This invention relates to a method of increasing the immunomodulatory effect, for example anti-inflammatory effect, of certain bacterial strains of Lactobacillus spp., by the use of specific growth conditions, product formulations, products and methods using such bacteria for immunomodulatory purposes in a host, such as treatment and prophylaxis of inflammation caused by inflammation-causing agents.
The Food and Agricultural Organization of the United Nations define probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Nowadays, a number of different bacteria are used as probiotics for example, lactic-acid producing bacteria such as strains of Lactobacillus and Bifidobacteria.
Prebiotics are defined as “non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one, or a limited number of bacteria in the colon that can improve the host health” Targets for prebiotics are usually bifidobacteria and lactobacilli. However, the selectivity of prebiotics is not always fully established, and hence stimulation of beneficial genera alone may be difficult to achieve. To alleviate the limitations of the probiotic and prebiotic approach, a solution could be to combine both in the form of a synbiotic.
Synbiotics were defined around a decade ago by Gibson and Roberfroid (1995) as “mixtures of pro- and prebiotics, which beneficially affect the host, by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract (GT)”. The prebiotic should be a specific substrate for the probiotic, being able to stimulate its growth and/or activity while at the same time enhancing indigenous beneficial bacteria.
Lactic-acid producing bacteria are not only used for their beneficial effect on human or animal health, but they are also widely used in the food industry for fermentation processes. The effectiveness of probiotics is strain-specific, and each strain may contribute to host health through different mechanisms. Probiotics can prevent or inhibit the proliferation of pathogens, suppress production of virulence factors by pathogens, or modulate the immune response in a pro-inflammatory or an anti-inflammatory way. Use of different strains of the probiotic lactic-acid producing bacteria Lactobacillus reuteri is a promising therapy for the amelioration of infantile colic, alleviation of eczema, reduction of episodes of workplace illness, and suppression of Helicobacter pylori infection. L. reuteri is considered an indigenous organism of the human gastrointestinal tract and is present on the mucosa of the gastric corpus, gastric antrum, duodenum, and ileum. See, for example U.S. Pat. Nos. 5,439,678, 5,458,875, 5,534,253, 5,837,238, and 5,849,289. When L. reuteri cells are grown under anaerobic conditions in the presence of glycerol, they produce the antimicrobial substance known as reuterin (β-hydroxy propionaldehyde).
Monocytes leave the bone marrow and travel through the peripheral blood vessels until they reach the mucosa/serosa of the gastrointestinal tract. These putative macrophages are key to the interaction and propagation of the signals necessary to regulate the immune system. In the gastrointestinal tract for example, there is a constant level of immune response in the macrophages of the mucosal epithelium to the bacteria in the intestinal lumen and attached to the intestinal mucosa. In the normal state, this response involves the generation of cytokine signals to restrict and contain an unnecessary inflammatory response. However, when a pathogen or toxin is presented to these cells, they form the first line of defense and react by producing an increasing amount of pro-inflammatory cytokines, which propagate the inflammatory response until the threat is removed. The generation of cytokines relevant to the interactions with commensal (non-threatening) bacteria as well as those involved in the full inflammatory response to pathogens is subject to intervention by lactic acid bacteria themselves (including surface antigens) or by substances produced by these lactic acid bacteria and it is clear that the commensal flora has extensive interaction with the macrophages of the mucosa to maintain a balanced reaction to the gut flora and thereby maintain optimal health.
It is known that various pathogens can cause inflammation, for example in the gastrointestinal tract. Such inflammation, for example, in the stomach and gastrointestinal tract, is mediated by intercellular signal proteins known as cytokines, which are produced by macrophages and dendritic cells in the epithelium in response to an antigenic stimulus such as that produced by a pathogen. Upon contact between the epithelium and the antigen of a pathogen or endotoxins produced by it, such as lipopolysaccharides (LPS), antigen presenting cells (including dendritic cells) in the epithelium propagate the signal to naive macrophages which then respond in a so-called Th-1 type response where pro-inflammatory cytokines including TNFα, IL-1, IL-6, IL-12 are produced by the macrophages. These cytokines in turn stimulate natural killer cells, T-cells and other cells to produce interferon γ (IFNγ), which is the key mediator of inflammation. IFNγ leads to an escalation of the inflammatory response and the reactions described above that lead to cytotoxicity. Naive macrophages can also respond to antigens with a Th-2 type response. This response is suppressed by IFNγ. These Th-2 type cells produce anti-inflammatory cytokines such as IL-4, IL-5, IL-9 and IL-10.
IL-10 is known to inhibit the production of IFNy and thus dampen the immune response. The balance between Th-1 and Th-2 type cells and their respective cytokine production defines the extent of the inflammation response to a given antigen. Th-2 type cells can also stimulate the production of immunoglobulins via the immune system. Anti-inflammatory activity in the gastrointestinal tract, where there is a reduced TNFα level, correlates with enhanced epithelial cells (gut wall lining integrity) and thus to a reduction in the negative effects caused by gastrointestinal pathogens and toxins.
Inflammation can be involved in several diseases in mammals both externally, for example on skin and eyes, and internally for example on various mucous membranes, in the mouth, gastrointestinal (GI) tract, vagina etc. but also in muscles, bone-joints, cardio vascular organs and tissues, including blood vessels and in brain-tissue and the like. In the GI tract there are several diseases connected to inflammation, for example, gastritis, ulcers, and inflammatory bowel disease (IBD). The disease has been linked to imbalances in the gut microflora and an over-expressed inflammatory reaction to components of the normal gut flora and this reaction is currently treated with poor success using a series of different drugs, one of which is based on anti-TNFα therapy designed to reduce the levels of TNFα in the gastro-intestinal mucosa. There are also several other diseases associated with inflammation, such as gingivitis, vaginitis, atherosclerosis, and various cancer forms that are thought to be associated with the composition of the microflora in different localities of the body.
With the shortcomings of abovementioned anti-TNFα therapies in mind it was a positive surprise when the inventors of the invention herein demonstrated that the replacement of sucrose by glucose as the sole carbon source for the growth of specific anti-inflammatory strains of L. reuteri in a defined medium significantly inhibited the TNFα production in LPS-stimulated macrophages. Accordingly growing specific anti-inflammatory Lactobacillus strains with a defined carbon source such as glucose gives the opportunity to provide anti-inflammatory strains of L. reuteri with even more potent anti-inflammatory properties.
As can be understood from the invention herein, also pro-inflammatory bacterial strains can be modified in their immunomodular properties, by modifying the carbon source for the bacteria to grow in.
As mentioned before, anti-inflammatory activity has already been associated with various lactobacilli for example U.S. Pat. No. 7,105,336 B2 describes Lactobacillus strains selected for their ability to reduce gastrointestinal inflammation associated with H. pylori infection in mammals using a mouse macrophage assay for TNFα activity. Another patent application mentioning the anti-inflammatory activity of L. reuteri is U.S. 2008/0254011 A1 describing strains of lactic acid bacteria selected for their capability of increasing the BSH-activity and consequently lowering serum LDL-cholesterol, and simultaneously decreasing the pro-inflammatory cytokine TNF-alpha levels for the treatment of cardiovascular diseases.
U.S. 2006/0233775 A1 describes the selection of strains of lactic acid bacteria selected for their capability of reducing inflammation, such as intestinal bowel disease. However none of above mentioned inventions attaches an importance to the choice of carbon source for the decrease in TNFα production.
Regulating other different activities of Lactobacillus spp. by the choice of different sugars is already known in the art. For example, Ávila et al (2009) showed that the growth condition was important for the regulation of α-L-rhamnosidase, as the activity was down regulated by the addition of glucose.
Growth behavior on glucose and sucrose has been studied by Årsköld et al (2008). It was shown that the choice of sugar source clearly affected the growth performance of L. reuteri ATCC 55730. Growth on sucrose resulted in a high growth rate and an appropriate biomass yield, whereas growth with glucose was characterized by a maximum specific growth rate and low ATP levels.
However nobody has hitherto demonstrated that specific carbon sources in the growth media can regulate the TNFα production. It is therefore one object of the invention to enhance the anti-inflammatory effect of already anti-inflammatory strains of L. reuteri as seen for example by reduced TNFα production in a host. It is a further object of the invention to provide products containing said strains, including agents for treatment or prophylaxis of inflammation caused by inflammation-causing agents for administration to humans, including conditioned media in which said strains have grown and protein-containing extracts thereof.
Another object of the invention is to provide products containing said strains together with a specific carbon source, in order to have a synbiotic product. A further object of this invention is to provide specific carbon source, such as sugar for consumption to individuals already colonized with anti-inflammatory strains of lactic acid bacteria.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.
The invention herein provides a specific method of improving immunomodulatory properties of lactic acid bacterial strains using growth media with a specific carbon source, including a method of increasing the anti-inflammatory effect of nonpathogenic anti-inflammatory bacterial strains, by the use of specific growth conditions.
A primary object of the present invention is to increase the immunomodulatory effect in mammals, of certain bacterial strains of lactic acid bacteria, by the use of specific growth conditions.
It is another object to enhance the anti-inflammatory effect of anti-inflammatory strains of L. reuteri by the choice of carbon source in the growth media.
Another object of the invention is to enhance the anti-inflammatory effect, in mammals seen as a decreased TNF-α production, of an anti-inflammatory L. reuteri strain together with glucose, lactose, fructose, starch, 1,2 propanediol or a prebiotic such as fructooligosaccharides as a primary carbon source in the growth media.
It is a further object of the invention to provide products containing said strains.
It is a further object of the invention to provide products containing said strains together with a specific carbon source, in order to have a synbiotic product.
Another object of this invention is to provide specific carbon sources in such a way that they are not digestible in the gastrointestinal tract such as prebiotic for consumption to individuals already colonized with anti-inflammatory strains of L. reuteri.
Other objects and advantages of the present invention will become obvious to the reader and it is intended that these objects and advantages are within the scope of the present invention.
Lactobacillus reuteri is a heterofermentative lactic acid bacterial species that naturally inhabits the gut of humans and animals. Specific probiotic L. reuteri strains potently suppress human TNFα production while other probiotic L. reuteri strains enhance human TNFα production.
In order to show how anti-inflammatory strains of L. reuteri grown with different carbon sources effect TNFα production, L. reuteri (ATCC 5289) was grown anaerobically in a defined medium with different sugars as sole carbon source until late stationary phase. Surprisingly, growth on glucose as the carbon source significantly decreased the production of TNF-α compared to growth on sucrose. The results are shown in
A study was performed to identify how glucose and sucrose in the growth media of different strains of L. reuteri influence on TNF-α production. Strains of L. reuteri already known to be TNF-inhibitory (ATCC PTA 6475 and ATCC PTA 5289) or TNF-stimulatory (ATCC 55730 and CF483A) were grown anaerobically in a defined medium with glucose or sucrose as sole carbon source until late stationary phase (24-28 hours). These results show that growth using sucrose as the primary carbon in for example the strains L. reuteri ATCC PTA-6475 and L. reuteri ATCC PTA-5289 significantly increased the production of LPS-stimulated TNF-α in human cells (
The results showed in
The effect of other carbon sources was also studied. L. reuteri (ATCC PTA 6475, DSM 17938) was grown anaerobically in a defined medium with glucose, 1,2 propanediol or starch as sole carbon source until late stationary phase. These results showed that DSM 17938 becomes TNF inhibitory when grown on 1,2 propanediol or starch as sole carbon source and ATCC PTA 6475 showed similar results for all three carbon sources (
Further this increased TNF-inhibitory effect is also seen when L. reuteri is grown in defined medium with lactose or fructose as sole carbon source (data not shown).
The strains of L. reuteri grown in defined medium with glucose, lactose, fructose, starch or 1,2 propanediol that are capable of decreasing the TNFα production include but are not limited to ATCC PTA 6475, ATCC PTA 5289, ATCC 4659, JCM 1112, and DSM 20016. A list of carbons sources that will affect L. reuteri to decrease TNFα production can be seen in
Products containing strains or conditioned medium capable of decreasing TNF-α production can be supplemented with specific carbon sources for example glucose after freeze-drying, in order to have a synbiotic product.
Carbon sources that together with lactic acid bacteria are capable of decreasing the TNFα production are preferably but not limited to glucose, lactose, fructose, starch, 1,2 propanediol or a prebiotic such as fructooligosaccharides with different degree of polymerization for example Synergy 1® (mixture of fructooligosaccharides and inulin, Orafti).
The product is preferably formulated but not limited to a tablet or a capsule.
The carbon source is integrated in the product in such a way that it will not be digested in the gastrointestinal tract, for example the glucose can be encapsulated separately in microcapsules as known in the art, before integrated in the tablet or capsule, with the chosen Lactobacillus strain.
The specific carbon source such as encapsulated glucose can be consumed by individuals known to be already colonized with anti-inflammatory strains, for example L. reuteri.
Since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.
THP-1 cells were incubated together with conditioned media (CM) from the growth of L. reuteri ATCC 5289. The conditioned media are cell-free supernatants from 24-hour cultures of L. reuteri ATCC 5289 cultured in LDMIII (S. Jones and J. Versalovic, BMC Microbiol. 2009; 9: 35) supplemented with one specific sugar as sole carbon source. THP-1 cells were stimulated with either control medium or E. coli-derived LPS (which leads to the generation of TNF-α in a normal inflammatory response) or PCK during a 3.5 hour incubation after which the cells were removed and the supernatants assayed for TNFα levels using an ELISA technique as known in the art.
L. reuteri strains were grown in deMan, Rogosa, Sharpe (MRS; Difco, Franklin Lakes, N.J.) or LDMIII (pH 6.5) with a unique sugar source (see list of sugar source used below) for LDM medium composition). An anaerobic chamber (1025 Anaerobic System, Forma Scientific, Waltham, Mass.) supplied with a mixture of 10% CO2, 10% H2, and 80% N2 was used for anaerobic culturing of lactobacilli.
Biogaia AB (Raleigh, N.C.) provided L. reuteri strains ATCC PTA 5289. THP-1 cells (ATCC TIB-202) were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, Calif.) at 37° C. and 5% CO2. All chemical reagents were obtained from Sigma-Aldrich (St Louis, Mo.) unless otherwise stated. Polystyrene 96- and 24-well plates for biofilm and tissue culture studies were obtained from Corning (Corning, N.Y.). Filters with polyvinylidene fluoride membranes (0.22 mm pore size) (Millipore, Bedford, Mass.) were used for sterilization.
For immunomodulation studies, 10 mL of LDMIII with a unique carbon source was inoculated with L. reuteri cultures (grown overnight in MRS broth for 16-18 hrs) and adjusted to OD600=0.1. Bacteria were then incubated for 24 hours at 37° C. in anaerobic conditions. Final OD600 was measured and LDM was diluted to the same OD for all samples to take into account possible growth variation with different sugar source. Cells were pelleted (4000×g, RT, 10 minutes) and discarded. Supernatants were filter-sterilized (0.22 μm pore size). Aliquots were vacuum-dried and resuspended to the original volume using RPMI.
As previously described (Lin et al, 2008), cell-free supernatants of L. reuteri planktonic cell (5% v/v) and E. coli O127:B8 LPS (100 ng/mL) were added to human THP-1 cells (approximately 5×104 cells). Plates were incubated at 37° C. and 5% CO2 for 3.5 hours. THP-1 cells were pelleted (1500×g, 5 minutes, 4° C.), and TNF quantities in monocytoid cell supernatants were determined by quantitative ELISAs (R&D Systems, Minneapolis, Minn.). RPMI and Media are both used as controls (typically RPMI 95% and Media=LDM 5%).
Addition of LPS to the THP-1 cells in the presence of 1) Synergy1 (BENEO-Orafti Inc. 2740 Route 10 West Morris Plains, N.J. 07950, USA); 2) Glucose; 3) Raftilose; 4) Galactose; 5) Raffinose; and 6) Sucrose led to the generation of 1) 145.3 pg/ml TNFα, 2) 104.3 pg/ml TNFα, 3) 204.3 pg/ml TNFα, 4) 260 pg/ml TNFα 5) 517.8 pg/ml TNFα, and 6) 347.9 pg/ml TNFα, respectively, during the 3.5 hour incubation period. Addition of the growth medium (RPMI and LDM) which acts as a control for the CM additions, led to the generation of 396 and 352 pg/ml TNFα respectively. The results show that glucose and or Synergy1 as a sole carbon source inhibited the TNFα production more than 50% compared to the RPMI and LDM controls. This was not the case when the strain was grown with sucrose or raffinose. The results are shown in
THP-1 cells were incubated together with conditioned media (CM) from the growth of selected L. reuteri strains grown with glucose, L. reuteri ATCC PTA-6475, L. reuteri ATCC PTA-5289, L. reuteri ATCC 55730 and L. reuteri strain CF48-3A and the same strains grown with sucrose. THP-1 cells were stimulated with either control medium (LDMIII) or E. coli-derived LPS during a 3.5 hour incubation after which the cells were removed and the supernatants assayed for TNFα levels using an ELISA technique. LDMIII with glucose respectively sucrose was used as a control.
The materials and methods were as in Example 1.
The results show (see
Using the method in Example 1, the conditioned medium from one effectively TNF-α decreasing strain was selected, in this example the medium from L. reuteri ATCC PTA-5289 grown with glucose as a sole carbon source. This medium was produced in larger scale by growing the strain in de Man, Rogosa, Sharpe (MRS) (Difco, Sparks, Md.). Overnight cultures of lactobacilli were diluted to an OD600 of 1.0 (representing approximately 109 cells/ml) and further diluted 1:10 and grown for an additional 24 h. Bacterial cell-free conditioned medium was collected by centrifugation at 8500 rpm for 10 min at 4° C. Conditioned medium was separated from the cell pellet and then filtered through a 0.22 μm pore filter unit (Millipore, Bedford, Mass.). The conditioned medium was then lyophilized and formulated, using standard methods, to make a tablet.
The freeze-dried Lactobacillus reuteri was then supplemented with glucose and formulated, using standard methods, to make a tablet or capsule for example as described in Example 6.
This was done as described in Example 4 but with sucrose as a sole carbon source instead of dextrose during fermentation.
The freeze-dried Lactobacillus reuteri was then formulated, using standard methods, to make a tablet or capsule for example as described in Example 6 (but without the addition of glucose, paragraph 4).
In this example, L. reuteri (ATCC PTA-5289) is selected based on good anti-inflammatory characteristics in general and TNF-inhibiting properties in order to add the strain to a tablet. The L. reuteri strain is grown and lyophilized, using standard methods for growing Lactobacillus in the industry as can be read in Example 4.
The steps of an example of a manufacturing process of tablet containing the selected strain follow including encapsulated glucose, with it being understood that excipients, fillers, flavors, encapsulators, lubricants, anticaking agents, sweeteners and other components of tablet products as are known in the art, may be used without affecting the efficacy of the product:
The use of SOFTISAN™, a hydrogenated palm oil, enables the Lactobacillus cells to be encapsulated in fat and environmentally protected.
As stated above, the product of the invention may be in forms other than tablet, and standard methods of preparing the underling underlying product as are known in the art are beneficially used to prepare the product of the invention including the selected L. reuteri culture.
A female subject suffering from colitis is treated with the product produced in example 4. The subject is treated twice daily, in the morning and at night.
After two weeks, the inflammation of the colon is significantly decreased. On cessation of L. reuteri treatment the condition returns but is suppressed with regular administration of L. reuteri.
While the invention has been described with reference to specific embodiments, it will be appreciated that numerous variations, modifications, and embodiments are possible, and accordingly, all such variations, modifications, and embodiments are to be regarded as being within the spirit and scope of the invention.
This application claims priority from U.S. provisional application Ser. No. 61/337,277 filed Feb. 2, 2010.
Number | Date | Country | |
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61337277 | Feb 2010 | US |