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Cartoon showing why it is important for researchers to know exactly what is in the subjects' vegetarian diet and to examine the effects of specific food items

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Interplays Between the Immune System and the Gut Microbiota

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Interplays Between the Immune System and the Gut Microbiota

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Methods:

By studying the immunological and metagenomic data, the expression of certain Ig isotypes (IgD, IgM, IgG, and IgA), IGH and TRB V families of genes and TCR diversity  was measured in order to show its level of association to the microbiota in the different control and study groups.

Results:

There was a significant association to the gut microbiota. This figure shows the various bacterial strains and whether they are correlated positively or negatively and in how the groups related to each other.

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Impact of Diet Pattern on the Diversity of Immune System

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Impact of Diet Pattern on the Diversity of Immune System

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Methods:

38 samples from day 0 and day 91 taken from the study group and two control groups.

1. Diversity of BCR and TCR repertoires measured in all samples measured by finding unique CDR3s and dividing it by the total sequences (CDR3 is a complimentary determining region on the heavy chain of immunoglobulin).

2. D50 values were measured to find the distribution between B or T cell clones by finding the percent of unique B or T cell clones that account for half of the CDR3s in a sample.

3. Delta index was measured by calculating the percent of turnover within the repertoires.

4. V genes usage and frequencies was measured based on the CDR3-V-J combinations.

5. Isotype frequencies identified by BCR frequencies.

Results:

Generally, the change to vegetarian diet for 3 months did not have a major effect on overall diversity of immune repertoire.

No significant changes at the two time points in study and controls groups in terms of CDR3 uniqueness, length, and frequency.

Long term vegetarians which had a low d50 value for TCR repertoires and a high D50 value for the BCR repertoire which can be explained by the repertoire being unbalanced by large clonal expansions

No significant changes in delta index.

IGHV5 family and  IGHV3-64, IGHV3-74,  IGHV3-15,  IGHV3-33, and TRBV7-4 were altered in the study group.

IGHV5 and IGHV7 families, as well as IGHV3-9, IGHV3-d, IGHV4-39 and TRBV3-1, TRBV9, TRBV16, and TRBV7-4 were altered in control groups.

Study group showed higher expression of IgA at day 91 than day 0 (15.6 vs. 10.7%) not as significant. While all other Ig isotype frequencies were reduced.

Long term vegetarians had significantly higher levels of IgM but lower IgG and IgE compared to omnivores.

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Consumption of processed meat and red meat are linked to adverse effects on the immune system, predisposing humans to inflammation related disorders.

Plant-derived proteins, non-digestible carbohydrates, and limited fat consumption can increase number of beneficial bacteria like Bifodobacterium and Lactobacillus within the gut (Petta et al. 2018).

Vegetarian diets are associated with enhanced gut microbial diversity and increased conversion of non-digestible carbohydrates into SCFAs (acetate, butyrate, propionate), which may reduce the risk of inflammatory diseases, type 2 diabetes, obesity, and heart disease.

High-fat diet (HFD), such as meat-based diets, affects B cells to contribute to pro-inflammatory reactions in adipose tissues, where they secrete pro-inflammatory cytokines that interferes with CD4+ and CD8+ T cell functions (Petta et al. 2018).

In germ-free model organisms, immune functions were impaired. Intestinal lymphoid structures were poorly developed, along with IgA deficiency.

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So, what type of diet is most beneficial in creating a highly diverse, more healthy gut microbiota that can strengthen our immunity?

How long does it take to gain the benefits of switching to lacto-ovo vegetarianism?

How does the composition and duration of diet impact the balance between pro- and anti-inflammatory factors in the gut microbiota and influence the immune system?

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A diverse bacterial community inside the gut restricts pathogenic colonization.

Sum of functional B-cell receptor and T-cell receptor population is called the immune repertoire, which is influenced by both genetic and environmental (diet!) factors.

B cells differentiate into plasma cells, which produce IgA antibodies inside the lamina propria of the intestinal epithelium (Petta et al. 2018).

IgA prevents infection by fighting against bacteria and viruses, and prevent them from adhering to the gut layer.

IgA plays a role in neutralizing food-derived toxins and  maintaining immune tolerance against food antigens.

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Figure 4: Selected Kyoto Encyclopedia of Genes and Genomes orthologous (KO) modules associated with short- and long-term vegetarian diets.

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Short- and Long-Term Vegetarian Diet-Associated Functional Changes in the Microbiome

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Short- and Long-Term Vegetarian Diet-Associated Functional Changes in the Microbiome

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Methods:

Knockout groups were looked at to elucidate the impact of diet on microbiota and a reporter score was calculated to estimate the significance of any variation between groups.

Gene abundance related to lipopolysaccharide (involved in inflammation) synthesis among the experimental group and both control groups was looked at.

Results:
Several trends of enrichment/depletion were found in both the short-term and long-term vegetarians:

Long-Term Omnivores:

The osmoprotectant transport system, which controls the cell's uptake of choline, carnitine and beatine molecules found in red meat, was found to be highly abundant in long-term omnivores.

Modules of type II and IV general secretion systems which are linked to intracellular and cholera toxins were also highly enriched in long-term omnivores.

Long-Term Vegetarians:

Pyruvate:ferredoxin oxidoreductase, which is associated with the production of short chain fatty acids) was highly enriched in long-term vegetarian individuals.

Low abundance of lipopolysaccharide-related genes were seen in the long-term vegetarians but no notable differences were observed in the experimental group or long-term omnivores.

Bottom line: The long-term but not short-term vegetarian diet might have an anti-inflammatory effect based on analysis of the microbiome!

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Impact of the Short-Term Vegetarian Diets on Metagenome

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Impact of the Short-Term Vegetarian Diets on Metagenome

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Methods:

1. In order to gain a better understanding of the microbiota, an enterotype analysis was conducted at the level of bacterial genus.

2. The evenness and richness of the gut microbiota was then looked at across samples at the genus, species and gene levels.

3. Bacterial profiles before and after diet switch were compared in order to isolate biomarkers that might be influenced by an altered dietary pattern.

Results:

1. Three enterotypes were identified and were found to be comprised primarily of Alistipes, Bacteroides, Prevotella, and Akkermansia. However, no siginificant relationship was found between enterotype and diet pattern.

2. No significant difference was elucidated in terms of gene count and/or alpha diversity in any of the groups at either point in time (day 0 or day 91). The beta diversity was found to be reduced in experimental participants who switched to the short-term vegetarian diet and this reduction was especially significant at the genus and species level. The general gut microbiota composition did not seem to be related to diet. The genus and species levels of the microbiota were affected by a short-term vegetarian diet.

3. No significant changes were observed in the bacterial profiles before and after diet switch. However, seven bacterial genera were enriched in those who switched to the short-term vegetarian diet. Most notably, Alistipes, which is a bile-tolerant bacteria, was found to be enriched in these individuals and decreased in abundance again after the short-term vegetarian period was passed. Nine species were directly associated with the change in diet and the control groups showed major differences in their bacterial profiles. Bacteroides finegoldii, which grows in the presence of bile and has been linked to colon cancer, was found to be increased in the long-term omnivores. Peptoniphilus duerdenii, which produces the beneficial short chain fatty acid butyrate, was found to be enriched in long-term vegetarians as was Blautia hydrogenotrophica which assists in the digestion of fibre.

Bottom line: No significant changes were observed in the bacterial profiles before and after diet switch, however some beneficial bacterial genera were enriched in those who switched to the short-term vegetarian diet!

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Methods:

To elucidate the effect of diets on gut microbiota, metagenomic sequencing was performed on fecal samples taken from individuals on day 0 (before diet switch in experimental group) and on day 91 (after diet had been switched for 3 months in experimental group).

A DNA library was constructed and cluster generation, DNA template hybridization, isothermal amplification, linearization, blocking, denaturation and hybridization were performed on the sequencing primers.

A pair-end library with an insert size of 350 base pairs was constructed for each sample and high throughput sequencing was performed in order to define the DNA sequences.

Results:
The number of reads was found to be similar across all samples regardless of group.

Bottom line: The number of DNA sequences were not significant different and thus, DNA sequences needed to be looked at individually for each group!

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Lacto-ovo-vegetarianism is a dietary lifestyle which excludes red meat, poultry, and fish. It consists of grains, legumes, fruits and vegetables, and allows consumption of diary products and eggs. It is known to be associated with lower rates of obesity, heart disease, type 2 diabetes, decrease blood pressure, reduce blood cholesterol levels, and certain types of cancer.

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Participant Criteria:

15 healthy omnivorous individuals were recruited and then switched to a lacto-ovo-vegetarian diet for 3 months, meaning that they were allowed to eat eggs and dairy but not allowed to eat meat. These participants were also referred to as "short-term vegetarians".

7 healthy omnivorous individuals were named "control group 1" and 7 healthy long-term vegetarian individuals were named "control group 2".

Participant Details:

Participants were excluded if they took antibiotics at any point in the 3 months leading up to the study or during the study period.

Blood and fecal samples were taken and weight was measured for each participant at day 0 and day 91.

Fecal sample RNA was purified and DNA was isolated immediately after sampling and sample details were collected.

Participants were asked to sign consent forms and the study was carried out in Sweden at the Karolinska Institute.

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Diet can modulate the gut microbiota such that it can influence the response of the immune system, via direct and indirect pathways.

Appropriate nutrition is required for proper immune cell function, but balance is key. While undernutrition may cause immunodeficiency and prone to infections, overnutrition can promote chronic inflammation and immune disorders.

Nutrition affects the rate of protein synthesis and cell proliferation. Certain vitamins are essential for conducting immunological functions, and lipids and fatty acids have immune-regulatory effects.

Healthy gut microbiota is determined by the balance between pro- and anti-inflammatory signals, which tolerates beneficial bacteria and fight against pathogenic microbes.

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Introduction written by Kate

Study Design, and Results (DNA Library and Metagenome Sequencing; Impact of the Short-Term Vegetarian Diets on Metagenome; Short- and Long-Term Vegetarian Diet-Associated Functional Changes in the Microbiome) written by Alisha

Results (Impact of Diet Pattern on the Diversity of Immune System; Interplays Between the Immune System and the Gut Microbiota) written by Nathalie

Conclusions, Punch Line, Critiques, Discussion, and Outlook & Future Directions written by Raymond

All members involved in integrating relevant multimedia, compiling the References list, formatting the Padlet, and editing each other's work

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3-months switch to a lacto-ovo-vegetarian diet from an omnivorous diet did not affect immune system diversity or metagenome composition.

However, there was increased prevalence of beneficial bacteria including butyrate-producers and probiotic species.

Various bacteria associated with IgA expression, a major class of gut antibody, was also identified.

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Diets of subjects were not well characterized: The authors did not document the specific food items that subjects were eating before or during the study. Without knowing the levels of different macromolecules in the diets, we cannot be sure what is affecting the gut microbiome. In addition, interactions between different food items can also potentially play a role (Vernocchi et al., 2020).

Findings were correlational: The authors noted differences in bacterial and immune cell populations between the examined diets. However, a clear causation was not established. They suggested possible mechanisms on how the different diets could induce selective pressures, but experiments were not done to support these hypotheses.

No follow-up experiments conducted: Subjects in the short-term vegetarian diet treatment group were not reexamined at a later period to determine how long the effects on microbiome and immunity persisted after they returned to an omnivorous diet. This finding could influence the therapeutic implications of exploiting temporary vegetarian diets.

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First study to simultaneously examine the effects of a 3-months vegetarian diet on both the microbiota and the immune system.

Identified the involved specific bacterial and immune cellular players at a molecular level by employing high-throughput sequencing on the metagenome alongside the BCR and TCR repertoires.

Compared results with two separate control groups (long-term omnivorous diet and long-term vegetarian diet) to show that a temporary switch to vegetarian diets can have distinct effects on the gut and immune system.

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Brisou G, Verney A, Wenner T, et al. (2014). A restricted IGHV gene repertoire in splenic marginal zone lymphoma is associated with autoimmune disorders. Haematologica 99:e197-198.

Childs, C. E., Calder, P. C., & Miles, E. A. (2019). Diet and Immune Function. Nutrients, 11(8), 1933. https://doi.org/10.3390/nu11081933

Dietitians of Canada (2010). Eating guidelines for Lacto-Ovo Vegetarians. https://www.mountsinai.on.ca/care/fammed/patient-resources/nutrition/vegetarian.pdf

Fava F, Rizzetto L, Tuohy KM (2018). Gut microbiota and health: connecting actors across the metabolic system. Proc Nutr Soc 78:177-188.

Gutzeit C, Magri G, Cerutti A (2014). Intestinal IgA production and its role in host-microbe interaction. Immunol Rev 260:76-85.

Kashyap M, Thornton AM, Norton SK, et al. (2008). CD4 T cell-mast cell interactions alter IgE receptor expression and signaling. J Immunol 180:2039-2043.

Luca F, Perry GH, Di Rienzo A (2010). Evolutionary adaptations to dietary changes. Annu Rev Nutr 30:291-314.

Perlman RL (2016). Mouse models of human disease. Evol Med Public Health 2016:170-176.

Petta L, Fraussen J, Somers V, Kleinewietfeld M. (2018). Interrelation of diet, gut microbiome, and autoantibody production. Front Immunol doi:10.3389/fimmu.2018.00439

Platts-Mills TA, Schuyler AJ, Erwin EA, et al. (2016). IgE in the diagnosis and treatment of allergic disease. J Allergy Clin Immunol 137:1662-1670.

Tomova A, Bukovsky I, Rembert E, et al. (2019). The effects of vegetarian and vegan diets on gut microbiota. Front Nutr doi.org/10.3389/fnut.2019.00047

Vernocchi P, Chierico FD, Putignani L (2020). Gut microbiota metabolism and interaction with food components. Int J Mol Sci doi:10.3390/ijms21103688

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Zhang and colleagues provided at a molecular level crucial insight into how change from an omnivorous diet to a generic vegetarian diet might affect the gut microbiota and immune repertoire. Nonetheless, future studies should aim to characterize each participant's diet because the authors showed that microorganism species within the gut are under selective pressure depending on the type of food (i.e., the major biological macromolecules that are being ingested).

1. Broaden the implications by conducting follow-up studies to repeat the present experiments with different forms of vegetarian diets.

Can be based on factors such as geography, culture, or religion.

Experimenters should keep careful track of all food items that the subjects are eating for both short- and long-term, and then compare the microbiome / immune repertoire.

Doing so would make the results more translatable to communities worldwide.

Challenge is that strictly controlling a person's diet for long periods of time would prove difficult, and perhaps even unethical from certain perspectives given that we are not entirely sure on how the specific dietary components might influence overall human health.

2. Although not as translatable compared to studying in humans, using animal models would allow researchers to more readily regulate the subjects' diets. For instance, mice are opportunistic omnivores, and would be a suitable candidate for replicating the experiments done in this study (Perlman, 2016).

Give mice a controlled omnivorous diet over a sustained period before starting experiments (i.e., baseline levels).

Control group remains on this diet, while the treatment groups receive either a short- or long-term vegetarian diet.

Afterwards, examine the gut microbiota and immune repertoire using the techniques presented in this paper.

In summary, experimental approaches using animals would deepen our understanding on the effects of specific macromolecules and their interactions with gut bacteria.

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A 3-months vegetarian diet can indeed affect your gut microbiota and immune repertoire!

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Fava et al., 2018: Schematic summarizing the understanding on how vegetarian diets play a role in influencing our health prior to the present study

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Although this study was framed around examining the effects of a short-term vegetarian diet, the authors also collected data on how a long-term vegetarian diet might influence the immune system and microbiota, since it was used as one of the controls. Interestingly, some of the differences at the molecular level between short- versus long-term vegetarian diets warranted further discussion.

Expression of IGHV genes showed an opposite trend: Certain IGHV genes have been found in auto-antibodies and associated with autoimmune diseases (Brisou et al., 2014). Specifically, IGHV5-51 was increased in short-term vegetarians but was decreased in long-term vegetarians when compared to the omnivorous control. One possible explanation is that a short-term vegetarian diet might actually sensitize our autoimmunity. Alternatively, the changes in genes are due to the sudden drastic change in diet (Luca et al., 2010), and not necessarily because of taking on a short-term vegetarian diet.

Serum IgE expression decreases with longer vegetarian diet: The immunoglobin IgE is a major immune index mediating allergies (Platts-Mills et al., 2016). Subjects in the long-term vegetarian group had lower IgE expression versus the short-term group, which already showed decreased levels compared to omnivorous control. Because IgE production is regulated by CD4 T cells (Kashyap et al., 2008), the authors examined TCR diversity and found a reduction in long-term, but not short-term, vegetarians. Indeed, we see decreases in gut inflammation in long-term vegetarians (Tomova et al., 2019). The authors speculated that reduction of TCR diversity potentially dampens both proinflammatory and allergic responses.  However, this speculation should be interpreted carefully since further studies are needed to determine the specific T cell populations that are being suppressed with long-term vegetarian diets.

Certain bacterial species negatively associated with TCR diversity were positively associated with IgA expression: Findings from the present study suggested that IgA, which is the key gut Ig class and regulates commensal bacteria (Gutzeit et al., 2014), maintains the critical homeostasis by balancing a wide range of bacterial populations. Thus, it was fascinating to note that some bacteria were inversely associated with TCR diversity versus IgA expression. This links back to differences between short- versus long-term vegetarian diets because it continues from the point above and raises a possible mechanism underlying the prevalence of reduced gut inflammation when on a vegetarian diet for longer periods of time (Tomova et al., 2019). That is, long-term vegetarian diets cultivate bacteria that promotes IgA expression, which in turn keeps any particular species from growing out of control. As a result, a gut environment in homeostasis would experience less inflammation.