Targeting Primary Cilia Immune Receptor Proteins for the ...

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Pharmaceutical Sciences (MS) Theses Dissertations and Theses

Spring 4-19-2019

Targeting Primary Cilia Immune Receptor Proteins for the Targeting Primary Cilia Immune Receptor Proteins for the

Treatment of Polycystic Kidney Disease Mechanisms Treatment of Polycystic Kidney Disease Mechanisms

Nedaa Alomari Chapman University, [email protected]

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Recommended Citation Recommended Citation Alomari N. Targeting Primary Cilia Immune Receptor Proteins for the Treatment of Polycystic Kidney Disease Mechanisms. [master's thesis]. Irvine, CA: Chapman University; 2019. https://doi.org/10.36837/chapman.000046

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Targeting primary cilia immune receptor proteins for the treatment of polycystic kidney

disease mechanisms

A Thesis by

Nedaa S. Alomari

Chapman University

Irvine, CA

School of Pharmacy

Submitted in partial fulfillment of the requirements for the degree of

Master of Science In Pharmaceutical Sciences

April 2019

Committee in charge:

Surya Nauli, Ph.D., Chair

Aftab Ahmed, Ph.D.

Yang Liu, Ph.D.

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Targeting primary cilia immune receptor proteins for the treatment of polycystic kidney

disease mechanisms

Copyright © 2019

by Nedaa S. Alomari

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ACKNOWLEDGEMENTS

I am deeply grateful to my supervisor, Prof. Surya Nauli, for his guidance, encouragement and

advice. I have been extremely lucky to be one of his graduate students. I would also like to thank

all of my thesis committee members Dr. Aftab Ahmed and Dr. Yang Liu for their guidance,

support, and sufficient and productive comments. In fact, it was a real fortunate to work with them.

In particular I would like to thank Dr. Rajasekharreddy Pala who helped me from the beginning

until the end of the project.

I wish to express my unqualified thanks to my family and friends. I could never have accomplished

this without them. They have been a constant source for strength and care.

Finally, I would like to thank Biomedical and Pharmaceutical Sciences Department at Chapman

University and Saudi Arabian Culture Mission. I could not have gone through the Master program

overseas without their financial support.

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ABSTRACT

Targeting primary cilia immune receptor proteins for the treatment of polycystic kidney

disease mechanisms

by Nedaa S. Alomari

Background: Primary cilia are cellular organelles project from the cell surface of mammalian cell

and play important roles in vertebrate development, organogenesis, health, and others genetic

diseases. Primary cilium functions as a mechano-sensor and chemo-sensor. Defect in primary cilia

causes the progression of polycystic kidney disease (PKD) which further leads to the inflammatory

responses. We, therefore, investigated the role of Toll-like receptors 4 and 9 (TLR) in primary cilia

towards PKD.

Purpose: The main purpose of the proposed study is to identify and target the immune reactive

proteins i.e. TLRs in the primary cilia. By targeting those primary cilia immune reactive proteins

using suitable agonist and antagonists to study the control of cystic formation and their progression

mechanisms.

Methods: To target the ciliary immune TLR proteins (TLR4 and TLR9), we did immunostaining

to evaluate their localization on primary cilia. Cilia lengths were measured and compared using

differential interference contrast (DIC) and fluorescent imaging techniques. The in vitro 3D cyst

progression was monitored by adding agonists lipopolysaccharide (LPS) and

oligodeoxynucleotides (ODN) and antagonist 4-hydroxy chloroquine (HCQ).

Results: From our results we found that the TLR antagonist HCQ increases ciliary length in treated

scrambled control, Pkd2 knockout (KO) and TLR4 KO cells as an immune response, whereas

opposite results were observed with TLR9 KO. However, the selected agonists for TLRs

(LPS/ODN) increases cilia length in TLR9 KO cells and decreases scrambled control, Pkd2 KO

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and TLR4 KO. In our 3D cyst cultures, we used agonists and antagonist for both the TLRs and

observed that the cyst formations and progressions were inversely related to the cilia lengths. From

these observations, we speculated that the new ciliary TLR proteins have a role in cystic

progression. In conclusion, we found that the TLRs agonists/antagonist can modulate cilia length

and TLRs role in inflammatory actions. The primary cilium already has central roles throughout

cell biology, but here we propose, for the first time, that the cilium and the regulation of its

structural importance in inflammation of PKD.

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TABLE OF CONTENTS

CHAPTER 1 - INTRODUCTION ……………………………………………………...1

1.1 Cilia …………………………………………………………………………………...1

1.2 Cilia structure ………………………………………………………………………....1

1.3 Roles of primary cilia in cellular signaling …………………………………………...3

1.4 Polycystic kidney disease …………………………………………………………….4

1.5 Role of inflammation in Polycystic kidney disease …………………………………..6

1.6 Toll-like receptors……………………………………………….…………………….6

1.7 Role of Toll-like receptors in kidney disease................................................................8

1.8 Toll-like receptors recognition of agonists ...................................................................9

1.8.1 Lipopolysaccharide......................................................................................9

1.8.2 CpG oligodeoxynucleotides.........................................................................9

1.9 Toll-like receptors recognition of antagonists ............................................................ 10

1.9.1 4-hydroxychloroquine ............................................................................... 10

CHAPTER 2 - METHODOLOGY................................................................................ 11

2.1 Materials ..................................................................................................................... 11

2.2 Cell culture .................................................................................................................. 12

2.3 Cilia length analysis/ Immunofluorescence staining ................................................. 12

2.4 3D cysts culture in vitro ............................................................................................. 13

2.5 3D cysts measurements .............................................................................................. 13

2.6 Pharmacological agonist/ antagonist .......................................................................... 13

2.7 Immunoprecipitation ................................................................................................. 14

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2.8 Immunoblotting........................................................................................................... 15

2.9 CRISPER knockout for TLR4 and 9 ......................................................................... 16

2.10 Statistical Analysis .................................................................................................... 17

CHAPTER 3 - RESULTS .............................................................................................. 18

3.1 Immunolocalization of Toll-like receptors in primary cilia ....................................... 18

3.2 knockout of TLR4, TLR9 and PC2 proteins .............................................................. 19

3.3 Treatment with hydroxychloroquine and cilia length effect ...................................... 19

3.4 Treatment with lipopolysaccharide and cilia length effect ......................................... 23

3.5 Treatment with CpG oligodeoxynucleotides and cilia length effect .......................... 26

3.6 Effects of PBS on the formation of cyst structure in 3D cell culture ........................ 29

3.7 Effects of hydroxychloroquine on the formation of cyst structure in 3D culture ....... 30

3.8 Effects of lipopolysaccharide on the formation of cyst structure in 3D culture ......... 31

3.9 Effects of CpG oligodeoxynucleotides on the formation of cyst structure in 3D culture

..................................................................................................................................... 32

CHAPTER 4 – DISCUSSION & CONCLUSION ...................................................... 37

BIBLIOGRAPHY ........................................................................................................... 42

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LIST OF FIGURES

Figure 1. Structure of primary cilium. A cilium is a membrane-bound structure and composed of

multiple central pairs of microtubules (axoneme) originating from the basal body. A basal body is

a microtubule-based structure composed of mother and daughter centrioles. The ciliary membrane

and axoneme contribute to the upper part of the cilium. The ciliary membrane is continuous with

the cell membrane, but cilia have their own proteins, ion channels and/or receptors. The ciliary

skeleton may have 9 + 0 or 9 + 2 axoneme compositions. Most 9 + 0 cilia lack inner and outer

dynein arms, radial spokes, and central sheath and are commonly referred as non-motile primary

cilia. Some 9 + 0 cilia lack the central microtubule only and are motile. Between the cell membrane

and cilium, there is a transition-membrane at the junction of the basal body acting as a barrier for

molecules to enter or exit from the primary cilium………………………………………………..2

Figure 2. Kidney disease progression in autosomal polycystic kidney disease (ADPKD)……….5

Figure 3. Toll-like receptors (human) display specificity in their recognition of pathogen-

associated molecular patterns and/or synthetic compounds………………………………………..8

Figure 4. A schematic image showing the total immunoprecipitation process for the Western

blot sampling…………………………………………………………………………………….15

Figure 5. Schematic of CRISPR-Cas9 mechanism in gene knockout process………………….16

Figure 6. Immunostaining for TLR4 and TLR9 are shown in middle panels (red), left panels

showing the DIC images, and right panels showing the merged image with nuclear staining DAPI

(Blue). N=3 for all experiments…………………………………………………………………18

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Figure 7. Representative Western blot shows that TLR4 and TLR9 expressions are reduced in our

TLR4 and 9 KO cells compared to control LLCPK-1 and Pkd2 KO cells. In the third row of image

PC2 knockout was also confirmed. GAPDH used as a validation for equal loading. N=3 samples

per group in each study………………………………………………………………………….19

Figure 8. Representative immunofluorescence images show that effects of 4-hydroxychloroquine

at different concentrations (0 to 1 µM) on the cilia length. Green indicates cilia labeled to

acetylated--tubulin (FITC) and blue indicates cell nucleus (DAPI). Representative bar graphs

indicate the measurements of 50 cell on cilia lengths. The significant differences between no

treatment and each concentration of 4-hydroxychloroquine treatment were shown. Acquired from

5 preparations in each group; a minimum of 10 cilia were randomly selected from each preparation.

**, p<0.01; ***, p<0.001; and ****, p<0.0001 compared with the control group……………….21

Figure 9. Length of primary cilia is altered after 4-hydroxychloroquine treatment. After treatment

with increasing concentrations of 4-hydroxychloroquine the length of primary cilia was measured

from immunofluorescence slides. Length measurements were made from images taken at one

single plane in triplicate. Cilia length was grouped in a discrete range, and the % distribution was

represented in histograms for each 4-hydroxychloroquine concentration. The cilia length

measurements were shown on X-axis and the % distribution of cilia lengths was shown on Y-axis.

N=50 cilia in each study………………………………………………………………………….22

Figure 10. Representative immunofluorescence images show that different concentrations (0 to

1000 nM) of lipopolysaccharide (LPS) effects on the cilia length. Green indicates cilia labeled to

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acetylated--tubulin (FITC) and blue indicates cell nucleus (DAPI). Representative bar graphs

indicate the measurements of 50 cell cilia lengths. The significant differences between no

treatment and LPS treatment at different concentrations were shown. Acquired from 5 preparations

in each group; a minimum of 10 cilia were randomly selected from each preparation. *, p<0.05;

**, p<0.01; ***, p<0.001; and ****, p<0.0001 compared with the control group………………..24

Figure 11. Length of primary cilia is altered after treatment with lipopolysaccharide (LPS)

treatment. After treatment with increasing concentrations of LPS the length of primary cilia was

measured from immunofluorescence slides. Length measurements were made from images taken

at one single plane in triplicate. Cilia length was grouped in a discrete range, and % distribution

was represented in histograms for each LPS concentration. The cilia length measurements were

shown on X-axis and the % distribution of cilia lengths was shown on Y-axis. N=50 cilia in each

study………………………………………………………………………………..…………….25

Figure 12. Representative immunofluorescence images show that effects of ODN D-SL03 (ODN)

at different concentrations (0 to 5 µM) on the cilia length. Green indicates cilia labeled to

acetylated--tubulin (FITC) and blue indicates cell nucleus (DAPI). Representative bar graphs

indicate the measurements of 50 cell cilia lengths. The significant differences between no

treatment and ODN treatment at different concentrations were shown. Acquired from 5

preparations in each group; a minimum of 10 cilia were randomly selected from each preparation.

*, p<0.05; **, p<0.01; ***, p<0.001; and ****, p<0.0001 compared with the control group……27

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Figure 13. Length of primary cilia is altered after treatment with ODN D-SL03 (ODN) treatment.

After treatment with increasing concentrations of ODN the length of primary cilia was measured

from immunofluorescence slides. Length measurements were made from images taken at one

single plane in triplicate. Cilia length was grouped in a discrete range, and percent distribution was

represented in histograms for each ODN concentration. The cilia length measurements were shown

on X-axis and the % distribution of cilia lengths were shown on Y-axis. N=50 cilia in each

study…………………………………………………………………..………………………….28

Figure 14. Representative 3D cyst DIC images showing cyst formation in Matrigel in basal

medium at different days of 3, 6, 9 and 12 days in different cell systems (scrambled, TLR4 KO,

TLR9 KO and Pkd2 KO) when treated with PBS. The representative immunofluorescence

images showing that the lumen formation as cyst growth increasing. Green indicates both apical

and basolateral membranes. Blue -nucleus (DAPI). ………………………………………….…30

Figure 15. Representative 3D cyst DIC images showing cyst formation in Matrigel in basal

medium at different days of 3, 6, 9 and 12 days in different cell systems (Scrambled, TLR4 KO,

TLR9 KO and Pkd2 KO) when treated with 4-hydroxychloroquine (1 μM) The representative

immunofluorescence images showing that the lumen formation as cyst growth increasing. Green

indicates both apical and basolateral membranes. Blue -nucleus (DAPI)………………………...31

Figure 16. Representative 3D cyst DIC images showing cyst formation in Matrigel in basal

medium at different days of 3, 6, 9 and 12 days in different cell systems (scrambled, TLR4 KO,

TLR9 KO and Pkd2 KO) when treated with lipopolysaccharide LPS (1000 nM). The

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representative immunofluorescence images showing that the lumen formation as cyst growth

increasing. Green indicates both apical and basolateral membranes. Blue -nucleus (DAPI)……32

Figure 17. Representative 3D cyst DIC images showing cyst formation in Matrigel in basal

medium at different days of 3, 6, 9 and 12 days in different cell systems (Scrambled, TLR4 KO,

TLR9 KO and Pkd2 KO) when treated with ODN D-SL03 ODN (4 μM). The representative

immunofluorescence images showing that the lumen formation as cyst growth increasing. Green

indicates both apical and basolateral membranes. Blue -nucleus (DAPI)……………………….33

Figure 18. The representative dot plots showing the mean values of cyst diameters at 3, 6, 9 and

12 days for different cell systems (Scrambled, TLR4 KO, TLR 9 KO and Pkd2 KO) and also with

different treatments. N=50 measurements of cysts in each study. *, p<0.05; **, p<0.01; ***,

p<0.001; and ****, p<0.0001 compared with the control group………………………………….34

Figure 19. The representative line graphs showing the mean values of cyst diameters for 3 to 12

days when Scrambled, TLR4 KO, TLR9 KO and Pkd2 KO cells treated with phosphate buffered

saline, lipopolysaccharide, ODN D-SL03 or 4-hydroxychloroquine. N=50 measurements of cysts

in each study. **, p<0.01; ***, p<0.001; and ****, p<0.0001 compared to the control group……35

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LIST OF TABLES

Table 1. The pharmacological agents used in our study along with their optimal concentrations

used. The mode of actions for the same are also presented………………………………………14

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CHAPTER 1

INTRODUCTION

1.1 Cilia

Cilia are microtubule-based organelles that extend from the apical surface of many eukaryotic

cells. Around 800 individual proteins have been identified in the cilia (Gherman, Davis, &

Katsanis, 2006) (Ishikawa, Thompson, Yates, & Marshall, 2012). Cilia can be motile or non-

motile (primary cilia). Motile cilia are found on the epithelial surfaces of ear, respiratory tracts,

fallopian tubes and the ventricles of the central nervous system with different length in each organ.

Motile cilia play critical roles in organ development, mucous clearance and sperm motility

(O'Callaghan, Sikand, & Rutman, 1999).

Primary cilia are found on cells of the olfactory epithelium and act as sensory antennae responding

to the intracellular signaling (Yoder, 2007). Defect in primary cilia structure or function known

as ciliopathies. Ciliopathies include a group of diseases such as obesity, hypertension, cancer and

PKD (Li et al., 2015) (Aboualaiwi et al., 2014) (Hildebrandt, Benzing, & Katsanis, 2011). The

first “ciliopathic” disorder was attributed to Bardet-Biedl syndrome (Ansley et al., 2003).

Based on the cilia motility and structure, nodal cilia with unique hybrid type have been identified.

Nodal cilia are solitary motile organelles which play role in embryonic development. Defect in

nodal cilia could potentially result in internal thoracic and abdominal organ configuration from

normal (situs solitus) to mirror-image configuration (situs inversus), randomized configuration

(situs ambiguous), or configuration with duplication (situs isomerism).

1.2 Cilia structure

The structure of primary cilium contains of nine parallel pairs of microtubules called the axoneme

to support the long cilia structure. In primary cilia the microtubules are without central pair. In the

nodal cilia, the axoneme also lacks the central pair of microtubules. However, it requires for

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motility dynein arms and radial spokes (Shinohara et al., 2015) (Figure 1). The discoveries of a

“3+0” axonemal in a protozoan and “9+4” axoneme in Hensen’s node of a rabbit have made the

classification of cilia structure more complicated (Feistel & Blum, 2006).

Figure 1. Structure of primary cilium. A cilium is a membrane-bound structure and composed of

multiple central pairs of microtubules (axoneme) originating from the basal body. A basal body is

a microtubule-based structure composed of mother and daughter centrioles. The ciliary membrane

and axoneme contribute to the upper part of the cilium. The ciliary membrane is continuous with

the cell membrane, but cilia have their own proteins, ion channels and/or receptors. The ciliary

skeleton may have 9 + 0 or 9 + 2 axoneme compositions. Most 9 + 0 cilia lack inner and outer

dynein arms, radial spokes, and central sheath and are commonly referred as non-motile primary

cilia. Some 9 + 0 cilia lack the central microtubule only and are motile. Between the cell membrane

and cilium, there is a transition-membrane at the junction of the basal body acting as a barrier for

molecules to enter or exit from the primary cilium.

Adapted from “Primary Cilium-Dependent Signaling Mechanisms.” Pala, R.; Alomari, N.;

Nauli, S.M. Primary Cilium-Dependent Signaling Mechanisms. Int. J. Mol. Sci. 2017, 18,

2272.

The ciliary membrane composed of many receptors. Most of the ciliary protein functions are not

yet completely established. Some of the receptors located in the cilia are ion channel, sensory

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proteins and protein transporters to provide support and function of the cilia. Cilioplasm is

constituted within the soluble compartment of the cilia (Nauli, Jin, & Hierck, 2011) (X. Jin et al.,

2014). The studies have shown that cilioplasm involved with the calcium signaling compartment

in order to response to chemical and mechanical stimuli (X. Jin et al., 2014), (Masyuk, Gradilone,

& LaRusso, 2014) (Lee et al., 2015) (Atkinson et al., 2015). Cilioplasm also contains other many

more signaling proteins. However, ciliary dynamic compartment includes signaling and transport

proteins for example, intraflagellar transport (IFT) proteins. Moreover, transport proteins and

signaling moieties are essential for cilia structure and function. The basal body composed of the

mother centriole to which the ciliary axoneme is rooted. In addition to its important structural role,

the basal body contains different signaling proteins that provide several functions. The transition

zone composed of transition fibers and transition zone. The transition zone connects ciliary

axoneme and basal body which plays an important role in ciliary access and ciliogenesis (Garcia-

Gonzalo & Reiter, 2012).

1.3 Roles of primary cilia in cellular signaling

Receptors located in the primary cilia can serve a wide range of functions. Recent data sets about

the ciliary genomics and proteomics have shown the cilium function engaged approximately 1,000

different polypeptides (Gherman et al., 2006). The fundamental role of cilia is to sense and conduct

the signaling pathways from either chemical stimulus as ligand and growth factor or mechanical

stimuli as pressure and flow. The ciliary pathways include sonic hedgehog, calcium, mTOR,

MAPK, JAK/STAT and Wnt, and these signaling pathways play vital roles in cellular processes

for instance, development, differentiation, cell cycle and apoptosis (Satir, Pedersen, & Christensen,

2010).

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Primary cilia can also play an opposite role of chemosensory role by performing “chemosecretory”

function, sending information to the extracellular environment (Wood, Huang, Diener, &

Rosenbaum, 2013). The study has demonstrated that ciliary membrane protein polyductin

undergoes proteolytic cleavage and release of extracellular domain into the lumen (Kaimori et al.,

2007). Another studies, Barr and Sternberg showed the polycystin-1 and -2 (PC-1/-2) are involved

in the PKD and autosomal dominant polycystic kidney disease (ADPKD) in human and the loss

of these two proteins affect the cilia behavior and function (Barr & Sternberg, 1999). Moreover,

genetic studies have demonstrated the link between the cilia; cyst formation and fibrosis. In case

of the complete loss of cilia in mouse result in slow down the growth of cysts, while in case of

cilia-polycystin mutations result in increased cysts proliferation ADPKD models (Ma, Gallagher,

& Somlo, 2017). The idea of studying the ciliary protein functions is interesting and worth

investigating further.

1.4 Polycystic Kidney disease

ADPKD is one of the most common inherited disease. ADPKD affects around 1 in 500 to 1000

person in the worldwide (Ali et al., 2015) (Pei & Watnick, 2010). In the United States of America,

approximately 2,144 patients start renal replacement therapy annually. African Americans are less

common to have end-stage renal disease (ESRD) as complication to ADPKD comparing to the

white people. The rate of the ESRD caused by ADPKD is 7⋅8 and 6⋅0 cases per million in Europe

from 1998 to 1999 and 8⋅7 and 6⋅9 cases per million in USA from 1998 to 2001 per year (Stengel

et al., 2003).

ADPKD is characterized by fluid filled cysts that grow in the kidney and interfere with their ability

kidney function (filtration) and eventually cause kidney failure (Figure 2). The cysts may grow in

other organs like liver (Nadasdy et al., 1995). The gene involved in the disease is PKD1 discovered

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in 1994. PKD1 located on the short arm of the chromosome 16 and produces PC1. The PKD2 was

discovered in 1996. PKD2 is located on the long arm of chromosome 4 and produces a smaller

glycoprotein, PC2. PC2 protein involves in calcium transport. PKD1 mutation responsible for 85%

of ADPKD cases while PKD2 accounts for 15% (Ali et al., 2015) (Braun, 2014) (Chapman et al.,

2015). Although PKD1 and PKD2 mutations produce similar extrarenal and renal manifestations,

PKD2 patients diagnosed later in life with less complications and longer renal survival compared

with PKD1 patients (Hateboer et al., 1999).

Figure 2. Kidney disease progression in autosomal polycystic kidney disease (ADPKD).

Adapted from:

https://mosaiques-diagnostics.de/mosaiques-diagnostics/Polycystic_kidney_disease

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1.5 Role of inflammation in polycystic kidney disease

In general, ADPKD is not an inflammatory disease. However, subclinical inflammation has been

linked with ADPKD. Studies have indicated that the inflammation occurs in early stage of the

disease and an inflammatory component exist in ADPKD patients. For instant, in cyst fluid of

PKD patients contained tumor necrosis factor-α (TNF-α), IL-2 (proinflammatory cytokines) and

interleukin (IL)-1β (Gardner, Burnside, Elzinga, & Locksley, 1991) (D. Zheng et al., 2003). In

addition, ADPKD patients’ urine had monocyte chemoattractant protein-1 (MCP-1). The level of

MCP-1 was positively linked to the progression of ADPKD patients (Mrug et al., 2008).

Zhou and co-workers observed that in cystic mice, a non-orthologous mouse model of autosomal

recessive polycystic kidney disease (ARPKD) was reported to upregulate genes that involved in

the innate immune system. They also found a high level of expression of macrophages and

monocytes (Mrug et al., 2008). Specifically, they found highly upregulated and activated CD14,

which is a pattern recognition receptor involved in TLRs for their activation (Zhou et al., 2010).

Menon and co-workers found that in declining kidney function the inflammatory markers was

increased such as C-reactive protein (CRP) and IL-6 (Menon et al., 2011). Thus, the data show a

potential role of inflammation in the progression of ADPKD.

1.6 Toll-like receptors (TLRs)

The immune system can sense and eliminate any invading pathogen in our body by the ability to

distinguish between self and non-self. The immune system is classified into “adaptive immunity”

and “innate immunity”. The adaptive immunity protects the body in a specific manner from

infectious disease using antigen on B and T cells surface. However, the innate immunity is the first

line defense mechanism in all organisms and it is nonspecific (Hoffmann, Kafatos, Janeway, &

Ezekowitz, 1999). Toll receptor was first found in drosophila which is considered as

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transmembrane receptor (Hashimoto, Hudson, & Anderson, 1988). A mammalian homologue of

drosophila Toll was found one year after the discovery of the Toll receptor in the drosophila

(Medzhitov, Preston-Hurlburt, & Janeway, 1997).

Afterward, the family of TLRs was identified which consist of 10 receptors TLR1 to TLR10

(Medzhitov et al., 1997) (Du, Poltorak, Wei, & Beutler, 2000) and the microbial ligand for these

receptors has been identified (Figure 3). Recently, an analysis of 10 human and 12 mice TLRs

found that they can be grouped into two categories based on their localization or their activation

by microbial membrane lipids, microbial nucleic acids, or bacterial proteins (De Nardo, 2015)

(Lim & Staudt, 2013). The ligands found in microbes include bacterial cell wall. TLR4 responds

to bacterial LPS found in Escherichia coli and TLR2 responds to peptidoglycan found in

Staphylococcus aureus (Poltorak et al., 1998) (Yoshimura et al., 1999). Furthermore, TLR2 and

TLR1 responses to tri-acylated lipoproteins and TLR2 and TLR6 respond to di-acylated

lipoproteins (Brightbill et al., 1999) (Ozinsky et al., 2000) whereas TLR9 responds to

unmethylated CpG DNA (ODN D-SL03; ODN) found in bacterial genomes (Hemmi et al., 2000).

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Figure 3. Toll-like receptors (Human) display specificity in their recognition of pathogen-

associated molecular patterns and/or synthetic compounds.

Adapted from “J.E. McInturff, R.L. Modlin, J. Kim. The role of toll-like receptors in the

pathogenesis and treatment of dermatological disease J Invest Dermatol, 125 (2005), pp. 1-

8.

1.7 Roles of Toll-like receptors in kidney disease

TLRs are a group of innate immune receptors that detect pathogen and regulate inflammation and

immune responses. The innate response works as first defense mechanism which is usually

nonspecific. We can classify the innate immune interaction into three different classes. First, it is

by recognizing the infection and start local activation of inflammatory and immunity response.

Second, it is by activating innate immunity systemically which leads the kidney to release

inflammatory mediators. Third, it is the homeostatic interactions between environmental stimuli

and innate immune system. Toll-like receptors (TLRs), nucleotide-binding oligomerization

domain receptors (NLRs) and PYD domains-containing protein 3 (NLRP3) monitor the kidneys

repair mechanism (Smith, 2009). These receptors play an important role in immune-mediated

responses and inflammatory diseases. It has been shown that TLR2, TLR4 and TLR9 are involved

in different inflammatory kidney disorders, such as in diabetic nephropathy, renal transplant

rejection, lupus nephritis and urinary tract infections. (Leemans, Kors, Anders, & Florquin, 2014,

Smith 2009) (Patole et al., 2006).

There are no studies available on the role of localization of TLR4 and TLR9 in the primary cilia

and they are cause/progression of PKD. Thus, we aimed to investigate the potential role of TLRs

in the cyst formation and we also tried to identify their role of TLRs specifically by using cellular

knockout of TLR4 and TLR9 and using genetic pharmacological approach.

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1.8 Toll like receptors recognition of agonists

The structural studies on TLRs have shown how some of the agonists bind to the receptor and

activate it. For instant, TLR1 and TLR2 bind to triacylated lipopeptide and TLR3 to viral double-

stranded RNA. Unmethylated CpG motifs in viral DNA activates TLR9 and guanosine in virus

RNA activates TLR7 and TLR8 (Pichlmair & Reis e Sousa, 2007). Moreover, LPS from Gram-

negative bacteria, envelope protein from mouse mammary tumor virus (MMTV) and protein from

respiratory syncytial virus (RSV) can stimulate TLR4 (Kurt-Jones et al., 2000) (Rassa, Meyers,

Zhang, Kudaravalli, & Ross, 2002). The agonists bind specifically to the receptors and form a

bridge which brings together the glycan-free surfaces of the TLRs (M. S. Jin et al., 2007) (Liu et

al., 2008).

1.8.1 Lipopolysaccharide (LPS)

LPS is the principal component of Gram-negative bacteria that activates the innate immune

system. LPS recognition is predominantly mediated by TLR4 (Poltorak et al., 1998). This

recognition involves the binding of LPS with lipopolysaccharide-binding protein and subsequently

with CD14 which physically associates with a complex including TLR4 and MD2 (Shimazu et al.,

1999). Formation of the TLR4-centered LPS receptor complex induces the production of

proinflammatory cytokines through the MyD88 pathway. LPS signaling also involves a MyD88-

independent cascade that mediates the expression of IFN-inducible genes.

1.8.2 CpG ODNs (ODN)

CpG ODNs are short synthetic single-stranded DNA molecules containing unmethylated CpG

dinucleotides in particular sequence contexts (CpG motifs). CpG ODNs possess a partially or

completely phosphorothioated (PS) backbone, as opposed to the natural phosphodiester (PO)

backbone found in genomic bacterial DNA. Three major classes of stimulatory CpG ODNs have

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been identified based on structural characteristics and activity in human peripheral blood

mononuclear cells (PBMCs), in particular B cells and plasmacytoid dendritic cells (pDCs). These

three classes are Class A (Type D), Class B (Type K) and Class C; CpG-A ODNs are characterized

by a PO central CpG-containing palindromic motif and a PS-modified 3’ poly-G string. They

induce high IFN-α production from pDCs but are weak stimulators of TLR-9-dependent NF-κB

signaling and pro-inflammatory cytokine (e.g. IL-6) production. CpG-B ODNs contain a full PS

backbone with one or more CpG dinucleotides. They strongly activate B cells and TLR9-

dependent NF-κB signaling.

1.9 Toll like receptors recognition of antagonists

1.9.1 Hydroxychloroquine (HCQ)

The studies on TLRs have demonstrated an example of the agents that can inhibit or reduce the

activity of the receptors. HCQ the antimalarial drug has been shown to modulate the intracellular

TLR pathway by reducing TLR4 and TLR9 expression (Sperber, Kalb, Stecher, Banerjee, &

Mayer, 1993). Recently, a new mechanistic model of this antagonist drug has been proposed that

HCQ directly interacts with nucleic acids and consequently causes structural modifications of the

TLR ligand to prevent the ligand from binding to TLR (Kuznik et al., 2011) (Venuturupalli,

Gudsoorkar, & Wallace, 2012) (Wallace, Gudsoorkar, Weisman, & Venuturupalli, 2012).

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CHAPTER 2

METHODOLOGY

2.1 Materials

LLCPK-1 (ATCC® CL-101.1TM) porcine renal epithelial cells from proximal tubule were

obtained from American Type Culture Collection (ATCC; Manassas, VA). Dulbecco's Modified

Eagle Medium (DMEM) was purchased from VWR International (Brooklyn, NY); trypsin,

penicillin-streptomycin solution and phosphate buffered saline (PBS) were ordered from Corning

(Manassas, VA). Fetal bovine serum was obtained from Seradigm (Logan, UT). The

pharmacological agents were purchased from different chemical companies. 4-Hydroxycloroquine

was purchased from Cayman chemicals, and lipopolysaccharide/CpG oligodeoxynucleotides were

purchased from InvivioGen (San Diego, CA). Most LPS preparations on the market are

contaminated by other bacterial components, such as lipoproteins, thus activating TLR2 signaling

as well as TLR4 signaling. High concentration (1 µg/ml) of the standard preparation of LPS can

induce TLR2 activity, whereas LPS (InvivoGen) activates the TLR4 pathway specifically (0.1

nM).

Western blot visualization kit was obtained from ThermoScientific (Rockford, IL). Nonfat dry

milk was purchased from LabScientific (Livingston, NJ). Protein lysis (2X) sample buffer was

obtained from Bio-Rad (Hercules, CA). Primary antibodies, acetylated-α-tubulin and anti-TLR9

were ordered from Sigma (St. Louis, MO); anti-TLR4, anti-PC2 and anti-GAPDH were purchased

from Santa Cruz Biotechnology.

The secondary antibody fluorescein anti-mouse and mounting media with DAPI were purchased

from Vector Laboratories (Burlingame, AL). For immunofluorescence, paraformaldehyde (PFA)

from EMS (Hatfield, PA) was used. Sucrose and Triton-X were purchased from Fisher Scientific

(Fair Lawn, NJ). For 3D cysts culture, Corning Matrigel matrix was purchased from CORNING

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(Corning, NY). For Crisper Cas9, Crisper/Cas9 Knockout Kits were obtained from ORIGENE

(Rockville, MD).

2.2. Cell Culture

Porcine renal epithelial cells from proximal tubule (LLCPK-1), Pkd2, TLR4 and TLR9 knockout

cells were cultured as monolayer in Dulbecco’s Modified Eagle Medium (DMEM) supplemented

with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 C in 5% CO2 incubator.

Cells were trypsinized with 0.05% solution of trypsin for passaging. Once differentiated, HCQ,

LPS and ODN were added onto culture plates to with final concentrations of 1.0 μM, 1000 nM

and 4 μM, respectively. For control experiments, PBS was added to cells in the same manner and

volume.

2.3. Cilia length analysis/ Immunofluorescence staining

First, cells were fixed using 4% paraformaldehyde and 2% sucrose in PBS for 10 minutes then

wash it with PBS three times for 5 minutes. Second, cells were permeabilized using 10% triton

X-100 for 5 minutes and washed with PBS three times for 5 minutes. Third, cells were incubated

with primary antibody acetylated-α-tubulin (1:10,000 dilution, Sigma Aldrich, St. Louis, MO) for

24 hours at 37°C. Then wash it with PBS three times for 5 minutes. Forth, cells were incubated

with secondary antibody using fluorescein isothiocyanate (TexRed/FITC)-conjugated (1:1000

dilution, Vector Labs Burlingame, CA) for 1 hour at 37°C. Slides were then mounted with DAPI

(Southern Biotech, Birmingham, AL). Nikon Eclipse Ti-E inverted microscope with NIS-

Elements imaging software (version 4.30) was used to capture images of single cell with their

single primary cilium with 100× magnification fields.

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2.4 3D cyst culture in vitro

This protocol was done accordingly with Corning instructions. First, Corning® Matrigel®

basement membrane matrix was thawed overnight by submerging the vial in ice in a 4°C

refrigerator before use. Once Matrigel matrix is thawed, vial was swirled to ensure that the material

was dispersed. On Day 0, the Matrigel matrix was diluted to 5 mg/mL with ice-cold complete cell

culture medium (DMEM + 10% FBS). Using pre-chilled tips, the pre-chilled 24-well plate was

coated by adding 100μL of Matrigel matrix (5mg/mL) into each well, spread evenly with a pipet

tip, and incubated at 37°C for 30 min to form gel. Next, the cells were trypsinized from a

monolayer to make a single-cell suspension and then pelleted through centrifugation at 125 x g for

5 min at room temperature. After, re-suspend the cells with DMEM medium and adjust the cell

density to 5 × 106 cells/mL. 30 μL prepared cell suspension was added to 270 μL Matrigel matrix

solution (5 mg/mL) which was kept on ice for a final cell density is 5 x 105 cells/mL. The plate

was incubated at 37°C for 30 to 45 min. 500 μL DMEM complete media was gently added to glass

bottomed 8 well plate. Culture was kept for 12 days and medium was changed every 2 days.

2.5 3D cysts measurements

The cyst growth was observed by using Nikon live cell microscope. The DIC images were captured

for every 3 days. Finally, cysts were fixed and immunostained. The fluorescence imaging was

done with Nikon confocal microscope to see the cyst formation or morphology.

2.6 Pharmacological agonist/antagonist

Cilia length measurement studies and 3D cysts culture experiments were conducted separately. All

the pharmacological agents used for 24 hour treatment. HCQ, LPS and ODN were diluted and

prepared for desired concentrations and stored under 4°C until use. table below shows the

concentrations and modes of action of the agents (Table 1).

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Table 1. The pharmacological agents used in our study along with their final concentrations used.

The modes of action for the same are also presented.

2.7 Immunoprecipitation

The cell protein lysates were first pre-cleared with the protein A or G agarose beads. Pre-clearing

the lysate can help reduce non-specific binding by incubating 20 μl of protein A or G agarose

beads (20 μl of 50% bead slurry) for 1 hour. The lysates were incubated with 10 μg of primary

antibody for gentle rocking overnight at 4°C, following which 20 μl of protein A or G agarose

beads will be added for 1 hour. Beads were collected by centrifugation and the supernatant was be

removed. Agarose beads were washed five times in 500 μl of 1X cell lysis buffer supplemented

with protease inhibitor to remove any non-specific bonds to protein of interest. The collected beads

were resuspended in 20 μl of 2X SDS-PAGE sample buffer and the pelleted protein were stored

at -80°C.

Agonist/antagonist Concentration Mode of actions

4-Hydroxycloroquine 1 µM Inhibit TLR9 and TLR4

Lipopolysaccharide 1 µM Activate TLR4

CpG oligodeoxynucleotides 4 µM Activate TLR9

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Figure 4. A schematic image showing the total immunoprecipitation process for the western blot

sampling.

Adapted from: https://www.expedeon.com/resources/applications/immunoprecipitation/

2.8. Immunoblotting

Scrambled, TLR4 KO, TLR9 KO and Pkd2 KO cells were collected by scraping then lysed in lysis

buffer and sonicated to isolate the proteins. After centrifugation protein concentrations were

measured using Nanodrop instrument. Then protein normalization was done and each sample

contain 30 μg of protein within loading buffer. After sample preparation, proteins were loaded on

10% SDS-polyacrylamide gel and the proteins were transferred to a nitrocellulose membrane.

The membrane was blocked with 5% milk in TBST buffer and probed overnight at 4°C with

primary antibodies with anti-TLR4 (1:200 dilution), anti-TLR9 (0.5 µg/mL), anti-PKD2 (1:400

dilution and anti-GAPDH (1:400 dilution). For visualization, HRP-conjugated anti-rabbit (1:1000

dilution) used as secondary antibody. The protein bands were visualized and quantified by using

Bio-Rad imaging system.

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2.9. CRISPR knockout for TLR4 and TLR9

One day prior to transfection, seed approximately 1.0 x 105-3.0 x 105 cells in each well of a 6-well

plate containing 2 mL of complete growth medium. Cells were grown overnight to approximately

90%-95% confluence. To transfect, initially equilibrate DNA with Turbofectin reagent and Opti-

MEM® I reduced serum medium to room temperature. 2.5 μg plasmid DNA was diluted with 125

μL Opti-MEM® I. Later, the mixture for 5 minutes was incubated at room temperature. Once the

transfection reagent was diluted, it was combined with the DNA within 30 minutes. The diluted

DNA was combined with the diluted transfection reagent, which was incubated at room

temperature for 5 to 20 minutes to allow DNA-transfection reagent complexes to form. The DNA-

transfection reagent complexes were added directly to the well and mix gently by rocking the plate

back and forth. The cells were incubated at 37°C in a CO2 incubator for a total of 24-48 hours.

Figure 5. Schematic of CRISPR-Cas9 mechanism in gene knockout process.

Adapted from: https://labiotech.eu/features/crispr-cas9-review-gene-editing-tool/

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2.10. Statistical Analysis

All quantifiable data are reported as the mean ± standard error of the mean (SEM). Statistical

analysis was performed using ANOVA followed by Bonferroni post hoc test. The statistical

analyses were performed with GraphPad Prism (version 7.0 Sample sizes are included in figure

legends. “∗” represent statistically significant differences at various probability levels (P).

Significance was designated as follows: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Page 33

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CHAPTER 3

RESULTS

3.1. Immunolocalization of Toll-like receptors in primary cilia

We used immunostaining technique to look for functional receptor proteins TLR4 and TLR9 in

primary cilia. TLR4 and TLR9 proteins were detected by immunofluorescence analysis of primary

cilia in LLCPK-1 cells with monoclonal antibodies against human TLR4 and TLR9 after 24 hours

serum starvation. Figure 6 shows the single cell single cilium from the side view and strong

staining was observed on the primary cilia for both TLR4 and TLR9 (red). The DIC (brightfield)

and nuclear staining (blue) was also observed. In contrast, no staining was found in these cells

when tested with the irrelevant (nonspecific) antibody.

Figure 6. Immunostaining for TLR4 and 9 is shown in middle panel (red), left panel showing the

DIC images. Right panel showing the merged image with nuclear stain DAPI (Blue). No

counterstain was done for cilia. N=3 for all experiments.

TLR-9

TLR-4DIC

DIC

Merged

Merged

Page 34

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3.2. TLR4, TLR9 and PKD2 knockout

To test the effects of TLR on primary cilia structure, we further KO the TLR4 and TLR9 using

CRISPR-Cas9. The absence of TLR4 and TLR9 protein expression in LLCPK-1 cells was

confirmed by Western blot analysis using anti-TLR4 and TLR9 antibodies (Figure 7). We further

knockout the PKD2 and confirmed by Western blot (Figure 7).

Figure 7. Representative Western blot data shows that TLR4 and TLR9 expressions are reduced

in our TLR KO cells compared to control and Pkd2 KO cells. In the third row of image PC2

knockout was also confirmed. GAPDH used as a Western blot validation control. N=3 samples

per group in each study.

3.3. Effects of hydroxychloroquine on cilia length

Figure 8 shows the increase in cilia length when we used different concentrations of HCQ in

different cells. For this experiment we used different cell lines, i.e., control cells (vehicle), TLR4

KO, TLR9 KO and Pkd2 KO cells. From the results it is observed that the very long cilia (10-15

µm) were grown when scrambled and Pkd2 KO cells treated with different concentrations.

PC2

TLR9

GAPDH

TLR4

scra

mb

led

con

tro

l

Pkd2

TLR9

TLR4

Page 35

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However, little increase in cilia length (7 µm) was observed when the TLR4 KO cells treated with

HCQ. The TLR9 KO cells treatment with HCQ showing the decrease in cilia length (3 µm) when

increasing the HCQ concentrations. In 2003 a novel mechanism was described that HCQ inhibits

stimulation of the TLR family receptors (Takeda, Kaisho, & Akira, 2003). Our results suggest that

this inhibition may cause the decrease in cilia length which has never been studied before.

Statistical analysis showed significant differences in cilia length between control and HCQ-

treatment in both normal and KO cells. All independent cilia length measurement data were also

shown (Figure 9); this distribution indicates the variability between cells and their cilia length.

Page 36

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0

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** **** **** ****

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5 µm

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0 1

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*******

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ia L

ength

(µ

m)

5 µm

0 0.10 0.25

0.50 0.75 1.00

Page 37

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Figure 8. Representative immunofluorescence images show that effects of 4-hydroxychloroquine

at different concentrations (0 to 1 µM) on the cilia length. Green indicates cilia labeled to

acetylated--tubulin (FITC) and blue indicates cell nucleus (DAPI). Representative bar graphs

indicate the measurements of 50 cell on cilia lengths. The significant differences between no

treatment and each concentration of 4-hydroxychloroquine treatment were shown. Acquired from

5 preparations in each group; a minimum of 10 cilia were randomly selected from each preparation.

**, p<0.01; ***, p<0.001; and ****, p<0.0001 compared with the control group.

.

Figure 9. Length of primary cilia is altered after 4-hydroxychloroquine treatment. After treatment

with increasing concentrations of 4-hydroxychloroquine the length of primary cilia was measured

from immunofluorescence slides. Length measurements were made from images taken at one

single plane in triplicate. Cilia length was grouped in a discrete range, and the % distribution was

represented in histograms for each 4-hydroxychloroquine concentration. The cilia length

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TLR4 TLR9

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Page 38

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measurements were shown on X-axis and the % distribution of cilia lengths was shown on Y-axis.

N=50 cilia in each study.

3.4. Treatment of lipopolysaccharide and cilia length effect

Figure 10 shows the immunofluorescence images of cilia lengths with the treatments of different

concentrations (0 to 1000 nM) of LPS. LPS decreased primary cilia length in control, TLR4 KO

and Pkd2 KO cells, but had an opposite effect in TLR9 KO cells. Unfortunately, there were no

previous reports available on the localization of TLR4 or TLR9 receptors on primary cilia and

their effects on cilia (structural effects). Figure 11 shows the histograms for cilia lengths when

different cells were treated with different concentrations of LPS. A significant increase in cilia

length was observed when the TLR9 KO cells treated with LPS, whereas decreasing cilia length

was observed when the control, TLR4 KO and Pkd2 KO cells treated with LPS in a similar fashion.

Our results are in consistent with the previous results (Baek et al., 2017). Statistical analysis

showed significant differences in cilia length between control vs. HCQ-treatment in both normal

and KO cells.

Page 39

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Page 40

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Figure 10. Representative immunofluorescence images show that different concentrations (0 to

1000 nM) of lipopolysaccharide (LPS) effects on the cilia length. Green indicates cilia labeled to

acetylated--tubulin (FITC) and blue indicates cell nucleus (DAPI). Representative bar graphs

indicate the measurements of 50 cell cilia lengths. The significant differences between no

treatment and LPS treatment at different concentrations were shown. Acquired from 5 preparations

in each group; a minimum of 10 cilia were randomly selected from each preparation. *, p<0.05;

**, p<0.01; ***, p<0.001; and ****, p<0.0001 compared with the control group.

Figure 11. Length of primary cilia is altered after treatment with lipopolysaccharide (LPS)

treatment. After treatment with increasing concentrations of LPS the length of primary cilia was

measured from immunofluorescence slides. Length measurements were made from images taken

at one single plane in triplicate. Cilia length was grouped in a discrete range, and % distribution

was represented in histograms for each LPS concentration. The cilia length measurements were

shown on X-axis and the % distribution of cilia lengths was shown on Y-axis. N=50 cilia in each

study.

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Cilia Length (µm)

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Page 41

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3.5 Treatment of CpG oligodeoxynucleotides and cilia length effect

After treatment with 0 to 5 µM ODN, ciliary length was measured through immunofluorescence

staining (Figure 12). Compared to control, TLR4 KO and Pkd2 KO cell treatment with ODN, cilia

length was increased in TLR9 KO cells. In TLR9 KO cells, the cilia length was increased up to 10

µm compared with control, TLR4 KO and Pkd2 KO cells (3 µm). Our knockout experiments

suggest that a significant difference in cilia length was observed in TLR9 KO cells compared with

control scrambled cells when treated with ODN. Figure 13 shows the histograms of cilia lengths

when different cells treated with different concentrations of ODN. Statistical analysis showed

significant differences in cilia length between control vs. ODN-treatment in both normal and KO

cells.

Page 42

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0 5

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*** **** **** **** ****

Page 43

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Figure 12. Representative immunofluorescence images show that effects of ODN D-SL03 (ODN)

at different concentrations (0 to 5 µM) on the cilia length. Green indicates cilia labeled to

acetylated--tubulin (FITC) and blue indicates cell nucleus (DAPI). Representative bar graphs

indicate the measurements of 50 cell cilia lengths. The significant differences between no

treatment and ODN treatment at different concentrations were shown. Acquired from 5

preparations in each group; a minimum of 10 cilia were randomly selected from each preparation.

*, p<0.05; **, p<0.01; ***, p<0.001; and ****, p<0.0001 compared with the control group.

Figure 13. Length of primary cilia is altered after treatment with ODN D-SL03 (ODN) treatment.

After treatment with increasing concentrations of ODN the length of primary cilia was measured

from immunofluorescence slides. Length measurements were made from images taken at one

single plane in triplicate. Cilia length was grouped in a discrete range, and percent distribution was

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Page 44

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represented in histograms for each ODN concentration. The cilia length measurements were shown

on X-axis and the % distribution of cilia lengths were shown on Y-axis. N=50 cilia in each study.

3.6 Effects of TLR4, TLR9 and PKD2 KO on the formation of cyst structure in 3D culture

The LLCPK-1 cell line has not generally been used in 3D tissue models due to its inability to

reliably form large numbers of cysts over long periods of time. In one case, isolation of a specific

clone was required to facilitate cyst formation in collagen gels (Wohlwend, Montesano, Vassalli,

& Orci, 1985). Therefore, to use the LLCPK-1 and their KO model cell lines to consistently induce

cyst growth in 3D, we used hormonal media composition previously shown to produce structural

growth of immortalized human renal proximal tubule cells and primary human cortical cells in 3D

tissues (DesRochers, Suter, Roth, & Kaplan, 2013) (Neufeld et al., 1992).

To observe if the scrambled, TLR4 or TLR9 KO and their antagonists/agonists can play any role in

the cyst formation, cyst growth/progression was evaluated with 3D cyst culture assays. Figure 14

shows that the representative DIC images of cyst culture with different cell types for 3, 6, 9 and

12 days. From the images we can clearly observe that the scrambled/control cells treated with

scrambled shown no cyst formation. On the other hand, with the PKD2, TLR4 and TLR9 KO cells

a clear cyst formation and progression was observed in a time dependent manner. In Another case,

the scrambled cyst cultures treated with PBS did not show any sign of cyst formation. The

immunofluorescence results indicate a clear lumen formation in consistent with the brightfield

imaging results.

Page 45

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Figure 14. Representative 3D cyst DIC images showing cyst formation in Matrigel in basal

medium at different days of 3, 6, 9 and 12 days in different cell systems (scrambled, TLR4 KO,

TLR9 KO and Pkd2 KO) when treated with PBS. The representative immunofluorescence

images showing that the lumen formation as cyst growth increasing. Green indicates both apical

and basolateral membranes. Blue -nucleus (DAPI).

3.7 Effects of hydroxychloroquine on the formation of cyst structure in 3D culture

To observe if and how the HCQ (1 µM) affected cilia length in Pkd2, TLR4 or TLR9 KO cells and

further leads to the cyst formation, cyst growth/progression was evaluated with 3D cyst culture

assays. Figure 15 shows that the representative DIC images for cyst culture in different cell types

for 3, 6, 9 and 12 days. From the images we can observe a surprising result that the all cyst cultures

showing the formation of small to medium range cyst when treated with HCQ except TLR4 KO

cultures. The immunofluorescence results are also in consistent with the brightfield imaging

results.

TLR4/PBS

TLR9/PBS

3 6 9 12 12Days:

Scrambled/PBS

Pkd2/PBS

Page 46

31

Figure 15. Representative 3D cyst DIC images showing cyst formation in Matrigel in basal

medium at different days of 3, 6, 9 and 12 days in different cell systems (Scrambled, TLR4 KO,

TLR9 KO and Pkd2 KO) when treated with 4-hydroxychloroquine (1 μM) The representative

immunofluorescence images showing that the lumen formation as cyst growth increasing. Green

indicates both apical and basolateral membranes. Blue -nucleus (DAPI).

3.8 Effects of lipopolysaccharide on the formation of cyst structure in 3D culture

To observe if the LPS (1 nM), affected cilia length in Pkd2, TLR4 or TLR9 KO cells and further

leads to the cyst formation, cyst growth/progression was evaluated with 3D cyst culture assays.

Figure 16 shows that the representative DIC images of cyst culture with different cell types for 3,

6, 9 and 12 days. Figure 16 indicates that there was no cyst formation in the TLR4 cultures and

treatment with LPS, whereas a clear cyst formation and progressions were observed in the

scrambled and Pkd2 cyst cultures. This cyst formations were confirmed by immunostaining of the

TLR4/HCQ

TLR9/HCQ

Pkd2/HCQ

Scrambled /HCQ

3 6 9 12 12Days:

Page 47

32

3D cysts, where the larger cysts showed a clear lumen formation apparently by dead cells and

partial hollowing out of the center of the cysts. The cyst formation results were consistent with our

cilia length measurements, i.e. cilia lengths are inversely proportional to cyst sizes.

Figure 16. Representative 3D cyst DIC images showing cyst formation in Matrigel in basal

medium at different days of 3, 6, 9 and 12 days in different cell systems (scrambled, TLR4 KO,

TLR9 KO and Pkd2 KO) when treated with lipopolysaccharide LPS (1000 nM). The

representative immunofluorescence images showing that the lumen formation as cyst growth

increasing. Green indicates both apical and basolateral membranes. Blue -nucleus (DAPI).

3.9 Effects of CpG oligodeoxynucleotides on the formation of cyst structure in 3D culture

To observe if the ODN (4 µM) effect, TLR9 KO, TLR4 KO and Pkd2 KO cells can play any role

in the cyst formation, cyst growth/progression was evaluated with 3D cyst culture assays. Figure

Scrambled /LPS

TLR4/LPS

3 6 9 12 12Days:

TLR9/LPS

Pkd2/LPS

Page 48

33

17 shows that the representative DIC images of cyst culture with different cell types for 3, 6, 9 and

12 days. Figure 17 indicates that there was no cyst formation in the TLR9 cultures and treatment

with LPS, whereas a clear cyst formation and progressions were observed with the scrambled,

TLR4 and Pkd2 cyst cultures. This cyst formations were also confirmed by immunostaining of the

3D cysts, where the larger cysts showed a clear lumen formation apparently by dead cells and

partial hollowing out of the center of the cysts. The cyst formation results were consistent with our

cilia length measurements with TLR4 KO and TLR9 KO cells.

Figure 17. Representative 3D cyst DIC images showing cyst formation in Matrigel in basal

medium at different days of 3, 6, 9 and 12 days in different cell systems (Scrambled, TLR4 KO,

TLR9 KO and Pkd2 KO) when treated with ODN D-SL03 ODN (4 μM). The representative

immunofluorescence images showing that the lumen formation as cyst growth increasing. Green

indicates both apical and basolateral membranes. Blue -nucleus (DAPI).

Scrambled /ODN

Pkd2/ODN

TLR9/ODN

3 6 9 12 12Days:

TLR4/ODN

Page 49

34

In all our cyst culture experiments, a consistent 50 numbers of cysts were taken in to consideration

for the cyst size and progression measurements. The representative dot plot data (Figure 18) shows

that the measurements of 50 individual cysts for each independent group. We evaluated the cyst

progression for 3, 6, 9 and 12 day’s time periods. The cysts of all three KO lines (TLR4, TLR9 and

PKD2) remained larger than the scrambled control (LLCPK-1) for the remainder of the time

course. This relative increase in cyst size was due to both an actual change in dead cell count/lumen

formation for each knockdown cell line. and to the fact that the cysts of the scrambled control did

not change in size after 12 days in 3D culture. The TLR4 and TLR9 KO cell lines showed reduced

cyst growth from 3 to 12 days when treated with LPS and ODN, respectively.

Figure 18. The representative dot plots showing the mean values of cyst diameters at 3, 6, 9 and

12 days for different cell systems (Scrambled, TLR4 KO, TLR 9 KO and Pkd2 KO) and also with

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PBS (Vehicle) LPS (1 nM) ODN (4 µM) HCQ (1 µM)

TLR9 Pkd2 Scrambled TLR4 TLR9 Pkd2Scrambled TLR4 Pkd2

12 days

Scrambled TLR4 Pkd2

**** ********

**** ****

****

********

****

**** ****

****

****

******

*

**

**

*

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TLR9TLR9

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Page 50

35

different treatments. N=50 measurements of cysts in each study. *, p<0.05; **, p<0.01; ***,

p<0.001; and ****, p<0.0001 compared with the control group.

Figure 19 shows the cyst progression over time period. Throughout the experiment, scrambled

cells treated with agonists/antagonists had the most dramatic increase in both cyst size and rate of

growth, whereas TLR4 and TLR9 KO cells treated with their corresponding agonists showed

reduced cyst growth and number over time. Taken together, these data indicate that loss of TLR4,

9 or PC2 leads to increased cyst size over time and that loss of TLR9 has the most significant

impact upon cyst size.

Figure 19. The representative line graphs showing the mean values of cyst diameters for 3 to 12

days when Scrambled, TLR4 KO, TLR9 KO and Pkd2 KO cells treated with phosphate buffered

Page 51

36

saline, lipopolysaccharide, ODN D-SL03 or 4-hydroxychloroquine. N=50 measurements of cysts

in each study. **, p<0.01; ***, p<0.001; and ****, p<0.0001 compared to the control group.

Page 52

37

CHAPTER 4

DISCUSSION & CONCLUSIONS

Toll-like receptors (TLRs) constitute the body's primary defense system because they detect and

rapidly respond to foreign substances or endogenous signals and initiate inflammatory cascades.

This study, for the first time, was undertaken to further understand the potential role of TLRs

localization in primary cilia and their role in PKD. The role of TLRs in innate immunity and their

ability to recognize microbial products have been well characterized. TLRs are also able to

recognize endogenous molecules which are released upon cell damage and necrosis and have been

shown to be present in numerous autoimmune diseases. Therefore, the release of endogenous TLR

ligands during inflammation and consequently the activation of TLR signaling pathways may be

the mechanism initiating and driving autoimmune diseases (Drexler & Foxwell, 2010). The main

function of TLRs is considered to be the recognition and response to microbial pathogens. More

recently, however, they have also been reported to recognize endogenous ligands, so called

“danger signals”, which are released during tissue damage, infections and cell necrosis (Anderson,

Jurgens, & Nusslein-Volhard, 1985).

The primary cilium is a cellular organelle present in the majority of mammalian cell types where

they function as coordinating centers for mechanotransduction, Wnt and hedgehog signaling. The

length of the primary cilium is proposed to modulate cilia function, governed in part by the activity

of intraflagellar transport. For example, in articular cartilage, primary cilia length is increased and

hedgehog signaling activated in osteoarthritis (Wann & Knight, 2012). In this study, we present

the effect of TLRs agonists/antagonist for the first time, on the potentially fundamental role of

IFT-mediated primary cilia elongation in the progression of inflammation. Our immunostaining

showed that, the two major TLRs, i.e. TLR4 and TLR9 were identified in the primary cilia;

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38

however, TLR4 showed stronger expression than TLR9 when we did immunostaining and Western

blotting. In accordance with our results, TLRs have been shown to be expressed throughout the

cell including cilia. Here, we examine primary cilia length with exposure to TLRs inflammatory

antagonists and agonist substances. Observations of renal epithelial control (vehicle) cells, TLR4

KO and Pkd2 KO cells when treated with HCQ the cilia length was increased except for TLR9 KO

cells. We speculate that the HCQ mainly affect TLR9 KO cell inflammatory action than TLR4 KO.

When we use specific agonists for TLR4 and TLR9 the cilia lengths were changed significantly.

The use of LPS as an agonist for TLR4 KO cells the cilia length was decreased significantly,

whereas when we used ODN as agonist for TLR 9 KO cells the cilia length was increased

significantly. This indicates that TLR 9 plays a major role in cilia length control.

We show that this phenomenon of cilia elongation causes decreased cyst formation in response to

the inflammatory action is clearly visible. Our results clearly disclose that cilia length is inversely

proportional to the cyst formations. When we used pharmacological agents or genetic approach to

increase cilia length the cyst formation or growth was significantly reduced, whereas shorter cilia

promote cysts. TLRs and inflammations are taken into high consideration because, under normal

circumstances inflammatory reaction is tightly controlled to restrict cell growth until infections are

resolved and tissue recovery process completed. In contrast, chronic inflammation establishes an

environment rich in inflammatory cells, which produce growth factors. In recent studies, specific

inflammatory conditions have been associated with certain cancers (Balkwill & Coussens, 2004)

(Balkwill & Mantovani, 2001) which is more dangerous. On the other hand, constant intake of

anti-inflammatory drugs can reduce the risk of developing certain types of cancer. Numerous

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39

reports have also described an association of certain TLRs with the risk of developing certain

cancers (S. L. Zheng et al., 2004).

Several researchers hypothesized that TLR involvement in tissue repair as well as inflammation is

supported by the increasing amount of evidence for endogenous TLR ligands (Rifkin, Leadbetter,

Busconi, Viglianti, & Marshak-Rothstein, 2005) (Brentano, Kyburz, Schorr, Gay, & Gay, 2005).

However, further studies are necessary to investigate possible endogenous targets on primary cilia

for TLRs. The recognition of so called “danger signals”, released following injury and cell

necrosis, would enable TLRs to survey their environment and induce an inflammatory response in

case of infection as well as a proliferative response subsequent to injury (Jiang et al., 2005).

ADPKD is one of the hereditary and progressive systemic disorders that results in end stage renal

disorder (Gabow, 1993). Due to its systemic and genetic pattern, many studies were conducted on

the early stage of the disease to find measurements for halting the disease growth (Santoro et al.,

2015) (Chapman, 2008). Through recent studies it is understood that the systemic inflammation is

evident in early phase ADPKD patients and this inflammatory action may contribute to both heart

and kidney function deterioration (Menon et al., 2011) (Kocyigit et al., 2012) (Swenson-Fields et

al., 2013). It is thought that the activation of systemic and local immune responses contributes to

renal dysfunction, but the mechanisms of activation are poorly understood.

Primary cilia have been proposed as a link between the development of kidney cysts and the

polycystin proteins. To assess the downstream functionality of cilia elongation in an inflammatory

context, we employed a CRISPR approach to knockout TLR4 and TLR9 whilst assessing

agonists/antagonists stimulated cyst formation (3D cyst cultures) classic inflammatory results of

PKD in many cell types. When we used LPS and ODN in TLR4 KO and TLR9 KO cells,

respectively no cyst formation/progression was observed. This may explain the downstream

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inflammatory signaling. This is also consistent with our cilia length increase and shows the

potential proof between cilia length and cyst formation through downward inflammatory response.

These results are in consistent with the previous results (Baek et al., 2017), in this TLR4 mediated

inflammatory response in hippocampal neurons were reported. In contrast, the cilia length was

decreased and surprisingly the cyst size also decreased in TLR4 KO cells with treatment of HCQ,

it tells us another independent mechanism is responsible for this action.

4.1 Conclusions

In summary, our study provides new understanding about TLR on cilia length and cyst formation.

TLR4 and TLR9 gene expression may be associated with rapid progression in our 3D cyst culture

studies. We find that the TLRs agonists/antagonists can modulate cilia length and their role in

inflammatory actions. The primary cilium already has central roles throughout cell biology, but

here we propose, for the first time, that the cilium and the regulation of its structure are of

fundamental importance in inflammation of ADPKD. Further studies are needed to investigate the

potential role of ciliary TLRs via inflammation and immune pathways in ADPKD animal models.

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