Periodic Reporting for period 1 - LactoVES (Immunomodulatory properties of L. plantarum extracellular vesicles in airway allergy)
Periodo di rendicontazione: 2022-07-01 al 2024-07-31
Sintesi del contesto e degli obiettivi generali del progetto
Allergic rhinitis (AR) is a common condition affecting up to 30% of European adults. It significantly reduces quality of life by disrupting daily activities, sleep, and work. AR is closely linked to early-onset asthma, a chronic lung condition. AR occurs when the immune system overreacts to allergens like pollen, which can also cause cross-reactions to certain fruits and vegetables, worsening symptoms. AR leads to substantial economic costs, with the U.S. alone spending around $3.4 billion annually on direct costs, including medication, and an estimated $10 billion in indirect costs. Current treatments involve long and painful desensitization processes that can risk severe allergic reactions, highlighting the need for better alternatives.
One promising area of research involves probiotics—live bacteria that, when taken in the right amounts, offer health benefits. Some studies suggest probiotics can reduce allergic reactions, but there are risks, especially for people with weakened immune systems. A new approach focuses on postbiotics, which are non-living byproducts of probiotics. These may offer similar benefits without the risks connected with the usage of live bacteria. One of the postbiotics are bacterial extracellular vesicles (EVs), tiny structures released by bacteria. Research is now exploring the potential of EVs from a specially engineered strain of bacteria, L. plantarum, to prevent or treat allergies, especially through nasal application.
This project aims to test the possibility of bacterial EVs intranasal usage in the therapy or prevention of allergy. The results could align with cutting-edge scientific research on microbial therapies and immune tolerance.
From the scientific point of view this project will answer the following questions: Do EVs from probiotic bacteria behave like the bacteria they come from, or do they have entirely different characteristics? Could they be used in therapies instead of whole bacteria? The results of this research will align with cutting-edge scientific trends and significantly contribute to our understanding of both microbiology and immunology. The knowledge gained could one day lead to new allergy therapies based on EVs from probiotic bacteria. Additionally, the protocols developed in this project will be applicable to studying vesicles from other probiotic strains that are planned to be used in the clinics. From the economical point of view, given that up to 30% of the population suffers from allergies, the potential impact of this research is vast. Allergies are a growing public health concern, and any new insights could be crucial in developing better, more effective and less costly treatments.
One promising area of research involves probiotics—live bacteria that, when taken in the right amounts, offer health benefits. Some studies suggest probiotics can reduce allergic reactions, but there are risks, especially for people with weakened immune systems. A new approach focuses on postbiotics, which are non-living byproducts of probiotics. These may offer similar benefits without the risks connected with the usage of live bacteria. One of the postbiotics are bacterial extracellular vesicles (EVs), tiny structures released by bacteria. Research is now exploring the potential of EVs from a specially engineered strain of bacteria, L. plantarum, to prevent or treat allergies, especially through nasal application.
This project aims to test the possibility of bacterial EVs intranasal usage in the therapy or prevention of allergy. The results could align with cutting-edge scientific research on microbial therapies and immune tolerance.
From the scientific point of view this project will answer the following questions: Do EVs from probiotic bacteria behave like the bacteria they come from, or do they have entirely different characteristics? Could they be used in therapies instead of whole bacteria? The results of this research will align with cutting-edge scientific trends and significantly contribute to our understanding of both microbiology and immunology. The knowledge gained could one day lead to new allergy therapies based on EVs from probiotic bacteria. Additionally, the protocols developed in this project will be applicable to studying vesicles from other probiotic strains that are planned to be used in the clinics. From the economical point of view, given that up to 30% of the population suffers from allergies, the potential impact of this research is vast. Allergies are a growing public health concern, and any new insights could be crucial in developing better, more effective and less costly treatments.
Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti
Performed scientific actions:
1. Design of production and purification protocol of LplEVs. We started the project by developing a protocol for obtaining vesicles from L. plantarum. As part of this task, the appropriate type, time and volume of bacterial culture were selected, as well as an effective method for vesicle purification. An analysis of the quality of the obtained EVs (size and particle concentration) and the repeatability of the method (by measuring EVs from multiple batches) were also carried out. Two methods of EV purification were compared: size exclusion chromatography (SEC) and density gradient ultracentrifugation (DG-UC). It was decided to use the SEC method due to the effectiveness and repeatability of the method.
The main findings of this action:
- the time of the culture influences the profile of isolated EVs, longer times of culture result in a significant variability in EVs sizes and the possible contamination of samples with bacteria debris
- the production is replicable in the terms of the size of the purified EVs and the final amounts of EVs
The effect of this task is the preparation of a protocol for the isolation and purification of EVs.
2. Analyzing the allergen content in the LpEVs. In our project, we planned to use two strains of L. plantarum, one producing the Bet v1 allergen and one wild type (WT) strain not producing the allergen. We isolated EVs from both strains and determined the allergen content using the Western Blot technique and specific anti-Bet v 1 antibodies. It turns out that purification of vesicles (with both methods SEC and DG-UG) causes the loss of allergen from the sample. The result of the task - for such applications, a bacterial strain should be developed that will produce allergen covalently linked to EVs so that it is not removed in the purification process. This work is ongoing but we were not able to finish it within the given time. Due to the fact that our vesicles are to be used in the clinic in the future, a high degree of purification is required and therefore we decided not to further study unpurified EVs but to focus on further characterization of EVs from the wild strain.
The main finding of this action is that purification methods are effectively removing the allergen that is present in crude EVs.
Effect of this task – the selection of WT strain EVs for further characterization and experiments.
3. Visualization of EVs. One of the methods to prove that EVs are actually isolated and not contamination from the culture is their visualization using one of the available microscopic methods. As part of the project and established cooperations, microscopic analysis of LpEVs was performed using TEM, cryo-EM and AFM methods.
The main finding of this action is that we are effectively isolating LpEVs with the stablish methods and their sizes measured with microscopy are similar to the ones measured by Dynamic Light Scattering (Zetasizer) in our Lab.
Effect of this task - images showing the morphology of LpEVs.
4. Characterizing of LpEVs cargo. Multiple methods were used to analyze the cargo and surface properties of LpEVs like: zeta potential measurement, SDS-PAGE analysis, proteomics, lipidomics, isolation and Bioanalyzer analysis of nucleic acids (DNA and RNA), lipoteichoic acid analysis with Western Blot and specific anti-LTA antibodies and peptidoglycan measurement with chemical methods. This qualitative and quantitative methods were supplemented with biological evaluation of the cargo using human reporter cell lines that express receptors specific for the structures mentioned above.
The main findings of this action:
-purification of the LpEVs is significantly reducing the protein and peptidoglycan content of EVs. There is small RNA in the EVs but no detectable DNA. Also,
- we have found LTA associated with EVs, which is suggested to be the EV marker for G+ bacteria EVs.
- EVs zeta potential is lower than bacteria ZP, which might be connected with the distinct surface properties.
Effect of this task – fully characterized LpEVs.
5. In vitro analysis of LpEVs’ biological activity. Due to the fact that bacterial EVs could be used in nasal preparations in the future, their safety and their effect on epithelial cells and immune cells should be investigated. These studies were conducted in vitro using cell lines. The cytotoxicity of LpEVs and the profile of induced cytokines were examined in human respiratory epithelial cell lines and mouse macrophage lines. Furthermore, we investigated how LpEVs affect inflammation in an in vitro model of inflammation using lipopolysaccharide.
The main findings of this action:
- LpEVs do not show cytotoxicity in human cell lines
- LpEVs do not induce pro-inflammatory response in human cell lines
- LpEVs lower the inflammation in an inflammatory in vitro model
Effect of this task – we show in vitro that LpEVs are safe to use.
6. Analysis of the EVs long-term stability. One type of preclinical study is to show that a given formulation is stable over an extended period of time. We tested the stability of LpEVs by storing them in five different buffers (PBS, PBS-A, HEPES, HEPES-N, HEPES-NA) and at three different temperature conditions (4, -20 and -80 deg.). We examined the size and number of particles at baseline and after 6 and 12 months.
Main finding of this action is that LpEVs are stable for at least a year when frozen. Also, it is not necessary to freeze them in -80 deg. since -20 deg is enough, which is important from the point of view of future usage.
Effect of this task – pre-clinical analysis of LpEVs long-term stability.
7. Animal allergy experiment (OVA). The antiallergic properties of LpEVs were studied in intranasal administration in allergic mice sensitized with ovalbumin (OVA). Mice received LpEVs 30 min before each dose of OVA. The effect of LpEVs was studied in three doses. In the case of the highest dose, a visible but statistically insignificant improvement was achieved in such parameters as the number of eosinophils in the lungs or the level of pro-allergic cytokines (IL-5, IL-13) in re-stimulated lung cells.
Main findings of this action:
- LpEVs are safe to use in mice since we did not observe any signs of distress.
- We have initially demonstrated the feasibility of using LpEVs for allergy prophylaxis, but it appears that larger doses or EVs with a larger surface area are needed.
Effect of this task – we show that EVs might have similar activity as their parent bacteria, but the effect is dependent on the right dose.
8. EVs and bacteria stability in physiological conditions. In the next step, we examined the stability of LpEVs in physiological conditions. To reproduce these conditions, we used buffers with different pH, salt concentration and detergent content (such as can be found in the small intestine) and then measured the size and number of particles. We also tested the viability of bacteria in the same conditions.
Main findings of this action:
- LpEVs are extremely stable in the physiological conditions
- In turn, bacteria seem to be sensitive to pH and salt change as well as to low amounts of SDS
Effect of the task – we show that some physiological conditions are stressors for the bacteria but they do not affect EVs.
9. Production of bile-induced EVs, characteristics. Based on the above we decided to produced stressed-EVs
Main findings of this action:
- stressed EVs are different than native EVs
- bacteria are able to produce different EVs depending on the environment and this might be one of their coping mechanism with changing environment
Effect of this action – bigger “stressed” LpEVs that might have different/better immunomodulatory EVs than “native” EVs.
1. Design of production and purification protocol of LplEVs. We started the project by developing a protocol for obtaining vesicles from L. plantarum. As part of this task, the appropriate type, time and volume of bacterial culture were selected, as well as an effective method for vesicle purification. An analysis of the quality of the obtained EVs (size and particle concentration) and the repeatability of the method (by measuring EVs from multiple batches) were also carried out. Two methods of EV purification were compared: size exclusion chromatography (SEC) and density gradient ultracentrifugation (DG-UC). It was decided to use the SEC method due to the effectiveness and repeatability of the method.
The main findings of this action:
- the time of the culture influences the profile of isolated EVs, longer times of culture result in a significant variability in EVs sizes and the possible contamination of samples with bacteria debris
- the production is replicable in the terms of the size of the purified EVs and the final amounts of EVs
The effect of this task is the preparation of a protocol for the isolation and purification of EVs.
2. Analyzing the allergen content in the LpEVs. In our project, we planned to use two strains of L. plantarum, one producing the Bet v1 allergen and one wild type (WT) strain not producing the allergen. We isolated EVs from both strains and determined the allergen content using the Western Blot technique and specific anti-Bet v 1 antibodies. It turns out that purification of vesicles (with both methods SEC and DG-UG) causes the loss of allergen from the sample. The result of the task - for such applications, a bacterial strain should be developed that will produce allergen covalently linked to EVs so that it is not removed in the purification process. This work is ongoing but we were not able to finish it within the given time. Due to the fact that our vesicles are to be used in the clinic in the future, a high degree of purification is required and therefore we decided not to further study unpurified EVs but to focus on further characterization of EVs from the wild strain.
The main finding of this action is that purification methods are effectively removing the allergen that is present in crude EVs.
Effect of this task – the selection of WT strain EVs for further characterization and experiments.
3. Visualization of EVs. One of the methods to prove that EVs are actually isolated and not contamination from the culture is their visualization using one of the available microscopic methods. As part of the project and established cooperations, microscopic analysis of LpEVs was performed using TEM, cryo-EM and AFM methods.
The main finding of this action is that we are effectively isolating LpEVs with the stablish methods and their sizes measured with microscopy are similar to the ones measured by Dynamic Light Scattering (Zetasizer) in our Lab.
Effect of this task - images showing the morphology of LpEVs.
4. Characterizing of LpEVs cargo. Multiple methods were used to analyze the cargo and surface properties of LpEVs like: zeta potential measurement, SDS-PAGE analysis, proteomics, lipidomics, isolation and Bioanalyzer analysis of nucleic acids (DNA and RNA), lipoteichoic acid analysis with Western Blot and specific anti-LTA antibodies and peptidoglycan measurement with chemical methods. This qualitative and quantitative methods were supplemented with biological evaluation of the cargo using human reporter cell lines that express receptors specific for the structures mentioned above.
The main findings of this action:
-purification of the LpEVs is significantly reducing the protein and peptidoglycan content of EVs. There is small RNA in the EVs but no detectable DNA. Also,
- we have found LTA associated with EVs, which is suggested to be the EV marker for G+ bacteria EVs.
- EVs zeta potential is lower than bacteria ZP, which might be connected with the distinct surface properties.
Effect of this task – fully characterized LpEVs.
5. In vitro analysis of LpEVs’ biological activity. Due to the fact that bacterial EVs could be used in nasal preparations in the future, their safety and their effect on epithelial cells and immune cells should be investigated. These studies were conducted in vitro using cell lines. The cytotoxicity of LpEVs and the profile of induced cytokines were examined in human respiratory epithelial cell lines and mouse macrophage lines. Furthermore, we investigated how LpEVs affect inflammation in an in vitro model of inflammation using lipopolysaccharide.
The main findings of this action:
- LpEVs do not show cytotoxicity in human cell lines
- LpEVs do not induce pro-inflammatory response in human cell lines
- LpEVs lower the inflammation in an inflammatory in vitro model
Effect of this task – we show in vitro that LpEVs are safe to use.
6. Analysis of the EVs long-term stability. One type of preclinical study is to show that a given formulation is stable over an extended period of time. We tested the stability of LpEVs by storing them in five different buffers (PBS, PBS-A, HEPES, HEPES-N, HEPES-NA) and at three different temperature conditions (4, -20 and -80 deg.). We examined the size and number of particles at baseline and after 6 and 12 months.
Main finding of this action is that LpEVs are stable for at least a year when frozen. Also, it is not necessary to freeze them in -80 deg. since -20 deg is enough, which is important from the point of view of future usage.
Effect of this task – pre-clinical analysis of LpEVs long-term stability.
7. Animal allergy experiment (OVA). The antiallergic properties of LpEVs were studied in intranasal administration in allergic mice sensitized with ovalbumin (OVA). Mice received LpEVs 30 min before each dose of OVA. The effect of LpEVs was studied in three doses. In the case of the highest dose, a visible but statistically insignificant improvement was achieved in such parameters as the number of eosinophils in the lungs or the level of pro-allergic cytokines (IL-5, IL-13) in re-stimulated lung cells.
Main findings of this action:
- LpEVs are safe to use in mice since we did not observe any signs of distress.
- We have initially demonstrated the feasibility of using LpEVs for allergy prophylaxis, but it appears that larger doses or EVs with a larger surface area are needed.
Effect of this task – we show that EVs might have similar activity as their parent bacteria, but the effect is dependent on the right dose.
8. EVs and bacteria stability in physiological conditions. In the next step, we examined the stability of LpEVs in physiological conditions. To reproduce these conditions, we used buffers with different pH, salt concentration and detergent content (such as can be found in the small intestine) and then measured the size and number of particles. We also tested the viability of bacteria in the same conditions.
Main findings of this action:
- LpEVs are extremely stable in the physiological conditions
- In turn, bacteria seem to be sensitive to pH and salt change as well as to low amounts of SDS
Effect of the task – we show that some physiological conditions are stressors for the bacteria but they do not affect EVs.
9. Production of bile-induced EVs, characteristics. Based on the above we decided to produced stressed-EVs
Main findings of this action:
- stressed EVs are different than native EVs
- bacteria are able to produce different EVs depending on the environment and this might be one of their coping mechanism with changing environment
Effect of this action – bigger “stressed” LpEVs that might have different/better immunomodulatory EVs than “native” EVs.
Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)
Results beyond the state of the art
Our project allowed for the first such preclinical analysis of bacterial EVs for intranasal use. We determined stability in physiological conditions and long-term stability, confirmed the safety of their intranasal use in vitro and in vivo. We also showed that bacteria produce altered vesicles in stressful conditions. Perhaps this opens the way to the production of "stress" vesicles with specialized immunomodulatory properties.
Further actions needed:
It seems that native EVs obtained in stable laboratory conditions may not have sufficiently strong immunomodulatory properties. The next step would be, based on the knowledge obtained in this project, to produce stress EVs in inflammatory conditions, they should be characterized and used in an allergy model. Another possibility would be to genetically modify the strain by introducing genes of the SpyTag/Catcher system, thanks to which it would be possible to decorate EVs with any allergens and such EVs could also be tested in an allergy model.
Our project allowed for the first such preclinical analysis of bacterial EVs for intranasal use. We determined stability in physiological conditions and long-term stability, confirmed the safety of their intranasal use in vitro and in vivo. We also showed that bacteria produce altered vesicles in stressful conditions. Perhaps this opens the way to the production of "stress" vesicles with specialized immunomodulatory properties.
Further actions needed:
It seems that native EVs obtained in stable laboratory conditions may not have sufficiently strong immunomodulatory properties. The next step would be, based on the knowledge obtained in this project, to produce stress EVs in inflammatory conditions, they should be characterized and used in an allergy model. Another possibility would be to genetically modify the strain by introducing genes of the SpyTag/Catcher system, thanks to which it would be possible to decorate EVs with any allergens and such EVs could also be tested in an allergy model.