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 Table of Contents  
Year : 2020  |  Volume : 9  |  Issue : 3  |  Page : 261-267

Liposomes derived from Mycobacterium smegmatis promote immune activation of mice bone marrow-derived dendritic cells

1 Department of Immunology, School of Medical Sciences, Universiti Sains Malaysia, Malaysia
2 Production Assurance and Control Department, Instituto Finlay, Havana, Cuba
3 Institute of Pharmacy and Food (IFAL), University of Havana, La Habana, Cuba
4 Biomedical Science Programme, School of Health Sciences, Universiti Sains Malaysia, Malaysia

Date of Submission26-Apr-2020
Date of Decision20-May-2020
Date of Acceptance14-Jun-2020
Date of Web Publication28-Aug-2020

Correspondence Address:
Armando Acosta
Biomedical Science Programme, School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan
Ramlah Kadir
Department of Immunology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmy.ijmy_82_20

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Background: Tuberculosis (TB) is the leading cause of mortality due to infectious diseases. The development of new generation vaccines against TB is of paramount importance for the control of the disease. In previous studies, liposomes obtained from lipids of Mycobacterium smegmatis (LMs) demonstrated their immunogenicity and protective capacity against Mycobacterium tuberculosis in mice. To characterize the immunomodulatory capacity of this experimental vaccine candidate, in the current study, the stimulatory capacity of LMs was determined on bone marrow-derived dendritic cells (BMDCs) from mice. Methods: LMs were obtained and incubated with mature BMDCs. The internalization of LMs by BMDCs was studied by confocal microscopy, and the LMs immune-stimulatory capacity was determined by the expression of surface molecules (CD86 and MHCII) and the cytokine production (interleukin [IL]-12, interferon-Υ, tumor necrosis factor-α, and IL-10) 24 h after exposure to LMs. Results: The interaction of LMs with BMDCs and its internalization was demonstrated as well as the immune activation of BMDCs, characterized by the increased expression of CD86 and the production of IL-12. The LMs internalization and immune activation of BMDCs were blocked in the presence of cytochalasin, filipin III and chlorpromazine, which demonstrated that internalization of LMs by BMDCs is a key process for the LMs induced immune activation of BMDCs. Conclusions: The results obtained support the further evaluation of LMs as a mycobacterial vaccine, adjuvant, and in immunotherapy.

Keywords: Dendritic cells, liposomes, Mycobacterium smegmatis

How to cite this article:
Mat Luwi NE, Kadir R, Mohamud R, A Garcia-Santana Md, Acevedo R, Sarmiento ME, Norazmi MN, Acosta A. Liposomes derived from Mycobacterium smegmatis promote immune activation of mice bone marrow-derived dendritic cells. Int J Mycobacteriol 2020;9:261-7

How to cite this URL:
Mat Luwi NE, Kadir R, Mohamud R, A Garcia-Santana Md, Acevedo R, Sarmiento ME, Norazmi MN, Acosta A. Liposomes derived from Mycobacterium smegmatis promote immune activation of mice bone marrow-derived dendritic cells. Int J Mycobacteriol [serial online] 2020 [cited 2021 Aug 3];9:261-7. Available from: https://www.ijmyco.org/text.asp?2020/9/3/261/293551

  Introduction Top

Liposomes are microscopic spherical vesicles made of bilayered phospholipid membranes, which were described by Bangham et al. in 1965.[1] Generally, nano or micro-sized liposomes are in the range of 25–2500 nm. The lipid bilayers in the liposomes are usually made of natural or synthetic phospholipids.[2] Liposomes were first used as drug carriers by Gregoriadis et al. in 1974,[3] and their applications are well established in several areas such as oral delivery of vaccines, insulin, and peptides among others.[2] It has also found application in chemotherapy, antibiotic therapy for bacterial infections, and the treatment of parasitic and viral infections.[2] The interest of liposomes is being steadily increased due to their versatility. They can be loaded with vastly different types of compounds while their physicochemical properties are easily customized, for example, to enhance their inherent immunological adjuvanticity.[4] Furthermore, they are biocompatible and biodegradable, which makes them suitable candidates for use as carriers.[5] Among several new drug delivery systems, liposomes are considered as an advanced technology to deliver active molecules, and at present, several formulations are in clinical use.[6],[7],[8] Hence, it is interesting to evaluate the use of liposomes as potential drug/vaccine delivery against targeted diseases.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs), central to the immune response. DCs initiate the primary immune responsesin vivo and are key linkers between innate and adaptive immunity.[9]

Liposomes have potent immunostimulatory properties and are efficiently taken up by human DC.[4] Several studies have demonstrated that liposomes derived from attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) are powerful in activating DCs as well as inducing strong and sustained immune responses.[7] Liposomes derived from Archebacteria (archeosomes), Escherichia coli (eschriosomes) and Mycobacterium bovis BCG (mycosomes), used as vaccine adjuvants or delivery vehicles, induced both humoral and cellular immune responses with minimum toxic effects to the host.[10],[11],[12]

The importance of including lipid-mycobacterial pathogen-associated molecular patterns in liposomes to induce immune responses, was demonstrated by the induction of a Th1 immune response and protection against tuberculosis (TB) in guinea pigs immunized with liposomes containing Mycobacterium tuberculosis (Mtb) cell wall lipids.[13]

Previous studies showed that liposomes obtained from polar lipids of Mycobacterium smegmatis (Ms) demonstrated the capacity to act as a potent-adjuvant in experimental vaccines against leptospirosis in a hamster model and the induction of activation of APCs.[14]

Our group reported that liposomes from Ms (LMs) induced cross-reactive responses and protection in mice in challenge studies against Mtb.[15],[16] The immunopotentiating effect of the expression of Mtb antigens in recombinant Ms was also reported.[17]

Taking into consideration the antecedents of the potential of LMs as adjuvant and vaccine candidate against Mtb, the present study had, as its main objective, the study of the interaction of LMs with bone marrow-derived-DCs (BMDCs), in particular, the importance of internalization of the LMs for the immune activation of BMDCs.

  Methods Top


Production of LMs

Ms mc2 155 strain was grown in a medium containing 1% (w/v) yeast extract, 0.5% (v/v) glycerol, 0.4% (v/v) Tween 80, in 8% nutrient broth at 37°C and agitated at 200 rpm for 48 h. The purity of culture was determined by Ziehl-Neelsen staining.

Total lipids from Ms were obtained by the method of Bligh and Dyer,[18] and the liposomes were produced by the dehydration-rehydration method.[19] After the production, the morphology and size of LMs were determined by scanning electron microscopy (SEM).

Bone marrow-derived dendritic cells

Generation of bone marrow derived dendritic cells

BALB/c mice (6–8 weeks old) were obtained from the Animal Research and Service Centre, Universiti Sains Malaysia. All procedures were carried out according to the international, National, and Institutional regulations of laboratory animal experimentation and have been approved by the USM Animal Ethics Committee-USM/Animal Ethics Approval/2016/(104) (802).

BMDCs were generated as described by Matheu et al.[20] Briefly, bone marrow cells from femurs and tibia of 5 individual naïve BALB/C mice were collected and treated with red blood cell lysis buffer (Gibco, USA) for 3 min at RT to lyse erythrocytes. Cells were washed and cultured at 1 × 106 cells/ml with complete media [RPMI 1640 (Gibco, USA), 100 μg/ml of Penicillin-Streptomycin (Nacalai Tesque, USA) and 10 μg/ml of heat-inactivated Foetal bovine Serum (Capricon Scientific, Germany)] at 37°C, 6% CO2. GM-CSF (Gold Biotechnology) (20 ng/ml) and interleukin (IL)-4 (Gold Biotechnology) (30 ng/ml) were added to induce BMDCs generation for 7 days. After 7 days of incubation, LMs (50 μg/ml) or Lipopolysaccharides (100 ng/ml) (Sigma-Aldrich, USA) were added into the cultures for 24 h. Chlorpromazine (Sigma-Aldrich, USA) (10 μg/ml), filipin III (Sigma-Aldrich, USA) (10 μg/ml), and cytochalasin (Sigma-Aldrich, USA) (10 μg/ml) were added to BMDCs and incubated with LMs for 24 h. After incubation, the cells were used for the morphological study by SEM to determine the uptake of LMs by BMDCs by confocal microscopy and to study the expression of surface activation markers by Flow cytometry FACSCanto II from BD Biosciences (New Jersey, USA). The culture supernatant was used for cytokines determination.

Scanning electron microscopy

For SEM, the cells were processed by primary fixation with McDowel Trump fixative (Electron Microscopy Sciences, USA) at 4°C for 24 h and underwent secondary fixation with cold 1% osmium tetroxide (Sigma-Aldrich, USA) at RT for 2 h. The cell was washed with ddH2O twice for 10 min and dehydrated sequentially with graded acetone (35%, 50%, 75%, 95%, and 100%). After that, the cells were soaked in Hexamethyldisilazane (Electron Microscopy Sciences, USA) for 15 min and dried overnight and mounted onto the sample stub and viewed under SEM Quanta FEG 450 (FEI, USA).

Confocal microscopy

Briefly, LMs were labeled by conjugation with FITC (green, 488 nm) (Biolegend, USA) as followed: FITC were loaded into LMs and shacked up at RT for 1 h. The mixture was washed away with a washing buffer and stored at 4°C. Then, BMDCs were stained with Phalloidin–Rhodamine (red, 543 nm) (Invitrogen, USA), and after mixing, BMDCs and LMs were incubated in the dark at 4°C overnight. After incubation, the cells were washed with phosphate-buffered saline (PBS) to remove unbound antibody. Cells were directly imaged on coverslips by a Nikon A1r Confocal microscope, operated by the Cells were directly imaged on coverslips by a Nikon A1r Confocal microscope, operated by the NIS Elements Viewer software (Nikon, Confocal microscope, Leica microsystems, Teban Gardens, Crescent, Singapore). Data were analyzed with FIJI software” to “Cells were directly imaged on coverslips by confocal laser scanning microscopy (Leica TCS-SP2, Leica Microsystems GmbH, Wetzlar, Germany).

Study of the immune activation

Flow cytometry

To verify the maturation and activation status of BMDCs upon exposure to LMs and the three inhibitors, flow cytometry were carried out. Briefly, BMDCs were stained with a combination of antibodies (AF-700 anti-mouse MHCII [Biolegend, USA], PE-anti mouse CD86 [Biolegend, USA] and PerCP anti-mouse CD11c [Biolegend, USA]) on ice for 20 min. BMDCs were washed with staining buffer (3% fetal bovine serum in 1X PBS) to block any nonspecific antibody binding, resuspended in PBS in the dark on ice for 30 min before acquisition by Flow Cytometer FACS CANTO II. BMDCs were acquired with FACS Diva™ software from BD Biosciences (New Jersey, USA) and analyzed by FlowJo® software from BD Biosciences (New Jersey, USA).

Study of the production of cytokines

Cytokines IL-12 (IL-12p70), interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-10 were determined from culture supernatants of BMDCs incubated with LMs and the three inhibitors by Multiplex assay (PrimePlex Mouse 4-plex Proteomics Assay Kit, Merck, Germany) according to the manufacturer's specifications. The analysis was performed by Luminex 200 (Luminex Corporation, USA).

Statistical analysis

Statistical analyses were performed using parametric methods with GraphPad Software Inc., La Jolla CA, USA (v 5.0). The variance between multiple groups was calculated with one-way analysis of variance, and the differences among groups were determined by Tukey's posttest. A P < 0.05 was considered statistically significant.

  Results and Discussion Top

Production of LMs

The SEM study showed the presence of spherical structures ranging between 20-80 nm in size, which were compatible with liposomes obtained in previous studies [Figure 1].[21]
Figure 1: Structural study of LMs. Scanning electron microscopy. Presence of spherical structures with size between 20 and 80 nm (×50,000)

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Liposomes can be classified based on the size and the number of bilayers.[2] Based on the lamellarity, they can be classified as unilamellar vesicles and multilamellar vesicles (MLVs).[2] According to their size, the unilamellar vesicles are classified as small unilamellar vesicles (SUVs) (0.02–0.05 μm), large unilamellar vesicles (> 0.06 μm) and giant multilamellar vesicles (10–100 μm)[2]. The results of the characterization by SEM suggest according to the size that it could be SUV, but further analysis to fully characterize the lamellarity, need to be made to complete the characterization.

It has been reported that liposomes obtained from total polar lipids from M. smegmatis (Ms), encapsulating ovalbumin, were classified as MLVs with a size of 225 ± 121 nm, and a similar size was obtained after the encapsulation of three different leptospiral antigens.[14]

Structural of bone marrow derived dendritic cells

The morphological characteristics of BMDCs were studied by SEM. After incubation, bone marrow cells without the addition of growth factors showed a round shape with the absence of dendrites [Figure 2]a. However, bone marrow cells cultured with growth factors acquired typical characteristics of BMDCs showing long and branched dendritic protrusions with irregular shape [Figure 2]b, as have been reported with the use of similar conditions in mice.[22] In the presence of LPS, BMDCs exhibited increased numbers of dendritic protrusions [Figure 2]c, compatible with activation and maturation as have been described by Zeng et al.[23] After incubation with LMs, it was found that LMs were attached to the surface of BMDCs and also promoted the maturation of BMDCs since peripheral cellular protrusions in BMDCs were visualized in this stage [Figure 2]d.
Figure 2: Morphological study of bone marrow-derived dendritic cells incubated with LMs. Scanning electron microscopy. (a) Bone marrow cells showed the typical round shape (×40,000). (b) Bone marrow-derived dendritic cells Presence of dendritic protrusions (×5000). (c) Bone marrow-derived dendritic cells incubated with LPS showing dendritic protrusions (×10,000). (d) Bone marrow-derived dendritic cells incubated with LMs showing abundant dendritic protrusions and round structures compatible with LMs located on the surface of bone marrow-derived dendritic cells (×10,000)

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In order to explore the participation of different endocytic pathways in the internalization of LMs on BMDCs immune activation, we used known inhibitors of the different pathways, such as chlorpromazine clathrin-mediated endocytosis (CME), filipin III clahtrin independent endocytosis (CIE) and cytochalasin (general endocytosis inhibitor).[24]

In this regard, we studied the influence of the use of said inhibitors on the structural characteristics of BMDCs by SEM. The use of the different inhibitors changed markedly the structure of BMDCs with a more marked effect in the use of chlorpromazine and filipin III [Figure 3]a, [Figure 3]b, [Figure 3]c. Regarding APC, It has been reported structural alterations in human macrophages in the presence of cytochalasin, characterized by retraction of cytoplasm, changes in cell thickness, and wrinkled appearance.[25]
Figure 3: Morphological study of bone marrow-derived dendritic cells incubated with three different inhibitors. Scanning electron microscopy. (a) chlorpromazine (10000x magnification), (b) Filipin III (20000x magnification) and (c) Cytochalasin (10000x magnification)

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Study of LMs uptake by bone marrow derived dendritic cells

The incubation of bone marrow cells with LMs showed the localization of LMs only at the surface of the cells, which suggests that bone marrow cells are unable to internalize the LMs [Figure 4].
Figure 4: Study of LMs uptake by bone marrow. Confocal microscopy. (a) LMs stained with FITC. (b) Bone marrow stained with Phalloidin- Rhodamine. (c) LMs (green) are located on the surface of bone marrow cells (red) without evidence of internalization, producing a yellow overlap region or peripheral green stain in the merge image. Scale bars: 40 μm

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In contrast, after incubation of BMDCs with LMs, the confocal microscopy study showed the localization of LMs inside BMDCs, which demonstrates the uptake and internalization of the LMs [Figure 5]. The size of LMs is a key factor in the interaction with the immune cells; several studies demonstrated that small particle liposomes are endocytosed by DCs, whereas particles of size higher than 500 nm are more readily cleared by the mononuclear phagocytic system.[26] Similar results have been reported with different liposomes after interaction with BMDCs from mice and humans.[27],[28]
Figure 5: Study of LMs uptake by bone marrow-derived dendritic cells. Confocal microscopy. (a) LMs stained with FITC. (b) Bone marrow derived dendritic cells stained with Phalloidin-Rhodamine. (c) LMs (green) are located inside bone marrow-derived dendritic cells (red), demonstrating the internalization of LMs by bone marrow-derived dendritic cells. Scale bars: 40 μm

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There was no evident interaction of LMs with the surface of BMDCs or internalization after incubation with the inhibitors (chlorpromazine, filipin III, and cytochalasin) (data not shown), suggest the main endocytic pathways involved in the internalization of LMs by BMDCs.

Some studies advocated the participation of specific endocytic pathways in the cellular internalization of liposomes,[29] whereas others reported the participation of all the main pathways in the internalization process,[30] showing a blockade of the internalization using the same inhibitors that were used in this study.

Study of the immune activation

Study of surface markers

There was a significant increase of the expression of CD86 in LMs stimulated BMDCs compared to nonstimulated BMDCs (P < 0.01). Significant inhibition of the expression of CD86 in LMs stimulated BMDCs was demonstrated with the use of cytochalasin (P < 0.001), filipin III (P < 0.01), and chlorpromazine (P < 0.001). BMDCs stimulated with LPS (positive control) showed a significant increase of the expression of CD86 compared with all the culture conditions (P < 0.01) (P < 0.001) [Figure 6]. In our previous studies, partial characterization of total lipids of Ms demonstrated the presence of mycolic acids and phospholipids, which may have an impact on BMDCs stimulation.[31] Particularly, the mycolic acid derivatives, like trehalose dimycolate stimulates innate, early adaptive and both humoral and cellular adaptive immunity.[32]
Figure 6: Study of bone marrow-derived dendritic cell immune activation. Surface markers. CD86 expression in different experimental conditions. Results are presented as mean ± standard error of the mean. Different letters mean significant differences (Pairs with significant differences (P < 0.01) are: bone marrow-derived dendritic cell + LMs >with bone marrow-derived dendritic cells and with bone marrow-derived dendritic cells + LMs + filipin III, The rest of the significant different pairs had P< 0.001. MHC II did not show an increase of the expression (data not shown)

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Regarding the other activation surface marker studies of MHCII, there was no statistical increase of its expression after the stimulation with LMs compared with nonstimulated BMDCs (data not shown).

Mice BMDCs stimulated with liposomes obtained from total polar lipids of M. smegmatis increased the expression of CD86 and CD80 but not MHCII.[14] Liposomes containing phosphatidylinositol mannosides from BCG increased the expression of CD80 and CD86 in a lesser extent.[10]

Different surface markers increase their expression during DCs activation and maturation, such as CD86, CD80, and MHCII.[33],[34] CD86 is considered a marker of DCs maturation, so its induced expression in BMDCs by LMs suggests the capacity of LMs to induce immune activation and maturation upon interaction with BMDCs.[9],[34]

Study of the production of cytokines

Immune cells produce a great variety of cytokines, which act as signaling molecules that play major roles in the immune response. In this study, the production of different cytokines upon the stimulation of BMDCs with LMs was evaluated.

The stimulation of BMDCs with LMs induce a significant increase in the production of IL-12 compared with nonstimulated BMDCs (P < 0.001) and other experimental conditions except LPS stimulated BMDCs (P < 0.001). LPS induced a significant increase of the production of IL-12 compared with the other culture conditions. There was a significant inhibition of the production of IL-12 in LMs stimulated BMDCs in the presence of inhibitors (P < 0.001) compared to LMs stimulated BMDCs [Figure 7]. The stimulation of BMDCs with LMs do not induce changes in the production of IFN-γ, TNF-α, and IL-10 (data not shown).
Figure 7: Study of bone marrow-derived dendritic cell immune activation. Cytokine production. Interleukin-12p70 production in different culture conditions. Results are presented as mean ± standard error of the mean. Different letters have significant differences (P < 0.001)

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Activated APCs, especially DCs, are the most important producers of IL-12, which is a heterodimeric cytokine made up of two chains (p35 and p70), which has important roles in innate and adaptive immune response.[35] The most important biological effect of IL-12 is the induction of a polarized Th1 immune response and different immunomodulatory effects with great impact against intracellular pathogens and tumors.[35]

During the process of activation and maturation of DCs, and depending on the microenvironment and type of stimulation DCs produce an array of different cytokines such as IL-1 β, TNF-α, IL-6, and IL-12 among others, which are of great of importance for the polarization of the T cell immune response.[36] In our experiments, the statistical increases in the production of IL-12 in BMDCs stimulated with LMs, compared with nonstimulated BMDCs together with the structural changes induced upon interaction with LMs and the increase of the expression of CD86 argue in favor of the immune activation induced by LMs as have been reported in other studies with liposomes containing mycobacterial lipids.

The immune stimulatory properties of polar lipids vary according to chain length and other factors. Dao et al. demonstrated that different mutants of mycolic acids were able to stimulate DC to induce IL-12 and TNFα whereas other only induce IL-12.[37]

The use of liposomes as adjuvants, particularly containing mycobacterial components, such as trehalose 6,6×-dibehenate, which is a synthetic analogue of mycobacterial cord factor trehalose di-mycolate, has been reported, with good results in terms of induction of TH1 immune response and protection.[34] The use of this kind of liposomes, in vaccine formulations for TB, induced potent Th1 immune responses when combined with different protein antigens and attenuated strains.[38]

Lipid components from BCG combined with DDA liposomes induced Th1 immune responses against different antigens.[39] Liposomes produced from total polar lipids of BCG induced strong immune responses in mice and the production of IL-6 and IL-12 in mice BMDCs.[10]

Liposomes obtained from Ms total lipids or by mixing commercial and Ms lipids were immunogenic in mice, inducing cross-reactive responses against Mtb.[15],[16] The formulations obtained from total lipids of Ms were protective either alone or adjuvanted with alum in an intratracheal challenge experiment with Mtb in mice, producing a decrease of the bacterial burden and the pneumonic area in lungs which were comparable to BCG vaccination.[40] Ms contains a great variety of lipid components, which could regulate in different ways the immune response by unknown mechanisms.

The interaction of liposomes obtained from total lipids of Ms with mice BMDCs induced a significant increase of IL-6 and IL-12 but not TNF-a.[14] Previous studies also reported that the production of cytokines from mature DCs was reduced in the presence of Phosphodiesterase 4 inhibitor, which resulted in the inhibition of naïve T cells, causing a reduction in cytokine secretion (IL-12p70, TNF-α, IFN-ϒ).[41]

The inhibition of the activation of BMDCs in the presence of the inhibitors employed in this study could be related to the blockage of internalization of LMs by BMDCs, which was demonstrated by confocal microscopy. It has been reported that cytochalasin blocks both actin-dependent phagocytosis and micro and macropinocytosis,[24],[42] in this way affecting most of the endocytic pathways.[24] Filipin III is a sterol-binding agent that attach to cholesterol, which disrupts lipid raft formation, including caveolin-containing rafts.[43] At higher concentration, it inhibits CIE and CME.[42] Chlorpromazine is an amphiphatic drug that causes the assembly of adaptor proteins and clathrin on endosomal membranes, which blocks CME and extracellular vesicles internalization.[42],[43]

Summarizing, the use of the different endocytosis inhibitors blocked the uptake of LMs by BMDCs, which suggest the participation of the different pathways of endocytosis in the internalization process, which is linked to the inhibition of the activation of BMDCs through different mechanisms. Thus, it could interfere with the uptake of LMs by BMDCs with the consequent lack of activation of BMDCs.

  Conclusions Top

After the interaction of BMDCs with LMs, their internalization by BMDCs was documented. BMDCs' exposure to LMs induced the immune activation, demonstrated by the increase of the expression of CD86 and the production of IL-12. The internalization of LMs by BMDCs is suggested to be a key element for the immune activation, which was blocked by inhibitors of the main endocytic pathways.


The authors acknowledge the financial supports under Short-term Research Grant Scheme (304/PPSP/6315005) and Long-term Research Grant Scheme (203/PPSK/67212002), Department of Higher education, Ministry of Education, Malaysia. Fundamental Research Grant Scheme (203.PPSP.6171217).

Financial support and sponsorship

Short-term Research Grant Scheme (304/PPSP/6315005) and Long-term Research Grant Scheme (203/PPSK/67212002), Department of Higher Education, Ministry of Education, Malaysia. Fundamental Research Grant Scheme (203.PPSP.6171217).

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]


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