The gut-brain axis (GBA) consists of bidirectional communication between the central and the enteric nervous system, linking emotional and cognitive centers of the brain with peripheral intestinal functions. Recent advances in research have described the importance of gut microbiota in influencing these interactions.

1. Microbiota-Gut-Brain Axis: New
Therapeutic Opportunities
Dr Mohit Kher
Pharmacology
LHMC

2. INTRODUCTION
MICROBIOTA – A collection of microbes.
MICROBOME – A collection of microbial genomes.
The microbiota inhabiting our bodies outnumbers our human cells by
approximately 1.3:1
The exact composition of the gut microbiota present in each
individual is different.
These commensal gut microbes largely have a more symbiotic
relationship with their host.
Important roles in digestion and immune system development.

3. Stomach &
Duodenum-
Helicobacter
Streptococcus
Jejunum & Ileum-
Bacteroides
Streptococcus
Lactobacillius
Bifidobacteria
Fusobacteria
Colon-
Bacteroides
Prevotella
Facaelbacterium
Ruminococcus
Roseburia
Clostridium
Bifidobacteria
Collinsella
Desulfovibrio
Bilophila
Akkermansia
Methanobrevibacter

4. Gut Microbiota Profile
Bidirectional gut-brain communication may seem obvious with respect to the
central control of hunger and satiety signals.
More widely implicated in cognition, social behaviour, fear expression, and stress
response.
Changes in behaviour are associated with both GI illness and functional GI
disorders.
Colonization of the gut is considered by most researchers in the field to primarily
occur at birth.
Birth via Caesarean-section results in a very different seeding of the gut
microbiota than that from vaginal delivery.

5. Infants born through CS will have a lower colonization of
Bifidobacterium, Bacteroides, and Lactobacillus.
Latter profile more closely resembles the maternal vaginal
microbiome.
Functional effects of the differential seeding may be linked to the
increased relative risk for childhood obesity and asthma.
Composition of the gut microbiome is further affected by antibiotic
use, Breastfeeding, diet, geography and exercise levels.

6. Signaling Mechanisms
From the Gut Microbiota
to the Brain

7. • Bottom-up modulation of the CNS by the microbiome occurs
primarily through Vagus nerve.
• This communication is mediated by short-chain fatty acids
(SCFAs), secondary bile acids (2BAs) and tryptophan metabolites.
• These molecules propagate signals primarily through interaction
with enteroendocrine cells (EECs), enterochromaffin cells (ECCs),
and the mucosal immune system.
• Microbiota can independently produce or contribute to the
production of a number of neuroactive molecules including
GABA, 5-HT, norepinephrine and dopamine.

8. Bacteria & Neurotransmitters
Neurotransmitter Genus
GABA Lactobacillus,
Bifidobacterium
Norepinephrine Escherichia, Bacillus,
Saccharomyces
Acetylcholine Lactobacillus
Serotonin Candida, Streptococccus,
Escherichia, Enterococcus

10. Neuroendocrine and Enteroendocrine Signaling
Pathways
• EECs are interspersed between gut epithelial cells throughout the length of the
gut and contain more than 20 different types of signaling molecules.
• These molecules can reach CNS through systemic circulation and involved in
ingestive behavior or act locally and activate closely adjacent afferent vagal
terminals in the gut or liver to generate brain signals.
• A series of receptors involved in the regulation of satiety and hunger have
been identified on these cells.
• Ileal expression of farnesoid X receptor (FXR) is activated by bile acids leading
to production of FGF19.

11. • Activation of the arcuate nucleus of the hypothalamus by the
FXR/FGF19 (Neuropeptide Y) has been implicated in improved
central regulation of energy and suppression of hypothalamic
pituitary adrenal axis activity.
• Some intestinal L cells express surface receptor G protein-
coupled bile acid receptor (TGR5), which is activated mostly by
secondary bile acids.
• SCFAs stimulate L cells located at the distal ileum to secrete
peptide YY and GLP-1, which induce satiety and behavioral
changes.
• SCFA modulate the expression and secretion of GLP-1 via FFAR2
and FFAR3 on L cells.

12. Enterochromaffin Cell Signaling

13. Neuroimmune Signaling
• Mouse models of multiple sclerosis and stroke have identified
substantial roles for gut microbial regulation of autoimmunity,
inflammation and immune cell trafficking.
• Gut microbiota influence the development and function of
microglia.
• GF mice have compromised microglia maturation and
morphology, leading to weakened early responses to pathogen
exposure.
• This phenotype can be normalized by postnatal SCFA
supplementation.

14. Direct Neural Signalling
• Vagal receptors can sense regulatory gut peptides, inflammatory
molecules, dietary components and bacterial metabolites to
relay signals to the CNS.
• Toll-like receptors 2 and 4 can recognize peptidoglycan and
lipopolysaccharide
• L rhamnosus (JB-1), B fragilis, and isolated polysaccharide A & B
fragilis all have been shown to activate intestinal afferent
neurons.

15. Signaling From the Brain
to the Gut Microbiota

16. GI Motility
• Both branches of ANS regulate gut functions.
• Regional intestinal transit times affect water content,
nutrient availability and bacterial clearance rates.
• Impaired migrating motor complex regularity can
reduce the flow rate, leading to small intestine
bacterial overgrowth.
• Bristol Stool Scale - Strongly correlates microbial
richness and composition with intestine transit time.

17. Intestinal Barrier
• Mechanisms: Direct modulation of epithelial permeability and
alterations in the properties of the intestinal mucosal layer.
• Rodent models – Increased jejunal and colonic permeability in
response to both acute and chronic stress.
• Increased leakiness facilitates the translocation of bacteria, such
as Escherichia coli, and their products (LPS), leading to a
proinflammatory environment in the gut.
• Hypersensitivity, psychological stress and brain injury leads to a
less-protective mucus layer.

18. Direct Modulation of Gut Microbiota by
Luminal Release of Neurotransmitters
• Host neuroendocrine system can communicate with the
microbiota more directly via intraluminal release of host
signaling molecules, including catecholamines, 5-HT,
dynorphin and cytokines.
• Epinephrine and norepinephrine are shown to increase
the virulence properties of several enteric pathogens.
• Gut microbiota show circadian rhythmicity.

19. ALTERED GUT MICROBIAL
PROFILE IN BRAIN DISORDERS
Noted in Alzheimer’s disease, Parkinson’s disease and in patients
with depression, autism, and post-traumatic stress disorder.
Fecal microbiota transplant (FMT) from patients with depression
resulted in a transfer of the depressive phenotype from patient
into preclinical models.

20. Acute stress – Appropriate HPA axis activation
Chronic stress - Inappropriate HPA axis activation.
Changing the early-life microbial profile through antibiotic
, lack of breastfeeding, birth by C-section, host genetics
and other environmental factors, can impact stress-
related physiology and behaviour.
Prenatal stress exposure in women lead to infant GI
symptoms and allergic reactions.
STRESS

21. Depression and Anxiety
• Anxiety and depression often are comorbid conditions in patients
with IBS.
• Preclinical studies have shown the microbiota’s capacity to
modulate emotional behaviors.
• Depressed human-to-rodent fecal microbial transplants have
induced depressive behaviors in the animal models.
• Coprococcus and Dialister species - Positively associated with
quality of life scores.

22. Parkinson’s Disease
• Risk of Parkinson's disease development increases
with infrequency of bowel movement and
constipation severity.
• FMT from PD patients into mouse models of PD
resulted in both motor deficits and
neuroinflammation.
• A full truncal vagotomy for treatment of peptic ulcer
disease have a diminished risk of PD.

23. Autism spectrum disorder
• ASD patients frequently present with gut-related comorbidities
and unusual dietary patterns.
• Bifidobacterium – Reduced abundance
• Clostridia – Increased abundance
• Treatment with vancomycin resulted in positive alterations in
behavioural symptoms.
• FMT from healthy individuals into ASD patients resulted in
improvements in both gut comorbidities and behaviours.

24. Schizophrenia
• Limited studies on the impact of gut microbiota.
• Presence of Candida albicans is associated with worse
psychiatric symptoms in males.
• Specific taxa, including Veillonellaceae and
Lachnospiraceae were associated with schizophrenia
severity.

25. Obesity/Food Addiction
• Gut microbiota and its metabolites play an crucial role in the
modulation of satiety signals and eating behaviors.
• Preclinical studies - Fecal transplantation from hyperphagic
obese mice to germ-free mice; induce hyperphagic behavior
and weight gain.
• Gut microbiome has been associated with changes in brain
microstructure in obesity.
• Dramatic change in gut microbial composition after bariatric
surgery.

26. Functional Intestinal Disorders
• Significant microbial shifts in fecal microbial
community composition between healthy people and
IBS patients.
• Dysbiotic IBS subgroup also differed in regional brain
volumes from the eubiotic group.
• Decreased relative abundance of the
genera Bifidobacterium and Lactobacillus and an
increased Firmicutes:Bacteroidetes ratios.

27. THERAPEUTICALLY TARGETING THE MICROBIOTA-GUT-
BRAIN AXIS
• The term psychobiotic was coined in 2013 by Dinan et al. as a
novel class of psychotropic, defined as a “live organism that,
when ingested in adequate amounts, produces a health
benefit in patients suffering from psychiatric illness.”
• Psychobiotics includes probiotics, prebiotics, synbiotics, and
postbiotics.
• These substances can be delivered through supplements,
functional foods and improvements to dietary intake.

28. Probiotics: Live Biotherapeutics
• First described over 100 years ago in the work of Elie Metchnikoff -
Consumption of lactic acid bacteria in fermented milk in a population of
poor Bulgarians.
• Probiotics are defined as “live microorganisms that, when administered in
adequate amounts, confer a health benefit on the host”.
• Health benefits of probiotics are entirely strain-dependent.
• Strains of Lactobacillus and Bifidobacterium have well-defined safety
profiles.
• Require daily consumption to maintain positive effects.

29. Probiotics studied with respect to microbiota-gut-
brain axis signalling
Human cohort studies Probiotic treatment Treatment Duration Effect
Stress Multibiotic: L.
acidophilus Rosell-52,
B. longum Rosell-175
3 weeks ↓ stress-associated GI
symptoms
L. plantarum DR7
12 weeks ↓ stress and anxiety
measures
-Improvements in memory
and cognition
-Enhanced serotonergic
signaling
↓ plasma cortisol
↓ proinflammatory
cytokines
Pregnancy L. rhamnosus HN001 <6 months ↓ postpartum depression
and anxiety Scores

30. Students during
exam stress
L. casei Shirota YIT
9029
8 weeks ↓ salivary cortisol
levels
↓ physical symptoms
of stress/anxiety
IBS patients B. longum NCC3001 6 weeks -↓ in depression scores
-No effect on anxiety
-↑ quality of life
MDD patients Multibiotic: L.
acidophilus, L. casei,
B. bifidum
8 weeks ↓ depression scores
Multibiotic: L.
helveticus R0052,
B. longum R0175
(CNCM strain I-3470)
8 weeks ↓ Depression score
L. plantarum 299V
(adjunctive to
antidepressants)
8 weeks -Improved attention
and verbal learning
-↓ Salivary cortisol

31. C. butyricum MIYAIRI
588 (adjunctive to
antidepressants)
8 weeks Improvements in
depression scores
Chronic fatigue
syndrome
L. casei Shirota 8 weeks ↓ anxiety symptoms
AD patients Multibiotic: L.
acidophilus, L. casei, B.
bifidum, L. fermentum
12 weeks -↑ cognition (MMSE)
-Changes in blood lipid
profile and
carbohydrate
metabolism factors
HIV patients Multibiotic: L. plantarum
DSM 24730,
S. thermophiles DSM
24731,
B. Breve DSM 24732,
L. paracasei DSM 24733,
L. delbrueckii subsp.
bulgaricus DSM 24734,
L. acidophilus DSM
24735,
B. longum DSM 24736, B.
6 months ↑ neurocognitive
performance

32. Schizophrenia
patients
B. breve A-1 4 weeks Improvements in
anxiety and
depressive scores
Bipolar
patients
Multibiotic: L.
rhamnosus GG,
B. lactis Bb12
14 weeks -Improvements in gut
symptoms
-No effect on
psychiatric symptoms
ASD Multibiotic: L.
acidophilus,
L. rhamnosus,
B. longum
3 months Improvements in
autism severity

33. Prebiotics
• Prebiotics are defined as a “substrate that is selectively
utilized by host microorganisms conferring a health
benefit”.
• Prebiotics consist of fibers such as inulin, fructo-
oligosaccharides, galacto-oligosaccharides (GOSs) and
resistant starch.
• Prebiotics are found in fruits, vegetables, grains and
human milk.

34. Synbiotics
• Synbiotics are a combination of probiotics and prebiotics.
• Mild to moderate MDD - GOS and a dual-strain probiotic (L.
helveticus and B. longum) ; reduction in depression scores and
positively impacted tryptophan signalling.
• Positive results of a fermented soy drink in a PTSD cohort.
• Functional GI symptoms were improved in a PD cohort.
• Synbiotic (Bifidobacterium infantis plus oligosaccharides)
reduced gut-related comorbidities related to ASD.

35. Postbiotics
• Postbiotics are nonviable entities that are metabolites of bacterial
fermentation and include bioactives such as SCFAs.
• General impacts of the gut microbiome and SCFAs on metabolic
traits and disease have recently been described.
• Inulin-proprionate ester; increases proprionate production in the
colon, brain imaging (via functional MRI) showed a reduction in
anticipatory reward responses in the striatum.
• Gut peptides: Role in stress, anxiety and depression.

36. • Paraprobiotics, or nonviable probiotics, e.g., heat-killed
probiotics, may exert biological activity in the host.
• Heat-killed, washed, paraprobiotic CP2305: Reduces stress
due to university exams in medical students.
• Heat-killed Lactobacillus paracasei PS23: Benefits in a
corticosterone-induced depressive phenotype, reversing
hippocampal and prefrontal cortex reductions in dopamine
levels.
• Distinct marketing advantage over probiotics.

37. Fermented Foods and Diet
• Lactobacillus delbreuckii subsp. bulgaricus and S. thermophilus.
• Dairy products - Bifidobacterium and Lactobacillus are commonly
used.
• Fermented milk drinks - Positive benefits in emotional processing
and stress.
• Mediterranean diets - Decreased risk of depression and cognitive
impairment.
• SMILES trial - Benefit in moderate to severe depression, with an
economic evaluation.

38. MEDICATIONS AND THE MICROBIOME
• A study on T2DM determined that metformin was responsible
for much of the alterations in the gut microbiota profiles of
T2DM patients.
• A small, recently published study described increases in
microbial alpha diversity in a cohort of depressed patients who
were treated with the SSRI (escitalopram) for 6 weeks.
• PPIs have a well-described effect on gut microbiota.

39. SCREENING THE MICROBIOME FOR
PSYCHOBIOTICS
• First step - Fresh faecal sampling, in conjunction with fresh plating of resultant bacteria
in a specific agar medium.
• Strains are then identified using genome sequencing.
• In the European Union, the group of microorganisms must meet certain criteria.
• Selected strains can be used in a battery of assays, such as enzyme assays, in addition
to in vitro cell testing.
• Depending on the cell type of interest, the cells can be monitored for growth;
morphology; and the production of bioactives.
• GPCRs within the gut can be directly targeted by microbially produced bioactives.

40. CONCLUSION

41. THANK YOU

42. Signalling Mechanisms Behind Microbiota-Gut-Brain
Axis Communication
Gut microbes can produce bioactive peptides, including
neurotransmitters; transformation of secondary bile acids; short-
chain fatty acids (SCFAs); branched-chain amino acids; and gut
hormones.
Modulating serotonin signalling in some cases.
Dopamine, noradrenaline, GABA, and acetylcholine can also be
synthesized by gut microbes.
Gut also contains a dense concentration of immune cells, which
provide a second line of défense to pathogens .

43. Vagus nerve is heavily implicated in microbiota-gut-brain axis
signalling.
Vagotomy was commonly used to treat peptic ulcer disease which
has been associated with a decreased risk of developing
Parkinson’s disease.
Recently, optogenetic stimulation was performed to show
communication between vagal gut-to-brain axis pathway and CNS
reward neurons.

44. • Microbiota can affect metabolism of drug and
its response.
• Gut microbiota represents an exciting
reservoir of new therapeutic opportunities.
• Targeting bacterial gut microbiota for the
effective treatment of CNS disorders.

45. Findings (Breast cancer)

46. • Current users of HRT at recruitment were more likely than
never users to develop breast cancer (relative risk 1.66 )
• Past users of HRT were, however, not at an increased risk of
incident (1.01)
• Incidence was significantly increased for current users of
preparations containing
Oestrogen only (1.30),
Oestrogen-progestagen (2.00)
Tibolone (1.45),
Others (1.44)

47. • The relative risk were increased for equine estrogen at both doses <0.625 mg
and >0.625 mg as compare to ethinyloestradiol doses < 1 mg and > 1mg(1.25,
1.36 vs 1.25, 1.19 respectively)
• The relative risks were significantly increased separately for oral, transdermal,
and implanted oestrogen-only formulations (1.32; 1.24; and 1.65 respectively)

48. • Results varied little between type of progestagens used;
or between continuous and sequential regimens
• Therapy for more than five years was associated with
higher risk irrespective of the constituent and regimen
followed
< 5 years >5 years

49. • In current users of each type of
HRT the risk of breast cancer
increased with increasing total
duration of use.
• 10 years' use of HRT is
estimated to result in 5
additional breast cancers per
1000 users of oestrogen-only
preparations
• 19 additional cancers per 1000
users of oestrogen-
progestagen combinations
Current use of HRT is associated with an increased risk of incident and fatal breast cancer; the
effect is substantially greater for oestrogen-progestagen combinations than for other types of
HRT.

50. Ovarian cancer
9,48,576 postmenopausal women from the UK Million Women Study who
did not have previous cancer or bilateral oophorectomy were followed up
for an average of 5.3 years for incident ovarian cancer and 6.9 years for
death.
Outcomes
• 2,273 incident of ovarian cancers and 1,591 deaths from the
malignancy were recorded.
• Current users were significantly more likely to develop and die from
ovarian cancer than never users (RR 1.20 for incident disease and
1.23 for death).
• For current users of HRT, incidence of ovarian cancer increased with
increasing duration of use, but did not differ significantly by type of
preparation used, its constituents, or mode of administration.

51. • Past users of HRT were not at an increased risk of ovarian
cancer (0.98 and 0.97 respectively, for incident and fatal
disease).
• Over 5 years, the incidence rates for ovarian cancer in current
and never users of HRT were 2·6 and 2.2 per 1000,
respectively—ie, one extra ovarian cancer in roughly 2500
users; death rates were 1.6 (1.4–1.8) and 1.3 (1.2–1.4) per
1000, respectively—ie, one extra ovarian cancer death in
roughly 3300 users.
Interpretation
• Women who use HRT are at an increased risk of both incident
and fatal ovarian cancer.

52. Endometrial cancer
7,16,738 postmenopausal women in the UK without previous cancer or previous
hysterectomy were recruited and were followed up for an average of 3.4 years,
during which time 1,320 incidence of endometrial cancers were diagnosed.
Findings
• 3,20,953 women reported at recruitment that they had used HRT, among whom
• 69,577 last used continuous combined therapy (progestagen added daily to
oestrogen),
• 1,45,486 last used cyclic combined therapy (progestagen added to oestrogen,
usually for 10-14 days per month),
• 28,028 last used tibolone, and
• 14,204 last used oestrogen-only HRT.

53. These HRT types had sharply contrasting effects on the overall risk of
endometrial cancer. Compared with never users of HRT,
o risk was reduced with last use of continuous combined preparations (0.71)
o not significantly altered with last use of cyclic combined preparations (1.05)
o increased with last use of tibolone (1.79) and
o oestrogen only (1.45)
A woman’s body-mass index significantly affected these associations, such
that the adverse effects of tibolone and oestrogen-only HRT were greatest
in lean women, and the beneficial effects of combined HRT were greatest
in obese women.
Interpretation
• Oestrogens and tibolone increase the risk of endometrial cancer.
• Progestagens counteract the adverse effect of oestrogens on the endometrium,
the effect being greater if they are added to oestrogen for more days every
month and more obese the women are.

54. 2. Osteoporosis
Postmenopausal osteoporosis is a major public health problem
Estrogen deficiency is a key factor in the pathogenesis of
postmenopausal osteoporosis
Evidence has accumulated that HRT leads to an increase in bone
mineral density (BMD).
Heart estrogen/progesterone replacement study (HERS) and HERSI
had shown no benefit of HRT on prevention of osteoporosis.
However, the published results of WHI report a decrease in hip
fracture risk in HRT users

55. • Reports from a meta-analysis (Torgerson D,2001), a 27%
decrease in the incidence of non-vertebral fractures has been
reported in women under 60 years showing greater risk
reductions
• Reports from another study using bisphosphonates, have shown
benefit in BMD and estrogen preservation and a decrease in the
hip fracture risk in 40-50% of women with osteoporosis
• Decrease in spinal fractures had also been displayed by
raloxifene
• Considering the data supporting the use of HRT for a reduction
in vertebral and non-vertebral fractures is limited, FDA has
approved HRT use only for prevention and not for treatment of
osteoporosis.

56. 3. Gall Bladder Disease
The Nurses Health Study (NHS), one of the earliest study to identify risk of
HRT and gall bladder disease
• prospective cohort study, performed with follow-up every 2 years
• Participants were 54,845 postmenopausal United States nurses
• Cholecystectomy was reported by 1750 women during 8 years of follow-
up
After adjusting for confounding factors,
• women currently using postmenopausal hormones were at an increased
risk of cholecystectomy (RR 2.1) compared to never-users
• For current users, the risk of cholecystectomy increased with increasing
duration of hormone use (RR 2.6, for 10 years or more) and higher
doses of estrogen (RR 2.4, for users of 1.25 mg or more)

57. • HERS study demonstrated an increased rate of biliary tract surgeries in
HRT users. However, WHI results have reported no such association
• A recent trial in European population (Hart AR ,2008) reported similar
results that’s were of NHS
Interpretation
Women using postmenopausal hormones are at an increased risk of gall
bladder disease and cholecystectomy.

58. 4. Cognition
• The Cache Country Study, a observational study observing
prevalence and incidence of Alzheimer’s disease, reported a
decreased risk of Alzheimer’s disease with HRT use for more than
10years [Zandi PP, 2002]
• Two large meta-analysis combining the data from the 35 years
regarding the effects of HRT on cognition, reported a decreased
risk of developing dementia in HRT users [Le Blanc E,2001]
However, the evidence based studies differed
• WHI Trial reported that estrogen and progestin therapy increased
the risk of probable dementia in women aged >65 years
• Estrogen alone therapy also led to an increase in risk for dementia
and mild cognitive impairment [Shumaker SA,2004].

59. Cochrane database systemic review
• A meta analysis report of 16 trials showed no effects of either
estrogen replacement therapy (ERT) or hormone replacement
therapy (HRT) in prevention of cognitive impairment after five
and four years of treatment, respectively
• Analyses assessing the effects of treatment over time found that
both ERT and HRT did not maintain or improve cognitive function
and may even adversely affect this outcome
• Negative effects were found for ERT after one year and HRT
after three and four years of therapy
• There is strong evidence that both ERT and HRT do not prevent
cognitive decline in older postmenopausal women when given as
short term or longer term (up to five years) therapy

60. • Ambiguity still exists that whether factors such as
- younger age (< 60 years of age),
- type of menopause (surgical or natural)
- type of treatment (type of estrogen with or without a
progestagen),
- mode of delivery (transdermal, oral or intramuscular)
- dosage
have positive effects on cognitive improvement or
maintenance at a clinically relevant level
• Based on the available evidence, HRT (estrogen alone or
in combination) cannot be recommended for overall
cognitive improvement or maintenance

61. Provisional guidelines for prescription of HRT
British Menopausal Society and North American Menopause
Society consensually prepared guidelines

62. Summary
 Primary indication for HRT prescription is vasomotor and other symptoms
 It should be used in smallest effective dose for shortest duration
 Young women with premature menopause clearly deserve HRT
 Hysterectomized women should receive estrogen alone while those with
intact uterus be given the combination therapy
 HRT does not afford cardioprotective effects, conventional combined therapy
may increase the risk of Stroke , MI and venous thromboembolism
 Combined HRT increase the risk of breast cancer and gall stones
 HRT is not the best option to prevent osteoporosis and fractures
 HRT does not offer protection against cognitive impairment, may increase
the risk of dementia
 Transdermal HRT may be advantageous over oral HRT
 The need for HRT should be assessed individually and not prescribed on routine
basis