Immunostimulant-induced maturation of monocyte-derived dendritic cells in respiratory infection prophylaxis

Abstract

Background

Immunostimulants were established many years ago to improve the capacity of the immune system to react to exogenous noxae. Immunostimulants can be synthesized in the lab or extracted from bacterial cultures. For example, bacterial lysates have been shown to have a significant effect on the number of infectious episodes, duration of fever and use of antibiotics in patients affected by recurrent respiratory tract infections. The capacity of bacterial lysates to induce the maturation of human monocyte-derived dendritic cells (DCs) has been indicated as the first and fundamental effect focused on the activation of an efficient immune response. Synthetic drugs, used in clinics in the prophylaxis of recurrent respiratory tract infections, have also been described to induce the maturation of DCs. Pidotimod, one of the most representative immunostimulant drugs, has been proposed to induce DC maturation. However, an optimal maturation of DCs is needed to achieve an efficient immune response.

Methods

We evaluated the capacity of pidotimod in inducing the maturation of DCs using a prototypic bacterial lysate, Lantigen B, as a control. This maturation was detected using a monocyte-derived DC maturation assay, measured as an increase in the expression of the CD83 and CD86 and the secretion of IL-1β, IL-12 and IL-23, three pivotal cytokines for the activation of an efficient immune response.

Results

In the current experimental conditions, 10, 1 and 0.1 μg of pidotimod had no effects on DCs, whilst Lantigen B was able to induce complete maturation of DCs in vitro. Additionally, the same doses of pidotimod used in conjunction with Lantigen B also had no further effect on DC maturation induced by the bacterial lysate.

Conclusion

These findings seem to support the concept that bacterial lysates are more effective than other synthetic immunostimulants in inducing an efficient immune response able to affect the incidence and severity of airway infections.

Plain language summary

PLAIN LANGUAGE SUMMARY: It has been claimed that chemically synthesized immunostimulants can induce the maturation of dendritic cells (DCs), thus mediating an efficient immune response suitable to control the number and the severity of respiratory tract infections. Bacterial-derived immunostimulants, obtained either by chemical or mechanical lysis, have been shown to enhance the immune system’s response against recurrent airway infections by directly promoting the activation of DCs thought specific receptors (such as the so-called pattern recognition receptors) on the cell surface. Optimal DC maturation is essential to drive an efficient and long-lasting immune response, but it is also fundamental to avoid the generation of a tolerogenic immune response, associated with a defective maturation of DC. While immunostimulants of bacterial origin, as stated in many different experiments and related references, induce complete activation of DCs, the extent of activation induced by synthetic ones seems lower, at least as described in specific literature. For this reason, the capacity of a prototypic synthetic immunostimulant to induce the maturation of monocyte-derived DCs in a well-established experimental protocol has been evaluated in vitro.

The ability of pidotimod, alone or in combination with the prototypic bacterial lysate Lantigen B, to induce the maturation of DCs derived from monocytes of peripheral blood was evaluated. We not only by quantified the expression of surface markers (CD83 and CD86, widely expressed on mature DCs) but also measured the secretion of IL-1β, IL-12 and IL-23, which are soluble mediators involved in the activation of an efficient immune response.

Pidotimod failed to induce the complete activation of DCs, unlike Lantigen B, which was used as positive control; notably, an additional effect of pidotimod on Lantigen B was found. These findings were observed both on the modification of the expression of CD83 and CD86, and on the secretion of IL-1β, IL-12 and IL-23. Synthetic immunostimulants, such as pidotimod, seem to trigger a less efficient immune response than do bacterial lysates. This finding could impact on the strategy used to attempt a reduction of the number and severity of acute episodes in patients with recurrent upper respiratory tract infections.

Introdution

The prophylaxis of recurrent respiratory tract infections is based on a multidisciplinary approach. Vaccines for influenza viruses, pneumococcal antigens and COVID-19 significantly reduce the number of infectious episodes during the cold season in temperate countries.1 In addition, hygienic and environmental controls (especially in patients with inhalant allergy due to molecular components of mites) represent a simple and efficient resource to reduce environmental allergens, thus decreasing the number of asthma or rhinitis exacerbations and allergy-related infections.2 Finally, pharmacological treatments with inhaled corticosteroids (frequently inappropriate),3 NSAIDs4 and, when needed, antibiotics,5 further prevent complications or improve the outcomes in patients affected by recurrent respiratory tract infections.

A heterogeneous family of drugs, characterized by the capacity to stimulate the immune system, has been used for decades in the prophylaxis of recurrent respiratory tract infections. Within this group of drugs, bacterial lysates were introduced in the early 1970s based on the idea that a potentiation of the immunological surveillance may be relevant for controlling recurrent respiratory tract infections.6 Of note, despite the first evidence suggesting that the specific activation of an adaptive immune response might be responsible for the clinical efficacy, more recent data demonstrated that a combination of both adaptive and ‘natural’ immunity co-operates in the control of respiratory infections.7 Other drugs have been reported to be equally as effective in improving locoregional defences with several studies stating pidotimod (PDT) as one of the most effective amongst them.8,9PDT or (4R)-3-[(2S)-5-oxopyrrolidine-2-carbonyl]-1,3-thiazolidine-4-carboxylic acid, is a synthetic dipeptide drug with a molecular weight of 244.27. Its half-life, after oral administration, is 4 hours.8 Although the natural receptor of PDT remains unclear at present, it has been reported that the administration of this drug can induce the maturation of dendritic cells (DCs) both in animal models10–12 and in humans.13 Other authors have observed that different effects are induced in human DCs after contact with PDT, as, for example, a significant decrease in the synthesis of key pro-inflammatory mediators compared to the production observed in Toll-like receptor-stimulated cells not treated with PDT14 or their expression of toll-like receptor 2.13,15

Nevertheless, by analysing the experimental results of Fogli et al.,14 it is evident that DC maturation, evaluated by flow cytometry, shows an extremely modest increase in the expression of costimulatory molecules, such as CD80 and CD86, as well as of the classical DC activation marker CD83. This is particularly relevant because, in other experimental models, the maturation of DCs induced by bacterial lysates assessed as the expression of these costimulating surface molecules appears much more evident.16

For these reasons, we compared the capacity of the doses of PDT used in these previous studies12,13 to induce the maturation of human monocyte-derived DCs with that of a sublingual bacterial lysate. Maturation was evaluated using the expression of both CD83 and CD86. CD83 is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily and is considered a surface hallmark of mature DCs. Its expression is low or absent on immature DCs and becomes markedly upregulated upon activation and maturation.17 CD86 is a costimulatory molecule expressed on antigen-presenting cells, including DCs, macrophages and B cells. It binds to CD28 and CTLA4 on T cells, delivering critical secondary signals required for T cell activation or inhibition, respectively. CD86 expression is rapidly upregulated during DC maturation in response to microbial products, cytokines or danger signals.18

In addition, the release of cytokines associated with DC maturation, that is,. (IL)-1β, IL-12 and IL-23, was also evaluated. IL-1β is an early-response pro-inflammatory cytokine primarily produced by activated monocytes, macrophages and epithelial cells. IL-1β signals through the IL-1 receptor type I (IL-1R1), leading to activation of the NF-κB and MAPK pathways; it promotes the expression of adhesion molecules and chemokines enhances antigen presentation by DCs and amplifies local inflammatory responses.19

IL-12 is a heterodimeric cytokine (p35/p40) produced mainly by DCs and macrophages in response to microbial stimulation. It binds to the IL-12 receptor (IL-12Rβ1/β2), leading to STAT4 activation in T cells and natural killer (NK) cells. IL-12 is essential for T helper 1 (T_H1) polarization, enhancing IFNγ production and cytotoxic activity. It bridges innate and adaptive immunity by promoting the differentiation of naive CD4⁺ T cells into IFNγ-producing T_H1 cells, thereby supporting cell-mediated immune responses.20

Finally, IL-23 is a heterodimer composed of the p19 and p40 subunits and is secreted by DCs and macrophages upon activation. It acts primarily through the IL-23 receptor complex (IL-23R/IL-12Rβ1), activating the JAK–STAT3 signalling pathway. IL-23 is crucial for the maintenance and expansion of T_H17 cells, which in turn secrete IL-17 and contribute to neutrophilic inflammation and antimicrobial defense.21

Methods

Preparation of human monocyte-derived DCs

Human DCs were derived from peripheral blood monocytes as previously described.22,23 Briefly, anonymized peripheral blood buffy coats were obtained from healthy donors by the blood bank of the San Martino Teaching hospital (Genova, IT), in the context of the signed Agreement #63683/22 for the Transfer of Buffy Coat for Laboratory Use.

PBMCs from buffy coats were diluted 1:2 in sterile phosphate buffer saline and were separated over Ficoll density gradient medium (Lympholyte®-H) by centrifugation (25 minutes, 25°C, 2000 rpm). PBMCs were collected and washed twice in sterile RPMI. Supernatants were then discarded, and cells were resuspended in complete RPMI (10% FBS). Cells were counted, and vitality was checked by trypan blue exclusion. Cells were then resuspended in 50 mL of separation buffer containing CD14 microbeads (MACS, Miltenyi) for the separation of CD14⁺ cells (monocytes) from PBMCs. After incubation, these cells were washed, and the pellet was resuspended in 0.5 mL of separation buffer. The magnetic separation was achieved with the appropriate MACS column (LS or MS) and MACS separator (MACS separation unit Miltenyi) by adding the cell suspension onto the column and rinsed three times with the separation buffer to elute the unlabelled cells. The column was removed from the magnetic separator and placed into a 15 mL collection tube. Buffer was then added to the column, and the magnetically labelled cell fraction (CD14⁺ monocytes) was immediately eluted by firmly flushing the column with a plunger. The procedure was performed twice to further improve the purification step, and finally, the isolated monocytes were counted with trypan blue and centrifuged at 4°C for 6–8 minutes at 1500 rpm. The purity of the separated cells was checked by labelling with lineage markers (CD14, CD3, CD56 and CD19). For this, cells were incubated for 15 minutes at 4°C and, after a washing cycle, cells were analysed using a cytofluorimeter (CytoFLEX LX Flow Cytometer, Beckman Coulter, Milan, Italy).

Experiments were performed using cells obtained from three independent donors with technical measurements carried out in triplicate for each donor. For each experimental condition, three different stimulus doses (10, 1 and 0.1 μg/mL of PDT) were tested to characterize the dose–response relationship. For cytokines secretion, three more different doses (from 0.01 to 0.0001 μg/mL) were also added. The experimental design allowed estimation of both technical variability and inter-donor biological variability. As predefined criteria for intra- donor reproducibility and inter-donor consistency were met across the tested dose range, no additional donors or technical replicates were included.

The purified CD14⁺ monocytes were cultured at a concentration of 500,000 cells/well in a 24-well plate (Costar or Falcon) in 1 mL well of complete RPMI 1640 medium or at a concentration of 3 × 10⁵ cells/well in 0.5 mL/well in a 48-well plate in triplicate. For differentiation-growth, IL-4 (20 ng/mL of complete RPMI) was added in 10% RPMI, and GM-CSF (25 ng/mL of complete RPMI) was used. Cells were cultured at 37°C + CO₂ 4% for 4 days. The culture was monitored using an inverted microscope.

After 4 days, the monocytes differentiated to immature DCs in suspension and were ready for the maturation procedure. For this, 0.5 mL of complete medium supplemented with IL-4 (20 ng/mL (1 μl/mL), GM-CSF (25 ng/mL (1 μl/mL) was added to each well. Maturation was induced by the test drug (PDT, Cat SC-204846, Santa Cruz Biotechnology, Inc., obtained from DBA, Lot n. C2415), diluted to 10, 1 and 0.1 μg/mL. These doses were chosen because these were the concentrations of PDT used in previous published experiments.13 A positive control, represented by diluted Lantigen B (1:100/mL, human use pharma-grade, obtained from a commercial source)24 and a negative control without stimulation were provided. Finally, a mixture of Lantigen + PDT was also prepared.

To evaluate the maturation of DC under the different experimental conditions, the expression of costimulatory molecules able to efficiently prime T lymphocytes (CD40, CD80, CD86) and other classical markers of human DC activation, such as CD83 and upregulation of HLA-DR molecules, were analysed upon 48 hour of culture, a time period suitable to evaluate complete DC maturation, essential for the activation of an efficient immune response.22,23,25,26

Evaluation of the expression of the CD83 and CD86 costimulatory molecules on the cell surface of DCs

Flow cytometric analyses were performed using a CytoFLEX LX Flow Cytometer (Beckman Coulter, Milan, Italy). The monoclonal antibodies to CD83 and CD86 were FITC labelled and purchased from Beckman Coulter, Milan, Italy. A pre-titred dilution of anti-CD83 and anti-CD86 was incubated for 30 minutes at room temperature in the dark. Cells were then washed in RPMI and immediately analysed. For this, 5000 events were acquired per tube. The primary gating strategy was based on a dot plot of forward scatter versus side scatter, allowing the identification and selection of the main cell population on the basis of physical characteristics (cellular size and internal complexity). Subsequently, the frequency of CD83⁺ and CD86⁺ DCs was determined as a percentage of gated cells and used for downstream analysis.

Evaluation of the secretion of cytokines in different experimental conditions

DC culture supernatants were also collected and analysed for the content of relevant pro-inflammatory cytokines, namely human IL-12 subunit-α (Kit ELISA, BT Lab, Zhejiang, Chine, range 10–2000 pg/mL, limit of detection 4.7 pg/mL), human IL-1β – Kit Elisa (Abcam Ltd, range 0.41–100 pg/mL, limit of detection <0.03 pg/mL) and human IL-23 subunit-α (IL-23A, AFG Bioscience LLC, range 62.5–4000 pg/mL, limit of detection 15.6 pg/mL), all purchased from Prodotti Gianni, Milano, Italy. A titration curve was prepared, and supernatants were assayed according to the producers’ instructions.

Statistical analysis

Tests were conducted on all the described conditions. Results from cytofluorometric data were analysed using the R statistical environment (version 4.5.0). One-way ANOVA was applied to compare the experimental conditions for each surface antigen, assuming approximate normality of the data distributions. When appropriate, Tukey’s post hoc test was used for pairwise comparisons. Considering that the magnitude of the differences observed in the experiments performed greatly exceeded intra-group variability as well as the presence of an established positive control, the statistical analysis was intended to be exploratory rather than confirmatory, and the conclusions are supported by the consistency of the biological effects observed across independent experiments. In figures, bar plots with error bars (mean +_ SEM) and significance annotations were generated using the ggplot2, dplyr and multcompView packages. A p value of <0.05 was considered statistically significant.

Results

This study aimed to verify whether the expression of CD83 and CD86 on monocyte-derived DCs, observed by other authors,10–13 could be considered suitable for demonstrating that an efficient immune response can be achieved via the administration of PTD. To rule out potential interfering effects of experimental conditions that could weaken the expression of costimulatory molecules, we introduced Lantigen B, a pharma-grade bacterial lysate, as a control for another strategy of immune stimulation.

The addition of PDT, at three different concentrations, to immature DC cultures did not induce any detectable (or better significant) changes in CD83 and CD86 expression compared to the negative control (unstimulated cells) (Figure 1). Despite formal statistics performed using ANOVA, for pure descriptive reasons, median values and ranges are described. Thus, the median percentage of CD83⁺ cells in immature, non-treated DCs was 12% (range 7.2–17.6). After stimulation with 10, 1 and 0.1 μg/mL of PDT, the results were 10% (6–23), 12% (8–21) and 9% (8–22), respectively. Similar results were obtained for CD86: 42% (26–44) of immature DCs expressed CD86 and the addition of PDT did not significantly modify the expression of CD86 on the surface (40%, 41.4% and 39% using 10, 1 and 0.1 μg/mL of PDT, respectively).

Figure 1.

Dendritic cell (DC) maturation was assessed by flow cytometric analysis of surface expression of CD83 (left panel) and CD86 (right panel). A, Immature DCs (served as control of the maturation process). B, C and D, DCs treated with 10, 1 and 0.1 μg/mL of PDT. E, DCs treated with the bacterial lysate alone. F, G and H, DCs treated with 10, 1 and 0.1 μg/mL of PDT and the bacterial lysate. Columns represent then mean % of DCs expressing the relevant surface antigen, CD83 and CD86. ***p<0.01; n.s., not significant.

In contrast, stimulation with a single dose of Lantigen B induced nearly complete maturation of immature DCs: the median stimulation for CD83 was 80% (72–92) and the addition of PDT to Lantigen B did not alter CD83 expression in mature DCs (80%, 86% and 88%, ranging from 70% to 89%) for the three different concentrations of PDT. Similarly, CD86 expression was maximal after stimulation with Lantigen B alone (99%, ranging from 98.8% to 99.8%) and virtually unmodified by the addition of PDT (99% in all conditions, ranging from 96.4% to 99.8%).

Similarly, regarding cytokine secretion, no effect could be observed by the administration of different doses of PDT, while the bacterial lysate, used as a positive control, induced a significant secretion of cytokines directly involved in the activation of a powerful immune response (Figure 2). Indeed, IL-1β, IL-23 and IL-12 were significantly secreted by the addition of Lantigen B (52 pg/mL (range 47–57), 565 pg/mL (range 550–572) and 710 pg/mL (range 605–790), respectively). The addition of six different doses of PDT (ranging from 10 to 0.0001 μg/mL) did not produce the same effect. For IL-1β, the secretion was one-third of that obtained by Lantigen B, without any dose–effect detectable. Of note, IL-1β secretion was not statistically different from that detected in immature DC cultures. For IL-23, there was no dose–effect and no difference between immature and PTD-treated DCs was detected. Finally, IL-12 showed no dose effect or differences between immature DCs and PDT-stimulated DCs, whilst we observed a significant difference in the amounts of cytokines detected in immature DC cultures and in the ones with the positive control, represented by those treated with Lantigen B.

Figure 2.

Pidotimod (PDT) fails to induce pro-inflammatory cytokine secretion by dendritic cells (DCs). Supernatants from DC cultures treated with decreasing concentrations of PDT (10 to 0.0001 μg/mL) or bacterial lysate (LB) were harvested after 48 hours and analysed by ELISA for IL-1β (top), IL-23 (middle) and IL-12 (bottom). ***p<0.001; n.s., not significant. I, immature DCs.

Discussion

Several studies have suggested that PDT may enhance DC maturation, representing the main mechanism of action for its potential to stimulate the immune system.10–13,16 Maturation of DCs is a critical step for the activation of an effective immune response, whereas impaired maturation may contribute to the establishment of a tolerogenic environment.25,26 In this context, NK cells play a key role in monitoring DC maturation status.27 Mature DCs typically exhibit high surface expression of HLA class I molecules, which serves as a ‘self-protective’ signal that prevents NK cell-mediated cytotoxicity. In contrast, poorly matured DCs express lower levels of HLA class I and are selectively eliminated by NK cells.23 Notably, DCs can also activate resting NK cells, contributing to a bidirectional regulatory mechanism within the immune system.

Thus, any molecule – whether natural or synthetic, including pharmacological agents – that effectively promotes DC maturation is expected to exert a beneficial effect in clinical settings where a robust immune response is required, such as in recurrent respiratory tract infections. Conversely, stimuli that only partially induce DC activation may be ineffective or, worse still, may contribute to the establishment of a tolerogenic environment, rendering them unsuitable for controlling infectious diseases.

The results of this study strongly suggest that the induction of DC maturation by PDT is not complete. Under the present experimental conditions, a commonly used bacterial lysate (Lantigen B) effectively promoted the maturation of nearly all DCs. In contrast, PDT showed scarce effects, similar to those previously described (+15% and +10% for CD83 and CD86),13 a modification that does not seem to be particularly effective in terms of upregulation of both surface molecules and cytokine secretion, especially when other stimuli achieved a much more evident result under the same experimental conditions. These findings are also consistent with those of a separate study16 that also failed to detect any biological activity of PDT under similar experimental conditions.

These negative results warrant careful consideration. Discrepancies between expected and observed outcomes in experimental settings may result from various factors, the first of which is reagent performance. Nevertheless, in this study, monocytes and monocyte- derived DCs were all derived from the peripheral blood of healthy volunteers, the culture media and stimuli were identical across conditions, and flow cytometric analysis of DC maturation was performed using the same monoclonal antibodies and the same cytometer settings. The only variable was the presence of different concentrations of PDT in specific tubes. PDT was sourced from a commercial supplier and, as an additional control, the formulation used in clinical practice (powder form) was also tested (data not shown). Nevertheless, neither preparation induced DC maturation. Importantly, the range of PDT concentrations tested was selected based on previously published studies in humans,13 as well as on studies employing polyvalent mechanical bacterial lysates as a positive control.16 Notably, in our experiment, the different stimuli added as a positive control in the same experimental conditions resulted in the complete maturation of DCs.

An in vitro study may only mimic what happens in vivo. Therefore, drawing conclusions based on merely in vitro studies could be inappropriate. However, DC maturation is an on-off phenomenon: in the presence of complete maturation, an efficient immune response may be expected. Conversely, incomplete maturation results in tolerogenic DCs,25,26 an effect that should be accurately avoided when the control of infection is expected. Further, although the study was conducted in vitro, a well- documented mechanism of action (DC maturation) of drugs that have a well-established immunostimulant effect on immune cells was observed only in the presence of the positive control, that is, the prototypic bacterial lysate. Under all other experimental conditions, where PDT was used alone in a 100-fold range of concentrations (difficult concentrations to achieve in vivo), no significant difference compared to immature DCs were observed, both in terms of CD83 and CD86 expression of cytokine secretion. Although it could be argued that in vitro results cannot be directly equated with in vivo outcomes, in vivo effects are often substantiated by in vitro evidence, whereas the converse relationship is less consistently observed.

Conclusion

In conclusion, this study shows that DC maturation – a critical step in initiating both innate and adaptive immune responses – occurred only in the presence of protein components derived from bacterial cultures, and the chemically synthesized compound PDT did not induce DC maturation under the tested in vitro conditions. Therefore, the potentiation of the immune response through this activation pathway remains debatable and requires further investigation.

Availability of data and materials

All data are available from the corresponding author upon reasonable request.