Increased expression of Fas receptor and Fas ligand in the culture of the peripheral blood mononuclear cells stimulated with Borrelia burgdorferi sensu lato

Ticks and Tick-borne Diseases 6 (2015) 189–197

Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis

Original article

Increased expression of Fas receptor and Fas ligand in the culture of the peripheral blood mononuclear cells stimulated with Borrelia burgdorferi sensu lato a ´ ´ Sambor Grygorczuk a,∗ , Joanna Osada b , Anna Moniuszko a , Renata Swierzbi nska , a a a b ˛ Maciej Kondrusik , Joanna Zajkowska , Justyna Dunaj , Milena Dabrowska , Sławomir Pancewicz a a b

˙ Department of Infectious Diseases and Neuroinfections, Medical University in Białystok, ul. Zurawia 14, 15-540 Białystok, Poland Department of Hematologic Diagnostics, Medical University in Białystok, ul. Waszyngtona 15A, 15-269 Białystok, Poland

a r t i c l e

i n f o

Article history: Received 25 July 2014 Received in revised form 1 December 2014 Accepted 2 December 2014 Available online 22 December 2014 Keywords: Lyme borreliosis Lymphocyte apoptosis Lymphocyte activation Fas receptor

a b s t r a c t Apoptosis of the lymphocytes plays an essential role in the regulation of inflammatory/immune responses and its abnormalities may contribute to a chronic infection, persistent inflammation and autoimmunity. Its role in the pathogenesis of the late Lyme borreliosis manifestations has not been studied so far. We have measured Th lymphocyte apoptosis rate, membrane expression of pro-apoptotic Fas receptor, and supernatant concentrations of selected soluble pro- and anti-apoptotic mediators in cultures of peripheral blood mononuclear cells from 16 patients with disseminated Lyme borreliosis (6 with osteoarticular symptoms, 7 with neuroborreliosis and 3 with acrodermatitis chronica atrophicans) and 8 healthy controls. The cultures stimulated for 48 h with live Borrelia burgdorferi sensu stricto, B. garinii or B. afzelii spirochetes. Fraction of the apoptotic Th (CD3+CD4+) lymphocytes and expression of Fas in this cell population was measured cytometrically and concentrations of soluble Fas, soluble Fas ligand, IL-10, IL-12 and TGF-␤ in culture supernatant with ELISA assays. The expression of IL-10, soluble and membrane Fas and soluble Fas ligand was increased under stimulation and higher in the presence of B. burgdorferi sensu stricto than the other species. Apoptosis rate was not affected. There was no difference between Lyme borreliosis patients and controls. IL-10 concentration correlated negatively with the membrane Fas expression and apoptosis under stimulation with B. afzelii and B. garinii. Expression of Fas/FasL system is up-regulated under stimulation with B. burgdorferi, but without corresponding increase in lymphocyte apoptosis. Variable responses observed with different B. burgdorferi species may reflect differences in the pathogenesis of the infection in vivo. © 2014 Elsevier GmbH. All rights reserved.

Introduction Lyme borreliosis (LB) is a multi-system disease caused by a tick-borne spirochete Borrelia burgdorferi sensu lato (B. burgdorferi), presenting with a wide spectrum of clinical manifestations. Although the disease is typically self-limited, in a minority of patients B. burgdorferi is able to evade inflammatory and immune response, resulting in an infection persisting for years (Steere et al., 1987; Steere et al., 2004; Wormser et al., 2006). Neurologic and osteoarticular symptoms may also persist after the antibiotic treatment and spirochete elimination (Steere and Angelis, 2006;

∗ Corresponding author. Tel.: +48 85 7409514/519; fax: +48 85 7409515. E-mail address: [email protected] (S. Grygorczuk). http://dx.doi.org/10.1016/j.ttbdis.2014.12.004 1877-959X/© 2014 Elsevier GmbH. All rights reserved.

Shin et al., 2007). Different mechanisms, including permanent tissue damage and autoimmune reactions, have been postulated to explain these late manifestations of the disease (Steere et al., 2004; Steere and Angelis, 2006; Shin et al., 2007; Wormser et al., 2006; Kalish et al., 2001). Three B. burgdorferi species responsible for systemic infections in humans (B. burgdorferi sensu strict – B. burgdorferi s.s., B. garinii and B. afzelii) cause the disease with different clinical features and dynamics. The more invasive early infection and the potential for precipitating autoimmune arthritis as a late complication is attributed to B. burgdorferi s.s. The initially milder disease with a weaker inflammatory response, but also with the extremely long survival of the spirochete in the skin, resulting in a chronic inflammation (acrodermatitis chronica atrophicans, ACA) is related to the infection with B. afzelii (Steere et al., 2004; Strle et al., 2009; Jones

190

S. Grygorczuk et al. / Ticks and Tick-borne Diseases 6 (2015) 189–197

et al., 2008). These differences may result from different tissue affinity and different immunogenic and inflammatory properties of B. burgdorferi species (Strle et al., 2009; Jones et al., 2008; Drouin et al., 2004). Apoptosis of activated T lymphocytes is essential in the regulation and timely resolution of inflammatory and immune responses (Akbar et al., 1993; Uehara et al., 1992; Alderson et al., 1995). Apoptosis not only removes activated lymphocytes, but by itself is an anti-inflammatory process, as phagocytes ingesting apoptotic cells release immunomodulatory factors (IL-10, TGF-␤) and downregulate the synthesis of pro-inflammatory cytokines (Fadok et al., 1998; Chen et al., 2001; Voll et al., 1997). In activated T lymphocytes, apoptosis is initiated by a binding of Fas death receptor by its ligand (FasL) – a transmembrane protein expressed by various cell types, including activated lymphocytes themselves (Alderson et al., 1995; Li-Weber and Krammer, 2003). Naive T cells do not present Fas, but are able to up-regulate it within hours after activation (Miyawaki et al., 1992). Recently activated T cells remain resistant to Fas stimulation and their sensitivity to apoptosis increases gradually, regulated by a number of interrelated factors, including availability of FasL-presenting cells and the intracellular balance of pro- and anti-apoptotic factors, itself dependent on the type of stimulation, type and age of the cell and the presence of cytokines (Alderson et al., 1995; Li-Weber and Krammer, 2003; Miyawaki et al., 1992; Krammer et al., 2007; Schmitz et al., 2003). Inflammatory cytokines (IL-2, IL-5, IL-6, IL-12) tend to protect Th1 cells from apoptosis, while immunomodulatory/anti-inflammatory ones like IL-10 have a pro-apoptotic effect (Akbar et al., 1993; Uehara et al., 1992; Estaquier et al., 1995; Roth et al., 2003). In the mononuclear cells culture, susceptibility of T lymphocytes to Fas-mediated apoptosis increases significantly 5–6 days after activation, which seems to reflect events occurring in vivo in the response to acute infection (Akbar et al., 1993; Uehara et al., 1992; Schmitz et al., 2003; Irmler et al., 1997). Because Th2 lymphocytes, in contrast with Th1 cells, are generally resistant to Fas-mediated apoptosis, it not only suppresses the inflammation, but may also influence the balance between Th1 and Th2 populations, and thus the character and course of the inflammatory response (Roth et al., 2003; Devadas et al., 2006). An impairment of the Fas-dependent apoptosis results in a lymphoproliferation and a tendency to autoimmunity (Sneller et al., 2003). On the other hand, increased apoptosis of Th1 lymphocytes could facilitate pathogen survival and contribute to the establishment of a chronic infection. The induction of apoptosis is a common mechanism used by pathogens to escape the immune response (Alderson et al., 1995; Hirsch et al., 2002; Labbé and Saleh, 2008). In the late stage of LB, both the persistent inflammation after the eradication of the pathogen (as in the antibiotic-resistant Lyme arthritis) and the long-lasting infection evading host immunity (as in ACA) has been observed. We have hypothesized that alterations in lymphocyte apoptosis, both on the side of the decreased and increased susceptibility to the apoptotic cell death (possibly dependent on the host factors or infecting species), could be involved in the pathogenesis of these manifestations. Previously we have observed an increased expression of membrane Fas receptor on T lymphocytes under stimulation with B. burgdorferi, with a strongest response to B. burgdorferi s.s. in comparison with B. garinii and B. afzelii. However, there was no simultaneous increase in apoptosis rate and differences between healthy donors and Lyme borreliosis patients (Grygorczuk, 2010). Here we present complementary data on the concentrations of soluble factors related to apoptosis in the supernatant of the PBMC culture incubated with B. burgdorferi. As cytokines may influence cell susceptibility to apoptosis, and, on the other hand, their synthesis may be affected by the apoptosis of the immune cells, we have also measured concentrations of TGF-␤, IL-10 and IL-12 together

with the soluble forms of Fas and its ligand, to investigate the relation between the apoptosis and cytokine response.

Material and methods Patients Study group consisted of 16 patients with disseminated LB (age 19–76 years, mean 51.2 ± 16.5), including 6 with osteoarticular symptoms (LA, age 49.3 ± 15.3 years), 7 with neuroborreliosis (NB, 50.3 ± 15.2 years) and 3 with acrodermatitis chronica atrophicans (ACA, 56.0 ± 26.2 years). All patients came from the endemic area, had a history of a tick bite and/or were frequently exposed to ticks during work or recreational activities. At the moment of admission patients with osteoarticular symptoms had either asymmetrical arthritis (edema, pain on motion and palpation) or asymmetrical arthralgia involving large joints. Patients with other diseases of the musculoskeletal system, elevated inflammatory parameters (leukocytosis, C-reactive protein, elevated erythrocyte sedimentation rate), any other data pointing to non-infectious etiology (e.g. detectable rheumatoid factor) or other infectious diseases were excluded. Neuroborreliosis presented with subacute meningitis in 2 patients, VIIIth cranial nerve involvement in one and with late neurologic symptoms (paresthesia, paresis) in 4. All these patients were consulted by a neurologist or referred from neurology unit after differential diagnostic had excluded other etiology. ACA was diagnosed on the basis of clinical picture and confirmed with histopathologic examination. Three patients in the NB group (with meningitis and VIIIth cranial nerve palsy) had onset of symptoms 2–3 weeks before sample collection (early disseminated infection). In 13 remaining patients, the symptoms persisted or recurred for from 9 months up to a few years. Of these, 9 patients had already undergone some antibiotic treatment without a resolution or with the recurrence of symptoms after an interval of several months to 2–3 years. The reasons for the clinically ineffective treatment could have been persistent infection because of non-optimal treatment scheme (e.g. macrolide treatment) or poor adherence, re-infection in individuals highly exposed to tick bites or, finally, permanent or slowly resolving residual symptoms without active infection at the moment of inclusion. Nevertheless, all these patients had a history of a recent, long lasting and symptomatic borrelial infection, meaning significant exposure of the immune system to B. burgdorferi, which should be reflected in PBMC response to stimulation. We excluded patients in whom the clinical picture was suggestive of “post-Lyme disease syndrome” (chronic subjective symptoms refractive to multiple antibiotic treatment, dubious serologic findings) or in whom symptoms could be explained by co-existing pathology. Serum sample obtained on admission was tested for antiBorrelia burgdorferi s.l. IgM and IgG antibodies with ELISA assay from Biomedica (Boston, USA). The result in IgG class was positive in all the patients except one with recent onset subacute meningitis, in whom it was borderline with accompanying very high positive result in IgM class. The positive results were confirmed either with Western-blot assay from DRG (New Jersey, USA) or immunoblot (EcoLine) from Genzyme Virotech (Rüsselsheim, Germany). The intrathecal antibody production in patients with NB was confirmed with the later test. Control group (C) consisted of 8 seronegative persons with no clinical suspicion of Lyme borreliosis, no symptoms of infection or inflammation (age 19–67 years, mean 48.1 ± 5.7). All subjects gave informed consent for participation and the research was approved by the Ethics Board of the Medical University in Białystok.

S. Grygorczuk et al. / Ticks and Tick-borne Diseases 6 (2015) 189–197

191

Fig. 2. Apoptosis of Th lymphocytes stimulated with B. burgdorferi. Fraction of the apoptotic (annexin V-positive, propidium iodide negative) CD3 + CD4 + Th cells in the PBMC culture after stimulation for 48 h with live B. burgdorferi spirochetes (MOI 10:1). Data from 16 experiments with PBMC from patients with Lyme borreliosis. (-) – negative control, B.a. – stimulation with B. afzelii, B.g. – stimulation with B. garinii, B.ss – stimulation with B. burgdorferi s.s. Showing median, quartiles (box), minimum and maximum values (whiskers). There was no significant difference between the non-stimulated and stimulated cultures.

Fig. 1. Expression of Fas on Th lymphocytes stimulated with B. burgdorferi. 1. Expression of the Fas receptor on CD3 + CD4 + Th cells in the PBMC culture after stimulation for 48 h with live B. burgdorferi spirochetes (MOI 10:1). Data from 16 experiments with PBMC from Lyme borreliosis patients. (-) – negative control, B.a. – stimulation with B. afzelii, B.g. – stimulation with B. garinii, B.ss – stimulation with B. burgdorferi s.s. Showing median, quartiles (box), minimum and maximum values (whiskers). A. Increased Fas expression calculated as MFI in B.a., B.g. and B.ss in comparison with the non-stimulated control; * p < 0.05 in comparison with (-); B. The difference between cultures stimulated with the different B. burgdorferi species. The percentage of Fas-presenting cells is expressed as the net value above the baseline level in the non-stimulated culture. Statistically significant difference between B.ss and B.g. is shown on the plot.

cultured in sterile tubes in 5% CO2 at 37 ◦ C in concentration of about 7.5 × 106 cells/ml. B. burgdorferi suspension containing 108 live bacteria/ml was obtained from the culture maintained at the Department of Rickettsiae, Chlamydiae and Zoonotic Spirochetes of the National Institute of Hygiene in Warsaw, kept in −70 ◦ C and thawed in room temperature directly before use. The preparation is standardized and the concentration and viability of the spirochetes is confirmed by the culture-providing laboratory, where the suspension prepared and frozen in a similar manner is routinely used for re-establishment of culture, and when assessed microscopically after de-freezing >90% of spirochetes are motile. PBMC from each subject were cultured: (1) in pure medium; (2) with addition of the spirochete strains representing three pathogenic species of B. burgdorferi: B. afzelii VS 46110 (B.a.), B. garinii 20047 (B.g.) and B. burgdorferi s.s. B-31 (B.ss.). Two hundred microliter of PBMC suspension (1.5–2 × 106 PBMC) and 200 ␮l of B. burgdorferi suspension (2 × 107 bacterial cells) was added to 600 ␮l of culture medium to obtain spirochete to PBMC ratio (multiplicity of infection – MOI) on the order of 10:1. PBMC from some donors were not stimulated with all three strains, with the omission of either B. garinii or B. afzelii, but most of samples were incubated with all three strains in parallel. After 48 h of culture, supernatant was pippeted, frozen at −70 ◦ C and stored for immunoenzymatic examinations, while PBMC were rinsed twice with PBS and suspended in PBS with 0.1% azide, in a concentration of 2–4 × 105 cells/ml.

PBMC isolation and culture

Immunoenzymatic measurements

Nine milliliter of venous blood were drawn to heparin-coated tubes and processed within 30 min. The samples were centrifuged in Gradisol L (Aqua Medica, Poland) at 400 g for 30 min, peripheral blood mononuclear cell (PBMC) fraction was pippeted and suspended in culture medium RPMI 1640 (Biomed, Poland), centrifuged again and re-suspended in 1 ml of RPMI 1640 with 10% inactivated bovine serum, streptomycin and penicillin. PBMC were

All the supernatant samples were stored and then thawed and tested simultaneously, with commercial enzyme-linked immunosorbent (ELISA) assays, strictly following manufacturers’ instructions. Assays for the detection of sFas, sFasL and TGF-␤ were purchased from Bender Medsystems (Vienna, Austria), and for IL-10 and IL-12 (p70) from BD Biosciences (San Jose, CA, USA). The detection limits were: for sFas 13.2 pg/ml, for sFasL 70 pg/ml, for TGF-␤

192

S. Grygorczuk et al. / Ticks and Tick-borne Diseases 6 (2015) 189–197

Fig. 3. Concentration of sFas in the PBMC culture stimulated with B. burgdorferi. Increased concentration of sFas in the supernatant of the PBMC culture after stimulation for 48 h with live B. burgdorferi spirochetes (MOI 10:1), expressed in pg/ml. (-) – negative control, B.a. – stimulation with B. afzelii, B.g. – stimulation with B. garinii, B.ss – stimulation with B. burgdorferi s.s.. Showing median, quartiles (box), minimum and maximum values (whiskers). A. Patients with Lyme borreliosis. Data from 16 experiments for B.ss, 15 for B.a. and 13 for B.g.; *** p < 0.001 in comparison with (-) in B.a. and B.ss; ** p < 0.01 in comparison with (-) in B.g.; the significant difference between B.g. and B.ss shown directly on the plot. B. Healthy controls. Data from 8 experiments for B.ss, 5 for B.a. and 3 for B.g.; * p < 0.05 in comparison with (-) in B.a. and B.ss, the difference between B.a. and B.ss shown on the plot (p = 0.07). C. The study population analyzed as a whole: *** p < 0.001 in comparison with (-) in B.a. and B.ss; ** p < 0.01 in comparison with (-) in B.g.; the significant difference betwen B.ss and B.g. (p < 0.01) and the difference between B.ss and B.a. with p = 0.08 are shown directly on the plot.

0.1 ng/ml, for IL-10 – 2 pg/ml and for IL-12 – 4 pg/ml. Concentration of IL-10 was measured in ten-fold dilution.

which is a commercial assay based on the annexin-V binding principle. Annexin-V-positive, propidium iodide–negative cells were considered apoptotic.

Flow cytometry Statistical analysis The fraction of T cells expressing Fas and the fraction of apoptotic cells was measured cytometrically on a FACSCalibur cytometer directly after PMBC retrieval from the culture. All the monoclonal antibodies and isotype controls were obtained from BD Biosciences (USA). Cells were gated with the mouse monoclonal IgG2a ␬ anti-CD3+ antibody stained with allophycocyanin (APC) and the mouse monoclonal anti-CD4+ antibody conjugated with biotin and incubated with the streptavidin-PE complex. Fas expression was measured with the use of a standardized pre-diluted solution of the fluoresceine (FITC) stained mouse monoclonal IgG1 ␬ and a recommended isotype control from the same manufacturer, following the manufacturer’s instruction. As recommended, 20 ␮l of a monoclonal antibody solution or of the isotype control per 106 cells were added and incubated in darkness at 2–8 ◦ C for 15 min before the analysis. Fas level was expressed as a fraction of the Fas-positive cells in the CD3+CD4+ population and as a mean fluorescence index (MFI). Fraction of CD3+CD4+ lymphocytes undergoing apoptosis was measured with Apoptosis detection kit II from BD Biosciences,

The data were analyzed with Statistica 9.0 software. Nonparametric tests were used because of the small numbers of cases and deviations from the normal distribution. In stimulated cultures, both absolute values and net values above the baseline level in the non-stimulated control were analyzed, with consistent results. To assess the differences between the groups of subjects, including healthy controls and sub-groups of patients with LB dependent on clinical manifestation (LA, NB, ACA) and stage of the disease (early disseminated and late) we used non-parametric Kruskal–Wallis ANOVA. For comparing the cultures non-stimulated and stimulated with distinct spirochete species the Friedmann ANOVA test was used and significant differences were verified with the Wilcoxon pair test. The analysis was originally performed in controls and LB patients separately. If the results did not differ between the groups, they were then analyzed in the study population as a whole as well. Correlations were assessed with the Spearman test. p < 0.05 was considered significant.

S. Grygorczuk et al. / Ticks and Tick-borne Diseases 6 (2015) 189–197

193

Fig. 4. Concentration of sFasL in the PBMC culture stimulated with B. burgdorferi. Increased concentration of sFasL in the supernatant of the PBMC culture after stimulation for 48 h with live B. burgdorferi spirochetes (MOI 10:1), expressed in pg/ml. (-) –negative control, B.a. – stimulation with B. afzelii, B.g. – stimulation with B. garinii, B.ss – stimulation with B. burgdorferi s.s.. Showing median, quartiles (box), minimum and maximum values (whiskers). A. Patients with Lyme borreliosis. Data from 16 experiments for B.ss, 15 for B.a. and 13 for B.g.; ** p < 0.01 in comparison with (-) in B.ss; * p < 0.05 in comparison with (-) in B.g.; # p = 0.08 in comparison with (-) in B.a.; significant difference between B.a. and B.ss shown directly on the plot. B. Healthy controls. Data from 8 experiments for B.ss, 5 for B.a. and 3 for B.g.; Friedmann ANOVA for four cultures gave p = 0,039, but no significant difference was revealed in direct comparisons between the cultures; difference between B.ss and B.a. with p = 0.07 is shown on the plot. C. The study population analyzed as a whole: ** p < 0.01 in comparison with (-) in B.g. and B.ss; difference between B.ss and B.a. has been shown directly on the plot.

Results

Cytokine synthesis

Th lymphocyte apoptosis and Fas expression

The stimulation had no effect on TGF-␤ expression. IL-10 concentration increased about 10-fold under stimulation both in LB patients and in controls and was higher under stimulation with B. burgdorferi s.s. and B. afzelii than with B. garinii (Fig. 5). There was no difference between the LB and C groups as well as between the subgroups of LB patients. The concentration of IL-10 under stimulation tended to correlate inversely with the cytometric parameters in LB patients, but not in controls (Fig. 6). The correlation was significant for net Fas expression under stimulation with B. afzelii, and for both the Fas expression and fraction of the apoptotic cells under stimulation with B. garinii, but was absent in cultures stimulated with B. burgdorferi s.s.

Fas expression on CD3+CD4+ cells was individually variable, but tended to increase under stimulation with additional 5–15% cells expressing Fas in most of the LB patients and controls. The response to B. burgdorferi s.s. was significantly stronger than to B. garinii (Fig. 1). The fraction of CD3+ CD4+ cells undergoing apoptosis did not change under stimulation (Fig. 2). Soluble Fas and soluble FasL Both sFas and sFasL were detected in the supernatant of nonstimulated cultures and were up-regulated under stimulation. The median levels of sFas increased 3–4-fold and were higher under stimulation with B. burgdorferi s.s. than B. garinii (Fig. 3). The concentration of sFasL was increased by 10–20% by B. garinii and B. burgdorferi s.s., but not B. afzelii (Fig. 4). There was no significant difference between LB patients and controls, as well as between sub-groups of LB patients. The concentrations of soluble factors did not correlate with the fraction of apoptotic Th cells or membrane Fas expression.

Discussion We have found that the rate of the T lymphocyte apoptosis in the presence of B. burgdorferi did not change, which differs from the results of a previous study, in which increase of Fas-mediated apoptosis was detectable as early as 4 h after the exposure to B. burgdorferi, suggestive of a specific pro-apoptotic effect supporting the spirochete survival (Perticarari et al., 2003). That was, however,

194

S. Grygorczuk et al. / Ticks and Tick-borne Diseases 6 (2015) 189–197

Fig. 5. Concentration of IL-10 in the PBMC culture stimulated with B. burgdorferi. Increased concentration of IL-10 in the supernatant of the PBMC culture after stimulation for 48 h with live B. burgdorferi spirochetes (MOI 10:1), expressed in pg/ml; (-) – negative control, B.a. – stimulation with B. afzelii, B.g. – stimulation with B. garinii, B.ss – stimulation with B. burgdorferi s.s.. Showing median, quartiles (box), minimum and maximum values (whiskers). A. Patients with Lyme borreliosis. Data from 16 experiments for B.ss, 15 for B.a. and 13 for B.g.; *** p < 0.001 in comparison with (-) in B.a. and B.ss.; ** p < 0.01 in comparison with (-) in B.g.; significant difference between B.g. and B.ss shown directly on the plot. B. Healthy controls. Data from 8 experiments for B.ss, 5 for B.a. and 3 for B.g.; * p < 0.05 in comparison with (-) in B.a. and B.ss. C. The study population analyzed as a whole: *** p < 0.001 in comparison with (-) in B.a., B.g. and B.ss.; the difference between B.g. and B.ss and between B.g. and B.a. is shown directly on the plot. D. TGF-␤ concentrations in the supernatant from the same series of experiments as in A (Lyme borreliosis patients) with no difference between the cultures (ng/ml).

detected only with MOI of 20:1 and 50:1, exceeding the concentration used in our study. The increased membrane Fas expression not accompanied by an increased lymphocyte apoptosis may be interpreted as an unspecific result of activation, analogous to what has been described in response to other inflammatory stimuli (Miyawaki et al., 1992; Schmitz et al., 2003). Soluble FasL is cleaved from the cell surface by metalloproteinases and has no significant cytotoxic activity (Schneider et al., 1998; Hohlbaum et al., 2000). Its increased concentration in serum or in the microenvironment has been interpreted as a marker of the up-regulation of the membrane FasL, and thus of the increased apoptosis rate (Hirsch et al., 2002; Kern et al., 2000; Doughty et al., 2002). However, its generation may directly result in a decreased apoptosis due to down-regulation of the functional membrane FasL. Moreover, accumulating sFasL may act as a competitive antagonist of the membrane form (Schneider et al., 1998; Hohlbaum et al., 2000). Hashimoto et al. (1998) found high concentrations of sFasL in the synovial fluid of patients with rheumatoid arthritis, correlating with the disease severity, and hypothesized that sFasL acted as an antagonist of lymphocyte apoptosis and a pro-inflammatory factor. This might apply to the antibiotic-resistant Lyme arthritis, which resembles rheumatoid arthritis with its probable autoimmune background and a dominant Th1 response (Steere et al., 2004; Shin et al., 2007). We have observed a minor increase of sFasL concentration in the supernatant of PBMC culture both from LB patients

and healthy controls, but no detectable anti-apoptotic effect of sFasL on Th lymphocytes. Soluble Fas is synthesized as a truncated protein consisting of the Fas extracellular domain and is an antagonist of the functional receptor (Liu et al., 2002; Cheng et al., 1994; Stricker et al., 1998). Its serum concentration is increased in infections, both acute (sepsis, malaria) and chronic (type C hepatitis), where it was interpreted as a factor perpetuating inflammation through antiapoptotic activity (Doughty et al., 2002; Panasiuk et al., 2010; Jain et al., 2008). The increased sFas expression results in a decrease of lymphocyte apoptosis in systemic lupus erythematosus and in some forms of leukemia (Liu et al., 2002; Cheng et al., 1994; Knipping et al., 1995). We have detected a few-fold increase of sFas concentration in the PBMC culture challenged with B. burgdorferi, proving that it is vividly up-regulated in the response to this pathogen. No difference between the groups nor correlation with apoptosis rate could be observed, however, making the patophysiologic relevance of this finding uncertain. Still, sFas and/or sFasL might exert an anti-apoptotic effect in the inflammation focus in some groups of LB patients, for example in antibioticresistant Lyme arthritis. This could be confirmed only by studying a larger group of patients with different clinical forms of LB or the material obtained directly from the inflammation site, especially mononuclear cells from synovial fluid and/or tissue in late Lyme arthritis.

S. Grygorczuk et al. / Ticks and Tick-borne Diseases 6 (2015) 189–197

Fig. 6. Correlation between IL-10 and apoptosis under B. burgdorferi stimulation. Inverse correlation between the net IL-10 concentration under B. burgdorferi stimulation (increase above the baseline concentration in the non-stimulated culture) and the net membrane Fas expression on CD3+CD4+ lymphocytes and net fraction of apoptotic (annexine V-positive, propidium iodide negative) CD3+CD4+ lymphocytes assessed with flow cytometry, as described in Materials and methods, in the PBMC culture from patients with Lyme borreliosis. IL-10 concentration is plotted on vertical axis, cytometric parameters –on horizontal axis. A, C, E – Fas expression (MFI); B, D, F – apoptotic lymphocyte fraction; A, B – stimulation with B. afzelii, C, D – stimulation with B. garinii, E, F – stimulation with B. burgdorferi s.s.. The trend was significant (p < 0.05) for A, C and D, as shown on the plot. No significant correlation appeared in controls, as well as between IL-10 and the other studied parameters. NS – non significant.

IL-10 and TGF-␤ are expressed as a result of apoptosis, both by apoptotic cells themselves and by macrophages engulfing apoptotic bodies, and the first rationale to study their concentrations was to verify if they could be used as markers of PBMC apoptosis under B. burgdorferi stimulation (Fadok et al., 1998; Chen et al., 2001; Voll et al., 1997; Cheng et al., 1994; Gao et al., 1998). Additionally, apoptosis-related release of IL-10 and TGF-␤ is physiologically relevant, turning apoptosis into an actively anti-inflammatory and immunomodulatory process (Chen et al., 2001; Gao et al., 1998). We have previously found the increased TGF-␤ concentration in the supernatant of the PBMC culture from patients with early neuroborreliosis after 7-day stimulation with B. burgdorferi, which is

195

consistent with the time required for the apoptosis of activated lymphocytes in the course of a response to a pro-inflammatory stimulus (Grygorczuk et al., 2007). However, in a 48-h PBMC culture, a vivid IL-10 synthesis was not accompanied by any increase of TGF-␤ expression, suggesting that their expression was regulated distinctly and not depended on a common mechanism like apoptosis. B. burgdorferi and its surface lipoproteins induce a strong inflammatory response and the synthesis of a wide spectrum of cytokines (IL-1␤, IL-6, IL-10, IL-12, TNF-␣, INF-␥) in PBMC (Jansky´ et al., 2003; Giambartolomei et al., 2002; Dennis et al., 2006; Murthy et al., 2000). Of these cytokines, IL-10 provides a negative feedback, controlling the synthesis of the pro-inflammatory factors in an autocrine and paracrine manner (Giambartolomei et al., 2002; Dennis et al., 2006). IL-10 and IL-12 may be considered antagonists both in the context of Th1 cells apoptosis and of the control of inflammation in general: IL-10 is an immunomodulatory and Th2related cytokine, which increases Th1 lymphocyte susceptibility to Fas-mediated apoptosis, while IL-12 is a pro-inflammatory and Th1 cytokine with an anti-apoptotic effect on Th cells (Estaquier et al., 1995; Roth et al., 2003). Both shift toward Th2 and uncontrolled Th1 response have been postulated in the pathogenesis of different clinical manifestations of Lyme borreliosis, but it is probably a combination of a strong Th1/inflammatory and a directly following effective Th2/immunomodulatory response that accounts for the good clinical outcome (Shin et al., 2007; Widhe et al., 2002; Grusell et al., 2002; Gross et al., 1998; Diterich et al., 2001; Jones et al., 2008; Müllegger et al., 2007). The low expression of IL-12 in our study made it impossible to analyze its synthesis quantitatively and to investigate how it was related to IL-10 and Th cell apoptosis. However, we were able to observe an inverse correlation between the membrane Fas and IL-10 expression, which may be explained by assuming that Fas presence was a result of the lymphocyte activation and IL-10 reduced the extent of activation through its immunomodulatory activity. The negative correlation was present, although both membrane Fas and IL-10 were individually up-regulated by B. burgdorferi, pointing to an individual variability in the response to stimulation. There was also a negative correlation between an IL-10 concentration and a fraction of apoptotic cells under stimulation with B. garinii – an interesting finding, suggesting some subtle short-term effects of this species on lymphocyte apoptosis itself. However, in the lack of any significant change of the directly measured apoptosis rate it must be considered a preliminary result and treated with caution. The differences between the stimulation with different B. burgdorferi species were small, but statistically significant. Similar differences have been previously observed and might partially explain a variability of the clinical manifestations of LB. The expression of several cytokines (TNF␣, IL-6, IL-8 and other chemokines, IL-10, IL-12) is higher in the presence of B. burgdorferi s.s. than of B. afzelii and B. garinii, both in vitro and in the infected tissue in vivo (Strle et al., 2009; Jones et al., 2008). Of note, probably only B. burgdorferi s.s. is able to cause a persistent and self-perpetuating inflammation in the antibiotic-resistant Lyme arthritis (Steere et al., 2004; Drouin et al., 2004). Here and in the previously described preliminary experiments (Grygorczuk et al., 2010), we have confirmed and extended these observations to the expression of Fas/FasL system, to our knowledge not studied in this context before. Of the three species studied, B. garinii proved consistently less stimulatory than B. burgdorferi s.s. and, in the case of IL-10 synthesis, than B. afzelii. The differences between species were not only quantitative, but qualitatively different patterns of response were present. For example, relatively high expression of IL-10 but no detectable increase in sFasL synthesis was characteristic for B. afzelii stimulation. The inverse correlation between IL-10 and activation-induced membrane Fas expression was present in cultures stimulated with

196

S. Grygorczuk et al. / Ticks and Tick-borne Diseases 6 (2015) 189–197

B. afzelii and B. garinii, but not with B. burgdorferi s.s., suggesting that IL-10 was able to effectively moderate Th cell activation under stimulation with two previous, but not with the last species. This lack of negative feedback could contribute to the pathogenesis of the uncontrolled, Th1-dominated inflammation encountered in some patients with B. burgdorferi s.s. infection. There were no significant differences between LB patients and healthy controls. That suggests that the observed phenomena depended mostly on unspecific, innate response by naive PBMC population, and not on the specific immune recognition in Borreliainfected patients. Analogously, the in vitro proliferative T cell response to B. burgdorferi or its sonicate does not differ between Lyme borreliosis patients and healthy subjects, confirming the dominant role of the unspecific recognition in this setting (Bauer et al., 2001). However, in our study the inverse correlation between IL-10 and Fas expression was limited to LB patients and absent in healthy subjects, suggesting some difference in the interplay of inflammation- and apoptosis-related factors between these groups, which may warrant further study. In conclusion, we have found that in the moderate concentration B. burgdorferi spirochetes do not have a direct effect on the apoptosis of human CD3+CD4+ lymphocytes, either from Lyme borreliosis patients or healthy donors. B. burgdorferi up-regulates the expression of a pro-apoptotic Fas receptor and its ligand in PBMC culture, which, however, does not evidently change lymphocyte apoptosis rate and may be explained as a non-characteristic effect of stimulation. This effect is stronger under stimulation with B. burgdorferi s.s. in comparison with B. garinii and B. afzelii. Fas expression in the presence of B. burgdorferi s.s. is not reduced by endogenous IL-10, suggesting the lack of the efficient immunomodulation during the response elicited by that species. Conflict of interest statement The authors declare that they have no conflict of interest. Acknowledgements The standardized live spirochete suspension was kindly provided by Prof. Stanisława Tylewska-Wierzbanowska and Tomasz Chmielewski Ph.D. from the Department of Rickettsiae, Chlamydiae and Zoonotic Spirochetes of the National Institute of Hygiene in Warsaw. The study was funded by the Medical University in Białystok, grant no. 3-45702L. References Akbar, A.N., Borthwick, N., Salmon, M., Gombr, W., Bofill, M., Shamsadeen, N., Pilling, D., Pett, S., Grundy, J.E., Janossy, G., 1993. The significance of low bcl-2 expression by CD45RO T cells in normal individuals and patients with acute viral infections. The role of apoptosis in T cell memory. J. Exp. Med. 178, 427–438. Alderson, M.R., Tough, T.W., Davis-Smith, T., Braddy, S., Falk, B., Schooley, K.A., Goodwin, R.G., Smith, C.A., Ramsdell, F., Lynch, D.H., 1995. Fas ligand mediates activation-induced cell death in human T lymphocytes. J. Exp. Med. 181, 71–77. Bauer, Y., Hofmann, H., Jahraus, O., Mytilineos, J., Simon, M.M., Wallich, R., 2001. Prominent T cell response to a selectively in vivo expressed Borrelia burgdorferi outer surface protein (pG) in patients with Lyme disease. Eur. J. Immunol. 31, 767–776. Chen, W.J., Frank, M.E., Jin, W., Wahl, S., 2001. TGF-␤ released by apoptotic T cells contributes to an immunosuppressive milieu. Immunity 14, 715–725. Cheng, J., Zhou, T., Liu, C., Shapiro, J.P., Brauer, M.J., Kiefer, M.C., Barr, P.J., Mountz, J.D., 1994. Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science 263, 1759–1762. Dennis, V.A., Jefferson, A., Singh, S.R., Ganapamo, F., Philipp, M.T., 2006. Interleukin10 anti inflammatory response to Borrelia burgdorferi, the agent of Lyme disease: a possible role for suppression of cytokine signaling 1 and 3. Infect. Immun. 74, 5780–5789. Devadas, S., Das, J., Liu, C., Zhang, L., Roberts, A.I., Pan, Z., Moore, P.A., Das, G., Shi, Y., 2006. Granzyme B is critical for T cell receptor-induced cell death of type 2 helper T cells. Immunity 25, 237–247.

Diterich, I., Härter, L., Hassler, D., Wendel, A., Hartung, T., 2001. Modulation of cytokine release in ex vivo-stimulated blood from borreliosis patients. Infect. Immun. 69, 687–694. Doughty, L., Clarck, R.S.B., Kaplan, S.S., Sasser, H., Carcillo, J., 2002. sFas and sFasL ligand and pediatric sepsis-induced multiple organ failure syndrome. Pediatr. Res. 52, 922–927. Drouin, E.E., Glickstein, L.J., Steere, A.C., 2004. Molecular characterization of the OspA(161–175) T cell epitope associated with treatment-resistant Lyme arthritis: differences among the three pathogenic species of Borrelia burgdorferi sensu lato. J. Autoimmunol. 23, 281–292. Estaquier, J., Idziorek, T., Zou, W., Emielie, D., Farber, C.-M., Bourez, J.-M., Ameisen, J.C., 1995. T helper type 1/T helper type 2 cytokines and T cell death: preventive effect of interleukin 12 on activation-induced and CD95 (FAS/APO-1)-mediated apoptosis of CD4+ T cells from human immunodeficiency virus-infected persons. J. Exp. Med. 182, 1759–1767. Fadok, V.A., Bratton, D.L., Konowal, A., Freed, P.W., Westcott, J.Y., Henson, P.M., 1998. Macrophages that have ingested apoptotic cells in vitro inhibit proimflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-␤, PGE2 and PAF. J. Clin. Invest. 101, 890–898. Gao, Y.K., Herndon, J.M., Zhang, H., Griffith, T.S., Ferguson, T.A., 1998. Antiinflammatory effects of CD95 ligang (FasL)-induced apoptosis. J. Exp. Med. 188, 887–896. Giambartolomei, G.H., Dennis, V.A., Lasater, B.L., Murthy, P.K., Philipp, M.T., 2002. Autocrine and exocrine regulation of interleukin-10 production in THP-1 cells stimulated with Borrelia burgdorferi lipoproteins. Infect. Immun. 70, 1881–1888. Gross, D.M., Steere, A.C., Huber, B.T., 1998. T helper 1 response is antigen specific and localized to the synovial fluid in patients with Lyme arthritis. J. Immunol. 160, 1022–1028. Grusell, M., Widhe, A., Ekerfelt, C., 2002. Increased expression of the Th1-inducing cytokines interleukin-12 and interleukin-18 in cerebrospinal fluid but not in sera of patients with Lyme neuroborreliosis. J. Neuroimmunol. 131, 173–178. ´ ´ nska, R., Pancewicz, S., KonGrygorczuk, S., Chmielewski, T., Zajkowska, J., Swierzbi drusik, M., Tylewska-Wierzbanowska, T., Hermanowska-Szpakowicz, T., 2007. Concentration of TGF-␤1 in the supernatant of peripheral blood mononuclear cells cultures from patients with early disseminated and chronic Lyme borreliosis. Adv. Med. Sci. 52, 174–178. ´ ´ Grygorczuk, S., Osada, J., Swierzbi nska, R., Zajkowska, J., Kondrusik, M., Pancewicz, S., ˛ M., 2010. Expression of Fas receptor on human T lymphocytes under Dabrowska, stimulation with Borrelia burgdorferi sensu lato – preliminary results. Adv. Med. Sci. 55, 228–234. Hashimoto, H., Tanaka, M., Suda, T., Tomita, T., Hayashida, K., Takeuchi, E., Kaneko, M., Takano, H., Nagata, S., Ochi, T., 1998. Soluble Fas ligand in the joints of patients with rheumatoid arthritis and osteoarthritis. Arthritis Rheum. 41, 657–662. Hirsch, C.S., Toosi, Z., Johnson, J.L., Luzze, H., Ntambi, L., Peters, P., McHugh, M., Okwera, A., Joloba, M., Mugyenyi, P., Mugerwa, R.D., Terebuh, P., Ellner, J.J., 2002. Augmentation of apoptosis and interferon-␥ production at sites of active Mycobacterium tuberculosis infection in human tuberculosis. J. Infect. Dis. 183, 779–788. Hohlbaum, A.M., Moe, S., Marshak-Rothstein, A., 2000. Opposing effects of transmembrane and soluble Fas ligand expression on inflammation and tumor cell survivial. J. Exp. Med. 191, 1209–1219. Irmler, M., Thome, M., Hahne, M., Schneider, P., Hoffmann, K., Steiner, V., Bodmer, J.L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L.E., Tschopp, J., 1997. Inhibition of death receptor signals by cellular FLIP. Nature 388, 190–195. Jain, V., Armah, H.B., Tongren, J.E., Ned, R.M., Wilson, N.O., Crawford, S., Joel, P.K., Singh, M.P., Nagpal, A.C., Dash, A.P., Udhayakumar, V., Singh, N., Stiles, J.K., 2008. Plasma IP-10, apoptotic and angiogenic factors associated with fatal cerebral malaria in India. Malar. J. 7, 83–97. ´ L., Reymanová, P., Kopecky, ´ J., 2003. Dynamics of cytokine production in Jansky, human peripheral blood mononuclear cells stimulated by LPS or infected by Borrelia. Physiol. Res. 52, 593–598. Jones, K.L., Muellegger, R.R., Means, T.K., Lee, M., Glickstein, L.J., Damle, N., Sikand, V.K., Luster, A.D., Steere, A.C., 2008. Higher nRNA levels of chemokines and cytokines associated with macrophage activation in erythema migrans skin lesions in patients from the United States than in patients from Austria with Lyme borreliosis. Clin. Infect. Dis. 46, 85–92. Kalish, R.A., Kaplan, R.F., Taylor, E., Jones-Woodward, L., Workman, K., Steere, A.C., 2001. Evaluation of study patients with Lyme disease, 10–20-year follow-up. J. Infect. Dis. 183, 453–460. Kern, P., Dietrich, M., Hemmer, C., Wellinghausen, N., 2000. Increased levels of soluble Fas ligand in serum in Plasmodium falciparum malaria. Infect. Immun. 68, 3061–3063. Knipping, E., Debatin, K.M., Stricker, K., Heilig, B., Eder, A., Krammer, P.H., 1995. Identification of soluble APO-1 in supernatant of human B- and T-cell lines and increased serum levels in B- and T-cell leukemias. Blood 85, 1562–1569. Krammer, P.H., Arnold, R., Lavrik, I.N., 2007. Life and death in peripheral T cells. Nat. Rev. Immunol. 7, 532–543. Labbé, K., Saleh, M., 2008. Cell death in the host response to infection. Cell Death Differ. 15, 1339–1349. Liu, J.H., Wei, S., Lamy, T., Li, Y., Epling-Burnette, P.K., Djeu, J.Y., Loughran Jr., T.P., 2002. Blockade of Fas-dependent apoptosis by soluble Fas in LGL leukemia. Blood 100, 1449–1453. Li-Weber, M., Krammer, P.H., 2003. Function and regulation of the CD95 (APO-1/Fas) ligand in the immune system. Semin. Immunol. 15, 145–157. Miyawaki, T., Uehara, T., Nibu, R., Tsuji, T., Yachie, A., Yonehara, S., Taniguchi, N., 1992. Differential expression of apoptosis-related Fas antigen on lymphocyte subpopulations in human peripheral blood. J. Immunol. 149, 3753–3758.

S. Grygorczuk et al. / Ticks and Tick-borne Diseases 6 (2015) 189–197 Müllegger, R.R., Means, T.K., Shin, J.J., Lee, M., Jones, K.L., Glickstein, L.J., Luster, A.D., Steere, A.C., 2007. Chemokine signatures in the skin disorders of Lyme borreliosis in Europe: predominance of CXCL9 and CXCL10 in erythema migrans and acrodermatitis and CXCL13 in lymphocytoma. Infect. Immun. 75, 4621–4628. Murthy, P.K., Dennis, V.A., Lasater, B.L., Philipp, M.T., 2000. Interleukin-10 modulates proinflammatory cytokines in the human monocytic cell line THP-1 stimulated with Borrelia burgdorferi lipoproteins. Infect. Immun. 68, 6663–6669. ˙ Panasiuk, A., Parfieniuk, A., Zak, J., Flisiak, R., 2010. Association among Fas expression in leucocytes, serum Fas and Fas-ligand concentrations and hepatic inflammation and fibrosis in chronic hepatitis C. Liver Int. 30, 472–478. Perticarari, S., Presani, G., Prodan, M., Granzotto, M., Murgia, R., Cinco, M., 2003. Lymphocyte apoptosis co-cultured with Borrelia burgdorferi. Microb. Pathog. 35, 139–145. Roth, G., Moser, B., Krenn, C., Brunner, M., Haisjacki, M., Almer, G., Gerlitz, S., Wolner, E., Boltz-Nitulescu, G., Ankersmit, H.J., 2003. Susceptibility to programmed cell death in T-lymphocytes from septic patients: a mechanism for lymphopenia and Th2 predominance. Biochem. Biophys. Res. Commun. 308, 840–846. Schmitz, I., Krueger, A., Baumann, S., Schultze-Bergkamen, H., Krammer, P.H., Kirchhoff, S., 2003. An IL-2-dependent switch between CD95 signaling pathways sensitizes primary human T cells toward CD-95 mediated activation-induced cell death. J. Immunol. 171, 2930–2936. Schneider, P., Holler, N., Bodmer, J.L., Hahne, M., Frei, K., Fontana, A., Tschopp, J., 1998. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J. Exp. Med. 187, 1205–1213. Shin, J.J., Glickstein, L.J., Steere, A.C., 2007. High levels of inflammatory chemokines and cytokines in joint fluid and synovial tissue throughout the course of antibiotic-refractory Lyme arthritis. Arthritis Rheum. 56, 1325–1335. Sneller, M.C., Dale, J.K., Straus, S.E., 2003. Autoimmune lymphoprolipherative syndrome. Curr. Opin. Rheumatol. 15, 417–421.

197

Steere, A.C., Schoen, R.T., Taylor, E., 1987. The clinical evolution of Lyme arthritis. Ann. Intern. Med. 107, 725–731. Steere, A.C., Coburn, J., Glickstein, L., 2004. The emergence of Lyme disease. J. Clin. Invest. 113, 1093–1101. Steere, A.C., Angelis, S.M., 2006. Therapy for Lyme arthritis, strategies for the treatment of antibiotic-refractory arthritis. Arthritis Rheum. 54, 3079–3086. Stricker, K., Knipping, E., Bohler, T., Benner, A., Krammer, P.H., Debatin, K.M., 1998. Anti CD-95 (APO-1/Fas) autoantibodies and T cell depletion in human immunodeficiency virus type 1 (HIV-1)-infected children. Cell Death Differ. 5, 222–230. ´ ´ E., Strle, F., Strle, K., Drouin, E.E., Shen, S., Khoury, J.E., McHugh, G., Ruˇzic-Sablji c, Steere, A.C., 2009. Borrelia burgdorferi stimulates macrophages to secrete higher levels of cytokines and chemokines than Borrelia afzelii or Borrelia garinii. J. Infect. Dis. 200, 1936–1943. Uehara, T., Miyawaki, T., Ohta, K., Tamaru, Y., Yokoi, T., Nakamura, S., Taniguchi, N., 1992. Apoptotic cell death of primed CD45RO + T lymphocytes in Epstein-Barr Virus-induced infectious mononucleosis. Blood 80, 452–458. Voll, R., Herrmann, M., Roth, E.A., Stach, C., Kalden, J.R., 1997. Immunosuppressive effects of apoptotic cells. Nature 390, 350–351. Widhe, M., Grusell, M., Ekerfelt, C., Vrethem, M., Forsbergh, P., Ernerudh, J., 2002. Cytokines in Lyme borreliosis: lack of early tumor necrosis factor-␣ and transforming growth factor-␤1 responses are associated with chronic neuroborreliosis. Immunology 107, 46–55. Wormser, G.P., Dattwyler, R.J., Shapiro, E.D., Halperin, J.J., Steere, A.C., Klempner, M.S., Krause, P.J., Bakken, J.S., Strle, F., Stanek, G., Bockenstedt, L., Fish, D., Dumler, J.S., Nadelman, R.B., 2006. The clinical assessment, treatment and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clin. Infect. Dis. 43, 1089–1133.