Polybacterial immunomodulator Respivax restores the inductive function of innate immunity in patients with recurrent respiratory infections

International Immunopharmacology 9 (2009) 425–432

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Polybacterial immunomodulator Respivax restores the inductive function of innate immunity in patients with recurrent respiratory infections Maria Nikolova a,1, Draganka Stankulova a,⁎,1, Hristo Taskov a, Plamen Nenkov b, Vladimir Maximov c, Bogdan Petrunov a a b c

Department of Immunology and Allergology, National Center of Infectious and Parasitic Diseases (NCIPD), Sofia, Bulgaria BulBio-NCIPD Ltd., Sofia, Bulgaria Specialized Hospital for Active Treatment of Pulmonary Diseases “St. Sofia”, Sofia, Bulgaria

a r t i c l e

i n f o

Article history: Received 4 November 2008 Received in revised form 7 January 2009 Accepted 13 January 2009 Keywords: Respivax Polybacterial immunomodulator DCs Monocytes TLR Th1/Th2 balance

a b s t r a c t Respivax (BulBio-NCIPD Ltd.) is an oral polybacterial immunomodulator intended for treatment and prevention of non-specific respiratory tract infections. We studied for the first time its effects on the inductive mechanisms of innate immunity, in the course of 3-month immunoprophylaxis of 25 patients with recurrent and chronic respiratory infections. The expression of pattern-recognition receptors on peripheral blood (PB) monocytes and plymorphonuclear cells (PMNs), the antigen-presenting and co-stimulatory potential of peripheral blood monocytes and dendritic cells; and the stimulated Th1/Th2 cytokine production were determined by flow cytometry. As compared to healthy controls, patients were characterized with down-regulation of TLR2 and TLR4/ CD14 complex on PB monocytes (p b 0.01), decreased share of CD14+CD16+ DCs precursors (p b 0.01), decreased CD86 expression on PB DCs (p b 0.05) and a Th2 shift of cytokine profile. Respivax modulated differentially the surface expression of pattern-recognition receptors on PB monocytes, increasing TLR2 and CD14 without affecting TLR4 expression. Further on, Respivax enhanced the differentiation of mature CD86high dendritic cells (DCs). Importantly, Respivax promoted a Th1 shift of cytokine profile and restored the Th1/Th2 cytokine balance without pro-inflammatory effects. Noteworthy, Th1/Th2 ratios in the patient's group correlated positively with the levels of TLR2 (R = 0.5, p b 0.001) and CD14 expression (R = 0.4, p b 0.05). We conclude that Respivax treatment restores the inductive function of innate immunity at three key levels: antigen recognition and presentation, costimulation of naïve T cells, and Th1/Th2 balance. This results, at least in part, from a differential modulation effect on the expression of pathogen-recognition receptors. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The oral polybacterial immunomodulator Respivax is composed of killed whole bacterial cells and lysates from the most frequent causative agents of respiratory tract infections: Streptococcus pneumoniae, Neisseria catarrhalis, Streptococcus pyogenes group А, Haemophilus influenzae type B, Staphylococcus aureus, Klebsiella pneumoniae. The protective role of Respivax has been demonstrated in different animal models [1,2]. In human clinical trials, Respivax reduced the days of antibiotic treatment and hospitalization, and the incidence of recurrent upper and lower respiratory tract infections in children and adults [3–5]. Several studies have demonstrated the positive effect of Respivax on humoral immune components, i.e. increased concentrations of sIgA in saliva, of complement, IFN-α and specific antibacterial antibodies (IgG, IgA and IgM) in

⁎ Corresponding author. Central Laboratory of Immunology, Department of Immunology and Allergology, NCIPD, Sofia 1504, 26 Yanko Sakazov blvd, Bulgaria. Tel.: +359 2 943 5636; fax: +359 2 943 30 75. E-mail address: [email protected] (D. Stankulova). 1 The first two authors contributed equally to this paper. 1567-5769/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2009.01.004

sera as well as higher bactericide activity of sera [6–8]. However, the mechanisms of these beneficial effects were not studied in detail. Recently, innate immunity has drawn much attention as a major activating and directing force of adaptive immune responses [9]. TLRinitiated phagocytosis is not only a major innate effector mechanism [10], but also a prerequisite for the maturation of antigen-presenting cells and adequate induction of antigen-specific T cell responses. Mature dendritic cells (DC) express increased quantities of HLA-DR and CD86 molecules indispensable for the two-step activation of naïve T cells [11]. The induction and differentiation of antigen-specific T cell effectors further depend on the preexisting Th1/Th2 cytokine profile, which is largely determined by the functional activity of innate regulatory subsets [12,13]. In 2004, similar polybacterial preparations, Luivac, Biostim and Ribomunil were shown to have a strong stimulating in vitro effect on dendritic cells [14]. Up to now there is no data available about the direct ex vivo influence of polybacterial immunomodulators on innate immunity mechanisms. Here we present for the first time the effects of Respivax on the inductive mechanisms of innate immunity in patients with chronic respiratory infections. A 90 day course of

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Respivax treatment resulted in a differential modulation of TLR2 and TLR/CD14 expression, maturation of antigen-presenting cells and restoration of Th1/Th2 cytokine balance, thus leading to a better quality of adaptive immune responses. 2. Materials and methods 2.1. Study population and design

identified as lin-negative (CD3−CD14−CD15−CD16−CD19−CD20−CD56−) cells expressing HLA-DR. Peripheral PMNs were identified according to their SSC characteristics and expression of CD15 lineage marker. Monocytes were identified according to their SSC characteristics and expression of CD14 lineage marker. At least 10 000 events were collected for the PMN gate and 3000 events—for the monocyte gate. Mean channel fluorescence intensity (MFI) was used to study the levels of surface TLR expression. 2.4. Quantitation of Th1/Th2 cytokines

Twenty-five patients (10 male, 15 female; average age 47, range 14– 74 years) with chronic and recurrent non-specific respiratory infections (COPD, chronic bronchitis, rhinitis, sinusitis) referred to the National Center of Infectious and Parasitic Diseases (NCIPD) for immunoprophylaxis, were included in the study. A recurrent infection was defined as more than 3 recidives during the 12 preceding months. The study inclusion criteria were evidence of chronic and/or recurrent infection, and no fever or other clinical symptoms of acute infection during the 30 days preceding the immunotherapy. Respivax (BulBio-NCIPD Ltd., Sofia, Bulgaria) was taken orally in 3 consecutive cycles as follows: one tablet (50 mg) daily for 20 days, followed by a 10-day pause. Age and sexmatched healthy control subjects (n = 23) without any history of chronic or recurrent respiratory disease were recruited according to CLSI criteria. Whole blood samples were collected in heparinized vacutainers (BD) at baseline (day 0), in the end of the first cycle (day 20) and on the last day of immunotherapy (day 80). The study protocol was approved by the local ethical committee (NCIPD), and performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all subjects before study initiation.

Statistical analysis was performed with GraphPad Prism 4.0 statistical software. Within group significant differences at days 0, 20 and 80 were evaluated using the non-parametric Wilcoxon matched pairs test, significant differences between healthy controls and patients at days 0, 20 and 80 were evaluated using the nonparametric Mann–Whitney U test. A value of p b 0.05 was considered significant.

2.2. Immunophenotyping

3. Results

FITC-conjugated monoclonal antibodies (mAb) against CD3, CD14, CD15, CD16, CD19, CD20 and CD56 lineage (lin) markers, PEconjugated anti-CD16 and anti-CD86, and PerCP conjugated antiHLA-DR mAb were products of Becton Dickinson Biosciences (BD). PEconjugated monoclonal antibodies against TLR2 and TLR4 were products of Santa Cruz Biotechnology. Isotype-matched irrelevant antibody controls were used to detect non-specific staining. Aliquots of whole blood (100 µl) were incubated for 15 min at room temperature in the dark, red blood cells were lysed using 2 ml of 1× FACS lysing solution (BD), the samples were washed twice in 2 ml phosphate buffered saline (PBS) and fixed in 300 µl 1% p-formaldehyde (Sigma).

3.1. Respivax modulates differentially the expression of patternrecognition receptors

2.3. Flow cytometry Flow-cytometry analysis was performed using FACSCanto™ Flow Cytometer and DIVA software (BD Biosciences). All data were collected with identical instrument settings. Dendritic cells were

Samples of DMEM-diluted (1:1 ratio) whole blood at final volume 500 µl were incubated with 10 µg/ml PHA (Sigma) or culture media only (as negative control) for 24 h at 37 °C and 10%CO2 followed by centrifugation for 5 min at 1200 rpm. The supernatant was collected and stored at −80 °C until analysis. Human Th1/Th2 Cytokine Kit II (BD Biosciences) was used for quantitative measurement of IL-2, IL-4, IL-5 IL-10, TNF-α and IFN-γ according to manufacturer's instructions. 2.5. Statistical analysis

Recognition of pathogens is the first step of immune response initiation. Peripheral blood phagocytes recognize peptidoglycan of Gram-positive bacteria through TLR2, and lipopolysaccharide (LPS) of Gram-negative species through the TLR4/CD14 complex [9]. We investigated the surface expression of these molecules on peripheral blood monocytes and PMNs. Patients with chronic and recurrent non-specific respiratory infections had a significantly decreased expression of TLR2 on circulating monocytes (median MFI 1091) and PMNs (median MFI 662) in comparison to healthy donors (median MFI 1374, and 992 respectively, p b 0.05 for both values). Respivax treatment significantly increased the expression of TLR2 on both monocytes (median MFI 1410) and PMNs, (median MFI 1034) so that on day 80 this expression approached the healthy volunteers' levels (p N 0.05, for both values)

Fig. 1. Respivax enhances the expression of TLR2 in patients with chronic and recurrent respiratory infections. Flow cytometry analysis of the expression level (MFI) of TLR2 on PB monocytes (A) and PMNs (B) of patients (n = 25) at baseline (P0) and on day 80 of Respivax treatment (P80), in comparison to controls, C, n = 23. Median values are indicated on the graphs for each category.

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Fig. 2. Modulation of TLR4/CD14 expression in the course of Respivax treatment. Flow cytometry analysis of the expression level of TLR4 (A,C), and CD14 (B) on PB monocytes (A,B) and PMNs (C) in patients with recurrent non-specific respiratory infections (n = 25) at baseline (P0) and on day 80 of Respivax treatment (P80), in comparison to controls, C, n = 23. Median values are indicated on the graphs for each category.

(Fig. 1A and B). Further on, patients with chronic and recurrent nonspecific respiratory infections had a significantly decreased baseline expression of TLR4 (median MFI 1097) and CD14 (median MFI = 860) on circulating monocytes as compared to healthy donors (median MFI 1516 and 1116 respectively, p b 0.05 for both values) (Fig. 2A and B). In parallel, TLR4 expression on patients' PMNs (median MFI 327), did not differ significantly from healthy controls (median MFI 351, р N 0.05) (Fig. 2C). Noteworthy, Respivax restored the expression of CD14 towards day 80 (median MFI 1106, p N 0.05 as compared to healthy controls), and did not alter the expression of TLR4 on circulating monocytes (Fig. 2A and B). Furthermore, Respivax decreased significantly TLR4 levels on circulating PMNs towards day 80 (median MFI = 214) as compared to baseline and healthy control levels (р b 0.05 for both comparisons) (Fig. 2C). 3.2. Respivax promotes the differentiation of antigen-presenting cells Dendritic cells are professional antigen-presenting cells (APCs) that deliver effectively both the first (antigen-specific) and the second (co-stimulatory) signal to naïve T cells. APCs originate from antigenstimulated phagocyting subsets. Their differentiation is accompanied by increasing expression of HLA-DR molecules presenting the processed antigen, and by up-regulation of co-stimulatory receptors such as CD86 [15]. We next investigated the effects of Respivax on monocyte and DCs differentiation, and the expression of CD86 costimulatory receptors on their surface. Circulating monocytes comprise two major subsets: CD14++CD16− and CD14+CD16+ (Fig. 3A). The latter expresses a higher level of HLADR molecules and is considered as differentiated precursors of DCs [16]. At baseline, patients with recurrent non-specific respiratory infections displayed a significantly decreased proportion of DC precursors (median 9.2%), as compared to healthy controls (median 17.2%, p b 0.01) (Fig. 3B). In addition, patients' CD14+CD16+ subset displayed a decreased level of HLA-DR molecules (median MFI 1223 ) as compared to healthy controls (median MFI 1822 ), p b 0.01 (Fig. 3C). In the course of Respivax immunomodulation the average relative proportion of DC precursors increased to median 11.2% at day 80 (p b 0.01 as compared to baseline). At the same time, the expression of HLA-DR was restored to controls' level (median MFI 2165), p N 0.05 as compared with healthy controls (Fig. 3B and C). At baseline, patients were also characterized with a significantly decreased expression of CD86 on peripheral monocytes (median MFI 980) in comparison to control group (median MFI 1219), (p b 0.05). In the course of Respivax treatment the expression level of CD86 receptor significantly increased, and at the end of the period approached the level of healthy volunteers (median MFI 1137, p N 0.05) (data not shown). Peripheral blood DCs comprise well distinguishable subsets

Fig. 3. Changes in PB monocyte subsets in the course of “Respivax” treatment. Monocytes were gated on a SSC/CD14 dot-plot and further CD14highCD16− and CD14lowCD16+ subsets were defined by flow cytometry analysis (A). Percentage of CD14lowCD16+ monocytes (B), and expression level (MFI) of HLA-DR by CD14lowCD16+ monocytes (C) were studied at baseline, on day 20 and on day 80 of immunomodulation with Respivax in 25 patients, and compared to control levels, C, n = 23. Median values are indicated on the graphs for each category.

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M. Nikolova et al. / International Immunopharmacology 9 (2009) 425–432 Table 2 Stimulated expression of Th2 cytokines in patients with chronic and recurrent respiratory infections Th2

IL-10 (pg/ml)

IL-5 (pg/ml)

IL4 (pg/ml)

0 day 20 day 80 day Healthy controls

411 ± 215a 376 ± 187a 528 ± 151a 783 ± 289

110 ± 84a 111 ± 76a 93 ± 62 53 ± 39

185 ± 105 167 ± 97 136 ± 83a 187 ± 103

Concentrations (mean ± SD pg/ml) of Th2 cytokines IL-4, IL-5, IL-10 at baseline, on day 20 and on day 80 of “Respivax” treatment in 25 patients and 23 healthy controls, measured with “Human Th1/Th2 Cytokine CBA kit” (BD Biosciences), after 24 h in vitro PHA stimulation. a Statistically significant difference as compared to controls (C) nonparametric Mann–Whitney U test.

Fig. 4. Changes in circulating DC subsets in the course of “Respivax” treatment. DC were defined using flow cytometry by the simultaneous absence of lineage-specific markers CD3, CD14, CD16, CD19, CD20, CD56 (lin-) and expression of HLA-DR molecules, and were further analysed for the expression of CD86 co-stimulatory receptor (A). The ratio between CD86high and CD86low DC was determined at baseline, on day 20 and on day 80 of immunomodulation in 25 patients, and compared to healthy control levels, C, n = 23. Median values are indicated on the graphs for each category (B).

expressing CD86 with different intensity: CD86high (mature) and CD86low (immature) (Fig. 4A). The median baseline ratio CD86high/ CD86low DCs in patients was 1.94, which was significantly lower as compared to healthy controls median 3.48, (p b 0.01). While Respivax treatment did not change the percentage and AC of circulating DCs (data not shown), it promoted a significant increase of this ratio to a median of 2.65, p b 0.01 as compared to baseline, as an indicator of DC maturation (Fig. 4B).

day 80. Although there were no significant differences in the PMNs, monocyte and lymphocyte counts between the patients and healthy control subjects (data not shown), cytokine production was standardized to the total white cell count in each sample. The average baseline stimulated concentrations of the Th1 cytokines IFN-γ (16 461 pg/ml, SD ± 4322) and TNF-α (2471 pg/ml, SD ± 1136) were lower as compared to controls (19 470 pg/ml, SD ± 6422 and 6867 pg/ml, SD ± 2091, respectively), p b 0.05 for TNF-α (Table 1). On the other hand, the baseline concentration of the Th2 cytokine IL-5 exceeded twice the control level (mean 110 pg/ml, SD ± 84 vs. 53 pg/ml, SD ± 39, p b 0.05). The concentration of the regulatory IL-10 was also significantly reduced in patients' group (mean 411 pg/ml, SD ± 215) as compared to healthy controls (mean 783 pg/ml, SD ± 289) (Table 2). Respivax treatment enhanced the production of Th1 cytokines at day 80 to a mean of 18 276 pg/ml (SD ± 2749) for IFN-γ and 3 214 pg/ml (SD ± 1026) for TNF-α. The level of IL-2 production did not change (Table 1). In parallel, the concentrations of Th2 cytokines IL-5 and IL-4 decreased to a mean of 93 pg/ml (SD ± 62) and 136 pg/ml (SD ± 83) respectively at the end of the period. Finally, the concentration of IL-10 increased to 528 pg/ml (SD ± 151), but remained below control level.

Respivax modulates the Th1/Th2 cytokine profile in patients with recurrent non-specific respiratory infections

3.3

After the activation of T cells, the efficient differentiation of antigen-specific effectors depends on the pre-existing Th1/Th2 cytokine background [17].We investigated the effects of Respivax immunomodulation on the expression levels of basic cytokines. Concentrations of IFN-γ, TNF-α, IL-10, IL-5, IL-4 and IL-2 were studied after 24 h in vitro PHA stimulation of PMNC, at baseline, day 20 and Table 1 Stimulated expression of Th1 cytokines in patients with chronic and recurrent respiratory infections Th1

IFN-γ (pg/ml)

TNF-α (pg/ml)

IL-2 (pg/ml)

0 day 20 day 80 day Healthy controls

16 461 ± 4322 19 439 ± 5829 18 276 ± 2749 19 470 ± 6422

2471 ± 1136a 3096 ± 1444a 3214 ± 1026a 6867 ± 2091

205 ± 106a 203 ± 143a 186 ± 148a 352 ± 159

Concentrations (mean ± SD pg/ml) of Th1 cytokines IFN-γ, TNF-α, and IL-2 at baseline, on day 20 and day 80 of “Respivax” treatment in 25 patients and 23 healthy controls, measured with “Human Th1/Th2 Cytokine CBA kit” (BD Biosciences), after 24 h in vitro PHA stimulation. a Statistically significant difference as compared to controls (C), nonparametric Mann–Whitney U test.

Fig. 5. Respivax restores the Th1/Th2 balance. Changes in the ratio IFN-γ/IL-4 stimulated secretion in the patients' group in the course of “Respivax” treatment (A) Modulation of the stimulated secretion of IFN-γ in patients with high (group A, n = 11) and low (group B, n = 14) baseline values (B). Cytokine concentrations (mean ± SD) were determined by CBA flow cytometry analysis on days 0, 20 and 80 of “Respivax” treatment, and compared to healthy control values, n = 23 (presented with thick and dotted lines). ⁎ Statistically significant difference as compared to controls, C, nonparametric Mann–Whitney U test. ⁎⁎ Statistically significant difference as compared to baseline (P0), Wilcoxon matched pairs test.

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Further on, we studied the ratio between IFN-γ and IL-4, since they are considered as “profile-defining” for Th1 and Th2 responses [18]. The baseline IFN-γ/IL-4 ratio in the group of patients was decreased (mean 121, SD ± 26), as compared to healthy controls (mean 139, SD ± 24), Respivax increased significantly this ratio at day 80 to 158 (SD ± 21), (p N 0.05 as compared to healthy controls). Therefore, while Th2 stimulated expression prevailed before treatment, Respivax promoted a shift towards Th1 profile (Fig. 5A). It should be noted, that the baseline concentration of stimulated IFN-γ secretion in patients' group (median 10 672 pg/ml) varied in a large range (min = 1478, max = 52 520 pg/ml). Therefore, we analyzed separately two groups of patients according to the baseline level of stimulated IFN-γ secretion: group A (n = 11), with baseline levels above the median (mean 26 434 pg/ml, SD ± 3544) and group B (n = 14), with baseline levels below the median (mean 8543 pg/ml, SD ± 3260). Interestingly, Respivax provoked a significant decrease of the high baseline levels of IFN-γ in group A to and average of 21979 (SD ± 3587) pg/ml and an increase of the low baseline levels of IFN-γ in group B to an average of 15713 (SD ± 3176) pg/ml (Fig. 5B). In that way, Respivax modulated the extreme Th1 and Th2 levels in patients with recurrent respiratory infections towards the level characteristic of healthy controls: mean 19 470 (SD ± 4422) pg/ml and potentiated a Th1/Th2 equilibrium favorable for an efficient effector response of Th1 type. 3.4. Cytokine profile in Respivax treated patients correlates with TLR expression levels Cytokine background is a complex result of multiple signals and cellular interactions that may occur at any stage of the innate and acquired immune responses. We asked whether the observed effects of Respivax treatment on Th1/Th2 cytokine levels were associated with the modified expression of TLR recognition receptors. A detailed data analysis revealed a significant direct correlation between the expression of TLR2 on monocytes in the patients' group at all time points, and the IFN-γ/IL-4 ratio (Spearman's R = 0.5, р b 0.001) (Fig. 6A). In parallel, the

Fig. 6. Cytokine balance is related to TLR expression levels. Correlation between the expression level (MFI) of TLR2 on monocytes and the IFN-γ/IL-4 ratio (R = 0.5, р b 0.001) in the patients' group (n = 25) at all studied time points (A). Correlation between the expression level (MFI) of CD14 on monocytes and IFN-γ/IL-4 ratio (R = 0.4, р b 0.05) in the patients' group (n = 25) at all studied time points (B).

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IFN-γ/IL-4 ratio correlated with the expression of CD14 on monocytes (Spearman's R = 0.4, р b 0.05) (Fig. 6B). No significant correlations were established with the other studied parameters (%CD14+CD16+ monocytes, intensity of HLA-DR expression on CD14+CD16+ monocytes, ratio CD86hi/CD86lo dendritic cells). We concluded that the expression pattern of the recognition receptors for Gram positive (TLR2) and Gram negative bacteria (CD14/TLR4) was a major determinant for the cytokine background, and the modulation of this expression in the course of 3 months Respivax treatment directly affected Th1/Th2 cytokine balance. 4. Discussion According to WHO reports, non-specific respiratory infections impose an enormous burden on modern society and are among the leading causes of death worldwide. Tobacco use, bacterial polyresistance and population aging further increase the negative impact of chronic and recurrent respiratory diseases [19]. This conclusion evokes the urgent need of new strategies for immunoprophylaxis and immunotherapy of non-specific respiratory infections. It is well established that one respiratory infection, even in the absence of shared antigen epitopes, can modify immune response to the next for extended periods of time. Moreover, this “immune adaptation” may affect not only adaptive immune responses, but innate immune compartments as well [20]. Therefore, innate immune mechanisms represent a potential target for immune modulation [21]. The polybacterial immunomodulator Respivax (BB-NCIPD Ltd.) has a well documented beneficial clinical effect in chronic non-specific respiratory conditions [1–5,22]. The purpose of the present study was to investigate for the first time its ex vivo effects on the inductive function of innate immunity in man. Pattern-recognition TLRs constitute the first line of host defense, inducing both innate and adaptive immune responses after recognizing the corresponding pathogen-associated molecular patterns. We established a significantly decreased expression of TLR2 on the circulating monocytes and PMNs of patients with history of chronic and recurrent non-specific respiratory infections. In addition, the expression of both TLR4 and CD14 was decreased on monocytes. TLR2 recognizes peptidoglycan and lipoteichoic acid—major cell wall components of Gram-positive bacteria with leading etiological role in respiratory diseases (e.g. S. pneumoniae, S. pyogenes) [23,24]. In addition, TLR2 may be induced by other pathogens relevant to recurrent respiratory infections as influenza virus, Pneumocystis or Chlamidia [25–27]. On the other hand, TLR4 is indispensable for recognizing LPS containing Gram-negative bacteria as K. pneumoniae or H. influenzae, RSV fusion protein, Chlamydial Hsp60 or small m.w. fragments as fibronectin, fibrinogen and surfactant protein A, available in any respiratory tract inflammation process [28–31]. In 2004 Szalmas et al. demonstrated that CD14 molecules increase the sensitivity of monocyte TLR4 to low LPS concentrations [32]. Modulation of TLR surface expression has been documented in a number of pathologic conditions [33]. A recent study on acute uveitis reported a decreased TLR2 and TLR4 expression in systemic circulation, proposing that binding of TLRs by the respective microbial ligands induces their internalization and down-regulation [34]. Indeed, TLR2 and TLR4 down-modulation has been demonstrated after LPS stimulation in vitro or i.v. administration to volunteers in vivo [35,36]. Further on, the phenomenon of “desensitization” to bacterial TLR ligands after respiratory viral infection has been described as a protective mechanism to inflammatory response in lungs [20]. Finally, there is evidence that down-regulation of TLR may lead to inefficient clearance of subsequent bacterial infections [20,27,37]. According to our results, patients with chronic respiratory infections would have decreased sensitivity to the most important etiological agents of respiratory infections that would perturb both the immediate innate effector activity and the induction of adaptive responses. This

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reduced TLR expression most probably results from the excessive activation by microbial products in the course of repeating infections. In its own turn, it would lead to incomplete clearance, especially of low pathogen concentrations in the final stage of acute infection. Therefore, the reduced level of recognition receptors in the course of repeated infections may turn into a mechanism for their chronification. While reduced TLR expression has been associated with decreased efficiency of anti-bacterial responses, up-regulation of TLRs is observed in the course of acute inflammatory reactions like sepsis or autoimmune exacerbations [25,38,39]. In fact, a number of pharmacological substances with anti-inflammatory effect reduce TLR surface expression: adapalene decreases TLR2 expression on keratinocytes [40], statins suppress TLR4 on PB monocytes [41], and insulin down-regulates TLR1, 2, -4, -7 and -9 mRNA expression [42]. Therefore, modulation of decreased TLR expression appears to be a very delicate issue. In our study Respivax increased the expression of TLR2 on both PMNs and monocytes, without exceeding the level of healthy controls. Noteworthy, Respivax treatment increased the expression of CD14 on monocytes to healthy controls level, without concomitant upregulation of TLR4 expression. Moreover, Respivax decreased the expression of TLR4 on PMNs. It was shown that not CD14 but TLR4 is responsible for the induction of inflammatory reactions during immune response to Gram-negative bacteria, and blocking TLR4 might be beneficial in lung diseases [43]. We demonstrated the important ability of our preparation to modulate selectively patternrecognition receptors, therefore increasing the sensitivity of monocytes to both G-positive and G-negative pathogens, without proinflammatory effects. A similar differential effect has been reported for vitamin D3 [44]. The “natural” ability for selective regulation of TLR2 and TLR4 expression has been demonstrated in the course of acute infection as a transient LPS hyporesponsiveness or “endotoxin tolerance” that restrains excessive inflammatory reaction [20], and may be lost in the course of repetitive stimulations. “Respivax” restores this ability by finely and differentially regulating the expression of TLR2, TLR4 and CD14 on monocytes and PMNs. Professional antigen-presenting cells (APCs) are key inductive elements of innate immunity because of their capacity to stimulate efficiently naïve T cells, to influence the Th1/Th2 balance and to maintain immune tolerance through negative feed-back mechanisms [45]. A number of studies reveal perturbed maturation and expression of co-stimulatory receptors by DCs in the course of acute inflammatory responses [46–48]. In line with these studies, we show that patients with recurrent non-specific respiratory infections are characterized with lower expression of HLA-DR and CD86 on PB monocytes and prevalence of immature (CD86low) DCs. Another demonstration of disturbed inductive function was the significant decrease of CD14+16+ monocytes, precursors of DCs with a marked antigen-presenting function and a basic source of key cytokines for Th1 differentiation [16]. The effects of polybacterial immunomodulators on APCs have been a matter of specific attention lately. A recent study demonstrated that in vitro Uro-Vaxom activated DCs display mature phenotype with increased surface expression of MHC molecules and co-stimulatory receptors (CD80,CD86) [49]. Further on, the immunomodulatory Streptococcal preparation OK432 induced phenotypic and functional maturation of human monocyte-derived DCs [50]. In still another in vitro study, Luivac, Biostim and Ribomunyl-treated human DCs displayed activated phenotype, reduced phagocytic activity and increased potential for stimulating the proliferation of allogeneic Tlymphocytes [14]. We studied for the first time the effects of the polybacterial immunomodulator Respivax on the maturation/activation state of circulating APC ex-vivo. HLA-DR molecule has a basic role in presenting the processed antigen and delivering the first antigen-specific signal to resting T cells [51]. The expression of CD80/CD86 (B7.1 and B7.2) molecules is a prerequisite for the delivery of “second” co-stimulatory signal and activation of naïve T cells. CD86 is expressed by all APC types, inducible

upon inflammation, and preferentially involved in initiating of activation through the CD28 T cell receptor [15]. Oral treatment with Respivax enhanced HLA-DR and CD86 expression on circulating monocytes, increased the ratio mature/immature DCs, as well as the share of CD14+16+ monocytes and the expression of HLA-DR molecules on their surface. Our data provide the first ex-vivo demonstration that Respivax treatment compensates for the disturbed differentiation of APC in recurrent non-specific respiratory infections. A number of studies have demonstrated a direct link between TLRmediated signaling and DC maturation. Different TLR4 agonists as heparan sulfate and bacterial hsp were shown to induce DC maturation evidenced by increased expression of HLA-DR, CD40, CD80 and CD86 [52–54]. TLR2-binding induces the differentiation of epidermal Langerhans cells [55] and of monocyte-derived DC; human bone marrow CD34+ progenitors differentiate into CD86+ DC using TLR7/8 specific agonist [56]. On this basis, we may speculate that the maturation effects of Respivax exerted on circulating monocytes and DC follow from the earlier-described modulation of TLR expression. Accumulating data reveal the unequivocal role of Th1 cytokines in innate and adaptive immune response against viruses, extracellular and intracellular bacteria [17,57]. On the contrary, prevalence of Тh2 cytokines, in allergic states or recurrent parasitic infections potentiates severe viral and bacterial infections and non-efficient antigenspecific T cell responses [58,59]. The cytokine “profile” is a natural result of the activities and interactions of innate (Mo, NK, plasmacytoid DCs, NKT cells) and adaptive immune response cell types (T lymphocytes) [13,60,61]. The factors that influence the cytokine “network” are complex, therefore immunoregulation of cytokine “profile” is an extremely complex and challenging goal. According to our results, patients with recurrent respiratory infections produce decreased quantities of Th1 cytokines upon stimulation (IFN-γ, TNF-α), as opposed to an increased IL-5 cytokine response. Further on, we registered a lower IFN-γ/IL-4 ratio in comparison to healthy volunteers, demonstrating the prevalence of Th2 cytokine profile as one of the mechanisms for recurrence of the infection. It should be noted however, that our patients were not homogeneous according to the baseline stimulated IFNγ secretion, some of them exhibiting extremely high IFNγ “background”. Consequently, a pronounced inflammatory response associated with high Th1 levels is not necessarily associated with efficient pathogen clearance. Respivax enhanced the stimulated secretion of Th1 cytokines (IFN-γ, TNF-α) and down regulated Th2 IL-4, and IL-5 cytokines. In conformity with our results recent data revealed the Th1 stimulating effect of OM85-BV in mice [62]. Studies on the polybacterial preparations BronchoVaxom and Uro-Vaxom in man demonstrated increase of the in vitro stimulated production of IFN-γ, TNF-α and IL-2 in PBMC [63,64]. One very important result of our study was that Respivax erased the significant baseline variations in IFN-γ levels in the studied patients' group. This reflected a genuine modulating effect of enhancing the low and limiting the high stimulated secretion of IFN-γ in patients with recurrent non-specific respiratory infections. Previous studies on the related polybacterial preparations Urostim, Dentavax and Pharinostim have demonstrated a similar modulating effect on the humoral immune response, by increasing the lower and decreasing the higher concentrations of specific antibodies in patients sera [65]. It should be pointed out that Respivax treatment did not change the level of the proliferation inducing cytokine IL-2, in consensus with the results obtained for other similar preparations, Urostim and Dentavax [22,66]. Therefore, Respivax treatment does not induce a spontaneous proliferative response, which is an important requirement for any immunomodulating treatment. IL-10 is defined as Тh3 cytokine with anti-inflammatory effects, and specific T regulatory subset mediator [67]. The concomitant up-regulation of Th1 cytokine secretion and IL-10 production observed as a result of Respivax treatment, is another demonstration of the subtle modulating activity of the preparation. In line with the above results, we should note that

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in the course of the immunomodulation and the 6 months that followed no important secondary effects of Respivax have been reported by the patients. TLR-mediated signalling has a primary and well documented role in the shaping of cytokine background. Both in vitro and vivo studies have demonstrated direct effects of TLR agonists on the production of different interleukins: TLR4 activation is associated with increased production of IL-6, IL-12, TNFα, [53,54], TLR2 induces not only IL-6, TNF-α and IL-12 in Langerhans cells but, importantly, IL-10 [55,68]. The observed correlations between the expression of TLR2 and CD14 on monocytes on one side, and the stimulated IFN-γ/IL-4 ratio on the other show that the changes in recognition receptors (TLR2 and CD14) in Respivax treated patients lead to changes in Th1/Th2 cytokine balance. Moreover, it was shown that TLR2 agonists induce both maturation of DC and enhanced IL-10 secretion [69], and we achieved both effects with Respivax. Our study proposes that the differential regulation of patternrecognizing receptors as a result of Respivax treatment has an integral beneficial effect on the inductive function of innate immunity. Respivax (BulBio-NCIPD Ltd., Bulgaria) is a well-titrated complex mixture of extracts and whole bacterial bodies, providing a complete set of ligands for TLRs. It restores the subtle TLR2 and TLR4/CD14 balance on monocytes and PMNs, fine-tuning the effector and inductive functions of innate immunity, signals the differentiation of APCs and promotes a Th1 cytokine response, while preventing an exaggerated pro-inflammatory reaction. For the first time we demonstrate the ex-vivo modulating action of Respivax on the inductive mechanisms of innate immunity. Our results complete the existing data about the effects of Bulgarian polybacterial preparations on the immune system. Acknowledgement This study has been financially supported by the Bulgarian National Science Fund, Ministry of Science and Education, Grant L-1519/05. References [1] Dobrev P, Nikolova P, Stankova S. Prevention of bronchopulmonary infections with the Bulgarian immunomodulator Respivax. Vutr Boles 1989;28:40–2. [2] Petrunov B, Nenkov P, Tsvetanov I, Dragulev B. The action on the immune system of Respivax—a polybacterial vaccine for the peroral immunotherapy and immunoprophylaxis of nonspecific respiratory tract infections. Zh Mikrobiol Epidemiol Immunobiol 1991:34–9. [3] Petrovska I, Tsvetkova L, Petrova O. [The results of a clinico-immunological examination of patients with infectious-allergic bronchial asthma undergoing treatment with the polybacterial immunostimulant Respivax]. Ter Arkh 1990;62:72–5. [4] Илиев И, Раданова В, Георгиева П, Петрова П. Клинико-имунологични резултати от приложението на Респивакс при деца с различни белодробни заболявания. Педиатрия 1990;29:55–61. [5] Йосифов Й, Бакалова Св, Коларова М. Приложение на Респивакс при лечението на някои остри белодробни инфекции при деца в двойнно сляп опит. Пневмология и Фтизиатрия 1989;26:24–7. [6] Kojuharova M, Petrunova S, Panova N. Efficacy of immunostimulator Respivax in patients with chronic non-specific pulmonary diseases. The impact on the level of endogenous alpha-interferon. Infectology 1998;35:30–2. [7] Kojuharova M, Gatcheva N, Vladimirova N. Efficiency of immunostimulator Respivax in patients with chronic non-specific pulmonary diseases. The impact on TD-postvaccine immunoresponse. Infectology 1999;36:25–7. [8] Petrunov B. Polybacterial immunostimulators in medical practice. J Microbiol Epidemiol Immunobiol 2004;6:122–6. [9] Medzhitov R, Janeway Jr C. Innate immune recognition: mechanisms and pathways. Immunol Rev 2000;173:89–97. [10] Aderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 1999;17:593–623. [11] Tarte K, Fiol G, Rossi JF, Klein B. Extensive characterization of dendritic cells generated in serum-free conditions: regulation of soluble antigen uptake, apoptotic tumor cell phagocytosis, chemotaxis and T cell activation during maturation in vitro. Leukemia 2000;14:2182–92. [12] Kalinski P, Vieira P, Schuitemaker JH, Cai Q, Kapsenberg M. Generation of human type 1- and type 2-polarized dendritic cells from peripheral blood. Methods Mol Biol 2003;215:427–36.

431

[13] Kapsenberg ML. Dendritic-cell control of pathogen-driven T-cell polarization. Nat Rev Immunol 2003;3:984–93. [14] Spisek R, Brazova J, Rozkova D, Zapletalova K, Sediva A, Bartunkova J. Maturation of dendritic cells by bacterial immunomodulators. Vaccine 2004;22:2761–8. [15] Sansom DM, Manzotti CN, Zheng Y. What's the difference between CD80 and CD86? Trends Immunol 2003;24:314–9. [16] Ziegler-Heitbrock L. The CD14+ CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol 2007;81:584–92. [17] Becker Y. Respiratory syncytial virus (RSV) evades the human adaptive immune system by skewing the Th1/Th2 cytokine balance toward increased levels of Th2 cytokines and IgE, markers of allergy—a review. Virus Genes 2006;33:235–52. [18] Kimura M, Tsuruta S, Yoshida T. Unique profile of IL-4 and IFN-gamma production by peripheral blood mononuclear cells in infants with atopic dermatitis. J Allergy Clin Immunol 1998;102:238–44. [19] WHO, MNC., CRA. WHO strategy for prevention and control of chronic respiratory diseases. Report 2001:24–8. [20] Didierlaurent A, Goulding J, Patel S, Snelgrove R, Low L, Bebien M, et al. Sustained desensitization to bacterial Toll-like receptor ligands after resolution of respiratory influenza infection. J Exp Med 2008;205:323–9. [21] Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004;5:987–95. [22] Marinova S, Nenkov P, Markova R, Nikolaeva S, Kostadinova R, Mitov I, et al. Cellular and humoral systemic and mucosal immune responses stimulated by an oral polybacterial immunomodulator in patients with chronic urinary tract infections. Int J Immunopathol Pharmacol 2005;18:457–73. [23] Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 2003;85:85–95. [24] Guthrie R. Community-acquired lower respiratory tract infections: etiology and treatment. Chest 2001;120:2021–34. [25] Chang JH, Hampartzoumian T, Everett B, Lloyd A, McCluskey PJ, Wakefield D. Changes in Toll-like receptor (TLR)-2 and TLR4 expression and function but not polymorphisms are associated with acute anterior uveitis. Invest Ophthalmol Vis Sci 2007;48:1711–7. [26] Kajiya T, Orihara K, Hamasaki S, Oba R, Hirai H, Nagata K, et al. Toll-like receptor 2 expression level on monocytes in patients with viral infections: monitoring infection severity. J Infect 2008;57:249–59. [27] Wang SH, Zhang C, Lasbury ME, Liao CP, Durant PJ, Tschang D, et al. Decreased inflammatory response in Toll-like receptor 2 knockout mice is associated with exacerbated Pneumocystis pneumonia. Microbes Infect 2008;10:334–41. [28] Branger J, Knapp S, Weijer S, Leemans JC, Pater JM, Speelman P, et al. Role of Tolllike receptor 4 in gram-positive and gram-negative pneumonia in mice. Infect Immun 2004;72:788–94. [29] Lorenz E, Chemotti DC, Vandal K, Tessier PA. Toll-like receptor 2 represses nonpilus adhesin-induced signaling in acute infections with the Pseudomonas aeruginosa pilA mutant. Infect Immun 2004;72:4561–9. [30] Rallabhandi P, Bell J, Boukhvalova MS, Medvedev A, Lorenz E, Arditi M, et al. Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling. J Immunol 2006;177:322–32. [31] Puthothu B, Forster J, Heinzmann A, Krueger M. TLR-4 and CD14 polymorphisms in respiratory syncytial virus associated disease. Dis Markers 2006;22:303–8. [32] Antal-Szalmas P, Poppelier M, Sumegi A, Bruggen T. Spare CD14 molecules on human monocytes enhance the sensitivity for low LPS concentrations. Immunol Lett 2004;93:11–5. [33] Cristofaro P, Opal SM. Role of Toll-like receptors in infection and immunity: clinical implications. Drugs 2006;66:15–29. [34] Chang JH, Hampartzoumian T, Everett B, Lloyd A, McCluskey PJ, Wakefield D. Changes in Toll-like receptor (TLR)-2 and TLR4 expression and function but not polymorphisms are associated with acute anterior uveitis. Invest Ophthalmol Vis Sci 2007;48:1711–7. [35] Flo TH, Halaas O, Torp S, Ryan L, Lien E, Dybdahl B, et al. Differential expression of Toll-like receptor 2 in human cells. J Leukoc Biol 2001;69:474–81. [36] Marsik C, Mayr F, Cardona F, Derhaschnig U, Wagner OF, Jilma B. Endotoxaemia modulates Toll-like receptors on leucocytes in humans. Br J Haematol 2003;121: 653–6. [37] Wu Q, Martin RJ, Lafasto S, Efaw BJ, Rino JG, Harbeck RJ, et al. Toll-like receptor 2 down-regulation in established mouse allergic lungs contributes to decreased mycoplasma clearance. Am J Respir Crit Care Med 2008;177:720–9. [38] Harter L, Mica L, Stocker R, Trentz O, Keel M. Increased expression of toll-like receptor-2 and -4 on leukocytes from patients with sepsis. Shock 2004;22: 403–9. [39] Sabroe I, Parker LC, Dower SK, Whyte MK. The role of TLR activation in inflammation. J Pathol 2008;214:126–35. [40] Tenaud I, Khammari A, Dreno B. In vitro modulation of TLR-2, CD1d and IL-10 by adapalene on normal human skin and acne inflammatory lesions. Exp Dermatol 2007;16:500–6. [41] Methe H, Kim JO, Kofler S, Nabauer M, Weis M. Statins decrease Toll-like receptor 4 expression and downstream signaling in human CD14+ monocytes. Arterioscler Thromb Vasc Biol 2005;25:1439–45. [42] Ghanim H, Mohanty P, Deopurkar R, Sia CL, Korzeniewski K, Abuaysheh S, et al. Acute modulation of toll-like receptors by insulin. Diabetes Care 2008;31:1827–31. [43] Jeyaseelan S, Chu HW, Young SK, Freeman MW, Worthen GS. Distinct roles of pattern recognition receptors CD14 and Toll-like receptor 4 in acute lung injury. Infect Immun 2005;73:1754–63. [44] Sadeghi K, Wessner B, Laggner U, Ploder M, Tamandl D, Friedl J, et al. Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns. Eur J Immunol 2006;36:361–70.

432

M. Nikolova et al. / International Immunopharmacology 9 (2009) 425–432

[45] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245–52. [46] Fitch PM, Roghanian A, Howie SE, Sallenave JM. Human neutrophil elastase inhibitors in innate and adaptive immunity. Biochem Soc Trans 2006;34:279–82. [47] Roghanian A, Drost EM, MacNee W, Howie SE, Sallenave JM. Inflammatory lung secretions inhibit dendritic cell maturation and function via neutrophil elastase. Am J Respir Crit Care Med 2006;174:1189–98. [48] Zhang R, Becnel L, Li M, Chen C, Yao Q. C-reactive protein impairs human CD14+ monocyte-derived dendritic cell differentiation, maturation and function. Eur J Immunol 2006;36:2993–3006. [49] Schmidhammer S, Ramoner R, Holtl L, Bartsch G, Thurnher M, Zelle-Rieser C. An Escherichia coli-based oral vaccine against urinary tract infections potently activates human dendritic cells. Urology 2002;60:521–6. [50] Itoh T, Ueda Y, Okugawa K, Fujiwara H, Fuji N, Yamashita T, et al. Streptococcal preparation OK432 promotes functional maturation of human monocyte-derived dendritic cells. Cancer Immunol Immunother 2003;52:207–14. [51] Schamboeck A, Korman AJ, Kamb A, Strominger JL. Organization of the transcriptional unit of a human class II histocompatibility antigen: HLA-DR heavy chain. Nucleic Acids Res 1983;11:8663–75. [52] Aosai F, Rodriguez Pena MS, Mun HS, Fang H, Mitsunaga T, Norose K, et al. Toxoplasma gondii-derived heat shock protein 70 stimulates maturation of murine bone marrow-derived dendritic cells via Toll-like receptor 4. Cell Stress Chaperones 2006;11:13–22. [53] Ashtekar AR, Zhang P, Katz J, Deivanayagam CC, Rallabhandi P, Vogel SN, et al. TLR4-mediated activation of dendritic cells by the heat shock protein DnaK from Francisella tularensis. J Leukoc Biol 2008;84:1434–46. [54] Spirig R, Gajanayake T, Korsgren O, Nilsson B, Rieben R. Low molecular weight dextran sulfate as complement inhibitor and cytoprotectant in solid organ and islet transplantation. Mol Immunol 2008;45:4084–94. [55] Peiser M, Koeck J, Kirschning CJ, Wittig B, Wanner R. Human Langerhans cells selectively activated via Toll-like receptor 2 agonists acquire migratory and CD4+T cell stimulatory capacity. J Leukoc Biol 2008;83:1118–27. [56] Sioud M, Floisand Y. TLR agonists induce the differentiation of human bone marrow CD34+ progenitors into CD11c+ CD80/86+ DC capable of inducing a Th1type response. Eur J Immunol 2007;37:2834–46. [57] Kitagaki K, Businga TR, Racila D, Elliott DE, Weinstock JV, Kline JN. Intestinal helminths protect in a murine model of asthma. J Immunol 2006;177:1628–35.

[58] Truyen E, Coteur L, Dilissen E, Overbergh L, Dupont LJ, Ceuppens JL, et al. Evaluation of airway inflammation by quantitative Th1/Th2 cytokine mRNA measurement in sputum of asthma patients. Thorax 2006;61:202–8. [59] Brazova J, Sediva A, Pospisilova D, Vavrova V, Pohunek P, Macek Jr M, et al. Differential cytokine profile in children with cystic fibrosis. Clin Immunol 2005;115:210–5. [60] Cooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, Ghayur T, et al. Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset. Blood 2001;97:3146–51. [61] Curtsinger JM, Valenzuela JO, Agarwal P, Lins D, Mescher MF. Type I IFNs provide a third signal to CD8 T cells to stimulate clonal expansion and differentiation. J Immunol 2005;174:4465–9. [62] Huber M, Mossmann H, Bessler WG. Th1-orientated immunological properties of the bacterial extract OM-85-BV. Eur J Med Res 2005;10:209–17. [63] Wybran J, Libin M, Schandene L. Activation of natural killer cells and cytokine production in man by bacterial extracts. Immunopharmacol Immunotoxicol 1989;11:17–32. [64] Wybran J, Libin M, Schandene L. Enhancement of cytokine production and natural killer activity by an Escherichia coli extract. Onkologie 1989;12(Suppl 3):22–5. [65] Костадинова Р, Маринова С, Ненков П. Стимулиращ ефект на оралния полибактериален имуномодулатор qУростимq върху хуморалния системен и лигавичен имунитету пациенти с хронични уроинфекции. Съвременна медицина 2006;2:9–17. [66] Petrunov B, Marinova S, Markova R, Nenkov P, Nikolaeva S, Nikolova M, et al. Cellular and humoral systemic and mucosal immune responses stimulated in volunteers by an oral polybacterial immunomodulator “Dentavax”. Int Immunopharmacol 2006;6:1181–93. [67] Battaglia M, Roncarolo MG. The role of cytokines (and not only) in inducing and expanding T regulatory type 1 cells. Transplantation 2004;77:S16–8. [68] Chen LY, Lin YL, Chiang BL. Levamisole enhances immune response by affecting the activation and maturation of human monocyte-derived dendritic cells. Clin Exp Immunol 2008;151:174–81. [69] Hoarau C, Lagaraine C, Martin L, Velge-Roussel F, Lebranchu Y. Supernatant of Bifidobacterium breve induces dendritic cell maturation, activation, and survival through a Toll-like receptor 2 pathway. J Allergy Clin Immunol 2006;117:696–702.