Probiotics: helpful or harmful for leaky gut?

BY LUCY MAILING, MD/PHD STUDENT

Intestinal permeability, or “leaky gut”, is associated with a wide range of chronic conditions, including allergies, obesity, diabetes, inflammatory bowel disease, skin conditions, and more. People often turn to probiotic supplements to help heal a leaky gut. But is this always a good idea? New research suggests that certain probiotics might exacerbate inflammation if the gut is permeable. Read on to get the details and learn the implications for restoring gut health.

As usual, my carefully pre-planned blog article got derailed this week. Several weeks ago, I wrote about why probiotics aren’t useless, and discussed two new papers published in Cell.

Now, a fascinating study has just been published in the journal PNAS, titled:

Intestinal barrier dysfunction orchestrates the onset of inflammatory host–microbiome cross-talk in a human gut inflammation-on-a-chip.”1

This study identifies gut barrier dysfunction, or “leaky gut”, as the primary initiating factor contributing to gut inflammation, and calls into question whether probiotics should be used in people with severe gut permeability.

In this article, I’ll break down the details of this complex study, discuss other relevant research on probiotics and leaky gut, and share how this influences my recommendations to those trying to heal their gut moving forward. If you get lost in the details, I’ll provide a summary and takeaways section at the end.

Gut inflammation basics

Gut inflammation is typically accompanied by (1) impaired gut barrier function, (2) gut dysbiosis, and (3) a hyperactive gut immune system.

But which comes first: the chicken, the egg, or the other egg? Until now, scientists haven’t been able to figure out which of these factors seem to be the initiating factor of gut inflammation. The gut barrier, gut microbiota, and gut immune system are in constant cross-talk with one another, so it’s virtually impossible to isolate the effects of one of these factors in a living system.

Enter researchers Woojung Shin and Hyun Jung Kim from the University of Texas at Austin, who have now created a novel organ-on-a-chip model of gut inflammation.

 A true bioengineering marvel

Organs-on-chips are tiny microchips lined by living human cells. They are used to model everything from the heart to the kidneys, lungs, and bone marrow. In a previous study, the researchers had already developed a human gut-on-a-chip using a human intestinal cell line. Amazingly, this dynamic microenvironment is able to undergo epithelial villus growth and cellular proliferation, produce mucus, maintain barrier function, support long-term culture with a microbiome, and simulate pathogenic infection.

Like a human gut, the device has two compartments separated by a single layer of intestinal cells. The compartment on the left represents the gut lumen, where the gut microbiome and dietary components would normally reside, while the compartment on the right represents the submucosa, complete with tiny blood vessels called capillaries.

To create the first disease model on a chip, the researchers needed a way to manipulate several variables in the device:

  • To manipulate the integrity of the gut barrier, the researchers took inspiration from animal models of colitis, in which the chemical dextran sodium sulfate (DSS) is commonly used to disrupt the gut barrier.
  • To manipulate the gut microbiota, the researchers used nonpathogenic coli, a normal strain that inhabits the human gut, or the probiotic blend VSL#3 to the luminal compartment.
  • To manipulate the activation of the immune system, the researchers used lipopolysaccharide (LPS), a component of bacterial cells walls that binds to receptors on immune cells and initiates an immune response, and peripheral blood mononuclear cells (PBMCs) to represent the gut immune system in the submucosal compartment.

They tried several variations and combinations of these three factors.

Is barrier disruption the initiator of gut inflammation?

So what did they find? When no DSS was given and the gut barrier is intact, the gut did not show any significant signs of oxidative stress or inflammation, even when the immune cells were stimulated with LPS or nonpathogenic E. coli.

However, when DSS was given and the gut barrier was disrupted, the addition of LPS or E. coli resulted in increased production of reactive oxygen species (ROS), elevated markers of oxidative stress, and the aggressive release of pro-inflammatory cytokines.

The authors summarized their findings:

“Taken together, these findings indicate that the cytoplasmic ROS generation requires intercellular cross-talk through direct contact between immune cells and the barrier-compromised epithelium”

In other words, gut barrier dysfunction is required for gut inflammation to occur, at least in this simplified system. You can add bacteria or stimulate the immune system, but these alone are not sufficient to cause widespread gut inflammation:

“Control of epithelial barrier dysfunction is the key determinant for maintaining the homeostatic tolerance in the gut.”

This was fascinating stuff, but something didn’t feel quite right about the author’s conclusion, so I went back to a previous study that I had read back in 2016, titled:

Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip.”

Ironically, this was performed by the same researchers and published in the same journal. In this study, adding a pathogenic (entero-invasive) strain of E. coli to the luminal compartment was able to rapidly disrupt the intestinal barrier and the normal structure of the gut epithelium.2 Even non-pathogenic E. coli or LPS in the absence of normal gut motility resulted in a progressive loss of gut barrier function.

It was very surprising to me that there was no discussion of this anywhere in the researcher’s latest paper, so I emailed senior author, Dr. Kim, to try to make sense of it all. He said that they didn’t use entero-invasive E. coli in the latest study because they “have a separate paper to test it”, but that yes, this pathogenic E. coli could disrupt the gut barrier.

In other words, a leaky gut is required for widespread gut inflammation to occur, but pathogenic infection, disrupted gut motility, or other factors (diet, medications, stress, etc.) may be responsible for causing leaky gut in the first place. 

Probiotics help prevent gut permeability but may stimulate gut inflammation if the gut is already permeable

Dr. Kim and his team also wanted to see whether the state of the gut barrier determined the therapeutic efficacy of probiotic treatment.

When the gut epithelium was intact, adding the probiotic formula VSL#3 to the luminal compartment significantly improved gut barrier function. Even when it was later challenged with DSS, the barrier integrity was maintained.

However, when the gut epithelium was challenged with DSS (i.e. made permeable) before addition of the probiotic VSL#3, the epithelium progressively lost barrier function, regardless of the probiotic treatment. The VSL#3 bacterial cells were also able to pass through the disrupted barrier to the submucosal compartment, where they potently stimulated the release of pro-inflammatory cytokines.

The authors write:

“This result implies that probiotic administration may be risky when intestinal barrier function is notably compromised.”

However, when I looked at the data objectively, I had a hard time coming to the same conclusion.

Commensal microbes do this too: a closer look at the data

After manipulating different variables in the system, the researchers measured several pro-inflammatory cytokines, or signaling molecules, that were released by the gut epithelium. These include IL-1β, IL-6, and TNFα.

If you look at Figure 4A, adding nonpathogenic E. coli (at a density of 10 million CFU/mL) to the luminal side in the presence of DSS stimulated the release of approximately:

120 pg/mL IL-1β

600 pg/mL IL-6

100 pg/mL TNFα

Compare this to figure 5D, where adding VSL #3 at the same density to the luminal side in the presence of DSS stimulated the release of approximately:

80 pg/mL IL-1β

270 pg/mL IL-6

250 pg/mL TNFα

This suggests that any microbe, whether commensal or probiotic, is likely to cause inflammation in someone with gut permeability. To say that taking a probiotic is “risky” when non-pathogenic E. coli also crosses a damaged gut barrier and causes a similar immune response, seems a bit of a stretch to me. From the estimates I could glean from the figures, the probiotic actually seems to be stimulating lower total pro-inflammatory cytokines than the commensal E. coli at the same cellular density. (The statistical significance of this is unknown since the researchers did not directly compare these two groups.)

Moreover, VSL#3 was shown to reduce cellular oxidative stress, regardless of whether it was administered pre- or post-DSS. Though previous studies had shown that VSL #3 was able to modulate gene expression, the researchers did not assess this here.

When I asked Dr. Kim about this, he said that he “can’t say anything about whether probiotics are beneficial or not.”

IL-8: the missing piece of the puzzle?

Interestingly, the researchers also chose not to include any measures of the cytokine IL-8 in their most recent paper, and I’m surprised that this slipped through the peer-review process.

In their 2016 paper, Dr. Kim and colleagues tested various cytokines to determine if any of them were sufficient to induce damage to the gut-on-a-chip. They found that IL-1β, IL-6, and TNFα or any combination of these three cytokines did not induce significant damage. However, if these three cytokines were administered along with IL-8, they produced significant injury to the gut epithelium:

“…although our results confirm that IL-6, IL-1β, and TNF-α may contribute to development of intestinal inflammatory disease as suggested in the past based on in vivo studies, our ability to manipulate these factors independently revealed that these cytokines must act in the presence of high levels of IL-8 to exert these disease-promoting effects.”

This is incredibly important and suggests that controlling IL-8 production may be key to reducing gut permeability.

IL-8 is a specialized type of cytokine called a chemokine, meaning it attracts other immune cells to a specific area. IL-8 specifically releases molecules like histamine to facilitate the attraction of neutrophils. Notably, oxidative stress increases IL-8,3 so it follows that the lower oxidative stress seen in the probiotic group may have reduced IL-8.

However, there was no mention of this in the discussion section of the 2018 paper. When I asked Dr. Kim by email why they did not measure IL-8 in their most recent publication, he said that they “have plans to measure this in future research”.

I have to say that, after poring over these studies for several hours to try to make sense of the results, I was quite disappointed by this hole in the data.

Other limitations and implications of this research

This study has a few other limitations that are worth noting. While the gut-on-a-chip is an incredible tool for investigating the independent effects of different variables, we cannot necessarily extrapolate these findings to a living human gut. The intestinal cell line used for this model (Caco-2) was originally isolated from a human colorectal tumor, though it has since been shown to exhibit features similar to a human small intestine.

Moreover, the researchers only tested one probiotic formulation at a single dose. VSL#3 is a clinically studied mixture that contains Lactobacillus spp. Bifidobacterium spp., and Streptococcus thermophilus. In a previous article, I wrote about how researchers elucidated that supplementation with Lactobacillus spp. prevented the return of the native gut microbiome after antibiotics. It’s possible that some probiotic genera or species not tested here would promote gut barrier integrity or stimulate less of an immune response.

Overall, I do not think this particular study makes a strong case for avoiding probiotics in cases of intestinal permeability. However, there are likely some instances where certain probiotics can exacerbate leaky gut and prevent gut healing. So, let’s look at a few other studies that can shed some light on the use of probiotics in leaky gut.

Other studies that shed some light on the merits of taking probiotics with a leaky gut

We’ll begin with animal studies that have looked at the effects of probiotic administration in animal models of DSS-induced colitis, since this is perhaps the most similar to the DSS-induced inflammation in the gut-on-a-chip. Unfortunately, most of these studies provided the probiotics before or in conjunction with the DSS treatment. However, the one study I could find that looked at the effects of probiotic VSL #3 provided for two weeks after DSS administration (i.e. after the gut was permeable) found no difference in treatment outcomes – the probiotic was neither harmful nor helpful.4

In other animal models of colitis, VSL#3 supplementation after the onset of colitis and disruption of barrier function led to significant improvement in symptoms. Di Giacinto et al. found that VSL#3 administration during a remission period increased production of the anti-inflammatory cytokine IL-10 and resulted in milder colitis symptoms.5 Soo et al. found that VSL#3 upregulated expression of a mucosal enzyme important for cell turnover and reduced disease activity in both mice and humans with ulcerative colitis.6

Other human studies have shown that VSL#3 supplementation can be helpful in diseased states. A 2014 meta-analysis found that VSL#3 induced remission in 43.8% of patients with mild to moderately active ulcerative colitis, with no serious side effects reported.7 The probiotic E. coli Nissle 1917 has also shown to be beneficial for inducing or maintaining remission in ulcerative colitis.8,9

In searching for this information, however, I came across several studies that found that treatment with certain Lactobacillus strains led to a deterioration in DSS colitis, regardless of whether they were administered before or after barrier disruption. These include Lactobacillus rhamnosus GG and Lactobacillus plantarum NCIMB8826.10,11

This seems to hold true in humans as well. While L. rhamnosus GG has shown some promise in randomized, placebo-controlled trials of mild-to-moderate ulcerative colitis, it has been linked to nausea, vomiting, epigastric pain, and other adverse events in Crohn’s disease.12,13 Several published case reports also suggest that Lactobacillus rhamnosus GG can cause sepsis in hospitalized patients with short bowel syndrome or severe ulcerative colitis flares.14,15

Summary and takeaways

Hopefully, you stuck with me through all of that! Below is a brief summary of the major takeaways from this research, and how it will influence my work with clients moving forward:

1) Targeting gut barrier function is crucial. This study shows that maintaining the integrity of the gut barrier is both necessary AND sufficient to reduce gut inflammation. Treating pathogenic infections, supporting gut motility, and avoiding medications, inflammatory foods, and environmental toxins that disrupt the gut epithelium are key to subduing gut inflammation.

2) Reducing IL-8 may be the key to healing the gut barrier. The 2016 study I discussed above showed that pro-inflammatory cytokines were only damaging to the gut barrier in the presence of IL-8, suggesting that inhibitors of IL-8 may help mitigate gut damage, even when inflammation is present. (Moreover, IL-8 is known to be implicated in the pathogenesis of inflammatory bowel disease, irritable bowel syndrome, depression, and even skin conditions like psoriasis and eczema.16–20)

There are many ways to naturally reduce IL-8. Caloric restriction, ketogenic diets, or fasting are particularly potent inhibitors of IL-8.21 The short-chain fatty acid butyrate at low concentrations has been shown to dramatically reduce IL-8 production, but high concentrations can increase IL-8 production,22 yet another example of the paradoxical effects of butyrate.

Probiotics have also been shown to impact IL-8. Bifidobacterium infantis W52, Lactobacillus casei W56, and Lactococcus lactis W58 have been shown to inhibit IL-8 production.22 Bifidobacterium longum, Saccharomyces boulardii, and Bacillus subtilis B10 may also reduce IL-8.23 Other nutrients or supplements that may reduce IL-8 include quercetin, resveratrol, zinc, licorice, curcumin, black cumin seed oil, omega-3s, theaflavin, milk thistle, gingko, and sumac.24–30

3) Choose your probiotics wisely. If your gut is in a relatively healthy state, probiotics can help shore up the gut barrier and protect it against future damage. However, if your gut barrier is damaged, certain probiotic strains may exacerbate inflammation. For instance, Lactobacillus rhamnosus GG and Lactobacillus plantarum NCIMB8826 have been shown to exacerbate gut inflammation.

This is one of the reasons why I continue to emphasize how important it is to use clinically studied strains and, whenever possible, to choose strains based on the particular health condition you are trying to treat. Blindly taking whatever probiotic you find on the shelf at your local grocery store is likely to do more harm than good.

To make this simpler, I recently conducted an extensive analysis of the clinical studies for the most common soil-based probiotic species and am currently working on similar analyses for S. boulardii, E. coli Nissle, Lactobacillus, and Bifidobacterium. You can subscribe to my newsletter to be notified when they are released.

That’s all for now! Let me know what you thought of this research in the comments below.

 

Sources:

  1. Shin, W. & Kim, H. J. Intestinal barrier dysfunction orchestrates the onset of inflammatory host–microbiome cross-talk in a human gut inflammation-on-a-chip. PNAS 201810819 (2018). doi:10.1073/pnas.1810819115
  2. Kim, H. J., Li, H., Collins, J. J. & Ingber, D. E. Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip. Proc Natl Acad Sci U S A 113, E7–E15 (2016).
  3. Verhasselt, V., Goldman, M. & Willems, F. Oxidative stress up-regulates IL-8 and TNF-α synthesis by human dendritic cells. European Journal of Immunology 28, 3886–3890 (1998).
  4. Gaudier, E., Michel, C., Segain, J.-P., Cherbut, C. & Hoebler, C. The VSL# 3 probiotic mixture modifies microflora but does not heal chronic dextran-sodium sulfate-induced colitis or reinforce the mucus barrier in mice. J. Nutr. 135, 2753–2761 (2005).
  5. Di Giacinto, C., Marinaro, M., Sanchez, M., Strober, W. & Boirivant, M. Probiotics ameliorate recurrent Th1-mediated murine colitis by inducing IL-10 and IL-10-dependent TGF-beta-bearing regulatory cells. J. Immunol. 174, 3237–3246 (2005).
  6. Soo, I. et al. VSL#3 probiotic upregulates intestinal mucosal alkaline sphingomyelinase and reduces inflammation. Can. J. Gastroenterol. 22, 237–242 (2008).
  7. Mardini, H. E. & Grigorian, A. Y. Probiotic mix VSL#3 is effective adjunctive therapy for mild to moderately active ulcerative colitis: a meta-analysis. Inflamm. Bowel Dis. 20, 1562–1567 (2014).
  8. Matthes, H., Krummenerl, T., Giensch, M., Wolff, C. & Schulze, J. Clinical trial: probiotic treatment of acute distal ulcerative colitis with rectally administered Escherichia coli Nissle 1917 (EcN). BMC Complementary and Alternative Medicine 10, 13 (2010).
  9. Rembacken, B., Snelling, A., Hawkey, P., Chalmers, D. & Axon, A. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. The Lancet 354, 635–639 (1999).
  10. Claes, I. J. J. et al. Impact of lipoteichoic acid modification on the performance of the probiotic Lactobacillus rhamnosus GG in experimental colitis. Clin. Exp. Immunol. 162, 306–314 (2010).
  11. Mileti, E., Matteoli, G., Iliev, I. D. & Rescigno, M. Comparison of the immunomodulatory properties of three probiotic strains of Lactobacilli using complex culture systems: prediction for in vivo efficacy. PLoS ONE 4, e7056 (2009).
  12. Zocco, M. A. et al. Comparison of lactobacillus Gg and mesalazine in maintaining remission of ulcerative colitis and Crohn’s disease. Gastroenterology 124, A201 (2003).
  13. Bousvaros, A. et al. A Randomized, Double-blind Trial of Lactobacillus GG Versus Placebo in Addition to Standard Maintenance Therapy for Children with Crohn’s Disease. Inflamm Bowel Dis 11, 833–839 (2005).
  14. Kunz, A. N., Noel, J. M. & Fairchok, M. P. Two Cases of Lactobacillus Bacteremia During Probiotic Treatment of Short Gut Syndrome. Journal of Pediatric Gastroenterology and Nutrition 38, 457 (2004).
  15. Farina, C., Arosio, M., Mangia, M. & Moioli, F. Lactobacillus casei subsp. rhamnosus sepsis in a patient with ulcerative colitis. J. Clin. Gastroenterol. 33, 251–252 (2001).
  16. Daig, R. et al. Increased interleukin 8 expression in the colon mucosa of patients with inflammatory bowel disease. Gut 38, 216–222 (1996).
  17. Pearl, D. S., Shah, K., Brown, J., Shute, J. K. & Trebble, T. M. Active ulcerative colitis is associated with downregulation of the TH1, TH2 and TH17 cytokine response and elevated IL-8 levels in mucosal biopsies. Gut 60, A214–A214 (2011).
  18. Scully, P. et al. Plasma cytokine profiles in females with irritable bowel syndrome and extra-intestinal co-morbidity. Am. J. Gastroenterol. 105, 2235–2243 (2010).
  19. Sticherling, M., Bornscheuer, E., Schröder, J.-M. & Christophers, E. Immunohistochemical studies on NAP-1/IL-8 in contact eczema and atopic dermatitis. Arch Dermatol Res 284, 82–85 (1992).
  20. Baune, B. T. et al. Inflammatory biomarkers predict depressive, but not anxiety symptoms during aging: the prospective Sydney Memory and Aging Study. Psychoneuroendocrinology 37, 1521–1530 (2012).
  21. Forsythe, C. E. et al. Comparison of low fat and low carbohydrate diets on circulating fatty acid composition and markers of inflammation. Lipids 43, 65–77 (2008).
  22. Malago, J. J., Koninkx, J. F. J. G., Tooten, P. C. J., van Liere, E. A. & van Dijk, J. E. Anti-inflammatory properties of heat shock protein 70 and butyrate on Salmonella-induced interleukin-8 secretion in enterocyte-like Caco-2 cells. Clin. Exp. Immunol. 141, 62–71 (2005).
  23. Rajput, I. R. et al. Saccharomyces boulardii and Bacillus subtilis B10 modulate TLRs mediated signaling to induce immunity by chicken BMDCs. J. Cell. Biochem. 115, 189–198 (2014).
  24. Aneja, R., Odoms, K., Denenberg, A. G. & Wong, H. R. Theaflavin, a black tea extract, is a novel anti-inflammatory compound. Crit. Care Med. 32, 2097–2103 (2004).
  25. Storey, A., McArdle, F., Friedmann, P. S., Jackson, M. J. & Rhodes, L. E. Eicosapentaenoic Acid and Docosahexaenoic Acid Reduce UVB- and TNF-α-induced IL-8 Secretion in Keratinocytes and UVB-induced IL-8 in Fibroblasts. Journal of Investigative Dermatology 124, 248–255 (2005).
  26. Kloesch, B., Dietersdorfer, E., Broell, J., Kiener, H. & Steiner, G. The polyphenols curcumin and resveratrol effectively block IL-1β and PMA-induced IL-6, IL-8 and VEGF-A expression in human rheumatoid synovial fibroblasts. Annals of the Rheumatic Diseases 71, A90–A91 (2012).
  27. Bodet, C., La, V. D., Gafner, S., Bergeron, C. & Grenier, D. A licorice extract reduces lipopolysaccharide-induced proinflammatory cytokine secretion by macrophages and whole blood. J. Periodontol. 79, 1752–1761 (2008).
  28. Zhao, Y. et al. Zinc Deprivation Mediates Alcohol-Induced Hepatocyte IL-8 Analog Expression in Rodents via an Epigenetic Mechanism. Am J Pathol 179, 693–702 (2011).
  29. Weng, Z. et al. Quercetin Is More Effective than Cromolyn in Blocking Human Mast Cell Cytokine Release and Inhibits Contact Dermatitis and Photosensitivity in Humans. PLOS ONE 7, e33805 (2012).
  30. Hussain, S. A., Jassim, N. A., Numan, I. T., Al-Khalifa, I. I. & Abdullah, T. A. Anti-inflammatory activity of silymarin in patients with knee osteoarthritis. A comparative study with piroxicam and meloxicam. Saudi Med J 30, 98–103 (2009).

Probiotics: helpful or harmful for leaky gut?

BY LUCY MAILING, MD/PHD STUDENT

Intestinal permeability, or “leaky gut”, is associated with a wide range of chronic conditions, including allergies, obesity, diabetes, inflammatory bowel disease, skin conditions, and more. People often turn to probiotic supplements to help heal a leaky gut. But is this always a good idea? New research suggests that certain probiotics might exacerbate inflammation if the gut is permeable. Read on to get the details and learn the implications for restoring gut health.

As usual, my carefully pre-planned blog article got derailed this week. Several weeks ago, I wrote about why probiotics aren’t useless, and discussed two new papers published in Cell.

Now, a fascinating study has just been published in the journal PNAS, titled:

Intestinal barrier dysfunction orchestrates the onset of inflammatory host–microbiome cross-talk in a human gut inflammation-on-a-chip.”1

This study identifies gut barrier dysfunction, or “leaky gut”, as the primary initiating factor contributing to gut inflammation, and calls into question whether probiotics should be used in people with severe gut permeability.

In this article, I’ll break down the details of this complex study, discuss other relevant research on probiotics and leaky gut, and share how this influences my recommendations to those trying to heal their gut moving forward. If you get lost in the details, I’ll provide a summary and takeaways section at the end.

Gut inflammation basics

Gut inflammation is typically accompanied by (1) impaired gut barrier function, (2) gut dysbiosis, and (3) a hyperactive gut immune system.

But which comes first: the chicken, the egg, or the other egg? Until now, scientists haven’t been able to figure out which of these factors seem to be the initiating factor of gut inflammation. The gut barrier, gut microbiota, and gut immune system are in constant cross-talk with one another, so it’s virtually impossible to isolate the effects of one of these factors in a living system.

Enter researchers Woojung Shin and Hyun Jung Kim from the University of Texas at Austin, who have now created a novel organ-on-a-chip model of gut inflammation.

 A true bioengineering marvel

Organs-on-chips are tiny microchips lined by living human cells. They are used to model everything from the heart to the kidneys, lungs, and bone marrow. In a previous study, the researchers had already developed a human gut-on-a-chip using a human intestinal cell line. Amazingly, this dynamic microenvironment is able to undergo epithelial villus growth and cellular proliferation, produce mucus, maintain barrier function, support long-term culture with a microbiome, and simulate pathogenic infection.

Like a human gut, the device has two compartments separated by a single layer of intestinal cells. The compartment on the left represents the gut lumen, where the gut microbiome and dietary components would normally reside, while the compartment on the right represents the submucosa, complete with tiny blood vessels called capillaries.

To create the first disease model on a chip, the researchers needed a way to manipulate several variables in the device:

  • To manipulate the integrity of the gut barrier, the researchers took inspiration from animal models of colitis, in which the chemical dextran sodium sulfate (DSS) is commonly used to disrupt the gut barrier.
  • To manipulate the gut microbiota, the researchers used nonpathogenic coli, a normal strain that inhabits the human gut, or the probiotic blend VSL#3 to the luminal compartment.
  • To manipulate the activation of the immune system, the researchers used lipopolysaccharide (LPS), a component of bacterial cells walls that binds to receptors on immune cells and initiates an immune response, and peripheral blood mononuclear cells (PBMCs) to represent the gut immune system in the submucosal compartment.

They tried several variations and combinations of these three factors.

Is barrier disruption the initiator of gut inflammation?

So what did they find? When no DSS was given and the gut barrier is intact, the gut did not show any significant signs of oxidative stress or inflammation, even when the immune cells were stimulated with LPS or nonpathogenic E. coli.

However, when DSS was given and the gut barrier was disrupted, the addition of LPS or E. coli resulted in increased production of reactive oxygen species (ROS), elevated markers of oxidative stress, and the aggressive release of pro-inflammatory cytokines.

The authors summarized their findings:

“Taken together, these findings indicate that the cytoplasmic ROS generation requires intercellular cross-talk through direct contact between immune cells and the barrier-compromised epithelium”

In other words, gut barrier dysfunction is required for gut inflammation to occur, at least in this simplified system. You can add bacteria or stimulate the immune system, but these alone are not sufficient to cause widespread gut inflammation:

“Control of epithelial barrier dysfunction is the key determinant for maintaining the homeostatic tolerance in the gut.”

This was fascinating stuff, but something didn’t feel quite right about the author’s conclusion, so I went back to a previous study that I had read back in 2016, titled:

Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip.”

Ironically, this was performed by the same researchers and published in the same journal. In this study, adding a pathogenic (entero-invasive) strain of E. coli to the luminal compartment was able to rapidly disrupt the intestinal barrier and the normal structure of the gut epithelium.2 Even non-pathogenic E. coli or LPS in the absence of normal gut motility resulted in a progressive loss of gut barrier function.

It was very surprising to me that there was no discussion of this anywhere in the researcher’s latest paper, so I emailed senior author, Dr. Kim, to try to make sense of it all. He said that they didn’t use entero-invasive E. coli in the latest study because they “have a separate paper to test it”, but that yes, this pathogenic E. coli could disrupt the gut barrier.

In other words, a leaky gut is required for widespread gut inflammation to occur, but pathogenic infection, disrupted gut motility, or other factors (diet, medications, stress, etc.) may be responsible for causing leaky gut in the first place. 

Probiotics help prevent gut permeability but may stimulate gut inflammation if the gut is already permeable

Dr. Kim and his team also wanted to see whether the state of the gut barrier determined the therapeutic efficacy of probiotic treatment.

When the gut epithelium was intact, adding the probiotic formula VSL#3 to the luminal compartment significantly improved gut barrier function. Even when it was later challenged with DSS, the barrier integrity was maintained.

However, when the gut epithelium was challenged with DSS (i.e. made permeable) before addition of the probiotic VSL#3, the epithelium progressively lost barrier function, regardless of the probiotic treatment. The VSL#3 bacterial cells were also able to pass through the disrupted barrier to the submucosal compartment, where they potently stimulated the release of pro-inflammatory cytokines.

The authors write:

“This result implies that probiotic administration may be risky when intestinal barrier function is notably compromised.”

However, when I looked at the data objectively, I had a hard time coming to the same conclusion.

Commensal microbes do this too: a closer look at the data

After manipulating different variables in the system, the researchers measured several pro-inflammatory cytokines, or signaling molecules, that were released by the gut epithelium. These include IL-1β, IL-6, and TNFα.

If you look at Figure 4A, adding nonpathogenic E. coli (at a density of 10 million CFU/mL) to the luminal side in the presence of DSS stimulated the release of approximately:

120 pg/mL IL-1β

600 pg/mL IL-6

100 pg/mL TNFα

Compare this to figure 5D, where adding VSL #3 at the same density to the luminal side in the presence of DSS stimulated the release of approximately:

80 pg/mL IL-1β

270 pg/mL IL-6

250 pg/mL TNFα

This suggests that any microbe, whether commensal or probiotic, is likely to cause inflammation in someone with gut permeability. To say that taking a probiotic is “risky” when non-pathogenic E. coli also crosses a damaged gut barrier and causes a similar immune response, seems a bit of a stretch to me. From the estimates I could glean from the figures, the probiotic actually seems to be stimulating lower total pro-inflammatory cytokines than the commensal E. coli at the same cellular density. (The statistical significance of this is unknown since the researchers did not directly compare these two groups.)

Moreover, VSL#3 was shown to reduce cellular oxidative stress, regardless of whether it was administered pre- or post-DSS. Though previous studies had shown that VSL #3 was able to modulate gene expression, the researchers did not assess this here.

When I asked Dr. Kim about this, he said that he “can’t say anything about whether probiotics are beneficial or not.”

IL-8: the missing piece of the puzzle?

Interestingly, the researchers also chose not to include any measures of the cytokine IL-8 in their most recent paper, and I’m surprised that this slipped through the peer-review process.

In their 2016 paper, Dr. Kim and colleagues tested various cytokines to determine if any of them were sufficient to induce damage to the gut-on-a-chip. They found that IL-1β, IL-6, and TNFα or any combination of these three cytokines did not induce significant damage. However, if these three cytokines were administered along with IL-8, they produced significant injury to the gut epithelium:

“…although our results confirm that IL-6, IL-1β, and TNF-α may contribute to development of intestinal inflammatory disease as suggested in the past based on in vivo studies, our ability to manipulate these factors independently revealed that these cytokines must act in the presence of high levels of IL-8 to exert these disease-promoting effects.”

This is incredibly important and suggests that controlling IL-8 production may be key to reducing gut permeability.

IL-8 is a specialized type of cytokine called a chemokine, meaning it attracts other immune cells to a specific area. IL-8 specifically releases molecules like histamine to facilitate the attraction of neutrophils. Notably, oxidative stress increases IL-8,3 so it follows that the lower oxidative stress seen in the probiotic group may have reduced IL-8.

However, there was no mention of this in the discussion section of the 2018 paper. When I asked Dr. Kim by email why they did not measure IL-8 in their most recent publication, he said that they “have plans to measure this in future research”.

I have to say that, after poring over these studies for several hours to try to make sense of the results, I was quite disappointed by this hole in the data.

Other limitations and implications of this research

This study has a few other limitations that are worth noting. While the gut-on-a-chip is an incredible tool for investigating the independent effects of different variables, we cannot necessarily extrapolate these findings to a living human gut. The intestinal cell line used for this model (Caco-2) was originally isolated from a human colorectal tumor, though it has since been shown to exhibit features similar to a human small intestine.

Moreover, the researchers only tested one probiotic formulation at a single dose. VSL#3 is a clinically studied mixture that contains Lactobacillus spp. Bifidobacterium spp., and Streptococcus thermophilus. In a previous article, I wrote about how researchers elucidated that supplementation with Lactobacillus spp. prevented the return of the native gut microbiome after antibiotics. It’s possible that some probiotic genera or species not tested here would promote gut barrier integrity or stimulate less of an immune response.

Overall, I do not think this particular study makes a strong case for avoiding probiotics in cases of intestinal permeability. However, there are likely some instances where certain probiotics can exacerbate leaky gut and prevent gut healing. So, let’s look at a few other studies that can shed some light on the use of probiotics in leaky gut.

Other studies that shed some light on the merits of taking probiotics with a leaky gut

We’ll begin with animal studies that have looked at the effects of probiotic administration in animal models of DSS-induced colitis, since this is perhaps the most similar to the DSS-induced inflammation in the gut-on-a-chip. Unfortunately, most of these studies provided the probiotics before or in conjunction with the DSS treatment. However, the one study I could find that looked at the effects of probiotic VSL #3 provided for two weeks after DSS administration (i.e. after the gut was permeable) found no difference in treatment outcomes – the probiotic was neither harmful nor helpful.4

In other animal models of colitis, VSL#3 supplementation after the onset of colitis and disruption of barrier function led to significant improvement in symptoms. Di Giacinto et al. found that VSL#3 administration during a remission period increased production of the anti-inflammatory cytokine IL-10 and resulted in milder colitis symptoms.5 Soo et al. found that VSL#3 upregulated expression of a mucosal enzyme important for cell turnover and reduced disease activity in both mice and humans with ulcerative colitis.6

Other human studies have shown that VSL#3 supplementation can be helpful in diseased states. A 2014 meta-analysis found that VSL#3 induced remission in 43.8% of patients with mild to moderately active ulcerative colitis, with no serious side effects reported.7 The probiotic E. coli Nissle 1917 has also shown to be beneficial for inducing or maintaining remission in ulcerative colitis.8,9

In searching for this information, however, I came across several studies that found that treatment with certain Lactobacillus strains led to a deterioration in DSS colitis, regardless of whether they were administered before or after barrier disruption. These include Lactobacillus rhamnosus GG and Lactobacillus plantarum NCIMB8826.10,11

This seems to hold true in humans as well. While L. rhamnosus GG has shown some promise in randomized, placebo-controlled trials of mild-to-moderate ulcerative colitis, it has been linked to nausea, vomiting, epigastric pain, and other adverse events in Crohn’s disease.12,13 Several published case reports also suggest that Lactobacillus rhamnosus GG can cause sepsis in hospitalized patients with short bowel syndrome or severe ulcerative colitis flares.14,15

Summary and takeaways

Hopefully, you stuck with me through all of that! Below is a brief summary of the major takeaways from this research, and how it will influence my work with clients moving forward:

1) Targeting gut barrier function is crucial. This study shows that maintaining the integrity of the gut barrier is both necessary AND sufficient to reduce gut inflammation. Treating pathogenic infections, supporting gut motility, and avoiding medications, inflammatory foods, and environmental toxins that disrupt the gut epithelium are key to subduing gut inflammation.

2) Reducing IL-8 may be the key to healing the gut barrier. The 2016 study I discussed above showed that pro-inflammatory cytokines were only damaging to the gut barrier in the presence of IL-8, suggesting that inhibitors of IL-8 may help mitigate gut damage, even when inflammation is present. (Moreover, IL-8 is known to be implicated in the pathogenesis of inflammatory bowel disease, irritable bowel syndrome, depression, and even skin conditions like psoriasis and eczema.16–20)

There are many ways to naturally reduce IL-8. Caloric restriction, ketogenic diets, or fasting are particularly potent inhibitors of IL-8.21 The short-chain fatty acid butyrate at low concentrations has been shown to dramatically reduce IL-8 production, but high concentrations can increase IL-8 production,22 yet another example of the paradoxical effects of butyrate.

Probiotics have also been shown to impact IL-8. Bifidobacterium infantis W52, Lactobacillus casei W56, and Lactococcus lactis W58 have been shown to inhibit IL-8 production.22 Bifidobacterium longum, Saccharomyces boulardii, and Bacillus subtilis B10 may also reduce IL-8.23 Other nutrients or supplements that may reduce IL-8 include quercetin, resveratrol, zinc, licorice, curcumin, black cumin seed oil, omega-3s, theaflavin, milk thistle, gingko, and sumac.24–30

3) Choose your probiotics wisely. If your gut is in a relatively healthy state, probiotics can help shore up the gut barrier and protect it against future damage. However, if your gut barrier is damaged, certain probiotic strains may exacerbate inflammation. For instance, Lactobacillus rhamnosus GG and Lactobacillus plantarum NCIMB8826 have been shown to exacerbate gut inflammation.

This is one of the reasons why I continue to emphasize how important it is to use clinically studied strains and, whenever possible, to choose strains based on the particular health condition you are trying to treat. Blindly taking whatever probiotic you find on the shelf at your local grocery store is likely to do more harm than good.

To make this simpler, I recently conducted an extensive analysis of the clinical studies for the most common soil-based probiotic species and am currently working on similar analyses for S. boulardii, E. coli Nissle, Lactobacillus, and Bifidobacterium. You can subscribe to my newsletter to be notified when they are released.

That’s all for now! Let me know what you thought of this research in the comments below.

 

Sources:

  1. Shin, W. & Kim, H. J. Intestinal barrier dysfunction orchestrates the onset of inflammatory host–microbiome cross-talk in a human gut inflammation-on-a-chip. PNAS 201810819 (2018). doi:10.1073/pnas.1810819115
  2. Kim, H. J., Li, H., Collins, J. J. & Ingber, D. E. Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip. Proc Natl Acad Sci U S A 113, E7–E15 (2016).
  3. Verhasselt, V., Goldman, M. & Willems, F. Oxidative stress up-regulates IL-8 and TNF-α synthesis by human dendritic cells. European Journal of Immunology 28, 3886–3890 (1998).
  4. Gaudier, E., Michel, C., Segain, J.-P., Cherbut, C. & Hoebler, C. The VSL# 3 probiotic mixture modifies microflora but does not heal chronic dextran-sodium sulfate-induced colitis or reinforce the mucus barrier in mice. J. Nutr. 135, 2753–2761 (2005).
  5. Di Giacinto, C., Marinaro, M., Sanchez, M., Strober, W. & Boirivant, M. Probiotics ameliorate recurrent Th1-mediated murine colitis by inducing IL-10 and IL-10-dependent TGF-beta-bearing regulatory cells. J. Immunol. 174, 3237–3246 (2005).
  6. Soo, I. et al. VSL#3 probiotic upregulates intestinal mucosal alkaline sphingomyelinase and reduces inflammation. Can. J. Gastroenterol. 22, 237–242 (2008).
  7. Mardini, H. E. & Grigorian, A. Y. Probiotic mix VSL#3 is effective adjunctive therapy for mild to moderately active ulcerative colitis: a meta-analysis. Inflamm. Bowel Dis. 20, 1562–1567 (2014).
  8. Matthes, H., Krummenerl, T., Giensch, M., Wolff, C. & Schulze, J. Clinical trial: probiotic treatment of acute distal ulcerative colitis with rectally administered Escherichia coli Nissle 1917 (EcN). BMC Complementary and Alternative Medicine 10, 13 (2010).
  9. Rembacken, B., Snelling, A., Hawkey, P., Chalmers, D. & Axon, A. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. The Lancet 354, 635–639 (1999).
  10. Claes, I. J. J. et al. Impact of lipoteichoic acid modification on the performance of the probiotic Lactobacillus rhamnosus GG in experimental colitis. Clin. Exp. Immunol. 162, 306–314 (2010).
  11. Mileti, E., Matteoli, G., Iliev, I. D. & Rescigno, M. Comparison of the immunomodulatory properties of three probiotic strains of Lactobacilli using complex culture systems: prediction for in vivo efficacy. PLoS ONE 4, e7056 (2009).
  12. Zocco, M. A. et al. Comparison of lactobacillus Gg and mesalazine in maintaining remission of ulcerative colitis and Crohn’s disease. Gastroenterology 124, A201 (2003).
  13. Bousvaros, A. et al. A Randomized, Double-blind Trial of Lactobacillus GG Versus Placebo in Addition to Standard Maintenance Therapy for Children with Crohn’s Disease. Inflamm Bowel Dis 11, 833–839 (2005).
  14. Kunz, A. N., Noel, J. M. & Fairchok, M. P. Two Cases of Lactobacillus Bacteremia During Probiotic Treatment of Short Gut Syndrome. Journal of Pediatric Gastroenterology and Nutrition 38, 457 (2004).
  15. Farina, C., Arosio, M., Mangia, M. & Moioli, F. Lactobacillus casei subsp. rhamnosus sepsis in a patient with ulcerative colitis. J. Clin. Gastroenterol. 33, 251–252 (2001).
  16. Daig, R. et al. Increased interleukin 8 expression in the colon mucosa of patients with inflammatory bowel disease. Gut 38, 216–222 (1996).
  17. Pearl, D. S., Shah, K., Brown, J., Shute, J. K. & Trebble, T. M. Active ulcerative colitis is associated with downregulation of the TH1, TH2 and TH17 cytokine response and elevated IL-8 levels in mucosal biopsies. Gut 60, A214–A214 (2011).
  18. Scully, P. et al. Plasma cytokine profiles in females with irritable bowel syndrome and extra-intestinal co-morbidity. Am. J. Gastroenterol. 105, 2235–2243 (2010).
  19. Sticherling, M., Bornscheuer, E., Schröder, J.-M. & Christophers, E. Immunohistochemical studies on NAP-1/IL-8 in contact eczema and atopic dermatitis. Arch Dermatol Res 284, 82–85 (1992).
  20. Baune, B. T. et al. Inflammatory biomarkers predict depressive, but not anxiety symptoms during aging: the prospective Sydney Memory and Aging Study. Psychoneuroendocrinology 37, 1521–1530 (2012).
  21. Forsythe, C. E. et al. Comparison of low fat and low carbohydrate diets on circulating fatty acid composition and markers of inflammation. Lipids 43, 65–77 (2008).
  22. Malago, J. J., Koninkx, J. F. J. G., Tooten, P. C. J., van Liere, E. A. & van Dijk, J. E. Anti-inflammatory properties of heat shock protein 70 and butyrate on Salmonella-induced interleukin-8 secretion in enterocyte-like Caco-2 cells. Clin. Exp. Immunol. 141, 62–71 (2005).
  23. Rajput, I. R. et al. Saccharomyces boulardii and Bacillus subtilis B10 modulate TLRs mediated signaling to induce immunity by chicken BMDCs. J. Cell. Biochem. 115, 189–198 (2014).
  24. Aneja, R., Odoms, K., Denenberg, A. G. & Wong, H. R. Theaflavin, a black tea extract, is a novel anti-inflammatory compound. Crit. Care Med. 32, 2097–2103 (2004).
  25. Storey, A., McArdle, F., Friedmann, P. S., Jackson, M. J. & Rhodes, L. E. Eicosapentaenoic Acid and Docosahexaenoic Acid Reduce UVB- and TNF-α-induced IL-8 Secretion in Keratinocytes and UVB-induced IL-8 in Fibroblasts. Journal of Investigative Dermatology 124, 248–255 (2005).
  26. Kloesch, B., Dietersdorfer, E., Broell, J., Kiener, H. & Steiner, G. The polyphenols curcumin and resveratrol effectively block IL-1β and PMA-induced IL-6, IL-8 and VEGF-A expression in human rheumatoid synovial fibroblasts. Annals of the Rheumatic Diseases 71, A90–A91 (2012).
  27. Bodet, C., La, V. D., Gafner, S., Bergeron, C. & Grenier, D. A licorice extract reduces lipopolysaccharide-induced proinflammatory cytokine secretion by macrophages and whole blood. J. Periodontol. 79, 1752–1761 (2008).
  28. Zhao, Y. et al. Zinc Deprivation Mediates Alcohol-Induced Hepatocyte IL-8 Analog Expression in Rodents via an Epigenetic Mechanism. Am J Pathol 179, 693–702 (2011).
  29. Weng, Z. et al. Quercetin Is More Effective than Cromolyn in Blocking Human Mast Cell Cytokine Release and Inhibits Contact Dermatitis and Photosensitivity in Humans. PLOS ONE 7, e33805 (2012).
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By |2018-11-10T00:50:43+00:00November 10th, 2018|

4 Comments

  1. GEORGE November 10, 2018 at 10:44 am - Reply

    excellent and scientific work, bravo!!!

  2. Alyssa November 12, 2018 at 9:27 pm - Reply

    Super interesting! Kinda disappointed that the lead researcher didn’t have better answers to your questions, though. I’m wondering if you have any thoughts on how the dosage of probiotics might affect immune system activation in an already-compromised gut? Because most probiotic supplements are in the 5-100 billion CFU range, but VSL#3 double strength can be up to 3.6 *trillion* CFUs if you take the highest recommended dose of four packets per day (which I often have in the past). Not sure if that would still be a drop in the bucket compared to exposure to endogenous bacteria, or if it might be a high enough dose that it could notably increase immune activation.

    • Lucy Mailing November 16, 2018 at 4:09 pm - Reply

      Yes, that’s a great point! I think the dosage is really important. While 3.6 trillion CFU may still seem like a drop in the bucket compared to the total number of bacteria in the entire gut, it would certainly represent an extremely high dose to the small intestine, which only has about 100 million microbes per gram of gut contents. So for someone with severe small intestinal permeability, such a high dose of probiotics could cause a major immune response. Hopefully, we’ll see more studies in other model systems and even human clinical trials that assess different dosages of different types of probiotics for individuals with confirmed intestinal permeability!

  3. Sannyzew December 10, 2018 at 3:29 pm - Reply

    more new posts please 🙂
    ___
    Sanny

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