Elucidation of the functional consequences of NLRP7

By: Ebtihaj Bukhari

Department of Genetics McGill University, Montreal July 2009

A thesis submitted to McGill University in partial fulfillment of the requirement of the Master of Science degree in Human Genetics

© Ebtihaj Bukhari, 2009

TABLE OF CONTENTS

ABSTRACT...... 4

RÉSUMÉ ...... 5

LIST OF ABBREVIATIONS ...... 6

LIST OF FIGURES AND TABLES ...... 7

ACKNOWLEDGEMENTS ...... 8

CHAPTER 1...... 9

1.1 Introduction and Clinical Manifestations of Hydatidiform Moles ...... 9

1.1.2 Epidemiology of Hydatidiform Moles...... 10

1.1.3 Karyotype and Genotype of Moles...... 11

1.1.4 Imprinting and DNA Methylation in Moles ...... 12

1.2 Identification of NLRP7 and Genotype Phenotype Correlations...... 16

1.2.1 Expression of NLRP7...... 18

1.2.2 The Known Role of NLRP7 ...... 20

1.2.3 and Pregnancies ...... 22

1.3 The NLR Family ...... 24

1.3.1 Activation: Mechanism of Action ...... 26

1.3.2 NLRP and Autoinflammatory Diseases...... 29

1.3.2.1 NLRP1...... 30

1.3.2.2 NLRP2...... 32

1.3.2.3 NLRP 3...... 33

1.3.2.4 NLRP5...... 35

1.3.2.5 NLRP12...... 36

2 CHAPTER 2...... 39

2. Materials and Methods ...... 39

2.1 Isolation of Full-length and Mutated NLRP7 cDNA...... 39

2.2 Site-Directed Mutagenesis...... 40

2.3 Subcloning of Wild-type NLRP7 cDNA ...... 42

2.4 Subcloning of Mutated NLRP7 cDNA ...... 43

2.5 Cell culture and Transient Transfection ...... 46

2.6 Enzyme-linked Immunosorbent Assay (ELISA)...... 47

2.7 Western Blot Analysis ...... 47

2.8 Statistical Analysis...... 48

CHAPTER 3...... 49

3. Results...... 49

3.1 Site-Directed Mutagenesis...... 49

3.2 Subcloning of Wild-type and Mutated NLRP7 cDNA ...... 51

3.3 Optimizing the Transfection Conditions ...... 51

3.4 NLRP7 Inhibits IL-1β Secretion in HEK293 cells ...... 53

CHAPTER 4...... 56

Discussion ...... 56

Conclusions and Future Perspectives ...... 58

References...... 60

APPENDIX A :Published Abstract and Presentations...... 67

APPENDIX B :Published Abstract ...... 67

APPENDIX C: Ethics Approval and Certificates ...... 69

3

ABSTRACT

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by the absence of, or abnormal, embryonic development and hydropic degeneration of the chorionic villi. In both Canada and the United States, the incidence of HM is 1 in every

1000 pregnancies. Recently, NLRP7 has been found to be responsible for recurrent hydatidiform moles (RHM) after the identification of various mutations in this . To investigate the functional consequences of NLRP7 mutations on IL-1β maturation and secretion, I used site-directed mutagenesis to introduce the various mutations found in patients with RHM into WT-NLRP7. HEK293 cell lines were cotransfected with WT-

NLRP7, procaspase-1, and pro-IL-1β, as well as with or without Cardinal (CARD-8.).

My results demonstrate that WT-NLRP7 inhibits IL-1β secretion in a dose-dependent manner. Furthermore, I found that Cardinal (CARD8), an important component of other , has no impact on the inhibitory effect of WT-NLRP7. My findings strengthen the idea that NLRP7 may function as a feedback regulator of IL-1β secretion.

4

RÉSUMÉ

Une môle hydatidiform (MH) est une anomalie de la grossesse caractérisée par soit l'absence de l’embryo ou du développement embryonnaire ainsi que la dégénérescence des villosités choriales. Au Canada et aux États-Unis, l'incidence des

MH est 1 sur 1000 naissances. Récemment, NLRP7 a été trouvé responsable des môles hydatidiform à répétition, après l'identification de différentes mutations dans le gène.

Pour étudier les conséquences fonctionnelles des mutations de NLRP7 sur la maturation et la sécrétion de IL-1β, j'ai utilisé le site de mutagenèse dirigée pour introduire les différentes mutations trouvées chez les patientes atteintes de môles recurrentes. Des cellules HEK293 ont été cotransfectées avec la copie normale de WT-NLRP7, procaspase-1, pro-IL-1β, avec et sans Cardinal (CARD-8.). Mes résultats démontrent que WT-NLRP7 inhibe la sécrétion de IL-1β d'une manière dose-dépendante. De plus, j'ai trouvé que Cardinal (CARD8), une composante importante d’autres inflammasomes, n'a pas d'effet sur l’inhibition de WT-NLRP7. Mes constatations renforcent l'idée que NLRP7 peut fonctionner comme un régulateur de la sécrétion de l'IL-1β.

5

LIST OF ABBREVIATIONS

BiHM: Biparental hydatidiform moles

CHM: Complete hydatidiform mole cDNA: Complementary DNA

ELISA: Enzyme-linked immunosorbent assay

FBS: Fetal bovine serum

HM: hydatidiform mole

HEK293: Human embryonic kidney 293 cell line

IL-1β: Interleukin -1β

LB media: Luria-Bertani media

NLRP: Nucleotide- binding domain, leucine rich repeat

PAMP: Pathogen-associated molecular pattern

PBS: phosphate buffered saline

PHM: Partial hydatidiform mole

RHM: Recurrent hydatidiform mole

WT: Wild-type

6

LIST OF FIGURES AND TABLES

Figure1. NLRP7 structure, domains, and positions of some mutations………………..…….19

Figure 2 NLRP3 inflammasome …………………………………………………………...... 28

Figure 3 Schematic representation of the protocol used to in molecular cloning…………....45

Figure 4. The amplification of NLRP7 cDNA using four different primers……...... 49

Figure5. Site directed mutagenesis showing the sequences of the various mutations…….... 50

Figure 6. Cloning in PcDNA3.1+………………………………………………………….... 51

Figure7. Optimizing the transfection conditions……………………………………………. 52

Figure 8. NLRP7 inhibits IL-1β secretion in HEK293 cells…...... 54

Figure 9. NLRP7 inhibits IL-1β secretion in the presence or absence of Cardinal………..... 55

Table 1. Methylation at imprinted genes in BiHMs ………………………………………... 15

Table 2. NLRP7 mutations identified in males with Normal reproduction ……………….... 18

Table 3. NLR subfamily and associated autoinflammatory human diseases……………...... 38

Table 4. Primers used to amplify NLRP7 …...……………………………………………… 39

Table 5. Primers designed for site-directed mutagenesis…………………………………… 41

Table 6. Primers used in PCR amplification to add a flag-tag to NLRP7…..……………..... 43

Table7. List of NLRP7 mutations and restriction enzymes used for cloning into flag WT-NLRP7 ………………………………………………………………………...44

7 ACKNOWLEDGEMENTS

First, I would like to extend my utmost thanks to my loving husband, Mohamed, who supported me throughout this entire endeavor. Without your encouragement and motivation, the completion of this thesis would never have been possible.

To my parents: your loving encouragement throughout the years away from home has been invaluable. Thank you for believing in me.

Lana, my sweetheart, thank you for letting mommy spend time away from home.

To my fellow colleagues Catherine Devault and Waffaa Chebaro: thank you. Your help and support throughout the past years has been unending. It was a pleasure working with you and I wish you both continued success in the future.

My heartfelt thanks must also be extended to David Lab’s, specifically Hiba Kazk, as well as Rosenblatt’s lab, specifically Lama Yamani. Their expertise and helpful suggestions were very much appreciated. Also, I would like to thank Dr. Saleh's Lab for teaching me how to perform cell cultures and transfection.

In addition, I would like to thank Qingling Duan for her teaching, Andrea Blotsky for her editing assistance, and Rabia Khan for her support throughout the difficult times.

To my scholarship sponsor, the Ministry of higher education in Saudi Arabia, and the Saudi Bureau in Ottawa, thank you for your generosity and unending support.

I would like to thank my supervisory committee Dr. Aimee Rayen, Dr. Patricia Tonin, and Dr. Maya Saleh for their feedback on my project.

Finally, I would like to thank my supervisor, Dr. Rima Slim, for providing me with the opportunity to work in her lab. Your feedback has enabled me grow within myself and has allowed me to develop the strength and determination to confront any future obstacles which I may face.

8 CHAPTER 1

1.1 Introduction and Clinical Manifestations of Hydatidiform Moles

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by the

absence of, or abnormal, embryonic development and hydropic degeneration of the

chorionic villi. The clinical manifestations of HM are vaginal bleeding in the first

trimester of the pregnancy, abnormal growth of the uterus, anemia, severe nausea and

vomiting, hyperthyroidism, and high levels of human chorionic gonadotropin (hCG)

(Harrisons, 2008).

In Western countries, HM diagnosis is performed using ultrasonography (US)

during the first trimester, which often reveals the absence of cardiac activity or fetal

pole and the presence of echogenic structures. This preliminary diagnosis is followed

by hormone serology, which, in case of high hCG levels, indicates an excessive

trophoblast proliferation and is suggestive of a molar pregnancy. Because of high hCG

and the absence of fetus, the product of conception is evacuated by suction curettage

and sent for histopathological examination. After evacuation, hCG levels are monitored

until their return to baseline level (i.e. pre-pregnancy level). Persistently high hCG is

often indicative of choriocarcinoma, whereas a low level or a rise in hCG after a

previous decline is indicative of persistent trophoblastic disease, often necessitating

chemotherapy.

Based on histopathology, HMs can be categorized as complete hydatidiform

moles (CHMs) or partial hydatidiform moles (PHMs). CHMs are characterized by

complete cystic degeneration of all villi as well as the absence of fetal membranes

(amnion, chorion), cord, and other embryonic tissues. In CHMs, all villi are enlarged

9 with cisternae, are avascular (with no fetal vessels), and are surrounded by multiple

areas of trophoblastic proliferation. In contrast, PHMs are characterized by moderate,

focal trophoblastic proliferation with a mixture of both hydropic and normal-appearing

villi (Garner et al, 2007). The trophoblastic proliferation within these moles is less

pronounced than in complete moles. PHMs may contain extra embryonic membranes

(amnion, chorion), cord, and occasionally well-preserved embryonic tissues or embryos

with morphological defects.

In many cases, it is difficult to divide moles into the two aforementioned

histological categories. A study conducted by Fukunaga et al (2005), shows a wide

interobserver and intraobserver variability in the histological diagnosis of hydatidiform

moles by five placental pathologists. This study shows an agreement amongst 4 or 5

pathologists on the same histopathological diagnosis (CHM, PHM, or hydropic

abortion) in only 30 of 50 cases examined. This difficulty is attributed to the fact that

the histopathological criteria used to divide moles were established 30 years ago at a

time when moles were evacuated at a later gestational stage (around 12 weeks of

gestation) (Szulman et al, 1978). Today, with the availability of ultrasound in most

gynecology clinics, moles are evacuated at around 8 weeks of gestation based on the

absence of embryonic pole or fetal heart activity (Fukunaga et al., 2005).

1.1.2 Epidemiology of Hydatidiform Moles

In both Canada and the US, HM occurs in 1 in every 1500 pregnancies.

Unfortunately, a 2-10 times higher incidence is found in developing and

underdeveloped countries from Latin America, the Middle East and the Far East. The

common form of molar pregnancies is sporadic and not recurrent. It affects one woman

10 per family and in only one of her pregnancies. Nevertheless, moles have been found to

recur in up to 5% of women with one mole. After a prior mole, the risk of having a

second mole is 5-40 times greater than that of women from the general population.

When other forms of reproductive wastage such as spontaneous abortions and stillbirths

are included, it is found that 10-25% of women with one HM experience a second

reproductive loss. In rare cases, it has been observed that more than one woman from

the same family have RHMs (familial moles). In patients with familial and non-familial

RHM, moles usually alternate with other forms of reproductive wastage, mainly

spontaneous abortions (Garner et al, 2007).

1.1.3 Karyotype and Genotype of Moles

Studies have shown that eighty percent of sporadic complete moles are diploid

and androgenetic (Kajii and Ohama, 1977; Kovacs et al., 1991). With respect to

recurrent HMs (RHMs) with no family history, most analyzed cases were found to be

biparental, though a few cases of androgenetic RHMs have been reported (Zhao et al,

2006). In the familial cases of HM reported thus far, genotyping has demonstrated both

maternal and paternal contributions to their chromosomal make-up (Fisher et al, 2004).

PHMs are mostly found to be triploid, having two copies from the paternal genome and

one from the maternal. Although the majority of analyzed molar tissues from patients

with NLRP7 mutations have been found to be diploid and biparental, in a recent study

conducted by our lab, different genotypic types of moles were found in patients carrying

NLRP7 mutations: diploid biparental, diploid androgenetic, triploid moles and tetraploid

conceptions (Deveault et al, 2009).

11 1.1.4 Imprinting and DNA Methylation in Moles

Genomic imprinting is a phenomenon, where only one parental allele of a gene

is transcribed. The phenomenon of genetic imprinting is regulated by complex

mechanisms, of which the most widely studied is DNA methylation. Imprinted genes

are thought to have a very important role in prenatal growth and development

(Charalambous et al, 2007).

Imprinted genes are generally grouped in clusters and their expression is

controlled by upstream elements known as differentially methylated regions (DMRs).

These regions are regulated by imprinting control centers (Reik et al, 2001). When a

gene is paternally expressed, it is said to be “maternally imprinted”, whereas if it is

maternally expressed, it is thus said to be “paternally imprinted” The inactive copy of

the gene is hypermethylated on its DMR, resulting in gene silencing, while the copy

which is not methylated remains active and is transcribed. In somatic cells, the pattern

of imprinting is maintained throughout each round of DNA replication (Reik et al,

2001).

The fact that maternal and paternal genomes do not contribute equally to

embryonic development was shown by early pronuclear transplant experiments in mice

engineered to create androgenetic and gynogenetic embryos (Surani et al, 1984). These

experiments showed that androgenetic embryos had excessive proliferation of the

trophoblast, as well as increased rates of embryonic death, endpoints also observed in

the development of human androgenetic moles. Thus, proper genomic imprinting is

believed to be essential for proper embryonic development (Li et al, 2002).

As was already established in earlier studies, most sporadic moles are

androgenetic (Kajii and Ohama, 1977; Wake et al, 1978). However, additional studies

12 have also indicated that familial moles are diploid and biparental (Helwani et al, 1999).

Since both types of moles (androgenetic and biparental) are phenotypically identical, it was suggested that the causative defective gene for familial diploid biparental moles regulates the expression of several imprinted maternally expressed genes. To address this hypothesis, Judson et al., analyzed the DNA methylation of 9 imprinted DMRs in one biparental molar tissue from a familial case. Out of the 9 imprinted DMRs, seven are maternally methylated whereas two are paternally methylated (Judson et al, 2002).

This group demonstrated that six of the seven maternally methylated DMRs showed abnormal loss of methylation, with both copies showing a paternal methylation pattern.

The authors observed that not all the paternally methylated DMRs behaved similarly: one had a normal methylation level and another was hypermethylated, thus leading them to conclude that BiCHM are caused by a defect of imprinting marks or failed imprinting in the female germline.

Similarly, our laboratory analyzed the methylation of four DMRs in 2 biparental complete moles from two sisters (El-Maarri et al, 2003, 2004), two maternally methylated and two paternally methylated. The studies showed that the paternally expressed genes had a lower level of methylation, while the maternally expressed genes showed higher levels of methylation when compared to DNA from normal chorionic villi and total blood. This work confirmed similar trends of abnormal methylation reflecting what was previously reported by Judson and suggesting that some maternal alleles are assuming paternal methylation patterns.

Further studies to trace the grandparental origin of the abnormal gain of DNA methylation in moles showed that the gain of methylation occurred on maternal alleles that are not methylated in patients’ blood. These findings indicated that the abnormal gain

13 of methylation in molar tissues is acquired de novo, either in the maternal germline or during the postzygotic development and the proliferation of the moles.

Through the analysis of other CpG regions, satellite sequences, promoters of silenced genes on the inactive the X-, and promoters of three cancer related genes known to be abnormally hypermethylated in sporadic HMs, as well as through the examination of thirteen CpG-rich regions scattered in a 762.5 kb region around the PEG3

DMR, our laboratory demonstrated abnormal DNA methylation at all analyzed CpG regions in two biparental molar tissues (Djuric et al, 2006). Recently, two studies have analyzed the methylation of DMRs in several BiHMs conceptions in unrelated patients.

In most cases, multilocus maternal imprinting defects were present, while the paternal

H19 was unaffected (Hayward et al, 2009; Kou et al, 2008). A Summary of data describing the methylation of imprinted genes in BiHMs is provided in Table 1.

14

Table 1. Methylation of imprinted genes in BiHMs

Methylation pattern Maternally 1 BiCHM (Judson 2 BiCHMs from 2 BiCHMs 4 BiCHMs, 2 from the Methylated st et al, 2002) 2 sisters from 2 same patient in the 1 (El-Maarri et al, unrelated column and 2 from 2003) patients unrelated patients (Kou et al, (Hayward et al, 2009) 2008) KCNQ1OT1 Unmethylated n.a. Unmethylated Unmethylated

SNRPN Unmethylated Unmethylated Unmethylated n.a.

PEG1 Unmethylated n.a. n.a. n.a. PEG3 Unmethylated Unmethylated Unmethylated n.a.

GNAS-1A Unmethylated n.a. Absent n.a. methylation GNAS-xLα5 Normal n.a. Normal n.a. methylation. methylation GNAS-AS Unmethylated n.a. Normal n.a. methylation Zac1 n.a. n.a. n.a. Unmethylated

PEG10 n.a. n.a. n.a. 1 of 4 only showed little loss of methylation (no methylation defect) Paternal Methylated GNAS- Increased Increased Completely Acquisition of NESP55 methylation methylation in 2 methylated methylation on maternal BiCHMs and also in allele normal term placenta H19 Normal Increased Normal n.a. methylation methylation in 2 methylation BiCHMs

15 1.2 Identification of NLRP7 and Genotype Phenotype Correlations

The candidate region responsible for HM was mapped by Moglabey et al. after

linkage analysis of families with recurrent HM identified a region on chromosome

19q13.3-13.4 (Moglabey et al, 1999). Later studies from several groups narrowed down

the candidate region on (Sensi et al, 2000; Hodges et al, 2003) and led to

the identification of the causative mutations in NALP7 (now NLRP7) by our laboratory

(Murdoch et al, 2006). Thus, NLRP7 became the first maternal effect gene identified in

and responsible for RHM.

To date, approximately 38 mutations in NLRP7 causing RHM have been identified

including missense, stop codon, and splice site mutations. These mutations were found in

families from different populations including French, Italian, German, Moroccan,

Mexican, Egyptian, Lebanese, Pakistani, Indian, and Chinese (INFEVERS,

http://fmf.igh.cnrs.fr/ISSAID/infevers/). Recently, several other novel mutations were

identified including several protein-truncating mutations and small and large

rearrangements causing a spectrum of phenotypes including RHM, stillbirths, and

spontaneous abortions (Kou et al., 2008; Wang et al, 2009; Hayward et al., 2009).

Genotype-phenotype correlation studies conducted by our laboratory indicated that

the most severe phenotype of this pathology is the RHM, while the milder phenotype is

the other forms of reproductive wastage such as stillbirths and spontaneous abortions

(Qian et al, 2007). Qian et al. suggested that heterozygous women carrying a single

in NLRP7 are at increased risk for stillbirths and other forms of reproductive

wastage. However, Hayward et al. (2009) did not confirm the predisposition of

heterozygous female to pregnancy loss. Contrarily, this group found that the mothers of

affected women were highly fertile and had had several normal pregnancies, which is

16 also what we observed in the families studied by our laboratory despite the fact that some carriers had had reproductive wastage. The susceptibility of women with a single NLRP7 mutation to reproductive wastage remains to be investigated in future studies.

Though NLRP7 mutations have been linked to RHM in the female gender, current observations indicate that NLRP7 mutations do not affect male reproduction. Qian et al

(2007) reported 2 mutations in NLRP7 in two affected sisters and their brother with normal reproductive outcomes. In the same study, another male, homozygous for

Arg693Pro, was also shown to have normal reproductive outcome. These observations were also confirmed recently by another group (Wang et al, 2009) and in additional families from our laboratory (Slim et al, in press). Presently, these males have not reported any reproductive problems, with each of them having between one to three children (Table 2). It remains unclear whether those with fewer children have made personal choices to restrict their family size or whether they are actually affected by a form of subclinical infertility, thus reducing their ease of conception. Unfortunately, spermograms have not been performed to further assess their fertility. Of note that the highest level of NLRP7 RNA is observed in testis Additional research needs to be conducted in this field in order to investigate possible implications of NLRP7 in male infertility.

17

Table 2. NLRP7 mutations identified in males with normal children

Wang et al., Slim et al, in Qian et al, 2007 2009 press

p.Glu99X p.Arg693Pro p.R693P p.Arg693Pro Mutation p.Asp657Val homozygous homozygous p.Asn913Ser

Number of 1 1 1 1 Males examined

Number of 1 3 1 1 Children

1.2.1 Expression of NLRP7

NLRP7 is transcribed in many normal human tissues and cell lines of

hematopoietic and non-hematopoietic origins (Kinoshita et al, 2005). Our laboratory

documented the presence of NLRP7 transcripts in normal human uterus, endometrium,

fallopian tubes, ovaries, denuded oocytes at the germinal vesicle and metaphase I stages,

and early cleavage embryos (Murdoch et al, 2006 and unpublished data). In a recent

study, Zhang et al (2008) investigated the presence of different NLRP gene transcripts by

Real-Time PCR in normal and abnormal human oocytes and in pre-implantation embryos

at different developmental stages. In this analysis, NLRP7 showed a different expression

pattern than any other NLRP. NLRP7 transcripts were lowest at day 3 of normal pre-

implantation embryos, but highest by day 5. Interestingly, the opposite was observed in

arrested embryos where the highest levels of NLRP7 transcripts were observed in day 3

and the lowest at day 5 (Zhang et al, 2008).

18 NLRP7 (nucleotide binding oligomerization domain, leucine-rich-repeat family, containing 7), also known as (NALP7, PYPAF3 and NOD12) is a cytoplasmic protein consisting of 3 main domains: a pyrin domain (PYD) which is known to be involved in apoptotic and inflammatory signaling pathway, a central nucleotide-binding domain (NACHT) which promotes oligmerization, and a leucine rich repeats (LRRs) domain known to be involved in protein-protein interaction and ligand sensing (Kobe et al, 1995; Proell et al, 2008 ). NLRP7 is one of 14 NLRP , a subfamily of the NLR family protein family implicated in auto- inflammation, , and intracellular regulation of pathogen-induced inflammation.

Figure1. NLRP7 structure, domains, and some of the mutations reported by our laboratory at the time when I started my project.

19 1.2.2 The Known Role of NLRP7

The role of NLRP7 was elucidated through in vitro studies conducted in Japan, where HEK293 cells were transfected with a plasmid containining pro-IL-1β, pro-

Caspase-1 together with NLRP7. It was found that an increase in the levels of transfected NLRP7 decreased the secretion of IL-1β. Moreover, it was shown that

NLRP7 co-precipitates with -1 and IL-1β, indicating their direct or indirect physical interactions (Kinoshita et al, 2005). In another study, where cDNA microarrays were used to isolate novel molecular targets for treatment of testicular germ cell tumors, significantly elevated expression of splice isoform (NLRP7-V1) was found in testicular seminoma tumors compared to normal testis, thus indicating that

NLRP7 plays a role in cell proliferation and testicular tumorogenesis (Okada et al,

2004).

The exact role of NLRP7 in the pathology of RHM remains unknown. As a maternal defective gene, NLRP7 could cause RHMs by leading to defective oocytes or by creating an abnormal maternal environment hostile to early embryonic development and implantation.

NLRP7 has been suggested to be a maternal effect gene. Maternal effect genes are defined as genes whose products are required in oocytes to support early embryonic development until the activation of the embryonic genome. Such genes are not believed to affect ovulation and fertilization, but their absence could lead to early embryonic arrest. As NLRP7 is expressed in unfertilized oocytes, the potential role of NLRP7 in oogenesis would explain the absence of maternally methylated marks on paternally expressed genes analyzed in molar tissues from patients with a NLRP7 mutation.

20 In addition, NLRP7 is transcribed in the human endometrium, and has been shown to play a role in IL-1β secretion, a cytokine involved in decidualization and trophoblast invasion (Strakova et al, 2002). The expression of NLRP7 has been examined in human endometrial cancer tissue. It was concluded that there is a correlation between increased NLRP7 expression and the depth of endometrial tumor invasion (Ohno et al, 2008). This finding underlines the need for appropriate NLRP7 expression in preventing endometrial pathogenesis. Moreover, a study showed that

NLRP7 interacts strongly with Fas-associated factor 1 (FAF1), a component of the death-inducing signaling complex, whose high expression has been shown to induce apoptosis in certain cell types (Kinoshita et al, 2006).

In a recent study from our laboratory, early cleavage abnormalities were found in embryos from two unrelated patients with different NLRP7 mutations. In one patient, in-vitro fertilized embryos displayed a higher rate of mosaic embryos with haploid blastomeres for more than 3 (haploid-diploid and haploid-aneuploid) as compared to embryos from patients undergoing IVF for various medical reasons

(12.5% versus 3%). In a second patient, all of her in-vitro fertilized embryos by intra- cytoplasmic sperm injection (ICSI) fragmented and degenerated by the 6-cell stage, resulting in no embryos suitable for transfer (Deveault et al, 2009).

Another study in our lab was performed to assess ex vivo cytokine production by mononuclear blood cells from different patients with NLRP7 mutations in response to various stimuli. The results showed lower IL-1β and TNFα secretion in cells from patients with NLRP7 mutations as compared to controls after stimulated with LPS or other (PAMPs). This finding was consistent among different patients from the same family and among patients from two families with different NLRP7 mutations

(unpublished data). 21 Our lab proposed a two-hit mechanism at the basis of androgenetic mole generation. This mechanism consists of variable degrees of early embryo cleavage abnormalities, leading to the development of chaotic mosaic aneuploidies with haploid, diploid, triploid and tetrapolid blastomeres. Embryonic cells that survive and reach implantation are then subjected to the maternal immune response. As a result of these patients' impaired inflammatory response, androgenetic cells (complete allografts) are able to grow and proliferate (Deveault et al, 2009).

Additional studies are required to elucidate more about the precise mechanisms of NLRP7 function. The objective of my project was to assess in vitro the effect of

NLRP7 mutations and variants on IL-1β secretion and caspase-1 cleavage and therefore develop an in-vitro assay that will allow us to test for the effect of any variant in the gene.

1.2.3 Inflammation and Pregnancies

Inflammation, the basic process by which the human body responds to injury, was first described by Celsus and Galen in the 2nd century, who characterized the phenomenon as a manifestation of calor (warmth), dolor (pain), rubor (erythema or redness), tumor (swelling), and function laesa (loss of function) (Cone, 2001). Today, these five cardinal clinical signs are still used to describe inflammation. It is now known that these physical findings are due to the action of chemokines, cytokines, and other inflammatory mediators on local blood vessels and tissues.

Inflammation is central to reproductive success, as the processes of ovulation, menstruation, implantation and parturition are defined as true inflammatory events. For example, during ovulation, extracellular matrix (ECM) degradation and ovarian

22 architecture rearrangement involve vascular and inflammatory changes modulated by the expression of chemokines, cell adhesion molecules and integrins, as well as the recruitment of leukocytes. Localized inflammation is activated during menstruation, with shifts observed in the percentages of macrophages, dendritic cells, and mast cells present in the endometrium during the follicular phase. In addition, implantation is associated with inflammatory changes designed to assist with tissue remodeling, and, both the molecular and cellular components of inflammation have been linked to myometrial activation, cervical ripening and membrane activation during the process of parturition

(Romero et al, 2007; Weiss et al, 2009).

During a normal human pregnancy, the uterus is a sea of inflammatory cells

(cytokines and leukocytes). In order to protect the foreign, semi-allogenic embryo from destruction in the maternal environment, a series of elaborate mechanisms have evolved to down-regulate the maternal immune system. The theory of maintaining a fine balance between maternal immunostimulation and immunosuppression was first proposed by

Medawar in 1953. Medawar suggested that the fetus does not habitually provoke an immunological reaction from its mother for the following three reasons: 1) the anatomical separation of fetus from mother; 2) the antigenic immaturity of the fetus; and 3) the immunological indolence or inertness of the mother. Concerning maternal immunological inertness, it is now believed that a successful pregnancy may depend upon a Th2-biased cytokine profile. As originally proposed by Wegmann et al. (1993), pregnancy was regarded as requiring the dominance of Th2-regulated cytokines, particularly interleukin-

10, and also granulocyte-macrophage colony-stimulating factor, for placental and fetal growth control. Failure to achieve or to maintain the appropriate Th1-Th2 balance has been suggested to be an underlying cause of pregnancy loss (Raghupathy, 1997).

23 Aberrant maternal immune system functioning has also been recognized as a

mechanism behind pregnancy complications such as hydatidiform mole (HM). As

opposed to being semi-allogenic cell structures, HMs are complete allografts

(androgenetic). Studies have suggested that HM development is secondary to inadequate

fetal antigenic stimulation, due to HLA antigen sharing between patients with HMs and

their partners (Couillin et al, 1987). In addition, other studies have demonstrated that

patients with HMs have a weak cellular-mediated immunity in response to various

mitogens, further suggesting the presence of a unifying immunological deficit underlying

abnormal pregnancies (Sasaki et al, 1981).

Many studies have been conducted to investigate the involvement of HM and

various cytokines present in the chorionic villi of the molar tissue. Two cytokines, IL-1β

and TGF-B were found to be associated with the progression of HM into

choriocarcinoma (Balaram et al, 1999). In addition, Pang et al. (2003) used RT-PCR to

detect the expression of transforming growth factor TGF-B in CHM and normal first-

trimester villi. His studies demonstrated that the expression of TGF-B was notably higher

when compared to normal first-trimester villi. These findings led to the conclusion that

the change of TGF-B expression might be involved in the development of trophoblastic

diseases of pregnancies (Pang et al, 2003).

1.3 The NLR Family

The NLR family, also known as NOD/NACHT/ pyrin (NLR) or CATERPILLER

protein family, is a group of proteins important for regulating the inflammation and

innate immune responses (Zhengmao, 2008). HUGO Committee

approved the use of the common nomenclature for the above-mentioned family as the

24 NLR or NBD-LRR containing family (Wilmanski et al, 2008). In the past few years, bioinformatic analysis identified the existence of 22 genes of NOD-like receptors (NLRs) in the (Harton et al, 2002).

The NLR family members have similar structures characterized by three main domains: NACHT, LRRs, and an effector domain. The effector domain can be either a pyrin domain (PYD), a caspase recruitment domain (CARD) or a baculovirus inhibitor of protein apoptosis repeat domain (Bir).

The NLR proteins can be divided into two large subfamilies: The NOD subfamily composed of 5 members and the NLRPs subfamily, composed of 14 NLRPs. In addition,

CIITA, IPAF, and BIR-containing NAIP are members of the NLR family, but have not been classified into either the NLRP or NOD subfamily (Fritz et al, 2006; Mathews et al,

2008). Several studies demonstrated that NLRs are expressed in many cell types including immune cells and epithelial cells. A few members are also expressed in phagocytes, including macrophages and (Franchi et al, 2009).

To date, the mechanisms underlying NLR signaling are not completely understood. It is known, however, that NLRs are normally present in the cytoplasm in an inactive, self-repressed form. It has been suggested that the LRR domain folds back onto the NACHT domain and inhibits the oligomerization and activation of NLR proteins (Carneiro et al, 2008). Upon activation of NLR proteins through direct or indirect binding to the LRR domain, NLR undergoes a conformational rearrangement, thereby exposing the NACHT domain and permitting oligomerization and exposure of the NLR effector N-terminal domain. Once activated, NLR mediate pro-inflammatory signaling via two distinct pathways: 1) activation of the caspase-1 inflammasome and

2) activation of the transcription factor NF-κB (Martinon et al, 2005; Carneiro et al,

2008, Franchi et al, 2009). 25 The importance of the NLR family is further illustrated by the role of its

subfamily NLRP, which are genes involved in the regulation of Caspase-1 activation

and IL-1β processing. The mechanisms that control IL-1β maturation were discovered

with the help of an in-vitro assay that monitors the processing of pro-IL-1β after

incubation with extracts from a human monocytic cell line (Kostura et al, 1989).

Further studies have also demonstrated the association of many members of the NLRP

family with various inflammatory disorders described in Table 1 (for a complete

review, please see Ting et al., 2008).

In addition to their role in IL-1β processing, several NLRs have been found to

exert a regulatory effect on the NF-κB activation pathway (Zhengmao et al, 2008).

Many studies have also demonstrated that various members of the NLR family can

detect bacterial molecules through mechanisms that have yet to be characterized.

Moreover, studies have indicated that many NLRs are necessary sensors of specific

PAMPs (pathogen-associated molecular patterns) (Fritz et al, 2006).

1.3.1 Inflammasome Activation: Mechanism of Action

The challenges that face the innate immune system rely on its ability to detect

any pathogenic microbes as foreign and to eliminate them, in hopes of eliminating the

risks of disease development.

A highly important arm of the innate immune system is a caspase-activating

multi-protein complex known as the inflammasome, which promotes the maturation of

the inflammatory cytokines IL-1β and IL-18 (Dinarello 1998; Martinon et al, 2002). To

date the inflammasomes of three NLRs - NLRP1, NLRP3, and IPAF - have been

characterized. For example, the NLRP1 inflammasome is comprised of NLRP1, ASC,

26 caspase-1 and caspase-5, while the NLRP3 inflammasome contains in addition to

NLRP3, Cardinal, ASC and caspase-1 (Martinon and Tschop, 2005). The third inflammasome is composed of IPAF and caspase-1 (Ting and Davis 2005).

As an example of inflammasome assembly, the activation of the NLRP3 inflammasome begins with the recognition of different stimuli such as bacterial RNA,

ATP, and uric acid crystals through the LRR domain. Upon stimulation, NLRP3 binds through its pyrin domain (PYD) to the adaptor protein, ASC. The CARD domain within

ASC binds to Caspase-1 to form an active inflammasome complex, which in turn activates IL-1β through the cleavage of pro-IL-1β (Martinon et al, 2007; Ferrero-

Miliani et al, 2006) (Figure 2).

IL-1β is a pro-inflammatory cytokine, also defined as an alarm cytokine, which has a role in the innate immune response, inflammation, and in the defense against pathogens. IL-1β is produced as an inactive pro-IL-1β by immune cells such as macrophages, and dendritic cells (Martinon et al, 2007).

27

Figure 2: NLRP3 inflammasome The components of NLRP3 inflammasome include ASC (apoptosis speck –like protein containing a caspase recruitment domain (CARD), Cardinal, and procaspase-1 upon activation through the interaction with LRR domain, the assembly of the domains leads to the release of active caspase-1 which in turn activate IL-1β through the cleavage of pro-IL-1β. The figure was adapted from CHURCH et al, 2008.

28

1.3.2 NLRP Genes and Autoinflammatory Diseases

An understanding of the differences between autoimmune and autoinflammatory

disorders is crucial for understanding the inflammatory pathways mediating the

development of these diseases. In essence, the main difference between

autoinflammatory and autoimmune disorders is that neither autoantigens nor

autoantibodies are involved in the pathogenesis of autoinflammatory diseases. Thus,

episodes of inflammation occur spontaneously without any detectable cause.

The autoinflammatory disease concept was developed in 1999 by Kastner et al.

while studying hereditary periodic fever syndromes. Many of the hereditary periodic

fever syndromes are attributable to mutations in specific genes involved in innate

immunity. These mutations alter signaling pathways or cytokine activation pathways,

as well as the maturation of certain proinflammatory cytokines, such as IL-1β.

NLR has been linked to the development of autoinflammatory diseases. The NLR-

related disorders include various autoinflammatory diseases, such as the

aforementioned NLRP3 associated periodic fever syndromes, Crohn’s disease, gout and

pseudogout. The activation of NLR proteins leads to inflammatory responses, which are

mediated by NF-kB, Caspase-1, and cytokine IL-1β activation (Wilmanski et al., 2008).

We now know that these proteins play critical roles in maintaining host-pathogen

interactions and inflammatory responses.

Unlike the classical autoimmune diseases where immunopathogenesis is played

out primarily in lymphoid organs, NLR-related disorders occur primarily in tissues

where inflammation arises. It is believed that tissue-specific factors in the target organs

themselves contribute to autoinflammatory disease expression, resulting in each

disease’s predominant pattern of organ attack (ie: Crohn’s disease and the

29 gastrointestinal tract, gout and the large joints). Additional studies suggest that these

proteins are actively involved in the secretion of inflammatory cytokines, thus aiding

the regulation of innate immunity (Ting at el, 2006).

There are over 20 NLR proteins in humans, which have evolved to acquire

specificity to various pathogenic microorganisms. Various genetic studies have linked

autoinflammatory disorders to different members of the NLR protein family (NLRPs

and NODs). For example, mutations in NOD2 are associated with Blau syndrome, a

rare autosomal dominant disorder characterized by granulomatous arthritis, uveitis, and

skin diseases (Miceli-Richard et al, 2001). The full spectrum of diseases associated

with mutations in NLRP protein family members, NLRP1, NLRP2, NLRP3, NLRP5,

and NLRP12, will be further discussed in the following sections.

1.3.2.1 NLRP1

NLRP1 was the first NLRP protein to be identified on the basis of its sequence

homology to apoptotic protease-activating factor-1 (APAF-1) (Martinon et al, 2001).

The highest level of NLRP1 transcripts are found in immune cells, particularly in

Langerhans and T cells, which emphasizes their role in innate immunity (Kummer et al,

2007). It is thought that NLRP1 recognizes PAMPs using pathogen-recognition

receptors (PRRs) such as Toll-like receptors (TLRs), thereby stimulating the assembly

of the NLRP1 inflammasome, which initiates an inflammatory response and possibly

apoptosis (Faustin et al, 2007).

The link between NLRP1 and autoimmune disease was established by Jin et al.

(2007) through his study of vitiligo and related autoinflammatory disorders. Vitiligo, an

autoimmune disorder, is characterized by development of white patches of skin,

30 mucosa, and overlying hair due to the loss of melanocytes from the involved areas

(Fain et al, 2003). In his initial study, Jin et al., reported genetic linkage between a locus on chromosome 17q13 (linkage peak) and multiple autoimmune diseases associated with vitiligo, a result obtained via genotyping microsatellite markers across the genome in 51 extended families. It was deduced that the region likely containing the gene causing vitiligo spanned a region containing ~ 80 known or predicted genes. To determine which of 80 genes were most likely to contribute to vitiligo development, Jin et al. genotyped the families for the 177 SNPs spanning the previously identified linkage peak. Using family-based association analysis, it was shown that 23 SNPs were associated with the vitiligo phenotype or with an expanded autoimmune and autoinflammatory disease phenotype. Three SNPs were found in the coding region and the extended promoter region of NLRP1. Thus, this study demonstrated that DNA sequence variants in the NLRP1 region are associated with the risk of several autoimmune and autoinflammatory diseases, including vitiligo, implicating the innate immune system in the pathogenesis of autoinflammatory disorders.

Apart from vitiligo, other autoimmune diseases have been shown to implicate

NLRP1. In a study conducted by Magitta et al. in 2009, large patient cohorts from six different autoimmune diseases (1) Addison's disease, 2) type 1 diabetes, 3) multiple sclerosis, 4) rheumatoid arthritis, 5) systemic lupus erythromatosis and 6) juvenile idiopathic arthritis) were analyzed for SNPs in NLRP1. Major alleles of the coding SNP rs12150220 revealed significant association with all of the aforementioned diseases except for juvenile idiopathic arthritis. This result indicates that NLRP1 and the innate immune system may be involved in the development of autoimmune disorders, particularly organ-specific autoimmune diseases (Kummer et al, 2007).

31 1.3.2.2 NLRP2

NLRP2 is presently recognized as an important mediator in autoimmune

inflammatory disorders (Agostini et al, 2004), and is known to be involved in protein

complexes that activate proinflammatory (Tschopp et al, 2003). It was

proposed to function as modulator of the activation of NF-κB and procaspase-1 in

macrophages (Bruey et al, 2004).

A recent study demonstrated that a NALP2 defect in women is responsible for

Beckwith-Wiedemann syndrome (BWS) in her offspring. BWS, is an imprinted

congenital overgrowth syndrome characterized by prenatal and postnatal overgrowth,

macroglossia, anterior abdominal wall defects, organomegaly, hemihypertrophy,

neonatal hypoglycemia, urogential abnormalities and urogenital tumors (ie: Wilm’s

tumor) (Meyer et al, 2009). BWS is caused by mutations or altered expression of linked

genes in the 11p15.5 imprinted chromosomal region. Those genes include IGF2,

KCNQ1OT1 (LIT1), which are paternally expressed, and H19 and CDKN1C, which are

maternally expressed. It is known that the IGF2 gene product is prenatal growth factor

and that the CDKN1C protein is a candidate tumour suppressor that negatively

regulates the cell cycle (Grandjean et al, 2000). However, 50% of the BWS cases are

sporadic and caused by epigenetic defects involving loss of methylation at DMR2.

While 2-7% of the epigenetic subgroup involved gain of methylation at DMR1, 20%

involved paternal uniparental disomy (UPD) (Weksberg et al, 2005).

Further research into the methylation defects underlying BWS have been

conducted by Meyer et al. (2009). Using autozygosity mapping, Meyer et al., identified

an extended homozygous region on chromosome19q13.4 (containing NLRP2 and

NLRP7 genes) in the mother of children affected with BWS (2009). Using a positional-

32 candidate gene approach, Meyer et al. found that the mother of BWS children was

homozygous for a frameshift mutation in exon 6 of NLRP2. The mutation was not

detected in 542 ethnically matched controls. In this family, one affected child was

homozygous for the mutation and the other two, one affected and one unaffected, were

heterozygous. Surprisingly, methylation analysis showed one of the affected siblings

(and all controls) had normal methylation levels, while the other affected child

demonstrated partial loss of methylation. It is suggested that the loss of methylation

secondary to NLRP2 mutations in the mother may be associated with the incomplete

failure of imprinting establishment and/or a partial failure of maintenance methylation,

leading to BWS in offspring. While germline mutations in NLRP7 have previously been

associated with familial hydatidiform mole, this was the first description of NLRP2

mutation in human disease and the first report of a trans mechanism for disordered

imprinting in BWS.

1.3.2.3 NLRP 3

The most extensively studied member of the NLRP family is NLRP3.

NLRP3, also known as cryopryin, has been shown to be a pivotal component of the

inflammasome signaling platform by exerting a role in the regulation of caspase-1 and

IL-1β maturation (Ting at el, 2006). NLRP3 was identified by a positional cloning

approach during the search for the gene responsible for two autosomal dominant

autoinflammatory diseases: familial cold autoinflammatory syndrome (FCAS) and

Muckle–Wells syndrome (MWS) (Hoffman et al, 2001). In addition, more recently,

NLRP3 has been associated with a group of rare autosomal dominant diseases known as

CAPS: Cryopyrin Associated Periodic Syndromes (Mathews et al, 2008). Although

CAPS disorder are classified individually, they have overlapping symptoms

33 characterized by fevers, urticarial skin rashes, varying degrees of arthragias/arthritis, -mediated inflammation, and hyper-activation of the acute-phase response

(Mathews et al, 2008 ).

To date, more than 20 mutations affecting NLRP3 have been identified (Ferrero-

Miliani et al, 2006), many of which have been found to contribute to the development of the CAPS disorders. These syndromes are caused by gain-of-function (GOF) mutations sharing a common mechanism. Functional studies on macrophages from patients with Muckle-Wells syndrome have revealed a constitutive increase in the secretion of IL-1β and IL-18 without stimulation, suggesting that mutations in NLRP3 increase production of these proinflammatory cytokines and promote inflammation

(Agostini et al, 2004; Inokuchi et al, 2006).

The role of NLRP3 in inflammatory responses has also been illustrated via gene knock-out models. Kanneganti et al. (2006) investigated the role of NLRP3 in caspase-1-dependent IL-1β secretion via generation of Nlrp3-deficient mice by homologous recombination using a targeting construct to replace exons I and II of the

NLRP3 gene (Cias1). Deletion of Nlrp3 confirmed its requirement for Caspase-1 mediated IL-1ß secretion in response to various stimuli such as Staphylococcus aureus,

Listeria monocytogenes, bacterial RNA, LPS, as well as viral products. Secretion of IL-

1β and IL-18 was induced by Escherichia coli RNA in wild-type and Cias -/+ macrophages, but was abolished in Cias -/- macrophages, thus proving that Nlrp3 is essential for activation of caspase-1 in response to bacterial and viral stimuli.

The role of Nlrp3 was further elucidated by Mariathasan et al. (2006). Using knock-out (KO) mice, Mariathasan demonstrated that Nlrp3-deficient macrophages cannot activate caspase-1 in response to Toll-like receptor agonists and ATP.

In addition, the release of IL-1β in response to nigericin (a potassium ionophore) and 34 maitotoxin (a potent marine toxin) was also found to be dependent on NLRP3.

Macrophages exposed to Staphylococcus aureus or Listeria monocytogenes required

both ASC and NLRP3 to activate caspase-1 and secrete IL-1β. These studies thus

demonstrated that NLRP3 is essential for inflammasome activation in response to

signaling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L.

monocytogenes.

1.3.2.4 NLRP5

NLRP5, originally named MATER (maternal antigen that embryos require) is an

oocyte-specific maternal-effect gene required for early embryonic development.

MATER is expressed in germ cells during oogenesis as well as during early cleavage

development. In mice, oocytes from knockout female mice (Nlrp5-/-) were found to

fertilize normally, but their embryos fail to develop after the two-cell stage. This

finding led the authors to suggest that NLRP5 plays a role in activating the zygotic

genome (Tong et al, 2000). In addition, Chiharu et al. demonstrated that MATER

functions as an anti-apoptotic protein (2005). Disruption of the MATER gene leads to

altered mitochondrial function and accelerated ovarian aging. The analysis of ovulated

oocytes lacking MATER revealed a significant increase in the mitochondrial membrane

potential (the voltage difference between the interior and the exterior of the

mitochondrion) and an increased accumulation of reactive oxygen species (ROS) with

decreased glutathione content (Chiharu et al, 2005). These results thus demonstrate that

MATER decreases mitochondrial membrane potential and increases ROS production,

leading to DNA damage and concomitant inflammation. Furthermore, it has been

35 suggested that oocyte quality is impaired in MATER-null females, leading to failure of

early embryo development via alteration of mitochondrial activity.

In humans, NLRP5 is predominantly expressed in the cytoplasm of parathyroid

chief cells, but also minimally expressed in the ovary, the brain, the placenta, the

pancreas, and the spleen. NLRP5 expression is found to be up-regulated in parathyroid

cells subjected to increasing calcium concentrations (Alimohammadi et al., 2008). It is

thus believed that NLRP5 may be involved in the process of calcium sensing or

homeostasis in parathyroid chief cells.

NLRP5 has been correlated with the development of Autoimmune polyendocrine

syndrome type 1 (APS-1), an uncommon autoimmune disorder caused by mutations of

the autoimmune regulator gene (AIRE). APS-1 is frequently identified by the clinical

triad of hypoparathyroidism, mucocutaneous candidiasis, and Addison's disease.

Alimohammadi et al. (2008) has demonstrated that the presence of NLRP5-specific

autoantibodies in 49% of the patients with APS-1 and hypoparathyroidism. In addition,

a correlation between the presence of NLRP5-specific autoantibodies and autoimmune

ovarian insufficiency has been observed. Such correlations are highly suggestive of the

involvement of NLRP5 in conditions attributable to aberrant inflammatory pathways.

1.3.2.5 NLRP12

NLRP12 mutations are associated with the development of hereditary periodic

fever (HPFs), six Mendelian autoinflammatory disorders marked by the uniting features

of systemic inflammation and recurrent febrile episodes, occasionally complicated by

. Using a candidate gene approach, Jéru et al. (2007) looked for mutations

in NLRP3, NLRP12, MEFV, TNFRSF1A and MVK. Mutations in NLRP12 (nonsense

36 and splice site mutations) in two families with periodic fever syndromes were identified: these mutations were found to induce a clear reduction of the inhibitory properties of NLRP12 on NF-κB signalling, thus leading to increased NF-κB production and secondary systemic inflammation.

In addition, NLRP12 has been postulated to be involved in the development of atopic dermatitis (AD), a dermatologic condition characterized by excessive immune reactions to ubiquitous antigens. In order to investigate the role of variations in NLR genes in AD, Macaluso et al. genotyped 23 single nucleotide polymorphisms (SNPs) in seven selected NLR genes (CARD4, CARD15, CARD12, NLRP1, NLRP3, NLRP12,

MHC2TA) in 392 unrelated patients with AD and 297 controls (adults >40 years of age without asthma, allergies, or AD). Single-SNP analysis demonstrated significant associations of the CARD15_R702W polymorphism and the NALP12_In9T-allele with

AD. These findings suggest that variation in individual genes from the NLR family could play a role in AD pathogenesis, specifically NLRP12. (Macaluso et al, 2007). A summary of different NLRPs and the associated human disease is shown in Table 2.

37

Table 3. NLRP subfamily and associated auto-inflammatory human diseases.

NLRP Associated human disease Molecular basis Putative molecular effect/ Reference and function

• Generalized Vitiligo Function: Involved in activation variants in the • vitiligo-associated multiple of caspase-1 and caspase-5, (Jin et al, 2007; NLRP1 autoimmune disease type 1 promoter region Magitta et al, which leads to processing and (VAMAS1) 2009) release of IL1β and IL18 Frameshift (Tschopp et al, Beckwith-Wiedemann Syndrome mutation in exon 6 Function: Activate caspase- 2003; Meyer et NLRP2 (BWS) in the mother of leading to the secretion of IL-1β. al, 2009)

affected children.

• Muckle-Wells Syndrome( MWS) Mostly missense • Familial Cold autoinflammatory Patients with MWS have (Rosenstiel et NLRP3 syndrome type 1( FCAS1) variants in the • Chronic Infantile neurologic constitutive IL-Iβ secretion al, 2007) NACHT domain cutaneous and articular while normal subject do not. syndrome (CINCA/NOMID ) NLRP5-specific (Alimohammadi • Polyendocrine syndrome (APS-1) autoantibodies May regulate caspase activation et al,2008; C. NLRP5 present in APS and apoptosis in injured neurons Frederick Lo et al ,2007) patients

• Recurrent Hydatidiform Moles All types of In vitro transfection of WT- (Murdoch et al, NLRP7 mutations and in NLRP7 inhibits IL-1β secretion. 2006; Kinoshita various domains et al ,2005)

• Hereditary Periodic Fever nonsense and splice In vitro over expression of WT- (Jéru et al ,2007) NLRP12 (HPFs) site mutations in the NLRP12 activates NF-κB and (Macaluso et al, • Atopic dermatitis (AD) NACHT domain Caspase-1 2007) • Spermatogenic Failure and azoospermia 1 nonsense and 4 Oocyte development arrest (Westerveld et NLRP14 missense mutations al, 2006) in knock down mice in various domains

38 CHAPTER 2

2. Materials and Methods

2.1 Isolation of Full-length and Mutated NLRP7 cDNA

Full length WT NLRP7 cDNA inserted into pCR-BluntII-TOPO vector was

obtained from Open Biosystems, (IMAGE ID: 40036028, Accession BC109125) and

plated on selective media. Plasmid DNA was prepared from a single bacterial colony

using QIAprep Miniprep Kit (Qiagen). To confirm the identity of the purchased clone,

PCR was performed on the purified DNA using four different pairs of NLRP7 cDNA

primers (Table 1). These PCR fragments were also sequenced for further verification.

Table 4. Primers used to amplify NLRP7 gene

Primers sets Expected size Primer Sequences (5`-3`) M13 forward GTAAAACGACGGCCAG 3519bp M13 reverse CAGGAAACAGCTATGAC 4.4 forward GTGGGCGCAGATGTCCGTGTTC NLRP7 full length 1886bp TCCCGCTTCCTGTGTAATTCGTAGA cDNA reverse1 4.2 forward GACGACGTCACTCTGAGAAACCAAC 1135bp 4.3 reverse TTTGCTGAAGAGGAAGATGTTACCC NLRP7 RT exon 6 TCGAGTGGGAACGCACGATGAT 286bp NLRP7 RT exon 8 CCTTGCAACTGGCTTCTGTAAGACG

39 2.2 Site-Directed Mutagenesis

To introduce the various mutations found in the patients into the full-length wild- type NLRP7 cDNA, QuikChange Site-Directed Mutagenesis kit (Stratagene) was used.

Ten mutations found in our laboratory were introduced in the wild-type NLRP7. Primers containing the desired mutations were designed using the web-based QuikChange Primer

Design program (www.stratagene.com/qcprimerdesign) (Table 5). After PCR amplification, the products were then placed on ice for 2 min to cool the reaction to 37ºC and the PCR products were subjected to digestion by the restriction enzyme Dpn1, which digests the non-mutated plasmid DNA that is methylated. (Dpn1 cleaves GATC only when the A is methylated). The mixture was incubated for 1 hr at 37°C. The mutated

PCR products were then transformed into XL1-Blue super-competent cells, which repair nicks in the mutated plasmid. Transformed bacterial cells were plated on agar containing ampicillin (100 μg/mL). Five colonies were selected and purified using miniprep for each mutation. DNA from the miniprep was sent for sequencing and the sequences were analyzed using DNASTAR. Only colonies containing non-rearranged plasmids with the mutations were kept.

DNA for each mutation was digested using different pairs of restriction enzymes in order to subclone the desired mutation in the mammalian vector PcDNA3.1+ and will be discussed in section 2.4

40

Table 5. Primers designed for site-directed mutagenesis

Primer name Primer sequence 5'-3' G251A 5'-ATGAATCTCACGGAATTGTATAAGATGGCAAAGGCTGAG-3' G251A antisense 5'-CTCAGCCTTTGCCATCTTATACAATTCCGTGAGATTCAT-3' G1196A 5'-CTGCGTTTCCTCTACAGCCGGTTCCCG-3' G1196A 5'-CGGGAACCGGCTGTAGAGGAAACGCAG-3' antisense G355T 5'-GAGTTAGCAAAGCCAGGTTAAAAGGAAGGATGGAGAA-3' G355T antisense 5'-TTCTCCATCCTTTCTTTTAACCTGGCTTTGCTAACTC-3' T2474A 5'-TTCCTGCAGATGTTGTCGTAGGAAAAACTGTCGTC-3' T2474A 5'-GACGACAGTTTTCCTACGACAACATCTGCAGGAA-3' antisense G2078C 5'-GAGTGACTCTTCTGTGCCGATTCTTTGTGACCACG-3' G2078C 5'-CGTGGTCACAAAGAATCGGCACAGAAGAGTCACTC-3' antisense A2738G 5'-CACAAACCTGGACTTGAGTATCAGCCAGATAGCTCG-3' A2738G 5'-CGAGCTATCTGGCTGATACTCAAGTCCAGGTTTGTG-3' antisense C2077T 5'-TGAGTGACTCTTATGTGTGGATTCTTTGTGACCAC-3' C2077T 5'-GTGGTCACAAAGAATCCACACAGAAGAGTCACTCA-3' antisense A1970T 5'-CTGGGCTCGGCAGGTTCTTCGCTCTCTTC-3' A1970T 5'-GAAGAGAGCGAAGAACCTGCCGAGCCCAG-3' antisense G295T 5'-GAGGACGGACAGGTGCAATAAATAGATAATCCTGAGC-3' G295T antisense 5'-GCTCAGGATTATCTATTTATTGCACCTGTCCGTCCTC-3' G955A 5'-AGCAGCCGATCTACATAAGGGTGGAGGGC-3' G995A antisense 5'-GCCCTCCACCCTTATGTAGATCGGCTGCT-3'

41

2.3 Subcloning of Wild-type NLRP7 cDNA

Wild-type NLRP7 cDNA was generated into a flagged mammalian vector in order to monitor the expression of the transfected DNA, since at that time, no specific antibody for NLRP7 was available. First, PCR was performed using flag primers,

NLRP7-AS and Full NLRP7 flag (Table 6). The PCR products were purified using

Qiaquick PCR purification kit (Qiagen), and digested with AflII and XhoI restriction enzymes, then the vector and the insert were dephosphorylated with 4 µL of alkaline phosphatase (AP) and incubated for one hr at 37ºC. Prior to ligation, the product was purified using QiA-quick PCR purification and 5 µl was loaded on a 1% agarose gel.

The products of the digestion were then ligated using the protocol provided with the

Rapid DNA ligation kit (Roche). Different molar ratios of vector DNA to insert DNA were prepared 1:1, 1:3, and 1:5. The mixtures were then incubated for 30 min at room temperature and subjected to transformation. The ligation products were transformed into bacterial cells, DH5α (Invitrogen). Briefly, competent cells were thawed on ice for

5 min and at the same time polypropylene tubes were chilled on ice. Fifty microliters of the competent cells were added to 10 µl of each ligation sample. The mixtures were incubated on ice for 30 min and heat shock was performed for 45 s at 42ºC in a water bath. Then the mixtures were placed on ice for 2 min. Five-hundred µl of pre-warmed

LB were added to each tube and incubated for one hr at 37ºC with shaking at 250 rpm.

Each tube was plated on ampicillin-selective agar plates, to ensure the growth of only transformed bacteria. Several colonies were selected to check for the NLRP7 insert. To confirm whether the selected colony contained the right insert, miniprep was performed according to the manufacturer’s instructions to purify DNA using QIA prep Miniprep

42 kit (QIAGEN). Plasmid-DNA was subsequently re-digested with XhoI and AflII to check for the presence of the NLRP7 insert. The clones were sent for sequencing and were analyzed to check for any mismatch. Sequence analysis demonstrated the introduction of the desired change in WT flagged-NLRP7 in PcDNA3.1 plasmid. When the results were confirmed, a midiprep was performed to obtain larger-scale DNA samples according to the manufacturer’s instructions. In order to determine the concentration of DNA from the midiprep, the DNA solution was diluted 1:200 in dH2O and absorbance was measured by spectrophotometer at 260 nm.

Table 6. Primers used in PCR amplification to add a flag to NLRP7.

Expected Primers Primer Sequences (5`-3`) size

NLRP7-AS GCGGCTCGAGTCAGCAAAAAAAGTCACAGCACGGG 3001 bp NLRP7-Flag GCGGCTTAAGCCACCATGGACTACAAAGACGA CGATGACAAGGGTACCATGACATCGCCCCAG

2.4 Sub-cloning of Mutated NLRP7 cDNA

By introducing the various mutations in the WT NLRP7 using site directed mutagenesis (described above), double restriction enzymes digestion were used either with SacII & XhoI or Kpn1 & PfIMI (shown in table 7). In the first digestion, samples were incubated overnight at 37ºC and checked by running on a 1% agarose gel for the presence of a linear fragment. Then a second digestion with the second restriction

43 enzyme was performed as described previously and incubated for 3 hr. In order to cut

the desired fragment that contains the mutation, double digested samples were run on a

1% agarose gel and the band of interests were excised from the gel using a scalpel

visualized under the UV-light. Using QIAEX gel extraction kit from QIAGEN, bands

were eluted and purified according to the kit protocol. Prior to ligation WT-flag NLRP7

was double digested with either SacII & XhoI or Kpn1 & PfIMI. Each mutated

fragment was ligated separately to WT-flag NLRP7 in PcDNA 3.1 vector using Rapid

DNA ligation kit (Roche). A schematic representation of the protocol used to in cloning

the WT-NLRP7 and mutated NLRP7 in PCDNA 3.1 + vector are shown in Figure 3.

Table 7. List of NLRP7 mutations and restriction enzymes used for Cloning in Flag WT-NLRP7

Mutations Restriction Enzymes Expected Size c.1196G>A SacII & XhoI 2240 bp

c.1970A>T SacII & XhoI 2240 bp

c.2077C>T SacII & XhoI 2240 bp

c.2078G>C SacII & XhoI 2240 bp

c.2474T>A SacII & XhoI 2240 bp

c.2738A>G SacII & XhoI 2240 bp

c.251G>A Kpn1& PfIMI 807 bp

c.355G>T Kpn1& PfIMI 807 bp

c.295G>T Kpn1& PfIMI 807 bp

.

44

Figure 3 Schematic representation of the protocol used to in cloning of the WT-NLRP7 and various mutated NLRP7 in PCDNA 3.1 + vector

45 2.5 Cell culture and Transient Transfection

HEK293 transformed cell lines -which does not express NLRP7- were cultured in

T-75 flasks with DMEM (Invitrogen) media supplemented with 10% FBS and ~1% L- glutamine and incubated at 37°C with 5% CO2. One day prior to transfection, cells were counted using a hemacytometer, and 1.2x105 (primary conditions) and 6x104 cells

(established conditions) were seeded in 24-well tissue culture plates in a total volume of

500 µl DMEM supplemented with 10% FBS, as well as ~1% L-glutamine, without antibiotics. Cells were incubated at 37ºC for 24 hr or until ~80% confluency is reached

Transient transfection was performed on the HEK293 cell lines grown as previously described. The total amount of DNA was kept constant in all transfections

(0.4 μg) using PcDNA3.1+ vector. HEK293 were cotransfected with plasmids encoding proIL-1β, Flag-tagged procaspase-1 and Cardinal with or without WT-NLRP7. Twenty- five µL of the Opti-MEM (Invitrogen) was added to each condition, then 4 µL of Plus

Reagent were added to each mixture of DNA vectors and incubated for 15 min at room temperature. 1:26 µL of Lipofectamine (Invitrogen) in Opti-MEM was prepared then added to each mixture and incubated for 15 min at room temperature.

Media was removed from 24-well plate in which the HEK293 cells were growing.

Wells were washed with 250 µL of 1X PBS. To each well, 200 µL of warm Opti-MEM was added. Then the different DNA Lipofectamine mixtures were added each to its corresponding well and mixed gently by rocking the plates. Cells were then incubated for

3 hours at 37ºC in 5% CO2, and the transfection media was replaced by supplemented

DMEM and the cells were incubated again at 37ºC in 5% CO2 for 24 h. The supernatants were then collected and centrifuged for 30 sec at 13,000 rpm. The supernatant was transferred into a new tube and frozen at -80ºC, in order to be used later for the ELISA

46 assay. Also, the cells were washed with 50 µL of warm 1X PBS. Then each well was scraped in order to take off the cells. Two hundred µL of cold 1X PBS was added and the cells were collected and spun at 13,000 rpm for 1 min. The supernatant was discarded, and 75 µL of lysis buffer (0.05M Tris/HCl, 0.15M NaCl, 0.001M EDTA, 10X Triton in

3.6 mL H2O supplemented with protease inhibitor) was added to the cell pellet. The cell lysate was then frozen at -80ºC and kept for western blot preparations

2.6 Enzyme-linked Immunosorbent Assay (ELISA)

ELISA was used to quantify IL-1β concentration using the supernatant collected from the transfected cells according to the manufacturer’s protocol (BD Biosciences

Pharmingen).

2.7 Western Blot Analysis

Protein quantification of the cell lysates was performed using the Bradford protein assay from (Bio-Rad Kit) according to the manufacturer’s protocol. After quantification, 10 μg of total protein per lane was used, and these samples were loaded with 1X loading buffer on a 12% polyacrylamide gel to be separated by SDS-PAGE electrophoresis at 200 V for 45 min. The gel was then electrotransferred using

MiniTrans-Blot (Bio-Rad) to polyvinylidene difluoride (PVDF) membranes for one hr at

100V. The membrane was blocked for 1 hr with 5% nonfat dry milk in TBS-T (1X TBS and 0.1% Tween -20) and subsequently washed 3 times with TBS-T. Membranes were incubated with the appropriate antibodies at 4ºC overnight on a shaker. IL-1β was detected by a polyclonal IL-1β antibody, which was diluted 1:1000. Caspase-1 was detected by Anti-Flag monoclonal antibody diluted 1:2000. WT- NLRP7 flag was

47 detected by Anti-Flag monoclonal antibody diluted 1:2000. The membranes were washed

again by TBS-T and depending on the primary antibody used; secondary antibodies (anti-

mouse or anti-rabbit) were incubated with the membranes for 1 hr on a shaker at room

temperature. The signals were then detected using ECL revealing reagent for 1 min at

room temperature and exposed to X-ray film.

2.8 Statistical Analysis

ANOVA test followed by post hoc test Tukey were used for Statistical Analysis

to compare the results of secreted IL-1β with or without WT-NLRP7. All values of

P <0.05 were considered significant.

48 CHAPTER 3

3. Results

3.1 Site-Directed Mutagenesis

Successful isolation of the NLRP7 full-length cDNA from pCR-BluntII-TOPO vector was obtained. The identity of the cDNA was confirmed by PCR using four different pairs of NLRP7 cDNA primers (Figure 4). Sequence analysis was completed for further clarification. Mutations were introduced in the isolated WT-NLRP7 vector using site- directed mutagenesis. Sequence analysis confirmed the introduction of the mutation in the

NLRP7 cDNA (Figure 5).

Figure 4. The amplification of NLRP7 cDNA using four different primers. Lane 1: 1 Kb Ladder. Lane 2: M13 forward & reverse. Lane 3: 4.4 forward & NLRP7 full length cDNA reverse1. Lane 4: 4.2 forward & 4.3 reverse. Lane 5: RT6 & RT 8

49

Mutations p.Cys84Tyr p.Glu99X IVS3+1 p. Val319Ile p. Cys399Tyr c.251G>A c.295G>T c.355G>T c.955G>A c.1196 G>A

Sequences

ATTGTATAA TGCAATAAATAG A G G T T A A A A G C T A C A T AAG C T C T A C A G C C

Mutations p.Asp657Val p.Arg693Trp p.Arg693Pro Leu825X p.Asn913Ser c.1970A>T c.2077C>T c.2078G>C c.2474 T>A c.2738A>G

Sequences

C A GGTTC TT TGTGCCGATTCT GTCGTAGGAAA T G T G T G G A T T C T GAGTATCAGCCAGATA

Figure 5. Site directed mutagenesis shows the sequences of the various mutations that were introduced in the NLRP7 full- length cDNA

50

3.2 Subcloning of wild-type and Mutated NLRP7 cDNA

Cloning of flag WT-NLRP7 into PcDNA3.1 vector as well as cloning the mutation in the flag-WT-NLRP7 vector was performed. Successful introduction of the WT-flag NLRP7 in the pcDNA3.1 vector is shown in (Figure 6). In order to investigate the effect of NLRP7 expression on the IL-1β secretion, different inflammasome components procaspase-1, pro-IL-1β, and Cardinal were also co- transfected.

Figure 6. Cloning the WT-flag NLRP7 in PcDNA3.1+ Enzymatic digestion of the DNA purified from 12 colonies using XhoI and AflII, lanes 2 to 13. The colonies in lanes 8 and 12 contain the WT-flag NLRP7 insert. Lane 1 indicates (1 Kb DNA Ladder).

3.3 Optimizing the Transfection Conditions

To achieve the best results, first I had to optimize the transfection conditions in order to test WT-NLRP7 and their effect on IL-1β cleavage and secretion. Then compared the

WT-NLRP7 to the various mutated-NLRP7. Since cell numbers and amount of DNA concentration are important factors which effect the experimental results, I initially

51 measured the level of IL-1β secretion in HEK293 by plating 2X105 cells in 24-well plate and by cotransfecting the cells with 100 ng of procaspase-1, proIL-1β, WT- flag NLRP7.

The resultant levels of IL-1β secretion were undetectable. The above experiments were repeated at least three times. Subsequently, I began to change several factors in order to obtain a higher level of secreted IL-1β. I then tested a lower number of cells (1.2X105 and

6x104 cell/well) with different DNA concentrations of pro-IL-1β (60 ng, 120ng, 180 ng and 240 ng) and constant amount of procaspase-1 (10 ng) as shown in Figure 7. The lower number of cells gave the highest amount of secreted IL-1β and was adopted in later experiments (Figure 7).

Figure 7. Optimizing the transfection conditions Cell numbers were reduced to 1.2X105 and 6X104 cells/well, and different amount of pro-Il-1β (60ng, 120ng, 180ng, 240ng) were used with constant amount of procaspase-1 (10 ng).

52

3.4 NLRP7 Inhibits IL-1β Secretion in HEK293 cells

From the data of the previous experiments, we adopted the following conditions:

the cells were plated in 6X104 cells/well and cotransfected with 150 ng of IL-1β and 10 ng

of procaspase-1. Under these conditions, approximately 70 pg/ml of secreted IL-1β was

detected in the culture supernatant by ELISA. However, when 100ng of WT-NLRP7

plasmid was added, the amount of secreted IL-1β was around 20pg/ml, which indicates a

significant inhibition (p<0.05) of IL-1β. To confirm this result, western blot was

performed on cell lysates to detect the cleaved form of IL-1β (17 kDa). As shown in

Figure 8, a faint band was obtained upon addition of 100ng of WT-NLRP7, corresponding

to a decreased production of cleaved IL-1β. Our results thus suggest that NLRP7

expression effects the processing of the pro-IL-1β destined for cleavage. In addition the

expression of Caspase-1, and α-Tubulin were detected from the same gel (Figure 8). This

experiment was repeated 2 times with duplicated wells and the results were reproducible.

These results confirm previous finding by Kinoshita et al (2004) that NLRP7 inhibits

caspase-1 dependent IL-1β secretion.

In a subsequent phase of our research, we asked whether a lower dose of NLRP7

would have the same effect on IL-1β release. HEK293 cells were cotransfected with

plasmid encoding procaspase-1, pro-IL-1β and with 100 ng and 25 ng of WT-NLRP7 and

constant amount of Cardinal (100 ng). Our results show that the 100 ng of NLRP7 led to

greater inhibition of IL-1β, while the 25 ng of NLRP7 had minimal or no effect on IL-1β

secretion as shown in (Figure 8). My results suggest that NLRP7 decreases IL-1β secretion

in a dose-dependent manner. However, this result was obtained in a single experiment

with duplicate wells and remains to be confirmed in future studies. 53 In addition, my work also examined the effect of cardinal on NLRP7 inhibition. It is known that Cardinal is a protein that interacts with the NBD domain of NLRP3, thereby allowing for the activation of caspase-1 and subsequently leading to the cleavage of pro-

IL-1β to its mature form. In order to examine how cardinal influences the inhibitory function of NLRP7, we used the HEK293 transfection system, in which plasmids encoding pro-IL-1β, procaspase-1 and 100 ng of NLRP7 were cotransfected with or without 100 ng of Cardinal. Our results indicate that NLRP7 inhibits IL-1β secretion, and Cardinal has no effect on the inhibitory process as shown in Figure 9. Our data also suggests that cardinal may increase the secretion of IL-1β when transfected with pro-IL-1β and caspase-1

(Figure 9A & 9C).

Figure 8. NLRP7 inhibits IL-1β secretion in dose-dependent manner. HEK293 were cotransfected with plasmid encoding proIL-1β (150 ng), procaspase-1 (10 ng ) with or without NLRP7 (100 ng ) , with or without 100 ng cardinal , and with 25 ng NLRP7. Lysates were collected and analyzed for the presence of IL-1β, anti-flag NLRP7 and α-Tubulin by Western blot.

54 A) C)

B) D)

Figure 9. NLRP7 inhibits IL-1β secretion in the presence or absence of Cardinal (A HEK293 cells were cotransfected with plasmid encoding pro-IL-1β (150 ng), Pro- caspase- 1 (10 ng) and WT-NLRP7(100 ng ) Cell culture media were harvested after 24 h ,

and IL-1β was measured in the cell culture medium by ELISA. B) The cell lysates from the

experiment were subjected to western blot and confirm that NLRP7 expression inhibits Il-1β. (C) HEK293 cells were cotransfected with plasmid encoding pro-IL-1β (150 ng), Pro- caspase- 1 (10 ng) and Cardinal (100 ng) with or without 100 ng NLRP7. Cell culture media

were collected after 24 h, and IL-1β was measured in the cell culture medium by ELISA D) Cell lysates from the same experiment were subjected to western blot and confirm the presence of mature form of IL-1β. DNA concentration was kept constant by inclusion of an empty vector plasmid. (A-C) ELISA experiments were performed at the same time. (B-D) w.blot results obtained from the same polyacrylamide gel.

55 CHAPTER 4

Discussion

NLRP7 has been identified as the causative gene for familial hydatidiform mole

(FHM); an autosomal recessive disorder characterized by recurrent molar pregnancies and associated reproductive wastage. FHM appears to result from the inability to establish correct maternal epigenetic identity at imprinted loci during oogenesis. Several women affected with FHM have previously been shown to have either homozygous or heterozygous mutations in NLRP7.

Recently, our lab has demonstrated that cells from patients with NLRP7 mutations have an impaired inflammatory response against various stimuli such as LPS and microbial products, resulting in lower IL-1β and TNFα secretion when compared to controls (unpublished data). Contrarily, in vitro studies conducted by Kinoshita et al. have suggested that wild type NLRP7 inhibits caspase-1 dependent IL-1β secretion.

These findings thus serve as the rationale behind the development of my present study.

The aim of this project was to establish an in vitro system to further examine the functional consequences of NLRP7 mutations on IL-1β secretion. Using site-directed mutagenesis, I was able to introduce 10 mutations into the full length of NLRP7 cDNA.

The wild-type and mutant NLRP7 were then cloned in a flagged PcDNA 3.1 in order to assess their impact on NLRP7 interaction with caspase and IL-1β in HEK293T cell lines.

As only one previous in vitro study has been completed using NLRP7, optimizing the transfection conditions was complicated by the lack of understanding of the NLRP7 partners involved in caspase-1 dependent IL-1β processing pathways (such as Cardinal and Caspase-5).

56 When transfecting the WT-NLRP7 with IL-1β and Caspase-1, I showed that the

WT-NLRP7 inhibited IL-1β secretion, thus confirming the results of the previous in vitro study conducted by Kinoshita. My experiments also demonstrated that inhibition of IL-

1β occurs in a dose-dependent manner. In addition, Cardinal, an important component of the NLRP3 inflammasome, exerted no effect on this inhibitory function. However, as dose-dependent studies were performed only once, subsequent dose-dependent analyses are required to confirm and validate my findings. As my results only showed the effects of transfecting cells with WT- NLRP7, the effect of IL-1β production upon transfection of mutated NLRP7 remains to be explored.

It is interesting to note that the ex-vivo data demonstrates that stimulation of patient's cells with either homozygous or heterozygous NLRP7 mutations, as well as the over-expression of WT-NLRP7 in HEK293; both decrease the cellular secretion of

IL-1β. These similar finding between over expressed WT-NLRP7 and mutated NLRP7 is puzzling, but could be explained by the abnormal protein interactions within NLRP7 protein complex. This abnormality could result from disruption of the NALP7 inflammatory pathway which is part of the cellular immune response. It is important to investigate the validity of the above results, by studying NLRP7 protein partner and assaying IL-1β processing and cleavage in order to further elucidate the role of protein-protein interactions. The creation of additional assays to better qualify IL-1β processing and cleavage could allow for the development of a more thorough understanding of the role of NLRP7 in IL-1β production.

. In addition, the mechanism behind IL-1β secretion inhibition is poorly understood. However, the interaction between NLRP7, procaspase-1 and pro-IL-1β was reported by Kinoshita et al. in 2004. I confirmed that NLRP7 may function as a feedback regulator of IL-1β secretion, thereby inhibiting the action of procaspase-1 directly

57 involved in IL-1β production. The interactions of NLRP7 with other members of the

innate immune system leading to IL-1β production remain to be explored.

Conclusions and Future Perspectives

In my study, I have demonstrated that:

1) The number of transfected cells would affect the transfection in HEK293.

2) NLRP7 inhibits IL-1β secretion in HEK293 cell line.

3) Cardinal, an important part of the NLRP3 inflammasome has no effect on the

NLRP7 inhibitory effect and IL-1β secretion.

To date, little is known about the normal physiological role of NLRP7. It is still

unclear how NLRP7 defects lead to RHMs and associated reproductive wastage. The

identification of NLRP7 as a defective gene responsible for recurrent forms of molar

pregnancies has been pivotal in advancing studies of this rare autosomal disease. It has

been proposed that the defect in NLRP7 leads to defective oocytes by a mechanism that

is yet to be completely understood. Further work must be initiated to better comprehend

this mechanism.

This project has also allowed for the examination and characterization of the

effects of the WT-NLRP7 on the processing of IL-1β. Since we have generated various

mutations in the WT-NLRP7 in the mammalian vector PcDNA3.1+, it is still important

to investigate the impact of NLRP7 mutations on IL-1β processing when co-transected

with vectors expression pro-IL-1β, procaspase-1, and WT-NLRP7 or mutant NLRP7.

Recently, a study examined protein-protein interaction between various NLRP

proteins and the adapter protein ASC, which is known to be part of NLRP3

inflammasome. Interestingly no physical interaction was observed between NLRP7 and

58 adaptor ASC (Wagner RN et al, 2009). The authors suggested the presence of a new adaptor or effector protein that serves in connecting NLRP family members to downstream signaling pathways (Wagner RN et al, 2009). These implications remain to be explored.

Although NLRP7 interacts with caspase-1 and IL-1β, it is still crucial to identify other partners involved in IL-1β processing pathways, such as Cardinal and caspase-5.

It is suggested that further studies be undertaken in order to further illustrate protein- protein interactions. Using immunoprecipitation (IP) assays and Western blot would thereby facilitate the identification of co-precipitated proteins acting as NLRP7 partners.

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66

APPENDIX A

Published Abstract and Presentations

Published abstract: 1- E. Bukhari1, 2,C. Deveault1,2 , J.Qian1,2,5, A. Mehio3, L. Gilbert2, M. Seoud4, X. Xie5, and R. Slim1,2.McGill University Health Centre, Montreal, Canada, Dept of Human

Genetics1 Dept of Obstetrics and Gynecology2., Dept of Pathology3; Dept of Obstetrics and Gynecology American University of Beirut4. Women’s Reproductive Health

Laboratory, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou,

China5. American Society of Human Genetics (A.S.H.G) 58Annual Meeting.

Philadelphia, Pennsylvania .November 11-15, 2008. ASHG abstractst. Abstract 1631/F. page 322.

Oral Presentations 1- In vitro analysis to assess the functional consequences of NLRP7. Obstetrics and

Gynecology, Research Division, RVH, May 28, 2007.

2- PYPAF3, a PYRIN-containing APAF-1-Like Protein, Is a Feedback Regulator

of Caspase-1-dependent Interleukin-1B secretion, Lab meeting, MGH, June4, 2007

3- Age-associated alteration of patterns in mouse oocytes, Lab

meeting ,MGH, December 3,2007

4- CATERPILLER protein family and Autoinflammatory Diseases May 5, 2008

Obstetrics and Gynecology, Research Division, RVH, May 5, 2008.

5- SDS-PAGE and Western Blotting Techniques, Lab meeting, MGH, January 26, 09.

6- Common Variants in the NLRP3 Region Contribute to Crohn’s Disease

Susceptibility, Lab meeting, MGH, February 9, 09

67 APPENDIX B

Published Abstract

American Society of Human Genetics

Involvement of pathogens in recurrent hydatidiform moles caused by NLRP7 mutations

E. Bukhari1, 2,C. Deveault1,2 , J.Qian1,2,5, A. Mehio3, L. Gilbert2, M. Seoud4, X. Xie5, and R. Slim1,2. McGill University Health Centre, Montreal, Canada, Dept of Human Genetics1 Dept of Obstetrics and Gynecology2., Dept of Pathology3; Dept of Obstetrics and Gynecology American University of Beirut4. Women’s Reproductive Health Laboratory, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, China5.

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by the absence of, or abnormal, embryonic development and hydropic degeneration of the chorionic villi. Recently, NLRP7 has been found responsible for recurrent hydatidiform moles (RHM) by the identification of 11 mutations in this gene (Murdoch et al., 2006; Kou et al, 2008). In this study, we investigated the presence of microorganisms in several molar tissues from several patients with NLRP7 mutations by microscopy screening of sectioned tissues stained with Gram and Grocott’s methenamine silver and by PCR amplification with universal bacterial and fungal primers followed by cloning and DNA sequencing. Microscopy screening revealed Gram-positive (G+) cocci, Gram-negative (G-) bacilli, yeast cells and filamentous fungi, in most tissues from the patients, while only 10% of control tissues from elective first trimester abortions had a few G+ bacilli and G+ cocci.PCR amplification with universal bacterial and fungal primers followed by cloning and DNA sequencing confirmed the presence of several microorganisms in the patients and the pathogenic nature of some of them. Our data demonstrate the involvement of pathogens in RHMs caused by NLRP7 mutations and will have great impact on our current understanding of the pathology of moles and reproductive wastage.

68

APPENDIX C

Ethics Approval and Certificates

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