Anti-Inflammatory effect of phage: A new insight into

phage therapy

A project proposal submitted to

The Department of Microbiology and Biotechnology

Centre

The Maharaja Sayajirao University of Baroda

As a part of

Master’s degree in
Microbiology (2019-2021)

By

Ms. Manasa Balakrishnan

Department of Microbiology and Biotechnology Centre

Faculty of Science
The Maharaja Sayajirao University of Baroda
Vadodara- 390002 Gujarat, India

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 INDEX

Title Page number

Summary 3
Introduction 4
Rationale 6
Objectives 7
Methodology 8
Time Schedule 12
Budget 13
References 14

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 SUMMARY

In the twenty first century, the changing epidemiology of inflammatory bowel

disease (IBD) globally with increasing disease incidence across many countries
relates to the altered gut microbiota, due to a combinatorial effect of environmental
factors, human immune responses and genetics. IBD is a gastrointestinal disease
associated with a gut microbial dysbiosis. IBD is amongst the most extensively
studied of all inflammatory diseases that are closely associated with the gut
microbiome. Treatment regimens for IBD involve anti-inflammatory drugs,
immunomodulators which prevent the immune system from attacking the bowel and
causing infection. Biologic therapies include genetically designed drugs to block
tumour necrosis factor-a (TNF-a) and stop inflammation. Under severe conditions
surgical procedures can be used to treat IBD. Currently there are a large variety of
available therapies to treat IBD but none of them offer complete remission to every
patient. Also, frequent cases of treatment ineffectiveness are known to be reported.
Hence there a need for a novel approach to treat IBD. In recent times, “Phage
therapy” is one such method that can be used to treat IBD. Phages are known to
confer anti-inflammatory effects by binding to LPS. This anti-inflammatory and
immunomodulating properties of phages can be exploited to treat various
inflammatory diseases like IBD.

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 INTRODUCTION

The human body is known to harbour a vast and complex ecosystem comprising
microbes, bacteria, fungi, viruses, and other living organisms, collectively known as
the microbiome. Skin, urogenital, and respiratory tracts are the places known to
harbour different microbial populations. The most abundant microbial populations
can be found in the lower part of the gastrointestinal tract, the colon. The part of the
microbiome residing in the small intestine and the colon is termed as the “intestinal
microbiome”. Intestinal microbiome bestows multitude of effects upon the host
ranging from modulating digestion and food passage, to production of vitamins,
short-chain fatty acids, and various other metabolites, as well as education of the
immune system or helping to maintain a balanced state. The gastrointestinal tract is
the major line of microbe–host interactions with the highest microbiota abundance
in the body.

Inflammatory bowel diseases (IBDs) are a group of chronic autoinflammatory

diseases that are instigated and amplified by the confluence of multiple genetic and
environmental variables that perturb the immune–microbiome axis. The two major
types of inflammatory bowel diseases (IBD) are ulcerative colitis (UC) and Crohn’s
disease (CD). They are characterized by a long-lasting recurrent inflammation of
various parts of the gastrointestinal tract. UC is restricted to the colon whereas
mucosal inflammation in CD can involve the entire depth of the tissue and is
associated with complications such as strictures and fistulas. Dysbiosis in gut occurs
in case of IBD and a broad microbial alteration occurs including reduction in
appearance of bacterial taxa within the phyla Firmicutes and Bacteroides and
increase in Gamma proteobacteria. E coli especially adherent invasive E coli (AIEC)

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strains is found in the gut of patients suffering from CD and C difficile in patients
suffering from UC.

Anti-inflammatory drugs such as amino salicylates, corticosteroids are the first step
in treating IBD. Treatment regimens also include immunomodulators which prevent
the immune system from attacking the bowel and causing infection. Biologic
therapies include genetically designed drugs to block tumour necrosis factor-a (TNF-
a) and stop inflammation. Under severe conditions surgical procedures can be used
to treat IBD.

Although there is currently a large variety of available therapies to treat IBD, none
of them offer complete remission to every patient. Also, frequent cases of treatment
ineffectiveness are known to be reported thus forcing patients to either change the
medication or increase the dosage. High dosage of any medication carries the risk of
worsening any potential side effects. Keeping this in mind, finding new approaches
to treat chronic inflammation in IBD is imperative.

In recent times, “Phage therapy” is one such method that can be used to treat IBD.
Bacteriophages (phages) are viral particles that infect and replicate inside a bacterial
cell with a high selectivity for a particular host. Though the main the aim of phage
therapy (PT) is the clearance of pathogens by killing the bacteria through lysis,
phages also act as immunomodulators and control inflammation including those that
occur in the course of non-communicable diseases.
These anti-inflammatory and immunomodulating properties of phages can be
exploited to treat various inflammatory diseases including IBD.

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 RATIONALE
➢ Inflammatory bowel disease (IBD) is characterized by flares of inflammation
with a periodic need for increased medication and sometimes even surgery.
The aetiology of IBD is partly attributed to a deregulated immune response to
gut microbe dysbiosis. IBD is dynamic and various factors are involved in
causing the disease.
➢ Currently available therapies to treat IBD have limitations. Also, frequent
cases of treatment ineffectiveness are known. Hence phages need to be
investigated as potential anti-inflammatory therapeutic agents.
➢ Phages were considered as mere “bacteria eaters” with potential for use in
combating antimicrobial resistance until recently the real value of phage
therapy was assessed.
➢ In addition to eliminating bacteria, phage therapy might also be considered to
control inflammation including that occurring in the course of some non-
communicable diseases.
➢ Phages are known to act as immunomodulators by showing anti-inflammatory
effects that may be independent of their well-known antibacterial activities
and immunomodulating effects.
➢ The ability of phages to show anti-inflammatory effects can be used to treat
inflammation caused in various diseases like IBD, Parkinson’s disease, Type
1 and 2 diabetes.

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 OBJECTIVES

OBJECTIVE 1: Isolation, purification and enrichment of bacteriophages

against enteric pathogens which are relatively abundant
in IBD patients, from sewage water.

OBJECTIVE 2: Extraction of LPS and to check LPS binding ability of

phages isolated against enteric pathogens using
dynamic light scattering (DLS) method.

OBJECTIVE 3: Study the anti-inflammatory activity of isolated phages

using qRT-PCR.

OBJECTIVE 4: In vivo study in murine models, to check anti-

inflammatory activity of isolated phages.

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 WORK PLAN AND METHODOLOGY

OBJECTIVE 1: Isolation, purification and enrichment of bacteriophages against

enteric pathogens. which are relatively abundant in IBD patients,
from sewage water.

➢ Sewage sample to be collected.

➢ Double agar layer method will be used to isolate phages and enumerate plaque
forming units (PFUs).
➢ Enteric bacteria which are relatively abundant in the gut of IBD patients will
be used as host culture to isolate phages.
➢ Plaques will be observed after 24 hrs of incubation at 37oC.
➢ Bacterial cultures will be mixed with sewage samples and incubated for 24hrs
at 37oC, centrifuged for 20 mins at 10,000g and filtered through a 0.45 micro
m pore size membrane filter. Pure phage strain will be obtained by three serial
single-plaque isolations.
➢ Phage lysate will be enriched.

EXPECTED OUTCOME: Enteric host specific phage may be isolated from

sewage sample which needs to be purified and
enriched.

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OBJECTIVE 2: Extraction of LPS and to check LPS binding ability of phages
isolated against enteric pathogens using dynamic light scattering
(DLS) method.
a) Extraction of Lipopolysaccharide from enteric bacteria.
➢ Enteric bacteria will be grown for 48 h at 37oC in LB medium
vigorously aerated by shaking.
➢ Bacteria will be killed with 0.5% phenol and centrifuged.
➢ Bacterial pellet will be washed three times with distilled water, treated
with 90% phenol/water (1:1), and heated to 65oC.
➢ LPS will be extracted using phenol-water as a precipitate according to
Westphal and Jann method.
➢ LPS shall be finally suspended in PBS.
b) To check LPS binding ability of phages using dynamic light scattering
method.
➢ The ability of phage to bind to LPS will be tested in vitro by particle
size distribution measurement in LPS solution.
➢ Dynamic light scattering method will be employed which relatively
determines increase in average size of aggregating molecules in
solutions.

EXPECTED OUTCOME: Phage having the ability to bind to LPS of enteric

bacteria may be isolated and should be used for further experiments.

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OBJECTIVE 3: To study the anti-inflammatory activity of isolated phages using
qRT-PCR.
➢ Human colon cancer cells HT-29 will be cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with fetal calf serum.
➢ HT-29 cells will be treated with LPS for 5hrs.
➢ Post incubation, these cells will be stimulated with phages.
➢ Real-time quantitative PCR (qRT-PCR) will be used to investigate the anti-
inflammatory effects of the bacteriophage on LPS induced inflammation in
HT-29 cells using specific primers for TNF-α, IL-1β, IL-8, IL-6, IL-10.

EXPECTED OUTCOME: Phage mediated decrease in cytokine levels

suggest the anti-inflammatory effect of phage.

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OBJECTIVE 4: In vivo study in transgenic HLA-B27 mice models, to check anti-
inflammatory activity of isolated phages.
a) Phage lysate will be administered in DSS induced inflammation in
transgenic HLA-B27 mice models to check the anti-inflammatory activity.
b) Study of euthanized HLA-B27 mice colon will be carried out to see the
efficiency of phage lysate for anti-inflammatory effects.

EXPECTED OUTCOME: Dampening of inflammation in the mice models

receiving phages as a therapeutic agent.

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 TIME SCHEDULE

Work plan Time

Objective 1
Isolation, purification and enrichment of 4 months
bacteriophages against enteric pathogens which
are relatively abundant in IBD patients, from
sewage water
Objective 2
Extraction of LPS and to check LPS binding 3 months
ability of phages isolated against enteric
pathogens using dynamic light scattering (DLS)
method.
Objective 3
To study the anti-inflammatory activity of 4 months
isolated phages using qRT-PCR
Objective 4
In vivo study in transgenic HLA-B27 mice 5 months
models, to check anti-inflammatory activity of
isolated phages.

TOTAL DURATION = 16 Months

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 BUDGET
Non-Recurring Cost

Name of the item Quantity Price (INR)

Malvern zetasizer nano 1 15,35,000

HT-29 Cell lines 4 1,50,000

LightCycler 480 (Roche 1 17,48,000

Diagnostic)
RNA kit (OMEGA) 3 37,296

qRT-PCR Kit 1 25,000

HLA-B27 mice models 5 35,000

CO2 Euthanasia 1 60,000

Chamber
Total 39,25,696

Recurring Cost

Product Quantity Price (INR)

Contingency - 3,25,000
Travel and overheads - 2,00,000

Fine chemicals 75,000

JRF 1 5,12,000

Glassware, chemicals, - 1,50,000

reagents

Plasticwares - 90,000

Total 13,52,000
TOTAL COST = 52,77,696 INR
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