Immunoregulatory effects of Lactococcus lactis‐derived extracellular vesicles in allergic asthma

Abstract Background Probiotics have been shown to prevent various allergic diseases by producing extracellular vesicles (EVs). However, the role of EVs in allergic asthma has not yet been completely determined. Methods Gut microbial composition, mainly genera related to probiotics, was investigated in allergic asthmatic mice. Moreover, EVs were isolated from Lactococcus lactis (L. lactis, a selected bacterium) and EV proteins were identified by peptide mass fingerprinting. EV functions in immune responses were evaluated in vivo or ex vivo. Furthermore, the levels of specific IgG antibodies (an alternative marker for EV quantification) to L. lactis‐EVs were measured by ELISA in the sera of 27 asthmatic patients and 26 healthy controls. Results Allergic asthmatic mice showed a lower proportion of Lactococcus compared to healthy mice. L. lactis was cultured and its EVs abundantly contained pyruvate kinase. When allergic asthmatic mice were intranasally treated with EVs, airway hyperresponsiveness, eosinophil number, cytokine secretion, and mucus production were significantly decreased. Moreover, L. lactis‐EV treatment shifted immune responses from Th2 to Th1 by stimulating dendritic cells to produce IL‐12. In addition, significantly lower levels of serum specific IgG4 (but not IgG1) to L. lactis‐EVs were noted in asthmatic patients than in healthy controls. A positive correlation between the levels of EV‐specific IgG4 and FEV1 (%), but a negative correlation between the levels of EV‐specific IgG4 and IL‐13 were observed. Conclusion These findings suggest that L. lactis‐EVs may have immune‐regulating effects on airway inflammation mediated by dendritic cell activation, providing a potential benefit for allergic asthma.


| INTRODUCTION
Asthma is a chronic inflammatory disorder of the airways primarily associated with T helper type 2 (Th2) cell-dependent immune responses, eosinophilia and IgE production. [1][2][3] In asthma pathogenesis, Th2 cells produce various cytokines, including interleukin (IL)-5, IL-9, and IL-13, orchestrating immune responses. 4,5 Especially, IL-5 contributes to eosinophilia, whereas IL-13 is involved in mucus hypersecretion. 6 Traditionally, dendritic cells play an important role in antigen presentation and T-cell differentiation in lymphoid organs as a consequence of allergen exposure. 7,8 They can modulate a Th1/Th2 balance by producing IL-12, which is essential for directing the development of Th1 cells. 9,10 In addition, bacteria have been highlighted to interact with dendritic cells in order to regulate allergic airway inflammation.
Changes in the diversity and abundance of commensal bacteria can influence asthma exacerbation by contributing to inflammation and remodeling in the lungs. 11,12 In particular, probiotics (defined as live microorganisms which confer a beneficial effect on the host) have been suggested to prevent allergic responses through multiple mechanisms. 13 They are predominantly associated with the development of regulatory T cells to produce IL-10, which is also known as an immunosuppressive cytokine. 14 Among them, Lactobacillus rhamnosus and Bifidobacterium breve have been shown to induce IL-10-producing T cells, resulting in attenuation of airway inflammation. [15][16][17] Moreover, Lactococcus lactis has been proposed to have an immunomodulatory function in allergic asthma. 18,19 These non-pathogenic and non-invasive bacteria have already been used in biotechnological applications. 20 Extracellular vesicles (EVs; membrane-bound organelles carrying a cargo of proteins, nucleic acids, lipids, and metabolites) are released by all cell types, including eukaryotes and prokaryotes, under physiological and pathological conditions. 21 As these novel molecules have function in intercellular communication with a relevant effect on the immune system, EVs have been suggested to be involved in several human diseases such as cancer, metabolic disorder and allergic disease. [22][23][24][25][26] For example, Bifidobacterium longum has been demonstrated to suppress mast cell activation in food allergy by producing EVs. 27 In addition, EVs derived from Lactobacillus plantarum showed a protective effect against atopic dermatitis. 28 Although the mechanism of EVs involved in asthma pathogenesis is not clear, these EVs could be a possible therapeutic agent for asthma treatment.
This study aimed to investigate (1) the relative abundance of probiotics in healthy and allergic asthmatic mice, (2) the function of EVs derived from L. lactis against airway hyperresponsiveness and inflammation in mice and (3)

| Metagenomic analysis of gut microbial diversity and composition
Bacterial DNA from fecal contents of healthy or allergic asthmatic mice were analyzed by the Closed-reference operational taxonomic unit picking method of QIIME (v1.9) were used, and Greengenes DB was used as a reference.

| Peptide mass fingerprinting
All chemicals used in this study, including 4-sulfophenyl isothiocyanate, α-cyano-4-hydroxycinnamicacid, sodium bicarbonate, and ammonium bicarbonate, were purchased from Sigma-Aldrich. For protein identification by peptide mass fingerprinting, protein spots were excised, digested with trypsin (Promega, Madison, WI), mixed with α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid, and subjected to MALDI-TOF analysis (Microflex LRF 20, Bruker Daltonics). Spectra were collected from 300 shots per spectrum over m/z range 700-4000 and calibrated by two points internal calibration using Trypsin auto-digestion peaks (m/z 842.5099, 2211.1046). Peak list was generated using Flex Analysis 3.0. Threshold used for peak-picking was as follows: 500 for minimum resolution of monoisotopic mass and 6 for S/N. The search program MASCOT, developed by The Matrixscience (http://www.matrixscience.com), was used for protein identification by peptide mass fingerprinting. The following parameters were used for the database search: trypsin as the cleaving enzyme, a maximum of one missed cleavage, iodoacetamide (Cys) as a complete modification, oxidation (Met) as a partial modification, monoisotopic masses, and a mass tolerance of �0.2 Da.

| EV treatment, adoptive cell transfer, and IL-12 neutralization
For EV treatment, each OVA-sensitized mouse was intranasally treated with 10 μg EVs (total protein concentration) and 50 μg OVA in  Peripheral total eosinophil counts were conducted using a hematology analyzer (Beckman Coulter, Fullerton, CA, USA).

| Blood collection and cell isolation
Blood from patients with asthma was collected into vacutainer tubes containing acid citrate dextrose solution (BD Biosciences) by percutaneous venous catheter sampling. Blood was layered on LEE ET AL. Lymphoprep solution (Axis-Shield, Oslo, Norway), followed by centrifugation at 800� g at 20°C for 25 minutes. The layer containing peripheral blood mononuclear cells was separated and red blood cells were eliminated by hypotonic lysis. Dendritic cells were analyzed and isolated using flow cytometry. The antibodies used were as follows: lineage, HLA-DR, CD1c, and CD303 (Bio-Legend, San Diego, CA, USA).

| Serum cytokine and EV-specific antibody measurement
The levels of IL-13 in the serum of the study subjects were measured using the Quantikine ELISA kits (R&D Systems). To evaluate the

| Statistical analysis
All statistical analyses were performed using IBM SPSS software,

| Gut microbiota and functional-gene profiles in allergic asthmatic mice
In allergic asthmatic mice, microbial diversity in fecal contents was markedly reduced ( Figure 1A). Especially, higher proportions of Bacteroidetes, but lower proportions of Firmicutes, were shown at the phylum level ( Figure 1B). Moreover, relative abundance of Bacteroidaceae increased, whereas those of Lactobacillaceae and Ruminococcaceae decreased at the family level ( Figure 1C). Among genera related to probiotics, the prevalence of Lactococcus was significantly lower in asthmatic mice than in healthy mice. Although Lactobacillus and Bifidobacterium also tended to decrease, a statistical significance was not noted ( Figure 1D). Here, we further investigated functional-gene profiles and found that multiple genes were involved in cellular, environmental information, and genetic information processing as well as metabolism ( Figure 1E). In particular, gene expression associated with transporters and transcription factors was lower in allergic asthmatic mice ( Figure 1F).

| Isolation and characterization of EVs derived from probiotics
The present study cultured L. lactis and purified their EVs. Isolated EVs showed a spherical lipid bilayer with a diameter of 60-100 nm (Figure 2A,B). Moreover, EVs were composed of various proteins ( Figure 2C). Here, we further performed peptide mass fingerprinting to identify specific proteins in the EVs. As a result, pyruvate kinase as well as arginine deiminase and ornithine transcarbamylase were abundantly found in the EVs ( Figure 2D).

| Effect of L. lactis-EVs on airway resistance and immune responses in mice
The safety of EV treatment in vivo was confirmed by evaluating mouse survival rates and body weight changes ( Figure S1A,B).
When allergic asthmatic mice were treated with EVs or Dex, airway hyperresponsiveness, eosinophil counts, and mucus production decreased significantly. In addition, both EVs and Dex could decrease the levels of IL-5 and IL-13, while EVs increased IFN-γ in the BALF of allergic asthmatic mice ( Figure 3A-D). To clarify the significance of Th1/Th2 balance, adoptive T-cell transfer was performed ( Figure S1A). Proportion of IFN-γ-producing CD4 + T cells from mice (treated with the EVs) were confirmed by flow cytometric analysis ( Figure S1B). By receiving spleen T cells from mice (treated with the EVs), airway hyperresponsiveness and Th2 cytokine production were significantly suppressed in allergic asthmatic mice ( Figure S1C,D).

| IL-12-mediated immune modulation by EV treatment in vivo or ex vivo
When allergic asthmatic mice were treated with the EVs, expression of GATA-3 and phosphorylation of STAT6 were markedly decreased  Figure 4B-D). To demonstrate shifting from Th2 to Th1 immune responses mediated by IL-12 production, human peripheral dendritic cells were isolated and analyzed by flow cytometry ( Figure 5A). As a result, the EVs enhanced IL-12p70 secretion from dendritic cells ( Figure 5B), but the levels of IL-12p70 in the supernatants were significantly reduced by SB203580 ( Figure 5C). IL-12p70 production induced by p38 pathway in dendritic cells was shown by Western blot analysis ( Figure 5D,E). However, heated or ProK-treated EVs could not increase p38 phosphorylation in the cells ( Figure 5F).

| Serum specific IgG antibodies to L. lactis-EVs in humans
To investigate the significance of L. lactis-EVs in the study subjects (Table S1), the levels of EV-specific IgG antibodies were measured in the sera of study subjects, because direct EV quantification had some limitations. The levels of serum EV-specific IgG4 were significantly lower in asthmatic patients than in HCs, whereas those of EV-specific IgG and IgG1 were not different between the two groups ( Figure 6A-C). The receiver operating characteristic curve of the EV-specific IgG4 levels was able to discriminate asthmatic patients from HCs (r = 0.786, p = 0.001; Figure 6D). In addition, serum IL-13 levels were significantly higher in asthmatic patients than in HCs ( Figure 6E). A negative correlation was noted between the levels of the EV-specific IgG4 and IL-13 (r = −0.450, p = 0.001; Figure 6F). A positive correlation was shown between the levels of the EV-specific IgG4 and FEV 1 (%) in asthmatic patients (r = 0.497, p = 0.021; Figure 6G).

| DISCUSSION
This is the first study to demonstrate an immunomodulating effect of L. lactis-EVs (rather than immune suppression) in allergic airway inflammation. These EVs could shift immune responses from Th2 to Th1 by dendritic cell activation and IL-12p70 production, which is critical for Th1 cell differentiation. Moreover, asthmatic patients showed significantly lower levels of serum specific IgG4 to L. lactis-EV in association with increased IL-13 and decreased FEV 1 (%). Therefore, measurement of specific IgG antibodies to EVs may replace direct quantification of EV concentrations in asthmatic patients.
Taken together, these provide an insight into identification, characterization, and function of L.lactis-EVs in allergic asthma.
Emerging evidence has highlighted the microbiota as a key player of the host immune system by regulating local and systemic immune responses. 29 Especially, probiotics have been suggested to prevent allergic diseases. 30 Probiotics are bacterial species traditionally regarded as safe; the main strains include lactic acid bacteria such as lactis has been demonstrated to strongly protect against the development of childhood allergic disease in humans. 33 Moreover, a recent paper has revealed that this bacterium prevents airway inflammation and remodeling in allergic asthma in vivo. 34 Therefore, the present study was attempted to find the functional mechanism of L. lactis in the pathogenesis of allergic asthma.
This study proposed EVs derived from probiotics as key molecules having functional effects on immune regulation. Recently, the potential use of EVs as a therapeutic agent has been extensively highlighted, because these EVs could transfer biological information by an endogenous mechanism of intercellular communication. 35 Furthermore, bacterial EVs have been implicated in human health and disease due to their roles in a wide range of biological events. 36 To date, comparative proteomic and lipidomic analyses have identified various proteins and lipid species in EVs. 37 Here, we showed that pyruvate kinase, arginine deiminase, and ornithine transcarbamylase were dominantly found in L. lactis-EVs. Among them, pyruvate kinase has been revealed to contribute to ERK and p38 phosphorylation in cancer cells. 38 In addition, a recent paper has demonstrated that this enzyme promotes dendritic cell activation to enhance IL-12 expression. 39 Although, EVs have opened a new era in studying pathophysiological processes, their diverse components and functions need to be further investigated.
In the current study, L. lactis-EVs did not attenuate Th2 immune response through an IL-10-mediated mechanism (data not shown), although some studies have suggested that probiotics could stimulate regulatory T cells to produce an anti-inflammatory cytokine. 40,41 Among Th2 cytokines, IL-5 and IL-13 have multiple functions in the development of allergic asthma. IL-5 has been known to play a central pathogenic role in differentiation, recruitment, survival, and degranulation of eosinophils. [42][43][44][45] In addition, IL-13 is a pleiotropic cytokine involved in many biological responses relevant to asthma, such as generation of eosinophil chemoattractants, maturation of mucus-secreting goblet cells, and production of extracellular matrix proteins. 46 Here, L. lactis-EVs significantly reduced eosinophilia (IL-5mediated) and mucus production (IL-13-mediated) in allergic asthmatic mice by enhancing Th1 immune response. Furthermore, adoptive T-cell transfer from mice treated with L. lactis-EVs to allergic asthmatic mice has revealed the significance of Th1/Th2 balance in vivo. This mechanism differs from the pathway by which corticosteroids contribute to immune suppression.  Dendritic cells play a central role in naïve T-cell differentiation determined by the cytokine environment. 47 IL-12 released from dendritic cells is essential for the induction of Th1 immune response. 48 Moreover, these cells have been shown to be stimulated by several factors including bacteria, cytokines, and simple chemicals like haptens. 49 Here, we found that L. lactis-EVs could elevate IL-12p70 production from human peripheral dendritic cells ex vivo. Indeed, a previous study has demonstrated that L. lactis strains enhanced expression of IL-12 in dendritic cells, leading to Th1 polarization. 50 In this aspect, the development of Th1 response might control Th2-driven allergic immune responses. 51 Taken together, L. lactis-EVs could effectively regulate allergic reactions by inducing a shift of immune responses from Th2 to Th1 rather than by inhibiting immune responses.
To date, bacterial EVs have been suggested to be a novel biomarker for allergic diseases. 26,52 However, EV-specific antibody production or metagenomic analysis is required to detect bacterial EVs. As these processes are time consuming and expensive, we evaluated EV-specific antibodies to show the relative abundance of bacterial EVs. As a result, significantly lower levels of specific IgG4 to L. lactis-EV (but not IgG or IgG1) were observed in asthmatic patients than in HCs. Previously, asthmatic patients have been found to be more sensitized to EVs derived from bacteria, such as Enterobacter cloacae, Pseudomonas aeruginosa, and Staphylococcus aureus, with higher levels of EV-specific IgG in their sera. 53 Nevertheless, clinical implications of EV-specific IgG subclasses have not been determined. Here, we showed a negative correlation between serum levels of EV-specific IgG4 and IL-13 in asthmatic patients. In addition, these EV-specific IgG4 levels were positively correlated with FEV 1 (%) values in asthmatic patients.
These findings may provide a new approach to evaluating the role of each bacterial EV in allergic asthma.
The present study has some limitations. The one is that the relative abundance of EV composition derived from probiotics in human samples was not evaluated. The other is that direct evidence for the correlation between the concentration of bacterial EVs and the degree of bacterial exposure has not been clarified.
Once these issues are clarified, the significance of bacterial EVs may be further emphasized.
In conclusion, L. lactis-EVs could shift immune responses from Th2 to Th1 via stimulating dendritic cells to produce IL-12, providing potential benefits for allergic asthma as an immunomodulator.