BB592: M.Sc.

Project Stage II Report

METABOLIC PROFILING OF CYTOKINE

INDUCED KERATINOCYTES USING NMR
SPECTROSCOPY

Project report submitted by

Bhavuk Dhamija

165300006

In partial fulfillment of the requirements for the award of the

degree of

Master of Science (Biotechnology)

Under the supervision of

PROF. RAHUL PURWAR

Department of Biosciences & Bioengineering

Indian Institute of Technology Bombay,

Mumbai 400076

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 (APRIL, 2018)

LETTER OF ACCEPTANCE BY GUIDE

The work reported in this dissertation entitled "Metabolic profiling of cytokine induced
keratinocytes using NMR spectroscopy” has been carried out by Bhavuk Dhamija
(165300006) under my guidance in my laboratory. I hereby approve the submission of the
project report.

Name- Prof Rahul Purwar

Guide

(Date)

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 DISSERTATION APPROVAL SHEET

Dissertation entitled “Metabolic profiling of cytokine induced keratinocytes using NMR

spectroscopy” by Bhavuk Dhamija (165300006) is approved in partial fulfillment of
requirement for Master of Science (Biotechnology).

Guide

Examiner 1

Examiner 2

Examiner 3

Date

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 PLAGIARISM UNDERTAKING

I, Bhavuk Dhamija Roll No 165300006, understand that plagiarism is defined as any one or
the combination of the following:

1. Uncredited verbatim copying of individual sentences, paragraphs or illustrations (such as

graphs, diagrams, etc.).
2. Uncredited improper paraphrasing of pages or paragraphs (by changing a few words or
phrases, or rearranging the original sentence order)
3. Credited verbatim copying of a major portion of a paper (or thesis chapter) without clear
delineation of who did or wrote what.

I have made sure that all the ideas, expressions, graphs, diagrams, etc., that are not a result of
my work, are properly credited. Long phrases or sentences that had to be used verbatim from
published literature have been clearly identified using quotation marks.

I affirm that no portion of my work can be considered as plagiarism and I take full
responsibility if such a complaint occurs. I understand fully well that the other authors of this
paper, or guide of the thesis / dissertation may not be in a position to check for the possibility
of such incidences of plagiarism in this body of work.

Signature with date

Name- Bhavuk Dhamija

Roll no. - 165300006

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 ACKNOWLEDGEMENT

I would like to express my sincere thanks and deep regards towards my guide Dr. Rahul
Purwar for his constant support and guidance.

I would also like to thank Prof. Ashutosh Kumar for his technical and theoretical guidance
throughout the project.

I would also like to thank my mentor Nikita Jain for motivating me throughout the semester
and helping me in tough times, which has been invaluable to me. I would also like to thank
my lab mate Gargi Das and other seniors Atharva, Sreya, Alka, Sathya, Sushant, Soumitra
and Vishakha for their valuable suggestions, help and support.
Lastly, I would like to thank my parents for supporting me in all my endeavors.

Sincerely,
Bhavuk Dhamija

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CONTENTS

Figures and Tables………………………………………………………………………………7

List of abbreviations….………………………………………………………………………....8
Abstract…………….………………………………………………………………………….....9
1. Introduction...……………………………………………………………………………...10
1.1 Physiology and Anatomy of skin………… …………………………………………..10
1.2 Inflammation and Skin related disorders …………………………………...............11
1.3 Significance of T cell subsets in immune response .………………………………….12
1.4 Prevalence of T cells in healthy and diseased skin..….….…………………………...16
1.5 Immune system and Metabolism……………………………………………………...18
1.6 Effect of metabolism on skin inflammation…………………………………………..21
1.7 Metabolic profiling of keratinocytes………………………………………………….22
2. Hypothesis………… ………………………………………………………………………..23
3. NMR Spectroscopy………………………………………………...............……………….23
Basics about NMR……………………………………………………………………….....23
4. Metabolomics/Metabonomics using NMR………………………………………………...26
5. Objectives…………………………………….……………………………………………...28
6. Plan of Work………………………………………………………………………………..29
7. Material and Methods……………………………………………………………………...29
7.1 Preparation of media…………………………………………………………………..29
7.2 Isolation of primary human keratinocytes…………………………………………...30
7.3 Cytokine treatment on keratinocytes…………………………………………………30
7.4 IFN-γ dose escalation on primary human keratinocytes……………………………30
7.5 Sample preparation for extraction of metabolites…………………………………...31
7.6 NMR spectroscopy and spectral acquisition.………………………………………...31
7.7 Data analysis and metabolite identification……………………………..…………....31
8. Results……………………………………………………………………………………….32
8.1 Differential expression of metabolites in untreated, IFN-γ treated and IFN-γ + IL-9
treated samples…………………………………………………………………………32
8.2 Effect of increasing dose of IFN-γ on Glucose levels inside and outside the cell…..36
8.3 Effect of IL-9 on Glucose levels inside and outside the cell in the presence of IFN-γ
milieu……………………………………………………………………………………37
8.4 Modulation of metabolic profile of keratinocytes due to IL-9……………………....38
8.5 IL-17 affects the metabolic profile of primary human keratinocytes……………....39
9. Discussion…………………………………………………………………………………...41
10. Future Prospects……………………………………………………………………………43
11. References…………………………………………………………………………………...44

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 Figures
Fig1: Anatomy of skin showing the three layers- epidermis, dermis and subcutaneous tissue.

Fig2: T cell differentiation into various subsets from naïve T cell.

Fig3: Evolutionary conserved signaling mechanism and immune response in drosophila, human

Fig4: Role of lipids and cytokines in controlling expression of metabolic genes

Fig5: Elevated LTB4 levels present in diseased skin as compare to normal healthy control.

Fig6: Energy differences in two spin states of nuclei

Fig7: Chemical shift indicator present at 0 ppm

Fig8: Chemical shifts for various functional groups

Fig9: NMR spectra for intra and extra cellular metabolites

Fig10: Different cytokine treatment in a 12 well plate.

Fig11; NMR spectra of (a) untreated (b) IFN-γ treated and (c) IFN-γ+IL9 treated keratinocytes

Fig12: Statistical analysis: fold change and volcano plot

Fig13: Differentially expressed metabolites in untreated vs. IFN-γ treated sample

Fig14: Statistical analysis: Fold change and Volcano Plot

Fig15: Differentially expressed metabolites in IFN-γ and IFN-γ+IL9 treated sample

Fig16: IFN-γ dose escalation experiment and effect on glucose

Fig17: Il9 and IFN-γ effect on glucose concentration

Fig18: Statistical analysis: Fold change and Volcano Plot

Fig19: Differentially expressed metabolites in IL4TGF-Β vs. IL4TGF-Β+IL9 sample.

Fig20: Statistical analysis: Fold change and Volcano Plot.

Fig21: Differentially expressed metabolites in untreated and IL-17 treated sample.

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 LIST OF ABBRREVATIONS

AD - Atopic Dermititis
IgE - Immunoglobulin E
Th - T helper cells
IFN-γ - Interferon – γ
IL – Interleukin
CD – Cluster of Differentiation
STAT - Signal transducer and activator of transcription
RORγ - RAR-related orphan receptor gamma
FOXP3 – Forkhead Box P3
CCR – C-C Chemokine Receptor
DC - Dendritic Cell
NK – Natural Killer cell
NKT – Natural Killer T cell
TGF – Transforming growth factor
TNF – Tumor necrosis factor
ILP – Insulin like peptide
TLR – Toll like receptor
JNK – Janus Kinase
iNOS – inducible Nitric oxide synthase
IRS – Insulin receptor substrate
PPP – Pentose phosphate pathway
OXPHOS – Oxidative Phosphorylation
NMR – Nuclear Magnetic Resonance
HIF – hypoxia induced factor
FT – Fourier transform
CSI – Chemical Shift Indicator
Ker – keratinocytes
TEWL - Transepidermal Water Loss

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 ABSTRACT

Keratinocytes are the primary cells present in our skin which act as a barrier against various
agents – chemical, physical or biological. There has been an increase in population across the
globe suffering from various skin diseases. Out of several prevalent skin diseases, our focus
of study is mainly on inflammatory and autoimmune diseases – Atopic Dermatitis (AD),
Psoriasis and Vitiligo. Keratinocytes being the solemn victims are involved in various skin
inflammatory and autoimmune diseases. Inflammation which takes place in these cells is
caused by multiple factors that includes- genetic, environmental and immunological. Immune
system has a key role to play in progression of these diseases as T cells are the main culprits
which are responsible for its pathology. T cells in turn mediate its effects via some chemical
or proteinaceous molecules called cytokines. It has been reported that there is an increase in
levels of pro-inflammatory cytokines like IFN-γ, IL-9 and IL-17 within patients suffering
from AD and Psoriasis. Several studies have exhibited a connection between immune system
and metabolism. It has been reported that TLRs and certain inflammatory cytokines can
regulate the metabolic properties of a cell at both transcription and translation level. Studies
on hepatocytes and adipocytes have proven this immune system mediated effect which causes
inflammation, obesity and other diet related problems. Studies have also shown how various
metabolites like lipid and certain amino acids contribute towards the scaly and lesionous skin
in AD patients. Correlating with this knowledge, we hypothesize that there might be some
effect of these pro-inflammatory cytokines on the metabolism of keratinocytes which could
contribute towards the inflammation. So in order to validate this hypothesis, we need to do
metabolomics (whole cell metabolite analysis) of keratinocytes under different cytokine
treatments. We have chosen IFN-γ, IL-9 and IL-17 for our current study and demonstrated the
differential expression of metabolites in the given conditions. Several metabolites have been
identified, some of which play critical role in carbohydrate and protein metabolism and are
also involved in energy production. Functional and physiological relevance of this differential
expression needs to be looked upon. The study will provide us with new insights in the field
of Metabolomics and open several possible areas of research which could be explored in
future.

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1. INTRODUCTION
1.1 Physiology and Anatomy of skin

The skin being the largest tissue of the body, account for 16% of the entire body weight. It serves
many roles including protecting us against pathogens, dust as well as prevention of excess fluid
loss. It also plays a significant role in maintaining body temperature. The skin primarily
comprises of 3 layers: epidermis, subcutaneous tissue and dermis. The outermost layer, the
epidermis, is made up of a group of cells called keratinocytes, synthesizing an elongated,
protective, thread like protein called keratin. The middle layer dermis along with the basement
membrane is made up of the fibrillar protein called collagen. The dermis lies above the
subcutaneous tissue, or panniculus, containing small lobes of fat cells known as
lipocytes(Kolarsick, Kolarsick, & Goodwin, 2011).
The epidermis, a stratified and squamous epithelium layer, itself is composed of 4 layers-
Stratum corneum, Stratum lucidum, Stratum granulosum and Stratum spinosum. Different types
of cells are spread across these 4 layers. Epidermis is primarily composed of two types of cells:
keratinocytes and dendritic cells(Kolarsick et al., 2011). Along with these cells, it is also harbors
several other cells, such as the neuro-endocrine Merkel cells, the immune-competent Langerhans
cells and melanocytes.

Fig1: Anatomy of skin showing the three layers-epidermis, dermis and subcutaneous tissue.
(Image courtesy: Pinterest)

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Keratinocytes out of all these are the most prevalent cell type of epithelial tissues which
comprise skin and mucosa, including oral, oesophageal, corneal, and a variety of genital
epithelia(Kirfel & Herzog, 2004). As the cells travel from the basal layer to the surface of the
skin there is process that takes place which causes keratinization. This process leads to a set of
events in which keratinocytes first travels from a synthetic phase and finally reaches a
degradative phase.
Skin in fact is an ideal system if we wish to study responses at both tissue and organism level. It
serves as an important modulator of immunological functions during various pathological
abnormalities as well as during normal homeostasis(Nestle, Di Meglio, Qin, & Nickoloff, 2009).

1.2 Inflammation and Skin Related Disorders

Inflammation is an altered immune response of body to injury. Inflammation can be categorized

as acute or chronic. In the acute phase there is an increased blood flow and vascular permeability
along with the infiltration of leukocytes, fluids, and inflammatory messengers such as
cytokines(Feghali & Wright, 1997). On the other hand, chronic phase is characterized by the
augmentation of certain cellular & humoral immune responses against the antigen present at the
site of injury(Feghali & Wright, 1997).

Skin Inflammatory diseases have been the most critical problem in dermatology. Some of them
include occasional rashes, some causes redness and skin itching. Some leads to serious
conditions such as dermatitis (eczema), psoriasis and rosacea. As mentioned earlier,
inflammation could be categorized as acute and chronic. Acute inflammation can result from
contact with chemical irritants, allergens, ionizing radiation or exposure to UV radiation (UVR).
It is typically dealt within 1 - 2 weeks with a minor effect on cellular and tissue damage. On the
contrary, chronic inflammation is caused due to a sustained and robust immune cell mediated
inflammatory response within the skin itself. Chronic phase lasts for longer duration and can
cause serious and significant cellular damage.
(https://www.dermamedics.com/inflammation_id55.html)

Our interest lies mainly on two types of skin inflammatory diseases – Atopic Dermatitis and
Psoriasis.

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T cells are the major player in etiology and pathology of these diseases. They act in
different aspects and perform distinct functions in each of these disorders. These various
functions are achieved through the differentiation of naive T cells as they are stimulated by
their cognate along with some proteinaceous factors called cytokines, to become effector
and memory cells bearing specialized phenotypic properties.

1.3 Significance of T cell subsets in immune response

CD4+ T-cells called T-helper cells are the central immune cells involved in activation and
regulation of host defense against infection and pathogens also activating the cytotoxic CD8+ T-
cells. These Th cells are also seen to be involved in autoimmune and inflammatory diseases.
According to their patterns of cytokine production, transcription factor expression, and cell
surface marker expression, CD4+ T helper cells are currently subdivided into multiple lineages,
encompassing Th1, Th2, Th17, Th9, follicular T helper (Tfh), and regulatory T (Treg) cells(Zhu,
Yamane, & Paul, 2010).

Fig2: T cell differentiation into various subsets from naïve T cell.

Th1 cells
Th1 cells get differentiated from T naive cells upon the action of IFN-γ and IL-12. The TH1
subset is responsible for many classic cell-mediated functions, including activation of cytotoxic
T lymphocytes and macrophages. They produce IFN-γ as their key cytokine.
IFN-γ enhances MHC class I and II expression on all nucleated cells and is known to regulate
many of the effector functions of phagocytes (mononuclear) (Feghali & Wright, 1997).

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IFN-γ functions in maintaining the barrier integrity of the skin(Leung, 2013)(Wang & Xu
Landén, 2015). It regulates the expression of various genes for eg. It causes upregulation of
hBD-2/3 and CCL20 (a chemokine) in cultured keratinocytes. Both of these play an important
role in the maturity and differentiation of keratinocyte and thus serve as regulators of barrier
function(Wang & Xu Landén, 2015).
IFN-γ also increases ceramide synthesis by over-expressing enzymes like b-glucocerebrosidase
and sphingomyelin phosphodiesterase, and hence strengthening the lipid envelope. The initiation
of inflammatory process, due to increased responsiveness of keratinocytes to CD40 receptor and
tumor necrosis factor (TNF) occurs in the presence of IFN-γ(Wang & Xu Landén, 2015).
IFN-γ also causes Keratinocyte apoptosis, which is an important aspect of spongiosis and eczema
in patients with Atopic Dermatitis(Meyer et al., 2010).
IFN-γ also exerts its effects on other cytokines as well. IFN-γ is believed to suppress IL-9
production in mouse T cells(Nalleweg et al., 2015)(Schlapbach et al., 2014).
IFN-γ programs myeloid origin APCs to activate IL-17 T cells via induction of IL-23 and IL-1.
IFN-γ also stimulates chemokine production- CCL20 from APCs, helping in migration and
localisation of IL-17 T cells. It also synergizes with IL-17 for the production of an antimicrobial
and chemotactic protein - human B-defensin 2(“Induction of IL-17+ T Cell Trafficking and
Development by IFN-γ.pdf,” n.d.).

Th2 cells:

Th2 cells get differentiated upon the action of IL-4. Granulocytes like eosinophils, basophils and
mast cells are the probable cells which produce IL-4 that activates Th2 cells. According to recent
reports these cells can also be activated by chitin present in parasites and fungi. These cells
produce cytokines like IL4, IL5 and IL13.

Cytokines from Th2 cells acts on keratinocytes and suppresses the expression of terminal
differentiation proteins (loricrin, filaggrin and involucrin) and hence destabilize the function of
cutaneous skin barrier(Biedermann, Skabytska, Kaesler, & Volz, 2015).
IL-4 released from Th2 cells acts as an IgE isotype switch factor. It helps in promoting the
growth of mast cells, and also induces the expression of vascular cell adhesion molecule

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(VCAM-1). These VCAMS are involved in the migration of eosinophils and mononuclear cells
into the site of tissue inflammation(Hamid, Boguniewicz, & Leung, 1994).
IL-4 is also shown to reduce the expression and function of the genes that contribute towards the
skin barrier function and are involved in the epidermal differentiation complex (EDC). Most
critical among them is filaggrin (FLG). This gene is very important as the degradation producs of
fillaggrin, mainly the hygroscopic amino acids, act as “natural moisturizing factors” in the
epidermis and keep the upper stratum corneum hydrated. In apoptosized keratinocytes, keratin
filaments are aggregated into bundles by the action of FLGs and these aggregated bundles help
in the construction of the cornified cell envelope. IL4 causes increase in the expression and
function of kallikrein 7, a serine protease present in keratinocytes. This promotes skin
desquamation by degradation of corneo-desmosomal protein and increasing the transepidermal
water loss (TEWL)(Wang & Xu Landén, 2015).
IL-4 also inhibits the Anti Microbial Peptides which acts as a crucial antimicrobial barrier. Most
important of them being human b-defensins (hBD-3 and hBD-22); their absence leads to
microbial invasion(Wang & Xu Landén, 2015).
Allergic inflammation in the epidermis takes place due the over-expression of IL-4. This later
causes bacterial infection, intraepidermal edema, pruritus and cutaneous inflammatory cell
enrichment, the clinical symptoms of AD(Wang & Xu Landén, 2015).

IL-5, another Th2 cytokine increases vascular endothelial adhesion, enhances differentiation, and
survival of eosinophils. It also helps in an augmented release of histamine from basophils(Hamid
et al., 1994).
There is an increase in the expression of EDC gene in the presence of both IL13 and IL4. This
milieu also promotes skin desquamation and TEWL(Wang & Xu Landén, 2015).

Th17 cells:
Th17 cells get activated from naïve T cells in the presence of Il-6 and TGF-βeta. Absence of Il-4
and Il-12 is also critical for their development of Th17 cells. IL-6 amd IL-17 are the two main
cytokines secreted by these cells along with the signature transcription factor RORγT.

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TH-17 cells lead to the expression of IL17F, IL17A, CCL20 and IL22, which regulates the
immune functions in various epithelial cells. These cytokines causes up-regulation of genes
encoding antimicrobial species and proinflammatory cytokines(Wilson et al., 2007).

IL17A along with IL22 can be reason for the acute inflammatory tissue remodeling which
promotes neutrophilic infiltration(Cesare, Meglio, & Nestle, 2008).

In keratinocytes, IL17 is also known to regulate the expression of FLG and associated enzymes,
antimicrobial peptides (AMPs), Keratin 17 and some tight junction proteins like ZO-1 and ZO-
2(Auriemma, Vianale, Amerio, & Reale, 2013)(Cai, Fleming, & Yan, 2012). These proteins
along with certain epidermis associated adhesion molecules like integrins and E-cadherin are the
targets for IL17(Auriemma et al., 2013)(Wang & Xu Landén, 2015). IL17 also regulates the
production of VEGF.

Th9 cells:
Th9 cells are one of the more recently described subsets of T cells. They develop from naive T
cells in the presence of TGFβ and IL-4. Th9 cells are primed for the production of IL-9 and
require transcription factors like PU.1, IRF4 (interferon response factor 4), and GATA3(Chang
et al., 2011; V Dardalhon, Awasthi, H, G, & Gao, 2008; Gery, 2011; Veldhoen et al., 2008). In
addition to IL-9, Th9 cells have been shown to produce IL-10 and IL-21, although these are not
regulated coordinately with IL-9, and their role in Th9 cell function is unclear(Chang et al.,
2011; V Dardalhon et al., 2008; Gery, 2011; Veldhoen et al., 2008).
IL9, a regulator of haematopoietic cells(Auriemma et al., 2013), has a role to play mainly as a
growth factor of T cells and its function is to cause proliferation and survival of T cells. IL9 is
shown to stimulate keratinocytes to release VEGF in acute inflammation and AD lesions(Wang
& Xu Landén, 2015).
Th-9 cells play a crucial role in mediating anti-tumor immune responses(Miccheli et al., 2006).
Recently they have been discovered to be helpful against tumor due to their properties.
Nevertheless, it’s not easy to conclusively elucidate the role of IL-9 in inflammation, given that
no significant study has been described to validate the relevance and function of Th9 cells or IL9
secreted by them.

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1.4 Prevalence of T cells in healthy and diseased skin

Normal healthy skin harbors more than 2*1010 resident T cells, which is way more the total
number of T cells present in circulation(Nestle, Di Meglio, Qin, & Nickoloff, 2009).
These T cells are heterogeneous and most of them are memory T cells. T cells in epidermis are
mainly present in the basal and suprabasal layer of keratinocytes, in close proximity with
Langerhans cells. On the other hand, inside the dermis, they are mainly clustered around post-
capillary venulles. Most of the memory T cells present in skin express cutaneous lymphocyte-
associated antigen (CLA) are contains an equal proportion of both CD4+ and CD8+ cells(Nestle
et al., 2009).
Also Rachael et.al demonstrated that majority of these memory cells are effector cells (98%)(Pan
et al., 2017).
There are four main types of CD4+ Th cell that has been found in the skin during various
inflammatory diseases - Th1, Th2, Th9 and Th17 cells.

As compared with normal healthy skin, skin of patients with Atopic Dermatitis lesions had
significantly increased numbers of cells that are positive for mRNA, IL-4 (P < 0.01), and IL-5 (P
< 0.01). Majority of IL5-expressing T cells were prevalent in chronic and acute lesions(Hamid et
al., 1994).

According to the data published by Chuanli Su et.al, the distribution of Th1, th2 and Th17 cells
were dictated in the acute stage, subacute stage, and chronic stages of AD(Su et al., 2017).

Cells/Stage Acute Sub-Acute Chronic

Th1 7.55% (±0.405%) 12.45% (±0.005%) 19.2% (±0.08%)
Th2 7.0% (±0.02%) 4.4% (±0.08%) 1.8% (±0.02%)
Th17 3.063% (±0.653%) 0.775% (±0.211%) 0.195% (±0.151%)
Table1: List of T cell subsets and their abundance in different stages of inflammation.

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Presence of increased number of Th17 cells along with high abundance of IL-17 in AD patients
shows that they have a crucial role in development of the disease.
IL-17 expression was significantly higher in acute as compared to the chronic lesions(Cesare et
al., 2008).
This low level of IL17 explains the absence of AMPs which ultimately leads to recuurent
bacterial infections in AD patients.
An increase in expression of IL4 was observed in the AD patients in the epidermis which results
in the marked allergic inflammation(Wang & Xu Landén, 2015). High levels of IL-5 have also
been observed in AD patients that cause itching in them. Also, there was a market increased of
CD41 T cells, transcription factor PU.1, IL-9 and IL-9 receptors observed in lesional and
cutaneous AD skin as compared to the normal healthy skin(Wang & Xu Landén,
2015)(Auriemma et al., 2013).

Large amount of cytokines like IFN-γ along with DCs and T cells were seen in psoriatic
lesions(Wilson et al., 2007).
The ratio of proliferating to non-proliferating keratinocytes in normal skin is found to be 60%.
On the contrary, it is almost 100% in psoriasis; the mean cell cycle time is also drastically
decreased from 311 to 36 h. Keratinocyte hyperproliferation is not just restricted to the basal
compartment containing the stem cells, but it also shown to be present in supra-basal
cells(Gudjonsson, Johnston, Sigmundsdottir, & Valdimarsson, 2004).

A significant higher expression of IL17A, IL17F, IL22, IL26 and IFNG was observed in lesional
skin as compared to the healthy skin(Wilson et al., 2007).
In Psoriatic patients, human B-defensin 2, an antimicrobial and chemotactic protein is shown to
be highly expressed by keratinocytes(“Induction of IL-17+ T Cell Trafficking and Development
by IFN-γ.pdf,” n.d.). Additionally, an increase in CD4+ T cells producing IL-9 was found by
Schlapbach et al. in skin lesions from psoriasis patients(Valerie Dardalhon, Collins, & Kuchroo,
2015).

In both these inflammatory disorders, there has been a vital role of Th9 and Th17 cells, the
newly discovered T-cell subsets. They both are involved in secreting inflammatory
cytokines like IL-9 and IL-17 respectively. These cytokines, earlier thought to be a product

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of only Th1 and th2 cells, categorized these inflammatory diseases as Th1/Th2 mediated.
These study of Th9 and Th17 cells along with the expression of cutaneous lymphocyte
antigen (CLA) marker on their surface opens a new sight of T –cell mediated inflammatory
response. IL-9 and Il-17 can be used for further studies to better understand the actual
mechanism and physiology related to these disorders.

1.5 Immune system and Metabolism

This is a new field of study where research has been going on to find if some sort of correlation
exists between immune response generated by an individual and his metabolism. A new term has
been coined based on this study – Immunometabolism. This study segregates along two paths.
The first investigates the effects of immune cells on organs that regulate whole body metabolism,
such as adipose tissue and liver and the second explores the role of metabolic pathways within
immune cells and how this regulates immune response outcome(Pearce & Pearce, 2014).

Many layers of evolutionarily conserved interactions occur between immune response and
metabolism(Hotamisligil, 2017). Proper and efficient regulation of the nutrients and energy
generated via both anabolic and catabolic processes is essential for the cellular and organismal
development. Immune response plays a fundamental role in safeguarding an individual from all
sort of danger and this response requires a constant input in order to give a robust output. This
means that immune cells also require nutrition in order to function properly; they can also sense
the changes in nutrient availability and react to in a way that can influence the intrinsic metabolic
action within neighboring cells which is critical for metabolic homeostasis, for example by
inhibiting glucose uptake/utilization or by altering lipid metabolism(Hotamisligil, 2017).

An evolutionary relationship has been observed which demonstrates the conservation of

crosstalk occurring between immune system and metabolism. This representation suggests that
cytokines can act as metabolic hormones/regulators to provide adaptations to nutrient
fluctuations; in more complex organisms they pose a problem in the face of chronic nutrient and
energy exposure, as observed in obesity(Hotamisligil, 2017).

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 Fig 3: Evolutionary conserved signaling mechanism for metabolic and immune response in drosophila and
humans(Hotamisligil, 2017)

It has been observed that the Drosophila fat body not only sense and store nutrients but also
defend against pathogens; over the course of evolution a similar structure recognized in an
ancient mammalian ancestor has given rise to the distinct metabolic and immune organs
observed in modern mammals, including adipose tissue and liver(Hotamisligil, 2017). The
crosstalk between immune and metabolic pathways has shown to be conserved from
invertebrates to mammals. The principle architecture of this network is mainly regulated by three
integrated systems; Eiger (TNF in mammals) and its receptor Wengen (TNF receptor in
mammals), dILP (insulin in mammals) and its receptors, and the TLR signalling
pathways(Hotamisligil, 2017).

These three receptors work in such a manner that activation of one can lead to inhibition of the
other. TNF and TLR signaling block insulin signaling hence its production through JNK
activation, from flies to humans, and blocking this pathway causes metabolic defects, whereas
activation results in abnormal metabolic homeostasis(Hotamisligil, 2017).
The response that is mounted by immune cells usually involves differential expression of large
numbers of genes, initiation and attenuation of several metabolic pathways and results in the
acquisition of new functions, such as the high production of cytokines, tissue remodeling
enzymes, lipid mediators, and the ability to migrate through tissues/matrix and undergo cell
division(Pearce & Pearce, 2014). One such study by Hotamisligil et.al shed light on the

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regulation of various metabolic genes by the action of certain cytokines and other dietary
metabolites. The concept that lipid and other free fatty acids present inside the cell and
circulating through the blood vessels has an effect on insulin signaling has been prevalent for
over 50 years. It was believed and proved that lipids have an effect on glucose uptake mediated
by insulin. They block the insulin signaling pathway and do not allow the expression of Glut
transporter and several other genes necessary for glucose uptake. This is supposed to be done
mainly via TLR receptor-TLR4 which is also a receptor for polyunsaturated and saturated fatty
acids. It is seen that mice with loss of function of TLR4 or with deletion of MyD88 (TLR
adaptor molecule) is protected from the lipid mediated insulin resistance(Hotamisligil, 2017).
Other mediators causing this problem are inflammatory cytokines like IL-1 and TNF-alpha,
which also work in a similar manner to hamper metabolism. They function via Janus Kinase and
IKK pathway and block IRS1 (downstream molecule required for insulin signaling).

Fig 4: Role of lipids and inflammatory cytokines in regulating expression of metabolic genes(Hotamisligil, 2017) .

Another critical mechanism might also contribute to this diet induced metabolic effect for
example, many cytokines induce the production of ceramides which themselves have their effect
on metabolism by inhibiting anabolism and causing continuous breakdown(Bikman & Summers,
2011). They also play a vital role in diseases like diabetes, obesity and cardiovascular diseases.

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1.6 Effect of metabolism on skin inflammation.

Food products, supplements and various metabolites have been seen to play a major role in
regulating certain inflammatory disorders. Either their deficiency or excessive uptake causes
changes in metabolism, affect critical pathways and hence cause lesions or other inflammatory
symptoms. Several studies have been done which proves this diet/metabolite effect on
inflammation.

 Pellagra, a diseased characterized by dementia, dermatitis and diarrhea is caused due to the
deficiency of tryptophan. It can also take place due to various medications, excessive alcohol
and leucine intake (Basavaraj et.al, 2010).
 A study done in puppies and kittens shows that a diet severely deficient in methionine and
excessive cystine causes psoriatic and foot pad lesions(Basavaraj et.al, 2010).
 Vitamin B12 deficiency is shown to be associated with hair changes, vitiligo and skin
hyperpigmentation (Basavaraj et.al, 2010).
 Walter and Motta through their studies have observed that there is a decrease content of
Ceramide levels in psoriatic and atopic dermatitis as compared to the normal healthy
skin(Holleran, Takagi, & Uchida, 2006).
 Levels of a leukotriene, LTB4 were detected to be significantly higher in blister fluid from
AD patients as compared to the normal healthy skin(Ruzicka et.al). (Fig.6)

Fig 5: Elevated LTB4 levels present in diseased skin as compared to healthy control(Ruzicka et al., 1986)

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 A study done by Jennifer et.al. reported that lysine deficient diet causes facial skin lesions in
kittens(Larsen, 2014).
 Lipids are also shown to cause problem in skin epidermis. Deficiency of a linoleic acid from
diet leads to excessive epidermal water loss and a characteristic skin disorder(Ziboh, Miller,
& Cho, 2000).
 Metabolites present in carbohydrate metabolism are also involved in physiology of certin
skin diseases. High glucose levels in serum of diabetic patients are reported to affect skin of
systemic organs. Glutamine supplementation on the other hand is shown to be anti
inflammatory(Alkhawtani, 2016).

1.7 Metabolic profiling of keratinocytes

For enhanced functional characterization of keratinocytes, metabolic analysis of the cells can
provide some fruitful insights as to what is happening inside the cells at the molecular level. One
of the major concerns in the metabolic analysis can be determining the energy generation
pathway of these cells. It has been suggested that the metabolic fuelling processes provide the
distinct functional role to the cells based on requirements (Matarese, Colamatteo, & Rosa, 2014).
Healthy cells do not proliferate much; they just maintain their normal repair mechanism. Hence,
do not require much energy. However, upon activation or any diseased scenario, they tend to
rapidly proliferate, hence their energy requirements tend to increase switching their metabolic
processes. There are various techniques and metabolome estimation methods available in
literature for metabolic analysis. These are: whole transcriptome analysis microarray (Crompton
et al., 2015), estimation of OCR (Oxygen Consumption Rate), SCR (Spare respiratory capacity)
and ECAR (Extracellular Acidification Rate) by extracellular flux analysis (Cells et al., 2016),
MS or NMR. Mass spectrometry is a common technique to analyze metabolome but the use of
NMR in analysis of Keratinocyte metabolome is relatively unexplored. Though less sensitive
than MS, it gives an advantage over the fact that samples can be reused and recorder again if
there is some error.

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2. CONCLUSION FROM LITERATURE AND GENESIS OF
HYPOTHESIS

Inflammatory diseases are modulated by immune cells, primarily T cells, which act via certain
chemical mediators known as cytokines. Cytokines like IFN-γ, IL-9 and IL-17 produced by Th1,
Th9 and Th17 cells respectively play a vital role in skin inflammation. Inflammatory cytokines
are seen to dysregulate metabolism in hepatocytes and adipocytes. Also, several metabolites and
macromolecules have been reported to be differentially expressed in inflammatory disorders
causing lesions and erythema.
It is hypothesized that inflammation or proliferation of skin (keratinocytes) can be related to the
changes in their metabolism and hence there might be some effect of the inflammatory cytokines
like IFN-γ, IL-9 and IL-17 on metabolic profile of keratinocytes.
To validate this hypothesis, metabolomics (study of whole cell metabolite) of keratinocytes
under various treatments has to be performed using specialized instrumentation technique –
NMR spectroscopy.

3. NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY

NMR spectroscopy is an advanced analytical technique which has been widely utilized across
the globe for studying the molecular and structural properties of a large number of compounds
namely proteins, sugars, lipids etc. It is based on the magnetic properties of an atom and the
electronic environment around it. It gives a particular defined spectrum for each
molecule/group.NMR can analyze mixture of compounds present in a sample by matching its
spectra against the spectra library of several compounds present in a database.

Some basics about NMR-

A proton can be thought of a compass needle having a north and a south pole and having
magnetic field lines generating from North Pole to South Pole. Due to these field lines, a
magnetic moment will be generated from South Pole to North Pole. So every nucleus/proton will
have a magnetic moment associated with it. A proton in an external magnetic field, B0; will
experience a quantized interaction between the external magnetic field and the magnetic moment

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of the proton and then this moment aligns with or against the magnetic field. This alignment in
turn depends on the spin of the proton. Direction of spin defines the magnetic moment. There are
two states present depending upon the alignment of the magnetic moment. As the magnetic
moment gets aligned with the external magnetic field B0, it is defined as the α state and when it
is aligned against B0, it is called the beta state. There is a difference in energy between these two
states, with beta state having higher energy than the alpha state as more energy is required to
orient the magnetic moment against the field. Increase in the external magnetic field strength
causes an increase in the energy difference between the alpha and beta spin states. A proton
present in the alpha spin state absorbs energy of certain frequency and move to the higher beta
spin state. When this happen the nucleus is said to be in resonance with the applied magnetic
field and the phenomenon is called nuclear magnetic resonance. This difference in energy
corresponds to a frequency, which falls in the radio waves of electromagnetic spectrum.

Fig6: Energy difference in two spin states of nuclei by an external magnetic field , Source:BioNMR

We can have different set of signal frequencies for different protons depending on the energy
difference they have in their spin states which in turn depends on the environment in which they
are in. This environment which we talk about can change the overall effective magnetic field felt
by the nucleus. This phenomenon is known as nuclear shielding. In the presence of an external
magnetic field, there is a circulation of electron density around the nucleus which creates an
induced magnetic field opposing the applied magnetic field. This decreases the overall effective
magnetic field felt by the proton/nucleus. This shielding of magnetic field by electron density
decreases the energy difference between spin states and hence the frequency. This change in
frequency is represented in the NMR spectra as the signal/shifts vary. A shielded proton absorbs

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at a lower frequency (upfield) than a de-shielded proton (downfield). This upfield or downfield is
also called chemical shift in the spectra and is defined as the relative shift/change in frequency
of a particular proton with respect to the chemical shift indicator (CSI).
Chemical shift = observed shift from TMS in Hz * 106/spectrometer frequency in Hz

There are various choices of CSI according to the nucleus being studied. For example, 2,2’
dimethyl silapentane-5-sulfonate (DSS), tetra-methylsilane (TMS) and
trimethylsilylpropionic acid (TSP), are all 1H and 13C chemical shift references that are based
on the Si atom. The main differences between these three compounds are pH dependence,
solubility and interference. DSS and TSP are both water soluble compounds.

DSS offers a slight disadvantage. It has proton resonance near 3 ppm which can overlap with
resonances of many important metabolites(Lane, 2012). DSS is pH independent. TSP on the
other hand is pH sensitive near pH 4. As opposed to DSS and TSP, TMS is soluble in non-polar
solvents such as chloroform and can be used as an internal reference only for non-aqueous lipid
like extracts(Lane, 2012). The methyl group of all these three compounds is set to zero and the
chemical shift of other groups/compounds are adjusted based on this.

Fig7: Chemical shift Indicator (DSS) present at 0ppm. (Image courtesy: Glen Facey)

Chemical shift which occurs for various molecules on the NMR spectra are dependent on the
frequency with which they resonates which in turn is dependent on the nuclear shielding
experienced by that atom. The shielding is mainly because of the electronegative atoms in
proximity of the proton. The functional groups which are present in different electronegative
environment, experience varying overall effective magnetic field. Functional groups like alcohol,
carboxylic and aromatic hydrocarbon have an electronegative group attached to the proto which
withdraws the electron cloud and make the proton less shielded. Thus it experiences more fields
and a higher shift in the spectra.

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 Fig8: Chemical shifts for various functional groups w.r.t CSI (TMS)(Starkey, n.d.) .

4. METABOLOMICS/METABONOMICS USING NMR

Metabonomics is defined as ‘the study of quantitative measurement of a metabolic response of

an individual to a genetic, environmental or pathologic modification’(Everett, Loo, & Pullen,
2013). It is basically a comparative study between metabolic responses under different
conditions. It is emerging as a crucial technology in a number of fields such as chemistry,
biology and new areas, such as pharmaco-metabonomics for personalized
drugs/medicine(Everett, Loo, & Pullen, 2013). The alternative term for metabonomics i.e.
metabolomics which came little late is defined as – ‘ the study of qualitative and quantitative
analysis of all cell metabolites present at a particular time under given condition’(Everett, 2015).
Identification of metabolites through NMR spectroscopy has proven to be very significant lately.
It has been greatly facilitated by the formation of many databases. Some of these databases are:
the Birmingham Metabolite Library (BML), Biological Magnetic Resonance Data Bank
(BMRB) and Human Metabolome Database (HMDB). These libraries store data of hundreds of
metabolites which is available either directly in the form of a spectra or in the form of standard
peaks, which can be used to compare it with the test sample spectra and identify the known or
unknown metabolite(Everett, 2015).

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Various software packages are used for these studies and spectrum is first processed before it can
be used for analysis. Different parameters are being looked upon for processing of the spectrum
– Fourier transformation, data acquisition, phasing, baseline correction and referencing (CSI).
After this processing, the peaks are assigned either manually or automated, which are further
uploaded on databases to check the match hits of compounds. Some software offers the
identification by manually comparing and aligning the peaks of our spectrum over the spectra of
known metabolites. This helps in direct identification of metabolites. One such software is
chenomx which includes 338 known metabolites and their spectra.
In a recent study, 72 metabolites were identified from NMR analysis of mammalian
cells(Kostidis, Addie, Morreau, Mayboroda, & Giera, 2017a). BHP2-7 cell line was chosen for
this study. They isolated both intra and extra cellular metabolites. These were identified on the
basis of comparison with HMDB, BMRB and BML databases, and along with proton 1D, it also
involved 1H-1H COSY, TOCSY and HSQC comparison.

1-Methyl-nicotinamide Malate GTP Taurine

α-ketoglutarate Methionine Galactitol Threonine
2-Oxo-isocaproate Methyl-malonate Galactose Tryptophan
3-methyl-2-oxovalerate Methyl-succinate Glucose Tyrosine
ATP N,N-Dimethyl-glycinec Glutamate UDP-Gluc-NAc
Acetatec N-Acetyl-aspartate Glutamine UDP-glucuronate
Alanine N-Acetyl-glutamine Glutathione UDP-Glucose
Arginine NAD COA UDP-Galactose
Asparagine O-Phospho-choline Glycine UMP
Aspartate NAD+ Histidine Valine
Betaine Ornithine Hypotaurine Myoinositol
Choline Pantothenate Isoleucine Niacinamide
Citrate Phenylalanine Lactate Galactose-1-phosphate
Creatine Proline Leucine sn-Glycero-3-phosphocholine
Phospho-creatine Pyroglutamate Lysine trans-4-hydroxy-l-proline
Formate Pyruvate Cystine β-Alanine
Fructose Serine Hypoxanthine 2-hydroxy-isobutyrate
Fumarate Succinate Pyridoxine 3-hydroxy butyrate
Table2: List of metabolites extracted and identified from intra and extra cellular mammalian cell line
BHP2-7(Kostidis, Addie, Morreau, Mayboroda, & Giera, 2017b).

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 Fig 9: 1D NMR spectra for intra and extra cellular metabolites(Kostidis et al., 2017b).

This study shows that there are be numerous metabolites which are could be present within or
secreted outside the cell playing a significant role in the metabolic and energy pathway required
for serving basic functions like growth, structure, support, transport and division (reproduction).

5. OBJECTIVES

In order to test our hypothesis, the effect of inflammatory cytokine stimulation on the metabolic
profile of keratinocytes has to be analyzed.

 Optimizing protocol for metabolite extraction and spectral acquisition.

 Elucidating the effect of IFN-γ on metabolic profile of primary human keratinocytes.
 Comparative analysis of differentially expressed metabolites by primary human
keratinocytes under IFN-γ treatment.
 Examining the effect of increasing dose of IFN-γ on glucose levels inside the cells.
 Perform experiments to see how IFN-γ affects glucose uptake/utilization.
 Investigating the role of IL-9 in modulating the metabolic profile of primary human
keratinocytes
 Checking the effect of IL-9 on keratinocyte’s metabolism in presence of IFN-γ.
 Examining the role of IL-9 in regulating the metabolic profile of keratinocytes
under IL-4, TGF-β milieu.
 Comparative analysis of differentially expressed metabolites in normal and IL17 treated
primary human keratinocytes

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6. PLAN OF WORK

Cytokine treatment of primary human keratinocytes

Optimization of protocol for extraction of metabolites from cells.

Preparation of sample mix for NMR experiment

Analysis of spectra and identification of metabolites

7. MATERIALS AND METHODS

7.1 Preparation of media

EpiLife (Calcium free media) from GIBCO, Invitrogen was used for culturing the keratinocytes
(serum-free media). It did not contain the hormones, growth factors and antibiotics so it had to be
added separately using the human keratinocyte Growth Supplement kit. First, the media (without
supplements) was filtered, then the following supplements were added to it- Calcium chloride,
Bovine transferrin, gentamicin, epidermal growth factor, bovine pituitary extract and bovine
insulin to make the complete media. Incomplete media comprised of everything but EGF and
BPE, which is essential for synchronizing the cells and to get a better response to the cytokines.

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7.2 Isolation of primary human keratinocytes

Isolation was keratinocytes (ker) was done by Sreya (PhD senior) and cells were given for the
cytokine treatment.

7.3 Cytokine treatment on primary human keratinocytes

Day 0 – Seeding of equal number of cells (0.2 million) was done in 6 wells in complete media.
Day 1 – Media was removed and replaced with fresh complete media.
Day 2 – Complete media was removed and replaced with incomplete media along with the
cytokine treatments.
Day 3 – Media was not removed and treatments were given again (because of reduced half life of
cytokines). [48 hrs treatment]
12a 12b

Fig 10a: Different cytokine treatments (for 48 hours) Fig 10b: IFN-γ dose escalation experiment to check glc .

7.4 IFN-γ dose escalation on primary human keratinocytes

Day 0 – Seeding of equal number of cells (0.1 million) was done in 6 wells in complete media.
Day 1 – Media was removed and replaced with fresh complete media.
Day 2 – Complete media was removed and replaced with incomplete media along with increase
doses of IFN-γ (0, 5, 10, 25 and 50 ng/ml). [24 hrs treatment]
Day 3 – Media was not removed and treatments were given again. [48 hrs treatment]

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7.5 Sample preparation and extraction of metabolites.

Keratinocyte samples seeded in the 12 well- plate were taken and media (supernatant) was
removed from them and stored in aliquots. Cells were washed with Phosphate Buffer Saline
(PBS) to remove any leftover media or impurities. Cells were lysed and collected in microfuge
tubes. Cells were vacuum dried and stored at -80°C.

7.6 NMR Spectroscopy and Spectral acquisition.

Vacuum dried samples were dissolved in Phosphate Buffer (pH 7.4) containing D2O. 10mM TSP
was used as a chemical shift indicator for referencing such that all chemical shifts are on the δH
scales relative to TSP at 0. It was transferred to the NMR tube. NMR experiment was performed
on a Bruker spectrometer operating at 750.0 MHz for 1H NMR, at a fixed temperature of 298K,
in 5 mm NMR tubes. Pulse program used was zgesp with 1024 scans. 1D spectrum for the
samples were obtained and used for identification of metabolites through analytical softwares.

7.7 Data analysis and identification of metabolites

All spectra processing was done in Chenomx NMR suite 8.3 (processor) software. Processing
included baseline correction, phase correction, shim correction, CSI calibration and line
broadening. Analysis of the spectral data and information regarding the Keratinocyte’s
metabolites was done using Chenomx NMR suite 8.3 (profiler). Peak list of 338 metabolites
present in the software were compared with the test sample spectra and metabolites were
identified both automatically and manually.
Statistical analysis of the data, T-test, volcano plot and PCA analysis was done using
Metaboanalyst Software.

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8 RESULTS

8.1 Differential expression of metabolites in untreated, IFN-γ treated and

IFN-γ + IL-9 treated samples.

A total of 141 metabolites were identified after the analysis of 1D NMR spectra of the three
keratinocytes samples. This identification was performed manually using the chenomx NMR
suite software where a spectrum for each and every metabolite was compared with the standard
available spectra spectra in the database.
As an example, lactate and pyruvate, important end products of glycolytic cycle, were identified
in all the three samples. Metabolites like Lactate and pyruvate were identified by matching their
peaks at 4.1, 1.3 and 2.4 respectively from the reference spectra in Chenomx. Similar peak
alignment analysis was done for rest of the metabolites. The 750 MHz 1D 1H NMR spectra of
the three samples are shown in Fig11 a, b and c respectively.

Fig 11:1D NMR spectra of (a) Untreated (b) IFN-γ treated and (c) IFN-γ+ IL-9 treated keratinocyte sample

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Metabolites and their concentrations were determined using Chenomx software and a csv file
was created. Metaboanalyst software was used to do the statistical analysis of the metabolites
getting differentially expressed in the three samples. Fold change and Volcano Plot were
generated and a total of 10 metabolites were found to be differentially expressed in untreated vs.
IFN-γ treated sample (Fig.12, 13)(p < 0.1). The experiment was carried out for n=3.

Fig12 Statistical analysis (a) Fold Change (FC=1.5) (b) Volcano Plot (p < 0.05)

Metabolite Fold Change P value

N-methyl hydantoin 0.3245 0.0064
AMP 0.1138 0.0095
Arginine 1.6085 0.0117
Glucose-6-phosphate 0.4633 0.0297
Glucose 0.4606 0.0338
Pyroglutamate 0.3556 0.0362
Creatine Phosphate 0.4394 0.0554
Isocitrate 0.1151 0.0701
Glucose-1-phosphate 0.4901 0.0717
Leucine 0.5496 0.0856

Table3: Differentially expressed metabolites untreated vs. IFN-γ treated samples

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 n=3

Fig13: Differentially expressed metabolites in untreated vs. IFN-γ treated keratinocytes.

Similar analysis was done for the IFN-γ and IFN-γ, IL9 treated samples; fold change and volcano
plot were plotted; and a total of 7 metabolites were found to be differentially expressed (Fig.14,
15) (p < 0.1). Some of these metabolites were common in both the data (Untreated vs. IFN-γ and
IFN-γ vs. IFN-γ, IL9). The experiment was carried out for n=3.

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 Fig14: Statistical analysis (a) Fold Change (FC=1.5) (b) Volcano Plot (p < 0.05)

Metabolite Fold Change P value

4-hydroxy 3-methoxy mandelate 3.1364 0.015
Glucose 1.4906 0.037
3-methyl xanthine 0.5263 0.046
AMP 5.7308 0.046
N-acetyl ornithine 0.2222 0.083
Homoserine 0.1319 0.096
Glucose-6-phosphate 1.4027 0.097
Table4: Differentially expressed metabolites IFN-γ vs. IFN-γ+IL9 treated samples
n=3

Fig15: Differentially expressed metabolites in IFN-γ vs IFN-γ+IL9 treated keratinocytes.

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8.2 Effect of increasing dose of IFN-γ on Glucose levels inside and outside the
cell.

IFN-γ dose escalation experiment was performed to validate the change in levels of target
metabolites in the primary human keratinocytes. Experiment was carried out with 0, 5, 10, 25
and 50ng/ml doses of IFN-γ and cells were treated for both 24 and 48 hours. At both time points
metabolites were isolated and glucose concentration were estimated using Chenomx software. A
decrease in concentration of glucose was observed with increasing dose of IFN-γ. The trend was
same for both 24 and 48 hours treated sample (Fig. 16a &b). Supernatant collected from wells
before scrapping out cells was also used to check for the levels of glucose in order to estimate the
effect of IFN-γ on glucose uptake by the cells. Surprisingly an increase in concentration of
glucose was observed in the supernatant with increasing dose of IFN-γ, implying that IFN-γ
might be affecting the glucose uptake inside the cell (Fig. 16c).

24 hours 48 hours

a) Glucose concentration inside the cells b) Glucose Concentration inside the cells
0.007 0.025
0.0065
Glucose Conc. (mM)
Glucose Conc. (mM)

0.02
0.006
0.0055 0.015
0.005
0.01
0.0045
0.004 0.005
0.0035
0.003 0
Control 5 ng/ml 10 ng/ml 20 ng/ml 50 ng/ml Control 5 ng/ml 10 ng/ml 20 ng/ml 50 ng/ml

IFN-g dose
IFN-g dose

c) Glucose concentration in the Supernatant

5.9
Glucose concentration (mM)

5.8
5.7
5.6
5.5
5.4
5.3
5.2
5.1
5
4.9
Control 5 ng/ml 10 ng/ml 20 ng/ml 50 ng/ml
IFN-g dose

Fig16: IFN-γ effect on glucose conc. inside the cells (a) 24 hrs (b) 48 hrs (c) in supernatant (24 hrs).

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8.3 Effect of IL-9 on Glucose levels inside and outside the cell in the presence
of IFN-γ milieu.

Another IFN-γ dose escalation experiment was carried out at 24 hours along with IL-9 to check
the effect of IL-9 in presence and absence of IFN-γ. On treatment of cells with IL-9, glucose
concentration increased inside the cells. This rise was more prominent in the presence of IFN-γ
than IL-9 alone. Supernatant was also checked for the glucose levels to see if IL-9 also affects
the uptake. A decrease in glucose concentration was observed in the supernatant on treatment
with IL-9 which shows that it might be increasing the glucose uptake by reverting IFN-γ effect.
This validated our previous differentially expressed metabolite data in which Glucose levels
were reduced in IFN-γ but were enhanced by IL-9.

Fig17: IL-9 and IFN-γ effect on glucose conc. (a)&(b) inside the cells (c) in supernatant (24 hrs).

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8.4 Modulation of metabolic profile of keratinocytes due to IL-9.

To check the effect of IL-9 on metabolism of primary human keratinocytes, cells were first
treated with IL-4 and TGF-β to over-express IL-9 receptors on the cell surface. IL-9 treatment
was given henceforth to see the metabolic changes taking place in comparison with the cells
devoid of IL-9 treatment. Metabolites and their concentrations were determined using Chenomx
software and a csv file was created. Metaboanalyst software was used to do the statistical
analysis of the metabolites getting differentially expressed in the two samples. Fold change and
Volcano Plot were generated and a total of 11 metabolites were found to be differentially
expressed in IL-4, TGF-β vs. IL-4, TGF-β + IL-9 treated sample (Fig.18, 19)(p < 0.1).

Fig18: Statistical analysis (a) Fold Change (FC=1.5) (b) Volcano Plot (p < 0.05)

Metabolite Fold Change P value

Threonine 0.473 0.00005
N,N-dimethyl formamide 0.125 0.0003
N-nitrosodimethyl amine 0.377 0.002
ATP 1.506 0.007
Trimethylamine 4.147 0.026
O-phosphocholine 0.277 0.037
Glycerate 0.332 0.049
Ethylene Glycol 2.785 0.060
N,N dimethyl glycine 1.758 0.076
Cholate 0.420 0.076
Pyroglutamate 2.544 0.083

Table5: Differentially expressed metabolites IL4+TGFB vs. IL4+TGFB+IL9 treated samples

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 n=3

Fig19: Differentially expressed metabolites in IL4 TGF-Β vs IL4 TGF-Β IL9 treated keratinocytes.

8.5 IL-17 affects the metabolic profile of primary human keratinocytes.

IL-17 treatment was given to see the metabolic changes taking place in the cells in comparison
with the cells devoid of IL-17 treatment. Metabolites and their concentrations were determined
using Chenomx software and a csv file was created. Metaboanalyst software was used to do the
statistical analysis of the metabolites getting differentially expressed in the two samples. Fold
change and Volcano Plot were generated and a total of 6 metabolites were found to be
differentially expressed in untreated vs. IL-17 treated sample (Fig. 20, 21)(p < 0.1).

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 Fig20: Statistical analysis (a) Fold Change (FC=1.5) (b) Volcano Plot (p < 0.05)

Metabolites Fold Change P value

N,N dimethylformamide 0.369 0.045
Creatine phosphate 0.38 0.060
O-phosphocholine 0.332 0.069
Sarcosine 0.337 0.071
Pyridoxine 2.098 0.085
2-hydroxy valerate 0.301 0.090
Table6: Differentially expressed metabolites in untreated vs. IL-17 treated samples

n=3

Fig21: Differentially expressed metabolites in untreated vs IL-17 treated keratinocytes.

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9 Discussion
A crucial challenge in metabonomics is the optimization of sample preparation for metabolite
extraction. After a number of trials, we were successfully able to optimize the sample preparation
protocol. This was best suited with an initial cell count of approximately 0.1 million. The given
cell count was sufficient to get a good spectra of detectable metabolites. Metabolites like
pyruvate, glucose and lactate were detected with a good intensity suggesting that glycolysis
might be the major metabolic pathway carried out in keratinocytes.

After performing differential metabolite expression analysis in untreated vs IFN-γ treated

keratinocytes, several hits were observed which were statistically significant (n=3) and had good
fold change values. Among these several hits, some metabolites like glucose, glucose-1-PO4,
glucose-6-PO4, isocitrate and pyroglutamate were involved in the carbohydrate metabolism
which is the key pathway for energy production. Metabolite like arginine which is already shown
in literature to have an effect on inflammation was also detected. Out of these several metabolites
our interest was in the molecules involved in glycolysis, which can change the fate of the cell by
either forming lactate or undergoing TCA cycle. We focused on glucose and carried our further
experiment to validate our screening result. To validate the decrease in glucose level inside the
cell due to IFN-γ treatment, we carried IFN-γ dose escalation experiment; a significant decrease
in glucose levels were observed inside the cells on increasing dose of IFN-γ in both 24 hrs and
48 hrs experiment. This generates two hypotheses, that either IFN-γ is decreasing the glucose
uptake inside the cells or it is increasing glucose utilization. Glucose levels in the supernatant
were also checked to give some light to these hypotheses and interestingly an increase in glucose
level was observed with increasing dosage. This strengthens the glucose uptake theory and
shows that IFN-γ is somehow affecting the entry of glucose inside the cells by either
downregulating or blocking transporters (GLUT1) involved in uptake or by converting glucose
into some other metabolite. Further research needs to be done to validate this hypothesis and
deriving the functional aspect of this effect.

Treatment of keratinocytes with IL9 increases the glucose concentration inside the cells as
opposed to IFN-γ. This increase by IL9 was there both in presence and absence of IFN-γ. IL9 is
generally considered to be a proinflammatory cytokine but prior research done on this cytokine
does not provide any significant evidence which can comment about its physiological or

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metabolic function. Given from the data, we see that IL9 negates the effect of IFN-γ that is it
increases glucose uptake inside the cell. IFN-γ induces stress conditions for the cell by limiting
glucose for metabolism and IL9 is trying to save the cell from this metabolic exhaustion and thus
counteracts IFN-γ effect.

Effect of IL-9 on metabolism of keratinocytes was also analyzed using another set of
experiments in which the cells were treated with IL4 and TGF-Β to induce IL9 receptors and
cells were then treated with IL9 to see the changes which take place in the metabolome of
keratinocytes. Several metabolites involved in various pathways were seen to be either up or
down regulated on treatment with IL-9. Some of these metabolites like threonine, N,N
dimethylglycine and trimethylamine are somehow related with inflammation and some studies
have reported that dysregulation in these metabolites is linked with such immune response.
However, the exact mechanism behind this and what functions do these metabolites play is still
unknown. Also, how IL-9 modulates it, what is the signaling mechanism involved, is still a
question to be addressed upon.

Another study showing differential metabolite expression analysis was done using IL-17, a
signature pro inflammatory cytokine present in various inflammatory disorders. Metabolites like
sarcosine and creatine phosphate, involved in arginine metabolism, were found to be down
regulated in IL-17 treated keratinocytes. These metabolites are the end product of one of the
arginine degradation pathway. Another pathway by which arginine metabolizes is via iNOS
pathway in which it generates NO which is involved in inflammation. The data generated here
somehow correlates with the literature in a way that shuttling of arginine towards this iNOS
pathway might be causing inflammation and therefore there has been a decrease in flux for the
other pathway involving creatine phosphate and sarcosine. IL-17 might be the one responsible
for this shuttling by either deactivating/inhibiting any enzyme responsible for sarcosine
formation or upregulating enzymes involved in iNOS pathway. Further research needs to be done
to understand the true mechanism of IL-17 mediated metabolic changes.

Metabolic profiling of primary human keratinocytes under different stress conditions has made
us look at this inflammatory scenario from a new perspective. It has opened several possible
areas of research which are unanswered and could be explored in future. Getting a glimpse about
the pathways involved in cellular homeostasis and the mechanism by which they are regulated

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can provide us with new insights in the field of metabolomics and medicine. The changes in
level of cellular metabolites can in turn affect the proliferative properties of the cell which could
lead to an inflammatory response. Targeting such metabolites would be an exciting area of
research which could help in combating these deadly inflammatory skin diseases.

10 Future Prospects

 Perform experiments to check how IFN-γ is affecting glucose uptake.

 Repeat glucose uptake assay with increasing dose of IL-9 to validate its counteractive
nature against IFN-γ.
 Check AMP/ATP ratio inside the cell to see whether these cytokines cause metabolic
exhaustion.
 Perform functional assays to check the physiological relevance of this effect.
 Perform in-vitro assays for metabolites which are over or under expressed under IL-4
+TGF-Β and IL-17 treatment
 Finding a novel metabolite(s) which could serve as a crucial target(s) against
autoimmune and inflammatory diseases.

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