Engineering extracellular vesicles with macrophage membranes fusion ameliorated psoriatic skin inflammation in imiquimod‐treated mice

doi:10.21203/rs.3.rs-2351714/v1

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Engineering extracellular vesicles with macrophage membranes fusion ameliorated psoriatic skin inflammation in imiquimod‐treated mice

https://doi.org/10.21203/rs.3.rs-2351714/v1

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Backgrounds: Psoriasis is an autoimmune skin disease that is difficult to cure and easy to relapse after drug withdrawal. Innate macrophage polarization reprogramming has a considerable impact on psoriasis prognosis. Herein, we introduce a method to ameliorate inflammatory responses based on macrophage membrane-engineered extracellular vesicles.

Results: Engineered extracellular vesicles inherited the high stability of M2 macrophage membrane and retained the macrophage reprogramming potential of Annexin A1 overexpressing T cell-derived exosomes. In the psoriasis-like skin mouse model, engineered extracellular vesicles successfully ameliorated inflammatory responses in the skin and spleen with high biosafety.

Conclusions: Our findings indicated that the M2 macrophage-like fusogenic extracellular vesicle-delivery platform had high inflammation-regulating ability and provided new insights and potential strategies for immunotherapy.

T cells

engineered extracellular vesicles

psoriasis

Annexin A1

Macrophages

Psoriasis is a common chronic inflammatory disease characterized by skin lesions. According to the the National Skin epidemiology survey, China is ranked first in countries with the highest prevalence of psoriasis worldwide, corresponding to approximately 2.3 million people [1].

The treatments of psoriasis mainly include topical therapy, phototherapy, immunosuppressive, and biological drug therapy [2]. However, most of the existing treatments had significant drawbacks, such as intolerable side effects or disease recurrence after drug withdrawal. Therefore, it is urgent to develop new treatment methods.

Although the pathogenesis of psoriasis is still unclear, the infiltration of macrophages has been shown to be closely related to the formation of psoriasis tissue microenvironment. A large-scale transcriptome comparative analysis found that M0 and M1 macrophages were significantly enriched in the skin of psoriasis patients compared with those without this skin condition. Macrophages also exhibited reprogramming potential in psoriasis treatment. Yang et al. found that a circRNA (HSA_circ_0004287) can reduce the RNA stability of MALAT1 by competing for the binding site of a RNA-binding protein (IGF2BP3), which reduced the M1 macrophage polarization and finally alleviated the skin inflammation induced by psoriasis [3]. Thus, exploring the methods to intervene the polarization of macrophage in the pathogenesis of psoriasis can provide new targets and ideas for the clinical treatment of psoriasis.

Annexin A1 (ANXA1), also known as lipocortin I, belongs to the annexin family of calcium-dependent phospholipid-binding proteins. As an effector of glucocorticoid-mediated response, ANXA1 plays an important role in innate immune response and has potent anti-inflammatory activity. Our previous studies showed that ANXA1 derived from Treg cells is closely related to the prognosis of patients with breast cancer. Targeting ANXA1 can reduce Treg cell function and tumor volume [4]. Therefore, it is reasonable to apply the anti-inflammatory identity of ANXA1 in psoriasis treatment adequately.

Exosomes are extracellular vesicles secreted by all kinds of cells with a particle size distribution between 30–150 nm. The lumen contains different components (e.g., nucleic acids, proteins, lipids, and other substances) which may play different roles in intercellular communication [5–7]. As natural nanoparticles, exosomes have gradually entered the vision of researchers because of their low toxicity, high biocompatibility, tissue targeting capacity, plasticity, and in vivo stability [8]. Thus far, a large number of clinical trials have been conducted to assess the applicability of exosome therapy for various medical conditions, such as chronic kidney disease [9], non-small cell lung cancer [10], intestinal cancer [11], and COVID-19 [12–14]. All these studies indicated that exosomes could potentially improve those conditions. The potential application of exosome therapy in psoriasis has also been previously assessed. Zhang et al. co-cultured the exosomes derived from human umbilical cord blood mesenchymal stem cells with dendritic cells (DCs) and found that these exosomes can inhibit the maturation and activation of DCs by reducing the phosphorylation of STAT3, thereby alleviating the skin inflammation induced by psoriasis [2]. These results suggested that the inflammatory response in vivo can be regulated by exogenous exosomes.

Although exosome treatment is being carried out in full swing, several caveats must be considered: (i) Heterogeneity: all cells could produce exosomes. But the composition and packaging efficiency of exosomes may vary a lot according to cell sources [15]; (ii) Stability: the stability of exosomes cannot be maintained even at − 80 ℃. Long-term storage facilitates fusion between exosomes [16]; (iii) Targeting capacity: although exosomes shoulder the important tasks of intercellular communication, their tropism will also vary significantly due to different cell sources [17]. Therefore, the present project aims to address the critical scientific issue of how to use bioengineering technology to construct T cell-derived exosomes with high expression of anti-inflammatory factors, high stability, and a strong inflammatory capacity.

The main purpose of engineering exosomes is to give new characteristics to exosomes based on their original properties, such as increasing targeting, increasing drug loading, and reducing immunogenicity. According to the literature reviews, the methods of exosomes engineering can be mainly divided into three categories: (i) top-down nano vesicle analog extrusion technology, (ii) bottom-up supramolecular chemical synthesis technology, and (iii) exosome biofilm hybridization and fusion technology [18]. The exosome biofilm hybrid fusion technology is derived from the classical exosome separation technology, which is of great significance in maintaining the nature of exosomes to the greatest extent. The biofilm used for exosome hybridization and fusion can be either artificially modified liposome membrane or extracted complete cell membrane such as erythrocyte membrane, platelet membrane, neutrophil membrane, and monocyte macrophage membrane. As an important member of leukocyte, macrophage naturally have the capacity for leukocyte circulation stability, endothelial cell adhesion, and inflammation targeting [17, 19]. Recently, Wang et al. take advantage of the stability and inflammatory cytokines neutralizing characteristics of the M1 macrophage membrane and constructed an inhalable engineered microsphere aerosol through co-extrusion. They found that the severity of COVID-19 infection could be significantly reduced in mice inhaled the microsphere [20].

In this study, we fused the M2 macrophage membrane with ANXA1 high-expressing T cell-derived exosomes using the exosome biofilm hybridization fusion method with different proportions of 1:1, 1:2, and 1:3. The fusion effect was the best when the mass ratio reached 1:3. In vitro results showed that the engineered extracellular vesicles could be effectively engulfed by macrophages and regulate the macrophage polarization to M2 phenotype. In addition, in vivo results showed that psoriasis like skin inflammation was significantly improved in mice after receiving engineered extracellular vesicle treatment.

Macrophages accumulated in psoriatic skin

To identify whether inflammation could induce macrophage accumulation in psoriasis skins, the number of macrophages were counted on sections of the biopsy tissues. As shown in Fig. 1A and 1B, the number of CD68⁺ macrophage was significantly higher in patient group than that in the control group. Similar results were observed in imiquimod (IMQ)-induced psoriasis-like mouse model (Fig. 1C and 1D). These results demonstrated that macrophage accumulation was significantly upregulated in psoriatic skins. It inspired us to ameliorate psoriatic skin inflammation through directly regulating macrophage polarization based on extracellular vesicle engineering technology.

Fabrication and characterization of engineered extracellular vesicles

Figure 2A showed the preparation and optimization of engineered extracellular vesicles. T cells were firstly infected with ANXA1 overexpressing lentivirus, and exosomes were purified from conditioned media by ultracentrifugation. On the other hand, macrophage membrane vesicles were separated from M2 macrophages by repeated freeze–thaw method. The engineering extracellular vesicles were obtained by co-extruding ANXA-1 overexpressing exosomes with M2 macrophage membrane. Firstly, WB results revealed that ANXA1 was successfully overexpressed in Jurkat and EL4 exosomes (Fig. 2B and 2C).

In order to optimize manufacturing procedures, we co-extruded these 2 kinds of bio-membranes together with different proportion. The cell membrane was set as 1 µg, and exosomes were added as 1, 2 or 3 µg. The size distribution results showed that 1:1 weight ratio had 2 peaks, 1:2 weight ratio still had 2 peaks. These results implied that these 2 kinds of membranes cannot be well-fused together at high weight ratio. When the weight ratio reached 1:3, there was only 1 peak. So, we chose this ratio for further experiments. The nano size distribution of human engineered extracellular vesicles was about 144nm (Fig. 2D). We then prepared the mice engineered extracellular vesicles in the same way, and the nano size of it was about 169nm (Fig. 2E).

As for the stability of engineered extracellular vesicles, JAM and EAM were stored at -80 ◦C for more than 6 months. The size distribution of nanoparticles was slightly increased overtime (Fig. 2F and 2G). TEM photos showed that the size of these 2 kinds of nanoparticles were a little bit smaller than NTA (Fig. 2H). Possibly, the samples need to dried out and stained for further TEM scanning. Zeta potential is an electrochemical property which can be used to measure the stability of exosome. High zeta potential represents for more electrostatic repulsion between particles, leading to higher stability. In our study, the cell membrane, exosomes, and engineered extracellular vesicles were approximately − 10, -5, and − 11 millivolts (mV), respectively (Fig. 2I), which indicated that co-extrusion improved the stability of exosome.

To confirm the bio-membrane hybrid following co-extrusion, we labelled the M2 macrophage membrane with DiO (green) and the exosomes with DiD (red). Yellow fluorescence was observed in the engineered extracellular vesicles after co-extrusion, while mixing them simply couldn’t fuse the bio-membranes together (Fig. 2J). Moreover, the expression of exosome marker, CD63, inflammatory cytokine receptor, Type2 IL-1 receptor (IL-1R2), and ANXA1 were detected by WB (Fig. 2K and 2L). The result show that JAMs were loaded with high level of IL-1R2 and ANXA1. All these data indicated a successful construction of engineered extracellular vesicles.

Cellular uptake and Endocytic mechanism of exosomes by macrophages

To identify whether engineered extracellular vesicles could be taken up by macrophages, JC-EXO, JA-EXO, and JAM were labelled with DiI dye, which has a strong red fluorescence. As highlighted by the fluorescent microscopy images, these nanoparticles were observed to be uptaken by macrophages in a time-dependent manner (Fig. 3A and 3B). On the other hand, macrophages were also seeded in the 48-well plate and incubated with DiI labeled engineered extracellular vesicles with different dosage. As shown in Fig. 3C and 3D, mean fluorescence intensity (MFI) of the DiI dye significantly increased in JAM treated cells compared with other groups. To further investigate the endocytic mechanism of exosomes in macrophage, cells were pre-incubated with inhibitory reagents for 30min. The effects of clathrin-mediated endocytosis on the internalization of exosomes were evaluated using sucrose, a kind of clathrin-coated pits formation blocking agent. DMA, a microtubule-disrupting agent, was used to evaluate the effects of macropinocytosis on the internalization of exosomes in macrophages. The effects of caveolae-mediated endocytosis on the internalization of exosomes were evaluated using genistein, a kind of caveolae disrupting agent. Our results showed that both sucrose and genistein significantly decreased red fluorescence in the macrophages cytoplasm. While DMA could also partly reduce cellular uptake of exosomes by macrophages (Fig. 3E and 3F).

JAM Reprogramed macrophages to M2 Phenotype In Vitro

To confirm the effect of JAM on the polarization of macrophage, PBS, JC-EXO, JA-EXO, or JAM were added to macrophages, and IL-4 stimulated macrophages were served as a positive control. Macrophage phenotypic distribution was observed by immunofluorescence staining. Inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) were taken as the marker of M1 and M2 macrophage, respectively. The results indicated that the proportion of the M1 subpopulation was reduced, while the proportion of the M2 subpopulation significantly increased in the JA-EXO and JAM groups (Fig. 4A and 4B). Meanwhile, the regulatory effect of JAM was greater than that of JA-EXO (Fig. 4B). In addition, according to semi-quantitative analysis by real time RT-qPCR, JC-EXO, JA-EXO, and JAM could significantly downregulate the gene expression of M1 markers (CD86, IL-6, and iNOS), while upregulate the gene expression of M2 markers (transforming growth factor 𝛽 (TGF-𝛽)), and JAM performed better (Fig. 4C). These results revealed that JAM can effectively reprogram macrophage toward a reparative phenotype in vitro. In addition, CCK8 assay revealed that JAM slightly reduced the cell number of THP-1 derived macrophages after long-time (72h) treatment (Fig. 4D).

EAM ameliorated psoriasis-like skin inflammation in mice

To validate the in vitro data of engineered extracellular vesicles, we investigated the role of EAM in IMQ-induced psoriasis-like mouse model. After daily application of IMQ cream, mice were treated with PBS, EC-EXO, EA-EXO or EAM every other day (Fig. 5A). On day 8, lesion score and spleen length were evaluated (Fig. 5B and 5E).

We observed that IMQ treated mice had the highest psoriasis area and severity index (PASI) score and the largest spleen size which was larger than 2cm. While extracellular vesicle treatment could alleviate inflammatory responses in the skin and PASI score decreased efficiently. Besides, extracellular vesicle treatment dramatically relieved inflammation in the spleen and the length of spleen were shorter than 2cm. However, the spleen length had no significant difference among the EC-EXO, EA-EXO, and EAM groups. The effect of engineered extracellular vesicles on epidermal hyperplasia was further evaluated by H&E stained slides of skin lesion area. Our results showed that IMQ would induce a significant increase in epidermal thickness, while treatment with extracellular vesicles especially EAM significantly decreased the epidermal thickness (Fig. 5C and 5E).

Immunohistochemical staining with monoclonal antibodies against CD68 on skin sections demonstrated that EAM treatment dramatically reduced the macrophage abundance in skin lesions (Fig. 5F). Furthermore, we observed that IMQ induced a significant decrease in the number of iNOS⁺ and Arg-1⁺ cells in the skin, and it was restored to normal level by EAM treatment (Fig. 5G and 5H). Inflammatory factors, such as TNFα, IL-6, IL-1𝛽, and IL-17A, are strongly associated with psoriasis prognosis. In our study, the abovementioned inflammatory factors in EAM group was reduced by 70%, 37%, 64%, and 83% compared with PBS groups, respectively (Fig. 5I-5L).

Effects of extracellular vesicle treatment on major organs in mouse model

The toxicity of engineered extracellular vesicles to cells in vitro, and its biological safety in vivo were evaluated. In vitro, we have already noticed the effect of JAM on THP-1 derived macrophages. In vivo, regarding the immune responses induced by IMQ in the whole body, the histomorphological changes of major organs were evaluated by H&E and IHC stanning on D8 (Fig. 6).

After IMQ treatment, the tissue morphologies of spleen, liver, and kidney were significantly different from that in the control group, indicating that IMQ caused acute inflammation in the body. In detail, the number of lymphocytes increased significantly after IMQ treatment, leading to an intense inflammatory reaction in the spleen. However, histopathological examination confirmed that extracellular vesicles treatments decreased the lymphocyte accumulation in spleen, and EAM even exhibited a better anti-inflammatory ability compared with other groups (Fig. 6A).

In addition, the morphology of hepatocytes in the IMQ group indicated a severe organ injury, manifested by unclear cristae and damaged hepatic lobules; while the extracellular vesicle treatment groups especially EAM dramatically restored the integrity of hepatic lobules (Fig. 6B). Significant injury in the renal interstitial structure was also found in the IMQ group, while extracellular vesicle treatment efficiently rescued these structural anomalies (Fig. 6C).

In respect of toxicity to other organs, no histopathological changes were observed in lung, brain, and heart of mice treated with or without extracellular vesicles compared to control group (Fig. 6D-6F). Thus, EAM is generally biologically safe, which provide possibility for the potential clinical application.

In this study, we demonstrated that engineered T cell-derived extracellular vesicles efficiently ameliorated psoriasis-like skin inflammation by transferring ANXA1 to macrophages, regulating their polarization procedures and reducing inflammatory responses in the skin. To the best of our knowledge, the present study is the first to apply engineering methodology for T cell-derived extracellular vesicles modification in psoriasis treatment (Fig. 7).

The cellular composition of skin greatly affects dermal inflammatory process. Previous studies showed that macrophage is likely to play a vital role in inducing lymphocyte accumulation in psoriasis skin [21]. These cells keep the delicate balance of inflammatory responses in the skin by releasing cytokines in the microenvironment.

Nowadays, many studies are focusing on fusogenic nanoparticles-based inflammation therapy. One representative example is that ACE2-engineered microfluidic microspheres could neutralize COVID-19 and calm cytokine release storm (CRS) [20]. Wang et al. modified the microspheres with hybrid cell membranes and compared the effect of engineered microspheres with normal microspheres on COVID-19 induced CRS. Their histological analysis and transcriptional sequencing identified that M1 macrophage membrane, which highly expressed TNFR, IL-1R, and IL-6R, may neutralize inflammatory factors through different pathways.

IL-1R2 as a decoy for IL-1 is crucial for the regulation of inflammation. As a result of lacking Toll/IL-1 receptor (TIR) domain, IL-1R2 efficiently prevented IL-1 signaling transduction [22]. Considering the unique functions of IL-1R2 exerted in macrophages, we thus fused T cell-derived exosomes with M2 macrophage membrane for pro-inflammatory cytokines neutralization. Surprisingly, in addition to the macrophage membrane, we also confirmed the accumulation of IL-1R2 in T cell-derived extracellular vesicles, and this discovery well explained why EC-EXO alone could partially abolish IMQ-induced inflammatory reactions in mice.

Endocytosis, which occurs in most cells as pinocytosis, occurs by at least four basic mechanisms: (i) clathrin-mediated endocytosis, (ii) caveolae-mediated endocytosis, (iii) micropinocytosis, and clathrin and caveolae-independent endocytosis. To explore the underlying pathways that mediate engineered extracellular vesicles uptake, macrophages were pretreated with potential inhibitors, including sucrose, DMA, and genistein as previously described [23].

Compared to the control group, the signals of engineered extracellular vesicles (red) were decreased in all the groups, with the lowest signal of JAM was observed in the genistein and DMA treatment groups (Fig. 3E-F). These results revealed that the uptake of engineered extracellular vesicles in macrophages was regulated by multiple pathways, and macropinocytosis and caveolae-mediated endocytosis might be the main routes [24].

As a crucial anti-inflammatory regulator with broad intracellular distribution, Annexin protein family, can provide sustained therapeutic effect and fundamental alterations of the local microenvironment. We previously reported that ANXA1 is closely associated with human triple negative breast cancer survival. Moreover, ANXA1 blocking significantly decreased the tumor volume in tumor-bearing mice. Furthermore, Ferraro et al. reported that ANXA1 was essential for macrophage-mediated inflammatory regulation in myocardial infarction. ANXA1 knocking out in macrophages dramatically reduced the capacity to release vascular endothelial growth factor-A (VEGF-A), and macrophages polarized toward a proinflammatory phenotype [25].

Considering all the above, it seems reasonable to speculate that ANXA1-uploaded engineered extracellular vesicles should be potentially effective in macrophage-mediated anti-inflammatory regulation. However, little is known about whether ANXA1 could be efficiently uploaded into the engineered extracellular vesicles. Therefore, ANXA1 loading in engineered extracellular vesicles was detected by WB. Our results suggested a highly preserved protein loading after cell-membrane co-extrusion, engineered extracellular vesicles (JAM group) have preserved ANXA1 loading compared with JA-EXO group (Fig. 2B). Moreover, macrophage polarization detection in vitro further confirmed the function of engineered extracellular vesicles. All the compelling evidence suggested that we successfully built a bridge between construction of fusogenic nanoparticle-delivery system and inflammation therapy.

Even though the overall components in engineered extracellular vesicles were not evaluated in our study, there have been convincing results supporting those anti-inflammatory responses were deeply dependent on pre-loaded ANXA1 and M2 macrophage membranes in the nanoparticles. Statistical analysis of the PASI score suggested that extracellular vesicles could effectively prevent the deterioration of mouse skin inflammation after IMQ-treatment, while EAM exhibited a better anti-inflammation ability (Fig. 5).

After animal sacrifice, we noticed that extracellular vesicle treatment groups effectively ameliorated inflammatory responses in the spleen and relieved splenomegaly. H&E staining showed that engineered extracellular vesicles dramatically decreased the epidermal thickness while EC-EXO had no significant effect on epidermal thickness amelioration. To investigate the immune regulatory effects of extracellular vesicles, immunohistochemical staining was performed. Compared with the IMQ + PBS group, mice treated with EA-EXO or EAM exhibited excellent therapeutic effect, while EAM decreased the number of CD68⁺ cells in the skin most significantly (Fig. 5F). In vitro experiments also showed that the EAM could induce a slight decrease in the number of macrophages after a long time treatment. All these results suggested that the engineered extracellular vesicles could ameliorate inflammatory responses of psoriasis through gradually restoring the number of macrophages in the skin to normal level after injection.

Previous studies have revealed that iNOS and Arg-1 were tightly associated with pro- and anti-inflammatory cells. Goren et al. assessed the localization and identity of iNOS⁺ leukocyte subsets in acute wound healing in mice. They identified that the leukocytes with high iNOS expression were mature neutrophils (Ly6-B2⁺/Ly-6G⁺) and inflammatory monocytes (Ly-6B2⁺/Ly-6G⁻), and this study may broaden our horizon for further analysis of pro-inflammatory cell subsets [26].

On the other hand, considering the skin accumulation of Arg-1⁺ cells which represent the typical myeloid-derived suppressor cells (MDSCs), we could confirm that after treatment with extracellular vesicles, the number of anti-inflammatory cells increased significantly in the skin, and these cells play a determinant role in the skin repair [27].

Among many cytokines, the dramatic up-regulation of IL-1𝛽, TNF-𝛼, and IL-6 have been most frequently reported in psoriasis patients or psoriasis-like mouse model. IL-6 is a key regulator for the expression of many other cytokines [28]. TNF-α is an acute multifunctional phase pro-inflammatory cytokine that can amplify inflammatory cascade and impair immune-suppressive T cells function [29, 30]. IL-1𝛽, a secreted proinflammatory cytokine, is pivotal for leukocytic differentiation and inflammatory microenvironment regulation. Previous studies have shown that the expression of IL-1β in psoriasis patient was about 3.5–4.5 times higher than that in the normal skin biopsy sample [31]. In the present study, we found that IMQ priming induced a dramatic increase in TNF-α (~ 6-fold), IL-6 (~ 2-fold), and IL-1𝛽 (~ 5-fold) expression in the skin compared to the sham group (Fig. 5I-5K). Conversely, extracellular vesicle treatment significantly downregulated the cytokines accumulation in skin, and engineered extracellular vesicles performed better than any other groups.

According to previous studies, IMQ may cause multi-organ toxicity including liver, kidney, and spleen. Thus, we evaluated the histopathological changes in those major organs after applying different treatments. Surprisingly, we found that extracellular vesicle treatment, especially EAM, recovered liver sinusoid remodeling and alleviated renal injury induced by IMQ. We further noted the changes in the splenic morphology and the proportion of infiltrating leukocytes infiltration in the spleen. The H&E staining showed that there were extremely significant changes in the proportion of immune cells in the pathologically enlarged spleen, while the treatment of extracellular vesicles effectively abolished the adverse effect of IMQ. All these results indicated engineered extracellular vesicles’ therapeutic benefits in the more extensive immune system.

In conclusion, in this research article, to improve psoriasis prognosis through innate macrophage polarization reprogramming, we introduced a method to ameliorate inflammatory responses based on macrophage membrane-engineered extracellular vesicles. Our findings indicated that engineered extracellular vesicles inherited the high stability of M2 macrophage membrane and retained the macrophage reprogramming potential of Annexin A1 overexpressing T cell-derived exosomes, successfully ameliorating inflammatory responses in the skin and spleen in the psoriasis-like skin mouse model with high biosafety. Thus, the M2 macrophage-like fusogenic extracellular vesicle-delivery platform provided a promising therapeutic approach for psoriasis-associated skin inflammation.

Human samples

In this study, the psoriatic skin biopsies were obtained from the lesional skin of psoriasis patients, and normal skin biopsies were obtained from surgical discard specimens of healthy donors. Both patients and healthy donors were strictly screened and were free from medical treatment within 3 months before the recruitment. The study was approved by Ethics Committee of Shanghai Tenth People’s Hospital affiliated to Tongji University School of Medicine (Shanghai, China) and was performed in accordance with the Declaration of Helsinki. All patients and donors signed consent forms.

Animal experiments

Male C57BL/6 mice (8-12 weeks old; shanghai laboratory animal center) were maintained under pathogen‐free conditions with ad libitum food and water. The mice in the treatment group were treated every day with a topical IMQ cream (62.5 mg in 5%; Sichuan Mingxin Pharmaceutical Co., Ltd) on the backs, whereas the mice in the control group were treated with Vaseline Lanette cream. All mice were observed for the following 7 consecutive days as previously described [32]. As for exosome treatment, 50 µg exosomes derived from different cell groups were subcutaneously injected into mice on Day 0, 2, 4, and 6. PASI score was calculated based on erythema, epidermal thickness, and scaling (score range, 0‐4), as previously described [33]. At day 8, mice were sacrificed, and tissues were obtained and analyzed as described below.

The animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85 − 23, revised 1996) and were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals.

Cell culture

Human monocytic cell line THP-1, human peripheral leukemia T cell line Jurkat and mouse lymphoma cell line EL4 were cultured and maintained in complete RPMI 1640 (Gibco, Life Technologies), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin. THP-1 cells were differentiated into macrophages by treatment with 100 nM phorbol myristate acetate (PMA; Sigma–Aldrich, Poland) for 24 h, and then the adherent M0 type macrophages were polarized towards M2 macrophages by treatment with 20 ng/mL of IL-4 for 48h.

Mouse monocyte macrophage line RAW264.7 and human embryonic kidney epithelial cell line 293T were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Life Technologies) containing 10% heat-inactivated FBS and 1% penicillin/streptomycin. RAW264.7 macrophages were either treated (M2) or untreated (M0) with 20 ng/mL IL-4 for 48 h. For all experiments, cells were grown at 37°C in a humidified atmosphere containing 5% (v/v) CO2.

Lentivirus and cell infection

For overexpression assay, ANXA1 sequence was cloned into pHR lentiviral vector (pHR-ANXA1) with GFP gene. Empty pHR vector (pHR-NC) was used as negative control. pHR-ANXA1 or pHR-NC, delta8.9, and pMD2.G were co-transfected into 293T cells with PEI reagent. The supernatants containing viruses were harvested at post-transfection 24h, 48 h, and 72h. Before infection, T cells were expanded for 4 days in 48-well plate. Next, T cells were diluted into 1 million/well and infected with lentiviruses, in the presence of 8µg/mL polybrene (Sigma-Aldrich H9268) for 24h. 3 days post-infection, all the cells were washed with FACS buffer to sort the GFP-positive cells.

Isolation and identification of exosomes

For exosomes isolation, conditioned medium (CM) was prepared by incubating cells in media containing exosome-depleted FBS by ultracentrifugation at 100,000 × g at 4°C for at least 4 h, and then pre-cleared by centrifugation at 500 × g for 15 min and 10,000 × g for 20 min. Exosomes were isolated by ultracentrifugation at 100,000 × g for 210 min and washed in PBS using the same ultracentrifugation conditions. When indicated, DiI (1,1′-Dioctadecyl- 3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Sigma) was added into the PBS at 1 µM. After incubation for 20 min, the excess dye was removed by washing spin. The pelleted exosomes were resuspended in ~ 100 µL of PBS and subjected to further treatments.

We named JA- EXO or JC-EXO as the exosomes isolated from Jurkat cells infected with ANXA1 or negative control lentivirus. We named EA- EXO or EC-EXO as the exosomes isolated from EL4 cells infected with ANXA1 or negative control lentivirus. At last, we named JAM or EAM as the engineered extracellular vesicles which were derived from JA-EXO or EA-EXO co-extruted with M2 macrophage membrane.

Fabrication of engineered extracellular vesicles

To fabricate M2 macrophage membrane vesicle, macrophages induced by IL-4 for 48h were collected in cell membrane extraction buffer (Beyotime, China). Then the cells was repeatedly freeze-thawed, centrifuged at 8000 ×g for 15 min and sonicated for 2 min at 42 kHz frequency with a power input of 100 W [34]. To investigate the contribution of M2 macrophage membrane input to the integrity and stability of engineered extracellular vesicles, engineered extracellular vesicles with different protein ratios of exosomes to M2 macrophage membrane vesicles (m:m, 1:1, 2:1, 3:1) were prepared and serially extruded through 100 nm polycarbonate porous membranes for 10 times each using an Avanti mini extruder (Avanti Polar Lipids, Alabaster, AL, USA) at room temperature. Then, the size distribution of engineered extracellular vesicles was tested to determine the final amount of M2 macrophage membranes to be used.

Characterization of nanoparticles/extracellular vesicles

Transmission electron microscopy (TEM) (JEOL JMPEG- PTMC-1230, Japan) and Nano tracking analysis (NTA) were performed using NanoSight (Malvern, Malvern, UK) to measure extracellular vesicles’ size and zeta potential. For the stability of the extracellular vesicles, samples were stored at − 80 ℃ for 6 months and then measured by NanoSight. Proteins extracted from extracellular vesicles or cells were determined by western blotting (WB). The BCA protein assay kit was used to quantify the extracellular vesicles.

Cellular uptake and endocytic mechanisms in vitro

Macrophages were seeded at a density of 2 × 104 cells/well in six-well plates, incubated for 12h, checked under the microscope for confluency and morphology. After being pre-incubated with Hank’s balanced salt solution (HBSS) for 15min, macrophages were incubated with DiI-labeled JAM at the final concentration from 0 to 150 µg/mL at 37°C for 0–48 h.

For cellular uptake mechanism assay, macrophages were seeded in six-well plates. After checking the confluency and morphology, inhibitory agents including sucrose (300mM), 5-(N, N-dimethyl) amiloride hydrochloride (DMA, 100µM) and Genistein (200µM) were added into each well and incubated for 30min, respectively. Then the compounds were withdrawn from the wells, and DiI-labeled JAM was added at the final concentration of 120µg/mL. After incubation, the cells were visualized under fluorescent microscope (Leica, Germany).

Real-time q-PCR

Total RNA was extracted from cells by using TRIzol reagent (Thermo, Shanghai, China). The purity of the isolated RNA was determined by the optical density 260/280 ratio using the NanoDrop ND-2000 (Thermo Scientific). The isolated RNA was reverse transcribed by using the Hiscript^™ III 1st Strand cDNA Synthesis kit (Vazyme, China). qPCR was performed using the SYBR qPCR Master Mix Kit (Vazyme, China) following the manufacturer’s instructions. The relative expression levels of the genes were normalized to that of GAPDH by using 2 − ΔΔCt.

Macrophage polarization identification

The polarization state of macrophages in vitro was further confirmed by immunofluorescence assay. The details of the experimental procedures are the same as the previous studies. Briefly, after THP-1 cells were treated with 100nM PMA (Sigma– Aldrich, Poland) for 24h, the adherent macrophages were washed with PBS and co-cultured with PBS, JC-EXO, JA-EXO or JAM, respectively. 48h later, cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 20 min. Then, the cells were blocked with 3% BSA and incubated with rabbit-anti-iNOS or rabbit-anti-Arg-1 antibody overnight at 4°C. After washing three times with PBS, the samples were stained with the secondary antibody. Fluorescence signals were detected by a confocal laser scanning microscope (Leica, Germany).

Cell Counting Kit-8

Cell Counting Kit − 8 assay was adopted to test the influence of different exosomes on the number of macrophages. The cells were seeded onto 96-well flat-bottomed plates with a density of 2500 cells/well and then were incubated in 5% CO₂ atmosphere at 37°C, followed by samples treatment for different duration. After incubation, the medium was added with 10µL of CCK8 solution for each well. The absorbance value was measured at 450 nm using microplate reader (Thermo, USA).

Western blotting

Isolated exosome pellet or cultured cells were lysed in radio-immunoprecipitation assay (RIPA) buffer supplemented with complete protease inhibitor cocktail tablets (Roche, Basel, Swiss) and incubation at 95◦C for 5 min. Lysates of cells or extracellular vesicles were separated by 8–12% SDS-polyacrylamide gels, transferred to PVDF membranes. The membranes were blocked for 2 h in 5% no-fat milk buffer, then incubated with primary antibodies at 4°C overnight. Protein expression levels were semi-quantitatively analyzed using densitometry analysis.

Anti-CD63 (ab59479), anti-Hsp70 (ab2787), and anti-Alix (ab117600) were purchased from Abcam (Cambridge, MA). Anti-ANXA1 (D5V2T) and HRP linked anti-rabbit IgG secondary antibody were obtained from CST (Beverly, MA, USA). Anti-IL-1R2 (60262-1-Ig) and anti-GAPDH (60004-1-Ig) was purchased from Proteintech (Rosemont, IL 60018, USA). Secondary antibody (HRP conjugated anti-mouse IgG was purchased from Promega (Beijing, China).

Histological analysis

For histological analysis, patient biopsies and mouse tissue samples were fixed in paraformaldehyde at room temperature for 24 h and embedded in paraffin. Hematoxylin and eosin (H&E) and standard immunohistochemical staining were performed, according to the standard protocols. Six sections taken from the middle portion of tissue were examined. Epidermal thickness of skin was calculated by ImageJ. Cells positive for CD3, CD19, CD68 and IL-17A were quantified as the mean number of positive cells in five fields (original magnification, ×400).

Anti-CD3 (GB111337), anti-CD19 (GB11061-1), anti-TNF-𝛼 (GB13452), anti-IL-6 (GB11117), anti-IL-1𝛽 (GB11113), anti-CD68 (GB113109), anti-iNOS (GB11119), and anti-Arg-1 (GB11285), anti-IL-17Awere obtained from Servicebio (Wuhan, China).

Statistical analysis

All the results reported here are representative of at least three independent experiments, and the data are presented as the mean ± SEM (standard error of mean). All data were evaluated for normal distribution using the Kolmogorov–Smirnov test. When data were normally distributed, Student’s t-test was used for comparisons between two groups, and One-way analysis of variance (ANOVA) or Two-way ANOVA was used for multiple comparisons followed by Tukey’s post-hoc test; otherwise, Kruskal–Wallis test followed by the Dunn post-hoc test was used. P values < 0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism 5.0 (Graph Pad Prism Software Inc, San Diego, CA, USA) and SPSS software (version 17.0, SPSS Inc., Chicago, IL, USA) for Windows.

GraphPad Prism 5.0 (Graph Pad Prism Software Inc, San Diego, CA, USA) was used to analyze data by ANOVA with Tukey’s post-hoc test, Student’s t-test, or Kruskal–Wallis test followed by Dunn post-hoc test when appropriate. Data were first analyzed for normality and equal variance to determine whether applied parametric tests were appropriate using Kolmogorov–Smirnov test with SPSS software (version 17.0, SPSS Inc., Chicago, IL, USA) for Windows. Data were plotted as means ± SD, and individual data points were included as dot points.

Abbreviation	Definition
ANOVA	Analysis of variance
ANXA1	Annexin A1
Arg1	Arginase 1
CM	Conditioned medium
CRS	Cytokine release storm
DCs	Dendritic cells
DiI	1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate
DMA	5-(N, N-dimethyl) amiloride hydrochloride
FBS	Fetal bovine serum
H&E	Hematoxylin and eosin
IL-1R2	Type2 IL-1 receptor
IMQ	Imiquimod
iNOS	Inducible nitric oxide synthase
MDSCs	Myeloid-derived suppressor cells
MFI	Mean fluorescence intensity
NTA	Nano tracking analysis
PASI	Psoriasis area and severity index
PMA	Phorbol myristate acetate
RIPA	Radio-immunoprecipitation assay
TEM	Transmission electron microscopy
TGF-𝛽	Transforming growth factor 𝛽
TIR	Toll/IL-1 receptor
VEGF-A	Vascular endothelial growth factor-A
WB	Western blotting

Funding

Our research is supported by National Key R&D Program of China 2019YFA09006100; National Natural Science Founding of China grants 81830051, 31961133011 and 32130041; Shanghai Academic Research Leader 16XD1403800; Innovative research team of high-level local universities in Shanghai; and Shanghai Collaborative Innovation Center of Cellular Homeostasis Regulation and Human Diseases.

Competing interest

The authors declare that they have no competing interests.

Authors information

Authors and Affiliations

Zeng Wang^{1, *}, Zhizhen Qin^{1, *}, Jiadie Wang¹, Xinqi Xu¹, Mengxin Zhang¹, Yuyue Liang¹, Yukun Huang², Zengyang Yu³, Yu Gong³, Luxian Zhou⁴, Yiran Qiu⁵, Minglu Ma⁶, Dan Li^{1, #}, Bin Li^{1, #}

Center for Immune-Related Diseases at Shanghai Institute of Immunology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

Zeng Wang, Zhizhen Qin, Jiadie Wang, Xinqi Xu, Mengxin Zhang, Yuyue Liang, Dan Li & Bin Li

Department of Pharmacology and Chemical Biology, Faculty of Basic Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

Yukun Huang

Department of Dermatology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China; Institute of Psoriasis, Tongji University School of Medicine, Shanghai, China.

Zengyang Yu & Yu Gong

Research Centre, Shanghai Archgene Biotechnology Co.,Ltd, Shanghai, China.

Luxian Zhou

Department of Breast Surgery, Obstetrics and Gynecology Hospital, Fudan University School of Medicine, Shanghai, China.

Yiran Qiu

Division of Cardiology, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

Minglu Ma

Author contribution

Z.W., Z.Z.Q., and B.L. designed the experiment. Z.W. and Z.Z.Q. performed the major experiments. J.D.W., X.Q.X., M.X.Z., and Y.Y.L constructed the plasmids and isolated the exosomes. Y.K.H. conducted the NTA analysis. Z.Y.Y. and Y.G. collected the human and mice samples. Y.R.Q. and M.L.M. performed WB. D.L., Z.Z.Q. L.X.Z. analyzed the data and prepared the figures. Z.W. wrote the main text. All authors read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Dan Li or Bin Li.

Ethics approval

The present studies involving the use of human skin samples were reviewed and approved by the Ethics Committee of Shanghai Tenth People’s Hospital. All psoriasis patients and healthy volunteers signed informed consent before recruitment.

The reported experiments on animals are in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010).

Consent for publication

All authors have contributed significantly to the submitted work and have approved the submission of this work. We confirm that the paper is not under consideration elsewhere and none of the paper's contents have been previously published (except in form of abstract or thesis).

Acknowledgement

We thank the Shanghai Collaborative Innovation Center of Cellular Homeostasis Regulation and Human Diseases for providing constructive comments on our work and Shouyu Ke, Xiaoxia Wang, Hao Cheng and Qian-Ru Huang for their valuable suggestions.

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No competing interests reported.

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Engineering extracellular vesicles with macrophage membranes fusion ameliorated psoriatic skin inflammation in imiquimod‐treated mice

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