The Role of Nicotine in Exacerbating the Inflammatory Response in Macrophages Infected with Mycobacterium Avium Paratuberculosis in Crohn's Disease Smokers

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Electronic Theses and Dissertations, 2020-

2020

The Role of Nicotine in Exacerbating The Inflammatory Response in Macrophages Infected with Mycobacterium avium paratuberculosis in Crohn's Disease Smokers

Dania Alqasrawi University of Central Florida

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STARS Citation Alqasrawi, Dania, "The Role of Nicotine in Exacerbating The Inflammatory Response in Macrophages Infected with Mycobacterium avium paratuberculosis in Crohn's Disease Smokers" (2020). Electronic Theses and Dissertations, 2020-. 795. https://stars.library.ucf.edu/etd2020/795 THE ROLE OF NICOTINE IN EXACERBATING THE INFLAMMATORY RESPONSE IN MACROPHAGES INFECTED WITH MYCOBACTERIUM AVIUM PARATUBERCULOSIS IN CROHN’S DISEASE SMOKERS

DANIA ALQASRAWI B.S. JORDAN UNIVERSITY OF SCIENCE AND TECHNOLOGY, 2004 M.S. JORDAN UNIVERSITY OF SCIENCE AND TECHNOLOGY, 2008 M.S. UNIVERSITY OF CENTERAL FLORIDA, 2019

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences in the Burnett School of Biomedical Sciences in the College of Medicine at the University of Central Florida Orlando, Florida

Fall Term 2020

Major Professor: Saleh A. Naser

ABSTRACT

It is a fact that cigarette smoke (CS) has negative effects on patients with Crohn’s disease (CD), whereas CS seems to provide protection to patients with Ulcerative Colitis (UC). The mechanism of how CS ingredients or nicotine modulate inflammatory phenotypic response in IBD subsets remained unclear. Unlike UC, CD has been associated with genetic disposition, immuno- dysregulation and infection by pathogens mainly Mycobacterium avium paratuberculosis (MAP).

In this study, we investigated the cellular and molecular effects of pure nicotine and tobacco extracts from HLE-nicotine rich-plant and LAMD-nicotine less-plant in infected macrophages.

Unlike LAMD extracts, Nicotine (4ug/mL) and HLE extracts (0.18%) significantly favored M2 polarization and phenotypic response. While macrophages infected with MAP or treated with LPS promoted pro-inflammatory response, treatment of infected macrophages with nicotine/HLE extracts further elevated pro-inflammatory response, and enhanced MAP burden. Pre-conditioning macrophages with nicotine or blocking alpha-7nAChR with antagonist reversed the effect of nicotine in infected macrophages. We demonstrated that MAP infection in macrophages was mediated through TLR2/MyD88 signaling; blocking TLR2 and TLR4 with antagonists significantly reduced the inflammatory effect of MAP and LPS. Interestingly, nicotine in infected macrophages significantly downregulated TLR2/TLR4 expression, activated MyD88, and increased expression of pro-inflammatory cytokines. Surprisingly, dual treatment of MAP- infected macrophages with MyD88 antagonist and nicotine absolutely impaired immune response during infection. The data shows a role for TLR2/MyD88 signaling in elevated inflammatory response during infection in CD smokers. Overall, we conclude that in absence of infection in UC, nicotine activates the cholinergic anti-inflammatory pathway through alpha-7nAChRs. However,

iii in CD smokers with MAP infection, nicotine action is mediated through TLR2/MyD88 signaling leading to significant inflammatory response. The study is the first to unmask the mechanism involved in the contradictory effect of CS in IBD populations.

To Mam and Dad Who always picked me up on time and encouraged me to go on in every adventure in my life, especially this one! This humble work is a sign of my love to you!

ACKNOWLEDGMENTS

First of all, I would like to express my gratitude and appreciation to my mentor, Dr. Saleh A.

Naser, for his patience, support, and guidance. I am very thankful to him for giving me this great opportunity to be a member in his laboratory. I would also like to thank my dissertation committee members, Dr. Annette Khaled, Dr. Kai McKinstry and Dr. Shibu Yooseph for their assistance and guidance throughout this dissertation process.

From the bottom of my heart, I would like to say big thank you for all my lab mates for their positive energy, understanding, and help throughout my project. It truly has been exceptionally good time in the lab!. Special thanks to our postdocs, Dr. Latifa S. Abdelli and Dr. Ahmad Qasem, without your help and wise guidance this project would have not been the same.

Last but not the least, my biggest thanks to my family and friends back home in Jordan and here in US to their spiritual support and motivation. And for my mom and dad, who had put up with my stresses all over three years. You have been amazing!

TABLE OF CONTENTS

LIST OF FIGURES ...... xi

LIST OF TABLES ...... xiii

LIST OF ACRONYMS/ABBREVIATIONS ...... xiv

CHAPTER ONE: BACKGROUND ...... 1

Overview of Inflammatory Bowel Disease ...... 1

Cigarette Smoke is Detrimental in Crohn’s Disease...... 3

Cigarette Smoke is a Protective Factor in Ulcerative Colitis ...... 4

Nicotine Replacement: ...... 6

The Contradictory Effect of CS on IBD subsets is due to Nicotine-multifactorial interaction .. 6

Nicotine and the other factors ...... 6

Nicotine and Immune Response: ...... 7

Cytokines ...... 7

α7nAChR in Immune Cells ...... 8

Microbial Dysbiosis: ...... 8

Epigenetic Susceptibility: ...... 9

Differences in Immunological Response Profile between Crohn’s Disease and Ulcerative

Colitis ...... 11

Summary and Outlook ...... 13

Figures...... 14

Tables ...... 17

vii

References ...... 19

CHAPTER TWO: MYSTERY SOLVED: WHY SMOKE EXTRACT WORENS DISEASE IN

SMOKERS WITH CROHN’S DISEASE AND NOT ULCERATIVE COLITIS? GUT MAP! . 28

Introduction ...... 28

Materials and Methods ...... 31

Culture Conditions of THP-1 Macrophages ...... 31

Infection and Treatment of THP-1 Macrophages ...... 31

Measurement of Relative Gene Expression Using RT-PCR ...... 32

Measurement of Cytokines and Cell Surface Markers ...... 33

Bacterial Viability Assay ...... 33

Measurement of Macrophage Apoptosis ...... 34

Statistical Analysis ...... 34

Results ...... 35

Determination of Ingredient(s) in CS Extracts Active in Modulating Macrophages Response

...... 35

Uninfected Cells...... 35

Infected Cells ...... 36

In Absence of CS Extracts and Nicotine...... 36

In Presence of CS Extracts and Nicotine ...... 36

Effect of Nicotine on Macrophages Response is Dose-Dependent ...... 36

Effect of Nicotine on Macrophages is Mediated through α7nAChRs ...... 37

viii

In Absence of Infection ...... 37

During Infection ...... 38

Pre-Conditioning Macrophages with Nicotine Protects against Infection-Induced

Inflammation ...... 39

Prior to Infection ...... 39

Nicotine Increases MAP Burden in Macrophages and Decreases Cellular Apoptosis ...... 40

Discussion ...... 41

Figures...... 47

Tables ...... 54

Reference ...... 55

CHAPTER THREE: NICOTINE MODULATES MYD88-DEPENDENT SIGNALING

PATHWAY IN MACROPHAGES DURING MYCOBACTERIAL INFECTION ...... 62

Introduction ...... 62

Materials and methods ...... 65

Culture Conditions of THP-1 Macrophages ...... 65

Infection and Treatment of THP-1 Macrophages ...... 65

Measurement of Relative Gene Expression Using RT-PCR ...... 66

Measurement of Cell Receptors ...... 67

MAP Viability Assay ...... 67

Statistical Analysis ...... 68

Results ...... 69

TLR2/TLR4 Expression During Infection in Macrophages ...... 69

Nicotine Modulates TLR2/TLR4 Expression in Macrophages during infection ...... 69

Nicotine/TLR2 Interaction Modulates MyD88 Signaling in Macrophages During Infection

...... 70

Antagonism of TLR4 Abrogates Macrophages Response Against Gram Negative Bacterial

Infection ...... 71

Antagonism of MyD88 or TLR2 Alters The Level of Macrophages Response During

Infection ...... 71

Effect of Nicotine on MyD88 Signaling in Macrophages During Infection ...... 72

The Role of Nicotine and TLR-2 /MyD88 Signaling in MAP Survival in Macrophages .... 73

Discussion ...... 73

Figures...... 79

Tables ...... 85

References ...... 88

CHPTER FOUR: CONCLUSION/FUTURE DIRECTIONS ...... 94

APPENDIX: CONSENTS FOR PUBLICATION...... 97

LIST OF FIGURES

Figure 1:Effect of multi-factorial interaction and nicotine on IBD pathogenesis...... 14

Figure 2:Immune Recognition and Phagocytosis of MAP in CD Macrophages...... 14

Figure 3:Nicotine Activates the Anti-Inflammatory Response in UC Macrophages...... 15

Figure 4:Schematic Illustration of the Effect of Cigarette Smoke and Nicotine on Modulating

Macrophage Polarization in IBD Patients...... 16

Figure 5:Effect of cigarette smoke (CS) and pure nicotine on Mycobacterium avium subsp. paratuberculosis (MAP)-infected macrophages...... 47

Figure 6:Nicotine induces dose dependent M2 macrophage polarization in vitro...... 48

Figure 7:Mecamylamine (MEC) abrogates the immunosuppressive effect of nicotine in vitro. .. 49

Figure 8:Effect of nicotine on macrophage response and infection...... 50

Figure 9:Interplay between nicotine, α7nAChR, and mecamylamine (MEC, an nAchR antagonist) in macrophages...... 51

Figure 10:Effect of nicotine preconditioning on infected macrophages...... 52

Figure 11:Effect of nicotine on apoptosis in macrophages post infection...... 52

Figure 12:Schematic illustration of the role of cigarette smoke (CS) and nicotine in IBD...... 53

Figure 13: Effect of nicotine on the TLR2/TLR4 expression on infected macrophages...... 79

Figure 14: Effect of nicotine on MyD88 signaling and subsequent IL-8 expression in macrophages during infection...... 80

Figure 15: TLR4 antagonist abrogates the macrophage response against LPS but not MAP: M1,

M2, TNF-α, and IL-8 studies...... 81

Figure 16: TLR2 antagonist decreases the macrophage response against MAP infection: M1, M2,

TNF-α, and IL-8 studies...... 82

Figure 17: MyD88 antagonist cancels the macrophage response against MAP infection: M1, M2,

TNF-α, and IL-8 studies...... 82

Figure 18: Effect of nicotine/MAP infection on Inflammatory response following TLR2 and

MyD88 inhibition in vitro...... 83

Figure 19: Effect of nicotine on MAP survival in macrophages blocked with TLR2/MyD88

Antagonists...... 83

Figure 20: Schematic illustration the proposed pathway of MAP recognition and inflammatory gene regulation in macrophages during infection in presence of nicotine treatment ...... 84

xii

LIST OF TABLES

Table 1: List of Clinical Studies investigating the Effect of Tobacco Smoking on CD patients. 17

Table 2: List of Clinical Studies investigating the Effect of Smoking on UC patients...... 18

Table 3:Effect of nicotine on bacterial viability in macrophages...... 54

Table 4: Toll-Like Receptors (TLRs) and their signaling pathways ...... 85

Table 5: Receptor antagonists names, functions and their molecular structures ...... 86

Table 6: RT-PCR primer sequences for interested genes ...... 87

xiii

LIST OF ACRONYMS/ABBREVIATIONS

α7nAChR: α7-nicotinic acetylcholine receptor

Ach: Acetylcholine

AIEC: Adherent Invasive Escherichia coli

CD: Crohn’s Disease

CS: Cigarette Smoke

CFU: Colony Forming Units

DAMPs: Damage-Associated Molecular Patterns

DNA: Deoxyribonucleic Acid

FBS : Fetal Bovine Serum

HLE-nicotine rich: Havana-Leave Extract

IBD: Inflammatory Bowel Disease

IFN-γ: Interferon Gamma

IL-1 β: Interleukin 1β

IL-2: Interleukin 2

IL-6: Interleukin 6

IL-8: Interleukin 8

IL-10: Interleukin 10

IL-12: Interleukin 12

IL-23: Interleukin 23

JAK: Janus Kinases

K. pneumonia: Klebsiella pneumoniae

xiv

LAM: lipoarabinomannan

LAMD-nicotine less: germplasm line of Maryland tobacco

LPS: Lipopolysaccharide

MAP: Mycobacterium avium subspecies paratuberculosis

MEC: mecamylamine miRNA: microRNA

MIC: Minimum Inhibitory Concentration

M. tuberculosis: Mycobacterium tuberculosis

MyD-88: Myeloid differentiation primary response-88

NF-ҝB: Nuclear Factor kappa-light-chain-enhancer of activated B cells

NOD2: Nucleotide-binding oligomerization domain-containing protein 2

OR: Odds Ratio

PAMPs: Pathogen-Associated Molecular Patterns

PBS: Phosphate Buffer Saline

PknG: Protein Kinase G

PMA: Phorbol 12-Myristate 13-Acetate

PRRs: Pattern-Recognition Receptors

PTP-A: Protein Tyrosine Phosphatase-A

RA: Rheumatoid Arthritis

RPMI: Roswell Park Memorial Institute Medium

RT-PCR: Real Time PCR

STAT-3: Signal Transducer and Activator of Transcription 3

TB: Tuberculosis

TE: Tris-EDTA

TGF-β: Transforming Growth Factor beta

TLR: Toll-Like Receptor

TNF-α: Tumor Necrosis Factor Alpha

UC: Ulcerative Colitis

UCF4: University of Central Florida strain 4

UV: Ultraviolet

xvi

CHAPTER ONE: BACKGROUND

Note: This section has been published in part and the citation link is: AlQasrawi, D., Qasem, A., & Naser, S. A. (2020). Divergent Effect of Cigarette Smoke on Innate Immunity in Inflammatory Bowel Disease: A Nicotine-Infection Interaction. International Journal of Molecular Sciences, 21(16), 5801.

Overview of Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic condition that involves severe inflammation in the lining of the digestive tract [1]. IBD which includes Crohn’s disease (CD) and Ulcerative colitis

(UC) occurs more commonly in developed countries with an average incidence of 10 per 100,000 persons [1]. IBD general symptoms include weight loss, bloody diarrhea, anemia, fever, rectal bleeding, severe ulceration and loss of appetite, which ultimately lead to poor quality of life [2,3].

The pathogenesis of IBD is still not fully understood. However, there are compelling evidence that support the association of IBD in genetically susceptible persons with environmental triggers including infection [4]. There are at least 200 genetic loci that have been identified as targets and may increase susceptibility to IBD [4]. For instance, variants of HLA haplotypes as HLA class II allele DRBI*0103 which have been strongly associated with UC, whereas Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) polymorphisms have been linked to CD [5].

NOD2 is the primary receptor responsible for intracellular bacterial recognition and clearance in tissue [5]. Other factors have been specifically associated with CD pathogenesis, and not UC, such as environmental triggers including diet, UV light exposure, vitamin D deficiency, infection, and tobacco smoking. Infection is well established as key complication in CD and not in UC pathogenesis [6]. Opportunistic enteric infections and zoonotic-linked pathogens have been reported exclusively in CD patients [4,7,8]. Among the most investigated pathogens in CD

1 etiology is Mycobacterium avium subspecies paratuberculosis (MAP) [7,8]. This fastidious acid- fast bacillus is responsible for CD- like disease in ruminants with Johne’s disease [9]. On the other hand, tobacco smoking has been reported to have contradictory effects on IBD patients [10]. It alleviates the symptoms and provides protective effect in smokers with UC. The opposite is true in CD smokers; hence, it causes detrimental effect and exacerbates the symptoms [10,11]. Such contradictory effects among IBD patients was initially reported in a 1982 study by Harries et al., who observed a protective effects of tobacco smoking in UC patients and was harmful in patients with CD [10]. This observation has been confirmed and became well accepted by clinicians. The latter now recommend nicotine replacement therapy as a treatment option for some UC patients

[10,11]. Smoking tobacco not just always exacerbates symptoms in CD patients, it also causes higher risk of relapse than non-smokers with CD [10,11].

To understand this dogma in IBD subsets, our research team has been investigating the effects of cigarette smoke and nicotine on the cellular and molecular changes in CD- and UC-like macrophages. AlQasrawi et al., reported recently that they were able to mimic macrophages similar to those in CD and UC patients with history of prior or active cigarette smoking, and studied cellular response in presence and absence of active bacterial infection [12]. Among the findings, they elucidated how nicotine in UC active smokers activates the cholinergic anti-inflammatory pathway through α7-nicotinic acetylcholine receptor (α7nAChRs). This caused shift toward M2- macrophage polarization, which resulted in upregulation of anti-inflammatory cytokines

Interlukin-10 (IL-10), and downregulation of pro-inflammatory cytokines (IL-6 and tumor necrosis factor alpha (TNF-α)). They reported that decrease in caspase-3 activity was key factor in macrophage modulation toward anti-inflammatory phenotypic response [12]. On the other hand,

2 the effect of nicotine in macrophages infected with MAP, M. tuberculosis, or Klebsiella pneumoniae was contrary to that observed in UC-like macrophages [12]. These findings confirm the school of thought that bacterial infection plays a key role in CD pathogenesis. So, this study will focus more on interplay between infection and smoking and their effects on IBD patients.

Cigarette Smoke is Detrimental in Crohn’s Disease

Many studies strongly support smoking as an independent risk factor of CD and even may affect the severity of the disease [Table 1]. CD patients who smoke or start smoking after diagnosis have higher numbers of relapses requiring greater immunosuppression that can lead to hospitalization, in comparison to patients who quit smoking or never smoked [11,13]. This was reported first in

1984 by Holdstock et al. by analyzing 172 variables [14]. They reported that active CD smokers were more likely to be hospitalized due to a higher number of relapses and increased disease severity [14]. Another study reported the association of smoking with increased risk of relapsing in 152 CD patients (smokers vs. non-smokers) which suggested a twofold increased relapse rate among smokers as compared to non-smokers [15]. Other researchers investigated the effect of dose and duration of smoking in CD patients [16,17]. In a thirteen-years long study of almost 3000 CD patients, the harmful effect of smoking on CD patients was dose dependent causing heavy smokers to be at higher risk of needing immunosuppressants [16]. In this study, they divided CD patients into three groups: non-smokers, light smokers (1–10 cigarettes per day), and heavy smokers (> 10 cigarettes per day), then they determined the annual disease activity and immunosuppressant requirement for each group [16]. Lindberg et al. investigated 231 CD patients and reported that heavy smokers (> 10 cigarettes per day) had an increased risk for surgery as well as higher risk of

3 further surgeries 10 years after diagnosis compared to non-smokers [17]. Limited studies reported contradictory observations where active CD smokers showed no negative effects as a result of CS

[18].

Genetics and/or ethnicity factors have been reported in the literature to influence the degree of how smoking impacts CD severity. Aldhous and Satsangi reported that there was no association between smoking and CD susceptibility among Jewish population [19]. Another study reported a higher percentage of smokers with CD in French Canadian populations compared to other

Caucasian populations [19]. Most recently, researchers adapted different criteria for better CD stratification such as Vienna and Montreal classifications which depend on disease location and behavior [19]. Using these criteria, some suggested that smoking can progress CD stage toward fistulas or strictures formation while the location of the disease remains stable [20]. Another study reported that smoking has a strong association with the development of penetrating profile in non- colonic CD [21]. On the other hand, several studies reported a lack of association between smoking and CD location and behavior [22]. Overall, all studies that found a direct link between smoking and CD concluded that smoking is more related to non-colonic and severe CD cases, whereas non- smoking or light smoking may be associated with colonic and benign CD.

Cigarette Smoke is a Protective Factor in Ulcerative Colitis

Paradoxically, smoking has been a favorable and protective environmental association in patients with UC [10]. This protective association between smoking and UC was first noted in 1976 by

Samuelsson et al. who attributed this observation to interactions with medication [23]. Five years later, Harries et al. confirmed the protective effect of smoking in UC patients by analyzing data

4 from 230 UC patients and 192 CD patients. They concluded that only 8% of UC patients were current smokers compared to 42% of CD patients [24]. They reported that 44% of UC patients and

27% of CD patients were ex-smokers [24]. A British study in 1998 involving 51 UC patients established that smoking has a positive effect on UC [25]. Further supporting evidence of the beneficial effects of smoking on UC were revealed by a subsequent study which concluded that resuming smoking in low doses can be used as a medication instead of steroids in ex-smokers with refractory UC [26]. A recent Hungarian study of 1420 IBD patients, including 914 UC patients and 506 CD patients, found that the effect of smoking on IBD is linked to gender and age, and it is most prominent in young adults [27]. They also showed that the prevalence of smoking is 14.9% in UC patients and 47% in CD patients [27]. This study was in line with a meta-analysis by Mahid et al. who confirmed the protective role of smoking on UC, with a 0.85 odd ratio (OR) of current smokers, as compared to lifetime non-smokers [28]. Further studies investigating the relationship between smoking and UC observed that smoking tends to be dose-dependent in UC patients

[29,30]. A study examining 499 UC patients found that relatively heavy smokers with UC have healthier colons and less histological evidence of inflammation in colonoscopy examinations than light smokers [29]. Prior to that, a Japanese study also revealed a significant dose-response relation between smoking and UC among Japanese people [30]. Subsequent studies have confirmed the observation that current smokers with UC have less relapses, reduced need for immunosuppressant therapy, and fewer hospitalizations, compared to ex-smokers and non-smokers with UC [23–30]

[Table 2].

Nicotine Replacement:

Interestingly, different nicotine replacement forms have been recently recommended by clinicians to help UC patients. This includes transdermal patches, chewing gum, and nicotine- based enemas [31]. In a study where 72 active UC patients were treated with either transdermal nicotine patches or placebo patches for six weeks, they found that transdermal nicotine replacement was significantly more effective than the placebo at reducing the severity of UC. They also reported serious side effects such as headache, nausea, sleep disturbance, and acute pancreatitis [32]. However, using nicotine enema or oral capsule formulations significantly decreased these side effects [33]. Another study claimed that chewing nicotine gum exerted the same positive influence on UC severity as observed in current smokers [34].

The Contradictory Effect of CS on IBD subsets is due to Nicotine-multifactorial interaction

Nicotine and the other factors

Tobacco leave extracts contain roughly 4500 components, and around 150 of those have been labeled as harmful to human health such as dioxins which is known to be both carcinogenic and an immunomodulator [10]. Heavy metals such as cadmium and arsenic are also considered to be carcinogenic in addition to their role in the development of gastric ulcers [35]. Despite all the carcinogenic or toxic effects of these components in human health, nicotine remains the most active agent in tobacco which is thought to have the greatest effect. Nicotine is an alkaloid found naturally in the roots and leaves of the nightshade family of plants, such as Nicotiana tabacum.

Although nicotine itself is not toxic, it is considered highly addictive component of tobacco [12].

This may explain why most recent studies are mainly focused on establishing the role of nicotine in IBD pathogenesis.

Despite the extensive studies about the involvement of nicotine in IBD pathogenesis, the mechanisms by which nicotine modulates immune response in CD and UC require more elucidation. We propose that these mechanisms require a multi-factorial interaction between nicotine and the immune system, dysbiosis leading to infection, or epigenetic as illustrated in

Figure 1.

Nicotine and Immune Response:

Cytokines

Nicotine is known to have a suppressive effect on both innate and humoral immune systems [12].

Many studies showed that nicotine inhibits TNF-α release from mononuclear cells isolated from healthy volunteers, while it stimulates the secretion of IL-10, which is known as an anti- inflammatory cytokine [12]. The inflammatory process in CD has been looked at predominantly as a humoral response by activation of Th1/Th17 cells, which in turn converts the inflammatory response to be more harmful [3]. Moreover, smoking leads to an elevation of pro-inflammatory cytokines including TNF-α, interferon gamma (IFN-γ), IL-23, IL-6 and IL-1β, both in the gut and peripheral blood of CD patients [3]. Consequently, this exacerbates inflammation and triggers CD symptoms [3]. Meanwhile, the effect of nicotine on innate immunity (monocyte/macrophage) in

CD remains to be elucidated.

α7nAChR in Immune Cells

It is well-known that nicotine activates cells through α7nAChR, which is responsible for the activation of cholinergic anti-inflammatory pathway [12]. The presence of α7nAChR on the cell surface of monocytes and macrophages adds to the role of nicotine in innate immune response

[12,36]. Infection triggers acetylcholine (Ach) release by the vagal nerve, which binds with α7- nAChR on the surface of macrophages and subsequently interferes with the production of pro- inflammatory cytokines [37]. Surprisingly, nicotine has the same effect as that of vagal nerve stimulation leading to anti-inflammatory response by shifting the macrophage polarization toward

M2, weakening the production of pro-inflammatory cytokines and increasing the secretion of anti- inflammatory cytokines as well [12,37]. Moreover, activation of α7nAChR in CD4+ CD25+ regulatory T-cells is responsible for an immunosuppressive effect by producing IL-2 and downregulating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ҝB) [37]. To confirm this, other studies claimed that the presence of α7nAChR in endothelial cells may help in reduction of chemokines and adhesion molecules during inflammation [38]. These findings may explain again the cholinergic anti-inflammatory protective effects of nicotine among UC patients.

However, limited studies have looked at how the innate immune response interacts with the overall inflammatory process in IBD, and the role of nicotine in macrophage recruitment, proliferation, and differentiation in CD vs. UC.

Microbial Dysbiosis:

The gut microbial diversity and load have been studied in IBD. Dysbiosis occurs in IBD as defined by decrease in gut microbial diversity with shifting the balance between beneficial and

8 pathogenic bacteria, which may lead to aberrant immune response in genetically susceptible individuals [39]. In CD patients, enteric dysbiosis is common, which is represented by significant reduction in commensal phyla such as Firmicutes and Actinobacteria, with a higher presence of proteobacteria phyla [40]. Many studies have looked to the role of microbial dysbiosis in initiation of pathogenic infection such as Clostridium difficile in CD [41]. However, Further investigation is needed to elucidate the effect of disturbance of intestinal microbial equilibrium in inducing MAP infection in CD. Among UC patients, gut dysbiosis has been also reported, but the bacterial diversity was not disrupted as much as in CD [40]. Many recent studies have demonstrated the role of the gut microbiome as a link between smoking and CD [10,42,43]. Significant increases in

Bacteroides-Prevotella abundance in smoker CD patients, compared to non-smokers have been detected through fluorescent in situ hybridization using 16S rRNA sequencing [42]. On the other hand, smoking cessation resulted in a high abundance of Firmicutes (Clostridium coccoidos,

Eubacterium rectale, and Clostridium leptum), Actinobacteria (high guanine and cytosine content bacteria (Propionibacteriaceae & Bifidobacteria)), and a decrease in Bacteroidates (Prevotella spp. and Bacteriods spp.) [43]. This observation may prove that nicotine withdrawal plays a major role in the shifting of microbial composition towards the composition of healthy individuals.

Epigenetic Susceptibility:

The term “epigenetic” refers to heritable changes of gene expression events, which is caused independently of genetic information from the primary DNA sequence [44]. The main epigenetic mechanisms include DNA methylation, histone modification, as well as microRNA (miRNA) [44].

Recently, many studies proposed that epigenetic mechanisms could improve our understanding of

IBD, which may lead to new insights of treatment options [44].

Noncoding MicroRNAs (miRNAs) pathways such as miRNA-18b (miR-18b), miRNA-140

(miR-140), and miRNA-124 (miR124) explains one of the epigenetic mechanisms by which nicotine regulates gene expression of pro-inflammatory cytokines [45]. miR-124 was claimed to be a critical mediator of inflammation which negatively controls its progression [45]. In many cases, it has been shown that nicotine can specifically upregulate miR-124 levels via α7nAChR in macrophages during sepsis [45]. Subsequent studies demonstrated that nicotine could exert an anti- inflammatory response in DSS colitis (resembling UC) by elevating miR-124 levels [46].

However, elevation of mir-124 in TNBS-induced experimental colitis (resembling CD) was found to worsen the inflammation while blocking miR-124 could alleviate the symptoms [47].

Nicotine has been shown to be responsible for miR-124 elevation in samples in both UC and

CD, while the latest has contradictory effect in the two diseases [48]. Recently, a study by Qin et al., has also demonstrated that miR-124 might mediate the bivalent effect of nicotine on IBD by shifting the Th1/Th2 balance toward Th1 [48]. Consequently, miR-124 could be a suitable target for the contrasting treatment of IBD; up-regulation in UC and down-regulation in CD. Overall, a broad knowledge about the role of nicotine in epigenetic modifications in IBD patients is still lacking. However, this could offer novel treatment options for IBD patients, considering miRNAs as potential therapeutic targets.

Differences in Immunological Response Profile between Crohn’s Disease and Ulcerative

Colitis

One of the most debated explanation for the contradictory effect of nicotine is the association of

CD with microbial infection, which lacks in UC. Among the most discussed pathogens associated with CD is MAP [7,8,49–58].

MAP is an intracellular pathogen capably of modulating the innate immune response in CD leading to significant production of pro-inflammatory cytokines [7]. It was reported that monocytes and macrophages in CD patients express Toll-Like Receptor (TLR) at higher levels compared with UC patients [59]. TLRs are pattern recognizing receptors in innate immune cells where the cytoplasmic domain is linked to Myeloid differentiation primary response-88 (MYD-

88). The latter activates transcription factors such as NF- ҝB to produce pro-inflammatory cytokines. Although there are at least 10 types of TLR, TLR2 binds to MAP through the cell wall lipoarabinomannan (LAM) which initiate the activation of the NF- ҝB pathway and production of pro-inflammatory cytokines [60]. Different types of receptors are responsible for MAP uptake such as scavenger, mannose receptors as well as complement receptors, which are mainly responsible for internalizing opsonized MAP. TLRs are considered as the sole receptors for immune response and activation of the pro-inflammatory pathway by recognition of lipoarabinomannan in the surface of macrophages mimicking CD macrophages. Figure 2 shows illustration of how MAP and its cell wall components interact with TLR during phagocytosis and inflammatory response.

The effect of nicotine on IBD patients is clearly demonstrated through changes to gastrointestinal tissue. However, the effect of nicotine and smoking on respiratory tract is well established. The

11 similarity between intestinal inflammatory response in CD smokers and others with pulmonary inflammation is well studied and reported in the literature. Specifically, the effect of smoking on tuberculosis and exacerbating of infection has been well elucidated [40,12]. For example, TB smokers are known to have higher levels of alveolar macrophages compared to non-smoker and former TB smokers [61,62]. Bai et al. demonstrated that nicotine impairs anti-MTB defense leading to increase survival and burden of the bacterium in macrophages through α7nAchR by decreasing apoptosis and activation of the NF-ҝB family [63]. We reported similar finding when we studied the effect of nicotine on macrophages infected with related microorganisms such as

MAP [12]. We validated our findings in CD- and UC-like macrophages and our conclusion should help explain the contradictory effect of smoking on IBD subsets.

Several mechanisms may explain the protective effect of nicotine on innate immunity in UC.

First, nicotine’s effect is mediated by activation of the cholinergic anti-inflammatory pathway via

α7nAChR, on macrophages, monocytes and dendritic cells [40]. This causes a shift in macrophage polarization towards M2 and elevation of IL-10 and decrease pro-inflammatory cytokines levels such as IL-6, IL-12 and TNF-α [38]. Other studies reported that T cells possess α7nAChR, which can be activated by nicotine in Th2 cells and Treg cells, therefore inducing them to produce IL-10 and TGF-β [40]. Second, nicotine was also found to induce production of miR-124, which has an inhibitory effect on pro-inflammatory cytokine production in LPS-induced macrophages [45].

MiR-124 inhibits the translation of TNF-α converting enzyme, therefore preventing the conversion of pro-TNF-α to TNF-α [45] [Figure 3].

Summary and Outlook

Activation of α7nAChRs downregulates the production of pro-inflammatory cytokines, including TNFα, IL-6 and IL-1β [64]. The downstream signaling to these receptors in macrophages involves NF-kB pathway, whereas in T cells anti-inflammatory effect requires further investigation, although stimulation of α7nAChRs in T cells showed anti-inflammatory effects

[38,65]. Several epidemiological studies showed a clear lower correlation between smokers and the incidence of UC in particular [66]. Nicotine was found as the active ingredient in CS, which is responsible for this immunosuppressive mechanism [67]. On the other hand, CS was associated with higher incidence of numerous inflammatory disorders including Rheumatoid Arthritis (RA), atherosclerosis, peptic ulcer disease and CD [66]. This complex effect of nicotine in inflammation is not well understood, but we speculate that the presence of certain pathogens will shift the anti- inflammatory role of nicotine to pro-inflammatory factor.

In conclusion, CS plays a contradictory role in IBD subsets; it is a risk factor in CD development and provides protection in UC. Our most recent in vitro study was the first to provide significant insights toward understanding the cellular events involved in the mysterious divergent effects of

CS ingredients on IBD patients [12]. Nicotine exacerbates MAP infection in macrophages in CD patients causing shift toward M1 polarization and excessive production of pro-inflammatory cytokines. The opposite is true in UC; nicotine activates the cholinergic pathway providing anti- inflammatory effect which provides a treatment option for many of UC patients. Figure 4 illustrates the effect of nicotine on macrophage modulations in IBD smokers. Studying the molecular process and the crosstalk between TLR signaling and miR-124 will further help in understanding why CS is beneficial to UC patients and harmful to those with CD.

Figures

Figure 1:Effect of multi-factorial interaction and nicotine on IBD pathogenesis.

Figure 2:Immune Recognition and Phagocytosis of MAP in CD Macrophages.

Figure 3:Nicotine Activates the Anti-Inflammatory Response in UC Macrophages.

Figure 4:Schematic Illustration of the Effect of Cigarette Smoke and Nicotine on Modulating Macrophage Polarization in IBD Patients.

Tables

Table 1: List of Clinical Studies investigating the Effect of Tobacco Smoking on CD patients.

Study Reference & Published National Type of Study Retrospective/ Number of Main Conclusion

Year Origin Prospective Participants

Holdstock et al (1984) [14] UK Case-Control Study Retrospective 172 IBD patients Smoking leads to CD rather than UC

Somerville et al (1984) [13] UK Case-Control Study Retrospective 82 variables CD patients are more likely to smoke

Timmer et al (1998) [15] Canada Cohort Prospective 152 CD patients Increased rate of relapses in CD smoker patients

Lindberg et al (1992) [17] France Case-Control Study Retrospective 231 CD patients CD heavy smokers have an increased risk of surgery

Seksik et al (1995-2008) [16] France Cohort Prospective 3000 CD patients Smoking has a dose-dependent effect on CD patients

Van der Heide et al (2009) [18] Netherland Case-Control Study Retrospective 820 CD patients No unfavorable effects of active smoking on CD

Table 2: List of Clinical Studies investigating the Effect of Smoking on UC patients.

Study Reference National Type of Study Retrospectiv Number of Participants Main Conclusion

Origin e/Prospective

Samuelsson et al (1976) [23] Sweden Unknown Unknown Unknown Low rate of smokers among UC patients

Harries et al (1982) [24] UK Cohort Prospective 230 UC patients Low rate of smokers among UC patients

Nakarnura et al (1994) [30] Japan Case-Control Study Retrospective 384 UC patients The relationship between smoking and UC is dose-

dependent

Green et al (1998) [25] UK Cohort Prospective 51 UC patients UC is a non-smoker disease

Aldhous et al (2007) [29] UK Case-Control Study Retrospective 499 IBD patients UC heavy smokers have healthier colons than UC

light smokers

Calabrese et al (2012) [26] US Case-Control Study Retrospective 15 UC patients Low doses of smoking can be used as a medication

in ex-smoker refractory UC patients

Lakatos et al (2013) [27] Hangiri Cohort Retrospective 1420 IBD patients Smoking prevents colectomy in UC smokers

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CHAPTER TWO: MYSTERY SOLVED: WHY SMOKE EXTRACT WORENS DISEASE IN SMOKERS WITH CROHN’S DISEASE AND NOT ULCERATIVE COLITIS? GUT MAP!

Note: This section has been published in part and the citation link is: AlQasrawi, D., Abdelli, L. S., & Naser, S. A. (2020). Mystery Solved: Why Smoke Extract Worsens Disease in Smokers with Crohn’s Disease and Not Ulcerative Colitis? Gut MAP!. Microorganisms, 8(5), 666.

Introduction

Cigarette smoke (CS) is considered an independent risk factor in the development of Crohn’s disease (CD) [1–3]. It is associated with exacerbated inflammatory relapses, postoperative recurrence, and poorer response to medical therapy [1–3]. A standard clinical practice for newly diagnosed CD patients is to quit CS to avoid the aforementioned debilitating consequences [4,5].

Generally, CS causes CD smokers to suffer from exacerbated inflammatory symptoms, severe phenotypical presentation of the disease, and in many cases leads to the patient’s hospitalization and poor long-term prognosis [6–9]. On the contrary, CS seems to be surprisingly protective in patients with ulcerative colitis (UC), which is largely known as the disease of the non-smokers

[3,5]. Inflammatory symptoms in UC are significantly decreased when tobacco is consumed, while smoking cessation results in abrupt flares of inflammation and more frequent relapsing episodes in these patients [10]. UC patients with history of smoking usually acquire their disease few years after they stop smoking, suggesting that smoking protected them from acquiring the disease earlier

[11]. Unlike UC, the etiology of CD has been vigorously researched and debated and now it is generally accepted that CD is associated with microbial infection, immune-dysregulation, and mucosal dysfunction. Among the most debated pathogens that have been studied for possible roles in CD pathogenesis are Mycobacterium avium subsp. paratuberculosis (MAP), adherent-invasive

Escherichia coli (AIEC), and Klebsiella pneumoniae [12–14]. The mechanism of how CS ingredients induce pro-inflammatory or anti-inflammatory phenotypes in IBD remained unclear.

This study is not about what causes CD; it is about understanding the mechanism involved in how

CS affects macrophages infected with microorganisms associated with CD pathogenesis. The monocytic response against intracellular bacterial infection such as MAP is usually mediated by

TLR2 receptor through the recruitment of cytoplasmic adaptor protein and activation of intracellular signaling molecules, which lead to phagosome maturation and synthesis of pro- inflammatory cytokines like TNF-α, IL-6, IL-8, IL-12, and IL-23 [15,16].

Nicotine, an addictive immunosuppressant ingredient in CS, is known to decrease the level of pro-inflammatory cytokines, inhibit dendritic cells, and prevent cell apoptosis [17,18]. Nicotine also decreases the production of cathelicidin, anti-microbial peptide, and inhibits formation of granuloma [19,20]. The concentration of nicotine in active cigarette smokers ranges between 4 and

72 ng/mL [21]. Although smokeless tobacco products, including e-cigarettes and smoking cessation nicotine-replacement products, are labeled as somewhat safe, the level of nicotine in the user’s blood can exceed the level achieved by cigarette smoking [22]. Tobacco-use is the leading cause of preventable disease and death in the United States [23]. Nearly half a million Americans die prematurely due to smoking or secondhand smoking exposure [23]. Another 16 million

Americans live with serious illnesses caused by cigarette smoking [5]. Nicotine as a potent alkaloid is considered to be the most addictive and pharmacologically active substance among the many compounds present in tobacco products.

Limited studies have investigated how the innate immune response interplays in the overall inflammatory process involved in IBD and what effects nicotine has on macrophage recruitment,

29 proliferation, and phenotypic response in CD and UC subsets. One clear link between innate immune cells and CS is the presence of a nicotinic acetylcholine receptor α7 (α7-aAChR) on monocytes and macrophages cell surface [24]. Experimental studies have demonstrated that nicotine inhibits pro-inflammatory cytokine release from in vitro monocytes-based cell culture

[24]. However, this does not explain the conflicting effects of CS on UC and CD symptoms.

Generally, monocytes respond to direct stimuli by differentiating into classical pro-inflammatory

M1 cells or alternative anti-inflammatory M2 macrophages [25]. M1 releases IL-1b, MCP-1, iNOS, IL-6, IL-8, IL-12, IL-23, and TNF-α, while M2 macrophages release IL-10, IL-1Ra, and higher levels of arginase-1. The latter inhibits nitric oxide production and reduces inflammation

[25]. This study is designed to study the mechanism of how CS or its most abundant and active ingredient, nicotine, modulates α7aAChR on macrophages infected with intracellular pathogens and elicit inflammatory response. Specifically, the study focused on investigating the effect of CS and nicotine on the cellular processes in THP-1 infected with MAP, or LPS derived from AIEC, mimicking CD smokers associated with bacterial infection.

Materials and Methods

Culture Conditions of THP-1 Macrophages

THP-1 monocytes (ATCC TIB-202) were cultured as described earlier [26]. Briefly, cells were maintained in a RPMI-1640 growth medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Invitrogen) and 0.09% β-mercaptoethanol and maintained in a humidified 5% CO2 incubator at 37 °C until they reached 80% confluency. Then, cells were activated by adding 50 ng/mL of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St.

Louis, MO, USA) for 48 h. All experiments were performed in quadruplicates and done within 12 passages.

Infection and Treatment of THP-1 Macrophages

Infection: activated THP-1 cells (M0 macrophages) were plated at a density of 3 × 105 cells in

7 12 well plates. Cells were infected for 24 h at 37 °C in 5% CO2 with 1 × 10 CFU/mL of MAP strain UCF4 (a clinical strain isolated from CD patient), Mycobacterium tuberculosis ATCC

HR237, or Klebsiella pneumoniae ATCC13883. Bacterial Lipopolysaccharide (LPS) from

Escherichia coli strain O111.B4 (Sigma Aldrich, 1 µg/mL) and uninfected cells were used as a control.

Treatment with CS extracts and nicotine: activated THP-1 macrophages were treated with nicotine (0 to 4 µg/mL, Sigma-Aldrich), 0.18% CS extracts from HLE-Petit Havana (tobacco leaves rich with nicotine), or 0.18% germplasm line of Maryland tobacco (LAMD, tobacco leaves with nicotine-less) for 24 h at 37 °C in 5% CO2. HLE and LAMD tobacco leaves were generously provided by Professor Henry Daniel (University of Pennsylvania). 31

Nicotine Receptor Inhibitor: To determine the role of α7nAchR in the cellular uptake of CS and nicotine, macrophages were treated with mecamylamine (MEC), an α7nAChR antagonist (Fisher

Scientific). Macrophages were pretreated with 0, 25, 50 and 75 µM of MEC at room temperature for 30 min prior to nicotine or CS treatments. All experiments were performed in quadruplicates.

Measurement of Relative Gene Expression Using RT-PCR

RNA Isolation: cell pellets were collected in 2 mL-microcentrifuge and suspended in 500 µL of

TRIzol ® reagent (Invitrogen). A total of 125 µL of chloroform was added to each tube, mixed, and incubated at room temperature for 5 min. Following centrifugation at 10,000 rpm for 5 min, the aqueous phase was transferred to a new tube and mixed with 275 µL isopropanol. Following centrifugation at 14,000 rpm and 4 °C for 15 min, RNA pellets were isolated, washed in 500 µL of 75% ethanol, air-dried, and then dissolved in 15 µL of Tris-EDTA (TE) buffer. RNA concentration and purity were analyzed using Nonodrop OneC (ThermoFisher) and stored at –80

°C until further use.

cDNA Synthesis: Reverse transcription reaction was carried out by adding 800 ng of RNA in

0.25-mL microtubes containing 20 µL of PCR reaction, 4 µL iScript™ Reverse transcription (Bio-

Rad®, Hercules, CA, USA) and up to 0.2 mL RNAse-free water. The reaction was performed using MyGene Series Peltier Thermal Cycler under the following conditions: 5 min at 25 °C,

20 min at 46 °C, and 1 minute at 95 °C.

RT-PCR: Gene expression analysis was performed as described earlier [27]. Briefly, a total volume of 20 µL reaction mixture containing 1 µL of cDNA (30 ng/µL), 10 µL of Fast SYBR

Green Mastermix (Thermo Fisher Scientific, Waltham, MA, USA), 1 µL of either iNOS, CD206,

IL-6, TNF-α, or IL-10, PrimePCR SYBER Green Assay mixes (Bio-Rad®, California, USA), and

8 µL of RNAse-free water were prepared in a 96-well Microamp RT-PCR reaction plate and analyzed using 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, United

States). Housekeeping GAPDH primer (Bio-Rad®, California, USA) was used to obtain baseline

CT readings. Relative mRNA expression was calculated by using the equation 2(−∆CT), where ∆CT

= [(Sample RT-PCR CT value) − (GAPDH CT baseline value)] × 1000.

Measurement of Cytokines and Cell Surface Markers

For cell surface markers, cell pellets were incubated with ice RIPA buffer (Thermo-Fisher, Cat#

89901, Massachusetts, USA) for 15 min, then centrifuged at 14,000 rpm for 20 min. Supernatant containing cell lysates were analyzed using ELISA kits: iNOS (LS-Bio; Washington, USA, Cat#

LS-F39142), CD206 (Sigma-Aldrich; Cat# RAB1178, Missouri, USA) following manufacturer’s instructions. Cell culture supernatants were used to measure cytokine levels: IL-6 (LS-Bio; Cat#

LS-F9754, Washington, USA), TNF-α (LS-Bio; Cat# EH3TNFA, Washington, USA), and IL-10

(Sigma-Aldrich; Cat# KHC0101, Missouri, USA). All ELISA experiments were done in triplicates.

Bacterial Viability Assay

Following the infection, macrophages were washed twice with PBS to remove extracellular bacteria, then lysed using cold RIPA buffer (Thermo-Fisher, Cat# 89901, Massachusetts, USA) for 15 min. Cell lysates were centrifuged at 14,000 rpm for 20 min and the supernatant was collected to perform bacterial viability using the Live/Dead Baclight Bacterial Viability Kit

(Molecular Probes, Inc., Eugene, OR, USA), as described earlier [26]. All viability experiments were performed in triplicates.

Measurement of Macrophage Apoptosis

In order to examine the apoptotic pathway induced by nicotine, caspase-3 colorimetric assay

(Abcam, Cambridge, UK) was used as described earlier [26]. Briefly, following infection and treatment, THP-1 cells were washed twice with PBS, collected and lysed using chilled Cell Lysis

Buffer. Total protein levels were analyzed using DEVD-pNA as a caspase-3-specific substrate.

After incubation at 37 °C for 1–2 h, absorbance was measured at 400–405 nm in a SpectraMax i3x plate reader. All apoptosis experiments were done in duplicate (n = 2) with each sample loaded in triplicate and the average was calculated.

Statistical Analysis

All data collected in this study were pre-tested for normal distribution using the Kolmogorov–

Smirnov normality test followed by Unpaired Two-tailed t test. p < 0.05 and a 95% confidence interval (CI) were used for the assessment of differences in all experiments. Data were presented as Mean ± SD. p-values <0.001 were also mentioned when achieved.

Results

Determination of Ingredient(s) in CS Extracts Active in Modulating Macrophages Response

In order to determine the active ingredient(s) in tobacco leaves that modulate inflammatory response in macrophages, we measured M1 (iNOS) and M2 (CD206) markers, IL-6, TNF-α, and

IL-10 expression in PMA-activated THP-1, and macrophages infected with MAP following a treatment with CS extracts from HLE-Petit Havana (nicotine-rich) and LAMD (nicotine-less). We estimated that 0.18% of HLE-Petit Havana contains 4 µg/mL nicotine [28]. Accordingly, we used

0.18% HLE-Petit Havana extracts in parallel to pure nicotine (4 µg/mL) and 0.18% of LAMD tobacco. Experiments were also performed with LPS-derived from AIEC. MTB was included as control.

Uninfected Cells

Although the effect of CS extracts from both HLE and LAMD on iNOS, CD206, IL-6, and TNF-

α was mostly similar (Figure 5A–D), expression of IL-10 was significantly low in LAMD-treated cells (0.22 ± 0.021; p < 0.05) compared to HLE-treated cells (0.40 ± 0.033; p < 0.05; Figure 5E).

Likewise, the effect of nicotine on iNOS, IL-6, and TNF-α was not significant, but it increased the expression of CD206 (3.84 ± 0.06 vs untreated cells 2.46 ± 0.08; p < 0.05; Figure 5D) and IL-10

(0.64 ± 0.020 vs untreated 0.46 ± 0.020; p < 0.05; Figure 5E).

Infected Cells

In Absence of CS Extracts and Nicotine

Infection of macrophages with MAP increased iNOS expression (13.09 ± 0.94 vs 5.11 ± 0.8 in uninflected cells), IL-6 (38.43 ± 1.2 vs 18.5 ± 1.8 in uninfected cells), and TNF-α (0.54 ± 0.075 vs

0.27 ± 0.08 in uninflected cells).

In Presence of CS Extracts and Nicotine

We also evaluated the effect of CS extracts from both HLE and LAMD, as well as nicotine on macrophages infected with MAP, and the outcome was surprisingly unexpected (Figure 5). CS extracts from HLE and nicotine both further increased iNOS expression in MAP-infected macrophages compared to CS extracts from LAMD and uninfected cells (HLE: 20.49 ± 0.4; nicotine: 15.64 ± 1.05; LAMD: 11.76 ± 1; and uninfected: 13.09 ± 0.94; p < 0.05; Figure 5A). The outcome was validated by a measurement of corresponding cytokines. Expression of IL-6 and

TNF-α increased in MAP-infected macrophages and subsequently treated with CS extracts of HLE or nicotine compared to CS extracts of LAMD and untreated cells (HLE: 59.8 ± 1.4; nicotine: 44.5

± 1.5; LAMD: 28.5 ± 2; and uninfected: 18.5 ± 1.8; p < 0.05 for IL-6) and (HLE: 2.7 ± 0.04; nicotine: 2.4 ± 0.05; LAMD: 1.7 ± 0.07; and uninfected: 0.27 ± 0.0.08; p < 0.05 for TNF-α; Figure

1B–C). The effect on CD206 and IL-10 expression was not significant (Figure 5D,E).

Effect of Nicotine on Macrophages Response is Dose-Dependent

The effect of nicotine dosage on macrophage polarization and response was investigated.

Activated THP-1 cells were treated with ascending concentrations of nicotine (0, 2, and 4 µg/mL)

36 for 24 h followed by RT-PCR and ELISA evaluation of the expression and production of iNOS and CD206 markers. As shown in Figure 6 (A: gene expression, and B: protein levels), iNOS (M1) gradually decreased and CD206 (M2) increased in the dose-dependency of nicotine (p < 0.05).

Based on these two markers’ gene expression, the percentage of M1, M2, and undifferentiated M0 macrophages were calculated in presence and absence of nicotine. Untreated cells were differentiated into M1:M2 macrophages with a ratio of 9:1 of M1:M2 (calculated from black bars in Figure 2A). Figure 6C depicts how nicotine treatment shifts this ratio. In fact, nicotine treatment at 2 µg/mL starts to shift polarization with a ratio of 42% M1 vs 34.5% M2 and 23.5% M0 cells while 4 µg/mL nicotine further increased M2 macrophages to 50.09% against 32.34% M1 and around 17.57% undetermined M0 cells. Gene expression and protein levels of IL-6, TNF-α, and

IL-10 were also illustrated in Figure 6D, E, respectively. Data indicate that an ascending concentration of nicotine correlates with decreased IL-6 and TNF-α gene and protein expression, which is reflective of the decreased M1 polarization marker iNOS. Accordingly, anti-inflammatory

IL-10 gene expression and not protein significantly increased with 2 and 4 µg/mL nicotine treatment.

Effect of Nicotine on Macrophages is Mediated through α7nAChRs

In Absence of Infection

To determine if nicotine modulates macrophages and shifts polarization to anti-inflammatory response is mediated through α7nAChRs, we blocked nAchR with mecamylamine (MEC), an nAchR antagonist. Specifically, activated THP-1 macrophages were pretreated with 0, 25, 50 and

75 µM MEC, followed by 4 µg/mL of nicotine. As shown in Figure 7A, in absence of MEC,

37 nicotine significantly decreased the iNOS level (6.25 ± 0.32 compared to 9.5 ± 0.25 in macrophages not treated with nicotine; p < 0.05). Preconditioning cells with MEC (25 to 75 µM) gradually neutralized the effect of nicotine on iNOS and reached its optimum effect at 50 µM

(Figure 7A). A similar effect was also observed on TNF-α expression (Figure 7B). Likewise, pretreatment of macrophages with MEC has altered the effect of nicotine on CD206 (MEC 50 µM

+ nicotine 4 µg/mL: 29.97 ± 2.45 vs MEC 50 µM: 25 ± 1.81 vs MEC 0 µM control: 30.5 ± 2.82; p < 0.05), Figure 7C, and IL-10 (MEC 50 µM + nicotine: 6.47 ± 0.56 vs MEC 50 µM: 6.5 ± 0.56 vs MEC 0 µM control: 7.8 ± 0.84; p < 0.05), Figure 7D.

During Infection

To explain why smokers with CD experience heightened inflammatory episodes compared to non-smoking CD patients or UC patients, we infected activated THP-1 with MAP for 24 h followed by treatment with 4 µg/mL nicotine. MTB and LPS were also used as controls. As expected, all infections resulted in a significant increase in pro-inflammatory status as evidenced by increased expression of iNOS, IL-6, and TNF-α (Figure 8A–C). Interestingly, adding nicotine treatment to infected macrophages almost doubled iNOS readings across all infections (MAP + nicotine: 55.7 ± 2.02 vs MAP alone: 22.94 ± 2.87), (MTB + nicotine: 77.15 ± 1.6 vs MTB alone:

29.81 ± 0.81. p < 0.05), or (LPS + nicotine: 80.39 ± 1.83 vs LPS alone: 35.16 ± 2.04; p < 0.05).

The significance effect was also observed when compared with untreated and uninfected control cells; p < 0.001. A similar trend was observed for IL-6 and TNF-α gene expression (Figure 8B,C).

It is worth noting that the effect of MTB on IL-6 was far superior to that of MAP or LPS (MTB:

19 ± 0.5 vs MAP 10.14 ± 0.5 vs LPS 11.8 ± 0.6. p < 0.05), Figure 8B. However, the increase in

IL-6 level following treatment with nicotine of infected macrophages was similar among MAP,

MTB, and LPS. There was a significant decrease in the expression of the anti-inflammatory CD206 marker following nicotine treatment of infected macrophages (p < 0.05), Figure 8D. IL-10 showed a clear decrease in expression following nicotine treatment of infected macrophages, but it was not statistically significant (Figure 8E).

To validate that the effect of nicotine on infected cells is mediated through α7nAChRs, we blocked nAchR in macrophages with MEC. Treatment with MEC seems to cancel the nicotine effect with iNOS expression levels more aligned with those found with MAP infection only (MAP

+ MEC + N: 67.9 ± 1.42 vs MAP: 71.22 ± 1.54. p < 0.05, Figure 9A). Pro-inflammatory cytokines

IL-6 and TNF-α also reflected the iNOS results, with MAP infection enhancing their expression, nicotine to further increase their expression, and MEC to cancel the nicotine effect back to a MAP inflammatory status (Figure 9B,C for IL-6 and TNF-α, respectively). Anti-inflammatory marker

CD206 and IL-10 cytokine also demonstrated a significant increase in gene expression upon nicotine treatment of uninfected macrophages (nicotine: 191.16 ± 2.4 vs control: 116.81 ± 2.6. p

< 0.05) for CD206. Interestingly, MEC pre-conditioning prevented nicotine-induced CD206 and

IL-10 gene expression; (114.5 ± 2.2 vs 191.16 ± 2.4. p < 0.05) for CD206 and (79.8 ± 2.1 vs 148.53

± 3.2. p < 0.05) for IL-10 (Figure 9D,E, respectively).

Pre-Conditioning Macrophages with Nicotine Protects against Infection-Induced Inflammation

Prior to Infection

Due to the bivalent effect of nicotine in IBD, we wondered whether the timing of infection in

IBD smokers could determine the direction of the inflammatory shift. To address that, we

39 evaluated the inflammatory profile of macrophages pre-conditioned with nicotine before infection with MAP (nicotine exposure prior infection) versus macrophages that were infected with MAP.

MTB and LPS were used as controls. As shown in Figure 10A, the iNOS level was significantly lower in cells preconditioned with nicotine prior to infection (MAP: 22.9 ± 2.8 vs nicotine + MAP:

17.17 ± 1.5. p < 0.05), (MTB: 29.8 ± 0.8 vs nicotine + MTB: 11.35 ± 1.7. p < 0.05), or (LPS: 35.16

± 2.04 vs nicotine + LPS: 18.06 ± 1.4); p < 0.05). Similarly, IL-6 and TNF-α gene expression results (Figure 10B,C, respectively) corroborated those of iNOS with the lowest values consistently reported for cells preconditioned with nicotine prior to infection. M2 cell surface marker CD206 and IL-10 cytokine, on the other end, showed their highest expression levels (p < 0.05) in cells pre-exposed to nicotine prior to infection (Figure 10D,E, respectively).

Nicotine Increases MAP Burden in Macrophages and Decreases Cellular Apoptosis

To determine MAP viability and cellular apoptosis in macrophages treated with nicotine prior and post infection, activated THP-1 cells were infected with MAP for 24 h, then treated with nicotine (4 µg/mL), and vice versa. As listed in Table 3, nicotine-maintained viability and increased the burden of MAP and MTB regardless of the timing of exposure to nicotine prior or post infection. For MAP: nicotine + MAP 91.4 ± 5.5, MAP alone 69.4 ± 5.2, and MAP + nicotine

80.79 ± 4.8. For MTB: nicotine + MTB 92.5 ± 4.6, MTB alone 74.2 ± 3.1, and MTB + nicotine

85.2 ± 6.2. K. pneumoniae was used as a negative control and the viability was the minimum as expected.

For apoptosis, active caspase-3 activity increased in MAP infected macrophages when compared to control, which was similar to macrophages infected with MTB or treated with LPS (Figure 11).

The data also show that the activity of caspase-3 in macrophages was lower in nicotine treated macrophages after MAP infection (MAP: 4.81 ± 0.28 vs MAP + nicotine: 3.22 ± 0.05. p < 0.05).

A similar trend was also observed for MTB and LPS.

Discussion

More studies are needed to elucidate the role of innate immune response in the overall inflammatory process in IBD smokers. Limited studies have investigated the effect of CS or nicotine on macrophage recruitment, proliferation, and consequent immune response among smokers with CD or UC. Extensive studies reported a strong association between microbial infection and CD pathogenesis; the opposite is true for UC [29]. Infection of macrophages with intracellular microorganisms, such as MAP or AIEC, CD-associated pathogens, leads to pro- inflammatory response and increase in M1/M2 ratio [30]. On the contrary, nicotine binds to

α7nAChR on monocytes and the macrophages cell surface and inhibits pro-inflammatory cytokine release leading to a decrease in M1/M2 ratio [31,32]. Extensive studies have investigated the effect of CS in patients with tuberculosis. Most reports have demonstrated a strong link between smoking and increased susceptibility to tuberculosis (TB) infection [33]. For example, studies reported that

TB smokers have higher levels of alveolar macrophages compared to non-smoker and former smokers with TB, which may explain the abnormal innate immune response in these patients

[33,34]. Since there is strong homology between MAP and MTB, and due to the fact that MAP is associated with CD but not UC, we stipulated that the effect of CS on CD smokers might be similar to that of tuberculosis. The nicotine-rich HLE extracts induced macrophages to an anti- inflammatory response due to significant upregulation in IL-10. The opposite effect was observed

41 when macrophages where treated with the nicotine-less LAMD extracts. The finding supports earlier observations which suggested that nicotine is a potent agent in tobacco [34,35]. The data clearly confirm that the anti-inflammatory effect observed in CS is due to nicotine. We were astonished as to how macrophages infected with MAP, or MTB, or even treated with LPS responded to exposure to nicotine. We anticipated nicotine to reduce the inflammatory response caused by the bacterial infection or the LPS derived from AIEC. Instead, the inflammatory response increased as indicated by the upregulation of iNOS, IL-6, and TNF-α. On the other hand,

CD206 and IL-10 were downregulated (Figure 8D,E). The effect of nicotine, the most active and potent ingredient in CS, on infected macrophages is clearly opposite to that seen in uninfected macrophages. In absence of infection, nicotine acted as an immunosuppressive drug, increasing

M2 macrophages and IL-10 cytokine (Figure 10); however, upon MAP infection, the anti- inflammatory role of nicotine was lost to a robust pro-inflammatory response even higher than that observed with infection only (Figure 8). This data is reflective of what is observed in IBD patients.

Knowing that some CD patients might be suffering from infection with MAP, AIEC, or possibly other microorganisms, which is not the case in UC patients, we propose that the successive inflammatory response among some CD smokers is due to bacterial infection associated with the disease. Dysbiosis and microbiota shifts have been reported in CD patients, which lead to an alteration in the proportion of bacteroides, firmicutes, and enterobacteriaceae [14]. In this study, we included MTB as a positive control in order to confirm the expected effect of nicotine on macrophages in TB patients. We also included LPS in order to investigate the effect of gram- negative bacteria, including AIEC and K. pneumoniae that also have been associated with CD

42 pathogens. The data clearly confirm that the successive inflammatory response observed in CD smokers is related to the association of the disease with bacterial infection.

Unlike the common knowledge, also confirmed in our study, about the anti-inflammatory effect of nicotine, the true effect of nicotine on macrophages depends greatly on the presence or absence of infection. A lack of infection in patients with UC should explain why they benefit from CS exposure as reported extensively in the literature [35]. In fact, nicotine is currently offered as a therapeutic agent for UC; present in many forms, such as nicotine patches, chewing gum, as well as nicotine-based enemas, because of its anti-inflammatory proprieties [36,37]. The opposite is true in CD smokers who often suffer from bacterial infection.

Further evidence to support the outcome of this study is the use of MTB as a control. The effect of nicotine or HLE extracts on MTB infection was strongly similar to that of MAP infection. Data in our study strongly support published reports indicating TB smokers to suffer greatly from increased symptoms compared to TB non-smokers [38]. We cannot exclude that the presence of oxidative and highly reactive compounds other than nicotine in CS may contribute to the successive pro-inflammatory response observed in infected macrophages [39].

To confirm and validate our conclusion, we proceeded to determine the mechanism by which nicotine modulates macrophages. First, nicotine in this study increased the viability of MAP in

THP-1 cells (Table 3). This confirms earlier reports from MTB studies which demonstrated that nicotine impairs anti-MTB defense within macrophages through α7nAchR, by decreasing apoptosis via activation of the NF-ҝB family—this ultimately leading to an increased MTB burden, thereof, worsening TB symptoms [33,38]. Among the virulence factors in Mycobacteria that are involved in their survival and persistence include Protein Kinase G (PknG) (an enzyme which

43 inhibits phagosome–lysosome fusion) and Protein Tyrosine Phosphatase-A (PTP-A, an enzyme that inhibits phagosomal maturation) [40]. Based on the close relatedness between MTB and the fact that MAP data in this study are similar to MTB, we propose that MAP survival in macrophages follows a mechanism similar seen in MTB. On the other hand, the viability of K. pneumoniae in macrophages was poor and most likely due to the lack of these key virulence factors. Clearly, the impact of increase in MAP and MTB viability in infected macrophages has caused these immune cells to release pro-inflammatory cytokines, including IL-6 and TNF-α, as demonstrated continuously and exponentially in this study (Figure 8). Interestingly, we were able to link the upregulation in these cytokines to the polarization of macrophages into M1 as demonstrated by upregulation of iNOS marker. Second, we observed a significant decrease in macrophages apoptosis upon nicotine treatment, which further supports the idea that nicotine increases mycobacterial viability by reducing the host’s cell death (Figure 11). Third, nicotine exerts its immunosuppressive effect via α7nAChRs on macrophages and other immune cells. These receptors are responsible for the activation of cholinergic anti-inflammatory pathway leading to decreased secretion of pro-inflammatory cytokines, such as IL-6 and TNF-α in vitro and in vivo which confirms earlier reports [41]. Our study confirmed the immunosuppressive effect of pure nicotine in THP-1 cells by shifting the polarization to alternative M2 macrophages and increased the production of anti-inflammatory cytokine IL-10. Our study further established that abrogation of nicotine immune modulatory effect depends on the blockage of its receptor with MEC, a pharmacological inhibitor to nAchRs (Figure 3). This data supports an earlier report regarding the downregulation of α7nAChRs expression in inflamed CD tissues [32].

Moreover, we investigated whether pre-conditioning macrophages with nicotine would confer protection against infection. Our results indicate that pretreatment of infected macrophages with nicotine significantly inhibits the production of pro-inflammatory cytokines (Figure 10B,C).

Similar findings were reported by a previous study showing that nicotine pretreatment significantly decreased IL-6, TNF-α, and IL-12 in Legionella infected macrophages [42]. In addition, pre- exposing cells to nicotine induces M2 polarization, which may not be canceled or reversed back to M1 status following infection (Figure 10). We plan to investigate the ability of M2 to reverse to

M1 and vice versa.

One limitation of this study is that it is an in vitro culture system in which THP-1 cells, an immortalized monocyte-like cell line, was used to mimic CD-like macrophages and perhaps peritoneal macrophages. We plan to further validate our findings in cells from CD and UC patients including peritoneal macrophages and peripheral mononuclear cells. Additionally, using an in vitro model system as in this study may suppress the true effect occurring in vivo. Future studies should involve investigations of the effect of nicotine and CS and possibly vaping in animals infected with CD-associated pathogens.

In summary, using an in vitro cell culture system, we were able to mimic a macrophages setup similar to those in CD and UC patients with history of prior or active cigarette smoking and in correlation with active bacterial infection. The effect of CS on the cellular processes in macrophages similar to those in UC active smokers, CD active smokers, and CD non-smokers are illustrated in Figure 8. Nicotine in UC active smokers activates the cholinergic anti-inflammatory pathway through α7nAChRs leading to M2-macrophage polarization, upregulation of CD206 and

IL-10, downregulation of iNOS, IL-6, and TNF-α, and significantly decreases caspase-3 activity

(Figure 8A). Similar cellular changes are expected in CD active smokers who are not associated with infection (Figure 12A). On the contrary, nicotine in CD active smokers associated with bacterial infection induce M1-macrophage polarization, upregulation of iNOS, IL-6, and TNF-α, downregulation in CD206, IL-10, decrease in caspase-3 activity, and increase in infection burden

(Figure 12C). CD non-smokers with bacterial infection suffer also from pro-inflammatory response but at a lower level (Figure 12B) compared to CD smokers with bacterial infection

(Figure 8C). Cellular apoptosis is always reduced in macrophages exposed to nicotine.

Overall, this is the first study to provide significant insight toward understanding the cellular events involved in the mysterious contradictory effect of CS among IBD patients with different history of smoking. We plan to investigate the effect of CS ingredients on the molecular processes involved in bacterial infection and interaction with nicotine receptors in macrophages mimicking that of IBD patients.

Figures

Figure 5:Effect of cigarette smoke (CS) and pure nicotine on Mycobacterium avium subsp. paratuberculosis (MAP)-infected macrophages.

Figure 6:Nicotine induces dose dependent M2 macrophage polarization in vitro.

Figure 7:Mecamylamine (MEC) abrogates the immunosuppressive effect of nicotine in vitro.

Figure 8:Effect of nicotine on macrophage response and infection.

Figure 9:Interplay between nicotine, α7nAChR, and mecamylamine (MEC, an nAchR antagonist) in macrophages.

Figure 10:Effect of nicotine preconditioning on infected macrophages.

Figure 11:Effect of nicotine on apoptosis in macrophages post infection.

Figure 12:Schematic illustration of the role of cigarette smoke (CS) and nicotine in IBD.

Tables

Table 3:Effect of nicotine on bacterial viability in macrophages.

Treatment Group Viability Fold Change in Viability Prior/Post Mean ± SD Nicotine Treatment (%) K. pneumoniae ATCC 3.7 ± 4.5 13883 MAP UCF4 69.4 ± 5.2 N(4 µg/mL) + MAP UCF4 91.4 ± 5.5 1.31 MAP UCF4 + N(4 µg/mL) 80.79 ± 4.8 1.17 MTB HR237 74.2 ± 3.1 N(4 µg/mL) + MTB HR237 92.5 ± 4.6 1.25 MTB HR237 + N(4 µg/mL) 85.2 ± 6.2 1.15 N: nicotine. MAP: Mycobacterium avium paratuberculosis. MTB: Mycobacterium tuberculosis.

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CHAPTER THREE: NICOTINE MODULATES MYD88-DEPENDENT SIGNALING PATHWAY IN MACROPHAGES DURING MYCOBACTERIAL INFECTION

Introduction

Crohn’s Disease (CD) is one of the relapsing chronic diseases that affects the gastrointestinal tract. Although the etiology of CD is still vigorously debated, mounting evidence at the basic and clinical research levels has highlighted and strongly associated CD with microbial infection, immune-dysregulation, and mucosal dysfunction. Among the most debated pathogens that have been studied for their possible role in CD pathogenesis are Mycobacterium avium subspecies paratuberculosis (MAP), adherent-invasive Escherichia coli (AIEC), and Klebsiella pneumoniae

[1-4]. The immune response in patients with CD is unique with clear involvement of both Th1/Th2 signaling leading to humoral and innate immune response. Innate immunity is considered an early warning system against any pathogenic attachment where macrophages play a crucial role in recognizing bacteria through their own cellular receptors such as pattern-recognition receptors

(PRRs). PRRs are highly expressed in macrophages and are involved in recognition of both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns

(DAMPs) [5]. Furthermore, toll-like receptors (TLRs) are members of the PRR family that have been identified as a central hub of pathogen recognition which results in the immune response [6].

TLRs are found in most mammals and each of them has specific transmembrane proteins with their own recognition patterns [7].While most TLRs are located on cell surface which play important role in recognition of microbial derivatives, TLR3, TLR7, and TLR9 are localized in the endosomes and bind mainly to microbial nucleic acids [8]. TLRs recognize their ligands as heterodimers in TLR1/TLR2 and TLR6/TLR2, homodimers in TLR3, TLR4, TLR5 and TLR9,

62 and monomers in TLR7 and TLR8 (Table 4). Table 4 summarizes key TLRs and their location and functions.

Recently, TLRs have been causally linked to play a role in the pathogenesis of inflammatory bowel disease (IBD). Specifically, macrophages in CD patients seem to have high expression of

TLRs compared to those in patients with Ulcerative Colitis (UC). We believe the difference in macrophage TLRs expression level among IBD subsets is most likely due to the association of

CD with intracellular infection. MAP is the causative agent of CD-like disease in animals and has been widely reported to be involved in more than 50% of CD cases [2, 10]. It is still unclear which

TLRs are involved in Mycobacterial infection. However, mycobacterial cell wall components such as lipoarabinomannan (LAM), similar to LPS in gram-negative bacteria, have been suggested to interact with TLRs during infection [11].TLR9 has been reported to recognize and interact with mycobacterial DNA in phagosomes following phagocytosis [11-13]. On the other hand, TLR4 is confirmed to play a role in recognition of gram-negative bacteria through interaction with LPS

[12].

TLR activation affects downstream signaling pathways including major adaptor proteins such as myeloid differentiation primary response 88 (MyD88) [14]. All TLRs, except TLR3, require

MyD88 to mediate signal transduction pathways in response to inflammatory triggers in macrophages [14]. MyD88-dependent signaling pathway was reported to be similar to IL-1 receptor pathways in activation of IRAK-1, leading to activation of the MAP-kinase and NF-κB pathways [14-16]. We speculated that there is a link between the high expression of TLRs in macrophages with MyD88-dependent pathway and the pro-inflammatory response during

63 infection in Crohn’s disease. If confirmed, the outcome should support a microbial etiology in CD pathogenesis.

Autophagy is an effector mechanism used by immune cells to eliminate intracellular bacteria

[17]..And it has been reported to occur during Mycobacterium tuberculosis infection. Recent studies reported an interplay between vitamin D-dependent antimicrobial response, TLR2, and the autophagy pathway leading to enhanced macrophages response against M. tuberculosis infection

[17-20]. Specifically, they reported that 1,25D3 induces the production of cathelicidin and activation of the autophagy pathway in MTB-infected macrophages resulting in protective immune response against M. tuberculosis infection [11]. Given the 90% homology between MTB and MAP, we proposed that TLR2-signaling pathway play a critical role in the macrophage response during MAP infection in CD.

As we reported recently, cigarette smoke (CS) and nicotine exacerbate inflammation in CD smokers and provide a protective anti-inflammatory response in UC smokers [21]. Specifically, we demonstrated that successive inflammatory response in CD smokers is due to the presence of

MAP infection in these patients [21,22]. We also elucidated the role of α7nAChR in mediating nicotine effect in MAP-infected macrophages [21]. We reported that nicotine exacerbates MAP infection in macrophages by upregulation of iNOS, increasing M1/M2 ratio and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and IL-6 [21]. Despite the new findings by this group, there is a big gap in the literature about how nicotine modulates macrophage recognition patterns including TLRs and MYD88 signaling during mycobacterial infection. Specifically, it remains not understood how nicotine affects MAP recognition and infection in macrophages causing further inflammation and worsening symptoms in CD smokers. This study is focused on

64 the elucidation of the mechanistic role of nicotine in modulating TLR and MyD88-dependent signaling pathway in MAP-infected macrophages. We investigated the interplay between nicotine, and TLR2 and TLR4 for their reported role in infection by gram-negative and -positive bacterial infection, and subsequent effect on MYD88 signaling, inflammatory response, and bacterial load in infected macrophages.

Materials and methods

Culture Conditions of THP-1 Macrophages

…THP-1 monocytes (ATCC TIB-202) were cultured as described earlier [23]. Briefly, cells were maintained in RPMI-1640 (ATCC 30-2001) growth medium with 10% fetal bovine serum (FBS;

Sigma Life Science, St. Louis, MO). The cells then were maintained in a humidified 5% CO2 incubator at 37oC until they reached 80% confluency. Then, cells were activated by adding 50 ng/mL of phorbol 12-myristate 13-acetate (PMA; Sigma Life Science, St. Louis, MO) for 24 hours.

All experiments were performed in duplicates and done within eight passages.

Infection and Treatment of THP-1 Macrophages

Infection: activated THP-1 cells (M0 macrophages) were plated at a density of 3x105 cells in 12

o 7 well plates. Cells were infected for 24 hours at 37 C in 5% CO2 with 1x10 CFU/mL of MAP strain UCF4 (a clinical strain isolated from CD patient), M. tuberculosis ATCC HR237, or K. pneumoniae ATCC13883. Bacterial Lipopolysaccharide (LPS) from E. coli strain O111.B4

(Sigma Life Science, St. Louis, MO, 5µg/mL), and uninfected cells were used as a control.

Treatment with nicotine: activated THP-1 macrophages were treated with nicotine (Sigma Life

Science, St. Louis, MO, 4µg/mL). Cells were incubated for an additional 24 hours at the same conditions.

…Receptor Inhibitors: to determine the role of TLR and MyD88 in the cellular recognition of

MAP and it interplays with nicotine, activated THP-1 macrophages were pretreated with MMG11

(TLR2 antagonist; Bio-Techne, MN), TLR4-IN-C34 (TLR4 inhibitor; Sigma-Aldrich, St. Louis,

MO) and T6167923 (MYD88 inhibitor; Aobious, MA) (Table 5) separately at room temperature for 30 minutes before MAP infection. All experiments were performed in duplicates.

Measurement of Relative Gene Expression Using RT-PCR

RNA Isolation: After 24 hours treatment, cell pellets were collected in 2mL-microcentrifuge and then suspended in 500µL of TRIzol ® reagent (Invitrogen, Carlsbad, CA). A 125µL of chloroform was added to each tube, mixed and incubated at room temperature 5 minutes. Following centrifugation at 10,000 rpm for 5 minutes, the aqueous phase was transferred to a new tube and mixed with 275µL isopropanol. Following centrifugation at 14,000 rpm and 4°C for 15 minutes,

RNA pellets were isolated, washed in 500µL of 75% ethanol, air-dried, and then dissolved in 15µL of Tris-EDTA (TE) buffer. RNA was stored at -80oC until further use.

cDNA Synthesis: reverse transcription reaction was carried out by adding 800ng of RNA in 0.25- mL microtubes containing 20µL of PCR reaction; 4µL iScript™ Reverse transcription (Bio-Rad®) and up to 0.20mL RNAse-free water. The reaction was performed using MyGene Series Peltier

Thermal Cycler under the following conditions: 5 min at 25°C, 20 min at 46°C and 1 min at 95°C.

RT-PCR: gene expression analysis was performed as described earlier [22]. Briefly, a total volume of 20µL reaction mixture containing1µL of cDNA (30ng/µL), 10µL of Fast SYBR Green

Mastermix (Thermo Fisher Scientific, Waltham, MA), 1µL of forward primer , 1µL of reverse primer of either TLR2,TLR4,MyD88, IL-8, iNOS, MMR,TNF-α or IL-10 (Thermo Fisher Scientific,

Waltham, MA) (Table 6), and 7µL of RNAse-free water, were prepared in a 96-well Microamp

RT-PCR reaction plate and placed analyzed using 7500 Fast Real- Time PCR System (Applied

Biosystems, Foster City, CA). Housekeeping GAPDH primer was used to obtain baseline CT readings. Relative mRNA expression as fold change was calculated by using the equation 2(−∆∆CT), where ∆CT= [(Sample RT-PCR CT value) − (GAPDH CT baseline value)] whereas ∆∆CT=[

∆CTTreated - ∆CTUntreated ].

Measurement of Cell Receptors

For cell receptors, cell pellets were incubated with iced RIPA buffer (Thermo-Fisher, Waltham,

MA) for 15 minutes, then centrifuged at 14000 rpm for 20 minutes at 4°C . Supernatant containing cell lysates were analyzed using ELISA kits: TLR2 (RayBiotech, Norcross, GA) and TLR4

(RayBiotech, Norcross, GA) following manufacturer’s instructions. All ELISA experiments were done in duplicates.

MAP Viability Assay

To determine if TLR2 and MYD88 have a role in MAP viability in macrophages, THP-1 macrophages were blocked by either MMG (5µg/mL) or T6167923 (5µg/mL) followed by MAP infection. After 24 hours of infection, THP-1 macrophages were washed twice with PBS to remove extracellular bacteria, then THP-1 macrophages were collected at 3 time points: 0, 24, and 67

84 hours. The cells were lysed using iced RIPA buffer (Thermo-Fisher, Waltham, MA), and then the samples were centrifuged at 14,000 rpm for 20 min at 4°C. Supernatants were collected and the LIVE/DEAD™ BacLight™ Bacterial Viability Kit (ThermoFisher Scientific, Waltham, MA) was used according to the manufacturer’s protocol as described earlier [24]. Briefly, 100 µL of each sample was loaded into separate wells of a 96-well flat-bottom microplate in triplicate and mixed with 100 µL of fluorescent stain solution. The plate was then incubated for 15 min at room temperature (RT) in the dark. Fluorescence was measured using a microplate reader at 530 nm to determine the proportion of live bacteria (green), and at 630 nm to determine the proportion of dead bacteria (red). Data was analyzed by generating a standard plot using fluorescence measurements of five different proportions of live to dead MAP including 0:100, 10:90, 50:50,

90:10, and 100:0. A graph was generated, and the equation of the least-squares fit of the relationship between percent live bacteria (x) and green/red fluorescence ratio (y) was used to calculate bacterial viability of the MAP-infected macrophages. The experiments were done in triplicates.

Statistical Analysis

Statistical analysis was performed by using GraphPad Prism 7.02 software. Significance among experiments was assessed by Unpaired Two-tailed t test at p <0.05 and a 95% confidence interval

(CI). All data collected in this study were pre-tested for normal distribution using the Kolmogorov–

Smirnov normality test. Data is shown as (Mean ± SD). P-values <0.001 is also mentioned when achieved.

Results

TLR2/TLR4 Expression During Infection in Macrophages

In order to determine the effect of bacterial infection on TLR2/TLR4 expressions on macrophages, we infected PMA-activated THP-1 with MAP for 24 hours. MTB and LPS were also used as controls. Infection of macrophages with Mycobacteria increased TLR2 in gene and protein levels, respectively (MAP: 10.03 ± 0.64, MTB: 10.11 ± 1.02 vs uninfected: 1.20 ± 0.67; p

< 0.05 for gene; Figure 13A), (MAP: 10.42 ± 0.20, MTB: 11.71 ± 0.42 vs uninfected: 6.15 ± 1.06; p < 0.05 for protein; Figure 13B). It is worth mentioning that the effect of mycobacterial infection on TLR4 expression was not significant when compared with uninfected untreated cells (Figure

13C). On the other hand, LPS elevated the expression of TLR2/TLR4 on macrophage at the gene level. However, the significant effect was observed more in TLR4 when compared with the negative control (LPS: 14.54 ± 1.07 vs uninfected cells: 1.02 ± 0.23 ; p < 0.001) (Figure 13A and

C). Similar trend was also observed at the protein level (Figure 13B and D).

Nicotine Modulates TLR2/TLR4 Expression in Macrophages during infection

To determine if nicotine modulates the expression of TLR2/TLR4 in infected macrophages, we treated infected macrophages with 4µg/mL of pure nicotine for 24 hours followed by RT-PCR and

ELISA evaluation of the expression and protein levels TLR2/TLR4. As shown in Figure 13A, adding nicotine treatment to infected macrophages significantly decreased TLR2 expression across all infections (MAP + nicotine: 2.0 ± 1.04 vs MAP alone: 10.03 ± 0.64; p < 0.05), (MTB+ nicotine:

2.24 ± 1.14 vs MTB alone: 10.11 ± 1.02; p < 0.05), or (LPS + nicotine: 3.07 ± 1.40 vs LPS alone:

8.19 ± 1.01; p < 0.05). The findings were consistent with results from protein level analysis 69

(Figure 13B) (MAP + nicotine: 1.59 ± 1.13 vs MAP alone: 10.42 ± 0.20; p < 0.05), (MTB+ nicotine: 1.44 ± 1.10 vs MTB alone: 11.71 ± 0.42; p < 0.05), or (LPS + nicotine: 1.52 ± 1.10 vs

LPS alone: 20.45 ± 2.4; p < 0.05). Similarly, TLR4 expression and protein levels corroborated those of TLR2 with lowest values reported after nicotine treatment of infected cells (Figure 13C and D).

Nicotine/TLR2 Interaction Modulates MyD88 Signaling in Macrophages During Infection

To determine if MyD88 is the main adaptor protein in TLR2 signaling and to examine the effect of nicotine on MyD88 signaling and subsequent pro-inflammatory cytokine IL-8 in macrophage during infection, we measured expression of MyD88 and IL-8 following nicotine treatment of

MAP-infected macrophages. MTB and LPS were also used as controls. Compared with MAP infection alone, nicotine upregulated MyD88 and IL-8 expression, the latter showed more noticeable change by 2.5 folds (Figure 14A and B) (MAP + nicotine: 22.3 ± 2.09 vs MAP alone:

13.39 ± 1.80; p < 0.05, for MyD88 ), (MAP+ nicotine: 6.40 ± 0.52 vs MAP alone: 3.52 ± 0.62; p < 0.05, for IL-8). However, the significant elevation in MyD88 expression was observed in all mycobacterial infection with/out nicotine treatment compared to untreated uninfected cells, p <

0.05. Interestingly, there was no clear change in MyD88 and IL-8 expression when macrophages treated with LPS (Figure 14A and B).

Antagonism of TLR4 Abrogates Macrophages Response Against Gram Negative Bacterial Infection

To validate the role of TLR4 in macrophages response against bacterial infection, we blocked

TLR4 with TLR4-IN-C34, an TLR4 antagonist. Specifically, PMA-activated THP-1 macrophages were pretreated with 0, 2.5, 5 and 10 µg/mL of TLR4-IN-C34, followed by either treated with LPS or infected with MAP. In absence of TLR4-IN-C34, LPS significantly increased iNOS level (3.66

± 0.66 compared to 1.005 ± 0.125 in macrophages not treated with LPS; p < 0.05). Pretreated macrophages with (2.5 to 10 µg/mL) gradually neutralized the effect of LPS on iNOS level and reached its optimum effect at 5 µg/mL (Figure 15A). A similar trend was also observed on TNF-

α and IL-8 expressions (Figure 15C and D). Likewise, preconditioning cells with TLR4-IN-C34

(5 µg/mL) has increased M2-macrophages expression (MMR) (TLR4-IN-C34 (5 µg/mL) + LPS:

1.10 ± 0.14 vs LPS alone: 0.62 ± 0.06 vs TLR4-IN-C34 (5 µg/mL) alone: 1.12 ± 0.18 ; p < 0.05)

(Figure 15B). Surprisingly, there was a modest decrease in iNOS, TNF-α and IL-8 levels even with high concentration of TLR4-IN-C34 (10 µg/mL) with MAP infection (TLR4-IN-C34 (10 µg/mL)

+ MAP: 1.92 ± 0.30 vs MAP alone: 2.5 ± 0.25; p < 0.05, for iNOS) (TLR4-IN-C34 (10 µg/mL) +

MAP: 4.98 ± 0.45 vs MAP alone: 6.50 ± 1.90; p < 0.05, for TNF-α) (TLR4-IN-C34 (10 µg/mL) +

MAP: 2.67 ± 0.21 vs MAP alone: 3.58 ± 0.51; p < 0.05, for IL-8) (Figure 15A, C and D).

Antagonism of MyD88 or TLR2 Alters The Level of Macrophages Response During Infection

…We showed in the previous experiment that TLR4 has no role during MAP infection; thus, we examined the effect of TLR2 and its adaptor protein (MyD88) on macrophages response during

MAP infection. Activated macrophages were preincubated with 0, 2.5, 5, 10 or 20 µg/mL of either

MMG11 (TLR2 antagonist) or T6167923 (MyD88 inhibitor), followed by infected with MAP for

24 hours. Interestingly, MMG11 at 5 µg/mL attenuated the pro-inflammatory status in MAP- infected macrophages with greatest decrease was observed in TNF-α by almost 4 folds compared to MAP infected macrophages alone (MMG11 (5 µg/mL) + MAP: 2.33 ± 0.98 vs MAP alone: 6.50

± 1.90; p < 0.05) (Figure 16C).

On the other hand, 10 µg/mL of T6167923 is the optimum to cancel the macrophages immunity against MAP infection by decreasing the expression of TNF-α and IL-8 to levels similar to uninfected macrophages (T6167923 (10 µg/mL) + MAP: 1.15± 0.35 vs T6167923 (10µg/mL):

1.080 ± 0.27; p < 0.05, for TNF-α) (T6167923 (10µg/mL) + MAP: 1.10 ± 0.27 vs T6167923

(10µg/mL) : 1.25 ± 022; p < 0.05, for IL-8) (Figure 17C and D) and skewing macrophages toward alternative M2 phenotype (T6167923 (10 µg/mL) + MAP: 0.92 ± 0.35 vs MAP alone: 0.71 ± 0.10 vs T6167923 (10 µg/mL) alone: 0.90 ± 0.20 ; p < 0.05, for MMR) (Figure 17B).

Effect of Nicotine on MyD88 Signaling in Macrophages During Infection

To address the effect of nicotine on TLR2/MyD88 signaling in macrophages, we targeted macrophages with either 5 µg/mL of MMG11 (TLR2 inhibitor) or 10 µg/mL of T6167923 (MyD88 inhibitor) in presence and absence of MAP infection and then subjected the cells to nicotine treatment. While the synergy of MAP and nicotine exhibited the highest levels of iNOS (4.62 ±

0.59) and IL-8 (6.40 ± 0.51) compared to all other groups, blocking MyD88 or TLR2 subverted macrophages response against MAP by reduction iNOS and IL-8 expressions to level similar to uninfected macrophages regardless nicotine treatment (Figure 18A and B). The opposite effect was observed in anti-inflammatory profile, using TLR2/MyD88 antagonists before MAP infection leads to shift polarization toward M2 and increase IL-10 production (Figure 18C and D). It is worth

72 to mention that using MyD88 antagonist before MAP infection significantly boosted the immunosuppressive effect of nicotine on macrophages by increasing IL-10 production by 2.5 folds comparing with MAP infection alone.

The Role of Nicotine and TLR-2 /MyD88 Signaling in MAP Survival in Macrophages

We previously published that exposure of MAP-infected macrophages to nicotine maintains

MAP viability and increases its burden in macrophages [21]. Here we studied a possible role for

TLR2 or MYD88 in MAP survival in macrophages in presence and absence of Nicotine treatment.

MAP viability in macrophages treated with anti-TLR2 decreased to 63% but after 48 hours infection. Likewise, MAP viability in macrophages treated with anti-MyD88 decreased to 55% but after 48 hours infection (Figure 719). Nicotine treatment did not alter the viability of MAP after TLR2/MyD88 antagonists compared to no nicotine treatment (Figure 719).

Discussion

MAP, a slow growing intracellular bacterium capable of surviving within host macrophages, remains one of the most debated etiological agents of CD [10]. Macrophages play an essential role during the early immune response against MAP infection. Several pattern recognition receptors such as TLRs have been involved in Mycobacterial recognition in macrophages [17]. Among all cell surface TLRs, TLR2 and TLR4 have been identified to interact with pathogens through specific ligand binding [17]. On the other hand, nicotine, a lipophilic compound, is considered the most active ingredient in tobacco leaves [25]. We have reported recently that nicotine immunosuppressive properties in macrophages were lost when macrophages were infected with

MAP, M. tuberculosis or treated with LPS. In fact, in our study we showed that nicotine seems to exacerbate inflammatory response during infection [21]. These intriguing findings should explain the pro-inflammatory role of cigarettes smoking in patients with CD which is contrary to smokers with UC. This rational is based on the extensive literature that associates CD with microbial infection. Not much is found in the literature on the mechanism involved in how nicotine exacerbation of inflammatory response in infected macrophages. This led us to study a possible interaction between nicotine and TLR2/TLR4 in macrophages during infection.

It was estimated that each tobacco cigarette contains an average of 8.4 mg of nicotine and the average nicotine concentration in a smoker’s venous blood is approximately 2 µg/mL [26]. Of course, these estimates are subjective since smokers vary in how much cigarette smokes they use daily. Interestingly, 80-90% of nicotine is absorbed by most tissues that have high affinity for nicotine which results in several folds greater of nicotine concentration than in venous blood.

Moreover, the first-pass metabolism of nicotine in the liver contributes to 10 folds increase in nicotine concentration in atrial blood compared to venous blood [26]. In this study, we tested nicotine at different concentrations and up to 10 µg/mL. Our data demonstrated that the optimum activity was reached at 4 µg/mL in macrophages. The optimum concentration of nicotine was determined based on maximum activity in macrophages with least cytotoxic effect. We also studied the effect of nicotine at different time intervals ranged from 10 min to 48 hours. We determined that 24 hours of nicotine treatment to provide optimum response in macrophages especially when included in experiments that involve additional treatment steps such as infection or antagonists.

Recent reports suggested the involvement of TLR2 in MTB infection especially with cell wall derivatives [27, 28]. Other study showed that TLR2-deficient mice failed to develop immune response against MTB [29]. However, TLR4-deficient mice were active and responded properly during MTB infection [30]. In gram negative bacterial infection and due to LPS molecule, TLR4 was reported to play a role in recognition of enteric pathogens [28]. In this study, we investigated the interaction of MAP, a CD- associated pathogen, with both TLR2 and TLR4. We observed a significant increase in TLR2 expression and in consequent cytokines including TNF-α and IL-8 in MAP-infected macrophages, there was no change in TLR4 expression in the same cells (Figure

13). In macrophages treated with TLR-2 antagonist, we observed a significant decrease in expression of IL-8 and TNF-α (Figure 16). We concluded that MAP relies on TLR2 during infection at least in our macrophage system. In this study we also blocked TLR4 with TLR4-IN-

C34 antagonist which did not affect inflammatory response in MAP-infected macrophages which confirms that MAP interaction with TLR2 is key during infection. Similarly, there was limited immune response in LPS-treated macrophages blocked with TLR-4 antagonist which confirms that gram negative bacterial rely primarily on TLR4 during infection. This finding is consistent with observation by other group who reported that TLR-4 knockout mice are resistant to gram negative bacterial infection [31]. Together, we observed that blocking of TLR2 reduced macrophages response during MAP infection by decreased IL-8 and TNF-α in transcriptional level whereas there is a no role of TLR4 in MAP infection. (Figure 15 and 16).

In our efforts to understand the effect of cigarette smokes on CD patients, we investigated possible interaction between TLR2 and nicotine in CD-like macrophages. The latter included activated macrophages infected with MAP or MTB or treated with LPS to represent gram negative

75 bacteria. .As expected, we did not observe any change in the expression of either TLR2 or TLR4 in uninflected cells under nicotine exposure. However, in MAP-infected macrophages, nicotine has significantly decreased expression of both TLR2 and TLR4. Our results are consistent with similar observations in MTB-infected macrophages derived monocyte (MDMs) and in lung epithelial cells [28, 32]. The present study is the first to our knowledge to report the negative effect of nicotine on the expression of TLR2 and TLR4 in macrophages infected with MAP. The finding should provide significant insights toward understanding the effect of cigarette smoke on worsening inflammation symptoms in CD patients.

To understand how nicotine modulates TLR-2 in CD macrophages, we investigated TLR2 downstream signaling in infected macrophages under nicotine stress. Specifically, we characterized the effect of nicotine on MyD88 in association with TLR2 in presence and absence of infection. MyD88 was reported to play an essential role in the development of early innate immunity against acute MTB infection [33]. This was based on a defective immune response against MTB in MyD88-deficient mice [33]. In present study, MAP upregulated MyD88 expression and increased its specific correspondence of IL-8 (Figure 14). Surprisingly, nicotine has further increased MYD88 and IL-8 expression in MAP-infected macrophages. This suggest that other TLRs other than TLR2 or TLR4 may be involved in this response. We speculate that

TLR9 may be involved in this response based on earlier report in an MTB infection model [34].

Whether TLR9 is involved alone or in interaction with other factors remains unclear. We believe that TLR9 might interact with TLR2 because of a report by other investigators who observed a shared phenotypic characteristics of MTB infection in TLR2 and TLR9 double-deficient mice and of MyD88-deficent mice [35]. This study illustrates the crucial role of MyD88 in upregulation of

76 pro-inflammatory genes during MAP infection. Macrophages treated with MyD88 antagonist resulted in decreasing in IL-8 and TNF-α levels to level similar to uninfected cells (Figure 17). We also observed a synergistic effect between MyD88 antagonist and nicotine as illustrated by shifting macrophages polarization to M2 and increasing IL-10 during MAP infection. In the same manner nicotine decreased the iNOS and IL-8 levels to minimum after TLR2 blockade in the same cells

(Figure 18).

Collectively, these findings led us to propose that MAP-Nicotine interaction with TLR-2 during

MAP infection is MyD88-dependent, the opposite is true for TLR-4. Moreover, when TLR2 or

MyD88 were blocked with antagonists, there was no significant change in MAP load in macrophages even after 48 hours exposure to nicotine treatment (Figure 19). This observation suggests a key role for TLR2 and MyD88 in MAP recognition and cytokine production but not in

MAP uptake and internalization in macrophages.

While MAP and LPS represent most pathogens associated with CD pathogenesis, there is no doubt that individual microbiome content plays a major factor in CD episodes and faltering. CD have been intensively studied in regarding of gut microbiome-immune system interaction and it has been reported that CD patients have intestinal microbiome dysbiosis, representing by an increase of Enterococcus spp. and Bacteroides spp. with a decrease of Firmicutes (Clostridium coccoidos, Eubacterium rectale, and Clostridium leptum) and Actinobacteria (high guanine and cytosine content bacteria (Propionibacteriaceae & Bifidobacteria)) [36]. An interesting study observed a variation in TLR2 and TLR4 expression on lamina propria cells in CD patients.

Specifically, it showed an upregulation of TLR2 and TLR4 expression in inflammatory cells from lamina propria of active CD patients while during homeostasis, TLR2 and TLR4 are presented in

77 small amount suggesting that those receptors may be responsible of the balance between immunity and tolerance [37,38].

Overall, the data is intriguing about the role of TLR2 and MyD88 signaling in MAP infected macrophages in association with nicotine exposure ( Figure 20). More studies are needed to expand on our findings. Specifically, it is important to validate our findings in primary macrophages from

CD patients with and without MAP infection. Ultimately, it would be the most informative to validate our findings in mice during infection and under cigarette smoking conditions.

Figures

Figure 13: Effect of nicotine on the TLR2/TLR4 expression on infected macrophages.

Figure 14: Effect of nicotine on MyD88 signaling and subsequent IL-8 expression in macrophages during infection.

Figure 15: TLR4 antagonist abrogates the macrophage response against LPS but not MAP: M1, M2, TNF-α, and IL-8 studies.

Figure 16: TLR2 antagonist decreases the macrophage response against MAP infection: M1, M2, TNF-α, and IL-8 studies.

Figure 17: MyD88 antagonist cancels the macrophage response against MAP infection: M1, M2, TNF-α, and IL-8 studies.

Figure 18: Effect of nicotine/MAP infection on Inflammatory response following TLR2 and MyD88 inhibition in vitro.

Figure 19: Effect of nicotine on MAP survival in macrophages blocked with TLR2/MyD88 Antagonists.

Figure 20: Schematic illustration the proposed pathway of MAP recognition and inflammatory gene regulation in macrophages during infection in presence of nicotine treatment

Tables

Table 4: Toll-Like Receptors (TLRs) and their signaling pathways

TLR Functional architecture Location & their Ligands

TLR1 Heterodimer with TLR2 Extracellular binds with triacylated lipopeptide TLR2 Heterodimer withTLR1/6 Extracellular bind with mycobacterial components TLR3 Homodimer Intracellular binds with nucleic acids TLR4 Homodimer Extracellular binds with LPS or HSP TLR5 Homodimer Extracellular binds with bacterial flagellin TLR6 Heterodimer with TLR2 Extracellular with diacylated lipopeptides TLR7 Monomer Intracellular binds with viral nucleic acids TLR8 Monomer Intracellular binds with viral nucleic acids TLR9 Homodimer Intracellular binds with bacterial CpG DNA TLR: Toll Like Receptor, LPS: Lipopolysaccharide

Table 5: Receptor antagonists names, functions and their molecular structures

Antagonist Name Function Molecular Structure

TLR4-IN-C34 An aminomonosaccharide that inhibits TLR4 signaling by docking with the hydrophobic pocket of the TLR4 co-receptor

MMG11 TLR2 Antagonist: Exhibits selectivity for TLR2 over TLR4,5,7,8, and 9

T6167923 Novel Inhibitor of MYD88: disrupts MyD88 homodimeric formation, which Is critical for signaling function

Table 6: RT-PCR primer sequences for interested genes

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CHPTER FOUR: CONCLUSION/FUTURE DIRECTIONS

CS plays a contradictory role in IBD subsets; it is a risk factor in CD development and provides protection in UC. Our study was the first to provide significant insights toward understanding the cellular events involved in the mysterious divergent effects of CS ingredients on IBD patients.

Specifically, using an in vitro cell culture system, we were able to mimic a macrophages setup similar to those in CD and UC patients with history of prior or active cigarette smoking and in correlation with active bacterial infection. In order to study how the combination of MAP infection and nicotine could modulate macrophages, we used THP-1 differentiated macrophages, which are easy to obtain, differentiate and polarize. M1 and M2 THP-1 macrophages have the same expression profiles as polarized primary macrophages. Specifically, macrophages were differentiated from human monocytic cell line THP-1 in the presence of PMA. Consequently, they were polarized into M1 or M2 macrophages that express markers similar to polarized macrophages obtained from freshly isolated intestinal macrophages.

Nicotine in UC active smokers activates the cholinergic anti-inflammatory pathway through

α7nAChRs leading to M2-macrophage polarization, upregulation of CD206 and IL-10, downregulation of iNOS, IL-6, and TNF-α, and significantly decreases caspase-3 activity. Similar cellular changes are expected in CD active smokers who are not associated with infection. On the contrary, nicotine in CD active smokers associated with bacterial infection induce M1-macrophage polarization, upregulation of iNOS, IL-6, and TNF-α, downregulation in CD206, IL-10, decrease in caspase-3 activity, and increase in infection burden. CD non-smokers with bacterial infection suffer also from pro-inflammatory response but at a lower level compared to CD smokers with bacterial infection.

Moreover, our study was also the first to investigate the role of TLR2 and MyD88 signaling in

MAP infected macrophages in association with nicotine exposure. We showed that in MAP- infected macrophages, nicotine has significantly decreased expression of both TLR2 and TLR4.

MAP upregulated MyD88 expression and increased its specific correspondence of IL-8.

Surprisingly, nicotine has further increased MyD88 and IL-8 expression in MAP-infected macrophages. Thus, this study illustrates the crucial role of MyD88 in upregulation of pro- inflammatory genes during MAP infection. Collectively, we suggested that MAP-Nicotine interaction with TLR-2 during MAP infection is MyD88-dependent. Moreover, when TLR2 or

MyD88 were blocked with antagonists, there was no significant change in MAP load in macrophages even after 48 hours exposure to nicotine treatment.

There is no doubt that more studies are needed to investigate the effect of nicotine and CS on the microbiome in patients with UC or CD in presence and association of infection. This area of research is intriguing; more studies have reported a strong role for the microbiome in wellness and susceptibility to disease in healthy individuals and patients with underlying conditions. While our study focused on the effect of nicotine in macrophages infected with MAP or gram-negative bacteria (represented by LPS), more studies should be conducted with other microorganisms especially those associated with dysbiosis. We also plan to continue on our work investigating the effect of nicotine in the two arms of NF-ĸB pathways in MAP-infected macrophages following nicotine exposure. We propose that nicotine through α7nAChR induces NF- ĸB signaling pathway

(survival pathway). The latter induces the expression of the survival genes such as members of the

Bcl2 family of apoptosis regulator which ultimately reduces apoptosis and increase infection load.

This may support the idea that nicotine activates NF-κB pathway to escape apoptosis in tumor

95 cells, which has been identified as one of the essential hallmarks of cancer. Specifically, nicotine activates survival pathway causing a phosphorylation to p100, which is bound to RelB. After phosphorylation, a portion of p100 is degraded in proteosomes, resulting in transcriptionally active form, p52. Then p52: RelB heterodimers are free to enter the nucleus and induce anti-apoptotic genes.

APPENDIX: CONSENTS FOR PUBLICATION

We, the authors, give our permission to include data and materials described in AlQasrawi et. al.

2020 (below) in the dissertation contents of Ms. Dania AlQasrawi for Doctor of Philosophy in

Biomedical Sciences at the University of Central Florida.

Article Title: Mystery Solved: Why Smoke Extract Worsens Disease in Smokers with Crohn’s

Disease and Not Ulcerative Colitis? Gut MAP!.

Authors: Dania AlQasrawi, Latifa S. Abdelli and Saleh A. Naser

Journal: Microorganisms, 8(5), 666.

DOI: https://doi.org/10.3390/microorganisms8050666

Published: May 2nd, 2020

We, the authors, give our permission to include data and materials described in AlQasrawi et. al.

2020 (below) in the dissertation contents of Ms. Dania AlQasrawi for Doctor of Philosophy in

Biomedical Sciences at the University of Central Florida.

Article Title: Divergent Effect of Cigarette Smoke on Innate Immunity in Inflammatory Bowel

Disease: A Nicotine-Infection Interaction.

Authors: Dania AlQasrawi, Ahmad Qasem and Saleh A. Naser

Journal: International Journal of Molecular Sciences, 21(16), 5801.

DOI: https://doi.org/10.3390/ijms21165801

Published: August 13th, 2020