Tick sialostatins L and L2 differentially influence dendritic cell responses to Borrelia spirochetes
© Lieskovská et al.; licensee BioMed Central. 2015
Received: 28 November 2014
Accepted: 6 May 2015
Published: 15 May 2015
Transmission of pathogens by ticks is greatly supported by tick saliva released during feeding. Dendritic cells (DC) act as immunological sentinels and interconnect the innate and adaptive immune system. They control polarization of the immune response towards Th1 or Th2 phenotype. We investigated whether salivary cystatins from the hard tick Ixodes scapularis, sialostatin L (Sialo L) and sialostatin L2 (Sialo L2), influence mouse dendritic cells exposed to Borrelia burgdorferi and relevant Toll-like receptor ligands.
DCs derived from bone-marrow by GM-CSF or Flt-3 ligand, were activated with Borrelia spirochetes or TLR ligands in the presence of 3 μM Sialo L and 3 μM Sialo L2. Produced chemokines and IFN-β were measured by ELISA test. The activation of signalling pathways was tested by western blotting using specific antibodies. The maturation of DC was determined by measuring the surface expression of CD86 by flow cytometry.
We determined the effect of cystatins on the production of chemokines in Borrelia-infected bone-marrow derived DC. The production of MIP-1α was severely suppressed by both cystatins, while IP-10 was selectively inhibited only by Sialo L2. As TLR-2 is a major receptor activated by Borrelia spirochetes, we tested whether cystatins influence signalling pathways activated by TLR-2 ligand, lipoteichoic acid (LTA). Sialo L2 and weakly Sialo L attenuated the extracellular matrix-regulated kinase (Erk1/2) pathway. The activation of phosphatidylinositol-3 kinase (PI3K)/Akt pathway and nuclear factor-κB (NF-κB) was decreased only by Sialo L2. In response to Borrelia burgdorferi, the activation of Erk1/2 was impaired by Sialo L2. Production of IFN-β was analysed in plasmacytoid DC exposed to Borrelia, TLR-7, and TLR-9 ligands. Sialo L, in contrast to Sialo L2, decreased the production of IFN-β in pDC and also impaired the maturation of these cells.
This study shows that DC responses to Borrelia spirochetes are affected by tick cystatins. Sialo L influences the maturation of DC thus having impact on adaptive immune response. Sialo L2 affects the production of chemokines potentially engaged in the development of inflammatory response. The impact of cystatins on Borrelia growth in vivo is discussed.
KeywordsDendritic cells Borrelia burgdorferi Tick cystatin Signalling
Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to mammals through the bite of infected Ixodes ticks. In the skin, dendritic cells (DC) are among the first immune cells to come into contact with B. burgdorferi . B. burgdorferi elicits a potent cytokine/chemokine response through activation of multiple pattern recognition receptors on innate immune cells, including Toll-like receptor (TLRs), NOD-like receptors (NLRs), and C-type lectin receptors (CLRs) . TLRs have an essential role in the control of B. burgdorferi burden, because mice deficient in the common TLR signaling molecule myeloid differentiation primary response 88 (MyD88), have up to 250-fold more spirochetes than the wild-type controls [3, 4]. Among Toll-like receptors (TLRs), TLR-2 has been found to be the most important receptor for induction of pro-inflammatory mediators, whereas endosomal receptors TLR-7 and TLR-9 mediate type I interferon production [5–9]. All these TLRs utilize MyD88 as adaptor molecule, however, TLR-2 dependent inflammatory responses to B. burgdorferi can also be mediated by Toll-IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) . Borrelia spirochetes activate multiple signalling pathways through these adaptors, including nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPK) (extracellular matrix-regulated kinase (Erk) 1/2, p38, Janus N-terminal kinase (JNK)) [11–13], phosphatidylinositol-3 kinase (PI3K) , and Protein kinase C (PKC) pathways . The p38 MAPK and NF-κB are critically involved in the expression of pro-inflammatory cytokines [12, 16], whereas PI3K pathway is fundamental for optimal phagocytosis . Borrelia also strongly induces anti-inflammatory cytokine IL-10, which has overall suppressive effect on induction of pro-inflammatory mediators [17, 18].
Dendritic cells, as a part of innate immune system, produce several cytokines and chemokines which in autocrine and paracrine manner regulate the establishment of an innate immune response, including the recruitment of monocytes, macrophages, and neutrophils . In addition, DC upon sensing pathogens undergo the maturation process, characterized by increased expression of co-stimulatory molecules, which is necessary for proper presentation of antigen to naïve T-cells. In vitro, dendritic cells can be obtained by culturing of bone- marrow cells in the presence of two cytokines, granulocyte-macrophage colony-stimulated factor (GM-CSF) or Fms-like tyrosine kinase 3 ligand (Flt-3L), respectively. By GM-CSF, the myeloid subset of dendritic cells can be generated, while with the Flt-3 ligand, lymphoid- type of plasmacytoid dendritic cells (pDC) can be obtained [20, 21]. The pDC are characterised by robust production of type I IFN . These subsets of DC differ in the cytokine profiles they induce in T cells in vivo .
Dendritic cells are key players in host defense against tick-transmitted borreliae . However, many functions of DC are negatively influenced by tick saliva [24–26]. In addition to prostaglandin E2 , purine nucleoside adenosine  and Salp15 , tick cystatins are also involved in the effect of tick saliva on dendritic cells .
Sialostatins L and L2 are cysteine protease inhibitors of the hard tick Ixodes scapularis. Both are strong inhibitors of cathepsin L [31, 32], but sialostatin L also inhibits cathepsin S. Immunosuppressive effects of Sialo L have been demonstrated in T cell line CTLL-2  and lipopolysaccharide-activated DC . Expression of Sialo L2 is greatly enhanced by feeding and is necessary for tick feeding success . In addition to being able to enhance the growth of Borrelia burgdorferi in vivo , this sialostatin has been shown to inhibit the inflammasome formation during infection with Anaplasma phagocytophilum in macrophages through targeting caspase-1 activity .
In order to understand how Sialo L2, a tick salivary molecule, can support Borrelia establishment in the host, we studied the effect of tick cystatins on DC maturation and function. The effect on the production of chemokines, IFN-β and signalling pathways activated in dendritic cells by Borrelia spirochetes and relevant TLR ligands was analysed.
Female C57BL/6 mice (10 weeks of age) were obtained from Charles River Laboratories. All experiments were performed with permission from Local animal ethics committee of the Institute of Parasitology, Biology Centre ASCR České Budějovice, PID 167/2011.
The strain of Borrelia burgdorferi sensu stricto obtained from ATCC collection was grown in Barbour-Stoenner-Kelly-H (BSK-H) medium (Sigma) supplemented with 6 % rabbit serum at 34 °C. The fourth passage was used in the experiments.
Preparation of recombinant cystatins
Recombinant cystatins Sialo L and Sialo L2 were expressed in Escherichia coli followed by purification of active protein, as previously described [31, 35]. LPS contamination was removed by Arvys Proteins using the detergent-based extraction method. The presence of endotoxin was estimated with a sensitive fluorescent-based endotoxin assay (Lonza Biologics) and was <3 x 10−14 endotoxin g/μg protein for both cystatins. The endotoxin level did not exceed 2 pg/ml during testing the effect of cystatins on DC.
Generation of bone-marrow-derived dendritic cells
Bone-marrow derived conventional dendritic cells (DC) and plasmacytoid (pDC) dendritic cells were prepared as described before [20, 21], respectively, with minor modifications. Briefly, mice were sacrificed by cervical dislocation, intact femurs and tibias were removed, and bone marrow was harvested by repeated flushing with MEM. To derive conventional DC, bone marrow cells (106/ml) were cultured for 7 days in 6-well plate in RPMI 1640 medium supplemented with 10 % FCS, 50 mM HEPES, 2 mM glutamine, 50 μM mercaptoethanol, penicillin, streptomycin, amphotericin B, and 30 ng/ml of recombinant mouse GM-CSF (Sigma-Aldrich). Half of the medium was replaced with the fresh medium on day 3 and 5. On day 7, non-adherent cells were harvested and used as immature DC.
To analyse the effects of cystatins on DC differentiation, 105 bone-marrow cells were seeded in 96-well plate in the same medium as described above (including GM-CSF) and the Sialo L or Sialo L2 were added to the culture on day 3 to final concentration 3 μM. Cells were fed on day 5 and 7, and harvested on day 9. Surface expression of MHC class II was determined by flow cytometry within CD11c-positive population.
To derive plasmacytoid cells, bone marrow cells (1.5 × 106 / ml) were cultured for 8 days in 6-well plate in RPMI 1640 medium supplemented with 10 % FCS, sodium pyruvate, glutamine, penicillin, streptomycin, amphotericin B (PAA) and 100 ng/ml of recombinant human Flt-3L (R&D Systems). Half of the medium was replaced once after 4 days of culture. On day 8, non-adherent cells were harvested, washed in fresh medium and used in subsequent experiments.
Freshly derived pDC were seeded in 96-well plate at a concentration of 2 × 105 cells per well. Following 2 h incubation with Sialo L or Sialo L2 (each 3 μM) the cells were stimulated with spirochetes at MOI = 10 (10 spirochetes per 1 cell), imiquimod (R837, 2 μg/ml) (InvivoGen), or CpG (ODN1668, 50 nM) (Enzo Life Sciences). MOI = 10 was sufficient to activate DC as shown previously . IFN-β was determined in cell-free culture supernatants harvested 5 and 16 h after stimulation using LEGEND MAX™ mouse IFN-β ELISA Kit (BioLegend) following the manufacturer’s instructions.
BMDC were seeded at concentration 0.5 × 106 or 2 × 105 cells per well in 24-well plate or 96-well plate, respectively. Next day DCs were incubated 2 h with Sialo L or Sialo L2 (both 3 μM) and then B. burgdorferi was added at MOI = 10. After 24 h, cell-free supernatants were collected and analysed in Proteome Profiler™ antibody array according the manufacturer’s instructions (R&D). The chemokines were visualized by enhanced chemiluminescence and the abundance of signal was measured using CCD image system (ChemiDoc™ MP Imaging System) and Image Lab software, v. 4.1 (BIO-RAD). Alternatively, the amount of secreted chemokines (IP-10, MPC-1, MIP-1α, MIP-1β, and MIP-2) was determined in cell-free culture supernatants using ELISA kits (PeproTech) following the manufacturer’s instructions.
Bone marrow-derived pDC were seeded on 96-well plate at the concentration of 1 × 106 cells per ml of complete culture medium with Flt-3L and pretreated with either Sialo L or Sialo L2 (both 3 μM). After 2 h, cells were activated either with imiquimod (2 μg/ml), CpG (ODN1668, 50 nM) or B. burgdorferi spirochetes (MOI = 10). After 24 h incubation, cells were washed once in PBS with 1 % FCS and stained for flow cytometry analysis with anti-CD11c-PE mAb, anti-MHCII-AlexaFluor700 mAb, anti-CD86-APC mAb (all from eBioscience), anti-CD11b-FITC mAb, and anti-B220-PE-Vio770 mAb (both from Miltenyi Biotech). Dead cells were excluded from analysis using propidium iodide. Flow cytometry was performed on FACS Canto II flow cytometer and data were analysed using FACS Diva software, v. 5.0 (BD Biosciences). Plasmacytoid DCs were gated from living single cells as CD11c+, CD11b- and B220+. Levels of expression of CD86 were measured as MFI of APC.
BMDC were seeded at 0.5 × 106 cells per well in 24-well plate. Next day DCs were incubated 2 h with tick cystatins (each 3 μM) prior to the addition of LTA (2 μg/ml) for 15, 30, and 60 min or Borrelia spirochetes (MOI = 10) for 15, 30, 60, and 120 min. Afterwards, cells were lysed in a RIPA buffer (1 % Nonidet P-40, 0.25 % sodium deoxycholate, 1 mM EGTA, 150 mM NaCl, and 50 mM Tris-HCl (pH 7.5)) in the presence of protease inhibitors (10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin) and phosphatase inhibitors (25 mM sodium fluoride and 2 mM sodium orthovanadate). 20 μg of total proteins were separated by SDS-PAGE using an 8 % gel and then electro-transferred to Immobilon-P membranes. The blots were incubated overnight at 4 °C with the antibody recognizing phospho-NF-κB p65 (Ser536), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), phospho-p38 MAPK (Thr180/Tyr182), phospho-Akt (Ser473), total NF-κB p65, p44/42 MAPK (Erk1/2), p38 MAPK, Akt, and β-actin (all from Cell Signalling) followed by incubation with secondary antibody conjungated with horse radish peroxidase. The proteins were visualized using enhanced chemiluminescence (Pierce), and their abundance was analysed using CCD image system (ChemiDoc™ MP Imaging System) and Image Lab software, v. 4.1 (BIO-RAD).
One-way analysis of variance (ANOVA) followed by Bonferroni test in GraphPad Prism, version 5.0 was used to compare the differences between control and treated groups. P ≤ 0.05 was considered statistically significant and is marked by one star, P ≤ 0.01 is marked by two stars.
Sialostatin L2 decreases the MIP-α and IP-10 production by dendritic cells in response to Borrelia burgdorferi
The effect of sialostatin L2 on the signalling pathways activated by LTA and Borrelia burgdorferi in dendritic cells
Sialostatin L decreases production of IFN-β in plasmacytoid dendritic cells activated by Borrelia burgdorferi and TLR-7 ligand
Sialostatin L negatively affects TLR-7 and TLR-9 mediated maturation of DCs but does not influence Borrelia burgdorferi induced maturation
Sialostatin L reduces differentiation of bone-marrow DC
Sialo L2 and Sialo L are tick salivary cystatins, which are together with other salivary compounds released by the hard tick I. scapularis into the wound during tick feeding. During this process B. burgdorferi could be transmitted to the host. In response to Borrelia spirochetes, dendritic cells and other skin-resident immunocompetent cells become activated which leads to the production of proinflammatory mediators attracting further immune cells to the site of infection and activating them. These events can lead to clearing of most bacteria. It has been shown that Sialo L2, when injected intradermally into the mice, increased the burden of Borrelia spirochetes in the skin . We hypothesized that observed effect could result from Sialo L2 evoked changes in dendritic cells function. Therefore we analysed the effect of Sialo L2 and related cystatin Sialo L on the immuno-modulatory function and signal transduction of mouse bone-marrow derived dendritic cells (DC) activated by Borrelia and relevant TLR ligands. We found that these two tick cystatins differentially modulate the function of DC. While Sialo L2 inhibited the production of chemokines MIP-1α and IP-10 in response to Borrelia spirochetes and attenuated the activation of Erk1/2, PI3K/Akt, and NF-κB pathways in response to TLR-2 ligation (the major receptor activated by spirochetal lipoproteins), the related cystatin Sialo L suppressed the production of IFN-β and attenuated the maturation and differentiation of DC.
In our ex vivo experiments, Borrelia-stimulated bone-marrow dendritic cells secreted several chemokines, including neutrophil-, monocyte/macrophage-, and T cell-recruiting chemokines, similarly as was reported by other studies [18, 38]. Sialo L2 suppressed significantly production of two chemokines, MIP-1α and IP-10. MIP-1α is a chemotactic factor for mononuclear cells, T cells, and mast cells and plays a role in differentiation of type 1 Th lymphocytes. IP-10 is a CXC chemokine and attracts, in addition to monocytes and Th1 cells, also NK cells . We predict that the recruitment of these cells could be impaired by Sialo L2 in vivo.
Dendritic cells are among the first immune cells to come into contact with Borrelia in the skin . Phagocytosis of Borrelia spirochetes leads to production of various proinflammatory cytokines  including chemokines. Inhibitory effect of sialostatin L2 on the production of chemokines attracting inflammatory cells into tick feeding site can lead to reduced inflammation due to tick saliva effect . Reduced influx of inflammatory cells could facilitate establishment and proliferation of spirochetes in the skin .
Dendritic cells are equipped with several pattern recognition receptors (PRR), which sense Borreliae, including TLR, NLR, and LTR . To reveal the mechanism of Sialo L2 effect on chemokine production by Borrelia-activated DC, we analysed the activation of chosen signalling molecules first upon TLR-2 ligation. TLR-2 is robustly activated by Borrelia lipoproteins  and critically involved in production of pro-inflammatory mediators, including chemokines. Moreover, TLR have an essential role in the control of B. burgdorferi burden [2, 4], which is enhanced by Sialo L2 in vivo . The most pronounced effect of Sialo L2 on activation of tested signalling molecules in response to LTA was observed on phosphorylation of Akt, the downstream target of PI3K pathway. Interestingly, even the basal level of this kinase was decreased by Sialo L2. Consequences of PI3K pathway inhibition can be predicted. The inhibition of PI3K significantly impaired induction of chemokine and cytokine genes via TLR-2 in DC, including IP-10 . Of note, PI3K pathway plays an important role in phagocytosis of Borrelia spirochetes by macrophages . The inhibition of Akt phosphorylation was not observed by Sialo L2 in Borrelia-activated DC, possibly due to weak activation of this kinase.
The other pathway attenuated by Sialo L2 (in LTA and Borrelia activated DC) was Erk1/2 mediated cascade. Both, Erk1/2 and PI3K kinases are indispensable for induction of MIP-1α and MCP-1 in LTA stimulated murine macrophages . IP-10 induction is mediated by IFNs (often produced in response to microbial products) and its upregulation is associated with the activation of JAK1, JAK2/STAT1 and MAPK pathways [47–49]. The decline of MIP-1α and IP-10 production in Borrelia-activated DCs by Sialo L2 could be thus mediated via inhibition of the Erk1/2 and PI3K signalling pathways. Recently, we have found that Sialo L2 attenuates IFN signalling triggered by IFN-β or LPS which leads to the suppression of interferon stimulated genes like IRF-7 and IP-10 . The decrease of IP-10 production by Sialo L2 in response to Borrelia spirochetes could be in part also a consequence of impaired IFN/JAK/STAT signalling.
The third pathway influenced by sialostatin L2 upon LTA stimulation was NF-κB pathway. The involvement of NF-κB pathway in the induction of proinflammatory mediators was documented; e.g. TLR-2/NF-κB/MAPK signalling plays a key role in IL-8 induction in macrophage cell line THP-1 exposed to B. burgdorferi . We however did not detect any defect in the activation of this pathway in response to borreliae. Since dendritic cells sense borreliae by several PRR , the moderate effect of Sialo L2 on signals triggered through TLR-2 could be masked by signals triggered through other receptors.
In addition to chemokines, type I interferons are important cytokines modulating immune response to pathogens. B. burgdorferi is able to induce type I IFN and this induction is mediated through endosomal receptors TLR-7 and TLR-9 [6–9]. Plasmacytoid DC are major producers of type I IFN . We found out that in plasmacytoid dendritic cells, the amount of produced IFN-β in response to Borrelia spirochetes and TLR-7 activation was decreased by sialostatin L and only weakly or not at all by sialostatin L2. IFN is pleotropic cytokine which recruits NK cells, has a direct antiviral effect on cells, and links the innate and adaptive immunity.
The down-regulation of IFN-β production by Sialo L in Borrelia/TLR-7/TLR-9 stimulated cells may have further consequences for the development of adaptive immune responses. In general, type I interferon directly influences the fate of CD4+ and CD8+ T cells during the initial phases of antigen recognition contributing to Th1 commitment and negatively regulating Th2 and Th17 differentiation . Down-regulation of interferon can bring about an opposite effect. Moreover as sialostatin L inhibits production of IL-12 and TNF-α by DC as well as their differentiation , it probably leads to Th2 polarization of the immune response which is advantageous for Borrelia establishment in the skin . In addition to modulation of the Th differentiation, type I IFNs also positively influence DC maturation [55, 56].
Indeed, we show that the maturation of plasmacytoid DC induced by TLR-7 or TLR-9 ligands was also decreased by Sialo L (judged by expression of co-stimulatory molecule CD86). When the maturation of DC was initiated by borreliae, only statistically not significant decline in CD86 expression was observed in the presence of Sialo L, presumably due to the fact that Borrelia spirochetes are weaker inducers of maturation then TLR ligands. In agreement, it was previously published that Sialo L inhibits the maturation of DC induced by LPS; it negatively affects the expression of the costimulatory molecules CD80 and CD86 . Thus, Sialo L influenced function of dendritic cells in a different way in comparison to Sialo L2.
We did not investigate the mechanism which is behind the declined IFN-β production due to sialostatin L effect. However, since cathepsin L has been implicated in processing of TLR-9 , and sialostatins L and L2 are strong inhibitors of this protease , we could speculate that the decline of IFN-β is the result of impaired TLR-9 processing. Moreover, the amount of endogenously produced IFN-β was not affected by sialostatins in splenic DCs stimulated with TLR-4 agonist, where no processing had occurred .
Finally we examined the effect of tick cystatins on the differentiation/derivation of dendritic cells from bone marrow and found that Sialo L negatively affects the number of differentiated dendritic cells (MHC class II and CD11c positive cells). MHC class II molecule is necessary for the presentation of antigen to naive T-cells. As cathepsin S is implicated in the processing of the invariant chain within MHC class II antigens and sialostatin L strongly inhibits this protease , it seems likely that the decrease in MHC class II expression is mediated through inhibition of cathepsin S . The inhibitory effect on differentiation of BMDC (measured by expression of MHC class II molecules) was also reported for cystatin rHp-CPI from murine nematode parasite Heligmosomoides polygyrus .
We show here that two related tick sialostatins affect different functions of dendritic cells. While sialostatin L influences the maturation of DC in part through the inhibition of IFN-β having thus an impact on adaptive immune response, sialostatin L2 affects, through attenuation of several signalling pathways, the production of chemokines engaged in the development of inflammation.
This work was supported by the Czech Science Foundation, grants P302/12/2208 and 14-25799S.
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