquin, S., de Boer, O.J., Roth, J., Zeerleder, S., van’ t Veer, C., de Vos, A.F., van der Poll, T., 2015. Myeloid-related protein-14 deficiency promotes inflammation in staphylococcal pneumonia. Eur. Respir. J. 46 (2), 464–473. De Jong, H.K., Achouiti, A., Koh, G.C., Parry, C.M., Baker, S., Faiz, M.A., van Dissel, J.T., Vollaard, A.M., van Leeuwen, E.M., Roelofs, J.J., de Vos, A.F., Roth, J., van der Poll, T., Vogl, T., Wiersinga, W.J., 2015. Expression and function of S100A8/A9 (calprotectin) in human typhoid fever and the murine Salmonella model. PLoS Negl. Trop. Dis. 9 (4), e0003663. Ehrchen, J.M., Sunderkotter, C., Foell, D., Vogl, T., Roth, J., 2009. The endogenous Tolllike receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer. J. Leukoc. Biol. 86 (3), 557–566. Hovius, J.W., Bijlsma, M.F., van der Windt, G.J., Wiersinga, W.J., Boukens, B.J., Coumou, J., Oei, A., de Beer, R., de Vos, A.F., van’ t Veer, C., van Dam, A.P., Wang, P., Fikrig, E., Levi, M.M., Roelofs, J.J., van der Poll, T., 2009. The urokinase receptor (uPAR) facilitates clearance of Borrelia burgdorferi. PLoS Pathog. 5 (5), e1000447. Lusitani, D., Malawista, S.E., Montgomery, R.R., 2003. Calprotectin, an abundant cytosolic protein from human polymorphonuclear leukocytes, inhibits the growth of Borrelia burgdorferi. Infect. Immun. 71 (8), 4711–4716.
3.2. MRP8/14-deficient leukocytes are undiminished in their ability to respond to B. burgdorferi To investigate whether MRP8/14 is implicated in local inflammation in Lyme borreliosis, we examined the contribution of MRP8/14 to the response of monocytes and neutrophils to B. burgdorferi ex vivo. Whole blood of mrp14−/− or wt mice was incubated with B. burgdorferi for 16 h. Subsequently, flow cytometry was performed and neutrophils and monocytes were identified as Ly6G and F4-80 expressing cells 4
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Montgomery, R.R., Schreck, K., Wang, X., Malawista, S.E., 2006. Human neutrophil calprotectin reduces the susceptibility of Borrelia burgdorferi to penicillin. Infect. Immun. 74 (4), 2468–2472. Radolf, J.D., Caimano, M.J., Stevenson, B., Hu, L.T., 2012. Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat. Rev. Microbiol. 10 (2), 87–99. Steere, A.C., 2001. Lyme disease. N. Engl. J. Med. 345 (2), 115–125. van Zoelen, M.A., Vogl, T., Foell, D., Van Veen, S.Q., van Till, J.W., Florquin, S., Tanck, M.W., Wittebole, X., Laterre, P.F., Boermeester, M.A., Roth, J., van der Poll, T., 2009. Expression and role of myeloid-related protein-14 in clinical and experimental sepsis. Am. J. Respir. Crit. Care Med. 180 (11), 1098–1106.
Manitz, M.P., Horst, B., Seeliger, S., Strey, A., Skryabin, B.V., Gunzer, M., Frings, W., Schonlau, F., Roth, J., Sorg, C., Nacken, W., 2003. Loss of S100A9 (MRP14) results in reduced interleukin-8-induced CD11b surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants in vitro. Mol. Cell. Biol. 23 (3), 1034–1043. Mason, L.M., Veerman, C.C., Geijtenbeek, T.B., Hovius, J.W., 2014. Menage a trois: Borrelia, dendritic cells, and tick saliva interactions. Trends Parasitol. 30 (2), 95–103. Mason, L.M., Herkes, E.A., Krupna-Gaylord, M.A., Oei, A., van der Poll, T., Wormser, G.P., Schwartz, I., Petzke, M.M., Hovius, J.W., 2015. Borrelia burgdorferi clinical isolates induce human innate immune responses that are not dependent on genotype. Immunobiology 220 (10), 1141–1150.
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