Fecal Microbiota Transplantation Induces Social Fear by Samantha Analiese

Conclusions

samanthaanaliese — 2024-03-02 02:08:20 UTC

Gut composition in FMT SAD mice showed a reduction in beneficial strands, like bacteroides nordii and cellulosilyticus, and an increase in non-beneficial strands, in this context, like phoncaeicola massiliensis.
Mice with an FMT of SAD showed increased social fear, without any other behavioural changes.
Mice with an FMT of SAD showed an immediate decrease in stress levels, but at the three time points, showed no difference in stress when compared to healthy controls.
Mice with an FMT of SAD showed a decrease in peripheral and central immune function.
Mice with an FMT of SAD showed a decrease in blood-brain barrier integrity.
Mice with an FMT of SAD had a reduction in oxytocin and vasopressin peptides, two social behaviour-modulating hormones, their receptors and gene expression in the MeA, mPFC and BNST.

Limitations

samanthaanaliese — 2024-03-02 02:08:47 UTC

Though the researchers tested for changes in vasopressin and oxytocin in the mPFC, MeA, and BNST, they did not test for changes in the hippocampus
- Since oxytocin and vasopressin in the hippocampus create the social memories that are the basis of SAD, and the hippocampus regulates and transmits the memories into the fear or HPA axis/circuit, it would be important to study^19,20,21.
The study does not look into the long-term effects of the microbiota transplant, specifically blood stress levels, which may have contributed to the lack of change in corticosterone levels in the mice.
- Microbiota composition is unstable due to diet, lifestyle, and environmental fluctuation. Individuals with SAD tend to have higher cortisol levels, which causes hypervigilance. Lengthening the study would help verify if this occurrence is replicated in FMTs of SAD too²².
The sample size was small and although female and male individuals were used, only male mice were the recipients of the FMTs.
- Studies have found that social anxiety tends to affect males and females equally, with some stating that females are more likely to be affected by it. Testing in female mice would help elucidate if the FMTs show sex-related differences in composition and behaviour ²³.

Contributions

samanthaanaliese — 2024-03-02 02:09:22 UTC

Reduction in beneficial strains, when comparing the gut composition of healthy and SAD individuals, and lack of non-social fear behavioural changes, shows that the microbiome may be a specific target to treat SAD.
The physiological changes in the microbiome and all over the body show the biological manifestation of social anxiety.

Both changes help to differentiate SAD from being shy or reserved.

Discussion (Comparison)

samanthaanaliese — 2024-03-02 02:10:10 UTC

Human microbiota from healthy and SAD individuals was sequenced using 16S rRNA and found no change in alpha diversity, but a large difference in beta diversity with a decrease of bacteroides and an increase in a non-beneficial strand called aneromassilibacillus, similar to the paper we chose.
- Interestingly enough, they found that individuals who take medicine for SAD or are co-morbid for psychiatric disorders like depression or anxiety had comparable microbiota composition. This means the gut may be a multi-psychiatric target²⁴.
Infants and 1 year old's microbiota were sequenced and they found a reduction in bacteroides and a large difference in diversity among anxious-behaving children, who had an increase in non-beneficial strains like veillonella, dialister, and clostridiales, similar to the paper we chose.
- However, they also showed a decrease in investigatory behaviour and reduced amygdala volume, when compared to healthy controls. This could suggest that some individuals are naturally predisposed to developing a comparable microbiota to SAD individuals, and when combined with negative social scenarios, may develop the illness²⁵.

Next Steps

samanthaanaliese — 2024-03-02 02:10:27 UTC

Using bacteroides nordii and cellulositylus as a probiotic in mice with FMTs from SAD individuals to see if it can resolve physiological changes, social fear, and gut dysbiosis, and therefore be a potential treatment for SAD.
- Ethically and scientifically, it would not be right to market it as a cure because the response to probiotics varies between people and what may be helpful could be harmful to someone else.
- As well, it might only resolve the microbial dysbiosis in SAD, similar to how psychiatric drugs improve behavioural symptoms.
With mice with SAD FMTs, use an MRI scan to see if there are any changes in hippocampal volume, and take a biopsy to see if there are changes in oxytocin and vasopressin peptides, their receptors, and gene expression there too²⁵.

SAD FMT lowers basal stress levels

2024-03-17 18:16:29 UTC

The figure shows the corticosterone levels of the mice who received a SAD FMT compared to the healthy control FMT mice before the stressor at t=0 and post-stressor until t=90.

Basal corticosterone levels were lower pre-stress in the SAD group signifying the effect of the social anxiety fecal microbiota transplant in altering initial stress levels.
No differences post-stress were observed.

Results 7

2024-03-17 18:16:42 UTC

Methods: Measuring corticosterone levels

2024-03-17 18:24:16 UTC

The basal corticosterone levels were measured in mice as an indicator of stress. Corticosterone is a glucocorticoid stress hormone that induces the HPA axis in mice to help the body manage stress, reduce inflammation, and influence the immune system¹⁴.
Levels were measured using blood samples and collected every 30 minutes to determine the changes in levels over time pre-stress and post-stress.

SAD FMT alters immune system

lauretymen — 2024-03-17 19:07:31 UTC

Interleukin-17A levels are displayed under the four stimulation substances using boxplots. The blue boxplots indicate the healthy controls which were compared to the red boxplots being the SAD FMT individuals.

IL-17A cytokines in the SAD FMT group were lower than in the control group following stimulation of all substances.
However, statistical significance was only revealed with LPS and ConA stimulation.

These results are consistent with a previous study the authors highlighted which saw IL-17A rescue social deficits. This signifies that social anxiety disorder can alter the immune system when it is activated.

Methods: Investigating SAD FMT impact on immune function

lauretymen — 2024-03-17 19:31:44 UTC

lauretymen — 2024-03-17 19:31:55 UTC

Part 1: Illeal Tissue Explant Stimulation

Following euthanasia, the researchers took small intestinal illeal tissue samples to examine gut immune function. The researchers explanted the tissue and activated the samples using four independent substances: a buffer, lipopolysaccharide, concanavalin, and T-cell receptor clusters of differentiation 3 and 28.

The buffer was used as a control and the LPS, ConA, CD3/CD28 were used as experimental conditions.
The role of these substances is to elicit an immune response. For example, LPS surrounds gram-negative bacteria providing integrity to bacterial cells, and with the presence of pathogens, it will respond to the proinflammatory stimuli^15. Therefore, releasing cytokine factors such as interleukins and tumor necrosis factor¹⁵.

After activation of the immune system, the cell suspension from each substance was harvested and centrifuged. Then, the IL-17A levels were tested from the supernatant using ELISA.

IL-17A are inflammatory cytokines responsible for inducing various proinflammatory cytokines and for T-cell activation. Therefore, their function greatly influences immune function and can be used as a marker for testing.

lauretymen — 2024-03-17 19:47:50 UTC

Part 2: Mesenteric lymph nodes & blood collection

To further study immune function, the authors harvested mesenteric lymph nodes and blood to assess immune cell populations. Flow cytometry was the method of choice because of its quantitative advantage.

F4/80+ macrophages, T helper cells, and CD4+ T helper cells were of focus following the use of the flow cytometer

How a flow cytometer works

lauretymen — 2024-03-17 20:12:00 UTC

This video demonstrated the flow cytometer method, showing how the authors identified levels of immune cell populations¹⁷.

SAD FMT reduces immune function

lauretymen — 2024-03-17 20:39:53 UTC

The results of the flow cytometer are shown in the following boxplots. The healthy control FMT individuals (blue) were compared to the SAD FMT individuals (red).

F4/80+ macrophages and CD4+ T helper cells had reduced presence following SAD FMT compared to the control.
T helper cells increased following SAD FMT, however, showed no statistical significance.

Past literature has shown macrophages migrating away from MLNs due to microbiota perturbation and this experiment reinforces those findings^1. Additionally, the authors concluded the lowering of CD4+ T cells seen is consistent with inadequate immune memory due to social anxiety. Therefore, the authors concluded that social anxiety disorder reduces immune function as SAD impacts the microbiome.

Methods: Gene expression analysis

lauretymen — 2024-03-17 20:45:48 UTC

Frozen brain slices of the euthanized mice were collected and dissected to determine gene expression.

The authors examined the bed nucleus of the stria terminalis (BNST) region, medial amygdala (MeA), and medial prefrontal cortex (MePFC).

The total RNA was extracted from the regions of interest on the brain slices and quantified using a Nanodrop spectrophotometer. Then, the RNA was converted to cDNA by reverse transcription and RT-qPCR took place using SYBR green. This allows for gene expression to be analyzed.

The authors chose various genes of interest to determine if there would be an impact due to social anxiety disorder FMT.

The genes ranged from tumor necrosis factor-alpha, arginine, interleukins, Toll receptors, tight junctions, and many more. The authors mention they chose these genes due to past research that found alterations in these genes target the microbiome.

Result 1: SAD Leads to Alterations in the Bacteriome of Human Donors

2024-03-18 00:48:19 UTC

Method: Gut microbiota composition between patients with SAD and healthy controls (HCs) was analyzed using 16S rRNA sequencing.

Beta Diversity differs between healthy controls and patients with SAD

2024-03-18 00:50:47 UTC

Participants: 6 patients with SAD and 6 matched HCs, including both male and female.
Result: analysis confirmed statistically distinct Aitchison beta diversity between patients with SAD and HCs, p<0.05.

Method: Fecal Microbiota Transplantation

2024-03-18 01:03:07 UTC

Murine recipients: 36 male C57Bl/6J mice received FMT from patients with FMT; 36 male C57Bl/6J mice received FMT from HCs.
Before transplantation, all mice received antibiotic cocktail treatment to deplete their pre-existing microbiota.
All mice were randomized to a specific donor and received FMT via oral gavage, with 6 mice receiving from a same donor.

Bacteriome: Beta Diversity

2024-03-18 01:05:31 UTC

Beta diversity significantly differs between SAD mice and HC mice, p<0.001.

Result 2: SAD FMT Leads to Alterations in Bacteriome of Murine Recipients

2024-03-18 01:06:19 UTC

Method: Bacteriome and virome composition of murine recipients were analyzed using full metagenomic

Bacteriome: alpha diversity

2024-03-18 01:19:54 UTC

No group differences were found in alpha diversity (Chao1, Shannon, and Simpson).
Alpha diversity for both groups reduced at the end of the study.

Virome: alpha and beta diversity

2024-03-18 01:20:43 UTC

No group differences were found in alpha or beta diversity

Result 3: SAD FMT Increases Sensitivity to Social Fear

2024-03-18 01:24:59 UTC

Method: Social fear conditioning was conditioned on day 1, pairing electric shock with social interaction. Interaction time with social stimulus or non-social stimulus was measured on day 2 to infer social fear extinction level.

SAD Mice Showed Less Social Fear Extinction

2024-03-18 01:29:01 UTC

For non-social stimulus (ns1-3), no significant differences were observed between two mice groups in interaction time.
For social stimulus (s1-6), SAD mice showed significantly less social interaction time than HC mice, indicating decreased social fear extinction. This provides evidence for SAD FMT leading to increased sensitivity for social fear in mice.

lauretymen — 2024-03-18 17:41:26 UTC

The image comes from a video showing the steps taken to perform RT-qPCR and how the authors generated their gene expression results¹⁸.

How is the central immunity affected by SAD FMT

lauretymen — 2024-03-18 17:52:05 UTC

Immune function was established to be altered in the individuals who received the SAD FMT compared to the control in the previous experiment. Therefore, neuroinflammatory and blood-brain barrier gene expression was studied to understand the specific role of SAD FMT on immunity.

Here are the significant results found:

Neuroinflammatory gene expression

Arg1 expression was lowered in all three regions
Tumor necrosis factor-alpha, Interleukin 10, and Cxcl15 expression lowered in the MeA region
Toll-like receptor 4 expression was lowered in the MePFC region

Blood-brain barrier gene expression

Tjp 1 was the most significant as its expression was reduced in the MeA and MePFC region
Cldn5 expression was reduced in the MeA region

The findings demonstrate the ability of the SAD FMT to reach neuroinflammatory and blood-brain barrier markers. This concludes how social anxiety microbiota can reach the immune system to impact immunomodulatory effects.

Results 4

lauretymen — 2024-03-18 17:58:20 UTC

Results 5

lauretymen — 2024-03-18 17:58:26 UTC

Results 6

lauretymen — 2024-03-18 18:37:05 UTC

SAD FMT altered ocytocin and vasopressin gene expression

lauretymen — 2024-03-18 18:42:41 UTC

The results of the RT-qPCR investigation of oxytocin and vasopressin gene expression are described above. The bar graph splits each region studied, showing the gene expression levels in the healthy control FMT mice (blue) and the SAD FMT mice (pink).

No significant differences between groups for the expression of vasopressin and oxytocin genes and their receptor in the BNST region.
Reduction of oxytocin gene and oxytocin receptor expression in the MeA region in individuals who received the social anxiety disorder FMT.
Reduction of oxytocin receptor and vasopressin receptor 1a expression in the MePFC region in individuals who received the social anxiety disorder FMT.

With these results, the authors present evidence for social anxiety disorder reducing oxytocin and vasopressin gene expression and justifying its role in social behavior.

Methods: Oxytocin neuronal expression

lauretymen — 2024-03-18 18:45:08 UTC

The authors sectioned the paraventricular nucleus of the hypothalamus (PVN), the supraoptic nucleus (SON), and the bed nucleus of the stria terminalis (BNST). They stained these regions for oxytocin-containing neurons using immunohistochemistry and observed the regions under a microscope.

Oxytocin is a neuropeptide responsible for social behavior, leading the authors to choose oxytocin for studying its effect due to a SAD FMT. The PVN, SON, and BNST regions regulate social behavior through oxytocin neural signaling making the regions relevant to study the effect of oxytocin^1.

Methods: Oxytocin and vasopressin gene expression

lauretymen — 2024-03-18 18:46:27 UTC

The same method was performed in this experiment as the gene expression analysis experiment. Thus, RT-qPCR was performed as described above in the methods of results 6.

The total RNA was extracted from the regions of interest on the brain slices and quantified using a Nanodrop spectrophotometer. Then, the RNA was converted to cDNA by reverse transcription and RT-qPCR took place using SYBR green. This allows for gene expression to be analyzed.

However, the focus here was oxytocin and vasopressin genes as they authors found evidence for their effect in altering social behavior.

The authors examined the bed nucleus of the stria terminalis (BNST) region, medial amygdala (MeA), and medial prefrontal cortex (MePFC).

BNST is sensitive to SAD fecal microbiota transfer

lauretymen — 2024-03-18 20:31:59 UTC

The quantification of staining of oxytocin neurons is observed in the following boxplots. The graph only highlights the comparison of healthy control to SAD in the BNST region as its region is the only one expressing statistical significance.

BNST oxytocin neurons decreased in those with the SAD fecal microbiota transfer compared to the healthy control fecal microbiota transfer.
No differences in oxytocin neurons were found in the PVN or SON regions when comparing healthy control FMT mice to SAD FMT mice.

With these findings, the authors concluded that the BNST region is associated with social fear behavior.

Introduction: Social Anxiety Disorder (SAD)

triciaedada — 2024-03-20 17:19:16 UTC

SAD is a psychiatric disorder, where an individual experiences an intense feeling of fear or anxiety in scenarios where they may feel judged by others¹.
SAD impacts an individual’s quality of life, as it tends to appear early and may also persist throughout their lives¹.
Some of the symptoms include a rapid heart race, shaking, and sweating².
According to Statistics Canada, 8% - 13% of Canadians may experience SAD in their lifetime³.

Background Findings 1: Gut-Brain Axis

triciaedada — 2024-03-20 19:40:17 UTC

A previous article found that the gut microbiota plays a role in the brain and behaviour related to social processes throughout the animal kingdom⁵.
Similar to lectures in HMB385, this finding demonstrates the axis between the gut and the brain.
- There are 3 pathways that connect the brain and the gut⁶:
  - immune system
  - circulatory system
  - vagus nerve
- A study found that germ-free mice had social impairments illustrating that the gut plays a vital role in our social behaviour⁶.

Background Findings 2: Gut Microbiota Composition Differences

triciaedada — 2024-03-20 19:43:12 UTC

Using Beta Diversity, a previous article discovered the differences in gut microbiota composition in SAD patients compared to healthy, age-matched controls. In particular the differences include an increased abundance of genera Anaeromassillibacillus and Gordonibacter in SAD individuals, while Parasuterella was higher in abundance in healthy controls⁷.
However, the limitation is that it is unknown whether this difference plays a causal role in social fear or anxiety (characteristics of SAD)¹.

Background Finding 3: Immune Function and Oxytocin Signalling

triciaedada — 2024-03-20 19:48:57 UTC

In social fear scenarios, such as inducing psychological stress on rodent participants, numerous studies have observed:
- a hyperresponsive HPA-axis⁸
- increased immune-inflammatory response⁹
- changes in expression in genes relating to dopamine function⁹
- negative impacts on the central oxytocin system¹⁰
Thus, illustrating a possible role of the immune system and oxytocin in SAD.

Goals of the Study

triciaedada — 2024-03-20 19:54:55 UTC

There are 2 aims in this study:

To understand the role of gut microbiota in SAD
To find a new approach for SAD treatment

Faecal Microbiota Transplantation (FMT)

triciaedada — 2024-03-20 19:56:29 UTC

FMT is where faecal bacteria is transferred from one individual into another individual¹¹.
It has been known to be a good technique to understand how the microbiota can cause phenotypic changes in the recipient¹.
FMT has already been used in other disorders such as depression and schizophrenia¹.
- These studies have shown transferable psychological and physiological traits¹.
- For example, in a depression FMT, one of the transferable traits was anhedonia¹¹ while for a schizophrenia FMT, psychomotor hyperactivity was evident in the recipient¹².
However, FMT has yet to be used for SAD, which the researchers of this article are very interested in¹.

Research Question

triciaedada — 2024-03-20 20:13:46 UTC

How does the gut microbiome play a role in the behaviours seen in Social Anxiety Disorder after a Faecal Microbiota Transplantation?

Hypothesis

triciaedada — 2024-03-20 20:15:13 UTC

The microbiota may play a casual role in promoting host social fear behaviour.

SAD Dilemmas

triciaedada — 2024-03-20 21:30:24 UTC

Currently, there are ineffective and limited treatments for SAD¹. In particular, there are up to 40% of SAD patients who do not respond to treatment, whether it is drug therapy or psychotherapy⁴.
The causes of SAD are still unclear¹.

References

triciaedada — 2024-03-23 16:35:14 UTC

Ritz NL, Brocka M, Butler MI, Cowan CS, Barrera-Bugueño C, Turkington CJ, Draper LA, Bastiaanssen TF, Turpin V, Morales L, et al. Social anxiety disorder-associated gut microbiota increases social fear. Proceedings of the National Academy of Sciences. 2023;121(1). doi:10.1073/pnas.2308706120
Arlin Cuncic M. Signs that you may have social anxiety disorder. Verywell Mind. 2023 Dec 7 [accessed 2024 Mar 20]. https://www.verywellmind.com/social-anxiety-disorder-symptoms-and-diagnosis-4157219
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Ritz NL, Brocka M, Butler MI, Caitlin, Barrera-Bugueño C, Christopher, Draper LA, Thomaz F.S. Bastiaanssen, Turpin V, Morales L, et al. 2023. Social anxiety disorder-associated gut microbiota increases social fear. Proceedings of the National Academy of Sciences of the United States of America. 121(1).
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The method of ELISA

lauretymen — 2024-03-24 15:29:41 UTC

This video demonstrates how the researchers used ELISA to measure levels of IL-17A¹⁶.

Acknowledgements

lauretymen — 2024-03-24 21:16:54 UTC

Introduction: Tricia Edada

Results 1-3: Yihan Chen

Results 4-7: Laure Tymen

Discussion: Samantha Rahamatali

Discussion

samanthaanaliese — 2024-03-24 21:23:26 UTC