Aloe-derived vesicles enable macrophage reprogramming to regulate the inflammatory immune environment

Introduction: Bacterial pneumonia poses a significant global public health challenge, where unaddressed pathogens and inflammation can exacerbate acute lung injury and prompt cytokine storms, increasing mortality rates. Alveolar macrophages are pivotal in preserving lung equilibrium. Excessive inflammation can trigger necrosis in these cells, disrupting the delicate interplay between inflammation and tissue repair. Methods: We obtained extracellular vesicle from aloe and tested the biosafety by cell viability and hemolysis assays. Confocal microscopy and flow cytometry were used to detect the uptake and internalization of extracellular vesicle by macrophages and the ability of extracellular vesicle to affect the phenotypic reprogramming of macrophages in vitro. Finally, we conducted a clinical feasibility study employing clinical bronchoalveolar lavage fluid as a representative model to assess the effective repolarization of macrophages influenced by extracellular vesicle. Results: In our study, we discovered the potential of extracellular vesicle nanovesicles derived from aloe in reprograming macrophage phenotypes. Pro-inflammatory macrophages undergo a transition toward an anti-inflammatory immune phenotype through phagocytosing and internalizing these aloe vera-derived extracellular vesicle nanovesicles. This transition results in the release of anti-inflammatory IL-10, effectively curbing inflammation and fostering lung tissue repair. Discussion: These findings firmly establish the immunomodulatory impact of aloe-derived extracellular vesicle nanovesicles on macrophages, proposing their potential as a therapeutic strategy to modulate macrophage immunity in bacterial pneumonia.


Introduction
Bacterial pneumonia caused by Streptococcus pneumoniae, Staphylococcus aureus, Gram-negative rods, and Acinetobacter is a significant public health issue (Cilloniz et al., 2011;Torres et al., 2017;Braverman et al., 2022).This disease severely impacts the alveoli and distal bronchial tree in the lungs.Failure to eliminate the pathogens and the associated inflammatory response can lead to acute lung injury, resulting in a high mortality rate, especially among children, the elderly, and individuals with compromised immune systems (Shi et al., 2020).Alveolar macrophages (AMs) play a vital role in lung immunity and tissue repair (Lambrecht, 2006;Hussell and Bell, 2014).Currently, the primary treatment for bacterial pneumonia involves antibiotics (Alvarez-Lerma, 1996;Ott et al., 2012).However, the widespread development of antibiotic resistance due to their extensive use in clinical treatment has led to treatment failures and exacerbated inflammation (Magiorakos et al., 2012).The increased inflammatory response causes non-apoptotic death of AMs, disrupting the homeostasis provided by these macrophages in terms of immunity and tissue repair (Gonzalez-Juarbe et al., 2015).The release of pro-inflammatory substances from necrotic cells triggers a more severe innate immune response, recruiting inflammatory monocytes-macrophages and neutrophils to the damaged sites, resulting in the secretion of a significant amount of pro-inflammatory cytokines like TNF-α, IFN-γ, IL-6, and IL-1β (Wen et al., 2020;Monteith et al., 2021;Zhang et al., 2021).This can lead to complications such as sepsis and cardiovascular disease (van der Poll et al., 2017).Therefore, controlling the inflammation levels in lung tissue becomes crucial.
Extracellular vesicles (EVs) are nanosized particles (ranging from 30 to 120 nm) that can be released from any cell, including both animal and plant cells.They carry a variety of substances such as DNA, RNA, proteins, and lipids, facilitating the exchange of important biomolecules and genetic information between different cells (Colombo et al., 2014;Peng et al., 2020;Dad et al., 2021;Xu et al., 2021;Boccia et al., 2022).This exchange can establish communication and influence cellular behavior between the same or different organisms (Dad et al., 2021).Due to the low immunogenicity and resistance to clearance by immune cells, EVs are efficient at delivering biomolecules and influencing cellular behaviors (Ju et al., 2013;Wang et al., 2014).Mammalianderived EVs have been extensively studied and validated for intercellular communication, physical characteristics, and vesicle functions.In contrast, EVs from plant sources, although discovered earlier than their mammalian counterparts, have been relatively understudied in terms of their biological effects on the human body (Dad et al., 2021).Recent research has successfully demonstrated that EVs derived from grapes, grapefruits, ginger, and aloe contribute to tissue regeneration and inflammation relief (Ju et al., 2013;Wang et al., 2014;Zhang et al., 2016;Kim et al., 2021).Furthermore, plant-derived EVs have a lower immunological risk and fewer side effects than mammalian-derived vesicles, alleviating concerns related to potential animal or human pathogens (Dad et al., 2021).These characteristics suggest that plant-derived EVs hold significant potential for immune regulation.
In this research, we successfully isolated and purified EV nanoparticles from aloe.The analysis results indicate that aloederived extracellular vesicle nanoparticles exhibit typical extracellular vesicle morphology and size.They can polarize pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages, effectively mitigating the cytokine storm and lung alveolar tissue damage caused by the overactive immune response during pneumonia development.These results suggested that aloe-derived EV nanoparticles have significant potential for treating bacterial pneumonia.

Preparation and characterization of EV Aloe
To investigate the properties of aloe-derived EV nanoparticles, we isolated and purified EV Aloe from the aloe-homogenized juice (defined as EV Aloe ) by consecutive centrifugation and ultracentrifugation (Figure 1A), and subsequent transmission electron microscopy (TEM) examination (Figure 1B) and nanoparticle tracking analysis (NTA) (Figure 1C) revealed that the EV Aloe exhibited a classic cupshaped spherical structure, with an average diameter of 144.5 ± 2.8 nm.The purified EV Aloe was quantified using a microbicinchoninic acid (BCA) protein analysis kit.The results indicated a high abundance of EV nanoparticles in aloe (approximately 500 mg/kg), suggesting that aloe can generate a significant amount of EV nanoparticles.Furthermore, we conducted a duplicate analysis of the protein composition of the purified EV Aloe using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 1D).The findings revealed a plethora of proteins within the EV Aloe that potentially possess immune-modulating capabilities.

EV Aloe shows a favorable safety test
To evaluate the biosafety of EV Aloe , we conducted cell viability and hemolysis assays.The EV Aloe exhibited minimal toxicity to macrophages at dosages up to 200 μg/mL (Figure 2A).Hence, we set the EV Aloe concentration below 200 μg/mL for assessing macrophage uptake and polarization modulation in our cellular study, considering that higher concentrations might induce cellular toxicity and complicate immunological responses.Hemolysis tests conducted on red blood cells incubated with various concentrations of EV Aloe revealed no observable hemolysis within a wide range of EV Aloe concentrations (Figure 2B).

Uptake of EV Aloe by macrophages through phagocytosis
To evaluate the impact of EV Aloe on macrophage immune activation, we first assessed macrophage uptake and internalized EV Aloe .Employing DID-labeled EV Aloe , we visualized the phagocytosis and internalization of EV Aloe on macrophages.Immunofluorescence imaging revealed a dose-dependent increase in EV Aloe within macrophages (Figure 3A).Additionally, similar outcomes were obtained through flow cytometry analysis, where the fluorescence signal of EV Aloe was notably higher in the group incubated with 200 μg EV Aloe than in other groups, indicating a dose-dependent increase in EV Aloe internalization by macrophages (Figures 3B, C).Importantly, as mentioned earlier, varying concentrations of EV Aloe showed no evident cytotoxic effects on macrophages.Together, these results suggest that EV Aloe can be engulfed and internalized by murine macrophages.

Macrophage polarization induced by EV Aloe
Regarding the preceding experiments, we confirmed the internalization and uptake of EV Aloe by murine macrophages.Subsequently, we assessed the impact of varying EV Aloe concentrations on the polarization capacity and phenotypic alterations in macrophages.Bright-field microscopy showed a notable transformation in macrophage morphology following EV Aloe treatment, particularly treated with higher EV Aloe concentrations, inducing more pronounced alterations in cell shape (Figure 4A).Additionally, flow cytometry analysis of primary macrophages treated with diverse EV Aloe dosages revealed the most substantial percentage of M2-like tumor-associated macrophages (TAMs) within the group exposed to 200 μg of EV Aloe (Figures 4B, C).These observations suggest that EV Aloe exhibits remarkable and dosedependent immunomodulatory attributes, effectively steering macrophages toward an M2 immune-activating phenotype (Figure 4C).

Clinical bronchoalveolar lavage fluid treatment
To assess the translational viability of murine macrophages influenced by EV Aloe , we conducted a clinical feasibility study  employing clinical bronchoalveolar lavage fluid (BALF) as a representative model (Figure 5A).Compared with lung biopsy, BALF is safer and less invasive, with few complications, and the resulting sample is larger than the source bronchus and multiple lung lobes (Mondoni et al., 2022).The information gained from BALF-EVs is regarded to be a complement to lung biopsy pathology (Zareba et al., 2021).To delve into this, we gathered BALF samples from bacterial pneumonia patients (n = 7), supported by confirmed clinical images (Figure 5A).Our investigation focused on discerning the immune impact of EV Aloe on macrophage cells within BALF under ex vivo conditions.Flow cytometry analysis of macrophages after EV Aloe incubation showcased a substantial increase in the expression levels of M2-associated surface markers compared to the untreated BALF control (Figures 5B-D).Simultaneously, a correlated decrease in the expression of M1-related protein markers was observed (Figure 5E).These discernible alterations in polarization biomarkers were further authenticated by quantifying the M1/M2 ratio (Figure 5F), signaling the effective repolarization of macrophages due to EV Aloe treatment.Furthermore, employing enzyme-linked immunosorbent assay (ELISA) to evaluate the inflammatory cytokine profile changes in BALF revealed increased levels of inflammatory cytokines in pristine pleural effusion across all samples, aligning with previous clinical observations of immune BALF (Figure 5G).However, upon EV Aloe treatment, a significant increase in anti-inflammatory cytokines evidently indicated the efficacy of the treatment.In concert, these results validate the substantial potential of EV Aloe for clinical research and its profound impact on immune modulation.

Conclusion
In conclusion, we successfully isolated and purified EV Aloe with the capacity to reprogram the immune phenotype of macrophages by consecutive centrifugation and ultracentrifugation.Characterization of the prepared EV Aloe revealed its possession of typical features akin to conventional extracellular vesicles.Additionally, hemolysis and cytotoxicity assays validated the robust biosafety of our EV Aloe , demonstrating its ability to repolarize pro-inflammatory macrophages into an antiinflammatory phenotype.Clinical assessments further confirmed that EV Aloe effectively reduces inflammation levels and promotes tissue repair.Our findings demonstrate that EV Aloe , through cellular engulfment and internalization, can reprogram proinflammatory macrophages toward an anti-inflammatory phenotype, attenuating excessive inflammatory responses and facilitating tissue repair.We propose aloe-derived EVs as a highly efficient, safe, and immensely promising macrophage polarization agent for treating acute lung injury induced by bacterial pneumonia.

Preparation and characterization of EV Aloe
EV Aloe was isolated from aloe (bought from the Curacao aloe base of Kangyun Biological Company, Yunnan Province, China) juice by differential centrifugation and then purified using sucrose gradient centrifugation methods.In brief, the aloe was washed with deionized water and then homogenized using a blender.The mixtures were first consecutively centrifuged at 500 g for 10 min, 3,000 g for 10 min and 3,000 g for 30 min, and then, 10,000 g for 1 h to deplete large fibers and cell debris, and then, the supernatant was ultracentrifuged at 150,000 g for 2 h.We resuspended the obtained pellet of EV Aloe in PBS and stored the solution at −80 °C until further use.For characterization of EV Aloe , the particle sizes of EV Aloe were characterized by NTA (Particle Metrix ZetaView, Germany).After screening of size, EV Aloe was prepared for TEM imaging; 10 μL EV Aloe was deposited onto the surface of a formvar-coated copper grid, 1% uranyl acetate was then added for 15 s twice, and the sample was allowed to dry for subsequent imaging.The EV Aloe protein expression was analyzed by SDS-PAGE, the concentrations of which were quantified based on protein concentration using Bicinchoninic Acid Protein Assay (KeyGEN BioTECH) following the manufacturer's protocol.Loading samples were prepared with 20 µg of protein per well.After the proteins in the loading samples were denatured for 10 min at 95 °C, the loading samples were analyzed by SDS-PAGE in a Stain-Free ™ Precast Gel (Bio-Rad #4568094).Phase contrast images were captured using an inverted microscope (Olympus CX41, Japan), and fluorescent images were captured by laser confocal microscopy (FV1000MPE, Olympus).

Biosafety test
To measure the cytotoxicity of EV Aloe in vitro, RAW 264.7 cells were incubated with different concentrations of EV Aloe for 24 h.The cell viability was evaluated by using a CCK-8 assay kit (BS350B, Biosharp).Furthermore, to assess the blood compatibility of EV Aloe , it was evaluated by hemolysis assay.In brief, pure 0.3 mL red blood cells were dispersed in 6 mL normal saline.Then, 0.1 mL of blood red blood cells were co-incubated with different concentrations of EV Aloe (12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, and 200 μg/ mL) at 37 °C for 3 h.Distilled water and saline were regarded as the control.The mixtures were centrifuged, and then, the supernatant was measured at an absorbance of 540 nm.The hemolysis rate was calculated as follows: In vitro macrophage uptake of EV Aloe EV Aloe was stained with 0.5 µM DiD far-red fluorescent probe (C1039, Beyotime) according to the manufacturer's protocol.The RAW 264.7 macrophage cells were seeded into confocal dishes, and the different concentrations of EV Aloe (20 μg, 100 μg, and 200 µg) were added for 4 h at 37 °C.Then, the cells were stained with the nucleus with the Hoechst 33258 (C1011, Beyotime).Laser confocal microscopy was used to present the stained cells (FV1000MPE, Olympus).Furthermore, the cells were then centrifuged at 500 g for 3 min and resuspended in PBS for further flow cytometry analysis.
Fluorescent signals were assessed using a NovoCyte FACS flow cytometer (ACEA Biosciences, Inc.), and data were analyzed using FlowJo software.

Cytokine analysis
An ELISA kit was used to measure the concentrations of inflammatory cytokines and chemokines according to the manufacturer's instructions.The macrophages from BALF were co-incubated with EV Aloe for 24 h; then, the supernatant was collected for the detection of macrophage-related cytokines, such as the pro-inflammatory phenotype (IL-10, TNF-α, IFN-γ, and IL-6).

FIGURE 1
FIGURE 1 Fabrication and characterization of EV Aloe.(A) Isolation and preparation of EV Aloe.EV Aloe could be isolated and prepared by a series of centrifugations, including ultracentrifugation and sucrose gradient ultracentrifugation. (B) TEM image of EV Aloe .EV Aloe harvested from the sucrose density gradient (45%) was characterized by TEM.Scale bar: 100 nm.(C) Size distribution of EV Aloe was measured by NTA.(D) SDS-PAGE analysis of the protein components of EV Aloe .The proteins in EV Aloe were analyzed via 10% SDS-PAGE.

FIGURE 2
FIGURE 2 Biosafety evaluation of EV Aloe.(A) Macrophage viability against different concentrations of EV Aloe treatment.(B) Blood hemolytic test of different concentrations of EV Aloe .Red blood cells were treated with a series of concentrations of TEV Aloe .Erythrocytes treated with PBS (0% hemolysis) were used as positive controls, and deionized water (100% hemolysis) was used as negative controls.N = 3, biologically independent replicates.Representative images per treatment group are shown.

FIGURE 3
FIGURE 3 Cellular uptake analysis of EV Aloe by the macrophage cell.(A) Cellular uptake of DiD-loaded EV with different doses of EV Aloe after 4-h incubation with macrophages (Hoechst; blue), as assessed by confocal microscopy.White scale bars: 20 μm.(B, C) DiD-positive rates of macrophages cocultured with the DiD-labeled EV Aloe for 4 h, analyzed by flow cytometry (n = 3).Representative images per treatment group are shown.The data are presented as the means ± SD.Statistical significance was calculated by one-way ANOVA with Tukey's multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns denotes no significant difference.

FIGURE 4
FIGURE 4 EV Aloe facilitates macrophage phenotype reprogrammed.(A) Morphological changes in macrophages after different concentrations of EV Aloe treatments.Representative images per treatment group are shown.(B, C) Flow cytometry images (B) and the corresponding quantification analysis (C) of CD11b + CD206 + M2 macrophages after incubation with EV Aloe for 24 h.(D) Scheme of the EV Aloe stimulation in the polarization of macrophages.N = 3, biologically independent samples.Representative images per treatment group are shown.The data are presented as the means ± SD.Statistical significance was calculated by oneway ANOVA with Tukey's multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns denotes no significant difference.