ABSTRACT GULLEDGE, TRAVIS VANCE. the Immunomodulatory Effects of Echinacea Purpurea and Its Constituent Molecules

ABSTRACT

GULLEDGE, TRAVIS VANCE. The Immunomodulatory Effects of Echinacea purpurea and its Constituent Molecules. (Under the direction of Dr. Scott Laster).

The immune system is important for providing host protection and survival.

However, the immune system is also responsible for mediating debilitating and life- threatening pathologies associated with infectious, allergic, and autoimmune diseases.

Therefore, a common therapeutic strategy is to inhibit inflammatory mediator production from immune cells. Glucocorticoids, non-steroidal anti-inflammatory drugs, and biologics are current pharmaceuticals that utilize this approach. However, these options are marginally effective, are expensive to produce, or cause serious side effects. Additional compounds that can inhibit inflammatory mediator production are necessary to overcome these limitations.

Some of the most commonly used pharmaceuticals were originally discovered from plants and more are likely to be identified and developed into useful products. Several

Echinacea spp. were used medicinally by Native Americans including Echinacea purpurea.

Echinacea preparations were commonly used to treat a variety of conditions associated with inflammation and allergies including gastrointestinal disorders, oral pain, sore throats, and skin inflammation. Today, Echinacea preparations are used as herbal supplements to prevent or treat upper respiratory infections (URIs).

Excess production of cytokines by macrophages has been linked to many inflammatory conditions, therefore in the studies shown in Chapter 2, we evaluated the effects of an E. purpurea extract, and fractions chemically separated from it, on macrophage cytokine production in vitro. The E. purpurea extract displayed both cytokine-stimulatory and cytokine-suppressive activity, while the separated fractions displayed either cytokine- stimulatory or cytokine-suppressive activity. These findings suggest that E. purpurea

extracts contain molecules with opposing effects on cytokine production. Extracts made from E. purpurea grown from sterilized seeds did not stimulate cytokine production indicating that endophytic bacteria and fungi were likely the sources of cytokine-stimulatory activity. Cytokine-suppressive fractions contained alkylamides and non-alkylamide molecules. In an attempt to identify non-alkylamide cytokine-suppressive molecules, we isolated and indentified xanthienopyran, which had not been isolated from E. purpurea previously.

The results described in Chapter 3 show the structure-activity relationship between alkylamide structure and their inhibition of TNF-α production from LPS-stimulated RAW

264.7 cells. Synthetic analogs of the alkylamide dodeca-2E,4E-dienoic acid isobutylamide, or alkylamide 15 (A15) were created through a two-step chemical process. We found that the double bonds along the alkyl chain and the isobutyl head group were not important for activity. However, shortening the alkyl chain led to a loss of TNF-α suppression and converting the amide group into an amine group caused significant cytotoxicity.

Next, we investigated the mechanism of cytokine suppression from macrophages by

A15. A15 broadly inhibited cytokine and chemokine production from macrophages stimulated with TLR agonists. We found that, except for CCL5, A15 treatment led to decreased levels of cytokine mRNAs suggesting that A15 is acting at a step in the cytokine production pathway upstream from protein synthesis. However, we found that NF-B p65 phosphorylation and translocation was not inhibited by A15 suggesting that this molecule is not the target of A15 activity. A15 also inhibited ionomycin-, but not PMA-stimulated production of TNF-α, indicating that calcium signaling may be blocked by A15. However, our experiments to test this hypothesis were inconclusive. Pharmacological experiments

supported the hypothesis but we were unable to measure an actual LPS-induced calcium signal with the dye fluo-4. Overall, our experiments suggest that the broad cytokine suppression by A15 may be mediated by multiple mechanisms of action.

In the final chapter, we evaluated the effects of A15 and an E. purpurea ethanolic extract on mast cell activity, which has not been investigated previously. We found that A15 inhibited RBL-2H3 and bone marrow-derived mast cell degranulation and calcium influx.

The E. purpurea extract also inhibited RBL-2H3 degranulation and calcium influx, and inhibition may have been dependent on alkylamide activity. In addition, A15 inhibited de novo synthesis of TNF-α and PGE2 from RBL-2H3 cells. These studies describe a novel activity for A15 and E. purpurea extracts, which may help explain the traditional use by

Native Americans for treating inflammatory and allergic diseases.

The Immunomodulatory Effects of Echinacea purpurea and its Constituent Molecules

by Travis Vance Gulledge

A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Immunology

Raleigh, North Carolina

2017

APROVED BY:

______Scott M. Laster, PhD Michael J. Sikes, PhD Committee Chair

______Susan L. Tonkonogy, PhD Joshua G. Pierce, PhD

______Nadja B. Cech, PhD Inter-Institutional Member

DEDICATION

This work is dedicated to my family who has made me the person that I am today.

Thank you for your guidance, support, and encouragement. I also dedicate this work to my brilliant wife, Monika. Thank you for your endless patience, understanding, and love. I appreciate all of the sacrifices you have made and look forward to our future together.

BIOGRAPHY

Travis Vance Gulledge was born in Winston Salem, North Carolina to Perry Vance and Laura Lee Gulledge. He attended North Carolina State University from 2007-2010 to earn his Bachelor of Science degree in Biochemistry with a minor in Genetics. After graduation, he worked as a laboratory research technician near Research Triangle Park at

Charles River Laboratories where he conducted pre-clinical oncology research. At Charles

River, he discovered the potential of the immune system to survey and remove cancerous cells from the body. This inspired him to return to North Carolina State University to obtain his Doctorate of Philosophy in Immunology.

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ACKNOWLEDGEMENTS

I would first like to thank Dr. Scott Laster for his mentorship and guidance. I have learned how to think more critically and strategically when it comes to performing research and I have become a better scientist because of him. I have enjoyed our friendship and our many conversations over coffee or sushi. I would also like to thank the past and present members of the Laster lab including Dr. Bola Oyegunwa who welcomed me into the lab and offered me helpful advice. I would especially like to thank Nicholas Collette for his friendship, discussions, and assistance with many experiments. I truly would not have made it through the long, difficult weeks without him. Dr. Amy Devlin, Stephanie Johnstone,

Lindsey Stevenson, and Dr. Farah Alayli were great friends who I will always be thankful for. Thank you all for offering to lend supplies, talk about journal club, grab lunch, or discuss our research.

I would like to thank Dr. Susan Tonkonogy, Dr. Michael Sikes, Dr. Nadja Cech, and

Dr. Joshua Pierce for serving on my committee and for their guidance and encouragement with my research. Finally, I would like to thank my friends and family for their support, patience, and understanding when I had to put in long nights or work on the weekends. I had a much more enjoyable experience in graduate school because of you all.

TABLE OF CONTENTS

List of Tables ...... vii

List of Figures ...... viii

List of Schemes ...... x

Chapter 1. Literature Review ...... 1

1. Introduction ...... 1

2. Immunopathology and Inflammation ...... 1

3. Current Treatment Options ...... 12

4. Echinacea ...... 16

5. Conclusions ...... 25

6. References ...... 26

Chapter 2. Ethanolic Echinacea purpurea Extracts Contain a Mixture of Cytokine- Suppressive and Cytokine-Inducing Compounds, Including Some that Originate from Endophytic Bacteria ...... 40

1. Abstract ...... 41

2. Introduction ...... 42

3. Materials and methods ...... 43

4. Results ...... 48

5. Conclusions ...... 57

6. References ...... 58

Chapter 3. Synthesis and biological evaluation of a series of fatty acid amides from Echinacea ...... 61

1. Abstract ...... 62

2. Introduction ...... 62

3. References and notes...... 64

Chapter 4. The Mechanism of Inhibiting Cytokine Production from Macrophages by the Alkylamide Dodeca-2E,4E-Dienoic Acid Isobutylamide ...... 66

1. Introduction ...... 66

2. Materials and methods ...... 69

3. Results ...... 75

4. Discussion ...... 87

5. References ...... 92

Chapter 5. Mast Cell Degranulation and Calcium Influx are Inhibited by an Echinacea purpurea Extract and the Alkylamide Dodeca-2E,4E-Dienoic Acid Isobutylamide ...... 96

1. Abstract ...... 97

2. Introduction ...... 100

3. Materials and methods ...... 103

4. Results ...... 109

5. Discussion ...... 122

6. References ...... 127

Summary ...... 134

LIST OF TABLES

Chapter 2. Ethanolic Echinacea purpurea Extracts Contain a Mixture of Cytokine- Suppressive and Cytokine-Inducing Compounds, Including Some that Originate from Endophytic Bacteria

Table 1. Alkylamide content of an Echinacea purpurea extract, chloroform layer, and column fractions from the chloroform layer ...... 46

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LIST OF FIGURES

Chapter 2. Ethanolic Echinacea purpurea Extracts Contain a Mixture of Cytokine- Suppressive and Cytokine-Inducing Compounds, Including Some that Originate from Endophytic Bacteria

Figure 1. Partitioning scheme for the Echinacea purpurea extract ...... 44

Figure 2. Influence of 75% ethanol extract and liquid:liquid partitions from Echinacea purpurea extract on TNF-α production from RAW 264.7 cells ...... 49

Figure 3. Alkylamides from Echinacea purpurea ...... 51

Figure 4. The effects of Echinacea purpurea extract fractions on production of TNF- α ...... 52

Figure 5. Cytotoxicity of Echinacea purpurea extract fractions and chloroform layer ...... 53

Figure 6. Structure and activity of xanthienopyran...... 53

Figure 7. Echinacea purpurea fraction effects on CCL3 and CCL5 ...... 54

Figure 8. Echinacea purpurea plants grown from sterilized seeds ...... 56

Chapter 3. Synthesis and biological evaluation of a series of fatty acid amides from Echinacea

Figure 1. Structure of fatty acid amide 1 from Echinacea ...... 63

Figure 2. Analogs prepared to explore the impact of unsaturation and alkyl chain length...... 63

Figure 3. TNF-α production in the presence of alkyl chain analogs ...... 63

Figure 4. Analogs prepared to explore the impact of the amide head group ...... 64

Figure 5. TNF-α production in the presence of head group analogs ...... 64

Figure 6. Cytotoxicity of the alkylamide structural analogs ...... 43

Chapter 4. The Mechanism of Inhibiting Cytokine Production from Macrophages by the Alkylamide Dodeca-2E,4E-Dienoic Acid Isobutylamide

Figure 1. A15 suppresses cytokine and chemokine production from RAW 264.7 cells and peritoneal macrophages ...... 77

Figure 2. A15 effects on cytotoxicity, cell growth, and transcription in RAW 264.7 cells ...... 79

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Figure 3. A15 does not inhibit phosphorylation or nuclear translocation of NF-B p65 in LPS-stimulated RAW 264.7 cells ...... 80

Figure 4. A15 inhibits production of LPS-stimulated cytokine and chemokine protein and mRNA in RAW 264.7 cells ...... 82

Figure 5. A15 inhibits intracellular cytokine and chemokine accumulation ...... 84

Figure 6. The role of intracellular Ca2+ in LPS-stimulated cytokine production ...... 86

Chapter 5. Mast Cell Degranulation and Calcium Influx are Inhibited by an Echinacea purpurea Extract and the Alkylamide Dodeca-2E,4E-Dienoic Acid Isobutylamide

Figure 1. A15 inhibits BMMC and RBL-2H3 cell degranulation ...... 110

Figure 2. A15 inhibits granule release from BMMCs ...... 112

Figure 3. A15 inhibits calcium influx in RBL-2H3 cells and BMMCs ...... 114

Figure 4. A15 inhibits A23187-stimulated calcium influx in RAW 264.7 and Jurkat cells ...... 117

Figure 5. Alkylamide-containing Echinacea purpurea extracts and fractions inhibit RBL-2H3 cell degranulation and calcium influx ...... 119

Figure 6. A15 inhibits de novo production of TNF-α and PGE2 from RBL-2H3 cells. RBL-2H3 cells were plated and incubated overnight prior to stimulation ...... 121

Supplemental Figure 1. A15 is not cytotoxic to RBL-2H3 cells ...... 131

Supplemental Figure 2. A15 does not inhibit ß-hexosaminidase enzyme activity ..132

Supplemental Figure 3. A15 inhibits calcium influx in RBL-2H3 cells ...... 133

LIST OF SCHEMES

Chapter 3. Synthesis and biological evaluation of a series of fatty acid amides from Echinacea

Scheme 1. Synthesis of fatty acid amide 1 ...... 63

CHAPTER 1. LITERATURE REVIEW

1. Introduction Infectious diseases, hypersensitivity reactions, and autoimmune diseases often result in tissue damage due to excessive or inappropriate immune responses. These immune responses can cause inflammation of organs throughout the body such as hepatitis, nephritis, dermatitis, and gastritis, among others. If left untreated, inflammation in these organs can lead to permanent damage resulting in organ dysfunction or, in extreme cases, organ failure and death of the host. Therefore, a common therapeutic strategy is to limit production of inflammatory mediators from activated immune cells. Three well-known treatment options that utilize this approach include nonsteroidal anti-inflammatory drugs, glucocorticoids, and biologics. However, these treatments have limited efficacies and prolonged use can lead to serious side effects such as hypertension, thinning of the skin, and gastrointestinal bleeding.

Additional therapeutic options are necessary to effectively treat immunopathological conditions, which are widespread diseases. Research groups around the world are investigating plants for potentially useful anti-inflammatory compounds. Plant-derived pharmaceutical compounds have been used to treat inflammation and it is likely that additional important compounds remain to be discovered from plant sources. In this chapter, we review common immune-mediated pathological reactions and their treatments. In addition, we review the medicinal use of Echinacea spp., and their constituent molecules.

2. Immunopathology and Inflammation Infectious Disease

Microbial pathogens elicit immune responses that are necessary to control and eliminate the infection. However, pathologies associated with infectious diseases are often caused by excessive release of inflammatory mediators from immune cells. In many cases,

1 reducing pro-inflammatory mediator production limits inflammation and tissue damage, while still allowing effective clearance of the invading microbe.

Pathogens first encounter cells of the innate immune system including epithelial cells, macrophages, dendritic cells (DCs), mast cells, neutrophils, and natural kill (NK) cells.

Innate immune cells express a variety of pattern recognition receptors (PRRs) including toll- like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and C-type lectin receptors (CLRs) that detect pathogen-associated molecular patterns (PAMPs). PRR activation initiates intracellular signaling pathways leading to the expression of pro- inflammatory mediator genes including cytokines, chemokines, enzymes, and cytotoxic molecules. DCs, and to a lesser degree, macrophages, are antigen presenting cells (APCs) that process antigens and migrate to draining lymph nodes.

In the lymph nodes, APCs present antigen on major histocompatibility complex

(MHC) molecules to T cells, which are adaptive immune cells. T cells express receptors that recognize antigen:MHC complexes. B cells are also adaptive immune cells that express a B cell receptor capable of binding antigenic structures without MHC molecule participation. T cell and B cell receptor signaling stimulates clonal expansion and migration of lymphocytes out of the lymph nodes towards the site of infection. Circulating leukocytes upregulate expression of adhesion molecules to adhere to endothelial cells and extravasate into the infected tissue. T cells secrete additional cytokines, chemokines, and cytotoxic molecules. B cells secrete cytokines, chemokines and antibodies after differentiating into plasma cells.

Importantly, memory T cells and B cells differentiate from a subset of the responding cells to establish immunological memory.

Virus-Induced Immunopathology

Influenza A virus (IAV) is a well-studied example of a virus that can cause severe immunopathology. IAV is a seasonal respiratory virus that infects lung epithelial cells and can cause severe respiratory tract inflammation resulting in acute lung injury, or more severely, acute respiratory distress syndrome. IAV activates TLR3, TLR7/8, RIG-I, and

NOD-, leucine-rich repeat-, and pyrin-domain containing 3 (NALP3) expressed by lung epithelial cells, alveolar macrophages, mast cells and DCs (reviewed by (1)). After IAV infection, alveolar macrophages produce an array of cytokines including tumor necrosis factor-alpha (TNF-α), interleukins IL-6, IL-1ß, type I interferons IFN-α/ß, chemokines, reactive oxygen species (ROS), reactive nitrogen species (RNS), prostaglandins (including prostaglandin E2 (PGE2)), and proteases. The excessive production of these pro- inflammatory mediators has been referred to as the “cytokine storm” (reviewed by (1)). The importance of TNF-α in IAV-mediated pathology was shown in TNF-α receptor deficient

(TNFR1-/-) mice infected with H5N1, which displayed reduced morbidity compared to wild type mice (2). In addition, triple knockout TNFR1-/- TNFR2-/- IL1RI-/- mice infected with highly pathogenic H5N1 displayed significantly reduced morbidity and mortality, lung inflammation, and macrophage and neutrophil recruitment compared to wild type mice (3).

Furthermore, Walsh et al. demonstrated that by suppressing the cytokine storm, viral replication was controlled and mice were significantly protected from mortality after infection with the pandemic H1N1 2009 IAV (4).

Similarly to IAV, Ebola virus (EBOV) can cause excessive activation of the innate immune response resulting in a cytokine storm (5). EBOV initially infects and replicates within macrophages, but can also infect DCs, hepatocytes, endothelial cells and fibroblasts

(6). EBOV-infected human monocyte-derived macrophages produce TNF-α, IL-6, IL-1ß,

CCL2, CCL3, and CCL5 (7, 8). These chemokines recruit additional monocytes for EBOV to possibly infect and replicate within (9). In addition, EBOV-infected monocytes express tissue factor (TF), which triggers disseminated intravascular coagulation (DIC) (10).

Macrophage-mediated DIC combined with hypotension due to increased vascular permeability ultimately leads to multiple organ failure and death in ~50% of EBOV infected individuals (11).

Another example of virus-induced immunopathology is the liver damage caused by hepatitis B virus (HBV) infection. HBV infects hepatocytes leading to acute and chronic necroinflammation and mononuclear cell infiltrate (12). If chronic inflammation is left untreated, liver cirrhosis or hepatocellular carcinoma can develop (12). HBV infection has been shown to activate TLR2 and melanoma differentiation-associated gene 5 (MDA5) signaling to promote cytokine production (13, 14). DCs process and present HBV antigen to

+ + CD4 T helper (TH) and CD8 cytotoxic T lymphocytes (CTLs) in the lymph nodes (15).

HBV-specific CTLs recruited from the lymph nodes enter the liver to kill HBV-infected hepatocytes through Fas/Fas ligand interactions and by releasing the cytotoxic mediators, granzyme B and perforin (16, 17). CTLs have been shown to contribute significantly to the liver injury associated with HBV infection (18). However, CTL-mediated killing of hepatocytes leads to recruitment of neutrophils, macrophages, and NK cells, which augment liver damage (19). Liver-resident macrophages (known as Kupffer cells) secrete TNF-α, IL-

6, IL-1ß, and CXCL8, which promote recruitment of NK and NKT cells to the liver to clear

HBV-infected cells, but also cause liver damage (20, 21).

Another group of viruses that cause chronic infections are the herpesviruses, including herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). HSV-1 and HSV-2 establish latent infections in the cell bodies of the trigeminal or dorsal root ganglion to avoid detection and clearance by the immune system. Upon reactivation, HSV virions travel down neuronal axons from which they bud to infect neighboring epithelial cells and initiate lesion formation. HSV infections may result in keratitis, which can lead to permanent blindness, or encephalitis, which may result in death or permanent brain damage (22). HSV-mediated inflammation in the brain has been linked to TLR2 signaling in microglial cells, which release ROS that cause radical-induced tissue damage (23). HSV also activates plasmacytoid

DCs (pDCs) to secrete IFN-α/ß and upregulate costimulatory molecules by signaling through

TLR9 (24). NK cells and HSV-specific CTLs are recruited to the site of infection by CXCL9 and CXCL10 to destroy HSV-infected cells, which promotes viral clearance, but also contributes to the overall tissue damage (25). The importance of the immune response in driving HSV-mediated inflammation was further demonstrated in HSV-infected mice when administration of IL-10, an anti-inflammatory cytokine, inhibited corneal inflammation (26).

Bacteria-Induced Immunopathology

Bacterial infections are also well-known causes of immunopathology. Helicobacter pylori is a gram-negative bacteria that can cause chronic inflammation in the gastrointestinal

(GI) tract and is associated with an increased risk of developing peptic ulcer disease or gastric cancer (27). Mucosal tissue from H. pylori-infected patients produced elevated levels of TNF-α, IL-1ß, and IL-8 compared to uninfected individuals (28). GI epithelial cells, macrophages, and DCs infected by H. pylori signal through TLR2 and TLR4 to stimulate secretion of IL-1ß, IL-6 and IL-8 (29). Neutrophils, monocytes, and mast cells are recruited

5 to the site of H. pylori infection (30, 31). Activated macrophages and neutrophils produce

ROS and RNS which can cause DNA damage and increase the likelihood of developing gastric cancer (32).

Sepsis is a life-threatening systemic inflammatory response caused by the entrance of a pathogen into the bloodstream. Bacterial infections are the most common cause of sepsis, although severe viral and fungal infections can also result in sepsis. During sepsis, macrophages in the liver and spleen become activated through PRR signaling to secrete pro- inflammatory cytokines and chemokines such as TNF-α, IL-6 and IL-1ß (33). Systemic

TNF-α release causes vasodilation and increased vascular permeability resulting in hypotension and shock (34). TNF-α also causes expression of TF on endothelial cells leading to DIC, which can result in multiple organ failure and death (35, 36).

Mycobacterium tuberculosis (Mtb) is the pathogen responsible for causing tuberculosis. Tuberculosis is characterized by pulmonary inflammation with granuloma formations (referred to as “tubercles”) consisting of infected macrophages surrounded by B cells and CD4+ and CD8+ T cells to contain the spread of Mtb (37). Alveolar macrophages phagocytose Mtb, but Mtb can prevent fusion of phagosomes with lysosomes to avoid destruction (38). Mtb replicates within the cytosol of alveolar macrophages after escaping the phagosome (39). Intracellular NLRs, such as NOD-1, NOD-2, and NLRP3 detect Mtb and stimulate TNF-α, IL-6, IL-1ß, IL-12, IL-23, and IFN- production, which contribute to lung inflammation (40, 41). For example, although TNF-α is required for host control of

Mtb, excessive TNF-α levels increase lung inflammation and worsen lung pathology (42).

Mtb also activates TLR2 signaling to induce cytokine production and apoptosis in macrophages, which is important for caseation of the granuloma, where necrotic tissue forms

6 a dry mass (43, 44). Chemokines recruit additional monocyte-derived macrophages and

DCs, as well as CD4+ and CD8+ T cells, which aid in Mtb clearance, but also contribute to lung inflammation and damage (reviewed by (45)).

Staphylococcus aureus is a common cause of skin, respiratory and joint infections occurring mostly in immunocompromised individuals. At the site of infection, epithelial cells and macrophages signal through PRRs including TLR2 and NLRP3 to produce TNF-α,

IL-6, IL-1ß, IL-8, and CCL2 initiating inflammation and neutrophil recruitment (46, 47). In addition, S. aureus produces superantigens such as S. aureus enterotoxin B (SEB), which non-specifically activates T cells by binding to MHC class II on APCs and the Vß chain of the T cell receptor (TCR) stimulating T cell proliferation and cytokine production (48). The resulting inflammation can lead to extensive tissue damage at the site of infection such as dermatitis, pneumonia, arthritis, and in the case of sepsis, even death. The immunopathology caused by S. aureus infection is demonstrated by decreased lung damage in S. aureus- infected intercellular adhesion molecue-1 (ICAM-1)-deficient mice (49). ICAM-1 is an important adhesion molecule for migration of leukocytes to the site of infection.

Hypersensitivity Reactions

In contrast to the beneficial role of the immune response in clearing pathogenic infections, the immune response elicited by hypersensitivity reactions causes tissue damage unnecessarily. Atopic dermatitis (AD) is a chronic inflammatory skin disease that is increasing in prevalence (50). Pruritis, or itching of the skin, followed by mechanical injury and release of pro-inflammatory cytokines is an initial trigger for inflammation in AD (51).

These events reduce skin barrier integrity, which are usually compromised in AD patients due to genetic factors, allowing entry of the skin microbiota to the epidermis and activation

7 of local keratinocytes, macrophages, mast cells, and skin-resident DCs, known as Langerhans cells (52). T cells and eosinophils are recruited to the epidermis and further contribute to inflammation and tissue damage (53). Activated keratinocytes produce the cytokine thymic stromal lymphopoietin (TSLP) in a calcium-dependent manner (54). TSLP skews additional cytokine production by DCs and T cells towards a TH2 response characterized by IL-4, IL-5, and IL-12 (55). IL-4 promotes B cell immunoglobulin (Ig) class-switching to IgE.

Consistent with this cytokine environment, AD patients have been shown to have elevated levels of serum IgE, which increases the likelihood of developing allergen-specific IgE antibodies (53). In addition, AD patients have elevated levels of IgE specific for staphylococcal superantigens such as SEB (56). IgE binds to the high affinity IgE receptor,

FcRI, expressed on tissue-resident mast cells and bloodborne basophils. Upon crosslinking of IgE-bound FcRI by multivalent antigen, mast cells degranulate within minutes releasing inflammatory mediators including proteases, cytokines, and histamine. Mast cells are well- known to contribute to the immediate reaction and subsequent lesional inflammation and itch sensations experienced by AD patients (reviewed by (57)).

AD often precedes development of other allergic diseases such as asthma, allergic rhinitis, and food allergies, a phenomenon termed the “atopic march” (58). Asthma is characterized by chronic inflammation of the airways and can lead to airway hyperresponsiveness and severe bronchoconstriction. Allergic rhinitis causes nasal congestion, excess mucus production, itching and rhinorrhea. Food allergies cause swelling of oral tissue, diarrhea, vomiting, and in severe cases anaphylaxis. Mast cells, eosinophils, and TH2 cells drive allergic inflammation in patients with these IgE-mediated diseases (59,

60). IL-4, IL-5, and IL-13 recruit eosinophils to the lungs, nasal cavity, or GI tract of atopic

8 patients where eosinophils release cytotoxic molecules such as eosinophil peroxidase (EPO) and major basic protein (MBP) that contribute to tissue damage (61, 62). Additionally, IL-4 and IL-13 promote Ig class-switching to IgE, increasing the likelihood of developing type I hypersensitivities to inhaled allergens (63). Allergen-specific IgE can bind FcRI on lung- resident mast cells. Upon re-exposure to the allergen, mast cells degranulate to release preformed mediators such as histamine, chymase and tryptase, and also initiate de novo synthesis of leukotrienes (LTs), prostaglandins (PGs), cytokines, and chemokines (64, 65).

Allergic contact dermatitis (ACD) is a delayed type IV hypersensitivity reaction caused by exposure to contact allergens, such as urushiol oil (from the poison ivy plant) or small metal ions including nickel (66). ACD is characterized by pruritic, painful skin lesions. Contact allergens causing ACD cross the skin barrier, react with self-proteins forming haptenated self-proteins. Macrophages, mast cells, and DCs can be activated directly or indirectly by contact allergens through TLR2, TLR4, and NLRP3 signaling to stimulate ROS, cytokine, and chemokine production (66). Haptenated self-proteins are processed and presented on MHC molecules by Langerhans cells (67). Effector CD4+ T cells secrete cytokines, such as IFN-, to activate macrophages and recruit circulating neutrophils and monocytes to the site of contact allergen exposure (68). CTLs destroy cells displaying haptenated self-proteins by releasing cytotoxic effector molecules further contributing to tissue damage (69).

Autoimmune Diseases

Autoimmune diseases are caused by immune responses against self-molecules resulting in severe, life-threatening immunopathologies. Type 1 diabetes mellitus is a common autoimmune disease mediated by self-reactive CTLs. Peptides from insulin are

9 processed and presented on MHC class I molecules by ß-cells of pancreatic islets, and autoreactive CD8+ T cells target those cells for destruction (70). It has recently been proposed that changes in the microenvironment of pancreatic islet tissue could predispose ß- cells to CTL destruction. Alterations to the extracellular matrix, such as increased hyaluronan build up, might serve as an initial signal to recruit monocyte-derived macrophages and DCs to pancreatic islets and increase the likelihood of ß-cell destruction by autoreactive CTLs (71).

Systemic lupus erythematosus (SLE) is an autoimmune disease caused by the production of autoantibodies specific for self-antigens such as the ribonucleoprotein complex containing Ro and La, DNA, and the nucleosome subunits of chromatin (72). Upon release of self-antigens from dead or dying cells, autoantibodies form immune complexes, direct complement activation and recruit IgG receptor (FcR)-expressing phagocytic or lytic cells leading to further tissue damage and release of additional self-antigens (73). Immune complexes can become deposited in the kidneys, joints, skin, respiratory tract, and along blood vessel walls causing skin rashes, arthritis, glomerulonephritis, cardiovascular disease, and potentially death (74).

Another prevalent autoimmune disease, rheumatoid arthritis (RA), is characterized by chronic inflammation and pain of the joint synovium eventually leading to cartilage and bone damage. It is now recognized that seropositive RA patients (patients with antibodies associated with development of RA) are seropositive well before disease onsets. These findings suggest that an initial event, such as lung or intestinal inflammation, triggers production of these antibodies, including rheumatoid factor (RF) and antibodies to citrollenated protein antigens (ACPA), well before bone and joint destruction occur (75).

TH1 and TH17 cells within the synovium produce IFN- and IL-17 to activate macrophages, endothelial cells, and fibroblasts to secrete TNF-α, IL-1ß, IL-6, IL-12 and IL-23 which are present in the inflamed synovium of RA patients (76). Additionally, synovial macrophages and fibroblasts release matrix metalloproteases and receptor activator of nuclear factor B

(NF-B) ligand (RANKL) causing tissue destruction and osteoclast activation, respectively

(77, 78).

Inflammatory bowel disease (IBD) includes ulcerative colitis (UC) and Crohn’s disease (CD), which are caused by immune responses elicited by the normal gut microbiota.

Although IBD is not a traditional autoimmune disease since the gut microbiota is not self, for the purposes of this review, the immunopathology elicited by this condition will be discussed here. Patients with IBD experience diarrhea, intestinal pain, weight loss, and fever. Multiple factors result in development of IBD including loss of mucosal barrier integrity due to genetic or environmental factors, which leads to activation of innate immune cells by the intestinal microbiota (79-81). Researchers have developed a number of knockout mouse strains that display IBD-like pathology emphasizing the vast number of genes involved in maintaining intestinal homeostasis (reviewed by (82)). In mouse strains that develop spontaneous colitis, crossbreeding with RAG-/- mice prevents colitis, suggesting that the adaptive immune system is essential for development of IBD (83). In addition, the cytokine milieu is critical for mediating the immunopathology of IBD. Epithelial cells, macrophages,

DCs, T cells, and more recently, group 3 innate lymphoid cells (ILCs), are important sources of cytokines and chemokines within the GI tract. Regulatory T cells (Treg) and tolerogenic

DCs are also critical for maintaining intestinal homeostasis by secreting anti-inflammatory cytokines such as IL-10 and TGF-ß (84, 85). The importance of IL-10 and the intestinal

11 microbiota in IBD is particularly apparent in IL-10-/- mice which spontaneously develop colitis only when the intestinal microbiota is present (86).

3. Current Treatment Options Due to the prevalence of immunopathologies, some of which were described above, medications that suppress immune responses to relieve inflammation and pain have proven invaluable to modern medicine. Glucocorticoids (GCs), or corticosteroids, are some of the most commonly used anti-inflammatory medications. GCs can be administered as a pill, topical cream, nasal spray, or inhaler for a wide range of applications. GCs bind and activate glucocorticoid receptors to promote or repress gene expression (reviewed by (87)).

Hydrocortisone, or cortisol, is a GC produced by the adrenal glands in humans and has since been structurally modified to improve its activity (88). GCs tether to transcription factors such as NF-B p65, activator protein 1 (AP-1), and members of signal transducer and activator of transcription (STAT) that promote expression of pro-inflammatory genes in numerous cell types (89-91). Additionally, GCs promote gene expression of anti- inflammatory genes by binding to glucocorticoid response elements in gene promoter regions such as glucocorticoid-induced leucine zipper (GILZ) and IL-10 (92, 93). Despite the success of GCs, there are many serious side effects associated with prolonged use including thinning of the skin, hypertension, osteoporosis, and gastric ulcers. These adverse side effects are particularly problematic in cases of chronic inflammation, such as chronic infections, skin hypersensitivities, and autoimmune diseases (94). Additionally, some diseases become refractory to GC treatment as is seen with GC-resistant cases of asthma

(95).

Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of molecules used to treat a variety of inflammatory and pain conditions including headaches, RA, and ankylosing

12 spondylitis. NSAIDs include non-selective cyclooxygenase (COX) inhibitors and COX-2- selective inhibitors. COX enzymes convert arachidonic acid (AA) into PGH2, the common precursor for PGE2, PGD2, prostacyclin (PGI2), and thromboxane (TX) A2, which are involved in promoting inflammation, blood clotting, and nociception (reviewed by (96)). At sites of inflammation, peripheral sensory neurons are sensitized by PGI2 and PGE2 receptors

(97, 98). Inhibition of COX activity dampens PG-mediated vasodilation, smooth muscle contraction, immune cell recruitment, and sensory neuron sensitization (reviewed by (99)).

One of the first NSAIDs discovered was aspirin, which is the active molecule in willow tree bark extracts (100). Aspirin, naproxen and ibuprofen are all non-selective COX inhibitors, which have side effects including dyspepsia, gastric ulcers, and gastric bleeding due to inhibition of constitutive COX-1 activity in the GI tract. In contrast, COX-2 is an inducible gene and COX-2-selective inhibitors, such as celecoxib, were developed to improve the effectiveness and decrease side effects caused by non-selective COX inhibitors. However, studies have revealed that patients taking COX-2-selective inhibitors have an increased risk of heart attacks and strokes (101).

Cromolyn, and related compounds such as nedocromil and lodoxamide, are referred to as “mast cell stabilizers.” Cromolyn was developed using synthetic chemistry techniques from khellin, the active molecule produced by the medicinal plant Ammi visnaga (102).

Cromolyn has been used clinically to treat allergic conditions such as asthma, allergic rhinitis, and allergic conjunctivitis for over 50 years. The mechanism of action for cromolyn is not clear, although histamine release from rat and human mast cells is inhibited by cromolyn treatment (103). However, other activities may explain its anti-asthmatic and anti-

13 allergic effects. Additionally, its effectiveness and selectivity for stabilizing mast cells in mice has been questioned (104).

Antihistamines are another common treatment for allergy-associated immunopathologies due to the importance of histamine in facilitating allergic responses.

Histamine is stored preformed in mast cell and basophil granules and is released into the tissue upon FcRI crosslinking. During an allergic response, antihistamines bind the histamine H1 receptor to block histamine signaling and reduce vasodilation, vascular permeability, smooth muscle contraction, and pruritis (105). H1 is expressed by a number of cell types including neurons, smooth muscle cells, and endothelial cells (106). First- generation antihistamines (diphenhydramine) cause drowsiness due to activation of H1 in the central nervous system (107). Second-generation antihistamines (loratadine, fexofenadine, and cetirizine) do not easily cross the blood-brain barrier, which results in less drowsiness

(108). Antihistamines are commonly used to treat allergic rhinitis, allergic conjunctivitis, and chronic urticaria (105, 109). However, there are many cases in which antihistamines are ineffective in preventing allergic inflammation and itch sensations such as in AD (110).

Targeting the action of specific cytokines, receptors, or costimulatory molecules using biopharmaceuticals is another treatment strategy to limit inflammation caused by the immune system. Many biologics have been developed, or are being developed, and several will be discussed here. Anti-TNF-α therapies have become widespread due to the critical role of TNF-α in promoting inflammation. Neutralizing monoclonal antibody (mAb) therapies (adalimumab and infliximab) and a TNFR fusion protein (etanercept) are used to limit inflammation associated with RA, psoriasis, IBD, and ankylosing spondylitis (111-116).

However, anti-TNF-α therapies often lead to immunosuppression and an increased risk of

14 developing serious infections (117). Ustekinumab is a neutralizing mAb that binds IL-12p40 which is the common subunit to both IL-12 and IL-23. Ustekinumab is used to treat psoriasis and CD (118, 119). Another cytokine-specific biologic is anakinra, which binds to the IL-1 receptor to prevent signaling by IL-1. Anakinra is currently used to treat RA (120). The cytotoxic T lymphocyte antigen 4 (CTLA)-Ig fusion protein, abatacept, blocks costimulatory signals from CD80/CD86 on APCs to CD28 on T cells. Abatacept is currently being tested for the treatment of type I diabetes, multiple sclerosis, and vitiligo (Clinical Trials. Org.

Identifier NCT02281058), and is already used to limit inflammation in patients with RA who do not respond well to anti-TNF-α therapies (121-123). While most biologics have significantly improved treatment outcomes for patients with immunopathological conditions, inhibiting a key cytokine or signaling pathway often results in immunosuppression of the patient, and biologics are very expensive to produce (124).

Immunosuppressants are a broad class of therapeutics that inhibit immune responses causing inflammation and tissue damage. Methotrexate is a folate antagonist originally developed as a chemotherapeutic agent. However, at lower concentrations, methotrexate inhibits purine metabolism leading to increased amounts of adenosine at inflamed sites, which inhibits T cell activation and ICAM-1 expression (125). Methotrexate is used to treat

RA, psoriasis, and CD (126-128). Calcineurin inhibitors, such as tacrolimus (FK506) and cyclosporin A, prevent calcineurin activity including dephosphorylation of the nuclear factor of activated T cells (NFAT) family of transcription factors. NFAT is important for regulating gene expression in a number of cell types including T cells, macrophages, DCs, and mast cells although the role of NFAT in each cell type may vary (129). Calcineurin inhibitors are being used to treat AD, RA, psoriasis, CD, and vitiligo (130-132). The

15 sphingosine-1-phosphate receptor (S1PR) antagonist, fingolimod, is another important immunosuppressant. The S1PR helps direct chemotaxis of T cells out of lymph nodes and into the bloodstream (133). Binding of fingolimod to S1PR prevents T cells from exiting the lymph node and may also cause endothelial cell tight junctions to tighten and reduce permeability of the blood-brain barrier (134). Fingolimod is used for the treatment of multiple sclerosis, which is a T cell-driven autoimmune disease that destroys myelinated neurons in the brain (135).

4. Echinacea Traditional Use

Compounds that prevent inflammation, tissue damage, and pain are constantly sought after due the prevalence of immunopathological conditions. Natural products have proven useful for the development of effective therapies such as aspirin, cromolyn, and corticosteroids, as described above. Native Americans used Echinacea spp. (commonly known as purple coneflower) to treat a variety of inflammatory conditions including toothaches, sore throats, upper respiratory infections (URIs), skin inflammation, wound infections, and gastrointestinal disorders (136, 137). Echinacea purpurea, Echinacea angustifolia, and Echinacea pallida are the three species used medicinally. Echinacea spp. are herbs within the Asteraceae family and are indigenous to the Great Plains region of the

U.S. and Canada. Originally classified under the genus Rudbeckia by Carl Linnaeus, Conrad

Moench revised the genus name to Echinacea in 1794 (137).

In the late 1800’s, H. C. F. Meyer discovered the use of Echinacea by Native

Americans and began marketing Echinacea with exaggerated claims of its effectiveness and uses (136). With the help of Dr. John King and the pharmacist John Lloyd, Echinacea became one of the most widely used medicines in the early 1900’s (136). However, many of

16 the outlandish claims for Echinacea use were discredited and the popularity of Echinacea in the U.S. faded. Echinacea was introduced to Germany in the 1920’s and research continued there until the 1980’s when interest in natural remedies began to increase in the U.S. again

(137). Today, Echinacea extracts (made mostly from E. purpurea or E. angustifolia) are commonly used around the world as a dietary supplement, and are prescribed in Germany, to prevent or treat URIs such as the common cold or the flu. It is not clear why the other applications of Echinacea used by Native Americans have not continued and have rarely been studied in the scientific literature.

Proposed Activities

The medicinal effects of Echinacea have been hypothesized to work in several ways.

First, Echinacea preparations may limit replication of pathogenic viruses or bacteria through antimicrobial activities. For example, the commercial E. purpurea extract Echinaforce® displayed virucidal activity against HSV-1, IAV, and respiratory syncytial virus (RSV), but not against rhinovirus (RV) or adenovirus (AV) (138). In another study, three commercially available E. purpurea and E. angustifolia extracts displayed varying degrees of activity against Streptococcus pyogenes, Haemophilus influenzae, Legionella pneumophila,

Propionibacterium acne, and Clostridium difficile, but little to no activity against

Escherichia coli, Psuedomonas aeruginosa, Klebsiella pneumoniae, Mycobacterium smegmatis, Bacillus subtilis, Acinetobacter baumanii, and Enterococcus faecalis (139).

However, tests with an E. purpurea aerial extract in IAV-infected mice suggested there was no antiviral activity in vivo (140).

The most-advertised hypothesis for Echinacea extract activity is that Echinacea boosts immune function to prevent or overcome infections. The immunostimulatory activity

17 of Echinacea extracts has been supported by in vitro studies with a number of cell types displaying enhanced cytokine and chemokine production after treatment. For example, See et al. demonstrated that E. purpurea extracts stimulated NK cell function and antibody- dependent cell-mediated cytotoxicity by human PBMCs in vitro (141). Additionally, E. purpurea dose-dependently stimulated TNF-α and NO production from RAW 264.7 cells, a murine macrophage-like cell line (142). During the numerous investigations of Echinacea immunostimulatory activity, the possible contributions of bacterial and fungal endophytes living within Echinacea plants were often not accounted for. The majority of in vitro immunostimulatory activity of Echinacea extracts has since been linked to the presence of endophyte-derived PAMPs, such as LPS and lipoproteins (143-145). It is currently unclear what effect, if any, endophytic products from Echinacea extracts may have in vivo.

More recently, Echinacea extracts have been hypothesized to improve URI patient recovery through anti-inflammatory activity. As discussed previously, the majority of symptoms associated with URIs such as IAV are due to excessive inflammatory mediator production. Echinacea treatment may reduce production of cytokines, chemokines, and eicosanoids; therefore, alleviating symptoms and preventing lung damage, while still allowing viral clearance. Evidence to support this hypothesis has been shown in vitro where

E. purpurea extracts inhibited LPS and IAV-stimulated TNF-α production from RAW 264.7 cells (143, 146). In another study, Echinaforce® inhibited IL-6 and IL-8 production from

IAV, RSV, RV, and AV-infected BEAS-2B cells, a human lung epithelial cell line (138). In addition, low concentrations of an ethanolic E. purpurea extract inhibited IL-2 production from stimulated Jurkat cells, a human T cell line (147). More recently, an ethanolic E.

18 purpurea extract was found to reduce TH2 cytokine levels in the bronchoalveolar lavage fluid in a guinea pig model of asthma (148).

Clinical Trials

Numerous clinical trials have been performed evaluating the ability of Echinacea extracts to prevent or treat URIs, although certain trials have been performed more rigorously than others such as those that employed randomized, double-blind, placebo-controlled protocols. The lack of understanding of Echinacea extract activity has likely contributed to the contradictory results generated from these trials. In addition, instances of positive clinical trial results cannot be attributed to immunomodulatory properties of Echinacea because the appropriate clinical measurements and assays to support these claims were not performed (149-151). For example, Jawad et al. assessed the number and duration of cold episodes while also confirming the presence of respiratory viruses in nasal swabs from study participants (151). However, the effect of E. purpurea on the immune response, such as serum cytokine levels, prostaglandin levels, and immune cell numbers were not measured.

The difficulties in comparing Echinacea clinical trial results were described in a recent review (152). Several key factors were revealed including differences in Echinacea species used, portions of the plant extracted (aerial versus roots versus mixtures), the method of extraction, combinations with other plant material, dosing protocols, and parameters used to evaluate efficacy (152). In the same study by Jawad et al., the ethanolic extract Echinaforce® was used, which consists of aerial (95%) and root (5%) portions (151). However in another study, Taylor et al. used aqueous extracts of the aerial Echinacea portions only (153). The inconsistencies described above highlight the necessity for understanding how Echinacea

19 extracts may benefit patients with URIs so that well-designed clinical trials can be performed.

Constituent Molecules

The activity of Echinacea extracts has been attributed to several constituent molecules including caffeic acid derivatives (CADs), polysaccharides, glycoproteins, and alkylamides (154, 155). High concentrations of CADs, glycoproteins, and polysaccharides are present in aqueous extracts from aerial portions of Echinacea, while alkylamides are present at highest concentrations in ethanolic (70-95%) root extracts (156-158). CADs, such as caftaric acid, cichoric acid, and echinacoside, have been shown to have antioxidant activity (159, 160). Polysaccharides, including fucogalactoxyloglucan and arabinogalactan, were reported to stimulate macrophage cytotoxic and phagocytic activity and increase NO,

IL-6, and TNF-α production (154, 161, 162). Intravenous administration of the glycoprotein- containing fraction of E. purpurea to mice was found to increase serum TNF-α and IL-1 levels and treatment of murine splenocytes in vitro stimulated IFN-α/ß production (163). In contrast to these stimulatory effects, alkylamides have been shown to inhibit production of cytokines, chemokines, and prostaglandins from stimulated epithelial cells, macrophages, and T cells in vitro and will be discussed in more detail below (143, 146, 147).

Since Echinacea extracts are complex mixtures that potentially contain opposing cytokine-stimulatory and cytokine-suppressive molecules, we hypothesized that these activities could be separated using chemical separation techniques (143). We found that fractions generated from an ethanolic E. purpurea extract contained cytokine-stimulatory and cytokine-suppressive fractions. We also found that non-alkylamide molecules, or unidentified alkylamides, that suppress cytokine production are present in E. purpurea

20 extracts. The cytokine-suppressive molecule, xanthienopyran, and a new alkylamide, 8,11- dihydroxy-dodeca-2E,4E,9E-trienoic acid isobutylamide were identified for the first time in an E. purpurea extract (143, 164). Additionally, LPS and likely other PAMPs derived from endophytes were linked to the cytokine-stimulatory activity of Echinacea, as extracts made from sterilely grown E. purpurea did not stimulate cytokine production (143). Due to the potential for alkylamides and alkylamide-containing Echinacea extracts to limit inflammation, we decided to focus our efforts on evaluating these fatty acid-like molecules.

Alkylamides

Alkylamides are a class of small, lipophilic molecules produced by several species within the Asteraceae, Piperaceae, and Rutaceae families, including Echinacea angustifolia,

Echinacea purpurea, Echinacea pallida, Acmella oleracea, Heliopsis longipes, Anacyclus pyrethrum, Otanthus maritimus, Zanthxylum americanum, and Piper nigrum (reviewed in

(165)). Structurally, alkylamides consist of an amide group connected to a fatty acid and a head group. The fatty acid may vary in length, number of unsaturations, hydroxylations and/or methylations. The majority of alkylamides identified in Echinacea contain an isobutyl head group, but several contain a methylbutyl group instead. Alkylamide content varies between the three medicinal Echinacea species with E. angustifolia and E. purpurea containing significantly higher concentrations compared to E. pallida (157). Each alkylamide appears to have similar biological effects with varying degrees of activity, although specific targets or effects may be identified with further research. It has been hypothesized that the anti-inflammatory activity of Echinacea extracts is due to synergistic or additive effects of the alkylamides present (166, 167).

The reports of anti-inflammatory activity by Echinacea alkylamides are largely based on suppression of cytokine production from stimulated macrophages and T cells in vitro. For example, fourteen different alkylamides from Echinacea suppressed LPS-induced NO production from RAW 264.7 cells to varying degrees (168). In addition, we found that the alkylamides undeca-2Z,4E-diene-8,10-diynoic acid isobutylamide (A4), dodeca-

2E,4E,8Z,10Z/E-tetraenoic acid isobutylamide (A11a/b), dodeca-2E,4E-dienoic acid isobutylamide (A15), and undeca-2E-ene-8,10-dynoic acid isobutylamide (A16) inhibited

TNF-α and PGE2 secretion from IAV stimulated RAW 264.7 cells with A15 displaying the greatest level of suppression (146). A15 also suppressed CCL2, CCL3, CCL5, CCL9, and

G-CSF production after IAV stimulation in the same report. We also found that TNF-α production was dose-dependently inhibited from LPS-stimulated RAW 264.7 cells treated with A15 (143). In another study, PMA/PHA induced IL-2 production from Jurkat T cells was inhibited by A11a/b, A15, and A16, but not CADs (147, 169). These suppressive effects have consistently been found to be unrelated to cytotoxicity at the doses tested (170-172).

Furthermore, the alkylamide pellitorine from Anacyclus pyrethrum, Piper nigrum, and

Asarum sieboldii, inhibited release of high mobility group box 1 (HMGB1), TNF-α, IL-6, IL-

1α, and IL-1ß, reduced permeability, and decreased adhesion molecule expression from stimulated human umbilical vein endothelial cells (173). In the same study, pellitorine administration improved survival in a mouse model of sepsis (173). Additionally, in a mouse model of fulminant hepatitis, A11a/b injected intraperitoneally reduced serum TNF-α levels and hepatocyte damage in the liver (174). The safety of alkylamides has not been investigated extensively; however, no adverse effects from alkylamide administration in vivo have been reported (173-175).

In a recent structure-activity relationship study, we synthesized structural derivatives of A15 with variations in the head group or alkyl chain. We found that removing the double bonds had no significant impact on A15 activity, but that reducing the length of the hydrocarbon chain resulted in reduced inhibition of LPS-stimulated TNF-α production from

RAW 264.7 cells (172). In contrast, changes to the isobutylamide head group led to reduced inhibitory activity, although significant suppression was still observed (172). The modification of alkylamide structure through relatively simple chemical synthesis methods may lead to improvements in their specific activity and effectiveness for suppressing cytokine, chemokine, and prostaglandin production.

The mechanism of alkylamide-mediated cytokine and chemokine suppression has been investigated by several groups but remains unresolved. Raduner et al. demonstrated that A11a/b and A15, but not A16, bound the cannabinoid receptor type 2 (CB2) (176).

However, the suppression of TNF-α, IL-1ß, and IL-12p70 from LPS-stimulated human whole blood by A11a/b, A15, and A16 was mediated through CB2-independent effects (176).

In another study by Hou et al., A11a/b suppression of LPS-stimulated TNF-α production from RAW 264.7 cells was attributed to JNK-mediated upregulation of heme oxygenase-1

(174). Finally, the alkylamide spilanthol (also known as affinin), which is produced by

Acmella oleracea and Heliopsis longipes, inhibited LPS-stimulated TNF-α, IL-6, and IL-1ß secretion and iNOS and COX-2 expression in RAW 264.7 cells (171). In these experiments, the inhibitory effects were linked to decreased NF-B DNA binding after a 10 hour pre- treatment (171).

In addition to cytokine-suppression, alkylamides have been investigated for the tingling and numbing sensations they elicit on sensory neurons in the mouth. High quality

Echinacea extracts induce tingling/numbing effects when applied orally, and this property is due to the presence of alkylamides (177). Additionally, Zanthoxylum plants, which are commonly referred to as “toothache trees,” produce alkylamides including hydroxy-α- sanshool (sanshool), which elicit numbing, analgesic effects on the oral cavity (178).

Sanshool and its derivatives were originally shown to activate transient receptor potential vanilloid 1 (TRPV1)- and ankyrin 1 (TRPA1)-expressing dorsal root ganglion, but Bautista et al. later found that sanshool blocked two-pore domain potassium channels (KCNKs) 3, 9, and 18 (175, 179, 180). In addition, sanshool blocked the excitability of A nociceptive mechanosensitive sensory neurons by inhibiting voltage-gated sodium channels, with Nav1.7 being the most strongly inhibited (181). Spilanthol (mentioned above) is associated with the analgesic and antinociceptive effects of Acmella oleracea and Heliopsis longipes. The antinociceptive effect of spilanthol may involve the NO and K+ channel pathways (182).

Furthermore, pellitorine may inhibit capsaicin-induced pain by blocking calcium movement through TRPV1 (183). Finally, alkylamides have long been known for their insecticidal activity, which was determined to be mediated through effects on voltage-gated insect sodium channels (184, 185).

Overall, alkylamides appear to have broad, inhibitory effects on a number of different cell types including epithelial cells, endothelial cells, macrophages, mast cells, T cells, and neurons. Numerous biological targets, especially ion channels in neurons, appear to be impacted by alkylamides. However, the disruption of ion channel function by alkylamides in immune cells has only recently been explored. For example, Walker et al. linked the cytokine-suppressive effects of pellitorine to the activation of TRPV1 and TRPA1 in LPS- stimulated U937 macrophages (186). In addition, we recently reported that A15 blocked

24 increases in intracellular calcium in A23187-stimulated RBL-2H3 mast cells, Jurkat T cells, and RAW 264.7 macrophages (170). Calcium influx is critical for mast cell degranulation and T cell activation, but its role is controversial in macrophage cytokine production.

However, A15 inhibited RBL-2H3 and bone marrow-derived mast cell degranulation, in agreement with the observed suppressive effect on calcium mobilization (170). How do alkylamides inhibit movement of ions through ion channels? Are specific channels targeted by different alkylamides? Can the activity and selectivity of alkylamides be improved through structural modifications? These are important questions that need to be addressed moving forward if alkylamides are to be developed for treating inflammation and pain.

5. Conclusions Diseases resulting in immunopathologies continue to impose significant health burdens on the human population. Although treatments have improved significantly over the past 60 years, available options are still limited, and many have serious side effects that restrict their usage. Therefore, additional pharmaceutical options are necessary to effectively treat inflammation and pain caused by excessive or inappropriate immune responses. A number of commonly used pharmaceuticals on the market today were originally plant- derived molecules and additional compounds are likely to be discovered from natural sources. Echinacea spp. preparations were often used by Native Americans to treat different inflammatory conditions, and although clinical trials investigating Echinacea activity have produced contradictory results, molecules with useful anti-inflammatory activities may still be identified. For example, the effects of alkylamides on inflammatory mediator production and sensory neuron function may prove to be useful for treating inflammation and pain.

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CHAPTER 2. Ethanolic Echinacea purpurea Extracts Contain a Mixture

of Cytokine-Suppressive and Cytokine-Inducing Compounds, Including

Some that Originate from Endophytic Bacteria

Daniel A. Todd*, Travis V. Gulledge*, Emily R. Britton, Martina Oberhofer, Martha Leyte-

Lugo, Ashley N. Moody, Tatsiana Shymanovich, Laura F. Grubbs, Monika Juzumaite, Tyler

N. Graf, Nicholas H. Oberlies, Stanley H. Faeth, Scott M. Laster, Nadja B. Cech

*These authors contributed equally to this work.

TVG designed the experimental protocols for measuring cytokine and chemokine production from untreated and LPS-treated macrophages. TVG established appropriate conditions for cell treatment experiments including the concentrations of each extract, fraction, and xanthienopyran tested. TVG performed ELISAs and cytotoxicity assays, graphed the results, and performed statistical analyses shown in Figs. 2, 4, 5, 6, 7 & 8. TVG assisted with writing portions of the manuscript including the introduction, results and figure legends associated with the aforementioned figures, and the discussion.

Published: May 1, 2015

DOI: 10.1371/journal.pone.0124276

CHAPTER 3. Synthesis and biological evaluation of a series of fatty acid

amides from Echinacea

Yasamin Moazami*, Travis V. Gulledge*, Scott M. Laster, Joshua G. Pierce

*These authors contributed equally to this work.

TVG designed the experimental protocols for measuring cytokine production from LPS- treated macrophages to test activity of the structural analogs. TVG established appropriate conditions for cell treatment experiments including determining the range of activity for the analogs and interpreting the results. TVG performed ELISAs and cytotoxicity assays, graphed the results, and performed statistical analyses shown in Figs. 3, 5 & 6. TVG assisted with writing portions of the manuscript including introductory material, results and figure legends associated with the aforementioned figures, and discussion of the results.

Published: June 8, 2015

DOI: 10.1016/j.bmcl.2015.06.024

CHAPTER 4. The Mechanism of Inhibiting Cytokine Production from Macrophages

by the Alkylamide Dodeca-2E,4E-Dienoic Acid Isobutylamide

1. Introduction

Echinacea spp. including Echinacea purpurea, Echinacea angustifolia, and

Echinacea pallida, were used medicinally by Native Americans to treat inflammatory conditions such as sore throats, oral pain, upper respiratory tract infections, skin inflammation, and gastrointestinal disorders (1, 2). Today, Echinacea preparations are commonly used as dietary supplements to prevent and treat upper respiratory infections

(URIs). However, clinical trials evaluating the effectiveness of Echinacea for URIs have led to contradictory results (3, 4). It has been suggested that the anti-inflammatory effects of

Echinacea extracts may explain the beneficial effects on respiratory virus infections (5, 6).

Suppression of symptom-inducing, pro-inflammatory mediator production may explain the relief associated with the use of Echinacea extracts.

Echinacea spp. produce a class of small, lipophilic molecules known as alkylamides that inhibit cytokine, chemokine, and prostaglandin production from diverse cell types such as epithelial cells, endothelial cells, macrophages, mast cells, and T cells (7-11).

Alkylamides contain a hydrocarbon chain that can vary in length, number of unsaturations, hydroxylations, and methylations. Alkylamides also have an amide group linked to an isobutyl, methylbutyl, benzyl, or other small functional group (12). Individual plant species can produce a number of different alkylamides. For example, at least 18 alkylamides have been isolated from E. purpurea (12-14). It has been hypothesized that plants utilize alkylamides to inhibit the growth of larval insects or microbial pathogens and therefore protect themselves from predation and disease (15). We were interested in evaluating the

66 cytokine-suppressive effects of the alkylamide dodeca-2E,4E-dienoic acid isobutylamide which has been isolated from E. purpurea, Piper chaba, and Achillea formosa (16, 17). This compound is also referred to as alkylamide 15 (A15), consistent with a previously reported alkylamide numbering system (18).

Due to the inhibition of cytokine, chemokine, and inflammatory lipid production from mammalian immune cells, several authors have hypothesized that alkylamides may be useful clinically for the treatment of inflammatory disease (19-24). For example, Raduner et al. tested the alkylamide dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide (an isomeric mixture also referred to as A11a/b) and A15 and found reduced cytokine production from

LPS-treated human whole blood (23). Similarly, our laboratories have demonstrated reduced production of cytokines, chemokines, and PGE2 from RAW 264.7 macrophage-like cells infected with influenza A virus after treatment with A11a/b and A15 (24). Consistent with these in vitro effects, Hou, et al. demonstrated that alkylamides could suppress inflammation in vivo (25). These authors found that A11a/b was hepatoprotective when administered intraperitoneally in mice challenged with LPS/D-galactosamine, which induces an acute inflammatory response resulting in hepatic injury.

Information on the mechanism of alkylamide-mediated suppression of pro- inflammatory mediator production is limited. Structurally, we have shown that the 12-carbon alkyl chain, as well as the presence of the amide moiety, are important for inhibition of cytokine production without causing cytotoxicity (19). A cannabinoid-dependent mechanism has been proposed since A11a/b and A15 display high binding affinities for the cannabinoid receptor type 2 (CB2) in radioligand displacement assays (23). However, the inhibition of

TNF-α, IL-1ß, and IL-12p70 from LPS-stimulated human whole blood was mediated by

CB2-independent effects. Other studies have attributed alkylamide-mediated cytokine suppression to inhibition of NF-B activity (22), while Hou et al. proposed that A11a/b inhibits TNF-α production through JNK-mediated upregulation of heme oxygenase-1 (HO-1) expression (25).

We have recently reported a high yield, high purity process for the chemical synthesis of A15, an alkylamide found typically at very low levels in Echinacea extracts (19). In this chapter, we used synthetic A15 to characterize its activity and study its mechanism of action.

Our results show that A15 exerts broad, but not universal, suppressive effects on cytokine and chemokine production from macrophages stimulated with a number of different ligands.

These broad effects were not due to cytotoxicity or general inhibition of transcription, as HO-

1 mRNA levels were upregulated. Furthermore, A15 did not inhibit TLR4-stimulated nuclear localization or phosphorylation of NF-B. A15 had differential effects on TNF-α,

CCL2, CCL3, and CCL5 transcript levels. Finally, suppression of TNF-α, CCL3, and CCL5 intracellular protein levels with A15 treatment indicated secretion was not inhibited. Overall, these findings suggest that A15 has broad suppressive activity and may have multiple mechanisms of action.

2. Materials and methods

Reagents and antibodies

All chemicals were purchased from either Sigma-Aldrich (St. Louis, MO) or Thermo Fisher

Scientific (Waltham, MA). TLR agonists were purchased from InvivoGen (San Diego, CA) except for LPS from Salmonella minnesota R595, which was purchased from List Biological

Laboratories (Campbell, CA). Dodeca-2E,4E-dienoic acid isobutylamide (A15) was purchased from ChromaDex (Irvine, CA) or synthesized at North Carolina State University

(Raleigh, NC) as described previously (19). Primary rabbit anti-phospho-NF-B p65 (93H1) and rabbit anti-total-NF-B p65 (D14E12) were purchased from Cell Signaling Technologies

(Danvers, MA). Rabbit polyclonal anti-TNF-α was from PeproTech (Rocky Hill, NJ).

Monoclonal mouse anti-mouse CCL5/RANTES (R6G9) and polyclonal rabbit anti-mouse

CCL3/MIP-1α were from Thermo Fischer Scientific. Polyclonal rabbit anti-Lamin B1

(Abcam, Cambridge, MA) or monoclonal mouse anti-mouse ß-actin (AC15) (Sigma-Aldrich) served as a loading controls. Secondary antibodies were goat anti-rabbit (Cell Signaling

Technologies) or rabbit anti-mouse (Sigma-Aldrich) conjugated to HRP.

Mice

C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were housed at the Biological

Resources Facility at North Carolina State University. All experiments were approved by the

Institutional Animal Care and Use Committee (IACUC) at NC State University.

Cell isolation and culture

Macrophages were isolated from the peritoneum under non-elicited conditions from

C57BL/6 mice as described previously (26). RAW 264.7 were obtained from the American

Type Culture Collection (Manassas, VA). RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS. Media and supplements were obtained from Caisson

Laboratories (Logan, UT) and Sigma-Aldrich. FBS was obtained from Gemini Bio-Products

(Sacramento, CA).

Cell treatments and cytokine measurements

Ethanol was used as the drug vehicle in all cell culture experiments. The maximum ethanol concentration was 1% in all assays and was found not to affect cytokine production. RAW

264.7 cells were seeded at 7.8x104 cells/cm2 in tissue culture plates (Genesee Scientific, San

Diego, CA) and allowed to adhere for 24 hr. Cells were stimulated with each agonist alone or in combination with A15, hydrocortisone, verapamil, or 3,4,5-trimethoxybenzoic acid 8-

(diethylamino)octyl ester (TMB-8) for 16–18 hr. Supernatants were collected, centrifuged at

16,000 x g for 5 min and stored at -80°C until analysis. Murine TNF-α, CCL2/MCP-1,

CCL3/MIP-1α, and CCL5/ ELISA kits were purchased from R&D Systems (Minneapolis,

MN) or eBioscience (San Diego, CA). Optical density was determined using a Synergy HT microplate reader (BioTek Instruments, Inc, Winooski, VT). Cytokine concentrations were interpolated from standard curves.

RNA isolation and real-time qPCR

Total RNA was extracted from RAW 264.7 cells using the RNeasy mini kit (Qiagen,

Valencia, CA) according to the manufacturer's specifications. Cell lysates were immediately homogenized using QIAshredder spin-columns (Qiagen). Residual genomic DNA was eliminated using on-column DNase digestion with the RNase-free DNase set (Qiagen) and resulting RNA was resuspended in nuclease-free water. Amount and purity of RNA was

70 determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). RNA

(800 ng) was denatured and reverse transcription was performed with the Improm ll Reverse

Transcription Kit (Promega, Madison, WI) in a reaction mix containing oligo(dT) primers

(100 µM) for 60 min at 42°C. The QuantiTect SYBR Green PCR kit (Qiagen), was used for real-time PCR analysis. cDNA was amplified using primers specific for murine GAPDH,

TNF-α, CCL2/MCP-1, CCL3/MIP-1α, and CCL5/RANTES genes. Primer combinations are

GAPDH [sense: 5' ACT CCC TCA AGA TTG TCA GCA AT 3'; antisense: 5' ATG TCA

GAT CCA CAA CGG ATA CAT 3']; TNF-α [sense: 5' AGA AGA GGC ACT CCC CCA

AAA 3'; antisense: 5' TCT TTG AGA TCC ATG CCG TTG G 3']; CCL2 [sense: 5' AGC

TGT AGT TTT TGT CAC CAA GC 3'; antisense: 5' TGC TTG AGG TGG TTG TGG AA

3']; CCL3 [sense: 5' CCC AGC CAG GTG TCA TTT TC 3'; antisense: 5' GTG GCT ACT

TGG CAG CAA AC 3']; CCL5: [sense: 5' CCC CAT ATG GCT CGG ACA CCA 3'; antisense: 5' CTA GCT CAT CTC CAA ATA GTT GAT 3']. All primer pairs were purchased from Integrated DNA Technologies (Coralville, IA). PCR was performed in 96 well plates (Thermo Fisher Scientific). Samples were amplified for a total of 40 cycles, followed by a melt curve analysis to ensure the specificity of reactions. Final RT-qPCR data

-C is expressed as the fold change in gene expression using the 2 T method using GAPDH as the reference gene (27). Results were further analyzed relative to two additional reference genes, ß-actin [sense: 5' AGA GCT ATG AGC TGC CTG ACG GCC 3'; antisense: 5' AGT

AAT CTC CTT CTG CAT CCT GTC 3'] and 18S rRNA [sense: 5' GTA ACC CGT TGA

ACC CCA TT 3'; antisense 5' CCA TCC AAT CGG TAG TAG CG 3'], to ensure the consistency of the changes observed in target gene expression in accordance with the MIQE guidelines (28).

Nuclear fraction isolation and western blotting

RAW 264.7 cells were plated in 60 mm tissue culture dishes (Corning, Corning, NY) and incubated overnight. Cells were stimulated with 10 ng/mL LPS and either vehicle or 100 µM

A15. At the indicated times, cell monolayers were rinsed with ice cold phosphate buffered saline (PBS). Whole cell lysates were made by adding ice cold RIPA lysis buffer (Cell signaling Technologies) containing 1X protease inhibitor cocktail, 0.5 mM DTT, and 1 mM

PMSF. Nuclei were isolated using Nuclei EZ Lysis Buffer (Sigma-Aldrich) with added 1X

HaltTM Protease Inhibitor Cocktail (Thermo Fisher Scientific), 0.5 mM DTT, and 1 mM

PMSF. Intact nuclei were collected by scraping and transferring to a microcentrifuge tube on ice. Nuclei were pelleted by centrifugation at 4°C for 5 min at 500 x g. The supernatant containing the cytoplasmic fraction was collected and stored at -80°C. Nuclei were rinsed with Nuclei EZ lysis buffer and centrifuged to pellet again. This rinse was discarded, and ice cold RIPA lysis buffer with inhibitors was added to lyse the remaining nuclei. The nuclear lysate was centrifuged for 10 min at 14,000 rpm to pellet debris, and the supernatant was collected and stored at -80°C. The protein concentration for each nuclear and cytoplasmic lysate was determined using the PierceTM BCA Protein Assay kit (Thermo Fisher Scientific).

Equal amounts of protein (10-25 μg) were loaded in each well of 4–12% Bis–Tris gels

(Thermo Fisher Scientific) and subjected to electrophoresis using the Novex Mini-Cell

System (Thermo Fisher Scientific). Following transfer to PVDF membranes, (Millipore,

Billerica, MA) membranes were blocked for 2 hr with 5% BSA Fraction V in TBS/0.1%

Tween-20 (TBST). Membranes were probed with a primary and secondary antibodies diluted in 5% BSA TBST. Bands were visualized by chemiluminescence using AmershamTM

ECLTM Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Little

Chalfont, UK) and autoradiography film (Genesee Scientific). Film was developed on a

SRX-101A film processor (Konica Minolta, Tokyo, Japan). Films were scanned in black and white as a JPEG file using a Konica Minolta scanner and analyzed using ImageJ as described on the following website: (http://www.lukemiller.org/journal/2007/08/quantifying-western- blots-without.html).

Immunofluorescence

RAW 264.7 cells were seeded onto 8-well chamber slides and stimulated with 10 ng/mL of

LPS or co-stimulated with 10 ng/mL of LPS and 100 μM A15 for various amounts of time.

To visualize the intracellular localization of NF-κB, cells were briefly rinsed with PBS, fixed in 4% formaldehyde, and blocked (5% normal horse serum and 0.3% Triton™ X-100 in

PBS) for 1 hr. The cells were then rinsed with PBS and incubated overnight at 4°C with a rabbit monoclonal α-NF-κB (p65) antibody (D14E12) (Cell Signaling Technologies), followed by incubation for 2 hr at room temperature with α-rabbit IgG (H+L) F(ab')2 fragment conjugated to the fluorescent dye Alexa Fluor® 488 (Cell Signaling Technologies).

After staining, the chambers were removed and a 20% glycerol solution was added before mounting a coverslip. Fluorescence was viewed using a Zeiss Axioskop 2 Plus microscope

(Zeiss). Images were captured using a Spot Pursuit digital camera (Diagnostic Instruments,

Inc., Sterling Heights, MI).

Ca2+ assay

RAW 264.7 cells were plated in clear-bottom, black, 96-well plates and incubated overnight.

Cells were washed 1X with Hank’s balanced salt solution (HBSS) and loaded with 50 µL of

2X fluo-4 AM dye solution from the Fluo-4 Direct™ Calcium Assay Kit (Thermo Fisher

Scientific) for 30 min at room temperature. The dye solution was removed after incubating and cells were rinsed 1X with calcium assay buffer (provided with the kit) containing 2.5 mM probenecid. A final volume of 100 µL of calcium assay buffer with probenecid was added to each well prior to stimulation. Cells were treated with either 1 µM A23187 or 10 ng/mL LPS and fluorescence was measured at 5 sec intervals for 2 min. The average baseline fluorescence of each well was subtracted from the stimulated fluorescent values to calculate the change in

RFU’s.

Statistical analyses

Significant differences between means were determined using the Student’s T test with

GraphPad Prism software (GraphPad Software, La Jolla, CA). Levels of significance are indicated in individual figure legends.

3. Results

Inhibition of cytokine and chemokine production by A15

We previously surveyed several alkylamides isolated from E. purpurea and found that each one inhibited production of inflammatory mediators (24). A15 displayed the broadest inhibitory properties, and therefore, in this chapter we sought to characterize its activity and mechanism of action (24). Initially, a range of concentrations of A15 were tested for the ability to inhibit TNF-α production from LPS-stimulated RAW 264.7 cells (Fig.

1A) with hydrocortisone included as a positive control (Fig. 1B). Both molecules displayed dose-dependent inhibition of TNF-α production, with an IC50 of 11.5 ± 0.7 M for A15. The

IC50 displayed by hydrocortisone in these experiments was 583.8 ± 13.9 nM, consistent with published values (approximately 20 fold lower than A15) (29). Interestingly, the maximum level of TNF-α suppression by A15 was similar to that observed with hydrocortisone, 50.5% versus 41.6%, respectively, suggesting that even at high concentrations, each can only inhibit a component of the response to LPS. We next tested whether combining A15 with hydrocortisone would lead to enhanced suppression of TNF-α. A greater level of hydrocortisone-dependent suppression was observed at each point tested when hydrocortisone was combined with 50 µM A15 (Fig. 1B). In addition, greater suppression was observed with 63.7% inhibition at 1 µM hydrocortisone and 50 µM A15 compared to

40.5% suppression by 100 µM hydrocortisone alone (Fig. 1B). These results indicate that combining A15 and hydrocortisone leads to greater suppression than either molecule can achieve on its own.

Previously, A15 purified from an ethanolic Echinacea extract was found to inhibit influenza A-stimulated production of several different cytokines and chemokines from RAW

264.7 cells (24). To confirm this finding with LPS stimulation, levels of IL-6, CCL2/MCP-1,

CCL3/MIP-1α, and CCL5/RANTES were measured in culture supernatants from LPS- stimulated RAW 264.7 cells. As shown in Fig. 1C, A15 significantly inhibited production of each molecule, confirming that A15 displays broad inhibitory activity. Peritoneal macrophages from C57BL/6 mice were tested to ensure that this effect was not limited to the

RAW 264.7 cell line. As shown in Fig. 1D, A15 broadly suppressed cytokine and chemokine production after LPS treatment. Next, the TLR specificity of A15 was characterized. RAW 264.7 cells were treated with pI:C, R848, and Pam3CSK4, agonists specific for TLR3, TLR7/8, and TLR1/2, respectively. A15 significantly inhibited TNF-α production triggered by each TLR agonist indicating that its inhibitory effect is not specific for TLR4 (Fig. 1E). To test if A15 activity was specific for receptor-mediated signaling, the receptor-independent agonists PMA and ionomycin were used to stimulate TNF-α production. PMA is a diacylglycerol (DAG) analog and ionomycin is a Ca2+ ionophore. We found that TNF-α production after PMA treatment was not blocked significantly by A15, but that ionomycin production was strongly inhibited (Fig. 1F). Overall, these findings indicate that A15 inhibits cytokine and chemokine production from macrophages activated with a number of TLR agonists and Ca2+ ionophore, but not PMA. A15 may target Ca2+-dependent, but not Ca2+-independent pathways.

A15 inhibits cell growth, but does not cause cytotoxicity or inhibit all transcription

In order to determine if A15 inhibits cytokine production through cytotoxic effects,

RAW 264.7 cells were treated with A15, and LDH release was measured. LDH is a cytosolic enzyme that is released into culture supernatants after treatment with cytotoxic molecules such as cycloheximide. No cytotoxic effects were observed by A15 up to 100 µM

A 17500 B 15000 Hydro Hydro + A15 15000 12500 12500 10000

10000 (pg/mL)

(pg/mL) 7500  7500  5000

5000 TNF- 2500 TNF- 2500 0 0 10 0 . 0 10 0 . 5 10 1 . 0 10 1 . 5 10 2 . 0 10 2 . 5 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 A15 Conc. (µM) Hydro Conc. (µM)

C 1 1 5 0 0 0 V e h ic le D 2 0 0 0 V e h ic le

A 1 5 1 0 0 µ M A 1 5 1 0 0 µ M )

7 5 0 0 0 )

L L

* * * 1 5 0 0 m

m 3 5 0 0 0

/ g

g 2 5 0 0 0

p (

( 1 0 0 0

2 0 0 0 0

. .

c * * c

1 5 0 0 0 n

n * *

o o

1 0 0 0 0 5 0 0 * *

C C * * * 5 0 0 0 * * * * * * * * * * * 0 0 T N F -  I L - 6 C C L 2 C C L 3 C C L 5 T N F -  IL - 6 C C L 2 C C L 3 C C L 5

1200 E 20000 Vehicle F Vehicle A15 100 µM 1000 A15 100 µM 15000 NS 800

(pg/mL) 600

10000 (pg/mL)

 * *  ** 400

5000 TNF- TNF- 200 ** *** 0 0 pI:C R848 LPS Pam PMA Ionomycin Figure 1. A15 suppresses cytokine and chemokine production from RAW 264.7 cells and peritoneal macrophages. RAW 264.7 cells were plated and stimulated overnight with 10 ng/mL LPS in combination with A15, hydrocortisone alone, or hydrocortisone at the indicated concentrations with 50 µM A15 (A-C). C57BL/6 peritoneal macrophages were stimulated 24 hr with 1 µg/mL LPS and either vehicle or 100 µM A15 (D). Alternatively, RAW 264.7 cells were stimulated with pI:C, R848, Pam3CSK4, PMA or ionomycin at 20 µg/mL, 100 ng/mL, and 100 ng/mL, 50 ng/mL, or 1 µM, respectively (E-F) . Cytokines and chemokines were measured using commercial ELISA kits. Dashed lines represent cytokine concentration of vehicle treated cells. Results are presented as the mean ± SEM of 3 or more independent experiments (A-B, E-F) or the mean ± SD of a representative experiment from 2 independent experiments (C & D). Statistical analyses comparing treatments with vehicle controls were performed using the Student’s T test, *p<0.05, **p<0.01, and ***p<0.001.

77 compared to the lysis buffer positive control (Fig. 2A). Alternatively, A15 may impact cell growth without causing cytotoxicity. RAW 264.7 cells were treated with A15 or vehicle only and cell growth was measured over 72 hr. RAW 264.7 cell growth was slower in the presence of A15 compared to vehicle only (Fig. 2B). Finally, the effects of A15 on HO-1 mRNA production was measured since a structurally similar alkylamide, A11a/b, was previously reported to upregulate HO-1 expression in RAW 264.7 cells. We found that A15 treatment of LPS-stimulated cells led to increased levels of HO-1 mRNA (Fig. 2C). In addition, we found that A15 itself caused a slight increase in HO-1 expression after 2 hr.

Together, these findings indicate A15 does not inhibit cytokine and chemokine production by causing cytotoxicity or by inhibiting transcription of all cellular proteins.

A15 does not inhibit NF-B p65 nuclear translocation or phosphorylation

LPS-stimulated cytokine and chemokine production occurs through TLR4 dimerization and subsequent intracellular signaling pathways resulting in degradation of IB and translocation of NF-B p65/p50 heterodimers into the nucleus (30). Phosphorylation of NF-B p65 is required for optimal activation and transcriptional activity once inside the nucleus (31). In order to determine if A15 prevents NF-B p65 nuclear translocation or phosphorylation to inhibit cytokine and chemokine production, we treated RAW 264.7 cells with LPS in combination with A15 or vehicle only and measured total and phosphorylated NF-B levels in nuclear lysates by western blot. As shown in Fig. 3A, at 5 min after LPS treatment there was very little to no NF-B p65 or phospho-p65 levels detected in the nuclei. However, by

20 min after treatment, the amount of NF-B p65 increased dramatically. A slight decline in band intensity was noted 60 min post-LPS stimulation, followed again by a rise at 120 min,

A 110 A15 100 90 80 70 60 50 40 30

%Cytotoxicity 20 10 0 10 0 . 5 10 1 . 0 10 1 . 5 10 2 . 0 10 2 . 5 Concentration (µM)

Vehicle B 1.510 7 A15

1.010 7

5.010 6 Cell Number Cell

0 -24 0 24 48 72 Time (hr)

C 30 LPS LPS + A15 Vehicle A15 *** 20

***

10 *** *** HO-1/GAPDH *** Fold Expression Fold * ** *** 0 0 1 2 3 4 5 6 7 8 Time (hr)

Figure 2. A15 effects on cytotoxicity, cell growth, and transcription in RAW 264.7 cells. RAW 264.7 cells were treated with the indicated doses of A15 for 18 hr. Supernatants were collected and LDH release compared to the lysis buffer positive control (dashed line) was analyzed (A). Cells were plated in separate 35 mm dishes 24 hr prior to treatment with either vehicle or 100 µM A15 at time 0. Cells were harvested and counted every 24 hr over a 72 hr timecourse (B). RAW 264.7 cells were treated with vehicle or 100 µM A15 alone or in combination with 10 ng/mL LPS and then cells were rinsed, lysed, and total RNA extracted at the indicated time points. cDNA was synthesized and HO-1 and GAPDH transcript levels were determined using RT-PCR (C). Results are displayed as fold expression using the 2(- C ) T method.  Vehicle,  A15,  LPS + Vehicle,  LPS + A15. Results shown are the means ± SD from 2 independent experiments (A), means ± SD of a representative experiment from 2 independent experiments (B), or means ± SEM of triplicate wells of a representative experiment from 3 independent experiments (C). Statistical analysis comparing A15 treatment with the vehicle control of LPS-stimulated cells was performed using the Student’s T test, *p<0.05, **p<0.01, and ***p<0.001.

A LPS LPS + A15 Time (min) 5 20 60 120 5 20 60 120 Vehicle A15 p-NF-B p65

NF-B p65 D E

Lamin B1 ) 6 0 0 0 0 V e h i c l e B s

y A 1 5 - LPS

t i

u 5 0 0 0 0

s a

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y n

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r d

t 2 0 0 0 0

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T i m e ( m i n ) ) 6 0 0 0 0 V e h i c l e C s

y A 1 5

t i

u 5 0 0 0 0 l

s + LPS a

n 4 0 0 0 0

y n

r 3 0 0 0 0

r d

t 2 0 0 0 0

a b

r 1 0 0 0 0

A ( 0 5 2 0 6 0 1 2 0 T i m e ( m i n )

Figure 3. A15 does not inhibit phosphorylation or nuclear translocation of NF-B p65 in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were treated with 10 ng/mL LPS and either vehicle or 100 µM A15. Cells were washed with ice cold PBS, and lysed with Nuclei EZ extraction buffer to obtain intact nuclei. Nuclei were rinsed and then lysed with RIPA lysis buffer. Protein concentrations of the nuclear lysates were determined and 10 µg of total protein were loaded per lane. Gel electrophoresis and western blotting procedures were performed as described in the Materials and methods section. Representative blots shown in A were probed with antibodies specific for phospho-NF-B p65, NF-B p65, or Lamin B1 (loading control). Images of 3 separate blots from 3 separate experiments were analyzed using ImageJ to calculate the band intensity. RAW 264.7 cells plated in 8-well chamber slides were treated with 10 ng/mL LPS and either vehicle or 100 µM A15 for 20 min (D-G). Cells were washed with PBS, fixed, and stained prior to visualization on a fluorescence microscope as described in the Materials and methods section. Results are shown as the means ± SEM of 3 independent experiments. Statistical analysis comparing A15 treatment with the vehicle control was performed using the Student’s T test. Compared to LPS stimulation A15 treatment did not produce statistically significant differences.

80 which is consistent with the cycling of NF-B p65 into and out of the nucleus (32).

Interestingly, our experiments did not reveal a statistically significant impact of A15 on the translocation of NF-B p65 into the nucleus or its level of phosphorylation (Figs. 3B and C).

Immunofluorescence experiments at 20 min post-LPS stimulation supported these findings

(Fig. 3D-G). Together, these results indicate that A15 does not block LPS-TLR4 intracellular signaling pathways that lead to NF-B p65 nuclear localization and phosphorylation.

A15 inhibits transcription of pro-inflammatory cytokines and chemokines

To understand the basis for inhibition of cytokine and chemokine production by A15, we sought to understand its effects on mRNA accumulation. Fig. 4 shows a series of matched time course experiments, performed with RAW 267.4 cells, where accumulation of cytokine and chemokine proteins in culture supernatants (Figs. 4A-D) and the same mRNAs in cell lysates were measured (Figs. 4E-H). As shown in Figs. 4A-D, the proteins were initially detected in supernatants after 2 hr of stimulation with LPS (TNF-α was detected at 1 hr) with levels continuing to increase throughout the experiment. With A15, a similar pattern of suppression was noted for each response (Figs. 4A-D) with the strongest suppression seen with CCL2 (64.0%) and weakest with CCL5 (38.3%) at 8 hr. As shown in

Figs. 4E-G, the effects of A15 on mRNA accumulation were more complex. For TNF-α,

A15 did not inhibit mRNA accumulation early, but did display significantly reduced levels at the 4 and 8 hr time points (Fig. 4E). More strikingly, A15 completely blocked accumulation of CCL2 mRNA at all time points (Fig. 4F), while partially inhibiting the accumulation of

CCL3 mRNA (Fig 4G). In contrast, no significant effect of A15 on the accumulation of

CCL5 mRNA at any time point was detected (Fig. 4H). Two additional housekeeping genes

A E LPS 12000 100 LPS + A15 Vehicle 10000 A15 75

8000 /GAPDH

(pg/mL) 6000 50 ***   **

4000 **

25 TNF- TNF- * 2000 ** Expression Fold *** 0 0 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Time (hr) Time (hr) B F 400 40

300 30

200 20

** CCL2/GAPDH CCL2 (pg/mL) CCL2 100 10 ** *** Expression Fold * *** 0 0 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Time (hr) Time (hr) C G 25000 150

20000 125 100 15000 *** ** 75 ** 10000

50 CCL3/GAPDH CCL3 (pg/mL) CCL3 5000 ** Expression Fold 25 0 0 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Time (hr) Time (hr) D H 2000 450 1750 375 1500 ** 300 1250 1000 225

750 150 CCL5/GAPDH

CCL5 (pg/mL) CCL5 500

* Expression Fold 75 250 0 0 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Time (hr) Time (hr)

Figure 4. A15 inhibits production of LPS-stimulated cytokine and chemokine protein and mRNA in RAW 264.7 cells. RAW 264.7 cells were stimulated with 10 ng/mL LPS and either vehicle or 100 µM A15. Supernatants were collected at the indicated time points and analyzed for TNF-α (A), CCL2 (B), CCL3 (C), and CCL5 (D) production by ELISA. The remaining cells were lysed, total RNA extracted, cDNA synthesized and TNF-α (E), CCL2 (F), CCL3 (G), and CCL5 (H) transcript levels determined using RT-PCR. Results are (-C ) displayed as fold expression using the 2 T method.  Vehicle,  A15,  LPS + Vehicle,  LPS + A15. Results shown are the means ± SEM of triplicates of a representative experiment from 3 independent experiments. Statistical analysis comparing A15 treatment with the vehicle control of LPS-stimulated cells was performed using the Student’s T test, *p<0.05, **p<0.01, and ***p<0.001.

(ß-actin and 18s rRNA) were utilized in accordance with the MIQE guidelines (28) and the

CT values of each remained constant over the 8 hr time course indicating that A15 is not inhibiting mRNA accumulation in a non-specific manner. In conclusion, A15 displayed differential effects on accumulation of certain cytokine and chemokine mRNAs and these effects may contribute to its effects on protein production.

A15 effects on intracellular cytokine and chemokine accumulation

To determine if reduced levels of cytokines and chemokines in cell culture supernatants was due to reduced secretion from the cell, we analyzed intracellular TNF-α,

CCL3, and CCL5 protein accumulation by performing western blots on whole cell lysates from LPS-stimulated RAW 264.7 cells. We chose to analyze intracellular protein levels between 6 and 10 hr post-LPS treatment because levels of CCL3 and CCL5 were very low through the first 4 hr. We did not analyze intracellular CCL2 levels since it was produced in much lower amounts (Fig. 4B). Consistent with mRNA levels observed in Fig. 4E, intracellular TNF-α protein levels declined between 6-10 hr post-treatment (Fig. 5A). In contrast, CCL3 levels increased throughout the timecourse peaking at 10 hr. Intracellular

CCL5 protein was more difficult to detect, but appeared to remain constant between 6-10 hr.

A15 co-treatment with LPS led to decreased amounts of intracellular TNF-α, CCL3, and

CCL5 protein. Furthermore, after an 18 hr LPS treatment, most TNF-α was secreted from the cells and A15 treatment did not cause TNF-α to accumulate intracellularly (Fig. 5B).

These findings suggest that the reduced level of TNF-α, CCL3, and CCL5 in cell culture supernatants is not due to inhibition of secretion and accumulation within cells.

LPS A 6 hr 8 hr 10 hr B 40000 Vehicle A15 100 µM V A15 V A15 V A15 35000

30000

TNF-α 25000

20000

(pg/mL) *

CCL3  15000

10000

CCL5 TNF- 5000

0 ß-actin Extracellular Extracellular Intracellular Intracellular

Figure 5. A15 inhibits intracellular cytokine and chemokine accumulation. RAW 264.7 cells were treated with 10 ng/mL LPS and either vehicle or 100 µM A15. Cells were washed with ice cold PBS, and lysed with RIPA lysis buffer at the indicated time points. Protein concentrations of the nuclear lysates were determined and 25 µg of total protein were loaded per lane. Gel electrophoresis and western blotting procedures were performed as described in the Materials and methods section. Representative blots shown in A were probed with antibodies specific for TNF-α, CCL3, CCL5 or ß-actin (loading control). RAW 264.7 cells were plated in 24-well plates and treated with 10 ng/mL LPS and either vehicle or 100 µM for 18 hr. Supernatants were collected, cells were rinsed with PBS and lysed with RIPA lysis buffer. Extracellular and intracellular TNF-α levels were determined using a commercial ELISA kit (B). V = vehicle only. Results shown are the means ± SD from 2 independent experiments. Statistical analysis comparing TMB-8 treatment with the vehicle control of LPS-stimulated cells was performed using the Student’s T test, *p<0.05.

The role of Ca2+ mobilization in RAW 264.7 macrophages

We recently reported that A15 inhibited Ca2+ influx in RAW 264.7 cells stimulated with the Ca2+ ionophore A23187 (11). LPS-treatment of macrophages has been shown to increase intracellular Ca2+ levels (33-36). However, we did not detect a change in intracellular Ca2+ within the first 2 minutes after LPS treatment in fluo-4-loaded RAW 264.7 cells (Fig. 6A). We did detect a rise in intracellular Ca2+ after treatment with the Ca2+ ionophore A23187, which was blocked by co-treatment with A15. To further investigate a possible role for Ca2+ in the LPS-mediated response, we utilized functional assays with well- characterized pharmacological agents known to modulate Ca2+ responses. As shown in Fig.

6B, we found that 100 µM A15 inhibited ionomycin-induced TNF-α production from RAW

264.7 cells by 67.8% (IC50 = 17.4 ± 3.0 µM) (Fig. 6B). In addition, two well-characterized

Ca2+ antagonists, verapamil, an L-type Ca2+ channel inhibitor, and TMB-8, an inhibitor of intracellular Ca2+ store release (37, 38), were shown to strongly suppress LPS-induced production of TNF-α (Fig. 6C) suggesting that Ca2+ is necessary for TNF-α production.

TMB-8 also inhibited the LPS-induced accumulation of TNF-α mRNA (Fig. 6D), suggesting that Ca2+ is critical for TNF-α mRNA production. Overall, we found that direct measurement of intracellular Ca2+ with fluo-4 AM did not support a role for Ca2+ in the response of RAW

264.7 cells to LPS. In contrast, functional assays with pharmacological agents did support a role for Ca2+ in these responses, and that A15 may block cytokine production through its effects on the Ca2+ response.

A B 5000 A23187 1 µM 400 A23187 1 µM + A15 100 µM 4000 Vehicle (0.25% EtOH) LPS 10 ng/mL 300 3000

(pg/mL) 200

2000 

in RFU's 

1000 100 TNF-

0 0 20 40 60 80 100 120 10 0 . 5 10 1 . 0 10 1 . 5 10 2 . 0 10 2 . 5 Time (sec) A15 Conc. (µM)

C 16000 Verapamil D 200 LPS TMB-8 TMB-8 + LPS 14000 Vehicle 12000 150 TMB-8

10000 /GAPDH

(pg/mL) 8000 100   6000

4000 50 TNF- TNF- **

Fold Expression *** 2000 *** 0 0 10 0 . 5 10 1 . 0 10 1 . 5 10 2 . 0 10 2 . 5 0 1 2 3 4 5 6 7 8 Conc. (µM) Time (hr)

Figure 6. The role of intracellular Ca2+ in LPS-stimulated cytokine production. RAW 264.7 cells loaded with fluo-4 were stimulated with 1 µM A23187 alone or with 100 µM A15, 10 ng/mL LPS, or vehicle only and the change in fluorescence over 2 min was measured every 5 sec (A). RAW 264.7 cells were plated and stimulated with 1 µM ionomycin and A15 at the indicated concentrations (B) or 10 ng/mL LPS and verapamil or TMB-8 at the indicated concentrations (C). Supernatants were collected and TNF-α levels were measured using a commercial ELISA kit. RAW 264.7 cells were treated with 10 ng/mL LPS with or without 50 µM TMB-8 (D). Cells were lysed, total RNA extracted, cDNA synthesized and TNF-α and GAPDH transcript levels were determined by RT-PCR. RT-PCR results are displayed as (-C ) fold expression using the 2 T method. Dashed lines represent cytokine concentration of vehicle treated cells. Data shown are the means ± SEM of 3-4 independent experiments (B & C) or as the means ± SEM of triplicate wells of a representative experiment from 3 independent experiments (D). Statistical analysis comparing TMB-8 treatment with the vehicle control of LPS-stimulated cells was performed using the Student’s T test, *p<0.05, **p<0.01, and ***p<0.001.

4. Discussion

A15 is found at low quantities in ethanolic Echinacea extracts, compared with the concentration of other alkylamides, limiting experimentation with this molecule (18).

Recently, we reported a method for synthesizing large amounts of A15, sufficient to perform a broad investigation of its activity and mechanism of action (19). We found that A15 inhibited cytokine and chemokine production from RAW 264.7 cells and peritoneal macrophages with several different agonists. In RAW 264.7 cells, we linked this activity to an effect on cytokine and chemokine mRNA accumulation. Pharmacological experiments supported a role for intracellular Ca2+ in TNF-α production; however, we did not detect increases in intracellular Ca2+ after LPS treatment using fluo-4. For the most part, we found that the inhibitory effects of A15 on cytokine production correlated with effects on cytokine mRNAs suggesting that A15 acts upstream of protein synthesis and secretory steps in cytokine production pathways. An exception was noted with CCL5 production, where A15 inhibited protein production but not mRNA accumulation. Apparently with this chemokine, accumulation of the transcript can occur as normal through an A15-resistant pathway, while an additional effect(s) of A15 on protein production accounts for the reduced levels of CCL5 intracellularly and in culture supernatants.

Quantitatively, our studies showed that A15 was approximately 20-fold less potent than hydrocortisone at inhibiting TNF-α production (Fig. 1). In the future, structural alterations could be created to increase the effectiveness of A15. Dexamethasone, for example, was synthesized from hydrocortisone in a process that included a number of structural modifications, ultimately increasing its anti-inflammatory activity 30-40 fold (39).

Alternatively, since A15 and other alkylamides are readily synthesized, the lower specific

87 activity of these molecules could be overcome by their use at higher concentrations. It was also noteworthy that A15 and hydrocortisone were each only able to block approximately half of TNF-α production. When lower doses of A15 and hydrocortisone were combined, greater suppression was observed than either compound could achieve alone (Fig. 1B).

These findings indicate that A15 and hydrocortisone may target different components of the

LPS response. Chronic inflammatory conditions requiring long-term use of glucocorticoids, such as hydrocortisone, can lead to many serious side effects. Therefore, A15 may be useful in combinations with hydrocortisone, or other glucocorticoids, to lower the dose needed, increase the effectiveness, and possibly avoid or limit side effects.

Alkylamides have been linked to the therapeutic effects of many medicinal plants

(15), and a number of studies have addressed the mechanism of cytokine suppression in immune cells by alkylamides. Wu et al. attributed alkylamide suppression of cytokine production to inhibitory effects on NF-B activity (22). However, NF-B nuclear translocation was not directly measured in that study, and we did not observe an effect on

NF-B p65 translocation or phosphorylation in our investigations (Fig. 3). It is possible that when RAW 264.7 cells are treated with LPS in combination with A15, NF-B p65 translocates to the nucleus as usual, but is unable to efficiently promote transcription. This could be due to interference from other transcription factors that prevent assembly of the normal enhanceosome in the promoter region of these target genes. Finally, Hou et al. pre- treated RAW 264.7 cells with the alkylamide A11a/b for 6 hr to inhibit LPS-stimulated TNF-

α production after 1 hr (25). Suppression was linked to increased HO-1 protein levels through a JNK-mediated pathway, which was proposed to reduce TNF-α production.

Similarly, we also found that A15 induced a rise in HO-1 mRNA. However, the rise in HO-1

88 mRNA was not detected until 2 hr of treatment which makes it unlikely that HO-1 protein could produce changes in cytokine levels within 2 hr of LPS treatment (Fig. 2C).

Extracts of Echinacea and other plants that produce alkylamides are known to cause a tingling or numbing sensation in the mouth upon ingestion (1, 40). Investigations into the mechanism of this response with hydroxy-α-sanshool, an alkylamide found in Szechuan peppers, linked this sensation to inhibition of two-pore domain potassium channels (8) or activation of transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) ion channels resulting in neuron activation (41-45). In addition, alkylamides were previously studied for their insecticidal activity, which was found to be mediated through insect sodium channel activation (46, 47). We recently reported that A15 inhibits Ca2+ influx in mast cells

(11). Therefore, it is possible that the effects of A15 on macrophage cytokine production also occur through effects on ion channels. Ca2+ has been shown to be critical for signaling through TLRs (34, 35, 48, 49) and for translocation and activation of various transcription factors (50, 51). However, it is unclear which channels are important for Ca2+ mobilization in macrophages after LPS treatment. A15 inhibited TNF-α production after stimulation with

TLR4, TLR3, TLR7/8, and TLR1/2 agonists indicating its activity specific to a shared component of TLR signaling (Fig. 1E). A15 also suppressed ionomycin-stimulated TNF-α production indicating that A15 can inhibit Ca2+-driven cytokine production (Fig. 6B).

Furthermore, PMA-stimulated TNF-α production was not significantly reduced by A15 indicating that A15 does not block TNF-α production through a kinase-dependent pathway. .

These findings suggested that A15 may be inhibiting cytokine and chemokine production from macrophages by inhibiting Ca2+ mobilization.

With RAW 264.7 cells, we took two approaches to evaluate the effects of A15 on

LPS- and ionophore-induced Ca2+ responses: pharmacologic manipulation and direct measurement with fluo-4 AM. The results of our experiments with pharmacological agents were consistent with published reports which have used these agents with various macrophage cell types (33, 35, 48, 49). We found that TMB-8 and verapamil blocked LPS- induced production of TNF-α mRNA and protein, indicating the response of RAW 264.7 cells to LPS is Ca2+-dependent. Furthermore, because A15 blocked the ionomycin-induced production of TNF-α, our experiments support the hypothesis that A15 is interfering with the

Ca2+ response in RAW 264.7 cells.

Our attempts to use fluo-4 AM to measure levels of intracellular Ca2+ in RAW 264.7 cells were not productive. Macrophages have the ability to rapidly compartmentalize and/or efflux fluorescent indicator dyes making use of these dyes with macrophages problematic, especially with cellular responses that occur during a multi-hour period (52). While a number of authors using Ca2+ indicator dyes have reported increased levels of intracellular

Ca2+ following LPS treatment (33-36) a number of others have not (52-54). In an attempt to overcome these problems, Kim et al. stained RAW 264.7 cells with fluo-4 AM 18 hr after treatment with pI:C, and did report elevated levels of intracellular Ca2+ (55). Using this approach, we also found increased Ca2+ levels after 18 hr of treatment with LPS; however, the fluo-4 staining pattern was punctate, suggesting the dye and/or Ca2+ was being partitioned to subcellular compartments, and the measurements were not indicative of free intracellular

Ca2+ (data not shown). Further investigations using genetically encoded Ca2+ indicator proteins will be necessary to resolve these questions in macrophages.

In this study, we have characterized the scope of A15’s activity and found that it inhibits the production of several key inflammatory cytokines and chemokines from macrophages stimulated with multiple agonists. Many inflammatory conditions are associated with excessive cytokine production, and increases in intracellular Ca2+ help promote cytokine secretion. Therapeutic strategies directed at blocking Ca2+ influx should therefore reduce cytokine levels and help ameliorate symptoms associated with inflammation. Alkylamides are natural products and have been tested safely in vivo in a fulminant hepatitis mouse model (25). Additional models of inflammation such as skin and intestinal inflammation should also be investigated. Overall, our findings suggest that additional research into alkylamides and their anti-inflammatory activity may lead to useful therapeutics.

5. References

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CHAPTER 5. Mast Cell Degranulation and Calcium Influx are Inhibited

by an Echinacea purpurea Extract and the Alkylamide Dodeca-2E,4E-

Dienoic Acid Isobutylamide

Travis V. Gulledge, Nicholas M. Collette, Emily Mackey, Stephanie E. Johnstone, Yasamin

Moazami, Daniel A. Todd, Adam J. Moeser, Joshua G. Pierce, Nadja B. Cech, Scott M.

Laster

Published: In Press

1. Abstract

Ethnopharmacological relevance

Native Americans used plants from the genus Echinacea to treat a variety of different inflammatory conditions including swollen gums, sore throats, skin inflammation, and gastrointestinal disorders. Echinacea spp. produce many fatty acid amides referred to as alkylamides, which can inhibit cytokine, chemokine, and prostaglandin production from macrophages and T cells. Alkylamides are thought to contribute to the anti-inflammatory activity of E. purpurea (L) Moench extracts by inhibiting production of inflammatory mediators.

Aim of the study

The goal of this study was to evaluate the effects of an ethanolic E. purpurea root extract and the alkylamide dodeca-2E,4E-dienoic acid isobutylamide (A15) on mast cells, which are important mediators of allergic and inflammatory responses. Inhibition of mast cell activation may help explain the traditional use of Echinacea by Native Americans.

Materials and methods

A15 was evaluated for its effects on degranulation, calcium influx, cytokine and lipid mediator production using bone marrow derived mast cells (BMMCs) and the transformed rat basophilic leukemia mast cell line RBL-2H3. Methods included enzymatic assays, fluorimetry, ELISAs, and microscopy. A root extract of E. purpurea, and low and high alkylamide-containing fractions prepared from this extract, were also tested for effects on mast cell function. Finally, we tested A15 for effects on calcium responses in RAW 264.7 macrophage and Jurkat T cell lines.

Results

A15 inhibited ß-hexosaminidase release from BMMCs and RBL-2H3 cells after treatment with the calcium ionophore A23187 by 83.5% and 48.4% at 100 µM, respectively.

Inhibition also occurred following stimulation with IgE anti-DNP/DNP-HSA. In addition,

A15 inhibited 47% of histamine release from A23187-treated RBL-2H3 cells. A15 prevented the rapid rise in intracellular calcium following FcRI crosslinking and A23187 treatment suggesting it acts on the signals controlling granule release. An E. purpurea root extract and a fraction with high alkylamide content derived from this extract also displayed these activities while fractions with little to no detectable amounts of alkylamide did not. A15 mediated inhibition of calcium influx was not limited to mast cells as A23187-stimulated calcium influx was blocked in both RAW 264.7 and Jurkat cell lines with 60.2% and 43.6% inhibition at 1 min post-stimulation, respectively. A15 also inhibited the release of TNF-α, and PGE2 to a lesser degree, following A23187 stimulation indicating its broad activity on mast cell mediator production.

Conclusions

These findings suggest that A15 and other alkylamides may be useful for treating allergic and inflammatory responses mediated by mast cells. More broadly, since calcium is a critical second messenger, the inhibitory effects of alkylamides on calcium uptake would be predicted to dampen a variety of pathological responses, and may help explain the traditional uses of Echinacea.

Abbreviations

A15, alkylamide 15; ß-hex, beta-hexosaminidase; BMMC, bone marrow-derived mast cell; CRAC, calcium-release activated calcium; CB2, cannabinoid receptor type 2; ER, endoplasmic reticulum;

FcRI, fragment crystallizable epsilon receptor 1; IACUC, Institutional Animal Care and Use

Committee; IgE, immunoglobulin E; IP3, inositol 1,4,5-trisphosphate; LPS, lipopolysaccharide; PLC-

, phospholipase C-gamma; PGE2, prostaglandin E2; STIM-1, stromal interaction molecule 1; TNF-α, tumor necrosis factor alpha

Keywords

Alkylamide, Echinacea purpurea, allergies, inflammation, mast cell

Chemical compounds studied in this article:

Dodeca-2E,4E-dienoic acid isobutylamide (PubChem CID: 6443006)

2. Introduction

Echinacea purpurea (L) Moench, Echinacea angustifolia (DC) Hell., and Echinacea pallida (Nutt.) Nutt. are medicinal herbs indigenous to the United States and Canada. Native

Americans used primarily preparations of Echinacea roots to treat a variety of conditions associated with inflammatory and allergic disease, including swollen gums, inflamed skin, sore throats, and gastrointestinal disorders (reviewed by (1)). Today, various preparations of

Echinacea, including ethanolic root extracts, capsules with ground plant material, and teas are used primarily to prevent or treat upper respiratory infections (URIs) (reviewed by (2)).

It is currently unclear how preparations of Echinacea benefited Native Americans or provide relief to the people who use Echinacea to treat URIs. Most early studies focusing on the medicinal properties of Echinacea hypothesized that it provides an “immune-boost” (3,

4). However, as the pathological mechanisms underlying inflammatory and allergic disorders, and URIs became known (5-8) it became increasingly unclear how boosting the immune system would benefit individuals already suffering from excess immune activation.

Furthermore, much of the immunostimulatory activity in Echinacea extracts has been attributed to lipoproteins and lipopolysaccharides derived from either bacterial contaminants or bacterial endophytes (9, 10) and not a constitutive activity of the plant. Instead, most recent investigations of Echinacea’s medicinal activity have focused on anti-inflammatory effects since suppression of inflammation would provide relief from many of the disorders listed above, including URIs. In support of this hypothesis, a number of in vitro studies have confirmed that Echinacea extracts can suppress production of inflammatory mediators from macrophages and T cells while suppression of inflammatory mediator production has also been demonstrated in vivo in influenza A infected mice (11).

100

The inhibitory effects of Echinacea extracts on inflammatory mediator production with macrophages and T cells have been attributed to alkylamides (10, 12, 13). Alkylamides, also known as alkamides, are a class of fatty acid-like molecules produced by a number of medicinal plants including Echinacea spp., Acmella oleracea, and Zanthoxylum americanum

((14-16) and reviewed by (17)). Each alkylamide contains an amide group, an alkyl chain, and a functional group such as isobutyl, benzyl, or methyl group. The number of carbons in the alkyl chain can vary, as well as the number and position of double and triple bonds.

Alkylamide structures can be further diversified with modifications including hydroxylations and methylations (16). The alkylamide dodeca-2E,4E-dienoic acid isobutylamide, whose activity was examined in this report, is referred to as alkylamide 15 (A15) according to a numbering system reported previously (18). The complete relationship between alkylamide structure and function is not known. Certain alkylamides have been shown to bind the cannabinoid receptor type 2, and thereby modulate constitutive cytokine expression with human whole blood (19), while structure-activity relationship studies have shown that the length of the alkyl chain is important for alkylamide-mediated suppression of TNF-α production from macrophages (20).

Another key immune cell involved in allergic and inflammatory responses, including

URIs, is the mast cell (7, 21, 22). The effects of Echinacea preparations, or of specific alkylamides, on mast cell responses have not been examined. Mast cells, found in mucosal and serosal tissues, contain large, dense granules filled with pre-formed inflammatory mediators. Mast cells can be activated by Ag-specific IgE loaded into FcR1 receptors or by several toll-like and complement receptors (23-25). Within seconds after activation, mast cells and basophils (a circulating form of the mast cell) exocytose their granules releasing

101 histamine, cytokines, chemokines, proteases, and growth factors into the surrounding tissue.

Mast cells and basophils also produce cytokines, chemokines, prostaglandins, and leukotrienes by de novo biosynthetic pathways (26). Several recent reports suggest that mast cells may indeed be a target for Echinacea preparations. For example, an E. purpurea aerial extract reduced several parameters of allergy in ovalbumin-sensitized guinea pigs (27).

Similarly, an E. purpurea root extract was shown to modulate disease in a model of atopic eczema (28). For the first time, in this manuscript we examine the effects of the alkylamide

A15, and an ethanolic E. purpurea root extract, on the degranulation of mast cells and the ability of mast cells to mediate calcium signaling.

102

3. Materials and methods

3.1 Reagents

Dodeca-2E,4E-dienoic acid isobutylamide (A15) was synthesized at North Carolina State

University (Raleigh, NC) as described previously (20). In brief, a two-step oxidation of the commercially available diene-containing alcohol was performed to create the carboxylic acid

® followed by coupling with isobutyl amine (T3P ). Flash chromatography on SiO2 was used to purify the crude reaction mixtures and performed on a Biotage Isolera utilizing Biotage cartridges and linear gradients (Biotage AB, Uppsala, Sweden). This process provided A15 in good yield and proved identical to the natural product by 1H and 13C NMR analysis with a

Varian Mercury-VX 300, a Varian Mercury-VX 400, or a Varian Mercury-Plus 300 instrument in CDCl3. All other chemicals were purchased from either Sigma-Aldrich (St.

Louis, MO) or Thermo Fisher Scientific (Waltham, MA).

3.2 Mice

Male and female C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were housed at the Biological Resources Facility at North Carolina State University. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at NC State

University.

3.3 Cell isolation and culture

Bone marrow cells were isolated from the femurs of 6-8 week old C57BL/6 mice and cultured in RPMI-1640 containing 10% heat-inactivated FBS, 1X non-essential amino acids,

10 mM HEPES buffer, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin, 5 ng/mL mIL-3, and 5 ng/mL mSCF for 4-6 weeks until mature, bone marrow-

103 derived mast cells (BMMCs) were obtained, as described previously (29). Differentiation into >98% mast cells was confirmed through toluidine blue staining (1%, pH 1). RBL-2H3,

RAW 264.7, and Jurkat cells were obtained from the American Type Culture Collection

(Manassas, VA). RBL-2H3 cells were cultured in MEM supplemented with 15% heat inactivated FBS, 1X non-essential amino acids (Sigma-Aldrich), and 1 mM sodium pyruvate

(Sigma-Aldrich). RAW 264.7 and Jurkat cells were cultured in DMEM supplemented with

10% FBS. FBS was obtained from Gemini Bio-Products (Sacramento, CA). For TNF-α, prostaglandin, and histamine measurements, cells were stimulated with A23187 alone or in combination with A15 for 8 hrs or 1 hr for histamine release. Supernatants were collected, centrifuged at 16,000 x g for 5 min and stored at -80°C until analysis. TNF-α sandwich

ELISA kits, or PGE2 and histamine competitive direct EIA kits were purchased from eBioscience (San Diego, CA), Enzo Life Sciences (Farmingdale, NY), or Oxford Biomedical

Research (Rochester Hills, MI), respectively. Optical density was determined using a

Synergy HT microplate reader (BioTek Instruments, Inc, Winooski, VT). Concentrations of each analyte were interpolated from standard curves.

3.4 ß-hexosaminidase degranulation assay

RBL-2H3 cell and BMMC degranulation was measured using a ß-hexosaminidase (ß-hex) activity assay. RBL-2H3 cells were plated into 96-well flat-bottom plates at 100,000 cells/well and BMMCs were plated into 96-well v-bottom plates (to allow cells to pellet during centrifugation) at 200,000 cells/well. Cells were incubated for 4 hr before media was aspirated and replaced with vehicle or media containing 1 µg/mL mouse IgE anti-DNP (SPE-

7) (Sigma-Aldrich) and incubated overnight. Prior to stimulation, cells were rinsed with calcium-containing Tyrode’s buffer (1.8 mM CaCl2, 135 mM NaCl, 5 mM KCl, 5.6 mM

104 glucose, 1 mM MgCl2, 0.1% BSA, 20 mM HEPES, pH 7.4). All treatments were prepared in

Tyrode’s buffer. RBL-2H3 cells were stimulated with either A23187 (Sigma-Aldrich) or

DNP-HSA (Sigma-Aldrich) alone or in combination with A15 and incubated for 1 hr at

37°C. Ethanol was used as the vehicle for A15 and the final concentration was 1% in all degranulation experiments. Wells were then aspirated, and solutions containing A23187 alone or in combination with A15 were added and incubated for 1 hr at 37°C. After the incubation, 30 µL of supernatant was collected and incubated with 10 µL of 3.4 mg/mL p- nitrophenyl N-acetyl-ß-D-glucosaminide (p-NAG) at 37°C for 1 hr to measure the amount of

ß-hex released from the cells. Excess supernatant was removed and the remaining cells were lysed with 100 µL of 0.1% Triton™ X-100 in Tyrode’s buffer. 30 µL of the cell lysate was incubated with 10 µL of p-NAG to determine the amount of ß-hex remaining in the cells.

The reaction was stopped by adding 100 µL of 0.2 M sodium carbonate buffer. Absorbance was read at 405 nm (A405) on a BioTek Synergy HT microplate reader. The percent degranulation was calculated as the A405 of the supernatant divided by total absorbance (A405 of the supernatant + A405 of the lysate) x 100.

3.5 LDH cytotoxicity assay

RBL-2H3 cells were seeded at 100,000 cells/well into 96-well plates and incubated overnight prior to treatment with a range of A15 concentrations or 10 µg/mL cycloheximide.

Supernatants were collected after 18 hr and analyzed for lactate dehydrogenase (LDH) activity using the commercially available Pierce LDH Cytotoxicity Assay Kit according to the manufacturer’s instructions (Thermo Fisher Scientific). In a 96-well plate, 50 μL of supernatant were incubated for 30 min with 50 μL of reaction mixture containing a tetrazolium salt that is reduced to formazan in the presence LDH protected from light. 50 μL

105 of stop solution were added, and the A490 with A690 subtracted to remove background absorbance on a BioTek Synergy HT microplate reader. LDH release from vehicle treated cells was used to determine spontaneous LDH release. Additionally, lysis buffer was used to determine maximum LDH release.

3.6 Calcium assay

RBL-2H3, BMMC, RAW 264.7, or Jurkat cells were plated at 100,000, 200,000, 80,000, or

125,000 cells/well, respectively, into clear-bottom, black, 96-well plates (BioExpress,

Kaysville, UT) and incubated overnight with media only or media containing IgE anti-DNP.

Cells were loaded with the calcium-sensitive dye fluo-4 AM using the Fluo-4 DirectTM

Calcium Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions by incubating at 37°C for 30 min followed by 30 min at room temperature. Prior to stimulation, baseline fluorescent readings were measured from triplicate wells in 5 s intervals for 1 min using a BioTek Synergy HT microplate reader with 485/20 nm excitation and

528/20 nm emission filters. Cells were then treated with either 1 µM A23187 or 50 ng/mL

DNP-BSA prepared in calcium- and magnesium-containing or calcium- and magnesium-free

HBSS with 20 mM HEPES, and fluorescence was measured in 10 s intervals for 2 min. The final concentration of ethanol was 0.25% in these assays. The average baseline fluorescence of each well was subtracted from the stimulated fluorescent values to calculate the change

() in RFU’s and graphed as shown.

3.7 Fluorescence microscopy

RBL-2H3 cells plated and loaded with fluo-4, as described above, were treated and observed with an Axioskop 2 microscope (Zeiss, Oberkochen, Germany) using a fluorescent

106 illuminator HXP-120 light source and images were acquired at 30, 60, or 120 sec after stimulation with an AxioCam MRc5 camera (Zeiss) and AxioVision software (Zeiss).

3.8 Separation and fractionation of an Echinacea purpurea extract

Echinacea purpurea (L) Moench roots were purchased from Pacific Botanicals (Grants Pass,

OR). A voucher specimen (NCU 633811) was deposited at the North Carolina Herbarium.

An ethanolic extract (EE) was prepared and fractionated as described previously (10).

Briefly, roots were collected, dried, and ground mechanically with a Wiley Mill Standard

Model No. 3 (Arthur H. Thomas Co., Philadelphia, PA) to mesh size 2 mm. Roots were macerated in 75% ethanol (Pharmaco-AAPER, Shelbyville, KY) for seven days. A liquid- liquid partitioning scheme was used to generate an alkylamide-containing chloroform layer

(CL) (10), which was subsequently fractionated using normal-phase flash chromatography with an automated Isco CombiFlash RF system over a RediSep Rf silica gel column

(Teledyne Isco, Lincoln, NE). The eluent was collected in 13 different fractions (F1-13) based on LC-UV chromatograms with Galaxie Chromatography Workstation software

(version 1.9.3.2). Concentrations of the most abundant alkylamides were quantified in each sample by LC-MS with an Acquity ultra-high performance liquid chromatography (UHPLC) system (Waters Corporation, Milford, MA) coupled to a LTQ Orbitrap XL Hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA) and used to estimate the total alkylamide content (µg/mg extract). Results for each extract or fraction were: EE = 51 ± 8.2,

CL 140 ± 4.7, F2 = below limit of quantification, F6 = 310 ± 42, F8 = 0.14 ± 0.034 (Todd et al., 2015).

107

3.9 Statistical analysis

Significant differences between means were determined using a Student’s unpaired t-test, a one-way ANOVA, or a repeated measures one-way ANOVA with Dunnett’s post-test to compare the significance of each dose or treatment tested with the vehicle control (30) using

GraphPad Prism version 5.0 software (GraphPad Software, La Jolla, CA). Tests used and levels of significance are indicated in individual figure legends.

108

4. Results

4.1 A15 inhibits mast degranulation

In these experiments, we examined two populations of mast cells; primary bone marrow derived mast cells (BMMCs) from C57BL/6 mice, and the cell line RBL-2H3, a rat basophil-derived, transformed cell line whose degranulation pathways are similar to those of primary mast cells and basophils and frequently used to study mast cell biochemistry

(reviewed by (31)). With both cell types, degranulation was induced by treatment with either IgE anti-DNP and DNP-HSA or the calcium ionophore A23187. To monitor degranulation, we measured the release of two granule components, the enzyme ß- hexosaminidase (ß-hex) and the vasoactive amine, histamine. The alkylamide we used in these studies was A15 because in experiments with monocytes and macrophages it displayed potent and broad cytokine inhibitory activity (19, 32). As shown in Fig. 1A, A15 contains an isobutyl head group, an amide group, and a 12-carbon fatty acid chain with double bonds positioned at carbons 2 and 4. Instead of isolating A15 from E. purpurea, A15 was chemically synthesized and analyzed by 1H and 13C NMR to ensure quality, as described previously (20).

Fig. 1 panels B-F, summarize key findings for the effects of A15 on mast cell degranulation. As shown in Fig. 1B, we found that A15 inhibited the release of ß-hex from

A23187-stimulated BMMCs in a dose-dependent fashion with a maximum inhibition of

83.5% ± 2.8% at a concentration of 100 µM. A15 also inhibited the release of ß-hex from

A23187-stimulated RBL-2H3 cells although the level of inhibition was less than with

BMMCs (48.4% ± 3.7% at 100 M) (Fig. 1C). As shown in Fig. 1D, in experiments with

109

A B 20

15 Fatty acid Amide Isobutyl

11 9 7 5 3 10 *** 1 12 10 8 6 4 2 5 *** %Degranulation ***

0 Vehicle 25 50 100 A15 Conc. (µM) C D 50 60 Vehicle A15 40 * 50

** 40 30 *** 30 20

20 %Degranulation

10 %Degranulation 10

0 0 Vehicle 25 50 100 0 10 20 30 40 50 60 E A15 Conc. (µM) F Time (m) 10 250

8 200 ** ** 150 6 ***

4 100

***

%Degranulation 2 50 Histamine (ng/mL) Histamine *** 0 0 Vehicle 25 50 100 0 25 50 100 A15 Conc. (µM) A15 Conc. (µM) Figure 1. A15 inhibits BMMC and RBL-2H3 cell degranulation. The structure of alkylamide A15 is shown with different functional groups and carbons in the fatty acid chain labeled (A). BMMCs were plated and stimulated 4 hrs later with 1 µM A23187 and the indicated concentrations of A15 or vehicle only. The percent degranulation was measured after 1 hr (B). RBL-2H3 cells were treated with A23187 and the percent degranulation was determined 1 hr later (C) or every 10 min over a 1 hr time course (D). RBL-2H3 cells were plated and incubated for 4 hrs before the media was aspirated and replaced with media containing 1 µg/mL IgE anti-DNP. Cells were incubated overnight and washed prior to stimulation. The percent degranulation from RBL-2H3 cells was calculated after stimulation with 50 ng/mL DNP-HSA in combination with the indicated concentrations of A15 or vehicle only after 1 hr (E). RBL-2H3 cells were stimulated for 1 hr in Tyrode’s buffer before supernatants were collected and levels of histamine were measured using a competitive inhibition EIA kit (F). Data shown are the means ± SEM from 3-4 independent experiments (B,C and F) or the means ± SEM of triplicate wells from a representative experiment from 3 independent experiments (D and E). Statistical analysis was performed using a repeated measures one- way ANOVA with Dunnett’s post-hoc test, *p<0.05, **p<0.01, ***p<0.001.

110

A23187-treated RBL 2H3 cells, we found that inhibition of ß-hex release was apparent within 20 min after treatment began. Fig. 1E shows that A15 also inhibited degranulation from RBL-2H3 cells when they are activated by IgE anti-DNP and DNP-BSA. Finally, in

Fig. 1F, we show that in addition to ß-hex, A15 also inhibited the release of the vasoactive amine histamine from RBL-2H3 cells treated with A23187 (47.0% ± 7% at 100 µM).

The data presented in Fig. 1 suggest that A15 acts biochemically to block degranulation from BMMCs and RBL-2H3 cells; however, because alkylamides have not been tested with these cells previously, we sought to reveal other explanations for these results. Lactate dehydrogenase release assays were performed to ensure that the results reported in Fig. 1 did not arise from a cytotoxic effect. As shown in Supplemental Fig. 1, we did not measure significant cytotoxic activity of A15 towards RBL-2H3 cells, even after overnight treatment, in agreement with previous studies of A15 and RAW 264.7 macrophages (20). In addition, we tested whether A15 could inhibit the enzymatic activity of ß-hex itself. As shown in Supplemental Fig. 2, when RBL-2H3 cell lysates were incubated with the substrate (p-NAG) alone or in the presence of the indicated concentrations of A15, production of the chromogenic product was not inhibited. Finally, as shown in Fig.

2, we found that the inhibitory effects of A15 on degranulation were apparent after staining with toluidine blue. Toluidine blue is a metachromatic dye which stains nuclei blue and mast cell granules purple. Treatment of BMMCs with A23187 for 30 min noticeably reduced staining compared to control cells (Figs. 2A and C), which was blocked by A15 (Fig.

2D) confirming that A15 does indeed block release of granules from BMMCs. A15 treatment alone did not noticeably affect staining (Fig. 2B).

111

Vehicle A15 A B

- A23187

C D

+ A23187

Figure 2. A15 inhibits granule release from BMMCs. BMMCs were treated with 100 µM A15 or 0.25% ethanol only (vehicle) alone and in combination with 1 µM A23187 for 30 min. BMMCs were cytocentrifuged onto a glass slide, fixed, stained with toluidine blue (1%, pH<1) for 30 min, and washed before observing microscopically. Images were acquired at 400X magnification and are representative.

112

4.2 A15 blocks calcium influx in BMMCs and RBL-2H3 cells

Since A15 blocked both FcRI and A23187-mediated degranulation, its effects likely occur via an element common to both pathways. Calcium is a key second messenger in the activation pathways of mast cells and basophils (reviewed by (25)). Therefore, we hypothesized that A15 was acting to inhibit calcium mobilization in BMMCs and RBL-2H3 cells. To address this hypothesis, we utilized the calcium-sensitive fluorescent dye fluo-4

AM. Cells were loaded with the dye, stimulated, and a multichannel absorbance plate reader was used to quantitatively examine levels of intracellular calcium. As shown in Fig.

3A, we recorded a rapid rise in levels of intracellular calcium in RBL-2H3 cells treated with

A23187. The increase was noticeable after ~10 sec and tended to plateau after ~60-80 sec.

We found that treatment with A15 blocked this response in a dose-dependent fashion (Fig.

3A) with 83.2% ± 5.5% inhibition measured at the 100 M concentration of A15 (Fig. 3B).

A15 also blocked the calcium response in RBL-2H3 cells treated with IgE anti-DNP and

DNP-BSA (Fig. 3C and D) and in BMMCs treated with either A23187 (Fig. 3E and F) or IgE anti-DNP and DNP-BSA (Fig. 4G and H). Again, readily visible by microscopy, we found that treatment of RBL-2H3 cells with A23187 induced an increase in fluorescence, which was blocked by A15 (Supplemental Fig. 3). It should also be noted that A15 did not cause a significant change in the level of constitutive fluorescence, compared to vehicle, in unstimulated cells. For example, in Supplemental Fig. 3, analysis of pixel intensity did not reveal a significant change in fluorescence with A15 treatment (p>0.05, n=5 fields) compared with a significant 158.4% increase with A23187 treatment (p<0.0001, n=5 fields).

113

RBL-2H3 + A23187 A 5000 Vehicle B 5000 100 µM A15 50 µM A15 4000 25 µM A15 4000 Assay Buffer

3000 3000

in RFU's in 2000 2000

**  ** 1000 min) (1 RFU's in 1000  ***

0 0 0 20 40 60 80 100 120 AB Vehicle 25 50 100 Time (s) A15 Conc. (µM)

RBL-2H3 + DNP-BSA C 2500 D 3000

2000 2000 1500

in RFU's in 1000 **

 1000 ***

500 min) (1 RFU's in 

0 0 0 20 40 60 80 100 120 AB Vehicle 25 50 100 Time (s) A15 Conc. (µM) BMMC + A23187 E 1500 F 1500

1000 1000

in RFU's in 500

 500 ** ** **

in RFU's (1 min) (1 RFU's in  0 0 0 20 40 60 80 100 120 AB Vehicle 25 50 100 Time (s) A15 Conc. (µM) G 5000 BMMC + DNP-BSA H 4000

4000 3000 3000 2000

in RFU's in 2000 * **  ** 1000

1000 min) (1 RFU's in 

0 0 0 20 40 60 80 100 120 AB Vehicle 25 50 100 Time (s) A15 Conc. (µM) Figure 3. A15 inhibits calcium influx in RBL-2H3 cells and BMMCs. RBL-2H3 cells (A- D) and BMMCs (E-H) were plated and incubated overnight prior to treatments. For DNP- BSA treatments, overnight incubation also included 1 µg/mL IgE anti-DNP. Prior to stimulation, cells were washed and loaded with the fluorescent, calcium-sensitive dye fluo-4

114

AM using the Fluo-4 AM DirectTM Calcium Assay Kit and fluorescence was measured on a microplate reader. Calcium influx was triggered in all experiments using either 1 µM A23187 or 50 ng/mL DNP-BSA, as indicated. Bar graphs represent the means ± SEM of the  in RFU’s at 1 min post-A23187- or DNP-BSA-stimulation from 3 independent experiments (B, D, F, H). Statistical analysis was performed using a one-way ANOVA with Dunnett’s post-hoc test, *p<0.05, **p<0.01, ***p<0.001.

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4.3 A15 inhibits calcium influx in A23187-stimulated RAW 264.7 and Jurkat cells

Calcium influx occurs in many cell types other than mast cells via similar store- operated calcium entry-dependent mechanisms (reviewed by (33, 34)). To determine if the suppression by A15 was specific to mast cells or occurred with other cell types, we stimulated fluo-4 AM-loaded RAW 264.7 macrophage-like cells (Fig. 4A) or Jurkat T cells

(Fig. 4B) with 1 µM A23187 and/or 100 µM A15. A15 treatment significantly reduced calcium influx in both RAW 264.7 and Jurkat cells at 1 min with 60.2% and 43.6% suppression, respectively, suggesting that A15 is a general inhibitor of calcium influx.

4.4 An ethanolic E. purpurea root extract and high-alkylamide fractions display inhibitory activities

Since E. purpurea is a natural source for alkylamides including A15, we hypothesized that an E. purpurea extract would display mast cell inhibitory activity. To test this hypothesis, we examined an extract and fractions separated from that extract, which were characterized previously for their effects on macrophage TNF-α production (10). The extract was prepared originally from E. purpurea roots macerated in 75% ethanol. Liquid:liquid partitioning was then used to purify a high alkylamide chloroform layer that was then separated into fractions with varying levels of alkylamides by flash chromatography over a

C18 column (Fig. 5A). A total of 18 fractions were collected, 3 of which (6, 7 and 8) contained quantifiable amounts of alkylamides determined using LC-MS and the peak area of the six most abundant alkylamide ion-chromatograms, as described in detail previously (10).

The extract and fractions were then characterized for effects on LPS-stimulated production of

TNF-α by RAW 264.7 macrophage-like cells (10).

116

A RAW 264.7 + A23187 B 5000 Vehicle 5000 A15 Assay Buffer 4000 3750

3000 2500 2000 in RFU's in *  1250

in RFU's (1 min) (1 RFU's in 1000 

0 0 20 40 60 80 100 120 AB Vehicle A15 Time (s) Treatment C 800 Jurkat + A23187 Vehicle D 1500 A15 Assay Buffer 600 1000 400

* in RFU's in

 500

200

in RFU's (1 min) (1 RFU's in  0 0 20 40 60 80 100 120 AB Vehicle A15 Time (s) Treatment

Figure 4. A15 inhibits A23187-stimulated calcium influx in RAW 264.7 and Jurkat cells. RAW 264.7 (A) or Jurkat cells (B) were loaded with fluo-4 AM using the Fluo-4 AM DirectTM Calcium Assay Kit and fluorescence was measured on a microplate reader. Cells were stimulated with 1 µM A23187 in combination with vehicle only or 100 µM A15. Bar graphs represent the means ± SEM of the  in RFU’s at 1 min post-A23187 stimulation from 3 independent experiments. Statistical analysis was performed using an unpaired Student’s t- test, *p<0.05.

117

For these experiments, we tested the extract and a subset of fractions that contained distinct alkylamide content and displayed distinct activities toward TNF-α production. As shown in Fig. 5B and C, the extract (EE, 51 ± 8.2 µg/mg) did indeed inhibit both degranulation and calcium movement with A23187-treated RBL-2H3 cells. The chloroform layer (CL, 140 ± 4.7 µg/mg) also displayed similar inhibitory activities (Fig. 5B and C).

Fraction 6 (also with high alkylamide content, i.e. 310 ± 42 µg/mg extract) was the only fraction tested that produced statistically significant inhibition of degranulation or calcium influx. Fraction 8, with low alkylamide content, (0.14 ± 0.034 µg/mg extract) did not produce significant inhibition of degranulation or calcium influx (Fig. 5B). Together these results suggest that inhibition of degranulation and calcium influx in mast cells requires alkylamides, and that other compounds can impact TNF-α production by macrophages. It should be noted that a suppressive trend was noted for calcium influx with fraction 8, suggesting that the low level of alkylamides, or perhaps other molecules, are able to weakly reduce calcium influx (Fig. 5C and D). Also, fraction 2 enhanced degranulation and calcium influx (Fig. 5B-D). Fraction 2 did not contain alkylamides detected by LC-MS, and was included as a control since it was previously found to stimulate cytokine and chemokine production from RAW 264.7 macrophages and contained LPS and potentially other pathogen-associated molecular patterns (10). These compounds appear to exert similar effects on mast cells.

4.5 A15 inhibits de novo production of PGE2 and TNF-α in RBL-2H3 cells

In addition to the rapid degranulatory process, mast cells initiate synthesis of newly formed pro-inflammatory mediators, including TNF-α and PGE2, which contribute to the

118

A B Ethanolic E. purpurea Extract 50 * 40 Liquid: Liquid Partitioning 30 * ** *** Chloroform Layer 20

Flash Chromatography %Degranulation 10 0 Vehicle †EE CL F2 F6 F8 Fractions 1-13 Treatment (25 µg/mL)

C 12000 Vehicle 15000 EE D CL 10000 F2 F6 8000 F8 10000

6000 in RFU's in

 4000 5000 *

* in RFU's (1 min) (1 RFU's in

2000  *** 0 0 0 20 40 60 80 100 120 Vehicle EE CL F2 F6 F8 Time (s) Treatment (25 µg/mL)

Figure 5. Alkylamide-containing Echinacea purpurea extracts and fractions inhibit RBL- 2H3 cell degranulation and calcium influx. A flow chart summarizing how fractions with high and low alkylamide content were prepared from an ethanolic extract of E. purpurea roots (A). RBL-2H3 cells were plated and incubated overnight prior to stimulation. Percent degranulation was measured 1 hr after stimulation with 1 µM A23187 in combination with vehicle only or 25 µg/mL of the indicated E. purpurea samples. (B). RBL-2H3 cells were loaded with fluo-4 AM using the Fluo-4 AM DirectTM Calcium Assay Kit and fluorescence was measured on a microplate reader. Cells were stimulated with 1 µM A23187 in combination with the indicated E. purpurea samples (C and D). EE = ethanolic E. purpurea extract, CL = chloroform layer, F2, F6, F8 = Fraction 2, Fraction 6 and Fraction 8, respectively. Results are displayed as the means ± SEM from 3 independent experiments (B and D) or the means ± SEM of triplicate wells from a representative experiment from 3 independent experiments (C). Statistical analysis was performed using a repeated measures one-way ANOVA with Dunnett’s post-hoc test, *p<0.05, **p<0.01, ***p<0.001. †EE was tested at 50 µg/mL.

119 long-term inflammatory responses associate with allergy (26). To test if A15 blocked these responses, RBL-2H3 cells were treated with A23187 and/or A15, overnight supernatants collected, and analyte concentration determined by ELISA. As shown in Fig. 6B, A15 produced significant, dose dependent inhibition of TNF-α at all doses tested. In contrast, production of PGE2 was weakly inhibited and only at the 100 M concentration of A15.

120

A 80

(pg/mL) 40  *** ***

TNF- 20

0 Vehicle 25 50 100 A15 Conc. (µM)

B 7500

5000

(pg/mL) 2 2500

PGE

0 Vehicle 25 50 100 A15 Conc. (µM)

Figure 6. A15 inhibits de novo production of TNF-α and PGE2 from RBL-2H3 cells. RBL- 2H3 cells were plated and incubated overnight prior to stimulation. Cells were stimulated with 1 µM A23187 in combination with the indicated doses of A15. Supernatants were collected 8 hrs later and TNF-α (A) or PGE2 (B) levels were measured using ELISA kits. Data shown are the means ± SEM from 3 independent experiments. Statistical analysis was performed using a one-way ANOVA with Dunnett’s post-hoc test, *p<0.05, **p<0.01, ***p<0.001.

121

5. Discussion

Alkylamides have been linked to the therapeutic effects of a number of medicinal plants (reviewed by (17)). Although alkylamide suppression of cytokine production has been described previously, the mechanism by which alkylamide-containing medicinal plants mediate their effects remains controversial (17, 35). In this report, we evaluated the effects of the alkylamide A15 on mast cell degranulation and calcium influx and found that it inhibited degranulation from RBL-2H3 cells and BMMCs (Figs. 1 and 2), and this effect correlated with the suppression of calcium influx (Fig. 3). The effects of A15 on degranulation and calcium movement were not attributed to cytotoxicity (Supplemental Fig.

1). Additionally, inhibition of calcium influx was not specific to mast cells as A15 also inhibited A23187-stimulated calcium influx in RAW 264.7 macrophages and Jurkat T cells

(Fig. 4). Inhibition of mast cell degranulation and calcium influx was observed by E. purpurea extracts and fractions with high alkylamide content, but not by fractions with little to no detectable alkylamide levels (Fig. 5). Lastly, we found that A15 inhibited de novo production of TNF-α and PGE2 (Fig. 6).

In mast cells and basophils, FcRI crosslinking leads to activation of phospholipase

C- (PLC-, which causes release of ER calcium stores, followed by stromal interaction molecule-1 (STIM-1) translocation to the plasma membrane and association with Orai1, the pore-forming subunit of the calcium-release activated calcium (CRAC) channel, and influx of extracellular calcium (36, 37). In contrast, ionophore treatment can transport calcium ions directly across the plasma membrane to increase calcium levels and can also cause rapid depletion of ER calcium stores and subsequent CRAC channel formation in the plasma membrane, bypassing upstream FcRI- and PLC--mediated events (38). A possible

122 explanation for the effect of A15 is that it could chelate calcium ions to prevent influx, which would block both FcRI- and ionophore-mediated calcium influx. However, alkylamides have not been reported to bind divalent ions previously, and known calcium chelators, such as EGTA, contain a cavity with multiple carboxyl groups to bind a calcium ion, which A15 lacks (39). More likely, A15 exerts direct or indirect effects on the CRAC channel. For example, A15 could bind directly to the CRAC channel, perhaps inserting into the pore to block influx of extracellular calcium. Or, A15 could inhibit the association of STIM1 with

Orai1 thereby preventing channel formation and influx of extracellular calcium.

Translocation of STIM-1 to the plasma membrane occurs by movement along microtubules, and therefore, if A15 disrupts microtubule assembly, this could also explain the inhibition of calcium influx through the CRAC channel (40). Alternatively, A15 could have a disruptive effect on membranes causing membrane-spanning ion channels (in the plasma membrane or the ER) to change conformation and prevent ion movement. Several of these hypotheses could also explain the ability of other alkylamides to inhibit the movement of potassium and sodium ions in neurons (41-43).

In these experiments we found that A15 substantially blocked production of TNF-

αfrom RBL-2H3 cells, but its inhibitory effect on PGE2 production was less, similar to results we found previously with RAW 264.7 macrophage-like cells (32). Production of both

TNF-α and PGE2 both likely involve calcium-dependent processes (26, 44). In mast cell types, for example, TNF-α can be released preformed from granules, which is calcium- dependent, and synthesized through a de novo, transcription-dependent pathway which can involve calcium-dependent transcription factors such as nuclear factor of activated T cells

(NFAT) (45). The PGE2 pathway may also involve calcium-dependent transcription factors

123 and calcium is also required for activation of cytosolic phospholipase A2, the first enzyme in the biosynthetic pathway which cleaves arachidonic acid from membrane phospholipid (44).

At present, it is unclear why PGE2 production is relatively resistant to treatment with A15.

Calcium responses may recover after several hours of treatment with A15, which are sufficient to drive production of PGE2 in overnight assays. In contrast, the production of

TNF-α may be more highly dependent on the immediate calcium response which was strongly inhibited by A15. A complete analysis of the effects of A15 on these pathways will be necessary to more fully understand the effects of A15 on cytokine and lipid mediator production.

Alkylamides have been linked to the anti-inflammatory effects of E. purpurea and shown to inhibit cytokine, chemokine and prostaglandin production from influenza A- stimulated macrophages in vitro (32, 46). Although the role of alveolar macrophages in response to influenza infection is well-known, it was recently reported that mast cells are critical to the pathogenesis of influenza infection (22). These findings suggest that the beneficial effects of E. purpurea extracts for colds and flu could be due to inhibition of mast cell degranulation, a possibility that has not been explored previously. It is also possible that

E. purpurea extracts with high alkylamide content could be useful for treating other forms of allergic inflammation mediated by mast cells such as hypersensitive reactions of the skin

(28), asthma (27), or allergic gastrointestinal disorders (in accord with the original use of the plant by Native Americans).

In addition to their effects on cytokine and chemokine production, alkylamides, including spilanthol and sanshool, have been linked to the tingling, analgesic activity of extracts made from alkylamide-producing plants such as E. purpurea, A. oleracea and Z.

124 americanum (47-49). The numbing effects of alkylamides have been linked to activation of the capsaicin receptor, TRPV1 (50). In addition, alkylamides have been shown to inhibit potassium movement through two-pore domain potassium channels (KCNK3, KCNK9, and

KCNK18), which are anesthetic-targeted channels (41, 51, 52). Interestingly, Bautista et al. found that the treatment of neurons with 100 µM sanshool caused calcium influx that was dependent on extracellular calcium (41). In contrast, with RBL-2H3 cells or BMMCs, A15 itself did not cause a calcium response (Supplemental Fig. 3D-F) perhaps suggesting differences in the activity of A15 and sanshool, or varying effects of alkylamides on neurons versus mast cells.

In conclusion, the alkylamide A15 from E. purpurea inhibits mast cell degranulation and calcium influx. The beneficial effects of E. purpurea extracts to patients with upper respiratory tract infections may be due to the suppressive effects of alkylamides on mast cell activation. It remains to be determined if structural improvements to A15 could result in a molecule with increased activity. The combined effects of A15 on mast cell activation with previous reports of cytokine suppression from macrophages supports the use of A15 and E. purpurea extracts with high alkylamide content for limiting inflammation associated with infections as well as allergic responses.

125

Declaration of conflicting interests

The authors declare no conflicting interests in regards to the authorship, research, and/or publication of this manuscript.

Author’s contributions

TVG, NMC, EM, SEM, YM, DAT, JGP, NBC, and SML designed the experiments. TVG,

NMC, YM, DAT, EM, and SEJ performed the experiments and analyzed the results. TVG and SML wrote the manuscript. TVG, NMC, AJM, JGP, NBC corrected the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Dr. Susan D’Costa for her technical assistance. This work was supported by grants from the National Center for Complementary and Integrative Health, a component of the

National Institutes of Health (1R15AT007259), the National Institutes of Health (R01

HD072968 to AJM), the Research and Innovation Seed Fund at North Carolina State

University, the Departments of Biological Sciences and Chemistry at North Carolina State

University, and the Comparative Medicine Institute at North Carolina State University.

126

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100 ***

25 %Cytotoxicity

0 Vehicle 25 50 100 CHX A15 Conc. (µM) Supplemental Figure 1. A15 is not cytotoxic to RBL-2H3 cells. RBL-2H3 cells were plated and incubated overnight prior to stimulation. Cells were treated overnight with the indicated concentrations of A15 10 µg/mL cycloheximide as a positive control. Supernatants were collected and analyzed for LDH release using the Pierce LDH Cytotoxicity Kit. Data are displayed as the means ± SD of duplicate wells from a representative experiment out of 2 independent experiments. Statistical analysis was performed using a one-way ANOVA with Dunnett’s post-hoc test, ***p<0.001.

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1.0

0.8

0.6 405

A 0.4

0.2

0.0 0 25 50 100 A15 Conc. (µM)

Supplemental Figure 2. A15 does not inhibit ß-hexosamindase enzyme activity. RBL-2H3 cells were lysed with 0.1% Triton X-100 to release ß-hex from the cells. Lysates were incubated for 1 hr at 37°C with the substrate, p-NAG, in combination with the indicated concentrations of A15 or vehicle only. The absorbance at 405 nm was measured using a microplate spectrophotometer. Results shown are the means ± SEM of triplicate wells from a representative experiment out of 2 independent experiments. Statistical analysis was performed using a one-way ANOVA with Dunnett’s post-hoc test.

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30 sec 60 sec 120 sec A B C

Vehicle Only

D E F

A15 Only

G H I

A23187

J K L

A23187 + A15

Supplemental Figure 3. A15 inhibits calcium influx in RBL-2H3 cells. Prior to stimulation, cells were washed and loaded with the fluorescent, calcium-sensitive dye fluo-4 AM using the Fluo-4 AM DirectTM Calcium Assay Kit. Cells were treated with 100 µM A15 or 0.25% ethanol only (vehicle) alone and in combination with 1 µM A23187 as indicated. Images were acquired at 30, 60, and 120 seconds, post-stimulation. Images are representative of several experiments.

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SUMMARY

Native Americans used preparations of Echinacea to treat different inflammatory conditions. Today, Echinacea is used primarily as an herbal supplement to prevent or treat

URIs. Although Echinacea extracts were initially proposed to boost immune function and promote clearance of URIs, it has more recently been hypothesized that Echinacea inhibits inflammatory mediator production to alleviate symptoms associated with URIs. We sought to determine whether or not E. purpurea extracts might be useful for treating URIs as well as other forms of inflammation.

We found that an ethanolic E. purpurea root extract contained both cytokine- suppressive and cytokine-stimulatory molecules (Chapter 2). When extracts were made from

E. purpurea plants grown from sterilized seeds, the cytokine-stimulatory activity was lost indicating that endophytic bacteria and fungi are likely the source of cytokine-stimulatory molecules. This study also showed that cytokine-suppressive activity was displayed by E. purpurea fractions containing alkylamides and non-alkylamide molecules. Although we focused our subsequent studies on alkylamides, identifying and more fully understanding the activity of the non-alkylamide cytokine suppressive molecules will likely be important to fully understand the activity of an Echinacea extract in vivo. Using defined Echinacea plant material with a specific activity may improve the consistency of clinical trial results using

Echinacea extracts.

Since alkylamides are capable of suppressing production of inflammatory mediators, they could potentially be developed into anti-inflammatory pharmaceuticals. In structure- activity relationship studies, we determined that the alkyl chain length and the amide group were critical for cytokine-suppressive activity displayed by A15, but that the isobutyl head

134 group and the double bonds were not (Chapter 3). The primary differences in structure between the alkylamides produced by E. purpurea are the number of double and triple bonds and their position along the alkyl chain. Alkylamides display varying degrees of cytokine- suppressive activity in vitro suggesting that double and triple bonds may modify the level of activity, but do not change it completely, which was supported by our observations in

Chapter 3. It is possible that alkylamides require a certain number of carbons in the fatty acid chain in order to interact with a target molecule(s), which remains unidentified. Future studies based on these findings may help generate an alkylamide with improved activity that could be developed into a useful treatment for inflammation.

The investigations into the mechanism of alkylamide-mediated cytokine suppression from macrophages are described in Chapter 4. A15 appeared to have multiple mechanisms of action occurring pre- and post-transcriptionally. We found that A15 was capable of suppressing calcium-ionophore driven calcium mobilization and cytokine production; however, the role of calcium in TLR signaling was unclear. Attempts to visualize increases in intracellular calcium early after LPS stimulation were not successful, but pharmacological experiments indicated that calcium was important for LPS-induced cytokine production. It is possible that alkylamides inhibit the movement of ions other than calcium, and the agonist or cell-type determine which ion’s movement is impacted. Future work could determine if mobilization of calcium, or another ion such as potassium, is important for cytokine production after TLR signaling.

The traditional use of Echinacea preparations by Native Americans suggests that it was applied to treat allergic responses and their accompanying inflammation. Mast cells are important mediators of allergic responses. Furthermore, recent evidence has suggested that

135 mast cells contribute to the excessive inflammatory response caused by URIs. Therefore, in

Chapter 5, we evaluated the effects of an E. purpurea extract and A15 on mast cell activity.

We discovered that A15 inhibited mast cell degranulation and calcium influx. Increases in intracellular calcium are required for degranulation suggesting that the reduced influx of intracellular calcium likely explained the inhibition of degranulation. An E. purpurea extract also inhibited mast cell degranulation and calcium influx, and this activity was mediated by alkylamides. The effects of A15 and E. purpurea extracts on mast cells suggest that the original use of Echinacea to treat inflammatory conditions may have been justified.

Furthermore, the combined suppression of inflammatory mediators from mast cells and macrophages indicates that alkylamides may be useful molecules that could be developed into pharmaceutical compounds for suppressing allergic responses and their associated inflammation.

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