Human catestatin induces gut microbiota dysbiosis

US 10 463 712B2

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Treatment with catestatin changes the proportion of two major phyla Bacteroidetes and Firmicutes in the gut microbiota in an opposite manner observed in intestinal disorders like IBD, IBS or non-intestinal disorders like obesity. Specifically, administration of an effective amount of catestatin increased the relative percentage of Bacteroidetes and decreased the relative percentage of Firmicutes in the gut microbiota.

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Claims

1. A method for increasing levels of Bacteriodetes relative to levels of other bacteria in the gut of an individual in need of such treatment comprising administering to said individual an effective amount of catestatin (CTS).

Show 6 dependent claims

8. A method for decreasing relative levels of Firmicutes relative to other bacteria in the gut of an individual in need of such treatment comprising administering to said individual an effective amount of catestatin (CTS).

Show 3 dependent claims

12. A method of modulating gut microbiota composition comprising administering to an individual in need of such treatment an effective amount of catestatin (CTS).

Show 6 dependent claims

Description

PRIOR APPLICATION INFORMATION

The instant application is a 371 of PCT Application CA2016/050477, filed Apr. 25, 2016, which claims the benefit of U.S. Provisional Patent Application 62/155,572, filed May 1, 2015.

BACKGROUND OF THE INVENTION

Over the last 15 years, bacterial multi-drug resistance (MDR) has emerged and it has several socio-economical causes, from the use of surface antibacterial agents that are now available in many household products1 to antibiotic over-prescription or failing to complete a course of antibiotics1. Although due to MDR new lines of antibiotics are required, the development of new antibiotics has been reduced by pharmaceutical companies because of the cost and complexity of clinical trials2. Currently, there are relatively few new antimicrobials in development.

The gastrointestinal tract is heavily colonized with an average of 1014 microbes that represent thousands of species, which is 10 times more than the total number of cells in the human body3. More than 90% of this bacterial population falls under two major phyla: Bacteriodetes (a gram-negative phylum) and Firmicutes (a gram-positive phylum)4, 5, while the remaining belong to phyla such as Proteobacteria, and Actinobacteria4, 5. In healthy individuals, microbial diversity in the intestine is stable over time and demonstrates a symbiotic relationship with the host3, but a shift in microbial composition, named dysbiosis, targeting mainly Firmicutes and Bacteroidetes, has been described in several pathologies, including related and non-related gastrointestinal pathologies6-8. For example, microbial dysbiosis in gut is observed in intestinal disorders like intestinal bowel syndrome (IBS), intestinal bowel disease (IBD) and also non intestinal disorders like obesity and type 1 and type-2 diabetes. Specifically, gut microbiota helps to digest food items and various metabolites and chemicals are produced by the resident microbiota, which plays a significant role in host health or disease state. For example, Bacteroides thetaiotaomicron can activate the toll-like receptors (TLRs) in the gut epithelium, which in turn can affect the expression of antimicrobial peptides, such as angiogenins9, 10. In addition to the innate immune system, gut microbiota can also control the host's adaptive immune system through T cell receptor αβ-positive intraepithelial lymphocytes, regulatory T cells and T helper 17 cells5. Overall, gut homeostasis is largely dependent on the normal gut microbiome11.

At the mucosal level the epithelium plays a major role in limiting the passage of bacteria to the sub-mucosa and restricts the presence of bacteria to the gut lumen; cell division is an important factor when the epithelial cells are altered and the epithelium needs to be regenerated12,13. Antimicrobial peptides (AMPs) secreted by epithelial cells have a broad spectrum effect against bacteria and they are part of an ancient defense mechanism that is present in virtually all mammals14. In the gastrointestinal tract, specialized intestinal epithelial cells or circulating inflammatory cells are a major source of these AMPs14. Within the epithelium, Paneth cells are the main producer of AMPs but new data indicate that enterochromaffin (EC) cells can hypothetically also produce certain types of AMPs15.

The EC cells are the major source of chromogranin A (CgA)16, a family of highly acidic proteins. The CgA gene is localized at 14q32 in the human genome, consisting of 8 exons and 7 introns, and its 2-Kb transcript is translated into the 457-residue CgA protein. The overall homology for CgA in different vertebrates is approximately 40%, but the most highly conserved regions occur at the N- and C-termini, which show up to 88% sequence homology. Cell- and tissue-specific CgA processing has been described in the rat, mouse and human GI tract17-19. The CgA primary structure from its cDNA sequence shows the presence of numerous pairs of basic amino acids. These are potential sites for cleavage by prohormone convertases (PC) or 2, and carboxypeptidase E/H2, which is consistent with evidence that CgA may serve as a prohormone for shorter bioactive fragments21; this is also suggested by the high sequence conservation of CgA-derived peptides. But in the gut, peptides can be highly sensitive to enzymes present in the luminal environment. Proteolytic fragments of CgA-derived peptides exert a broad spectrum of regulatory activities on the cardiovascular, endocrine and immune systems. Among its highly conserved C-terminal regions, CgA gives rise to a peptide of biological importance: the antihypertensive peptide catestatin (human CTS; CgA352-372)22-24, which has restricted antimicrobial activity against Staphylococcus aureus in vitro25. Similar to other AMPs, CTS can interact with anionic components of fungi and viruses. As a result, the microbial membrane is permeabilized, leading to cell lysis26. In vitro studies have demonstrated that CTS is effective against gram-positive bacteria such as Staphylococcus aureus and group A Streptococcus, gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, yeasts such as Candida albicans and filamentous fungi such as Aspergillus niger, A. fumigates and Trichophyton rubrum26, 27. However, to date, there has been no indication that the in vitro data can be reproduced using an in vivo model, as due to the presence of several enzymes located in the gut lumen, CTS peptide can be rapidly inactivated. Moreover, there is no indication about the type of microblota affected, as the colonic mucosa associated population differs completely for the population present in the feces.

Despite the close association between CTS and Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa in vitro, the effects of in vivo CTS treatment on the different type of gut microbiota are unknown. Our aim was to assess the composition of fecal and colonic mucosa associated microbiota and functional alterations in mice that were exposed to CTS for 6 days.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a method for increasing levels of Bacteroidetes relative to levels of other bacteria in the gut of an individual in need of such treatment comprising administering to said individual an effective amount of catestatin (CTS).

According to a further aspect of the invention, there is provided a method for decreasing relative levels of Firmicutes relative to other bacteria in the gut of an individual in need of such treatment comprising administering to said individual an effective amount of catestatin (CTS).

According to yet another aspect of the invention, there is provided use of catestatin (CTS) for increasing levels of Bacteroidetes relative to levels of other bacteria in the gut of an individual in need of such treatment.

According to still another aspect of the invention, there is provided use of catestatin (CTS) for decreasing relative levels of Firmicutes relative to other bacteria in the gut of an individual in need of such treatment.

According to a yet further aspect of the invention, there is provided a method of modulating gut microbiota composition comprising administering to an individual in need of such treatment an effective amount of catestatin (CTS).

According to a still further aspect of the invention, there is provided use of catestatin (CTS) for modulating gut microblota composition.

According to another aspect of the invention, there is provided a method of treating or preventing or prophylactically treating type 1 diabetes, type 2 diabetes, obesity, IBS or IBD in an individual in need of such treatment comprising administering to said individual an effective amount of catestatin (CTS).

According to a further aspect of the invention, there is provided use of catestatin (CTS) for treating or preventing or prophylactically treating type 1 diabetes, type 2 diabetes, obesity, IBS or IBD in an individual in need of such treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Rarefaction analysis on Chao 1, a measure of species richness based on operational taxonomic unit (OTU), for the fecal samples; control and CTS. Control and CTS treated mice fecal samples are more diverse compared to DSS and DSS+CTS mice treated samples, which have a low diversity. Diversity is not significantly affected by CTS treatment.

FIG. 2: Rarefaction analysis on Chao 1, a measure of species richness based on operational taxonomic unit (OTU), for the colonic mucosa associated samples; Control and CTS. Diversity is not significantly affected by CTS treatment.

FIG. 3: Principal coordinate analysis (PCoA) based on the unweighted UniFrac distance metric. Each colored point represents a fecal sample obtained from one mice and it is shaded according to different treatment (CTS or Control). P values were calculated using PERMANOVA. Samples clustered according to treatment status of the mice (P<0.05), suggesting that CTS and Control mice fecal samples are composed of distinct bacterial communities.

FIG. 4: Principal coordinate analysis (PCoA) based on the weighted and unweighted UniFrac distance metric. Each colored point represents a colonic mucosa associated sample obtained from one mice and it is shaded according to different treatment (CTS or Control). P values were calculated using PERMANOVA. Samples did not cluster according to treatment status of the mice (P>0.05), suggesting that CTS treatment did not significantly change bacterial communities.

FIG. 5: Effect of CTS treatment on the abundant phyla (1%) present in the fecal samples. After quality filtering steps 10 phyla was identified in fecal samples. Among these 4 phyla were considered abundant within the population (1%), including Firmicutes, Bacteroidetes, Proteobacteria, and Deferribacteres. CTS treated mice had a significantly higher level of Bacteroidetes population compared to Control (P<0.05, t-test). On the other hand Firmicutes population were lowered significantly in the feces of CTS treated animals (P<0.01, t-test)

FIG. 6: Effect of CTS treatment on the abundant phyla (1%) present in the colonic mucosa associated samples. After quality filtering steps 19 phyla was identified in colonic mucosa associated samples. Among these 4 phyla were considered abundant within the population (1%), including Firmicutes, Bacteroidetes, Proteobacteria, and Deferribacteres. CTS treatment did not have a significant impact on these abundant phyla present in the colonic mucosa associated samples.

FIG. 7: Partial least square discriminant analysis (PLS-DA) of bacterial communities comparing taxa that were associated with the Control or CTS treatments in the mice fecal samples. All taxa are colored based on the phyla to which they belong. Some sequences could only be affiliated to phylum (P), order (O), family (F) or class (C) levels. Specific taxa were significantly associated with each treatment group, which may be an indicator of shifts in the physiological or metabolic processes that the taxa may influence.

FIG. 8: Partial least square discriminant analysis (PLS-DA) of bacterial communities comparing taxa that were associated with the Control or CTS treatments in the mice colonic mucosa associated samples. All taxa are colored based on the phyla to which they belong. Some sequences could only be affiliated to phylum (P), order (O), family (F) or class (C) levels. Specific taxa were significantly associated with each treatment group, which may be an indicator of shifts in the physiological or metabolic processes that the taxa may influence.

FIG. 9. Subsystems and pathways enriched or decreased within the CTS or (Control) mice fecal samples. Corrected P-values were calculated using the Storey FDR correction. Subsystems or pathways overrepresented in the CTS or Control mice fecal samples have a positive or (negative) difference between mean proportions and are indicated by purple or (orange) coloring respectively.

FIG. 10. Subsystems and pathways enriched or decreased within the CTS or (Control) mice colonic mucosa associated samples. Subsystems or pathways overrepresented in the CTS or Control mice colonic mucosa associated samples have a positive or (negative) difference between mean proportions and are indicated by Purple or (orange) coloring respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

The mammalian intestinal tract is heavily colonized with a dense, complex and diversified microbial population. In healthy individuals, host and gut microbiota enjoy a symbiotic relationship by maintaining intestinal homeostasis and an array of epithelial antimicrobial agents is secreted into the gut to promote intestinal homeostasis. Enterochromaffin cells in the intestinal epithelium are a major source of chromogranin A (CgA), which is a pro-hormone and can be cleaved into a shorter bioactive peptide called catestatin (CTS). This study was carried out to evaluate the possible impact of CTS on gut microbiota in vivo using a mouse model. The CTS treatment did not modify the richness of the bacterial species in the fecal and colonic mucosa associated samples; however, the treatment significantly modified the bacterial community composition between the groups. The PLS-DA analysis revealed an association between specific taxa and the CTS-treated group at lower taxonomic levels. The CTS-treated mice had a significantly lower relative abundance of Firmicutes and higher abundance of Bacteroidetes. No significant change at the phylum level was observed in CTS-treated mice colonic mucosa associated samples. However, at lower phylogenetic levels, some bacterial taxa were significantly associated with CTS-treated mice in both fecal and colonic mucosa associated samples. Differences in microbial functional pathways in both fecal and colonic mucosa associated samples were detected. These results support the hypothesis that CTS treatment modulates gut microbiota composition under physiological conditions. Accordingly, these data indicate that CTS can be used to induce gut homeostasis which in turn can prevent, treat or prophylactically treat diseases such as obesity, type 1 diabetes, type 2 diabetes, inflammatory bowel disease (IBD), inflammatory bowel syndrome (IBS) or other health conditions.

As used herein, CTS refers to the antihypertensive peptide catestatin (human CTS). The CTS is derived from amino acids 352-372 chromogranin A (CgA), a family of highly acidic proteins. In some embodiments, CTS has the amino acid sequence SSMKLSFRARAYGFRGPGPQL (SEQ ID NO:1) although variants of this sequence, both naturally occurring and recombinant, may be used within the invention.

Rabbi et al. (2014, Biochem. Pharma. 89: 386-398) teaches that CTS is increased during colitis and that CTS modulates intestinal inflammation via the macrophage population and through a STAT-3 dependent pathway. Specifically, treatment with full length CTS (amino acids 352-372), proximal CTS fragment (amino acids 352-366) and distal CTS fragment (amino acids 360-372) resulted in less severe induced colitis, which indicates that CTS and the fragments thereof can be used to treat intestinal inflammation.

As will be appreciated by one of skill in the art, this indicates that treatment with CTS or either the proximal or distal fragment thereof will reduce inflammation in an individual suffering from colitis. Specifically, it is believed that CTS influences macrophage production of cytokines, which in turn reduces inflammation. Antigen presenting cell like macrophages, are one of the main producer of proinflammatory cytokines. CTS blocks the intracellular pathway implicated in the regulation of proinflammatory cytokines (i.e. IL-1b, IL-6).

Consequently, Rabbi et al. (2014) teaches that CTS can be used to reduce intestinal inflammation in an individual, which in turn would be expected to reduce the severity of symptoms associated with a colitis attack and/or reduce the frequency of colitis episodes.

However, this anti-inflammatory activity is separate and distinct from the known antimicrobial activity of CTS, which had previously only been demonstrated in vitro.

Rabbi et al. (2014) further speculates that intrarectal infusion of CTS peptides might induce a beneficial gut microbiota dysbiosis, which subsequently can affect the development of colitis.

As discussed above, in vitro studies have demonstrated that CTS is effective against gram-positive bacteria such as Staphylococcus aureus and group A Streptococcus, gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, yeasts such as Candida albicans and filamentous fungi such as Aspergillus niger, A. fumigates and Trichophyton rubrum26, 27. However, previously, there had been no indication that the in vitro data can be reproduced using an in vivo model, as due to the presence of several enzymes located in the gut lumen CTS peptide can be rapidly inactivated. Specifically, there are a number of proteases which can act on serine, cysteine and aspartic amino acids28. As the human CTS sequence used in this study has serine and aspartic sequences (SSMKLSFRARAYGFRGPGPQL, SEQ ID No:1), it was anticipated that CTS could be inactivated in the gut.

Specifically, CTS is a cationic peptide and as such the inventors hypothesized that if CTS avoided degradation in the gastrointestinal environment, this peptide might exerts it's effect by electrostatic interactions with the negatively charged phospholipid microbial cell walls. As a result of these interactions, any suitable microbial membrane that CTS could interact with would be disrupted. It was further anticipated that CTS could have a general effect, for example, reducing levels of all bacteria.

However, as discussed herein, the gastrointestinal environment in vivo is incredibly complex and it is impossible to predict the exact effect that a general antimicrobial such as CTS would have in the gut, assuming that it was not degraded by the serine and aspartic add proteases. Furthermore, it is simply not possible to create a complex gastrointestinal environment in vitro that has the enormous microbial diversity to sufficiently mimic the in vitro gut.

Furthermore, as discussed below, in fact, S. aureus and E. coli levels in the gut were not affected by CTS administration in vivo, demonstrating the difference that can be found between in vitro studies and in vivo experiments. As such, it is clear that not all in vitro results can be extrapolated to in vivo analysis, especially in the context that in vitro, many bacteria are non-cultivable due to the lack of anaerobic condition and are present when the whole microbiome is studied.

Furthermore, as discussed herein, CTS treatment did not modify the richness of the bacterial species in fecal and colonic mucosa associated samples. Specifically, as shown in FIG. 1 and as discussed herein, control and CTS-treated mice treated samples were more diverse than DSS and DSS+CTS mice treated samples, which have a low diversity. These data demonstrated that bacterial diversity or richness was not significantly affected by CTS treatment.

Based on these results, one conclusion would have been that the in vitro anti-microbial activity could not function in vivo, possibly due to protease degradation.

The inventors theorized that coating CTS might prevent or delay degradation; however, there were concerns that the coating would interfere with the antibiotic activity of CTS which, as discussed above, interacts with anionic components of the microbial membrane and permeabilizes the membrane, leading to cell lysis. As will be appreciated by one of skill in the art, there was considerable concern that a suitably protective coating that would protect CTS from the proteases would also prevent CTS from interacting with the negatively charged components of the bacterial membranes.

Another possibility would have been that CTS was having a general or broad spectrum antimicrobial affect.

As shown in FIG. 4, CTS treatment also did not significantly change the bacterial communities in the colonic mucosa associated samples, as discussed in greater detail below.

However, surprisingly, as shown in FIG. 3, it was determined that CTS and control mice fecal samples were composed of distinct bacterial communities. More significantly, as shown in FIG. 5, CTS treated mice had a significantly higher level of Bacteroidetes compared to the control mice. The CTS treated mice also had a significantly lower level of Firmicutes compared to the control mice. Thus, in the fecal samples, CTS treatment significantly modified the bacterial community composition between the CTS treatment group and the control.

While CTS was not having a specific effect on diversity of the microbiota or in the colonic mucosa associated samples, CTS treatment was having a significant effect on distinct bacterial communities within fecal samples, indicating that coating was not necessary for rectal administration.

This modification or modulation of the gut microbiota for example to increase relative levels of Bacteroidetes Is significant as lower levels of Bacteroidetes may result in adverse health effects as these bacteria have important starch degrading enzymes and also help to establish normal gut immunity. For example, B. thetaiotaomcron prevents activation of the proinflammatory transcription factor NFkβ41. Bacteroidetes also help to prevent production of virulent factors from pathogenic E. coli42. Moreover, there is data showing that fecal transplantation from a healthy person to IBD patients helps to reduce the symptoms43, 44 and that administration of probiotics which modify the microbiota can improve IBS and IBD.

Described herein is a method for increasing levels of Bacteroidetes relative to levels of other bacteria in the gut of an individual in need of such treatment comprising administering to said individual an effective amount of catestatin (CTS).

REFERENCES

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