Investigating the Immune Function and Proteomic Proles of Plasmal Exosomes in Lactobacillus Plantarum-treated Immunosuppressive Broilers

Background: Exosomes are extracellular membranous nanovesicles that carry functional molecules, such as proteins, to mediate local and systemic cell-to-cell communication. Exosomes released by cells can present in the plasma and involved in the regulation of immunity. Probiotics play a benecial role in improving the immune function of host through many mechanisms. However, whether probiotics can increase the immune function of broilers by regulating plasmal exosomal cargo is unclear. Methods: Three hundred broilers were allocated to three treatments: control diet (CON group), control diet + dexamethasone (DEX) injection (DEX group), control diet containing 1 × 10 8 cfu/g Lactobacillus plantarum P8 + DEX injection (P8+DEX group). The immune function of broilers was detected by measuring the levels of inammatory cytokines and immunoglobulins in plasma and jejunal mucosa. Exosomes were isolated from the plasma via EIQ3 isolation kits and characterized via transmission electron microscopy, nanoparticle tracking analysis, and Western blot. Then, exosomal protein prole was determined by proteomic. At last, correlation analysis was performed to gure out the potential role of exosomal proteins in regulating immune function of P8-treated broilers. Results: P8+DEX treatment improved the immune function of DEX-induced immunosuppressive broiler through decreasing plasmal IL-1β, IL-6, TNF-α and jejunal IL-1β, as well as increasing plasmal IL-10 and jejunal IgM. The isolated extracellular vesicles had an average diameter of 125.8 nm, exhibited a cup-shaped morphology and expressed exosomal markers. A total of 784 proteins were identied in the exosomes. Among the 784 proteins, 126 differentially expressed proteins (DEPs) were found between DEX and CON groups, 102 DEPs were found between P8+DEX and DEX groups. Gene Ontology analysis indicated that DEPs between DEX and CON groups are mainly involved in metabolic process, cellular anatomical entity, cytoplasm, extracellular region and binding. DEPs between P8+DEX and DEX are mainly involved in multicellular organismal process, response to stimulus, cytoplasm, cell periphery, membrane, binding, protein binding and ion binding. Further, pathway analysis revealed that most of the DEPs between DEX and CON participated in the ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton, endocytosis and phagosome. Most of the DEPs between P8+DEX and DEX participated in the ErbB and PPAR signaling pathways. Moreover, many immunity-related DEPs were correlated with the altered immune parameters in plasma and jejunal in broilers fed with P8. Conclusions: Our ndings demonstrated that plasmal exosomes in immunosuppressive broilers fed with P8 carry proteins related to immune function, and may have immunomodulatory effects on the plasma and intestinal immunity. P8: Lactobacillus plantarum P8; CON: control diet; DEX: dexamethasone; IL: interleukin; TNF-α: tumor necrosis factor; Ig: immunoglobulin; DEP: differentially expressed protein; WB: western blot; NTA: nanoparticle tracking analysis; TEM: transmission electron microscope; GO: gene ontology; KEGG: kyoto encyclopedia of genes and genomes.

Probiotics have been reported to improve the immune health status in immunocompromised animals or patients. For example, Lactobacillus strains protected mice from cyclophosphamide-caused myelosuppression and improved phagocytic cell recruitment to C. albicans infectious sites [15]. Lactobacillus rhamnosus GR-1 could lead to an increase in CD4 positive T cells and a decrease in febrile episodes in HIV patients [16]. Probiotics administration also reduced tumor incidence in Marek's disease virus-infected chickens [17]. Lactobacillus plantarum P8 (P8) is a probiotic strain isolated from the natural fermented yogurt of the Inner Mongolian herder's family. It is suggested that P8 could alleviate the hyperlipidemic of rat [18] and reduce the stress of adults [19]. Our previous study demonstrated that 1×10 8 cfu/g P8 inhibited oocyst shedding and elevated the growth performance as well as the intestinal health of broilers infected with Eimeria (data now shown). But whether P8 can improve the immune function and alter the exosomal composition of broilers under immunosuppression is unclear. Thus, in the present study, the levels of cytokines and immunoglobulins in the plasma and jejunal mucosa of dexamethasone sodium phosphate (DEX)-induced immunosuppressive broilers were measured, moreover, the quantitative proteomic analysis and potential biological functions of exosomal proteins were determined. In addition, correlation analysis was performed to gure out if the exosomal proteins paly a potential role in regulating the immune function of immunosuppressive broilers.

Birds and diets
Three hundred one-day-old male Cobb broilers with similar initial body weights were purchased from Henan Academy of Agricultural Sciences. The basal diet was obtained from Henan Academy of Agricultural Sciences. The composition and nutrient levels of the basal diet is listed in Additional le 1 Table S1.

Purity and identi cation checks of bacteria
The culture and preparation of P8 was prepared by the Department of Animal Nutrition, Qingdao Agricultural University, China. P8 was cultured on Man Rogosa Sharpe media, kept at 37 ℃ for 24 h. Pure bacterial cells were collected after centrifugation at 5000 × g for 10 min at 4 ℃. Then, these cells were washed twice with sterile 0.85 % sodium chloride solution. Ultimately, the culture purity and identi cation were constantly checked by the spreading plate method [20].

Experimental design
A total of 300 broilers were equally divided into 4 treatments with 10 replicated cages of 10 birds each for a 21-day feeding period. The treatments were control diet (CON group), control diet + DEX intraperitoneal injection (DEX group), control diet containing 1 × 10 8 cfu/g P8 (P8 group), and control diet containing 1 × 10 8 cfu/g P8 + DEX intraperitoneal injection (P8+DEX group). At day 16, broilers in DEX and P8+DEX groups were injected with 3mg/Kg BW DEX, while broilers in the CON and P8 groups were injected with equal volume of saline. Fresh water and feed were provided ad libitum. The temperature of the room was set at 33-35 °C in the rst week, and then decreased 2 °C every week until 24 °C.

Sample collection
At day 21, blood samples from 1 broiler of each replicate were randomly collected by cardiac puncture into vacuum tubes containing anticoagulant and centrifuged for 10 min (3000 × g) at 4 ℃. Pure plasma samples were collected and stored in 1.5 mL Eppendorf tubes at -20 ℃. The segments of jejunum from 1 broiler of each replicate were collected. Mucosa was scraped from 10 cm of the jejunum using a glass slide (5 cm proximal to the Meckel's diverticulum). Jejunal mucosa samples were placed immediately in liquid nitrogen and then held at -80 °C.

Exosome Isolation
Exosomes were isolated from plasmal samples by Exosome Isolation Q3 kit (EIQ3-02001, Wayen Biotechnologies, Shanghai, China). The frozen plasma samples were thawed in a 25 ℃ water bath and then placed on ice. Four microlitre Reagent C was added into 200 μL plasma and mixed well by vortexing until obtain a homogenous mixture. The mixture was incubated at 37 ℃ for 15 min. After incubation, the samples turned into jellylibe status. The tubes were taped rmly to change the jellylibe status into liquid status and then centrifuged at 10000 × g for 10 min at room temperature. The supernatant was transferred into a new 1.5 mL tube and then placed on ice. Thereafter, 50 μL Reagent A was added in 200 μL pre-treated plasma and mixed. The mixture was incubated at 4 ℃ for 30 min. After incubation, the supernatant was centrifuged at 3000 × g for 10 min at room temperature. The pellet at the bottom of the tube was resuspended completely with 200 μL 1 × PBS and mixed well by vortexing until obtained a homogenous mixture. Fifty microlitre Reagent B was added into the re-suspension and mixed well by vortexing until obtained a homogenous mixture. The mixture was incubated at 4 ℃ for 30 min. After incubation, the mixture was centrifuged at 3000 × g for 10 min at room temperature. The exosomes pellet was obtained by removing the supernatant. The exosomes pellet completely in 50-120 μL 1 × PBS and mixed well to obtain a homogenous mixture. Once the pellet was re-suspended, the exosomes resuspension was aliquoted and stored at -80 ℃ till next experiments immediately.

Exosomal protein extraction
Exosomes samples were added the same value of protein lysis buffer (7 M Urea, 2 % SDS) containing 1 × protease inhibitor cocktail, followed by 1 min of sonication on ice using a ultrasonic processor (ultrasound on ice for 2 s, stop for 5 s), and rested on ice for 30 min. The lysate was centrifuged at 13000 rpm for 20 min at 4 ℃, then the supernatant was transferred to a new 1.5 mL tube. Four times volume of 100 % acetone was added and the mixture was precipitated overnight at -20 ℃. The sample solutions were centrifuged at the next day. The pellet at the bottom was collected and washed twice with 500 μL pre-cooling washing buffer (ethanol: acetone: acetic acid = 50: 50: 0.1). Finally, after centrifugation at 13000 rpm for 15 min at 4 ℃, the precipitates were dissolved in buffer containing 6 M guanidine hyfrochloride and 300 mM TEAB, and the protein concentration was quanti ed with BCA assay.

Transmission Electron Microscope (TEM)
Five microlitre exosome sample was deposited on Formvar-carbon-coated copper grids for 5 min at room temperature. The excess liquid was removed using Whatman lter paper. Add a drop of 2 % uranyl acetate and incubated for 1 min at room temperature. The excess liquid was removed using Whatman lter paper. After drying, samples were observed under a Tecnai G2 Spirit BioTwin TEM at 80 kV. The acquisitions were made with Gatan Orius SC200D camera. Nanoparticle Tracking Analysis (NTA) The frozen exosomal samples were thawed in a 25 ℃ water bath and then placed on ice. 1 × PBS was used to dilute exosomes for NTA. NTA was performed using a NanoSight instrument (PARTICLE METRIX Malvern Panalytical, Ltd., Malvern, United Kingdom) with a 488 nm laser and automated syringe pump as previously described [21]. The ZetaView 8.04.02 software was used to process the recorded movies.
Western Blot Analysis (WB) Equal amounts of exosomal proteins from each group were subjected to SDS-PAGE, then proteins on the gel were transferred to nitrocellulose membrane. Membranes were blocked by 5 % skimmed milk and then incubated with the primary antibodies (anti-CD63, anti-TSG101, and anti-Calnexin) overnight at 4 °C. After washing with Tris Buffered Saline Tween, membranes were incubated with secondary antibody adjusted with Horseradish Peroxidase (Beyotime Biotechnology, China) [22].

Filter aided proteome preparation
Eighteen microgramme protein solution samples were taken from each sample, and the volume was determined to 100 μL with 25 mM ammonium bicarbonate. Then, 1 M DTT was added (terminal concentration 20 mM) and incubated at 57 •C for 1 h. Then, 10 μL 1 M iodoacetamide was added (terminal concentration 90 mM) and incubated for 40 min at room temperature under dark conditions.
The sample solution was centrifuged on a 10 kDa ultra ltration tube at 12,000 rpm, and dissolution buffer (ammonium bicarbonate) was added into the ultra ltration tube to wash four times. The sample was digested with trypsin which was diluted with dissolution buffer at 37 •C overnight. Next day the peptides were collected by centrifugation, and dried by centrifugal concentration.

Desalination
The dried peptides were desalted on a Monospin desalting column for mass spectrometry analysis. Dissolution the dried mixed peptide using 0.1 % tri uoroacetic acid (TFA) solution. The 100 % acetonitrile was used to activate the desalting column. Then, the 0.1 % TFA solution was used to equilibrate the desalting column. The re-dissolved samples were added to the desalting column and centrifuged.
Desalting column was cleaned using 0.1 % TFA solution. Thereafter, 50 % acetonitrile solution was added to collect the elution solution in a new tube. The elution solution was concentrated and dried by centrifugation to remove acetonitrile.

Liquid Chromatography Tandem Mass Spectrometry (LC-MS)
The dried samples were re-dissolved in 0.1 % uoroacetic acid (FA) solution and 1-2 μg sample was taken for mass spectrometry analysis. The on-line Nano-RPLC liquid chromatography was performed by Easy-nLC 1000 system (Thermo Scienti c, USA). The trap column was home-made C18 (C18, 5 μm, 100 μm * 2 cm) and the analytical column was C18 reversed-phase column (C18, 1.9 μm, 75 μm × 200 mm). The peptides results were subjected to nano electrospray ionization source followed by tandem mass spectrometry in Orbitrap Fusion Lumos (Thermo Scienti c, USA). The mass spectrometer was operated in the data-dependent mode. For MS scans, the scan ranged from 350 to 1,600 m/z. Intact peptides were detected at a resolution of 60,000 and peptides were then selected for MS/MS at a resolution of 15,000. Collision energy: 30% HCD [21].

Proteomic Analysis
The MS/MS data were analyzed with MaxQuant software (version 1.5.8.3, Max-Planck Institute for Biochemistry, Germany), and proteins were identi ed by comparing the peptide spectra against the Swissprot databases. The Trypsin was speci ed as the cleavage enzyme, and up to two missed cleavages were allowed. The mass tolerance value for the fragment ions was set to 0.05 Da. The FDR was set to < 1 %. Proteins were quanti ed using label-free quanti cation, and the relative protein abundances are presented as the mGC/HC ratios. The differential expression threshold was set to a 2fold change. Data analysis was contract service offered by Wayen Biotechnologies (Shanghai), Inc. (Shanghai, China).

Statistical data analysis
One-way ANOVA was used for single factor analysis by SPSS 20.0 for windows (SPSS Inc. Chicago, IL). Spearman's correlation coe cient was calculated using SPSS Version 20.0 (SPSS Inc., Chicago, IL) and GraphPad Prism 8 (GraphPad Software, Inc.) software and used to assess bivariate relationships between variables. Results were expressed as means and the differences were considered signi cant at P < 0.05.

Results
Effects of P8 on the levels of cytokines in the plasma and jejunal mucosa in broilers In the plasma, compared to the CON group, DEX signi cantly increased the level of IL-1β (P < 0.01), and signi cantly decreased the level of IL-10 (P < 0.01). Besides, compared to the DEX treatment, P8+DEX led to lower levels of IL-1β (P < 0.01), IL-6 (P < 0.05), TNF-α (P < 0.01), and higher level of IL-10 (P < 0.01) ( Table 1).

Effects of P8 on the levels of immunoglobulins in the plasma and jejunal mucosa in broilers
In the plasma, the levels of IgM, IgG and IgA were not altered signi cantly by different treatments. However, in the jejunal mucosa, DEX treatment led to a lower level of IgM (P < 0.01), which was reversed by the treatment of P8+DEX (P < 0.01). But there were no signi cant differences of the IgG and IgA secretions among groups ( Table 2).

Characterization of exosomes
The characterization of exosomes was performed by TEM, NTA and WB. TEM analysis demonstrated cup-shaped vesicles with a size range from 100-150 nm in diameter (Fig. 1A). NTA showed that the mean size of puri ed exosomes was 125.8 ± 3.6 nm, and the primary peak size was 129.3 nm (Fig. 1B).
Moreover, WB analysis revealed that exosomal marker proteins (TSG101 and CD63) were obviously expressed in the exosome samples. However, calnexin, which generally represents contamination by intracellular proteins, was absent (Fig. 1C).

Proteomic analysis of exosomes
A total of 784 proteins were identi ed in plasma exosomes by label-free quantitative proteomic analysis, indicating that the exosomes contained abundant exosomal proteins (Additional le 1 Table S2). Through exploration, we found that 126 differentially expressed proteins (DEPs) (P < 0.05) between DEX group and CON group were screened from the results based on the differential expression threshold (fold change > 2 times) (Fig. 2). Among the 126 DEPs, 58 proteins were up-regulated (Table 3), while 68 proteins were down-regulated (Table 4) in plasmal exosomes isolated from broilers injected with DEX relative to those isolated from the control ones. Moreover, 102 DEPs were screened between the exosomes from the P8+DEX group and DEX group (Fig. 2). Among the 102 DEPs, 40 proteins were upregulated (Table 5), while 62 proteins were down-regulated (Table 6) in plasmal exosomes isolated from broilers receiving P8+DEX relative to those isolated from broilers receiving DEX.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of DEPs
GO and KEGG analysis were conducted to understand the functional signi cance of DEPs. The results of GO enrichment analysis were classi ed into three sections: cellular component (CC), molecular function (MF), and biological process (BP). Compared to the CON group, DEPs in exosomes from the DEX group mainly participate in organic substance metabolic process (BP), nitrogen compound metabolic process (BP), macromolecule metabolic process (BP), cellular anatomical entity (CC), cytoplasm (CC), extracellular region (CC), binding (MF), protein binding (MF) and ion binding (MF) (P < 0.05) (Fig. 3).

Discussion
In recent decades, more and more reports have proved the effective roles of exosomes involved in immunomodulation [13,23,24]. However, most of the research were done in human or murine models, little is known about the biofunction of exosomes in chickens. Limited reports on chicken exosomes have suggested that chicken biliary exosomes possess the capacity to in uence the immune responses of Numerous reports demonstrated that probiotics can enhance the immune function of hosts through non immune mechanisms (stabilization of the gut mucosal barrier, competition for adhesion, secretion of antimicrobial substances, etc.) and the modulation of the mucosal and systemic immune responses [28]. A recent study also reported that the serum exosomes isolated from Lactobacillus plantarum No.14treated mice reduced in vitro cytokine production [29]. Thus, we hypothesized that probiotics may elevate the immune function of broilers through the circulating exosomes with functional biomolecules, such as proteins, lipids and nucleic acids.
In the present study, we used DEX to induce the immunosuppression of broilers [30] and we found that P8 could improve the immune function of DEX-treated broilers, re ected by the decreased plasmal IL-1β, IL-6, TNF-α and jejunal IL-1β, as well as the increased plasmal IL-10 and jejunal IgM. Then, we isolated exosomes from plasma samples by using EIQ3 exosome isolation kit. The isolated plasmal exosomes were identi ed by morphological observation and biochemical analysis. We observed that the ultrastructure and size of plasma exosomes complied with the typical morphology of exosomes [31,32]. The surface markers of exosomes mainly included CD9, CD63, CD81, CD82, HSP27, HSP90, TSG101 and ALIX [33]. Here, the presence of exosomes was con rmed with the detection of CD63 and TSG101, and the purity of exosomes was con rmed by the absence of Calnexin.
In the past decades, the proteomic cargo of exosomes under immunosuppression have been investigated. Osteosarcoma exosomes contained immunosuppressive proteins including TGF-β, α fetoprotein and heat shock proteins [34]. Collagen type V alpha 2 chain (COL5A2) and lipoprotein lipase (LPL) were signi cant higher in ovarian cancer cells derived exosomes than ovarian surface epithelial cells [35]. In the present study, proteomic analysis uncovered that a total of 784 proteins were present in plasmal exosomes. Out of the total 784 proteins, DEX induced 126 DEPs compared to the CON group, while P8 + DEX induced 102 DEPs compared to the DEX group. Unfortunately, no other studies using DEX or probiotics have reported data on exosomal proteomic to serve for comparison with our results. Further, we explored the general trends in functional changes of exosomal proteins identi ed in the present study via GO analysis. Most of the DEPs between DEX and CON groups were in the organic substance metabolic process, nitrogen compound metabolic process, cellular anatomical entity, binding, protein binding and ion binding. Besides, most of the DEPs between P8 + DEX and DEX were in the multicellular organismal process and response to stimulus, cytoplasm and binding, indicating their critical roles in the metabolism, stimulation and cell differentiation, yet their veri cations merit further evaluating.
Furthermore, the proteins were analysed using KEGG database. DEPs between DEX and CON groups were mainly included in endocytosis (RABEP1/VPS37C/HSPA2/CHMP5/BF2/CHMP1A), phagosome (ITGB2/BF2) signaling pathway and so on, which might be involved in in ammation [36]. Besides, DEPs between P8 + DEX and DEX groups were mainly involved in ErbB signaling (PAK3/KRAS/CAMK2D), PPAR (ILK/FABP6) signaling pathway and so on. The ErbB signaling pathway is related to the development of cancer [37]. PAK3, KRAS and CAMK2D are genes involved in the ErbB signaling pathway. The decreased abundances of KRAS and CAMK2D in P8 + DEX group implied the attenuation of immunosuppression [38,39]. PAKs are important regulators of the in ammatory response. As reported by Taglieri et al. [40], only PAK1 and PAK2, but not PAK3, have been thus far associated with in ammation, immunity, and infective disease. Thus, the increased PAK3 expression in the present study may paly other biological roles rather than regulating the immunosuppression. Moreover, PPAR signaling pathway has antiin ammatory effects [41]. ILK and FABP6 are genes involved in the PPAR signaling pathway. The activation of PPAR upregulates ILK gene expression [42]. The elevated ILK abundance might indicate the decreased in ammation. In addition, FABP6 was high expressed in patients with cancer [43,44]. Hence, the decreased FABP6 level implied the alleviation of immunosuppression. This investigation offers insight into a potential role for circulating exosomes in regulation and function during immunosuppression.
To further con rm the effect of exosomal proteins on the immune function of broilers, the correlation analysis was performed between exosomal proteomic and immune parameters in plasma and jejunal mucosa. Among the DEPs that correlated with the immune parameters, we found that the expressions of protein E1C007 (PACSIN2), P35062 (HIST1H2A3), A0A1D5PMA3 (NELL2), A0A1L1RMF4, A0A3Q2U540, Q5W9C5 (BF1), R4GLT1 (CST3), E1C3Y3 (TSPAN8) and F1NLE7 (AIMP1) in the DEX group was higher than those of the CON and were lower than those of P8 + DEX group. Moreover, the expressions of protein R4GKL8 (C1QTNF3), Q9DER4 (ZP1), Q90WD0 (ACTR3) and A0A3Q2U3V9 in the DEX group were lower than those of the CON group and were higher than those of the P8 + DEX group. Reports have suggested that some of the aforementioned proteins, including E1C007 (PASCSIN2), A0A1D5PMA3 (NELL2), Q5W9C5 (BF1), R4GLT1 (CST3), E1C3Y3 (TSPAN8), were associated with the impairment of immune function, leading to immunosuppression [45][46][47][48][49], whereas, C1QTNF3 and ACTR3 were crucial for the normal immune function [50,51]. Results of the correlation analysis revealed that E1C007 was positively correlated with plasmal IL-1β, and E1C007, A0A1D5PMA3, Q5W9C5, R4GLT1, E1C3Y3 as well as F1NLE7 were negatively correlated with jejunal IgM, besides, R4GKL8 and Q90WD0 was negatively correlated with the jejunal IL-1β, indicating that the P8-induced plasmal exosomal proteins play an important role in improving the immune function of broilers.

Conclusion
In summary, we demonstrated that P8 effectively improved the immune function of DEX-induced immunosuppressive broilers. Moreover, a remarkable number of proteins involved in various biological processes, including ErbB and PPAR signalings are packed with plasmal exosomes from P8-treated immunosuppressive broilers. Correlation analysis indicated that the exosomal cargo of immunosuppressive broilers fed with P8 were involved in the improvement of immune function. These ndings shed some light on the bene cial role of probiotic in regulating immune function of broilers through plasmal exosomal proteins.      KEGG pathway analysis of proteins in plasmal exosomes.

Figure 5
Spearman correlation analyses of exosomal proteins and immune parameters.

Supplementary Files
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