Activation of Host Immune Cells by Probiotic-Derived Extracellular Vesicles via TLR2-Mediated Signaling Pathways

Abstract

Since probiotic-derived extracellular vesicles (EVs) are capable of activating innate immunity, they are expected to be useful as novel adjuvants. To elucidate the mechanisms underlying the immunostimulatory effects of EVs released from probiotic cells, we newly investigated the role of Toll-like receptor 2 (TLR2) and immune cell downstream signaling in the generation of proinflammatory cytokines. Isolated Bifidobacterium- and Lactobacillus-derived EVs expressed peptidoglycan, one of the major pathogen-associated molecular patterns. EVs particle diameter were approximately 110–120 nm with a negative-zeta potential. The generation of proinflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in TLR2-expressing mouse macrophage-like RAW264.7 cells and mouse dendritic DC2.4 cells treated with Bifidobacterium- and Lactobacillus-derived EVs decreased after the addition of T2.5, a TLR2 inhibitory antibody. Furthermore, we showed that the signaling pathways of c-Jun-NH2-terminal kinase (JNK)/mitogen-activated protein kinases (MAPK) and nuclear factor-kappaB (NF-κB) were also involved in the production of proinflammatory cytokines from EV-treated immune cells. These results provide valuable information for understanding of the host biological function induced by probiotic-derived EVs, which is helpful for developing an EV-based immunotherapeutic system.

INTRODUCTION

Extracellular vesicles (EVs) are membrane-covered particles of diverse sizes and shapes released from cells.¹⁾ They serve critical functions in cell-to-cell communication by transporting their cargoes, which include proteins, lipids, and nucleic acids.²⁾ Bacterial cells have also been shown to generate EVs that function as bacteria-to-bacteria and bacteria-to-host contact systems.^3,4)

In addition to extensive research on investigating the host biological response after incorporating pathogenic bacteria-derived EVs, development of new therapeutic applications using EVs from beneficial bacteria have been reported.^5,6) We have recently characterized EVs released by several types of probiotics which provide a health benefit.⁷⁾ Our findings showed that EVs from probiotics enhanced innate immunity by promoting the secretion of proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from host immune cells. This finding proposed the usability of EVs secreted from probiotic cells as attractive adjuvants formulation to develop immunotherapeutic systems. To establish an EV-based therapeutic system, it is necessary to understand the molecular mechanisms contributing to the EV-mediated biological function. However, the mechanisms by which EVs from probiotics enhance the proinflammatory cytokines generation by immune cells are not fully elucidated.

Immune cells are shown to recognize preserved bacterial structures known as pathogen-associated molecular patterns (PAMPs) using their pattern recognition receptors (PRRs), which activate intracellular signaling cascades, such as mitogen-activated protein kinases (MAPK) and nuclear factor-kappaB (NF-κB) for producing proinflammatory cytokines.^8–10) PAMPs such as peptidoglycan (PGN), lipoteichoic acid, and lipoproteinis are found in bacterial cell walls and have been shown to promote the inflammatory cytokines generation when they are recognized by PRRs, such as Toll-like receptor 2 (TLR2).¹¹⁾ Although our previous study also revealed the expression of PGN as a component in probiotic-derived EVs, it is unclear whether the probiotic-derived EVs stimulate proinflammatory cytokine production via TLR2-dependent signaling.

The aim of this research was to reveal how TLR2 and its downstream signaling are involved in host immune cells activation by EVs from probiotics. We selected Bifidobacterium longum and Lactobacillus plantarum WCFS1 as model probiotic cells and collected EVs. The production levels of IL-6 and TNF-α released from mouse immune cells in the presence of TLR2 antagonistic antibody (T2.5) and signaling pathway inhibitor after the addition of probiotic-derived EVs were measured.

MATERIALS AND METHODS

Isolation and Characterization of Probiotic-Derived EVs

Isolation of EVs released from Bifidobacterium longum and Lactobacillus plantarum WCFS1, PGN measurement, atomic force microscope (AFM) observation, and physicochemical properties analysis of EVs were performed according to our paper as reported previously.⁷⁾

Detection of TLR2 on Immune Cells

We cultured RAW264.7 (macrophage-like cells) and DC2.4 (dendritic cells) according to the method of our previous report.⁷⁾ After inoculation at a density of 9 × 10⁴ cells per well into 48-well plates, the cells were collected and stained for 30 min at 4 °C in the dark with PE anti-mouse CD282 (TLR2) antibody (BioLegend, San Diego, CA, U.S.A.) or isotype-matched control antibody. A FACS Calibur (BD Bioscience, Irvine, CA, U.S.A.) was used to detect antibody-labeled cells after the reaction.

Evaluation of Cytokine Secreted by Immune Cells

Immune cells were seeded into 48-well plates and incubated for 24 h at 37 °C. Thereafter, collected EVs (0.5 µg/well) were added to well in the presence of 1 µg/well TLR2 blocking monoclonal antibody (T2.5) (Invivogen, San Diego, CA, U.S.A.). After 6 h of incubation, release of TNF-α and IL-6 were quantified by enzyme-linked immunosorbent assay (ELISA). For the investigation of the roles of signaling pathways in the activation of immune cells treated with EVs, cells were pretreated for 2 h at 37 °C with 25 M of the c-Jun-NH2-terminal kinase (JNK)/MAPK inhibitor SP600125 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) or the NF-B inhibitor BAY11-7082 (Tokyo Chemical Industry Corp., Tokyo, Japan). After removing supernatants, EVs were treated and further incubated for 6 h. Thereafter, proinflammatory cytokines were measured by ELISA.

Analyses of EVs Cellular Uptake Efficiency by Immune Cells

The cells were incubated for 6 h with fluorescent dye PKH67-labeled EVs (0.5 µg/well) under T2.5 (1 µg/well). The intensity of the cell’s fluorescence was measured using a FACS Calibur flow cytometer, after which the mean fluorescence intensity (MFI) was calculated to estimate the cellular uptake efficiency.

Statistical Analyses

The Student’s t-test was used to determine statistical significance. p-Values less than 0.05 were regarded statistically significant.

RESULTS

Characterization of EVs from Probiotics and Detection of TLR2 on Mouse Immune Cells

Firstly, EVs released from Bifidobacterium longum (B-EVs) and Lactobacillus plantarum WCFS1 (L-EVs) were isolated and PGN levels were quantified. We confirmed that amounts of PGN (ng) per EV protein (µg) were approximately 0.46 ng/µg (B-EVs) and 0.15 ng/µg (L-EVs), respectively (Table 1). Evaluation of physicochemical properties of EVs indicated that their particle diameter were approximately 110 to 120 nm and EVs had a negative-zeta potential (Figs. 1A, B, Table 1, Supplementary Figs. 1A–D). Furthermore, we confirmed the expression of TLR2 on RAW264.7 and DC2.4 cells (Figs. 1C, D) selected as immune cells.

Table 1. Physicochemical Characteristics of Probiotic-Derived EVs

	PGN amount per EVs protein amount (ng/µg)	Diameter (nm)	PDI	Zeta potential (mV)
B-EVs	0.46 ± 0.28	126 ± 3.78	0.26 ± 0.02	−10.8 ± 1.53
L-EVs	0.15 ± 0.05	108 ± 0.80	0.27 ± 0.004	−27.2 ± 2.03

Fig. 1. Characterization of Probiotic-Derived EVs and Immune Cells

Atomic force microscopic observation of (A) B-EVs and (B) L-EVs. Scale bar = 100 nm. Flow cytometry was used to examine TLR2 expression in (C) RAW264.7 cells and (D) DC2.4 cells using PE anti-mouse CD282 (TLR2) antibody (black line). The gray line indicates cells that have been tagged with an isotype-matched control antibody.

Contribution of TLR2 on Immune Cells to Probiotic-Derived EV-Mediated Cytokine Production

Since the probiotic-derived EVs contained PGN, we next quantified the EV-mediated cytokine production from immune cells after the addition of TLR2 antagonistic antibody T2.5. Figure 2 shows the proinflammatory cytokine production from RAW264.7 and DC2.4 cells after incubation of EVs released by probiotics. TNF-α and IL-6 secretion by B-EV-treated RAW264.7 cells were significantly diminished by T2.5 (Figs. 2A, B). Similar to the result obtained from RAW264.7 cells, proinflammatory cytokine production from B-EV-treated DC2.4 cells were significantly decreased when the cells were incubated with T2.5 (Figs. 2C, 2D). Moreover, production of these cytokines by L-EV-treated RAW264.7 cells also declined in the presence of T2.5 (Figs. 2E, F). As for the L-EV-mediated cytokine secretion by DC2.4 cells, amounts of TNF-α were diminished by T2.5 (Fig. 2G). We also confirmed that addition of T2.5 reduced IL-6 production in L-EV-treated DC2.4 cells (Fig. 2H).

Fig. 2. Cytokines Generated from Immune Cells by Adding EVs under TLR2 Antagonistic Antibody (T2.5)

In the presence of T2.5 (1 µg/well), RAW264.7 cells were incubated with collected EVs from Bifidobacterium longum and Lactobacillus plantarum WCFS1 (0.5 µg/well). As a positive control, PGN (1 µg/well) was prepared. DC2.4 cells were also incubated with collected EVs from Bifidobacterium longum and Lactobacillus plantarum WCFS1 (0.5 µg/well). As a positive control, PGN (10 µg/well) was prepared. ELISA was used to assess the amounts of cytokines in the medium after 6 h of incubation. RAW264.7 producing tumor necrosis factor-α (TNF-α) (A) and interleukin-6 (IL-6) (B) after the incubation with B-EVs. DC2.4 producing TNF-α (C) and IL-6 (D) after the incubation with B-EVs. RAW264.7 producing TNF-α (E) and IL-6 (F) after the incubation with L-EVs. DC2.4 producing TNF-α (G) and IL-6 (H) after the incubation with L-EVs. Each value represents the mean + standard deviations (n = 4). ** p < 0.01, compared with the PGN. ^†† p < 0.01, compared with the B-EVs or L-EVs.

Effect of T2.5 Treatment on EVs Uptake by Immune Cells

To investigate whether addition of T2.5 influence the EVs cellular uptake efficiency, we then carried out FACS analysis using macrophage and dendritic cells after treatment of fluorescently-labeled probiotic-derived EVs. We confirmed that MFI of RAW264.7 cells incorporating EVs were hardly affected by treatment with T2.5 (Figs. 3A, B, Supplementary Figs. 2A, B). Furthermore, EVs uptake efficiency of DC2.4 cells was comparable even after the T2.5 treatment (Figs. 3C, D, Supplementary Figs. 2C, D).

Fig. 3. Effect of the T2.5 Treatment on Cellular Uptake of EVs from Probiotics

Flow cytometric measurement using RAW264.7 cells after 6 h of incubation with (A) fluorescently-labeled B-EVs and (B) fluorescently-labeled L-EVs under T2.5. Flow cytometric measurement of DC2.4 cells after 6 h of incubation with (C) fluorescently-labeled B-EVs and (D) fluorescently-labeled L-EVs under T2.5. Results are expressed as means + standard deviations (n = 4). n.s. stands for not significant.

Investigation of EV-Mediated Immune Cell Activation by JNK/MAPK and NF-κB Signaling Pathways

To further clarify the mechanisms of immune cell activation by probiotic-derived EVs, proinflammatory cytokine production from EV-treated RAW264.7 and DC2.4 cells after pretreatment with JNK/MAPK inhibitors (SP600125) and NF-κB inhibitors (BAY11-7082) was measured. As shown in Figs. 4A and 4B, pretreatment of JNK/MAPK inhibitor significantly suppressed the amounts of cytokines generated from RAW264.7 cells after incubation with B-EVs and L-EVs. These cytokines released from EV-treated DC2.4 cells also declined by the inhibitor of JNK/MAPK (Figs. 4C, D). Moreover, we also evaluated the contribution of NF-κB on EV-mediated cytokine release from immune cells. After adding EVs derived from Bifidobacterium longum and Lactobacillus plantarum WCFS1 to RAW264.7 cells, pretreatment with NF-κB inhibitors (BAY11-7082) significantly reduced TNF-α and IL-6 production. (Figs. 5A, B). Regarding DC2.4 cells, these cytokines production after EVs treatment were also reduced by the BAY11-7082 (Figs. 5C, D).

Fig. 4. Proinflammatory Cytokine Release from EV-Treated Immune Cells by a JNK/MAPK Signaling Pathway

SP600125, a JNK/MAPK inhibitor, was applied to RAW264.7 or DC2.4 cells for 2 h. After removing the supernatants, EVs (0.5 µg/well) were added and incubated for 6 h. ELISA was used to measure the amounts of cytokines in the supernatant. (A) Tumor necrosis factor-α (TNF-α) and (B) interleukin-6 (IL-6) generated from RAW264.7 by addition of B-EVs and L-EVs. (C) TNF-α and (D) interleukin-6 (IL-6) generated from DC2.4 by addition of B-EVs and L-EVs. Results are expressed as means + standard deviations (n = 4). * p < 0.05, ** p < 0.01, when compared to the control group.

Fig. 5. Proinflammatory Cytokine Release from EV-Treated Immune Cells by a NF-κB Signaling Pathway

NF-κB inhibitor BAY11-7082 was applied to RAW264.7 or DC2.4 cells for 2 h. After removing the supernatants, EVs (0.5 µg/well) were added and incubated for 6 h. ELISA was used to measure the amounts of cytokines in the supernatant. (A) TNF-α and (B) interleukin-6 (IL-6) generated from RAW264.7 by addition of B-EVs and L-EVs. (C) TNF-α and (D) interleukin-6 (IL-6) generated from DC2.4 by addition of B-EVs and L-EVs. Results are expressed as means + standard deviations (n = 4). * p < 0.05, ** p < 0.01, when compared to the control group.

DISCUSSION

In this study, we found that TLR2 on immune cells is an important molecule in probiotic-derived EV-mediated host’s immune activation. Furthermore, JNK/MAPK as well as NF-κB signaling pathways play important roles in EV-treated immune cell cytokine production. To the best of our knowledge, this is the first study to show that TLR2 and its downstream factors are involved in host immune cells activation by probiotic-derived EVs.

Given that PGN, lipoteichoic acid and lipoproteinis are major ligand for TLR2, it has been suggested that probiotic-derived EVs stimulated secretion of inflammatory cytokine from immune cells through the recognition of these components by TLR2 on immune cells. TLR2 is expressed by dermal dendritic cells (DDC), and antigen-adjuvant delivery to DDC results in a potent antigen-specific immune response.¹²⁾ Since the present study demonstrated that the adjuvanticity of probiotic-derived EVs occurred via TLR2, transdermal delivery of probiotic-derived EVs modified with a targeting ability to DDC will be expected as an EV-based immunotherapeutic system. Meanwhile, distinct subpopulations of EVs released from the same cell type have been identified based on their shape, size, density, and cargo, which exert diverse biological functions.^13,14) In this study, AFM observation revealed that B-EVs and L-EVs also showed different subpopulations with respect to morphology and size. Furthermore, in accordance with the preceding study reporting heterogeneity of EVs, some B-EVs exhibited pleomorphic and incomplete vesicle structures.¹⁵⁾ Since the extent of TLR2-mediated immune cell activation would differ among subpopulations of probiotic-derived EVs, application of helpful methods which enable purification of morphologically and functionally homogenous EVs will be desired.^16,17) Moreover, PGN amount per EVs and the uptake amount of EVs were correlated with the result obtained from cytokine responses (Table 1, Figs. 2, 3). Since an interesting finding about a cell surface receptor for PGN endocytosis has been indicated, EV-expressing PGN is an essential factor for the characterization of probiotic-derived EVs.¹⁸⁾ Therefore, defining probiotic-derived EVs based on their subpopulation will provide a better understanding of the development of a new EV-based immunotherapy.

Despite the fact that cytokine production by B-EV and L-EV treatment was reduced by TLR2 inhibition, a certain amount of cytokines were detected even after treatment with T2.5. We added 1 µg/well of TLR2 blocking antibody (T2.5) corresponds to approximately 3 µg/mL, which is a higher concentration than in other previous study evaluating TLR2-mediated immune activation.¹⁹⁾ As another subset of PRRs, nucleotide-binding oligomerization domain-2 (NOD2) has been known to induce innate immune response by recognizing PGN.²⁰⁾ Since the RAW264.7 cells used in this study have been reported to express NOD2, interaction of PGN contained in EVs with NOD2 would partially contribute to the EV-mediated host immune activation.^21,22) Additionally, bacterial nucleic acids are sensed by endosomal PRRs including TLR3, TLR7, and TLR9, resulting in the generation of inflammatory cytokines by immune cells.²³⁾ It has been demonstrated that EVs reflect the cargoes of their cell origin and EVs from bacterial cells contain bacteria-derived nucleic acids.²⁴⁾ Therefore, an investigation to elucidate the role of nucleic acids incorporated into probiotic-derived EVs in cytokines response will be helpful for further understanding of the mechanisms by which the cargoes of probiotic-derived EVs are recognized by immune cell sensors and result in host immune activation.

Regarding the possible factors contributing to probiotic-derived EV-mediated immune activation, internalization of EVs by target cells should be considered since the uptake efficiency of EVs by target cells highly impacts the subsequent biological response.²⁵⁾ In this study, we treated immune cells with TLR2 antagonistic antibody T2.5, that have been widely used to evaluate the TLR2 dependence.^26,27) As the T2.5 used in this study has been reported to inhibit binding of ligand to TLR2 expressed on the immune cell surface, it is possible that uptake efficiency of EVs released from probiotics by immune cells might be affected by the treatment of T2.5, which may also contribute to the decrease in cytokine production.²⁸⁾ However, uptake of EVs was not influenced under T2.5 treatment (Fig. 3), indicating that reduction of EV-mediated cytokine production by T2.5 treatment is not due to a change in uptake efficiency of EVs by immune cells, but rather to the inhibition of TLR2 signaling.

Finally, our findings offer a promising perspective for the understanding of the host biological function induced by probiotic-derived EVs, which will aid in the establishment of an EV-based therapeutic system.

Acknowledgments

This work was supported by JSPS KAKENHI (Grant No. 20K20205).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Maas SLN, Breakefield XO, Weaver AM. Extracellular vesicles: unique intercellular delivery vehicles. Trends Cell Biol., 27, 172–188 (2017).
• 2) Nederveen JP, Warnier G, Di Carlo A, Nilsson MI, Tarnopolsky MA. Extracellular vesicles and exosomes: insights from exercise science. Front. Physiol., 11, 604274 (2021).
• 3) Macia L, Nanan R, Hosseini-Beheshti E, Grau GE. Host-and microbiota-derived extracellular vesicles, immune function, and disease development. Int. J. Mol. Sci., 21, 107 (2020).
• 4) Bose S, Aggarwal S, Singh DV, Acharya N. Extracellular vesicles: an emerging platform in gram-positive bacteria. Microb. Cell, 7, 312–322 (2020).
• 5) Seo MK, Park EJ, Ko SY, Choi EW, Kim S. Therapeutic effects of kefir grain Lactobacillus-derived extracellular vesicles in mice with 2,4,6-trinitrobenzene sulfonic acid-induced inflammatory bowel disease. J. Dairy Sci., 101, 8662–8671 (2018).
• 6) Huang W, Shu C, Hua L, Zhao Y, Xie H, Qi J, Gao F, Gao R, Chen Y, Zhang Q, Li W, Yuan M, Ye C, Ma Y. Modified bacterial outer membrane vesicles induce autoantibodies for tumor therapy. Acta Biomater., 108, 300–312 (2020).
• 7) Morishita M, Horita M, Higuchi A, Marui M, Katsumi H, Yamamoto A. Characterizing different probiotic-derived extracellular vesicles as a novel adjuvant for immunotherapy. Mol. Pharm., 18, 1080–1092 (2021).
• 8) Veerman RE, Güçlüler Akpinar G, Eldh M, Gabrielsson S. Immune cell-derived extracellular vesicles—functions and therapeutic applications. Trends Mol. Med., 25, 382–394 (2019).
• 9) Januzi L, Poirier JW, Maksoud MJE, Xiang YY, Veldhuizen RAW, Gill SE, Cregan SP, Zhang H, Dekaban GA, Lu WY. Autocrine GABA signaling distinctively regulates phenotypic activation of mouse pulmonary macrophages. Cell. Immunol., 332, 7–23 (2018).
• 10) Lu Y, Suh SJ, Kwak CH, Kwon KM, Seo CS, Li Y, Jin Y, Li X, Hwang SL, Kwon O, Chang YC, Park YG, Park SS, Son JK, Kim CH, Chang HW. Saucerneol F, a new lignan, inhibits iNOS expression via MAPKs, NF-κB and AP-1 inactivation in LPS-induced RAW264.7 cells. Int. Immunopharmacol., 12, 175–181 (2012).
• 11) Dziarski R. Recognition of bacterial peptidoglycan by the innate immune system. Cell. Mol. Life Sci., 60, 1793–1804 (2003).
• 12) Takeuchi J, Watari E, Shinya E, Norose Y, Matsumoto M, Seya T, Sugita M, Kawana S, Takahashi H. Down-regulation of Toll-like receptor expression in monocyte-derived Langerhans cell-like cells: implications of low-responsiveness to bacterial components in the epidermal Langerhans cells. Biochem. Biophys. Res. Commun., 306, 674–679 (2003).
• 13) Lässer C, Jang SC, Lötvall J. Subpopulations of extracellular vesicles and their therapeutic potential. Mol. Aspects Med., 60, 1–14 (2018).
• 14) Ramasubramanian L, Kumar P, Wang A. Engineering extracellular vesicles as nanotherapeutics for regenerative medicine. Biomolecules, 10, 48 (2020).
• 15) Höög JL, Lötvall J. Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy. J. Extracell. Vesicles, 4, 28680 (2015).
• 16) Brouwers JF, Aalberts M, Jansen JWA, van Niel G, Wauben MH, Stout TAE, Helms JB, Stoorvogel W. Distinct lipid compositions of two types of human prostasomes. Proteomics, 13, 1660–1666 (2013).
• 17) Huang G, Lin G, Zhu Y, Duan W, Jin D. Emerging technologies for profiling extracellular vesicle heterogeneity. Lab Chip, 20, 2423–2437 (2020).
• 18) Clarke TB, Weiser JN. Intracellular sensors of extracellular bacteria. Immunol. Rev., 243, 9–25 (2011).
• 19) Dunne A, Mielke LA, Allen AC, Sutton CE, Higgs R, Cunningham CC, Higgins SC, Mills KHG. A novel TLR2 agonist from Bordetella pertussis is a potent adjuvant that promotes protective immunity with an acellular pertussis vaccine. Mucosal Immunol., 8, 607–617 (2015).
• 20) Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, Thomas G, Philpott DJ, Sansonetti PJ. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem., 278, 8869–8872 (2003).
• 21) Li ZZ, Tao LL, Zhang J, Zhang HJ, Qu JM. Role of NOD2 in regulating the immune response to Aspergillus fumigatus. Inflamm. Res., 61, 643–648 (2012).
• 22) Lee KH, Biswas A, Liu YJ, Kobayashi KS. Proteasomal degradation of Nod2 protein mediates tolerance to bacterial cell wall components. J. Biol. Chem., 287, 39800–39811 (2012).
• 23) Kumar H, Kawai T, Akira S. Toll-like receptors and innate immunity. Biochem. Biophys. Res. Commun., 388, 621–625 (2009).
• 24) Schwechheimer C, Kuehn MJ. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat. Rev. Microbiol., 13, 605–619 (2015).
• 25) Takenaka T, Nakai S, Katayama M, Hirano M, Ueno N, Noguchi K, Takatani-Nakase T, Fujii I, Kobayashi SS, Nakase I. Effects of gefitinib treatment on cellular uptake of extracellular vesicles in EGFR-mutant non-small cell lung cancer cells. Int. J. Pharm., 572, 118762 (2019).
• 26) Blanc L, Castanier R, Mishra AK, Ray A, Besra GS, Sutcliffe I, Vercellone A, Nigou J. Gram-positive bacterial lipoglycans based on a glycosylated diacylglycerol lipid anchor are microbe-associated molecular patterns recognized by TLR2. PLOS ONE, 8, e81593–8 (2013).
• 27) Jankute M, Alderwick LJ, Noack S, Veerapen N, Nigou J, Besra GS. Disruption of mycobacterial aftB results in complete loss of terminal β(1→2) arabinofuranose residues of Lipoarabinomannan. ACS Chem. Biol., 12, 183–190 (2017).
• 28) Meng G, Rutz M, Schiemann M, Metzger J, Grabiec A, Schwandner R, Luppa PB, Ebel F, Busch DH, Bauer S, Wagner H, Kirschning CJ. Antagonistic antibody prevents toll-like receptor 2-driven lethal shock-like syndromes. J. Clin. Invest., 113, 1473–1481 (2004).