The impact understanding of exosome therapy in COVID-19 and preparations for the future approaches in dealing with infectious diseases and inflammation

Cytokine storms, which result from an abrupt, acute surge in the circulating levels of different pro-inflammatory cytokines, are one of the complications associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. This study aimed to assess the effect of exosomes on the release of pro-inflammatory cytokines in patients with coronavirus disease 2019 (COVID-19) and compare it with a control group. The cytokines evaluated in this study were TNF-α, IL-6, IL-17, and IFN-γ. The study compared the levels of these pro-inflammatory cytokines in the peripheral blood mononuclear cells (PBMCs) of five COVID-19 patients in the intensive care unit, who were subjected to both inactivated SARS-CoV-2 and exosome therapy, with those of five healthy controls. The cytokine levels were quantified using the ELISA method. The collected data was analyzed in SPSS Version 26.0 and GraphPad Prism Version 9. According to the study findings, when PBMCs were exposed to inactivated SARS-CoV-2, pro-inflammatory cytokines increased in both patients and healthy controls. Notably, the cytokine levels were significantly elevated in the COVID-19 patients compared to the control group P-values were < 0.001, 0.001, 0.008, and 0.008 for TNF-α, IL-6, IL-17, and IFN-γ, respectively. Conversely, when both groups were exposed to exosomes, there was a marked reduction in the levels of pro-inflammatory cytokines. This suggests that exosome administration can effectively mitigate the hyperinflammation induced by COVID-19 by suppressing the production of pro-inflammatory cytokines in patients. These findings underscore the potential safety and efficacy of exosomes as a therapeutic strategy for COVID-19.


Electron microscopy
Images obtained through field emission scanning electron microscopy (FESEM) confirmed that the exosomes derived from MSCs were small, spherical, and less than 100 nm in size.The morphology of these exosomes was further analyzed using transmission electron microscopy (TEM) with negative staining.The TEM images offered a more intricate view of the exosomes derived from MSCs.These images revealed that the particle pellets were vesicles resembling a cup shape, with membranes attached (Fig. 3a,b).
The bicinchoninic acid assay protein assay (BCA) Additionally, the protein concentration was quantified using the BCA method.The resulting value, determined from the protein concentration standard curve depicted in Fig. 4, was measured to be 3482 µg/mL.

Dynamic light scattering technology (DLS)
The size distribution of the isolated exosomes was determined using the DLS.The DLS analysis revealed that the average size of the exosomes was 89.65 nm, with measurements taken at a constant temperature of 25 °C (Fig. 5).

Western blot analysis
The Western blot analysis detected the presence of CD9, a surface marker typically found on exosomes derived from MSCs.The protein content of the exosomes can be assessed by flow cytometry and Western blot, and the combination of these two methods results in an investigation of both the membrane-bound (CD9, CD63, and CD81) and internalized proteins (Tsg101 and Alix) of the exosomes.Detection of proteins enriched in exosomes, such as CD9, Tsg101, and Alix, and the absence of proteins, such as the endoplasmic reticulum protein calnexin, is an indication that the exosome-enriched pellet is indeed exosomes and not contaminating vesicles from other Figure 1.hUC-MSC morphology in culture conditions (× 100).hUC-MSC, Human umbilical cord-derived mesenchymal stem cells.compartments of the cell as is presented.The exosomes harvested from hUC-MSCs are devoid of any cellular components, as evidenced by the absence of the endoplasmic reticulum chaperone protein, calnexin (Fig. 6).

Determination of the dose
The assay was employed to identify the optimal dose of the isolated exosomes after 72 h (Fig. 7).Various doses of 10 µg/ml, 20 µg/ml, 40 µg/ml, and 60 µg/ml were evaluated for this purpose.Despite the differences in the  www.nature.com/scientificreports/calculated viability of the samples not being statistically significant, a dose of 40 µg/mL (the highest dose) was selected as the optimal dose for subsequent experiments.

Statistical society
Five patients were admitted to the hospital's intensive care unit (ICU), all of whom tested positive for COVID-19 via the PCR test, with cycle threshold (CT) values ranging between 15 and 25.The healthy controls consisted of five individuals who neither contracted the virus during the epidemic, nor received a vaccine for prevention.
In the patient group, three individuals (60%) were men, and two (40%) were women.The healthy group also comprised five individuals, including one (20%) man and four (80%) women.

Investigation of hematology, biochemistry, and coagulation tests in people with COVID-19
Upon admission to the ICU, the patients were subjected to laboratory tests to determine their white blood cell (WBC) and platelet (PLT) counts, as well as their blood erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and D-dimer levels (Table 1).

Efficacy of exosome treatment in reducing pro-inflammatory cytokines
The levels of four cytokines were measured in the culture supernatants of PBMCs.These conditions include 1. exposure of PBMCs to the RPMI 1640 medium as a negative control, 2. exposure of PBMCs to the inactivated virus, 3. treatment with exosomes after exposure to the negative control (RPMI 1640 medium), and 4. treatment with exosomes after exposure to the inactivated virus in both patients and healthy participants.To begin with, due to our small sample size, the Shapiro-Wilk test was used to assess the distribution of cytokines' levels in each group in patients and healthy participants.An independent T-test was performed for variables with normal distribution, and for others Mann-Whitney was used to compare the cytokine levels.Results are shown in Tables 2 and 3.All our results demonstrated a significant difference between patient and healthy participants for each condition except for TNF-α, IFN-γ and IL-17 levels in PBMCs exposed to inactivated virus and treated by exosome P-values were 0.055, 0.327, and 0.627 respectively.We used paired T-test and Wilcoxon Signed Ranks Test to compare cytokines' levels before and after PBMCs exposure to the inactivated virus and before and after PBMCs treatment with exosome.We observed a notable decrease in the levels of cytokines among the patient  group.Additionally, we found a significant reduction in cytokine levels among healthy controls whose PBMCs were stimulated with inactivated virus.P-values and effect sizes for these observations are available in Tables 4  and 5.The findings suggested that when PBMCs were stimulated with the inactivated virus for 24 h, there was a significant increase in the release of pro-inflammatory cytokines, such as IL-6, IFN-γ, TNF-α, and IL-17.Infection with SARS-CoV-2 resulted in the production of pro-inflammatory cytokines.Individuals with symptomatic COVID-19 had higher levels of four cytokines (IL-6, IFN-γ, TNF-α, and IL17) than healthy controls.In Fig. 8, the expression levels of cytokines (IL-6, IFN-γ, TNF-α, and IL17) were displayed for patients and healthy individuals under four different conditions mentioned above.

Discussion
This study aimed to investigate the impact of exosomes on the secretion of pro-inflammatory cytokines.Based on our findings, a therapeutic strategy for SARS-CoV-2 infection could effectively involve immunomodulatory treatment to regulate cytokine responses, in conjunction with antiviral treatment.Current evidence suggests that administering exosomes is beneficial for patients with hyperinflammation induced by COVID-19.This is due to the inhibition of the production of several cytokines, specifically IFN-γ and TNF-α, by activated T lymphocytes.
The results revealed that the levels of these cytokines were elevated in the culture supernatants of PBMCs from COVID-19 patients compared to those from healthy controls; this suggests that these cytokines could contribute to the pathogenesis of the disease.Furthermore, increased levels of IFN-γ and TNF-α have been linked to the severity of COVID-19, reinforcing their potential role in disease progression.Several clinical studies have found a correlation between the severity and mortality of COVID-19 and hyperinflammation.This hyperinflammation is marked by increased serum levels of pro-inflammatory cytokines and chemokines.Postmortem analyses have shown that high concentrations of these pro-inflammatory cytokines are associated with the infiltration of cells into organs, such as the kidneys, heart, and lungs [40][41][42] .Furthermore, Han et al. conducted a study where they observed cytokine storms, characterized by elevated serum levels of TNF-α and IL-6 43 .These storms were suggested to be indicators of the severity of the disease.In a similar vein, a retrospective observational study involving hospitalized COVID-19 patients revealed that if the serum level of IL-6 exceeded 30 pg/mL, it was a predictor of the need for invasive mechanical ventilation 44 .Notably, a comparable pattern of cytokine storms

Ethics approval and consent to participate
All participants in the study had the option to participate voluntarily, and their privacy was highly valued.Prior to entering the survey, all participants provided their informed consent for research participation.The participants were guaranteed that their personal information would be kept confidential and not be disclosed.All methods used in the study were in compliance with relevant guidelines and regulations.All methods were conducted in accordance with the Declaration of Helsinki and relevant guidelines and regulations.All experimental protocols were approved by an institutional and/or licensing committee.All experiments followed the guidelines of the Laboratory Ethical Commission of the Faculty of Medical Sciences, Tarbiat Modares University.After receiving Approval No. IR.TMU.REC.1400.017, the study complies with the rules and regulations.Ethical standards were strictly adhered to during all phases of this research.

Cell isolation and culture
In this study, umbilical cords were obtained from the Private Royan Umbilical Cord Blood Bank and transferred to the laboratory in phosphate-buffered saline (PBS, Gibco, Germany), containing 100 mg/mL of penicillin and streptomycin antibiotics (Gibco, Germany) under sterile conditions.After being washed, the cords were sectioned into 5-cm pieces each.The blood vessels, which included one large vein and two smaller arteries, were removed, and the Wharton's jelly was subsequently extracted.The cells were isolated using the explant method.For this purpose, the Wharton's jelly was formed into small spheres measuring 3-5 mm.They were then cultured in T-25 flasks filled with Dulbecco's modified eagle medium (DMEM, Gibco, Germany), 20% FBS, and 1% Pen-Strep.The cultures were then placed in a CO 2 incubator at a temperature of 37 °C.The culture media were renewed every three days.After observing tiny clumps of cells around the pieces on days 5-7, the culture medium was renewed every other day for up to seven days 23 .The passage was iterated three times to achieve a uniform cell population.It was then sent to the laboratory to confirm the presence of CD34, CD44, CD45, and CD90 surface markers.Cell surface marker expression in hUC-MSCs was determined from the third passage using a FACSCalibur flow cytometer (BD Biosciences, USA).The anti-human antibodies used for staining included CD34, CD44, CD45, and CD90 (all from eBioscience).

Isolation and purification of hUC MSC-derived exosomes
After reaching 80-90% confluency, MSCs at passage 2 were adapted to serum-free culture by gradually reducing serum concentrations over two weeks.After 48 h, the cell supernatants were collected and filtered through 0.22 μm filters.Exosomes were then extracted using an extraction kit (Exosib, Iran) with two reagents (A and B), according to the manufacturer's instructions.The culture supernatant was mixed with reagent A at a ratio of 5:1, vortexed for five minutes, and incubated at 4 °C overnight.It was then centrifuged at 3500 rpm for 40 min, and the resulting supernatant was discarded.The exosome sediment was mixed with 100 µL of reagent B and stored at − 80 °C for future studies.

Electron microscopy
The morphology and size of the exosomes were evaluated via FESEM (MIRA3 TESCAN) and TEM (Zeiss, EM10C).For FESEM imaging, 1 µg/mL of exosome solution was dried on a glass slide for 24 h and covered with a thin layer of gold.For TEM, exosomes derived from MSCs were fixed in paraformaldehyde and glutaraldehyde.www.nature.com/scientificreports/Subsequently, they were loaded on a formvar/carbon-covered mesh and contrasted with 2% uranyl acetate.Multiple fields of view were examined for both FESEM and TEM imaging.Representative images were selected based on their ability to accurately allocate the overall characteristics observed in the various fields of view.

The bicinchoninic acid assay protein assay
The total exosome content was extracted for quantification with a Bicinchoninic (BCA) Acid Protein Assay Kit (DNA Biotech, Iran), consisting of a standard solution, copper, and BCA reagents.The standard curve was plotted at different levels (50-250 µg/mL) against bovine serum albumin (BSA) as the standard solution.The exosomes and standard solutions were separately mixed with a mixture of copper and BCA reagents at a ratio of 1:50 and incubated at 60 °C for 60 min.Finally, the absorbance of the samples was read with a spectrophotometer at 562 nm (MPR4 + ; Hyperion, Roeder mark, Germany).

DLS technology
The size of the exosomes was determined using DLS with a Zeta device (Malvern Instruments, UK).PBS, with a refractive index of 33.1 and viscosity of 1.08, was used as the solvent.These parameters are essential for the device software to analyze the data.This simple, rapid, and nondestructive method, can measure particles ranging from a few nanometers to micrometers.The exosomes were diluted five times, and the sample was then read and analyzed using the Zetasizer APS.

Western blot analysis
The protein production was confirmed using the Western blotting analysis.For this purpose, the samples were centrifuged at 14,000 rpm for 20 min at 4 °C to eliminate the lysate.The protein concentration was measured using the BCA Protein Quantification Kit, following the instructions provided by the manufacturer.Equal volumes of lysate and Laemmli 2X sample buffer were combined, and then, 20 µg of this mixture was boiled for five minutes.Subsequently, it was subjected to SDS-PAGE and transferred to a 0.2-µm membrane (Immun-BlotTM, PVDF).Next, the membranes were blocked with 5% BSA in 0.1% Tween 20 for one hour and incubated with anti-CD9 (Cat.No.: ab223052, Abcam) and anti-calnexin control antibodies (Cat.No.: ab133615, Abcam) to show the purity of the extracted exosome from contaminated cellular components, at room temperature for one hour.The membranes were washed with TBST three times and incubated with goat anti-rabbit IgG (H&L) secondary antibody.They were then incubated with enhanced chemiluminescence immunoassay for 1-2 min.

MTT assay
The cytotoxicity of exosomes was evaluated using the colorimetric MTT assay.For this purpose, 100 µL of DMEM culture medium containing 10 6 PBMCs was added to each well of a 96-well plate.The MTT assay was performed after 72 h by adding 100 μL of MTT solution (5 mg/mL in PBS) into each well.The cells were then incubated for four hours.The MTT solution was removed, and 100 µL of dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA) was added to each well to dissolve the purple formazan crystals.The cytotoxic activity of exosomes was then evaluated by a standard MTT assay.The optical absorbance at 570 nm was measured using a microplate reader (ELISA reader, ELX808, BioTek).The results were reported as the rate of viability based on the concentration curve.All tests were performed in three iterations.The relative cell viability was calculated as follows: Relative cell viability (%) = (ODs/ODc) × 100.

Patients and sample collection
The relative cell viability was calculated as follows: Blood samples were collected from five COVID-19 patients admitted to the ICU, who were selected between July and October 2021 in Tehran, Iran.The inclusion criteria were a positive real-time PCR result with a CT value of 15-25, an age range of 20-40 years, and no history of any underlying diseases.Tests for CBC, CRP, ESR, D-dimer, and IL-6 were conducted to ensure that the patients were in similar conditions in terms of disease severity.The exclusion criteria were any dissatisfaction with sampling on the part of the patients and any deterioration in the patient's condition.

Isolation and culture of PBMCs
Three milliliters of blood, treated with heparin, was collected from the patients and then diluted with an equal volume of PBS.This mixture was then added to 2 mL of Ficoll ® (Innotriane, Germany) and centrifuged at 2400 rpm for 20 min.The PBMCs, which appeared as a cloudy layer between the Ficoll ® and the diluted blood, were carefully collected using a Pasteur pipette.The collected cells were suspended in 2 mL of PBS and centrifuged at 2000 rpm for 10 min to remove Ficoll ® ; this step was repeated at 1000 rpm to remove platelets.

PBMCs exposed to inactivated virus
Following a 3-hour incubation period, the PBMCs from each patient were cultivated in six wells.The titer of SARS-CoV-2 was determined to be 10 7.66 ID 50 /mL.Three of the PBMC culture wells were exposed to 0.3 µL of inactivated SARS-CoV-2, while the remaining three wells, which contained PBMCs but were not exposed to the virus, served as negative controls (RPMI 1640 medium).

PBMCs exposed to exosomes
After 24 h, the cell supernatant was collected, centrifuged, and stored at − 80 °C until further evaluation.Subsequently, 40 µg/mL of exosomes was added to the wells containing the control sample from a healthy individual, as well as to the other wells containing patient samples.They were then incubated for 72 h.www.nature.com/scientificreports/

Measurement of cytokines
Following exposure to the inactivated SARS-CoV-2 virus and exosomes, the supernatant from the cell culture was used to measure the levels of IL-6, IFN-γ, IL-17, and TNF-α.This was accomplished using the ELISA method, with the Human Cytokines Measurement Kit.

Statistical analysis
The gathered data was analyzed using SPSS Version 26.0 and GraphPad Prism 9.An independent sample t-test was employed to compare the mean values between patient and healthy participants.For data with a nonparametric distribution, the Mann-Whitney U test was used for analysis.A P-value of less than 0.05 was considered statistically significant.For data with normal distribution and for non-parametrical data Paired t-tests and Wilcoxon were respectively performed to evaluate effects of exosome treatment in cytokines levels variations.Continuous variables were expressed as either the mean with standard deviation or the median with interquartile range.Categorical variables were represented as frequency percentages. Vol

Figure 2 .
Figure 2.Histogram of a flow cytometric analysis: hUC-MSCs were positive for CD44 and CD90 with percentages of 99.8% and 97.3% of total cells, respectively, and negative for CD34 and CD45 with percentages of 1.85% and 2.05% of total cells, respectively.

Figure 4 .
Figure 4.The BSA calibration curve was determined by utilizing the Protein Assay BCA Kit and analyzing the protein concentration.BSA, Bovine serum albumin; BCA, The bicinchoninic acid assay protein assay.

Figure 5 .
Figure 5. Results of DLS analysis for exosome size distribution.DLS, Dynamic light scattering technology.

Figure 6 .
Figure 6.Western blot analysis: (a) the presence of the surface marker CD9 (Molecular weight: 24 kDa) in exosome and MSC cells as negative control.(b) the Western blot analysis for Calnexin (Molecular weight: 69 kDa) as a negative control in exosomes and positive in MSC cells.

Table 2 .
Comparing cytokine levels after interventions between patients and healthy controls using independent t-test.SD, Standard deviation; α = 0.05, P-values < 0.05 are significant, n = 10 (5 participants in each group).