Macrophages are key modulators of immune response and are widely distributed throughout the body, particularly the gut [1]. These cells form a crucial line of defence between the environment and the host and provide a target for the investigation of host-microbe immunological interactions. Macrophages exhibit significant plasticity and can alter their phenotype to suit their environment and function [2]. Macrophage polarisation typically produces two phenotypes; a classical pathogen-clearing pro-inflammatory ‘M1’, or, an alternatively-activated anti-inflammatory ‘M2’ phenotype [3,4]. To enhance pathogen clearance, macrophages can utilize the IL-1β and IL-18-producing inflammasome, an intracellular multiprotein complex essential for innate immune response [5,6]. Numerous inflammasome complexes can form, each with distinct properties that allow the host cell to elicit antibacterial and antiviral responses accompanied by cytokine production [7-12]. Although the classical nucleotide-binding oligomerization domain-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) inflammasome is typically implicated in driving IL-1β secretion, Caspase-1 is also required for maturation and secretion of IL-1β, in addition to non-NLRP3 inflammasomes that can form which can also regulate IL-1β [6].

The human gut houses the most densely populated bacterial ecosystem in the body with around 1x10¹⁴ bacterial cells and these bacteria outnumber host human cells by over 10:1 [13,14]. These microorganisms can significantly impact metabolic diseases, inflammation and infection and modulation of the gut microbiota is being targeted for the prevention and/or treatment of gut dysbiosis with probiotics being widely used to restore imbalance [15-21]. Probiotics are classified as ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host’ and a plethora of in vivo evidence now supports their beneficial immunomodulatory potential [22-28]. Recent emphasis has been placed on delineating these benefits at a mechanistic level and it is emerging that non-pathogenic commensal organisms can activate distinct inflammatory and inflammasome pathways in immune cells to impart health benefits [6,29,30].

The beneficial effects of the Lab4 and Lab4b probiotic consortia are well-reported [22,27], however, the potential mechanisms by which they have been able to reduce the incidence of upper respiratory tract infections (URTI) and atopic sensitization during childhood have remained unclear [22,27]. Therefore, the aim of this study was to assess the regulatory roles of Lab4 and Lab4b on the innate immune system using cultured macrophages.

Bacterial culture

All bacterial preparations were provided by Cultech Ltd. DeMan Rogosa Sharpe (MRS) broth (Oxoid) was inoculated with freeze-dried preparations of a consortium comprising of Lactobacillus acidophilus CUL21 (NCIMB 30156), Lactobacillus acidophilusCUL60 (NCIMB 30157), Bifidobacterium bifidum CUL20 (NCIMB 30153) and Bifidobacterium animalis subsp. lactis CUL34 (NCIMB 30172) (Lab4), or, a consortium comprising of Lactobacillus salivarius CUL61 (NCIMB 30211), Lactobacillus paracasei CUL08 (NCIMB 30154), Bifidobacterium bifidum CUL20 (NCIMB 30153) and Bifidobacterium animalis subsp. lactis CUL34 (NCIMB 30172) (Lab4b) and was incubated anaerobically (10% carbon dioxide, 10% hydrogen and 80% nitrogen) at 37°C for 18 hours, diluted to 1x10⁹ CFU/mL and harvested by centrifugation at 2,500 g for 10 minutes. Bacterial pellets were then resuspended in phosphate-buffered saline (PBS), centrifuged, and resuspended in RPMI-1640 medium containing no serum or antibiotics. Cell suspensions were incubated for 5 hours under anaerobic conditions at 37°C,centrifuged, and supernatants were passed through a 0.22 µm polyethylene syringe filter. The pH of the supernatant was then adjusted to 7.4 and supplemented with 50 µg/mL penicillin and 50 U/mL streptomycin. Cell-free supernatants containing either Lab4 or Lab4b metabolites were applied directly to cultured macrophages during experiments. Hereafter, unless stated otherwise, Lab4 and Lab4b cell-free supernatants containing metabolites will be referred to as just Lab4 and Lab4b.

Macrophage cell culture

THP-1 monocytes were maintained in RPMI-1640 supplemented with 10% (v/v) FCS, 50 µg/mL streptomycin and 50 U/mL penicillin at 37°C in 5% (v/v) CO₂ (reagents from Life Technologies, UK). THP-1 monocytes were plated at 1x10⁶ cells per well in 24-well plates and differentiated into macrophages by incubation with 160 nM phorbol 12-myristate 13-acetate (PMA) for 24 hours [31]. PMA, poly I:C, LPS and ATP were all prepared in PBS, pH was adjusted where appropriate to 7.4 and PBS was used as a vehicle control. For bacterial challenge studies, THP-1 monocyte-derived macrophages were primed with 1 µg/mL LPS for 4 hours, before also stimulating with 5 mM ATP for 30 minutes while co-incubating with control, Lab4 or Lab4b cell-free supernatant. For the assessment of IL-12 expression, either metabolites, poly I:C viral challenge (50 µg/mL) or a combination of the two were incubated with cells for 4 hours. Viability of THP-1 monocyte-derived macrophages was maintained in all conditions tested (Supplementary Figure S1B). All reagents and cells, unless otherwise stated, were purchased from Sigma Aldrich, UK.

RT-qPCR

RNA was extracted using RiboZol (VWR, UK) following manufacturer’s instructions and was quantified using QuBit Fluorometric Quantification (Thermo Fisher, UK). cDNA (500 ng) was generated using a High Capacity cDNA Reverse Transcription Kit (Life Technologies, UK). For RT-qPCR, 10 ng cDNA was used in each 25 µL SYBR green (BioRad) reaction. Primer sets were designed, or, primer sets were obtained from the literature (for sequences and PCR cycling conditions, see Supplementary Table S1). Negative control RNA samples not treated with reverse transcriptase were also analysed alongside RT-qPCR samples to monitor primer-specificity and ensure reactions were contaminant-free. Custom primers were designed in-house using BLAST^® (NCBI) with primer storage and oligonucleotide analysis performed using oliGO (https://oligo.co). Quantification was carried out on a CFX96 (Bio-Rad) and data are expressed as fold changes according to the 2^{-(ΔCt1–ΔCt2)} method. Primers used were: For M1 and M2 polarisation mRNA expression analysis (Figure 1): Interleukin (IL)-1β, IL-18, cluster of differentiation (CD)-80 and CD206. For inflammasome mRNA expression analysis (Figures 2 and 3): NLRP3, Caspase-1, NLRP1, NLR family caspase activation and recruitment domain (CARD) domain-containing protein 4 (NLRC4) and absent in melanoma-2 (AIM2). For antiviral response mRNA expression analysis (Figures 4 and 5): IL-12p35 and IL-12p40.

Figure 1: Lab4 and Lab4b drive the mRNA expression of M1 markers in human macrophages. The mRNA expression levels of IL-1β, IL-18, CD80 and CD206 in human THP-1 macrophages incubated with control medium (black bars) or treated with Lab4 (grey bars) or Lab4b (hashed bars) for 4 hours. Data (mean ± SEM) are from five independent experiments expressed as fold changes from the control arbitrarily set to 1.0. Statistics: One-way ANOVA with Tukey's multiple comparisons. Significance: *=p ≤ 0.05, **=p≤ 0.01.

Figure 2: Lab4 and Lab4b activate the inflammasome in human macrophages. (A) The mRNA expression levels of IL-1β, and (B) IL-1β protein secretion, and (C) mRNA expression levels of NLRP3, Caspase-1, NLRP1, NLRC4 and AIM2 were determined in human THP-1 monocyte-derived macrophages that were treated with control medium (black bars), Lab4 (grey bars) or Lab4b (hashed bars) over 4 hours. Data (mean ± SEM) are from four independent experiments expressed as fold changes from the control arbitrarily set to 1.0. Statistics: One-way ANOVA with Tukey's multiple comparisons. Significance: *=p ≤ 0.05, **=p ≤ 0.01, ***=p ≤ 0.001.

Figure 3: Lab4 and Lab4b enhance inflammasome activation in LPS- and ATP-challenged human macrophages. (A) The mRNA expression of IL-1β, and (B) protein secretion of IL-1β, and (C) mRNA expression of NLRP3, Caspase-1, NLRP1, NLRC4 and AIM2 were determined in human THP-1 monocyte-derived macrophages. Conditions are: Control medium-treated cells (white bars) with a PBS vehicle control for 4 hours 30 minutes (duration of experimental period), LPS (1 μg/mL, 4 hours) followed by ATP (5 mM, 30 minutes)-treated cells (black bars), Lab4-treated cells with the same LPS and ATP conditions (dotted bars), Lab4b-treated cells with the same LPS and ATP conditions (hashed bars). Data (mean ± SEM) are from four independent experiments expressed as fold change compared to the LPS- and ATP-treated cells arbitrarily set to 1.0. Statistics: One-way ANOVA with Tukey's multiple comparisons. Significance: *=p ≤ 0.05, **=p ≤ 0.01, ***=p ≤ 0.001.

Figure 4: Lab4 and Lab4b promote IL-12 production in human macrophages. (A) The secreted protein levels of IL-12, and (B) mRNA expression of IL-12p35, and (C) mRNA expression of IL-12p40 were determined in human THP-1 monocyte-derived macrophages. Conditions are: Control medium-treated cells (black bars), Lab4-treated cells (grey bars), Lab4b-treated cells (hashed bars). Data (mean ± SEM) are from four independent experiments. Statistics: One-way ANOVA with Tukey's multiple comparisons. Significance: *=p ≤ 0.05, ***=p ≤ 0.001.

Figure 5: Lab4 and Lab4b promote IL-12 production in poly I:Cchallenged human macrophages. (A) The secreted protein levels of IL-12 in response to 50 μg/mL poly I:C over 4 hours. Conditions are: Control medium with a PBS vehicle-treated cells (black bars) and poly I:C treated cells (grey bars). (B) The secreted protein levels of IL-12, and (C) mRNA expression of IL-12p35, and (D) mRNA expression of IL-12p40 in human THP-1 monocyte-derived macrophages. Conditions are: control medium-treated cells coincubated with poly I:C (black bars), Lab4-treated cells coincubated with poly I:C (grey bars) and Lab4b-treated cells coincubated with poly I:C (hashed bars). Data (mean ± SEM) are from four independent experiments. Statistics: One-way ANOVA with Tukey's multiple comparisons. Significance: *=p ≤ 0.05, **=p≤ 0.01, ***=p ≤ 0.001.

IL-1β and IL-12 quantification by ELISA

Cell culture supernatants were centrifuged at 5,000 g for 5 minutes and the resultant cell-free supernatant was assayed for IL-1β or IL-12 concentration using human IL-1β or IL-12 standard 3,3’,5,5’-Tetramethylbenzidine (TMB) ELISA development kits (Peprotech), respectively, in accordance with manufacturer’s instructions.

Phagocytosis assay

Phagocytosis assays were performed using the Vybrant^® assay kit (Thermo Fisher, UK). 1x10⁵ THP-1 macrophages per wel were platedl in 96-well plates and incubated in the presence of either control medium or Lab4 or Lab4b cell-free supernatant for 4 hours. These supernatants were diluted 1:10 to be compatible with the kit. Cells were washed with PBS before incubating with FITC-labelled E. coli bioparticles for 2 hours and the plate was read with excitation/emission at 480/520 nm.

Statistical analysis

Prior to statistical significance testing, normality of the data was assessed using D’Agostino-Pearson and Shapiro-Wilk tests. For multiple comparisons, values of P were determined using one-way ANOVA with Tukey’s post-hoc analysis when data were normally distributed. For data not normally distributed, non-parametric one-way ANOVA (Kruskal-Wallis) with Dunn’s post-hoc analysis was performed. For single comparisons between two groups, values of P were calculated using a two-tailed unpaired student’s t-test. Statistical outliers were excluded from analysis. All statistical testing was performed using GraphPad 7.0 (Prism) and data are presented as the mean ± SEM of the defined number of experiments. Significance was defined when P<0.05 and P values are stated unless they fall below P<0.001.

Promotion of the M1 phenotype in human macrophages

The presence of Lab4 or Lab4b induced M1 marker mRNA expression in THP-1 macrophages of: IL-1β (5.2-fold, P=0.022 and 7.2-fold, P=0.0014), IL-18 (2.0-fold, P=0.0082 and 2.1-fold, P=0.0021) and CD80 (36.3-fold, P=0.081 and 56.7-fold, P=0.0050) for Lab4 and Lab4b, respectively, compared to the control. Lab4 and Lab4b reduced the mRNA expression of M2 marker CD206 (0.79-fold, P=0.37 and 0.31-fold, P=0.022, respectively) compared to the control.

IL-1β activation in human macrophages

The effects of Lab4 and Lab4b on inducing IL-1β and IL-18 mRNA expression (Figure 1) suggested the involvement of the inflammasome [32]. In separate experiments, IL-1β mRNA expression was induced by Lab4 (12.1-fold, P=0.013) and Lab4b (28.2-fold, P<0.001). Lab4b also induced IL-1β mRNA expression significantly more than Lab4 (28.2-fold vs. 12.1-fold, P<0.001, Figure 2A). IL-1β protein secretion was 9,586 pg/mL for Lab4 and 4,459 pg/mL for Lab4b compared to a control of 43.0 pg/mL (P<0.001 and P=0.022, respectively) with Lab4 producing more of a response than Lab4b (P=0.0060, Figure 2B).

Inflammasome activation in human macrophages

Inflammasome activation was determined by measuring mRNA expression of five different inflammasome pathway components: NLRP3, Caspase-1, NLRP1, NLRC4 and AIM2. Both Lab4 and Lab4b induced the expression of NLRP3 (3.0-fold, P<0.001 and 2.5-fold P=0.0010, respectively) and Caspase-1 (3.6-fold, P<0.001 and 3.8-fold, P<0.001, respectively) compared to the control. Lab4 induced mRNA expression of NLRP1 (2.6-fold, P=0.040) and AIM2 (4.0-fold, P<0.001). Lab4b attenuated mRNA expression of NLRC4 (0.3-fold, P<0.001, Figure 2C). Lab4 induced significantly more NLRP1 mRNA expression compared to Lab4b (2.6-fold vs. 1.0-fold, respectively, P=0.040). Lab4b attenuated significantly more NLRC4 mRNA expression compared to Lab4 (0.7-fold less vs. 0.1-fold less, respectively, P<0.001, from the control arbitrarily set to 1.0). Lab4 induced significantly more AIM2 mRNA expression compared to Lab4b (4.0-fold vs. 0.8-fold, respectively, P<0.001).

Enhanced IL-1β activation in challenged human macrophages

Bacterial challenge was simulated by the addition of LPS and ATP to macrophages as described in the methods. LPS and ATP were able to significantly induce IL-1β mRNA and protein (Supplementary Figure 2). With the co-incubation of Lab4 or Lab4b with challenge, IL-1β mRNA (2.9-fold, P=0.0030 and 3.3-fold, P<0.001, respectively) and IL-1β protein (15,756 pg/mL and 6,546 pg/mL vs.2,873 pg/mL LPS
and ATP challenge, P<0.001, Figure 3B) expression was enhanced further by both consortia. Differences between Lab4 and Lab4b were observed where Lab4 was able to induce more IL-1β protein than Lab4b (15,756pg/mL vs. 6,546pg/mL, P<0.001 Figure 3B).

Enhanced inflammasome activation in challenged human macrophages

In the presence of LPS and ATP challenge, NLRP3 and AIM2 mRNA expression was significantly induced, while NLRC4 mRNA expression was attenuated compared to the control (Supplementary Figure 2). Using these challenged conditions as a baseline, co-incubation of Lab4 or Lab4b was also able to further upregulate inflammasome mRNA expression. With challenge co-incubation, Lab4 was able to induce mRNA expression of NLRP3 (1.9-fold, P=0.0020), Caspase-1 (3.5-fold, P=0.0010), NLRP1 (3.4-fold, P=0.030), NLRC4 (1.7-fold, P=0.030) and AIM2 (2.0-fold, P=0.0050) when compared to challenge alone (Figure 3C). There were trends for Lab4b co-incubation with challenge to upregulate NLRP3, Caspase-1, NLRP1 and downregulate NLRC4 and AIM2 mRNA expression compared to challenge alone which were not significant (Figure 3C). The two consortia also demonstrated differential effects on inflammasome gene expression. Lab4 and Lab4b regulation of NLRC4 (1.7-fold vs. 0.5-fold, respectively, P<0.001) and AIM2 (2.0-fold vs. 0.6-fold, respectively, P<0.001) mRNA expression were significantly different.

Induction of an antiviral response in human macrophages

Viral infection triggers host innate immune responses indicated by cytokine production (such as IL-12) and inflammasome activation [33]. Both Lab4 and Lab4b induced the macrophage secretion of IL-12 protein. For Lab4, 34.2 pg/mL (P<0.001) and, for Lab4b 25.4 pg/mL (P=0.010) compared to 4.3 pg/mL detected in the control (Figure 4A). IL-12p35 mRNA expression was induced 2.6-fold (P=0.040) and 7.2-fold (P<0.001) in the presence of Lab4 and Lab4b, respectively (Lab4b induction significantly higher than Lab4 for IL-12p35, P<0.001, Figure 4B). Expression of IL-12p40 was not significantly different for either consortia, though there was a trend towards induction (Figure 4C).

Enhanced IL-12 activation in challenged human macrophages

Viral challenge was simulated by the viral mimic, poly I:C [34], and this challenge induced the mRNA expression of IL-12p35 and IL-12p40 compared to the control (Supplementary Figure 3). IL-12 protein expression was induced by stimulation with poly I:C (17.8 pg/mL for challenge vs. 5.21 pg/mL control, P<0.001, Figure 5A). Co-incubation of poly I:C with Lab4 enhanced IL-12 protein production compared to poly I:C challenge alone (39.1pg/mL vs. 15.9pg/mL, P<0.0020, Figure 5B). There was a trend for Lab4b to induce IL-12 protein which was not significant but did further highlight the different potential of these two consortia (Figure 5B).

IL-12p35 (4.8-fold, P<0.001, Figure 5C) and IL-12p40 (11.4-fold, P=0.030, Figure 5D) mRNA expression was induced in the presence of challenge and Lab4b. There was no significant induction of IL-12p35 or IL-12p40 by Lab4 with poly I:C challenge, however, Lab4b was able to induce more IL-12p35 mRNA compared to Lab4 (4.8-fold vs. 1.8-fold, P<0.001, Figure 5C).

Induction of phagocytosis by human macrophages

Macrophages were incubated with Lab4 or Lab4b prior to incubation with FITC-labelled E. coli bioparticles. Both Lab4 and Lab4b significantly induced phagocytosis of FITC-labelled bioparticles by the macrophages (141.2%, P=0.033, and 219.4%, P<0.001, respectively vs. 100% control, Figure 6).

Metabolites generated by the Lab4 and Lab4b consortia of probiotics, in vitro, are able to polarise cultured macrophages towards a classical M1 pro-inflammatory phenotype. Under conditions chosen to simulate bacterial and viral challenge in vitro, the Lab4 and Lab4b consortia enhanced the macrophage M1 pro-inflammatory responsiveness. mRNA expression data indicated that both Lab4 and Lab4b upregulated mRNA expression of IL-1β, IL-18 and CD80 with a greater response observed for Lab4b. mRNA expression of the M2 marker CD206 was dampened by both Lab4 and Lab4b (Figure 1). Lab4b has been found to reduce atopic eczema sensitization [22] and the ability of this consortium to skew towards the M1 phenotype could help explain this outcome.

Miettinen et al have previously shown that both live and heat-killed Lactobacillus rhamnosusGG are capable of activating NLRP3, Caspase-1, and IL-1β [6]. However, to our knowledge, the role of numerous alternatively non-NLRP3 activated inflammasomes by commensal bacteria (such as Lactobacillusand Bifidobacterium) has not yet been demonstrated. The induction of both IL-1β mRNA and protein by Lab4 and Lab4b suggested the involvement of the inflammasome. This IL-1β response was potentially regulated by the NLRP3 inflammasome, as indicated by induction of NLRP3 and Caspase-1 mRNA (Figure 2). However, in this study we show that the consortia can also differentially regulate NLRP1, NLRP3, NLRC4 and AIM2 mRNA expression (Figure 2). The Lab4 consortium reduced the incidence and duration of upper respiratory tract infection (URTI) in children attending preschool and the activation of different inflammasome pathways could provide a possible link [27]. The Lab4b strain, Lactobacillus paracaseiCUL08, has recently been shown to dampen IL-1β production via NF-κB inhibition in THP-1 macrophages [30]. In contrast, we show that Lab4b is able to induce an inflammatory response alone, and, is less pro-inflammatory when compared to Lab4. This could potentially be explained by Lab4b demonstrating a more specific response, regulating just NLRP3 and NLRC4, while still inducing IL-1β mRNA and protein when compared to Lab4. These data highlight the potential of these two consortia to regulate immune function in a species-specific manner and suggests the potential benefits of using different consortia.

To ascertain whether Lab4 and Lab4b were able to assist or enhance macrophage activation and immune response during bacterial challenge, LPS was used, in addition to ATP. It is well-documented that LPS provides the initial ‘priming’ signal required for inflammasome activation, and ATP represents the second ‘danger’ signal which is required for full IL-1β maturation and secretion [34-36]. Challenge of Lab4- or Lab4b-treated macrophages with these agents was able to induce a greater inflammasome response (Figure 3). Lab4 and Lab4b were significantly different in their potential to regulate the inflammasome. These data suggest that Lab4 may regulate NLRP3, NLRP1, NLRC4 and AIM2 inflammasomes during challenge, while Lab4b regulates just NLRC4 and AIM2, highlighting potential species-specific effects.

Viruses are also a major cause of URTI [37] and so the antiviral potential of Lab4 and Lab4b was investigated. IL-12 is a key mediator in viral response that is activated in response to toll-like receptor 3 (TLR3) stimulation with double-stranded viral RNA [38-40]. Both Lab4 and Lab4b were able to induce expression of IL-12 mRNA and protein (Figure 4). Both consortia were also able to enhance the IL-12 response in the presence of the TLR3 viral mimic agonist, poly I:C (Figure 5), which is commonly used to simulate viral challenge in macrophages [34], indicating the potential of Lab4 and Lab4b to prime macrophages towards antiviral responsiveness [40,41].

Phagocytosis is an essential clearance mechanism carried out by macrophages that ensures removal of bacteria, viruses and debris [42] and phagocytosis assays were performed to assess the functionality of Lab4 and Lab4b. Significant increases in phagocytosis by THP-1 macrophages were observed in response to exposure of Lab4 or Lab4b treatment of E. coli bioparticles indicating antipathogenic activity by these cells (Figure 6).

Figure 6: Lab4 and Lab4b promote phagocytosis in human macrophages. THP-1 macrophages were differentiated as described and incubated with control medium (black bars) or treated with Lab4 (grey bars) or Lab4b (hashed bars) for 4 hours. After this time, cells were washed briefly before the addition of Vybrant^® fluorescently-labelled bioparticles for 2 hours in 1 X HBSS. Data (mean ± SEM) are from five independent experiments and expressed as percentage change from the control arbitrarily set to 100%. Statistics: One-way ANOVA with Tukey's multiple comparisons. Significance: *=p ≤ 0.05, ***=p ≤ 0.001.

These studies indicate both antibacterial and antiviral responses of macrophages to both Lab4 and Lab4b- a characteristic recently observed in other commensal bacteria and supports the findings of reduced incidence of URTI and reduced atopic eczema sensitization [22,27].

To our knowledge, we show for the first time that different consortia of Lactobacillus and Bifidobacteria probiotic bacteria have differential effects on macrophage activation, phenotype, inflammasome and viral activation in cultured macrophages. These in vitro data suggest that the Lab4 and Lab4b consortia can (i) polarise macrophages towards an ‘M1’ phenotype, (ii) induce IL-1β secretion in human macrophages, (iii) enhance their response to bacterial challenge by differentially regulating a number of inflammasome components, (iv) induce IL-12 secretion by human macrophages and enhance their response to viral challenge, and (v) enhance the phagocytic activity of human macrophages. These in vitro findings provide novel insights into the potential immunomodulatory mechanisms underlying the actions of different strains of Lactobacillus and Bifidobacteria. This study provides a foundation and valuable basis for future investigation in primary cells, animal models and clinical trials.

TSD, DRM and SFP were responsible for the design of the study, Experiments were performed by TSD, DRM, AAJ and MDA. Data Analysis was performed by TSD and DRM. TSD prepared the manuscript and all authors contributed to the review of the manuscript.

We thankfully acknowledge the contributions of Professor Dipak P. Ramji at Cardiff University for reading the manuscript and for his helpful comments, together with Joe. W. E. Moss and Victoria O’Morain for their input.

This study was supported by Cultech Ltd., Port Talbot, UK. TSD, SFP, AAJ, MDA and DRM are employees of Cultech Ltd.