Toll-Interacting Protein Regulation of Low-Grade Non-Resolving

Toll-Interacting Protein Regulation of

Low-grade Non-resolving Inflammation

Elizabeth J. A. Kowalski

Thesis/Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

Doctor of Philosophy In Biochemistry

Liwu Li, Committee Chair David Bevan Bin Xu Richard Helm

Graduation Date: May 3, 2017 Blacksburg, VA

Keywords: Low-grade inflammation, TLR4, Tollip, LPS, Lysosome Fusion, Mitochondria

Academic Abstract

Innate leukocytes manifest dynamic and distinct inflammatory responses upon challenges with rising dosages of pathogen associated molecular pattern molecules (PAMPs) such as lipopolysaccharide (LPS). To differentiate signal strengths, innate leukocytes may utilize distinct intra-cellular signaling circuitries modulated by adaptor molecules. Toll-interacting protein

(Tollip) is one of the critical adaptor molecules in Toll-like receptor 4 (TLR4) signaling and potentially playing key roles in modulating the dynamic adaptation of innate leukocytes to varying dosages of external stimulants. While Tollip may serve as a negative regulator of NFκB signaling pathway in cells challenged with higher dosages of LPS, it acts as a positive regulator for low- grade chronic inflammation in leukocytes programmed by subclinical low-dosages of LPS. We aim to show recent progress of Tollip involvement in complex innate leukocyte dynamics and its relevance in the pathogenesis of resolving versus non-resolving chronic inflammatory diseases.

General Audience Abstract

White blood cells, or leukocytes, have a dynamic inflammatory response to rising doses

of bacterial cell wall components. Lipopolysaccharide (LPS) is a ubiquitous component of gram

negative bacteria that is recognized by Toll-like receptor 4 (TLR4) and can shed into the blood

stream, causing low-grade non-resolving inflammation. In order to differentiate between varying

signal strengths of LPS, leukocytes utilize signaling within the cell, which is often regulated by

adaptor molecules. Toll-interacting protein (Tollip) is one of the critical adaptor molecules in

TLR4 signaling and potentially plays key roles in modulating the dynamic adaptation of innate

leukocytes to varying dosages of external stimulants. While Tollip serves to inhibit the pro-

inflammatory NFκB signaling in cells challenged with higher dosages of LPS, it acts to increase

low-grade chronic inflammation in leukocytes programmed by low-dosages of LPS. In these

studies we show recent progress in elucidating the mechanism for Tollip involvement in low-

grade non-resolving inflammation in mouse fibroblast cells.

Acknowledgments I would like to thank my family, especially my mother and father. My mother has always taught me and showed me how to work hard and has always been one of my biggest supporters. My dad has always been my number one fan and showed the upmost interest in my work and for this I thank him. I am very thankful for the experience I have obtained here at Virginia Tech. Thank you to Dr. Li for taking a chance on me when I walked into his office to ask for a nonconventional rotation in a subject that inspired me. Thank you to the Biochemistry department, especially Dr. Bevan, for the support and encouragement during the difficult portions of my career. Thank you to my laboratory mates for supporting me and giving me someone to talk to when my goals seemed unattainable. And lastly thank you to my boyfriend, Luke, for all the support, love, and kindness he has shown me when I was at one of the busiest and stressful points in my life.

Abbreviations Bak, BCL2 antagonist/killer 1 Bax, BCL2 associated X BCL2, B-cell lymphoma 2 C2, Conserved 2 domain

CD, Crohn’s disease CD14, cluster of differentiation 14 CUE, coupling of ubiquitin to ER degradation domain Cyt c, Cytochrome c IFN, interferon IKK, IkB kinase IL-6, Interleukin-6 IL-12, Interleukin-12 IR, ischemia-reperfusion IRAK, interleukin-receptor-associated kinase LPS, lipopolysaccharide MAPK, mitogen-activated protein kinase MAVs, Mitochondrial antiviral-signaling protein MCL1, Myeloid leukemia cell differentiation protein MD2, Myeloid differentiation factor 2 MyD88, Myeloid differentiation primary response gene 88

NFκB, nuclear factor κ of activated B cells PAMPs, Pathogen-associated molecular pattern PI3K, phosphatidylinositol-3-kinase PRR, Pathogen recognition receptor ROS, Reactive oxygen species SOCS, Suppressor of cytokine signaling STAT, Signal transducer and activator of transcription TBD, Tom1 binding domain

TIR, Toll/IL-1R homology TGFβ, transforming growth factor β TLR, Toll-like receptor

TNFα, tumor necrosis factor α Tollip, Toll-interacting protein Tom1, Target of Myb protein 1 TRAF, tumor-necrosis-factor-receptor-associated factor TRIF, TIR-domain-containing adaptor protein inducing interferon-β UC, Ulcerative colitis.

Table of Contents Chapter 1. Literature Review…………………………………..……………………………….1 Current knowledge of low-grade inflammation and limitations 1 Molecular circuitries responsible for signal-strength dependent innate immunity responses 3 Tollip structure and subcellular localization 5 Lysosomal Tollip and its potential role in resolution of inflammation 7 Mitochondrial Tollip and its role in low-grade inflammation 8 Tollip Involvement in Disease 11 Chapter 2. Toll-interacting Protein facilitates low-level expression of TNFα and IL-6 while dampening the expression of interferon-induced genes in fibroblasts………………14 Specific Aims 14 Abstract 19 Introduction 20 Methods and Materials 21 Results 23 Discussion 32 Figures 36 Supplementary Material 52 Chapter 3. Tollip deficiency increases cell survival through mediation of key proteins in murine embryonic fibroblasts………………………………………………………………64 Specific Aims 64 Abstract 68 Introduction 68 Methods and Materials 70 Results 72 Discussion 79 Figures 82

References…………………………………………………………………………………….90

vii

Chapter 1. Literature Review: Toll-interacting Protein in Resolving and

Nonresolving Inflammation

Current knowledge of low-grade inflammation and limitations

The innate immune system plays a pivotal role in the immediate recognition of

Pathogenassociated molecular patterns through Pattern Recognition Receptors (PRRs) and the subsequent induction of the inflammatory responses (Medzhitov and Janeway, 2000). Upon PAMP recognition, cell surface PRRs will activate intracellular adaptor molecules, protein kinases, and transcription factors (Akira and Takeda, 2004). These molecules will trigger the subsequent inflammatory responses. The stimulation of PRRs and the signal transduction pathways associated with them ultimately result in gene expression of cytokines, chemokines, cell adhesion molecules,

and immune-receptors (Akira et al., 2006). This broad range of molecules together coordinate

the complex responses of the host to infection and other inflammatory stimulants.

Among the germ line-encoded PRRs, the Toll-like receptors (TLRs) play an intricate role in innate immune system regulation and the inflammatory response. These TLRs recognize a wide range of PAMPs such as viral components and invariant bacterial components. TLR7, TLR9, and

TLR3 are located in the endo-lysosomal compartment and are responsible for detecting viral nucleic acids (Alexopoulou et al., 2001; Diebold et al., 2004; Lund et al., 2004; Lamphier et al.,

2006). In contrast, TLR2, TLR5, and TLR4 detect different bacterial cell wall components and are localized on the cell surface. TLR7, TLR9, and TLR3 induce a robust type 1 interferon response, which is key for antiviral defense, while TLR2, TLR5, and TLR4 may preferentially induce proinflammatory cytokines, although TLR4 ligand can be more pleiotropic and induce both 1 inflammatory cytokines and interferon responses (Hayashi et al., 2001; Toshchakov et al., 2002;

Kawai and Akira, 2007a). Given the intriguing complexity of TLR4 responses, we have been focusing on the dynamic modulation of TLR4 signaling networks. Lipopolysaccharide (LPS) is a ubiquitous surface component of Gram-negative bacteria, and is recognized by innate immune cells through Toll-like receptor 4 (TLR4). It is well known that high dosages of bacterial endotoxin can induce a robust pro-inflammatory cytokine storm followed by a later refractory tolerant state with reduced cytokine expression (Morris et al., 2014b). The cause of endotoxin tolerance is likely due to the induction of a multitude of negative regulators including IRAK-M, PI3K/AKT, MKP1, and

SOCS (Morris et al., 2014a).

An often-ignored effect of Gram-negative bacteria is low-grade non-resolving inflammation. Gram-negative bacteria occur naturally within the mucosal system, and shed endotoxin may permeate through leaky mucosal layers into circulation contributing to low-level endotoxemia (Franceschi et al., 2000; Castro et al., 2016). In contrast to high doses of LPS, circulating concentrations of super low-dose LPS (1-100pg/mL) remain in humans with chronic infections, people with obesity, as well as individuals experiencing the natural process of aging. It may also occur in individuals with life styles that include excessive drinking and chronic smoking

(Goto et al., 1994; Wiedermann et al., 1999; Cani et al., 2008; Szeto et al., 2008; Rao, 2009; Lira et al., 2010). Low levels of endotoxin have been shown to cause persistent low-grade inflammation that is characterized by chronic low levels of pro-inflammatory mediators (Maachi et al., 2004;

Manco et al., 2010; Mehta et al., 2010; Sun et al., 2010; Terawaki et al., 2010; Laugerette et al.,

2011). Subclinical endotoxemia may program the host to a state of low-grade non-resolving inflammation, subjecting the host to more severe diseases (Chaudhry et al., 2013; Morris et al.,

2014b). Despite increasing recognition that host innate leukocytes cannot only recognize the nature

2 and identity, but also the signal strengths of external stimulants, mechanisms responsible for the signal-strength dependent leukocyte activation are not well understood.

Molecular circuitries responsible for signal-strength dependent innate immunity responses

As mentioned above, studies conducted with varying dosages of LPS led to the concept of innate immune programming dynamics and memory (Yuan and Li, 2016). Although extensive studies have revealed a large array of intracellular signaling molecules responsible for innate immune cell responses to LPS, their context-dependent modulations in response to varying dosages of LPS have just recently received attention. Both hematopoietic and non-hematopoietic cells express TLR4 on the cell surface (Kurt-Jones et al., 2004; Taylor et al., 2004). Like most surface receptors, TLR4 contains both an extracellular domain and an intracellular domain that has a highly regulated signaling cascade, which follows activation. Though TLR4 is important for LPS recognition it has been shown that TLR4 alone may not be sufficient to elicit an inflammatory response. Myeloid differentiation 2 (MD2) must have physical association with TLR4 in order to induce ligand activation (Nagai et al., 2002). Once LPS binding occurs TLR4 and MD2 form a complex that changes the conformation of the TLR4 homodimer that induces the recruitment of adaptor proteins such as TIR, MyD88, TRAF, TRIF and TRAM (Barton and Medzhitov, 2003).

Through the engagement of TLR4 receptor and possibly other less-defined co-receptors, varying dosages of LPS may selectively activate distinct intracellular adaptor molecules such as

TIRAP, TRAM, MyD88, TRIF, SARM, and Tollip (Kawai and Akira, 2007b) through poorly defined dynamics. MyD88 has been widely implicated in the robust responses of innate leukocytes to high doses of LPS (Laird et al., 2009). Recruitment of MyD88 stimulates the phosphorylation of IL-1R associated kinases (IRAKs). The pathway will then signal and activate many downstream molecules, which in turn phosphorylate and activate mitogen-activated protein kinases (MAPKs) 3 and IκB kinase complex (IKK). This leads to the activation of key transcription factors, NFκB and

AP-1, as well as robust induction of pro-inflammatory cytokines (Barton and Medzhitov,

2003). NFκB activation also induces the expression of inhibitor molecules such as IRAK-M,

Tollip, IκB and SOCS. MyD88 pathway may also activate the PI3K pathway that further contribute to the induction of negative regulators of inflammatory processes (Laird et al., 2009; Medina et al., 2010). Collectively, these negative regulators serve as negative feedback mechanisms to induce endotoxin tolerance.

In sharp contrast, super-low dose LPS does not induce robust activation of NFκB, and only mildly induce low-grade inflammatory responses (Fig. 1) (Maitra et al., 2011). Super-low dose LPS also fails to induce the expression of negative regulators, thus allowing the nonresolving low-grade inflammation to persist (Maitra et al., 2012a). Under such non-resolving inflammatory process, our recent study reveals that MyD88 is not the primary adaptor molecule being utilized in the signaling process. Rather, TRAM/TRIF and Tollip may step in and serve to propagate the low-grade inflammatory process (Yuan et al., 2016). Tollip deficient macrophages have reduced expression of pro-inflammatory cytokines only when challenged with a super-low dose LPS signal (Didierlaurent et al., 2006b; Yuan et al., 2016). These findings suggest that Tollip serves as a positive signal to propagate low-grade inflammation (Fig. 1). This is in contrast to the inhibitory effect of Tollip on high-dose LPS, which induces strong NFκB activation and cytokine storm.

Figure 1. Potential roles and modulations of Tollip in acute and chronic inflammation. Under strong and acute inflammatory conditions, cell membrane localized Tollip serves as a negative inhibitor for the NFκB signaling pathway facilitates the resolution of inflammation, and clearance of cellular debris as well as excessive lipids. However, under low-grade inflammatory conditions, Tollip undergoes translocation from cellular membrane to mitochondria. Dislocated Tollip loses its homeostatic function, and fails to facilitate lysosome fusion. Instead, mitochondrial Tollip may facilitate non-resolving low-grade inflammation.

Tollip structure and subcellular localization

At the structural level, Tollip has three distinct domains with the Tom1 binding domain

(TBD), the Conserved 2 domain (C2), and the coupling of ubiquitin to ER degradation (CUE) domain (Fig. 2) (Capelluto, 2012). The Tollip TBD is involved in protein sorting via association with target of Myb protein (TOM1), clathrin, and ubiquitin during early endosomal interactions

(Yamakami et al., 2003). The C2 domain is found in over 100 different proteins and is approximately 130 residues in size. The C2 domain has been shown to bind to phospholipids in both a calcium-dependent and independent manner (Sutton et al., 1995). In proteins, such as synaptotagmin, calcium binding will not induce a conformation change, but will affect the

5 electrostatic potential that augments phospholipid binding (Sutton et al., 1995). This suggests that the C2 domain functions primarily through electrostatic activation. As previously discussed the C2 domain of Tollip has been shown to bind specifically to phospholipids, thus enabling Tollip localization with cellular membranes rich in phospholipids such as the cell membrane, endosome and lysosome (Li, 2004; Katoh et al., 2006; Baker et al., 2015). The CUE domain is typically a much smaller domain of approximately 40 residues and performs a variety of functions, such as protein sorting and interacting with ubiquitinated proteins. The CUE domain is very similar to the ubiquitin-binding UBA domain, which contains a three-helix bundle. The CUE domain contains a conserved MFP and LL motif in the α−helix1 and α−helix3, respectively (Kang et al., 2003). These two motifs are well known for interacting with the hydrophobic patch of ubiquitin (Kang et al.,

2003). When stimulated with high doses of LPS Tollip may aggregate at cellular and/or lysosome membranes with IL-R1 and TLR4, contributing to the inhibition of TLR4 mediated immune response via the CUE domain. Tollip also negatively regulates IRAK-1 and IRAK-2 by directly binding to these proteins via the CUE domain and inhibiting auto-phosphorylation (Zhang and

Ghosh, 2002a) (Fig. 2).

On the other hand, Tollip translocates to mitochondria in cells challenged with super-low dose LPS (Baker et al., 2015). When the CUE domain is mutated at its MFP motif, causing an inability to interact with ubiquitinated proteins, the Tollip CUE domain mutant fails to translocate to mitochondria, and remains at the endosome-lysosome (Baker et al., 2015). The molecular mechanisms for Tollip translocation are not clear. Structural analyses suggest that ubiquitin binding via Tollip CUE and C2 domain may reduce its interaction with phospholipids (Mitra et al.,

2013a). Since phospholipids are primarily localized at cell membrane, endosome, lysosome, and

Golgi, but not on mitochondria (Suetsugu et al., 2014), enhanced ubiquitin interaction and reduced

6 phospholipid binding of Tollip may be responsible for Tollip’s translocation from lysosome membrane to mitochondria. These molecular and cellular studies suggest that Tollip may play distinct roles in modulating inflammation through its differential subcellular localization.

Figure 2. Tollip domains and known actions within the cell. All domains are labeled as indicated. Each domain of Tollip performs a specific action as labeled, giving Tollip an incredibly broad range of actions to both induce and inhibit the inflammatory response based on the stimulus and dosage of stimulus. Lysosomal Tollip and its potential role in resolution of inflammation

Tollip may associate with the cell membrane and/or other intracellular membrane structures such as endosomes, lysosomes, and Golgi, due to its affinity with phosphoinositide through its C2 domain. Tollip has been shown through kinetic studies to reversibly bind to PtdIns3P

(phosphatidylinositol 3-phosphate) and PtdIns(4,5)P2 (phosphatidylinositol 4,5bisphosphate), with low micromolar affinity (Ankem et al., 2011). Through its phospholipid interaction, Tollip may fulfil its homeostatic role by inhibiting IRAK-mediated robust NFκB signaling and cytokine storm under acute and severe inflammatory conditions (Piao et al., 2009). Indeed, Tollip was shown to be critically important during the development of endotoxin tolerance, by suppressing the robust

NFκB pathway and preventing cytokine storm (Piao et al., 2009). Tollip deficient cells or mice fail to develop endotoxin tolerance when challenged with higher dose LPS (Diao et al., 2016).

Furthermore, PtdIns(4,5)P2 has been shown to be necessary for vacuole fusion and it has been speculated that PtdIns(4,5)P2 plays a direct role in membrane fusion by binding and recruiting specific molecules to the vacuoles being fused (Mayer et al., 2000). PtdIns3P has also been shown to play an important modulatory role in autophagy (Devereaux et al., 2013). By interacting with these lipids, Tollip may fulfil its role in the fusion of the endosome/autophagosome with the lysosome. Proper fusion of autolysosome may enable efficient clearance of cellular stress molecules and restore cellular homeostasis (Deretic et al., 2013). Lysosomes are not only critical for autophagy completion, but also serve as major signaling platforms for innate immunity signaling by recruiting key signaling molecules such as Mitochondrial antiviral proteins (MAVs) and Signal Transducer and activator of transcription proteins (STATs) (Settembre et al., 2013).

Tollip may serve as a negative regulator to dampen innate stress signaling processes at the lysosome platform.

Mitochondrial Tollip and its role in low-grade inflammation

Under low-grade inflammatory conditions, however, Tollip was shown to be cleared away from lysosome, thus compromising its homeostatic function (Baker et al., 2015). In sharp contrast,

Tollip translocates to mitochondria through its CUE domain interaction upon stimulation with super-low doses of LPS. Mitochondrial Tollip, instead, is an important facilitator for the generation of mitochondrial reactive oxygen species (ROS), which drives the expression of pro-inflammatory mediators through the activation of selected transcription factors such as C/EBPδ (Hsu and Wen,

2002; Maitra et al., 2012a). Tollip deficient macrophages have been shown to be unable to induce

8 mitochondrial ROS (Maitra et al., 2012a). Along with ROS reduction in Tollip deficient cells, there have also been reports of significantly decreased Interleukin-6 (IL-6) and Tumor Necrosis

Factor α (TNFα) in Tollip deficient cells (Didierlaurent et al., 2006b; Maitra et al., 2012a). Under low-grade chronic inflammatory conditions, Tollip deficient mice have reduced levels of proinflammatory cytokines such as TNFα, IL-12, and elevated levels of anti-inflammatory cytokines such as TGF (Keqiang Chen, 2017). Potentially due to its translocation away from lysosome, Tollip deficient cells also express higher levels of interferon-induced genes. Together, these studies suggest that the mitochondria localization of Tollip may play an important role in the lowgrade inflammatory response of innate leukocytes.

The mitochondria also plays an important role in regulation of cell survival and necroptosis. Low-doses of LPS have been shown to cause necroptosis and mitochondrial fission in macrophages (Baker et al., 2014). The propagation of low-grade, non-resolving inflammation can be potentially accomplished by cell necroptosis instead of apoptosis (Günther et al., 2013).

Necroptosis may contribute to prolonged inflammation through the release of damage-associated molecular patterns (DAMPs), which can propagate inflammation (Vanlangenakker et al., 2012).

Mice deficient of IRAK1 do not respond to the effects of LPS on necroptosis (Baker et al.,

2014). Tollip is a known negative regulator of IRAK1 and may be involved in the regulation of this process. Due to the importance and nature of the balance between cell survival and apoptosis, many molecules tightly regulate the mechanisms. The BCL2 family of proteins contain both pro-apoptotic and pro-survival proteins that inter-regulate one another and the permeability of the mitochondrial membrane (Czabotar et al., 2014). The BCL2 family of protein homologs can be broken into three categories, but for sake of simplicity, two categories will be discussed. The pro-apoptotic proteins that will be discussed are BCL2 associated X (Bax) and

BCL2 antagonist/killer 1 (Bak), while the anti-apoptotic proteins are B-cell lymphoma-2 (BCL2) and Myeloid cell leukemia-1 (MCL1) (Cory and Adams, 2002). These proteins are subdivided based on their anti-apoptotic and pro-apoptotic action and the BCL2 Homology (BH) domains present.

In resting cells, the Bax protein is located in the cytosol as a monomer. When an apoptotic stimulus occurs, Bax will translocate to the mitochondria and undergo a conformational change to produce oligomers (Hsu et al., 1997; Antonsson et al., 2001). While

Bax roams the cytosol, Bak is continually bound to the mitochondrial membrane via a Cterminal transmembrane domain (Lazarou et al., 2010). The change in mitochondrial permeability via oligomeric Bax and Bak was unclear until the observation that Bax resembles bacteria toxins that puncture holes in the membrane. These bacteria toxins will induce cell death through these holes via a change in permeability of the membrane and it is believed that Bax utilizes this mechanism

(Muchmore et al., 1996). Though the biochemical nature of this process is still unclear, it has been well established that Cytochrome c (cyt c) release requires Bax oligomerization (Antonsson et al., 2000). Cyt C at resting state is bound to the inner membrane of the mitochondria via cardiolipin with both electrostatic and hydrophobic interactions. When apoptosis is initiated mitochondrial ROS is produced, this will oxidize cardiolipin via a peroxidase function and the cyt c detaches from the inner membrane (Orrenius and Zhivotovsky, 2005). Cyt c is then released from the mitochondria via the now permeable membrane caused by the Bax and Bak oligomer and binds to apoptotic protease activating factor-1 (Apaf-1) to initiate the apoptotic cascade (Li et al., 1997). Membrane bound BCL2 proteins such as the B-cell lymphoma-2 (BCL2) heavily regulate oligomerization of Bax and Bak. Proteins like BCL2 have the capability to interact with the BH3 domain of Bax or Bak and prevent oligomerization, thus preventing the apoptotic signal.

Myeloid-cell leukemia 1 (MCL1) will also regulate Bax and Bak indirectly by inhibiting negative regulators of BCL2 (Gomez-Bougie et al., 2004).

The regulators for differing pathways of mitochondrial induction of pro-inflammatory responses is not a well-understood topic. Mitochondrial ROS will induce inflammatory responses, though dosage and context play an important role in the chronicity, resolution of inflammation, or initiation of apoptosis. The mitochondria can induce multiple transcription factors and therefore the inflammatory response. The diversity of mitochondrial activity implicates other mediators, such as Tollip, and negative mediators in the pathways that need to be further studied both for inflammation and cell death pathways.

Tollip Involvement in Disease

Translational studies with both animal models and human clinical studies in the recent years have yielded compelling data that support the role of Tollip in inflammatory diseases. For example,

Tollip expression has been found to be significantly increased in ischemia-reperfusion (I/R)- challenged brain tissue of humans, rats and mice in vivo (Li et al., 2015). In this study, it was also discovered that Tollip deficient mice are protected against acute I/R injury by reducing neuronal apoptosis through decreased expression of pro-inflammatory and pro-apoptotic genes, while increasing anti-apoptotic genes (Li et al., 2015).

Recent genetic and mechanistic studies also reveal that Tollip is involved in the pathogenesis of gut mucosal inflammatory syndromes such as inflammatory bowel disease

(IBD),

Crohn’s disease (CD) and Ulcerative colitis (UC) (Steenholdt et al., 2009; Maillard et al., 2014;

Diao et al., 2016; Fernandes et al., 2016). These syndromes may be results of altered microbiome as well as altered mucosal immune environment. TLR4 expression is significantly increased in 11

IBD, while Tollip expression is significantly decreased in both active and inactive UC and CD

(Fernandes et al., 2016) (Diao et al., 2016). We recently reported that Tollip deficient mice suffer from more severe chemically induced acute colitis with unabated expression of pro-inflammatory cytokines (Diao et al., 2016).

Genetic variants in human TOLLIP gene have been associated with idiopathic pulmonary fibrosis (IPF) (Noth et al., 2013). IPF is a devastating disease and is characterized by an interstitial fibrotic process and high mortality, which has an unknown etiology. Although lung transplant may hold treatment potential, the immunosuppression associated with transplant therapies may cause severe side effects (Raghu et al., 2011). There were three TOLLIP single nucleotide polymorphisms (rs111521887, rs5743894, rs5743890) identified in the genome wide association study that were associated with protection against IPF. TOLLIP expression was decreased by 20% in patients carrying the rs5743890 allele. This allele showed protection from development of IPF, but once IPF was developed the patients had higher mortality rates (Noth et al., 2013). The other two variants, rs111521887 and rs5743894, showed decreased TOLLIP expression by 40% and

50%, respectively.

In a recent study from our group, we observed that Tollip deficient mice tend to develop larger yet stable atherosclerotic plaques with increased lipid deposition as well as increased plaque content of smooth muscle cells and collagen (Keqiang Chen, 2017). We reported that the increased lipid deposition may be due to disrupted lysosome fusion and compromised lipophagy due to Tollip deficiency (Keqiang Chen, 2017). On the other hand, Tollip deficient mice have reduced circulating levels of pro-inflammatory cytokines such as TNFα, and increased levels of antiinflammatory TGF . This may explain the stable atherosclerosis phenotype with increased smooth muscle cells and collagen. Together, these data reveal compound phenotypes associate

12 with Tollip variants and deficiency, and further suggest that Tollip may play pleotropic roles in a context dependent fashion.

As discussed above stimulation of TLR4 signaling and Tollip are involved in many facets of inflammation and disease. TLR4 singling and Tollip play a role in autophagy and mitochondrial induced inflammation, but also in the subsequent roles these processes play in apoptosis and cell survival. Many adaptor molecules, such as Tollip, are important for the proper regulation of all subsequent pathways TLR4 signaling is involved. Tollip negatively regulates and positively regulates the induction of some cytokines and is involved in the fusion step of macroautophagy.

Tollip acts as a positive regulator, co-localizes with the mitochondria, and is necessary for ROS production, which in turn activates many cellular responses. Understanding the mechanism by which Tollip exhibits these functions will lead to a better understanding of how to regulate and intervene in diseases associated with Tollip and TLR4 signaling, apoptosis, and autophagy.

Chapter 2. Toll-interacting Protein facilitates low-level expression of TNFα and IL-6 while dampening the expression of interferon-induced genes in fibroblasts. Specific Aim 1: Define the subsets of cytokines that are differentially induced in Tollip

deficient cells.

Rationale: It has been previously shown that Toll-interacting protein is involved in the differential induction of certain cytokines. Tollip deficient cells are unable to express IL-6 and

TNFα with low-doses of LPS and this has been observed, but the phenomenon has never been fully understood (Maitra et al., 2012a). Tollip is an adaptor protein and plays a role in negative regulation of TLR4 signaling and assists in protein trafficking, but Tollip’s role in context dependent positive regulation of inflammation is not well understood (Liew et al., 2005; Zhu et al., 2012). Tollip assists with lysosome fusion to the endosome/autophagosome, but it has also been shown that Tollip will co-localize with the mitochondria when stimulated with low doses of

LPS (Baker et al., 2015). TLR4 signaling induces the production of both pro-inflammatory cytokines through mitochondrial activity and interferon-induced cytokines through the endosome. The regulation of these two subsets of cytokines may be modulated through the subcellular localization of Tollip (Noppert et al., 2007). It is important to understand the mechanism by which Tollip regulates TLR4 signaling to better intervene and prevent lowgrade, non-resolving inflammation in patients.

Working Hypothesis: If Tollip is not present the cytokine profile of Tollip deficient cells will be differentially expressed. Since Tollip deficient cells have been shown to lack induction of IL-6 and TNFα with low-dose LPS treatment, one would expect to observe this in the Murine

Embryonic Fibroblast (MEF) model being used for this project. Since Tollip is involved in the fusion process in autophagy, it is expected that levels of cytokines involved in endosomal stress,

14 such as IFN-induced cytokines IFIT, IFNλ2 and IFNλR1 will be elevated (Kagan et al., 2008;

Diamond and Farzan, 2013).

Specific Aim 2: Test the hypothesis that a dysfunctional Tollip CUE domain will cause

differential mRNA levels of IFN-induced cytokines and pro-inflammatory cytokines when

compared to WT and Tollip -/- cells.

Rationale: As previously mentioned, Tollip will co-localize with the mitochondria when stimulated with low-doses of LPS. Tollip loses the ability to co-localize with the mitochondria when the CUE domain is dysfunctional. The CUE domain in this model has been mutated at

M240A/F241A, which leaves the Tollip CUE domain unable to interact with ubiquitin (Baker et al., 2015). It is unknown why this particular mutation specifically cannot interact with the mitochondria, but the CUE domain is key for ubiquitin binding and this mutation changes a MFP ubiquitin interaction patch (Shih et al., 2003). When cells are deficient of Tollip, the cell is unable to induce Reactive Oxygen Species (ROS) via the mitochondria. This would lead one to conclude that since the mitochondria interacts with Tollip via the CUE domain, that a dysfunctional CUE domain will cause decreased ROS (Maitra et al., 2012a); therefore, leaving the cell unable to induce certain pro-inflammatory cytokines induced by ROS or with other mitochondrial products. It is believed that the C2 domain of Tollip is involved in the binding of phosphoinositides, most notably with PtdIns3P (phosphatidylinositol 3-phosphate) and

PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), on the endosome-lysosome in order to assist with fusion (Ankem et al., 2011). The mutant being used in this model has an intact C2 domain, which should not show a change in lysosome-endosome fusion and the subsequent cytokines involved via this mechanism.

Working Hypothesis: If the CUE domain of Tollip is dysfunctional, then decreased proinflammatory cytokines such as IL-6 and TNFα will be observed. Because the C2 domain of the Tollip protein being expressed is still functional, the CUE mutant will express IFN-induced cytokines similar to the WT cells and not to the level Tollip deficient cells express these cytokines.

Specific Aim 3: Test the hypothesis that Tollip deficiency leads to differential expression of

key transcription factors, HIF1α and STAT1, thus changing the mRNA levels of

proinflammatory and IFN-induced cytokines involved in Tollip regulation of inflammation.

Rationale: Mitochondrial ROS has been shown to stabilize HIF1α (Jung et al., 2008). HIF1α is induced via LPS in non-hypoxic conditions and has been shown to be a key transcription factor for IL-6 and TNFα (Jeong et al., 2003; Blouin et al., 2004; Nishi et al., 2008). Therefore, it is possible that Tollip deficiency and lack of ROS to stabilize HIF1α in Tollip deficient cells causes decreased levels of IL-6 and TNFα. It has also been previously shown that endosomes will signal through STAT proteins, specifically STAT1 and STAT2 (Shoenfelt and Fenton, 2006). When

Tollip is deficient it is possible that the endosomal stress from incomplete autophagy causes an increase in STAT1 and STAT2 to induce IFN-induced cytokines, which would normally be inhibited via normal macroautophagy.

Working Hypothesis: If cells are deficient of Tollip, the induction of HIF1α will be significantly decreased and this will cause decreased induction of IL-6 and TNFα when stimulated with low doses of LPS. Tollip deficiency will also cause an increase in the transcription factors STAT1 and STAT2, which will mirror the increased induction of IFN-induced cytokines in Tollip deficient cells.

Specific Aim 4: Test the hypothesis that HIF1α degradation through chaperone mediated

autophagy activation will cause a decrease in IL-6 and TNFα.

Rationale: It has been previously shown that HIF1α is regulated via Chaperone mediated

Autophagy (CMA) (Hubbi et al., 2013). Since IL-6 and TNFα have normal induction in WT cells it may be possible that Tollip deficient cells have increased CMA in order to compensate for the decreased macroautophagy seen in Tollip deficient cells (Massey et al., 2006). This increase in CMA may be leading to the degradation of HIF1α even when inappropriate to do so.

It is also possible that HIF1α is destabilized by either reduced ROS induction or increased hydroxylase activity (Marxsen et al., 2004). The reduction in ROS to stabilize HIF1α or an increase in hydroxylase activity would cause a significant decrease in HIF1α presence in Tollip deficient cells. Both possibilities could lead to increased degradation through CMA.

Working Hypothesis: Chemically activated CMA is predicted to cause a decrease in HIF1α and therefore decrease the levels of pro-inflammatory cytokines IL-6 and TNFα.

Specific Aim 5: Test the hypothesis that increased macroautophagy will decrease the

induction of IFN-induced cytokines in WT and Tollip deficient cells.

Rationale: Macroautophagy plays a pivotal role in the resolution of inflammation within the cell and has been implicated in many diseases (Henderson and Stevens, 2012; Wolfe et al., 2013).

Tollip deficiency has been shown to decrease macroautophagy completion, thus causing cellular stress due to lack of clearance. Tollip is believed to assist in the fusion of the lysosome to the endosome/autophagosome via the C2 domain of this protein. The increased levels of IFNinduced cytokines in Tollip deficient cells may be due to the induction of STAT1 through the endosome caused by decreased completion of macroautophagy and cellular stress.

Working Hypothesis: If macroautophagy is chemically activated, it is expected that STAT1 will show decreased protein levels and/or activation and this will subsequently decrease IFN-induced cytokine mRNA levels.

Specific Aim 6: Test the hypothesis that inhibiting macroautophagy will increase STAT1

protein levels in WT and Tollip-/- cells.

Rationale: Tollip deficiency leads to a decrease in macroautophagy and a decrease in cellular homeostasis as a result. Tollip deficient cells have a distinct increase in IFN-induced cytokines, which the endosome can regulate when stressed. In order to mimic Tollip deficiency, a macroautophagy inhibitor, Bafilomycin A will be utilized. Bafilomycin blocks the fusion of the endosome-lysosome, much like Tollip deficiency does.

Working Hypothesis: If macroautophagy is inhibited, then WT cells will show an increase in activated STAT1 and therefore an increase in IFN-induced cytokines. It is also hypothesized that

Tollip deficient cells will show an increase in IFN-induced cytokines as well.

Abstract

Tollip is a unique adaptor for Toll-like receptor (TLR) signaling that may modulate diverse inflammatory functions. Tollip deficiency has been implicated in complex inflammatory and infectious disesaes with poorly understood mechanisms. Tollip may fulfil its context-dependent modulatory functions through differential subcellular membrane localizations with the lysosome and mitochondria. Consequently, Tollip may mediate the expression of distinct subsets of inflammatory genes through context-dependent modulation of transcription factors. In this study, we attempted to address molecular mechanisms for the complex functional phenotypes associated with Tollip. By comparing the gene expression profiles of wild type and Tollip deficient murine embryonic fibroblast cells, we observed that Tollip deficiency selectively compromised the expression of inflamamtory cytokines IL-6, IL-12, and TNFα. In contrast, Tollip deficiency increased the mRNA levels of interferon-induced genes IFIT, IFNλ2, and IFNλR1. We observed that HIF1α was drastically reduced, while STAT1 was elevated in Tollip deficient cells, correlating with reduced mRNA levels of acute pro-inflammatory cytokines and increased mRNA levels of interferon-induced genes. We demonstrated that HIF1α levels were reduced in wild type cells treated with geldanamycin. Consequently, geldanamycin-treated WT cells had reduced expressions of IL-6 and TNFα. We observed that Tollip CUE mutant cells had reduced HIF1α and pSTAT1 levels as well as reduced gene expression of both subsets of cytokines. Application of bafilamycin A increased, while the application of LiCl reduced cellular levels of pSTAT1 and interferon-induced genes. Taken together, our study reveals the differential modulation of HIF1α and STAT1 by Tollip, underlying the context-dependent regulation of inflammatory and interferon-induced genes.

Introduction

Host inflammatory processes are required to elicit context-dependent responses to cope with infection and/or inflammation. Dysregulated inflammation may serve as a critical risk factor for inflammatory and infectious diseases (Kim et al., 2000; Shoelson et al., 2006; Andreasen et al.,

2011; Chen et al., 2012; Wolfe et al., 2013). Toll-like-receptor (TLR) signaling pathways are important networks regulating cellular inflammatotory responses, capable of modulating the expression of diverse inflammatory cytokines and interferon-induced genes (Kawai and Akira,

2006). TLR signaling networks may regulate differential gene expression through activation of transcription factors such as NFkB, STATs, HIF1a, and IRFs. Although significant progress has been made in the last decade regarding the TLR signaling networks, context-dependent activation of these transcription factors as well as levels of cytokine and interferon-induced genes are not clearly defined.

Toll-interacting protein (Tollip) is an intra-cellular adaptor for the TLR signaling network with less-well defined functions than other adpator molecules in these pathways. Tollip contains a C2 domain capable of binding with phosphatidyl-inositol phosphate (PIP) and a CUE domain capable of interacting with ubiquitin or ubiquitinated proteins (Zhang and Ghosh, 2002b; Li et al.,

2004). Through its C2 domain with PIP, Tollip is involved in the fusion of lysosome with endosome, phagosome and/or autophagosome (Lu et al., 2014; Baker et al., 2015). Proper lysosome fusion may serve as a key mechanism for maintaining cellular homeostasis. Disrupted lysosome fusion may generate cellular stress and create signaling platforms for the activation of stress kinases. For example, JAK/STAT pathway has been shown to be assembled near the lysosome and may be a pathway activated in this manner (Spath et al., 2009; Chmiest et al., 2016).

Tollip CUE domain, on the other hand, may reduce the PIP binding capability of C2 domain

20 through ubiqutin binding and enable Tollip clearance from lysosome and shuttle to mitochondria

(Maitra et al., 2012a; Mitra et al., 2013a; Baker et al., 2015). Mitochondrial Tollip may be involved in the generation of reactive oxygen species and subsequent expression of inflammatory mediators (Maitra et al., 2012a). Previously, we reported that super-low doses of LPS cause Tollip to relocate and associate with the mitochondria rather than with late endosome/lysosomes (Baker et al., 2015). As a result, super-low doses of LPS induce mitochondrial reactive oxygen species

(ROS) and perpetuate low-grade inflammation (Maitra et al., 2012a).

HIF1α can be stabilized and therefore activated by reactive oxygen species and potentiate the expression of inflammatory cytokines (Hu et al., 2014). On the other hand, the instability and protein degradation of HIF1α will contribute to the inflammatory cytokine expression (Hubbi et al., 2013). STAT1 Signal transducer and activator of transcription 1 (STAT1) is a key mediator for the expression of interferon-induced genes, and may be associated with autophagy (Toshchakov et al., 2002). Based on these studies, we tested the hypothesis that Tollip may modulate the expression of inflammatory and interferon-induced genes through differential activation of HIF1α and STAT1. Utilizing wild type and Tollip deficient murine embryonic fibroblasts (MEFs), as well as MEFs carrying CUE-domain mutant Tollip, we examined the role of Tollip in the activation of HIF1α and STAT1, as well as the mRNA levels of inflammatory cytokines and interferon-induced genes.

Materials and Methods

Reagents—LPS (Escherichia coli O111:B4), Bafilomycin, LiCl, Geldanamycin, 3-

Methyladenine, and Wortmannin were obtained from Sigma-Aldrich. Anti-β-actin antibody was obtained from Santa Cruz Biotechnology. Anti-HIF1α, anti-STAT1, and anti-pSTAT1 (S727) were obtained from Cell Signaling Technology. Anti-STAT2 was obtained from Abcam.

Cell Culture

Cells were harvested using 0.05% trypsin and re-suspended in DMEM supplemented with 2%

(v/v) fetal bovine serum, 1% (v/v) Pen/Strep, and 1% (v/v) L-glutamine and allowed to equilibrate overnight before additional treatments were performed. WT and Tollip-deficient murine embryonic fibroblast (MEF) cells and Tollip mutant MEFs harboring the CUE domain

M240A/F241A mutation were cultured in DMEM supplemented with 10% (v/v) FBS, 1% (v/v)

L-glutamine, 1% (v/v) streptomycin/penicillin.

Cell Transfection

Murine Embryonic Fibroblasts were cultured in complete DMEM medium, as previously described. Transient transfection of GFP-Tollip and Tollip mutant plasmids with G418 selecting gene were performed using the Lipofectamine 2000 reagent (Invitrogen) at a DNA to

Lipofectamine ratio suggested by the manufacturer’s instructions. The cells were cultured in

DMEM reduced serum medium at 2x105 cells total. Twenty-four hours after transfection, cells were grown in G418 supplemented (400ug/mL) complete DMEM media for 4 weeks to select for transfected cells before use in the following experiments.

Immunoblotting

Cells were washed with DMEM containing no FBS, harvested in SDS lysis buffer containing protease inhibitor mixture (Sigma), phosphatase inhibitor cocktails 1 and 2 (Sigma), and subjected to SDS-PAGE. The protein bands were transferred to an Immun-Blot PVDF membrane

(Bio-Rad). Western blot analyses were performed with the specified antibodies according to manufacturer’s instructions. Graphical analyses were performed using the NIH ImageJ program.

Quantitative mRNA Expression Analysis

Total RNA was extracted after specified treatments using an Isol-RNA lysis reagent (Invitrogen) and cDNA was generated with a high-capacity cDNA reverse transcription kit (Applied

Biosystems). This was then followed by analysis using SYBR Green Supermix on an iQ5 thermocycler (Bio-Rad). The relative levels of mRNA levels were calculated using the DDCt method, and results were normalized based on the mRNA levels of β-actin within the same experimental setting. The relative level of mRNA in untreated WT MEF cells was adjusted to 1 and served as the basal reference value for all subsequent samples tested. Primers Sequence

5’ β-actin 5’-TCGTACCACAGGCATTGTGATGGA-3’ 3’ β-actin 5’-TGATGTCACGCACGATTTCCCTCT-3’ 5’ TNFα 5’-GGCAGGTCTACTTTGGAGTCATTG-3’ 3’ TNFα 5’-ACATTCGAGGCTCCAGTGAATTCG-3’ 5’ IL6 5’-ACAAGTCGGAGGCTTAATTACACAT-3’ 3’ IL6 5’-TTGCCATTGCACAACTCTTTTC-3’ 5’ IL12p40 5’-AGACCCTGCCCATTGAACTG-3’ 3’ IL12p40 5’-GAAGCTGGTGCTGTAGTTCTCATATT-3’ 5’ IFNλ2 5’-CAAGGATGCCATCGAGAAG-3’ 3’ IFNλ2 5’-GGAAGAGGTGGGAACTGC-3’ 5’ IFNλR1 5’-ACCAGGTGGAATTCTGGAAG-3’ 3’ IFNλR1 5’-CTGGAGAGGAATCTGCACTG-3’ 5’ IFIT 5’-GCGATCCACAGTGAACAACAG-3’ 3’ IFIT 5’-GCTGCTCCAGTATTTCCCTGTAA-3’ 5’ HIF1α 5’-TTCTGTGCGGTGACCAGTGT-3’ 3’ HIF1α 5’-GCACACCAGGAGGTCACAGT-3’ 5’ STAT2 5’-GCTGCAGAGCCATCTCCTCA-3’ 3’ STAT2 5’-CAGTCTGCGTTCCTGGTTGC-3’ 5’ STAT1 5’-GCCATGGCTGTACCTGTCCT-3’ 3’ STAT1 5’-GGCTCCAGGTGATCCGAGAC-3’

Results

Tollip differentially regulates the induction of inflammatory cytokines

The effect of Tollip deficiency on the induction of select inflammatory cytokines from the acute inflammatory response and interferon stimulated response was tested utilizing Tollip knock out (Tollip -/-) MEF cells. We first compared the mRNA levels of selected inflammatory cytokines such as IL-6, TNFα, and IL-12 as well as interferon-induced genes such as IFIT, IFNλ2, and

IFNλR1 in WT and Tollip deficient MEF cells treated with a low-dose LPS (10ng/mL) through

RT-PCR analyses. As shown in Fig. 1, the treatment of WT MEF cells with LPS led to a significant induction of acute inflammatory cytokines, Il6, Tnfα, and Il12 (Fig. 1A-C). In contrast, Tollip deficiency significantly decreased the mRNA levels of Il6, Tnfα, and Il12. The basal levels of Il6,

Tnfα, and Il12 in untreated Tollip deficient cells were also lower as compared to that in WT cells.

Our data demonstrate that Tollip positively regulates the induction of Il6, Tnfα, and Il12 in cells stimulated with low-doses of LPS.

Tollip differentially regulates the mRNA levels of interferon-induced genes.

The effects Tollip deficiency had on other subsets of cytokines was tested by analyzing interferon induced cytokine mRNA levels. These cytokines have been previously shown to be stimulated through endosomal signaling (Diamond and Farzan, 2013). Tollip deficient cells have reduced endosome-lysosome fusion that causes cellular stress, therefore endosomal stress may be present in these cells. Interferon-induced proteins with Tetratricopeptide repeats (IFIT) showed increased mRNA levels in Tollip-/- cells both at basal levels and with LPS stimulation (Fig. 2A).

The induction of Ifnλ2 mRNA levels were significantly increased in Tollip-/- MEFs with LPS stimulation, while WT MEF cells were non-responsive to LPS in the context of Ifnλ2 mRNA induction (Fig. 2B). Lastly, Ifnλr1 mRNA showed increased levels both with LPS stimulation and at basal levels compared to the WT MEF cells, much like Ifit mRNA levels (Fig. 2C). From this data it is clear that the absence of Tollip dysregulates interferon-induced cytokine mRNA

24 levels, possibly due to the dysregulation of macroautophagy in these cells. Taken together with

Figure 1, our data suggest that Tollip positively and negatively modulates two distinct subsets of cytokines in MEF cells.

Tollip CUE domain necessary for induction of pro-inflammatory cytokines

The role the Tollip CUE domain plays in the induction of select cytokine subsets was tested utilizing Tollip mutant fibroblasts harboring the CUE domain M240A/F241A mutation.

This mutation yields a dysfunctional CUE domain that is unable to interact with ubiquitinated proteins, which is important for its function. It has been previously shown that this particular mutant of Tollip causes Tollip to no longer interact and co-localize with the mitochondria as the

WT Tollip has been shown to do with low-dose LPS stimulation (Baker et al., 2015). Much like the Tollip deficient cells, the Tollip CUE mutant was unable to induce Il6, Tnfα, and Il12 mRNA levels with LPS stimulation (Fig. 3 A-C). This data taken together shows the necessity of the

Tollip CUE domain and its ubiquitinated protein interaction motif as crucial for induction of acute inflammatory mediators.

Tollip CUE domain not necessary to inhibit IFN-induced cytokine mRNA levels.

The role the Tollip CUE domain plays in the induction of interferon-induced cytokines was tested utilizing the TmCUE MEF cells previously described. Unlike the Tollip deficient cells the CUE domain does not induce significantly higher levels of Ifit mRNA (Fig. 4A). Ifnλ2 and

Ifnλr1 mRNA levels in TmCUE cells are higher than WT mRNA levels, but not as highly induced as in Tollip deficient cells (Fig. 4B, 4C). This is due to the still intact Tollip C2 domain that assists with fusion of the lysosome to the endosome/autophagosome. These results indicate that the CUE domain of Tollip plays a mild role in the inhibition of interferon-induced cytokines, but that the mutation does not mimic the same highly induced interferon stimulated cytokine

25 mRNA levels as Tollip deficient cells. This may be that the CUE domain is necessary for dimerizing or some other interaction that assists with the C2 domain and fusion, but the CUE domain does not play a large a role in interferon-induced cytokine inhibition as the C2 domain does. This data together with Figure 3 shows that the CUE domain of Tollip is necessary for the induction of IL-6, TNFα, and IL-12, but is not fully responsible for the inhibition of IFN-induced cytokine mRNA levels with LPS stimulation.

Tollip deficient cells show differential protein levels of HIF1α, STAT2, and STAT1.

Potential mechanisms for Tollip-mediated induction of cytokines and interferon-induced genes were tested by examining transcription factors HIF1α, STAT2, and STAT1. WT and T-/-

MEF cells were treated with LPS (10ng/mL) for a 1, 3, 6-hour time course. LPS treatment significantly induced HIF1α at 6 hours in WT fibroblasts, but not in Tollip deficient cells (Fig. 6A,

6B). Tollip deficient cells were unable to induce HIF1α protein levels, yet these cells showed greatly increased protein levels of STAT2 even at basal levels (Fig. 6A, 6C). To test whether the induction of HIF1α is at the RNA or protein levels, we tested the mRNA levels of Hif1α and observed higher levels of Hif1α mRNA in Tollip deficient cells (Fig. 6D). WT cells showed no change in Hif1α mRNA levels with LPS treatment (Fig. 6D). The mRNA levels of Stat2 were significantly higher at both basal levels and with LPS stimulation in Tollip deficient cells, which translated to the protein level (Fig. 6E). WT cells showed a slight but significant increase in mRNA levels of Stat2, though this did not translate to the protein levels (Fig. 6E). Together, our data suggest that the reduction of HIF1α in Tollip deficient cells is most likely due to protein posttranslational degradation rather than transcriptional induction.

We also examined the protein levels of STAT2, STAT1, and its active form pSTAT1

(S727) due to that fact that STAT1 and STAT2 often dimerize in order to translocate to the

26 nucleus and transcribe for IFN-induced cytokines. Both STAT1 and pSTAT1 were significantly increased in Tollip deficient cells when compared to WT cells (Fig. 6F). WT cells have inducible pSTAT1 with LPS treatment, but not at the levels that Tollip deficient cells exhibit (Fig. 6G).

Both WT and Tollip deficient cells appear to reach peak pSTAT1 protein levels at 3 hours of treatment. Thus, the trend of induction is identical, but Tollip deficient cells have consistently higher activated STAT1 protein levels both with and without LPS stimulation. LPS treatment induced STAT1 protein levels in Tollip deficient fibroblasts, but only slightly for WT cells in comparison (Fig. 6H). WT cells showed significant induction of Stat1 mRNA levels with LPS stimulation, though the mRNA levels were significantly lower than Tollip deficient cells (Fig.

6I).

Our data reveal that Tollip deficient cells have decreased HIF1α protein levels, which is a known transcription factor for acute inflammatory cytokines such as IL-6 and TNFα. Our study provides a potential mechanistic cause for decreased IL-6 and TNFα in Tollip deficient cells.

Interestingly, Hif1α mRNA levels are higher in Tollip deficient cells, which led to the hypothesis that decreased HIF1α at the protein levels, may be due to post-translational modification. On the other hand, Tollip deficient cells also have much higher protein levels of STAT1 and pSTAT1 even at basal levels. This gives a mechanistic explanation for increased IFN-induced cytokine mRNA levels, due to STAT1 involvement in the transcription of these types of cytokines. Our data suggest that Tollip may regulate these two unique subsets of cytokines through differential modulation of HIF1α and STAT1.

Tollip CUE domain necessary for induction of key transcription factors.

Previous studies suggest that the CUE domain of Tollip may be involved in the modulation of Tollip interactions with lipids, as well as its differential subcellular localization at

27 lysosome and mitochondria (Ankem et al., 2011; Baker et al., 2015). Since HIF1α can be activated and stabilized by mitochondrial ROS and STAT1 is involved in autophagy and endosomal signaling, we tested whether the Tollip CUE domain may be causally associated with the differential activation of HIF1α and STAT1. To test this, Tollip mutant MEFs harboring the

CUE domain M240A/F241A mutation (TmCUE) were utilized (Baker et al., 2015). We previously reported that Tollip CUE mutant constitutively resides at lysosome, enhances lysosome fusion, and fails to translocate to mitochondria in cells challenged with low-dose LPS

(Baker et al., 2015). By real-time RT-PCR analyses, we observed that low-dose LPS fails to induce pro-inflammatory cytokines such as Il6, Tnfα, and Il12 in the Tollip CUE mutant cells

(Fig. 3A-C). LPS increased the accumulation of HIF1α over the 6-hour treatment in WT MEF cells, while TmCUE MEF cells were unresponsive and showed a significant decrease of HIF1α levels with LPS stimulation (Fig. 6A, 6B). HIF1α protein levels appear to be higher at basal levels for TmCUE cells, but LPS reduced HIF1α levels similar to Tollip deficient cells (Fig. 6B).

Reduced HIF1α protein levels with LPS is consistent with reduced induction of Il6 and Tnfα mRNA in TmCUE cells. As previously mentioned, HIF1α is an important transcription factor in the transcription of these cytokines, thus the decreased levels in Tollip CUE mutant cells is also congruent with the presented data. STAT2 had increased protein levels in TmCUE cells both at basal levels and with LPS stimulation compared to WT fibroblast STAT2 protein levels (Fig. 6A,

6C). TmCUE cells showed lower protein levels of pSTAT1 both at basal levels and with LPS stimulation (Fig. 6A, 6D). Protein levels of STAT1 are significantly higher in TmCUE cells both at basal levels and with LPS stimulation, though the activated STAT1 was significantly less (Fig.

6E). Similar to Tollip deficient cells, TmCUE cells showed higher mRNA levels of Hif1α; further suggesting that HIF1α induction in WT cells may be due to protein stability (Fig. 6F).

TmCUE cells show significantly higher mRNA levels of Stat1 and Stat2 compared to WT cells

(Fig. 6G, 6H). This data indicates that HIF1α accumulation requires Tollip and a functional CUE domain, which are both also required for IL-6 and TNFα induction. Though STAT1 and STAT2 have higher protein levels with a dysfunctional CUE domain, the activated form of STAT1 is reduced when Tollip does not have a functional CUE domain. This work supports the claim that a dysfunctional CUE domain will not activate STAT proteins, though the overall protein levels of STAT1 and STAT2 are increased. This may be due to the intact C2 domain that assists with lysosome-endosome fusion and suppresses IFN-induced cytokines.

Chemical suppressor of autolysosome fusion elevates pSTAT1 and the induction of interferoninduced genes

The role of autolysosome fusion in the modulation of STAT1 and interferon-induced gene mRNA levels was independently assessed by utilizing bafilomycin, a late stage autophagy blocker that prevents maturation of endosome/autophagosome by blocking fusion between the endosome/autophagosome and the lysosome as well as preventing acidification of the lysosome.

Tollip assists in endosome/autophagosome-lysosome fusion, when Tollip is not present fusion is disrupted (Baker et al., 2015). Indeed, bafilomycin and LPS co-treatment led to an elevation of pSTAT1 in WT cells (Fig 7A, 7B). Although the pSTAT1 levels were constitutively high in

Tollip deficient cells, co-stimulation with LPS and bafilomycin further increased the levels of pSTAT1 (Fig. 7A, 7B). Overall STAT1 protein levels were slightly elevated by bafilomycin in

Tollip deficient cells, but not in WT cells (Fig. 7A, 7C). Bafilomycin treatment of WT cells showed very little change in the mRNA levels of Ifit, but Tollip deficient cells showed a slight increase with bafilomycin treatment alone (Fig. 7D). WT and Tollip deficient cells showed a significant increase in Ifnλ2 with bafilomycin treatment, and Tollip deficient showed an increase with co-stimulation of LPS and bafilomycin (Fig. 7E). Tollip deficient cells showed significantly 29 increased levels of Ifnλr1 with bafilomycin alone and with co-stimulation of bafilomycin and

LPS (Fig. 7F). Due to the low mRNA levels of Ifnλr1 in WT cells, the WT cells were analyzed separately (Fig. 7G). WT cells showed a significant induction of Ifnλr1 with bafilomycin treatment alone (Fig. 7G). This data taken together shows that the blocking of fusion between the lysosome-endosome causes increased induction of IFN-induced cytokine mRNA levels and increased the protein levels of pSTAT1 in both WT and Tollip deficient cells.

Chemical inducer of lysosome fusion reduced STAT1 activation and IFN-induced cytokines

In order to independently corroborate the contribution of lysosome fusion with the modulation of STAT1 and interferon-induced genes in WT and Tollip deficient cells, we applied the chemical inducer of lysosome fusion LiCl (Bertsch et al., 2011). As shown in Figure 8, the addition of LiCl reduced LPS-induced elevation of total STAT1 and pSTAT1 (Fig. 8A, 8B, 8C).

WT and Tollip deficient cells also showed a significant decrease in overall STAT1 protein levels with macroautophagy activation (Fig. 8A, 8C). WT and Tollip deficient cells showed no change in

Stat1 mRNA levels with macroautophagy activation (Fig 8D). WT and Tollip deficient cells showed significant decreases in Ifit mRNA levels with LiCl treatment and co-treatment with LPS

(Fig. 8E). Both Ifnλ2 and Ifnλr1 showed decreased mRNA levels with macroautophagy activation, though WT cells did not show the same trend (Fig. 8F, 8G). Functionally, addition of LiCl significantly reduced the induction of interferon-induced gene ifit in both WT and Tollip deficient cells. Collectively, our data suggest that Tollip may suppress STAT1 through facilitating lysosome fusion.

Chemical inhibition of autolysosome fusion increases Interferon-simulated cytokines in Tollip

CUE mutant, chemical activation of macroautophagy decreases Ifit mRNA levels.

Next, in order to test the effects of autophagy dysregulation when Tollip has a dysfunctional CUE domain, we utilized the Tollip CUE mutant previously described. The Tollip

CUE mutant appeared to have higher basal levels of Ifit and this increased with autophagy inhibition as seen in the Tollip deficient cells (Fig. 9A). Autophagy inhibition alone also increased Ifnλ2 and Ifnλr1 mRNA levels in the Tollip CUE mutant cells (Fig. 9B, 9C). Activation of macroautophagy had little effect on IFN-induced cytokine induction of TmCUE cells similar to WT cells. LiCl treatment decreased mRNA levels of Ifit in TmCUE cells similar to WT fibroblasts (Fig. 9D). LiCl treatment showed no significant differences in Ifnλ2 and Ifnλr1 mRNA levels in TmCUE cells (Fig. 9E, 9F). This data together shows that both the CUE domain and lack of endosome fusion will increase the levels of interferon induced cytokines. This implies a role that the CUE domain may play in assisting with the regulation of this specific subset of cytokines since the increase in IFN-stimulated cytokines is only observed when the

CUE domain is deficient and endosome-lysosome fusion is prevented. The C2 domain may play a larger role in the regulation of IFIT specifically due to the similar regulation of IFIT in both

WT and TmCUE cells.

Chemical inducer of HIF1α degradation reduces the induction of Il6 and Tnfα, mimicking Tollip deficiency in WT MEF cells.

Geldanamycin, a chemical inducer of HIF1α degradation, was utilized to independently confirm that HIF1α stabilization is responsible for the induction of Il6 and Tnfα. WT and Tollip deficient cells were co-stimulated with LPS and/or GA for 6 hours prior to protein and RNA analyses. It has been shown previously that CMA regulates HIF1α through degradation, therefore it may be a form of regulation in TLR4 signaling to regulate inflammation. WT and

Tollip deficient cells were co-stimulated with LPS and GA for 6 hours before protein and RNA

31 extractions were performed. Transcription levels of Il6 and Tnfα were tested as read outs for the inflammatory response. WT cells showed a significant decrease in HIF1α protein levels with

CMA activation, Tollip deficient cells also showed a decrease in HIF1α protein levels (Fig. 10A,

10B). WT cells showed markedly lower transcription levels of Il6 and Tnfα with GA treatment, which mimicked the Tollip deficiency mRNA levels (Fig. 10C, 10D). Tollip-/- cells were unaffected by the addition of GA into the system (Fig. 10C, 10D), which is expected because the mRNA levels are significantly lower and not inducible with LPS. In order to demonstrate that

HIF1α protein levels were decreased due to degradation and not less mRNA induction RT PCR was utilized to observe Hif1α mRNA levels. WT MEF cells showed significantly higher induction of Hif1α with CMA activation (Fig. 10E). This data suggests that CMA may be upregulated in Tollip deficient cells or HIF1α is destabilized and thus degraded, which is necessary for the transcription of IL-6 and TNFα. This data taken together, suggests a mechanistic explanation for low HIF1α protein levels in Tollip deficient cells and the regulation of chronic inflammation utilized by WT cells.

Discussion

Tollip modulates two distinct subsets of inflammatory mediators. Tollip deficient fibroblasts subjected to low-dose LPS were unable to express IL-6, TNFα, and IL-12. On the other hand, Tollip deficient cells had increased mRNA levels of IFIT, IFNλ2, and IFNλR1 at basal levels as well as upon LPS stimulation. We demonstrate that Tollip may facilitate the low-grade induction of IL-6, TNFα, and IL-12 through stabilizing HIF1α, while suppressing the induction of IFIT,

IFNλ2, and IFNλR1 through suppressing STAT1 and STAT1 phosphorylation (Fig. 11). Our structure function analyses reveal that the functional CUE domain of Tollip is required for the activation of HIF1α and suppression of STAT1.

Our study clarifies the divergent functions of Tollip in the innate immune responses of fibroblasts. Tollip can serve as either a positive or a negative regulator of LPS signaling in myeloid cells such as macrophages, dependent upon the signal strength of LPS. Higher LPS levels can induce compensatory endotoxin tolerance, and elevated Tollip levels may contribute to endotoxin tolerance by suppressing IRAK-1 mediated NFkB activation (Piao et al., 2009). In contrast, superlow dose LPS does not trigger robust NFkB activation nor endotoxin tolerance (Morris et al.,

2014b). Instead, super-low dose LPS triggers prolonged low-grade inflammatory gene expression through translocating Tollip from cellular and/or lysosomal membranes to mitochondria, and facilitating reactive oxygen species generation (Baker et al., 2015). Thus, Tollip serves as a context-dependent adaptor for either endotoxin tolerance (when localized at cellular/lysosome membrane in cells challenged with strong LPS signals) or non-resolving low-grade inflammation

(when translocated to mitochondria in cells challenged with weaker LPS signal). We also reported that the functional CUE domain is required for signal-dependent translocation to the mitochondria

(Baker et al., 2015). Tollip CUE mutant constitutively resides at the lysosome (Baker et al., 2015).

Although the role of cellular/lysosomal membrane-localized Tollip has been relatively well defined in the context of high dose endotoxin tolerance, the role of Tollip in mediating low-grade inflammation in cells challenged with low-dose LPS is not well defined. In order to properly address this issue, our current study utilizes fibroblast cells, which are known to be non-tolerizable to LPS. These fibroblasts provide a robust system for a focused study of the non-resolving inflammation without the complication of compensatory tolerance (Kalliolias et al., 2010). In agreement with studies conducted in macrophages (Didierlaurent et al., 2006a; Baker et al., 2015), we observed that Tollip is a positive regulator for the induction of inflammatory IL-6, TNFα, and

IL-12 in fibroblasts induced by LPS.

Our study reveals that Tollip fulfils its role of inducing inflammatory cytokines by stabilizing HIF1α. Although HIF1α is known to be induced by LPS from previous studies and is involved in the transcription of inflammatory cytokines, our current data provide evidence-linking

Tollip as a key mediator for LPS-induced HIF1α activation in fibroblasts under a low-grade nonresolving inflammatory condition. HIF1α is regulated by gene transcription and posttranslational stabilization/degradation (Kasivisvanathan et al., 2011; Ferreira et al., 2013). Our data reveal that

Tollip-mediated HIF1α activation in inflammatory fibroblasts is due to HIF1α protein stabilization instead of mRNA levels. HIF1α protein degradation occurs due to elevated chaperon-mediated autophagy (CMA) that is not only distinct from, but also may compete with the classical macroautophagy involving lysosome fusion with autophagosome (Ferreira et al., 2013). Under inflammatory condition, Tollip in fibroblasts may translocate away from lysosome and thus the traditional macroautophagy/lysosome fusion may be compromised (Keqiang Chen, 2017). This may allow for enhanced CMA and HIF1α degradation. Our current study provides supporting evidence that both Tollip CUE mutant continual lysosome localization, as well as the CMA activator geldanamycin blocks HIF1α activation by LPS.

Our study also reveals the less-recognized role of Tollip in modulating the expression of interferon-induced genes in fibroblasts. Interferon-induced genes are critical for viral defense and activation of adaptive immune responses (Ivashkiv and Donlin, 2014). Dysregulated expression and induction of these genes has been associated with viral infection risks as well as asthma. To this regard, human Tollip polymorphisms have been linked with elevated risks of asthma and

COPD (Ritter et al., 2005; Huang et al., 2016). Our current study complements these pathophysiological studies and reveal potential mechanistic cause of Tollip-mediated regulation of

34 interferon-induced genes. We observed that Tollip may dampen the activation of STAT1 in resting fibroblast cells, and this may help to prevent excessive activation of cellular mediators associated with the pathogenesis of asthma and other airway inflammation. Our data also suggest that Tollip dampens STAT1 activation through facilitating lysosome fusion. This is consistent with previous studies that proper lysosome fusions with endosomes/autophagosomes are critically important to maintain cellular homeostasis (Deretic et al., 2013; Jaber and Zong, 2013). Lowgrade inflammation conditions may elevate interferon-induced gene levels through CUE domain mediated Tollip translocation away from lysosome, causing disruption of lysosome fusion, and

STAT1 activation. Our data with CUE mutant cells as well as lysosome fusion inhibitor

Bafilomycin independently support this conclusion.

Further studies are required to observe the protein levels of the pro-inflammatory cytokines and IFN-induced cytokines. This will confirm the protein levels of these cytokines are congruent with the mRNA levels observed in this study. It may also be useful to add a control with Tollip deficient cells transfected with a WT Tollip plasmid to rule out transfection as the cause for differences in inflammatory cytokine expression of the TmCUE fibroblasts. The effects of ROS induction on HIF1α stabilization and protein levels should be tested in the future to examine the causal effect of HIF1α degradation in Tollip deficient and TmCUE fibroblasts. Taken together, our current study provides evidence-linking Tollip with the differential activations of HIF1α and

STAT1 in fibroblasts that contribute to the regulation of both IL-12, TNF, IL-6 as well as interferon-induced genes. Our data provide insight for future translational studies on inflammatory diseases related with Tollip variations or deficiencies.

Figures

Figure 1. Tollip deficient cells express decreased levels of pro-inflammatory cytokines. Wild Type (WT) and Tollip knockout (Tollip -/-) MEF cells were untreated (-) or treated (+) with 10ng/mL LPS for 6 hours prior to RNA isolation, and real time-PCR of selected inflammatory cytokines was performed and analyzed. A) Il6 mRNA levels in WT and T- /- MEF cells with LPS stimulation. B) Tnfα mRNA levels. C) Il12 mRNA levels. All values were normalized, using WT (-) as 1. Data are ± SEM of experiments performed in triplicate. Significance was determined by ttest; p-value<0.05 *, p-value<0.01 **, pvalue<0.001 ***.

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Figure 2. Tollip deficient cells show ( C ) increased mRNA levels of interferoninduced cytokines. WT and Tollip -/- MEF cells were untreated (-) or treated (+) with LPS (10ng/mL) for 6 hours before RNA isolation, and real timePCR of selected inflammatory cytokines was performed and analyzed. A) Ifit mRNA levels in both WT and T-/- MEF cells. B) Ifnλ2 mRNA levels. C) Ifnλr1 mRNA levels. Data are ± SEM of experiments performed in triplicate. All values were normalized, using WT (-) as 1. Significance was determined by t-test; p-value<0.05 *, p-value<0.01 **, pvalue<0.001 ***.

Figure 3. D ysfunctional CUE domain eliminates Tollip’s ability to induce IL6, TNFa, and IL12. WT and Tollip CUE domain mutant (TmCUE) MEF cells were untreated ( -) or treated (+) wit h 10ng/mL LPS for 6 hours prior to RNA isolation, and real time- PCR of select inflammatory cytokines was performed and analyzed. A) Relative Il6 mRNA levels of WT and TmCUE with LPS treatment. B) Tnfa mRNA levels. C) Relative Il12 mRNA levels. Data are ± SEM of experiment s performed in triplicate. All values were normalized, using WT ( -) as 1. Significance was determined by t -test; p -value<0.05 *, p -value<0.01 **, p - value<0.001 ***, p- value<0.0001 ****.

Figure 4. Dysfunctional CUE domain significantly reduces interf eron induced cytokine mRNA levels compared to Tollip defi cient cells. WT, Tollip -/ -, and Tollip CUE domain mutant (TmCUE) MEF cells were untreated ( -) or treated (+) with 10ng/mL LPS for 6 hours before RNA isolation, and real time - PCR of selected inflammatory cytokines was performed and analyzed. A) Ifit mRNA le vels. B) Ifnλ2 mRNA levels. C) Ifnλr1 mRNA levels. Data are ± SEM of experiment s performed in triplicate. All values were normalized, using WT (-) as 1. Significance was determined by t-test; p-value<0.05 *, p-value<0.01 **, p-value<0.001 ***, p-value<0.0001 ****.

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Figure 5. HIF1α, STAT2, and STAT1 are differentially expressed in WT and T - /- MEF cells. WT and T - /- MEF cells were treated with LPS (10ng/mL) for times indicated and the levels of HIF1α, STAT2, STAT1, pSTAT1, and β - actin controls were determined by Western blot analyses. Quantification of mRNA was performed for WT and T - / - MEF cells after 6 hour LPS (10 ng/mL) treatment utilizing RT - PCR. A) WT and T - / - MEF cells were treated for the indicated times with LPS and probed for HIF1α and STAT2 which were compared to the βactin controls via Western blot analyses. B) Graphical representation of HIF1α protein levels with 1, 3, and 6 hr LPS treatment. C) Graphical representation of STAT2 protein levels with 1, 3, and 6 hr LPS treatment. D) Hif1α relative mRNA levels in WT and T-/- cells treated with LPS for 6 hours and normalized to β-actin control mRNA levels. Levels of mRNA were quantified via RT-PCR as previously described. E) Stat2 relative mRNA levels in WT and T-/- cells. F) WT and T-/- MEF cells were treated for the indicated times with LPS and probed for STAT1 and pSTAT1 which were compared to the β-actin controls via Western blot analyses. G) Graphical representation of pSTAT1 protein levels with 1, 3, and 6-hour LPS treatment. H) Graphical representation of STAT1 protein levels with 1, 3, and 6hour LPS treatment. I) Stat1 relative mRNA levels in WT and T-/- cells. The blots were normalized to β-actin using ImageJ to create graphical data. Data represents three separate experiments, excluding STAT2 protein levels. Data are ± SEM of triplicate in each group. All values were normalized, using WT (-) as 1. Significance was determined by t-test; pvalue<0.05 *, p-value<0.01 **, p-value<0.001 ***, p-value<0.0001 ****.

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Figure 5. HIF1α, STAT2, and STAT1 are differentially expressed in WT and T-/- MEF cells. WT and T-/- MEF cells were treated with LPS (10ng/mL) for times indicated and the levels of HIF1α, STAT1, pSTAT1, and β-actin controls were determined by Western blot analyses. Quantification of mRNA was performed for WT and T-/- MEF cells after 6 hour LPS (10ng/mL) treatment utilizing RT-PCR. A) WT and T-/- MEF cells were treated for the indicated times with LPS and HIF1α were analyzed and compared to the β-actin controls. B) Graphical representation of HIF1α protein levels with 1, 3, and 6 hour LPS treatment. C) Hif1α relative mRNA levels in WT and T-/- cells treated with LPS for 6 hours and normalized to βactin control protein levels. Levels of mRNA were quantified via RT-PCR as previously described. D) WT and T-/- MEF cells were treated for the indicated times with LPS and STAT1 and pSTAT1 were analyzed and compared to the β-actin controls. E) Graphical representation of pSTAT1 protein levels with 1, 3, and 6 hr LPS treatment. F) Graphical representation of STAT1 protein levels with 1, 3, and 6 hr LPS treatment. G) Stat1 relative mRNA levels in WT and T-/- cells. The blots were normalized to β-actin using ImageJ to create graphical data. Data represents three separate experiments. Data are ± SEM of experiments performed in triplicate. All values were normalized, using WT (-) as 1. Significance was determined by t-test; pvalue<0.05 *, p-value<0.01 **, p-value<0.001 ***, p-value<0.0001 ****.

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Figure 6. Key transcription factors are differentially expressed in WT and TmCUE MEF cells. WT and TmCUE MEF cells were treated with LPS (10ng/mL) for amount of times indicated and the protein levels of HIF1α, STAT2, STAT1, pSTAT1, and β-actin controls were determined by Western blot analyses. A) WT and TmCUE cells were treated for the indicated times with LPS and probed for HIF1α, STAT2, pSTAT1, and STAT1. B) Graphical representation of HIF1α blot data with 1, 3, and 6 hr LPS treatment. C) Graphical representation of STAT2 protein levels. D) Graphical representation of pSTAT1 blot data. E) Graphical representation of STAT1 blot data. All blots were normalized to βactin. F) Hif1α relative mRNA levels in WT and TmCUE cells treated with LPS for 6 hours. G) Stat2 relative mRNA levels. H) Stat1 relative mRNA levels. Western blot data represents three separate experiments. RT-PCR Data are ± SEM of triplicate experiment in each group. All values were normalized, using WT (-) as 1. Significance was determined by t-test; pvalue<0.05 *, p-value<0.01 **, p-value<0.001 ***.

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Figure 7. Blocking late stage macroautophagy differentially alters mRNA levels of select cytokines and increase pSTAT1 protein levels in WT and T-/- MEF cells. WT and T-/- MEF cells were treated (+) or untreated (-) with a low dose of LPS (10ng/mL) and Bafilomycin (Baf; 10nM) for 6 hours before RNA isolation was performed. Real-time PCR was performed in order to detect mRNA levels of select cytokines. A) WT and Tollip-/- cells were treated for the indicated times with LPS and probed for pSTAT1, STAT1 and β-actin controls. B) Graphical representation of pSTAT1 protein levels with 1, 3, and 6-hour LPS treatment. C) Graphical representation of STAT1 blot data. All blots were normalized to β-actin. D) Ifit relative levels of mRNA. E) Transcription levels of Ifnλ2 with LPS and Baf treatment. F) Transcription levels of Ifnλr1. G) Transcription levels of Ifnλr1 in WT cells only. Data are ± SEM of experiment performed in triplicate for each group. All values were normalized, using WT (-) as 1. Significance was determined by t-test; p-value<0.05 *, pvalue<0.01 **.

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Figure 8. STAT1 and pSTAT1 protein and IFN-induced cytokine mRNA levels decreased with macroautophagy activation in WT and T-/- MEF cells. WT and T-/- MEF cells were treated with LPS (10ng/mL) and LiCl (10mM) for 6 hours prior to protein and RNA extraction. A) Protein levels of STAT1, pSTAT1, and β-actin controls were determined by Western blot analyses. B) Graphical representation of pSTAT1 protein levels. C) Graphical representation of STAT1 protein levels. Blots were normalized to β-actin. D) Stat1 mRNA levels in WT and T/- MEF cells treated with LPS and LiCl (10mM). E) Ifit mRNA levels with LiCl and LPS treatments as indicated. F) Ifnλ2 mRNA levels. G) Ifnλr1 mRNA levels. Western blot data represents three separate experiments. The blots were normalized to β-actin utilizing ImageJ. RT-PCR data are ± SEM of experiments run in triplicate for each group. All values were normalized, using WT (-) as 1. Each Western blot data represents three separate experiments. Significance was determined by t-test; p-value<0.05 *, p-value<0.01 **, p-value<0.001 ***.

Figure 9. Tollip CUE domain mutant shows increased interferon-stimulated cytokine mRNA levels with autophagy inhibition, and decreased Ifit mRNA levels with LiCL treatment. WT and TmCUE MEF cells were treated with LPS (10ng/mL) and Baf (10nm) or LiCl (10mM) for 6 hours before RNA isolation and subsequent RT-PCR was performed. A) Ifit mRNA levels in WT and TmCUE MEF cells with LPS and Baf treatment. B) Ifnλ2 mRNA levels with LPS and Baf treatment. C) Ifnλr1 mRNA levels with LPS and Baf treatments. D) Ifit mRNA levels in WT and TmCUE MEF cells with LPS and LiCl treatment. E) Ifnλ2 mRNA levels with LPS and LiCl treatment. F) Ifnλr1 mRNA levels with LPS and LiCl treatments. All experiments were performed in triplicate. Significance was determined by t-test; pvalue<0.05 *, p-value<0.01 **, p-value<0.001 ***.

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Figure 10. Activation of CMA decreases mRNA levels of Il6 and Tnfα in WT MEF cells. WT and Tollip -/- MEF cells were treated (+) or untreated (-) with a low dose of LPS (10ng/mL) and Geldanamycin (GA; 2μM) for 6 hours before Protein and RNA isolation was performed. Realtime PCR was performed in order to detect mRNA levels of select cytokines. Protein levels of HIF1α, STAT2, STAT1, pSTAT1, and β-actin controls were determined by Western blot analyses. A) WT and Tollip -/- cells were treated with the indicated reagents and probed for HIF1α protein levels. B) Graphical representation of HIF1α protein levels with indicated treatments. The blots were normalized to β-actin using ImageJ. C) Transcription levels of Il6 in WT and T-/- MEF cells treated with GA and LPS. D) Transcription levels of Tnfα. E) Transcription levels of Hif1α. RT-PCR data are ± SEM of experiments performed in triplicate. All values were normalized, using WT (-) as 1. Each Western blot represents three separate experiments. Significance was determined by t-test; p-value<0.05 *, p-value<0.01 **, pvalue<0.001 ***.

Figure 11. Proposed mechanism for Tollip regulation of Low-grade inflammation. 1) TLR4 is stimulated by LPS and adaptor proteins and other modulatory proteins signal to Tollip. 2) Tollip translocates to the mitochondria and interacts with the mitochondria to induce mitochondrial activity. This translocation also causes reduction of fusion between the endosome/autophagosome and lysosome. 3) HIF1α is activated and relocates to the nucleus to transcribe pro-inflammatory cytokines IL-6 and TNFα to induce low-grade inflammation. 4) The reduced fusion between the lysosome-endosome/autophagosome causes activation of STAT1 and the subsequent transcription of IFN-induced cytokines.

Supplementary figures

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Supplemental Figure 1. Tollip C2 domain mutant mimics Tollip deficiency with acute inflammatory responses, but does not show IFN-induced cytokine mRNA levels similar to Tollip deficiency. WT and Tollip C2 mutant (TmC2) MEF cells were treated (+) or untreated (-) with a low dose of LPS (10ng/mL) for the indicated times prior to protein extraction and for 6 hours before RNA isolation was performed. Real-time PCR was performed in order to detect mRNA levels of select cytokines. A) WT and TmC2 MEF cells were treated for the indicated times with LPS and protein levels of HIF1α and β-actin controls were determined by Western blot analyses. B) Graphical representation of HIF1α protein levels with 1, 3, and 6 hr LPS treatment. Blots were normalized to β-actin. C) Il6 relative mRNA levels. D) Tnfα relative mRNA levels. E) Ifit relative mRNA levels. Data are ± SEM of experiments run in triplicate for each group. All values were normalized, using WT (-) as 1. Significance was determined by t-test; p-value<0.05 *, p-value<0.01 **, pvalue<0.001 ***, p-value<0.0001 ****. 52

Tollip C2 domain mutant K162A expresses cytokines similar to CUE mutant.

The importance of the C2 domain on the mRNA levels of pro-inflammatory and

IFNinduced cytokines was tested utilizing a Tollip C2 mutant (TmC2). This C2 mutant contains a mutation at K162A, this residue is important for binding to phosphoinositides (Ankem et al.,

2011). This mutation was expected to cause increased IFN-induced cytokines and still induce pro-inflammatory cytokines. Similar to the Tollip deficient cells, the C2 mutant was unable to induce HIF1α protein levels to the degree WT cells do (Supplemental Figure 1A). There was a slight induction of HIF1α, which was the opposite of the CUE mutant that showed a decrease in

HIF1α with LPS stimulation (Supplemental Figure 1B). Due to the low protein levels of HIF1α, there was no significant induction of Il6 and Tnfα (Supplemental Figure 1C, 1D). The TmC2 mutant showed an increase in Ifit mRNA levels compared to WT cells, though it did not show mRNA levels comparable to Tollip deficient cells (Supplemental Figure 1E). This data taken together does not show the expected outcome. The mutation of the C2 domain also decreased the mRNA levels of Il6 and Tnfα, which was not expected. It is possible that the C2 domain is necessary for the CUE domain to fully interact and induce inflammation through the mitochondria. Interaction of ubiquitin with the C2 domain and the CUE domain inhibits binding of Tollip to phosphoinositides (Mitra et al., 2013b). It is possible that alterations in the C2 domain directly affect the CUE domain through structural change of the protein. It is also possible that the C2 domain also plays an important role in regulation of inflammation through the mitochondria. It is interesting that altering the C2 domain by one residue showed similar cytokine mRNA levels as the CUE domain. It is possible a different residue is responsible for the

C2 domain activity. This would require further studies of the C2 domain structure and function.

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Supplemental Figure 2. HIF1a accumulates with macroautophagy inhibition, while pro-inflammatory cytokines decrease with macroautophagy inhibition. WT and T-/- MEF cells were treated (+) or untreated (-) with a low dose of LPS (10ng/mL) and Bafilomycin (Baf; 10nM) for 6 hours prior to RNA and protein isolations were performed. Real-time PCR was performed in order to detect mRNA levels of select cytokines. A) Il6 mRNA levels. B) Tnfα mRNA levels C) WT and Tollip-/- cells were treated with LPS and Baf and protein levels of HIF1α and β-actin controls were determined by Western blot analyses. D) Protein levels of HIF1α. Blot was normalized to β-actin. E) Transcription levels of Hif1α with LPS and Baf treatment. Data are ± SEM of experiments performed in triplicate for each group. All values were normalized, using WT (-) as 1. Significance was determined by t-test; p-value<0.05 *, p-value<0.01 **.

Chemical Inhibition of macroautophagy increases HIF1α accumulation, but decreases proinflammatory cytokine mRNA levels.

The effect of autophagy inhibition on HIF1α protein levels and subsequent proinflammatory cytokine levels was tested utilizing bafilomycin. Bafilomycin blocks the fusion of the lysosome to endosome/autophagosomes, as well as prevents lysosome acidification. The blocking of acidification of lysosomes also inhibits CMA. It was expected that HIF1α accumulation increased with Bafilomycin treatment. HIF1α is regulated through CMA degradation, thus blocking the acidification of lysosomes would increase HIF1α accumulation.

WT and Tollip deficient cells showed HIF1α accumulation when co-stimulated with LPS and bafilomycin (Supplemental Fig. 2A, 2B). Bafilomycin treatment did not affect Hif1α mRNA levels in Tollip deficient cells, but showed an increase in WT cells when co-stimulated with LPS and Baf (Supplemental Fig. 2C). Though HIF1α accumulated, Il6 and Tnfα mRNA levels was decreased in WT cells (Supplemental Fig. 2D, 2E). IL-6 mRNA levels was decreased in Tollip deficient cells even further with bafilomycin treatment (Supplemental Fig. 2D). This data taken together suggests that the blocking of autophagy induces higher Hif1α mRNA in WT cells and causes a decrease in pro-inflammatory cytokines that mirror Tollip deficiency. Bafilomycin inhibits the fusion of the endosome and lysosome like Tollip deficient cells; therefore, it is not far-fetched to observe WT cells behaving like Tollip deficient cells with bafilomycin treatment.

The accumulation of HIF1α is due to the inhibited CMA, but macroautophagy inhibition may induce Hif1α. The question remains, why is there no induction of pro-inflammatory cytokines with this increase in HIF1α. A possible explanation is that blocking autophagy does not induce the relocation of HIF1α to the nucleus or may even inhibit the relocation, thus causing IL-6 and

TNFα induction prevention.

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Supplemental Figure 3. HIF1α protein levels increase with macroautophagy inhibition, while pro-inflammatory cytokine Il6 mRNA levels decrease with macroautophagy activation. WT and T-/- MEF cells were treated (+) or untreated (-) with a low dose of LPS (10ng/mL) and Lithium Chloride (LiCl; 10mM) for 6 hours prior to RNA and protein isolations were performed. Real-time PCR was performed in order to detect mRNA levels of select cytokines. A) WT and Tollip-/- cells were treated with LPS and LiCl and protein levels of HIF1α and β-actin controls were determined by Western blot analyses. B) Relative protein levels of HIF1α. Blot was normalized to β-actin. C) Transcription levels of Hif1α with LPS and LiCl treatment. D) Il6 mRNA expression levels. E) Tnfα mRNA levels Data are ± SEM of experiments performed in triplicate. All values were normalized, using WT (-) as 1. Significance was determined by t-test; p-value<0.05 *, p-value<0.01 **.

Chemical induction of macroautophagy increases HIF1α accumulation and decreases IL-6 mRNA levels.

The effects of macroautophagy activation on HIF1α protein levels and subsequent proinflammatory cytokines was tested utilizing LiCl, a strong activator of macroautophagy.

CMA degradation regulates HIF1α and it is possible that activating macroautophagy could affect the protein levels of HIF1α by competing with CMA. Macroautophagy activation increased

HIF1α accumulation in WT cells (Supplemental Fig. 3A, 3B). Tollip deficient cells showed no change in HIF1α protein levels with LiCl treatment (Supplemental Fig. 3A, 3B). The mRNA levels of Hif1α was also elevated with LiCl treatment in WT cells, while Tollip deficient cells were unaffected (Supplemental Fig. 3C). WT cells showed a significant decrease in Il6 mRNA levels in both WT and Tollip deficient cells (Supplemental Fig. 3D). Both WT and Tollip deficient cells showed no change in Tnfα mRNA levels with LiCl treatment (Supplemental Fig.

3E). This data taken together, shows that macroautophagy activation induces HIF1α. When combined with the previous figure this data shows that macroautophagy partially modulates

HIF1α. It is possible that the activation of macroautophagy competes with CMA and causes a decrease in CMA. This would lead to the slight accumulation of HIF1α with LiCl treatment. The

Il6 mRNA levels, curiously, decreases with any manipulation of autophagy and further studies are needed to determine this mechanism of pro-inflammatory regulation.

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Supplementary Figure 4. CMA activation decreases pSTAT1 in WT cells and decreases Ifit mRNA levels. WT and T-/- MEF cells were treated (+) or untreated (-) with a low dose of LPS (10ng/mL) and Geldanamycin (GA; 2μM) for 6 hours prior to RNA and protein isolations were performed. Real-time PCR was performed in order to detect mRNA levels of select cytokines. A) WT and Tollip-/- cells were treated with LPS and GA and protein levels of pSTAT1, STAT1, and β-actin controls were determined by Western blot analyses. B) Graphical representation of pSTAT1 protein levels. C) Graphical representation of STAT1 protein levels. Blots were normalized to β-actin. D) Transcription levels of Stat1 with LPS and GA treatment. E) Transcription levels of Ifit with LPS and GA treatment. Data are ± SEM of experiments run in triplicate for each group. All values were normalized, using WT (-) as 1. Significance was determined by t-test; p-value<0.05 *, p-value<0.01 **. Activation of CMA decreases pSTAT1 levels in WT cells and decreases IFIT mRNA levels. 58

The effects of activated CMA on IFN-induced gene mRNA levels were tested utilizing

Geldanamycin (GA). Geldanamycin is a strong and selective activator of CMA. CMA is not a known mechanism of regulation for STAT1, therefore, no change in STAT1 protein levels should have been observed with CMA activation. STAT1 overall protein levels were unchanged with GA treatment, but pSTAT1 showed a significant decrease both with and without LPS treatment when treated with GA in WT cells (Supplemental Fig. 4A, 4B). Tollip deficient cells showed an increase in pSTAT1 protein levels with GA treatment, which was the opposite effect seen on WT cells (Supplemental Fig. 4B). Overall STAT1 protein levels appeared to decrease with GA treatment (Supplemental Fig. 4C). CMA activation decreased Stat1 mRNA induction in both WT and Tollip deficient fibroblasts (Supplemental Fig. 4D). CMA activation alone is able to decrease Ifit mRNA levels in both WT and Tollip deficient cells (Supplemental Fig. 4E).

It was expected that GA would have no effect on the induction of IFN-induced cytokines. It is possible that CMA activation compensates for decreased macroautophagy through degrading debris and resolving inflammation. This would explain the decrease in pSTAT1 and Ifit mRNA levels observed.

Inhibitor and Activation of Macroautophagy Studies

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Supplemental Figure 5. Chemical inhibition of macroautophagy through PI3K inhibition decreases inflammatory response in WT cells and increases acute inflammation in Tollip deficient cells. WT and T-/- MEF cells were treated (+) or untreated (-) with a low dose of LPS (10ng/mL) and 3-Methyladenine (3-MA; 5mM) for 6 hours prior to RNA and protein isolations were performed. Real-time PCR was performed in order to detect mRNA levels of select cytokines. A) WT and Tollip-/- cells were treated with LPS and 3-MA and protein levels of HIF1α and β-actin controls were determined by Western blot analyses. B) Relative protein levels of HIF1α. Blot was normalized to β-actin. C) Relative mRNA levels of Ifit with LPS and 3-MA treatment. D) Transcription levels of Il6. E) Transcription levels of Tnfα. Data are ± SEM of experiment performed in triplicate for each group. All values were normalized, using WT (-) as 1. Significance was determined by t-test; p-value<0.05 *, p-value<0.01 **.

Inhibition of autophagosome formation differentially changes inflammatory response in WT and

Tollip deficient cells

The effects of macroautophagy inhibition were tested through blocking autophagosome formation utilizing 3-Methyladenine (3-MA). 3-MA inhibits autophagosome formation via the inhibition of type III and I Phosphatidylinositol 3-kinases (PI3K) (Seglen and Gordon, 1982).

This inhibitor was meant to block macroautophagy specifically, but had many unforeseeable effects on the expression of IFN-induced cytokines and pro-inflammatory cytokines.

Autophagosome formation decreased HIF1α protein levels in both WT and Tollip deficient cells

(Supplemental Fig. 5A, 5B). Autophagosome formation inhibition drastically decreased mRNA levels of Ifit in both WT and Tollip deficient cells (Supplemental Fig. 5C). Once again the manipulation of autophagy decreased Il6 mRNA levels with the co-stimulation of LPS in WT cells, but with 3-MA treatment alone Il6 induction was increased (Supplemental Fig. 5D). With

3-MA treatment Tollip deficient cells had significantly increased Il6 mRNA levels, both with and without LPS challenge (Supplemental Fig. 5D). WT cells showed a significant decrease in

Tnfα induction with 3-MA treatment, while Tollip deficient cells continued to show no induction

(Supplemental Fig. 5E). Much like the activation of CMA, 3-MA treatment decreased HIF1α protein levels. It is possible that blocking autophagosome formation causes the cell to depend on

CMA to degrade proteins and bring the cell back into homeostasis, thus increasing CMA.

Increasing CMA decreases HIF1α levels in the cell, therefore CMA activation compensates for inflammation through degradation of HIF1α. Once HIF1α becomes degraded, WT cells have reduced mRNA levels of Il6 and Tnfα. Curiously, 3-MA treatment restored Il6 induction in

Tollip deficient cells. IL-6 has many transcription factors that regulate its induction; it is possible that 3-MA induces another transcription factor that transcribes this cytokine. 3-MA treatment alone reinforced this assumption when Il6 mRNA levels increased in WT cells as well as Tollip deficient 61 cells. PI3K and other negative regulators are suppressed by low-doses of LPS and this augments low-grade non-resolving inflammation (Maitra et al., 2012b). Thus, the reduction of inflammation with 3-MA and LPS treatment needs further investigation. This, however, partially explains the increase in Il6 and Tnfα with 3-MA treatment alone in WT cells. The decrease in Ifit mRNA may be due to increased lysosome fusion to endosomes due to the lack of competition with autophagosomes for degradation of contents.

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Supplemental Figure 6. Treatment with wortmannin does not alter cytokine levels, but decreases HIF1α protein levels in WT and Tollip deficient cells. WT and T-/- MEF cells were treated (+) or untreated (-) with a low dose of LPS (10ng/mL) and Wortmannin (Wort; 0.25μM) for 6 hours prior to RNA and protein isolations were performed. Real-time PCR was performed in order to detect mRNA levels of select cytokines. A) WT and Tollip-/- cells were treated with LPS and Wort and protein levels of HIF1α, STAT2, and β-actin controls were determined by Western blot analyses. B) Relative protein levels of HIF1α. C) Relative protein levels of STAT2. Blots were normalized to β-actin. D) Relative mRNA levels of Il6 with LPS and Wort treatment. E) Transcription levels of Ifit. Data are ± SEM of experiments performed in triplicate for each group. All values were normalized, using WT (-) as 1. Significance was determined by t-test. Wortmannin treatment has no effect on induction of cytokines with LPS treatment

In order to test other potential macroautophagy blockers, we utilized wortmannin.

Wortmannin nonspecifically blocks PI3Ks, which can effect multiple pathways (Arcaro and

Wymann, 1993). It also blocks the recovery of calcium and potassium channels. Wortmannin slightly decreased HIF1α protein levels in both WT and Tollip deficient cells (Supplemental Fig.

6A, 6B). STAT2 protein levels were slightly elevated with Wortmannin treatment in WT cells, but Tollip deficient cells were unaffected (Supplemental Fig 6C). The mRNA levels of Il6 and

Ifit were unaffected by wortmannin treatment (Supplemental Fig. 6D, 6E). Though wortmannin has been shown to block autophagy similar to 3-MA, the effects were not the same in this fibroblast model. Wortmannin appears to have no effect on WT and Tollip deficient cells, therefore it was not a suitable inhibitor for this study.

Chapter 3. Tollip modulates cell survival through regulation of BCL2,

MCL1, and Bax in fibroblasts with chronic exposure to LPS.

Specific Aim 1: Test the hypothesis that Tollip deficient cells have disregulated cell survival

and necrosis compared to WT MEF cells with chronic exposure to LPS.

Rationale: It has been shown previously that Tollip interacts with the mitochondria via the CUE domain. A single nucleotide polymorphism (SNP) in Tollip has been associated with Idiopathic

Pulmonary Fibrosis (IPF) (Noth et al., 2013). Fibroids are characterized by increased survival and decreased cell death of smooth muscle cells and fibrous connective tissue. IPF is characterized as a disease in fibroblasts, increasing the validity of using this model to study this phenomenon. Mitochondria play a huge role in the regulation of cell death and cell survival via the BCL2 protein family (Gross et al., 1999). Tollip may play a role in the regulation of cell survival and cell death through its interaction with the mitochondria. IRAK1 deficient mice are not sensitive to LPS induced necroptosis (Baker et al., 2014). Tollip is a known negative regulator of IRAK1 and therefore may play a role inhibiting IRAK1. It is important to discover the mechanism behind Tollip regulation of cell survival in fibrobalsts because this may lead to a better understanding and treatments of this debilitating disease IPF.

Working Hypothesis: If cells are Tollip deficient, it is expected to observe higher cell survival compared to the WT cells withchronic exposure to low-dose LPS.

Specific Aim 2: Test the hypothesis that Tollip CUE and C2 fomain are important for

proper regulation of cell survival.

Rationale: As previously discussed, the Tollip CUE mutant protein is unable to interact with mitochondria leading to low induction of IL-6 and TNFα. It was also shown that

64 interferoninduced cytokines have decreased induction in Tollip CUE mutant cells. This may be due to the localization of the Tollip CUE mutant with the lysosome-endosome, which was previously decribed (Baker et al., 2015). The Tollip CUE mutant may be causing increased macroautopagy, which has been shown to induce apoptosis in neuroblastoma cells (Zheng et al.,

2011) and has been reviewed extensively in previous papers (Baehrecke, 2005). It is also possible that the CUE domain of Tollip plays a role in the proper regulation of apoptotic proteins via the mitochondria.

Working Hypothesis: If the Tollip CUE domain is unable to interact with the mitochondria, than cell survival will be decreased due to disregulated apoptotic proteins and possibly by the increase in macroautophagy.

Specific Aim 3: Test the hypothesis that key pro-survival and pro-apoptotic proteins are

differentially expressed in Tollip -/-, WT and TmCUE.

Rationale: Tollip deficient fibroblasts show increased cell survival. Because Tollip is not present to interact with the mitochondria, this may cause a dysregulation of key pro-survival proteins, such as MCL1 and BCL2. Tollip may normally inhibit the activation of BLC2 and MCL1 with low doses of LPS, thus Tollip deficieny will show an increase in phosphorylated MCL1 and

BCL2. The lack of Tollip may also cause a decrease in pro-apoptotic proteins such Bax and Bak, thus leading to the increased survival of these cells. Bax and BCL2 are expressed on the outer membrane of the mitchondria, thus Tollip may interact with these proteins when chronically exposed to low doses of LPS and contribute to the necroptosis.

Working Hypothesis: It is hypothesized that Tollip deficient cells will have higher levels of prosurvival proteins and show lower levels of pro-apoptotic proteins realtive to WT cells. These differences in protein levels of pro-apoptotic and pro-survival proteins may give a possible

65 explanation for the increased survival of these cells. It is also hypothesised that the Tollip CUE mutant will have increased pro-apoptotic and decreased pro-survival proteins, thus contributing to the increased cell death and necrosis observed.

Subaim 3A: Test the hypothesis that the increased induction of pro-survival proteins

is due to the increased protein levels of phosphorylated STAT3.

Rationale: STAT3 is a key transcription factor of pro-apoptotic proteins BCL2 and

MCL1. Increased protein levels of STAT3 and activated STAT3 have been assocaited

with increased cell survival and increased levels of BLC2 and MCL1. (Bhattacharya et

al., 2005)

Working hypothesis: Tollip deficient cells are expected to have increased phosphorylated

STAT3 at both basal levels and with LPS stimulation.

Specific Aim 4: Test the hypothesis that decreased autophagy will increase necrotic cell

populations in WT cells with chronic low-dose LPS challenge.

Rationale: It has been previously shown that decreased autophagy can increase cell death due to lack of clearance and increased inflammation, but it has also been shown that autophagy can induce cell death if dysregulated (Baehrecke, 2005). It is an unsolved paradox that Tollip shows increased cell survival, though macroautophagy completion is deficient in these cells. It is possible that the decreased autophagy and cell response to LPS stimulation in Tollip deficient cells actually leads to an increase in survival. It is curious though, that Tollip deficient cells show increased necrotic cell levels as well with 16-hour low-dose LPS exposure. This may be due to lack of clearance of debris, which will put the cell into a necrotic state rather than the highly regulated process of apoptosis. Therefore, WT cells with decreased macroautophagy may have increased necrosis with low-dose LPS stimulation. 66

Working Hypothesis: Chemically inhibited macroautophagy is expected to increase the necrotic cell populations similar to Tollip deficient cells in WT fibroblasts.

Specific Aim 5: Test the hypothesis that increased autophagy will decrease necrotic cell

populations in WT cells with chronic low-dose LPS challenge, but will increase necrosis

without chronic low-dose LPS challenge.

Rationale: Increased macroautophagy is generally beneficial while clearing infection; however, macroautophagy can also cause cell death if over activated. If the necrotic cell population increases with macroautophagy inhibition, it is possible that increasing macroautophagy can protect fibroblasts against necroptosis when chronically exposed to low-dose LPS. Autophagy can also activate an autophagy induced cell death pathway, thus over-activating macroautophagy could lead to increased necrosis (Baehrecke, 2005).

Working hypothesis: If macroautophagy is chemically activated and co-stimulated with LPS, then the necrotic cell population will decrease. If macroautophagy is activated absent of LPS stimulation, it is possible that necrotic cell populations will be increased.

Abstract

Innate leukocytes have highly regulated systems to control apoptosis and cell death upon challenges with rising dosages of pathogen associated molecular pattern molecules (PAMPs) such as lipopolysaccharide (LPS). Subclinical low-doses of LPS will shift cells into a state of necroptosis and cell death with chronic exposure. To regulate these processes, innate leukocytes utilize distinct intra-cellular signaling circuitries modulated by adaptor molecules. Toll-interacting protein (Tollip) is one of the adaptor molecules in the TLR4 signaling pathway and potentially 67 plays a key role in the regulation of apoptosis and cell death in response to low-doses of LPS.

Tollip acts as a positive regulator for low-grade inflammation through the mitochondria. Here, we show a novel mechanism in which Tollip is important for proper regulation of cell survival through modulation of BCL2 proteins and STAT3 when exposed to low-doses of LPS.

Introduction

The balance between apoptosis and cell survival plays a pivitol role in disease progression and infection. Cells with overactive survival can cause cancer progression, while cells with upregulated apoptosis caused by external stimuli can lead to cell death, eventual detrimental inflammation, and tissue damage. High-doses of lipopolysacharides (LPS), a surface component of Gram-negative bacteria, cause cells to induce an inflammatory response and will induce anti-inflammatory mechanisms to resolve inflammation. If high-doses of LPS persist this can cause cells to become apoptotic, which can contribute to the resolution of acute inflammation

(Medan et al., 2002; Gautier et al., 2013). Individuals with adverse health conditions and lifestyles tend to maintain mildly elevated levels (~1-100pg/mL) of circulating LPS, a phenomenon referred to as subclinical low-grade endotoxemia (Cani et al., 2008; Castro et al.,

2016). In contrast to high-doses of LPS, low-doses of LPS will cause cells to enter a state of necroptosis through TLR4 signaling (Baker et al., 2014). Different from apoptosis, necroptosis contributes to prolonged inflammation through the release of inflammationpropagating damage- associated molecular patterns (Vanlangenakker et al., 2012).

A key regulator in TLR4 signaling is Toll-interacting Protein (Tollip). Tollip has many roles within immune cells, not only having inhibitory effects on inflammation, but also a dynamic role in inflammatory activation depending on dosage of LPS and environmental stimuli.

Tollip is involved in protein trafficking and assistance of endosome-lysosome fusion (Capelluto,

2012; Baker et al., 2015). Tollip has also been shown to induce ROS response through mitochondrial activity with stimulation from low-doses of LPS (Maitra et al., 2012b).

Mitochondrial involvement in the survival and apoptosis pathways has been extensively studied.

Principally, BLC2 (B-cell lymphoma 2) family proteins, such as Bax, Bak, BCL2 and MCL1, mediate the mitochondrial apoptosis pathway. These BCL2 family proteins fall under three major categories: the pro-apoptotic BH3-only proteins; the pro-survival BCL2-like proteins; and the pore-forming Bax and Bak proteins (Westphal, 2011). BCL2 exerts a pro-survival signal and does this partially through forming a heterodimer with the Bax protein making it unable to exert its pro-apoptotic signals. Reactive oxygen species induction will decrease BCL2 protein levels and will increase the protein levels of Bax (Li et al., 2014). Many transcription factors also regulate the levels of these proteins including STAT proteins, such as STAT3 that transcribes

BCL2 (Choi et al., 2009).

Higher dosages of LPS are known to induce compensatory tolerance in innate immune cells, as well as increased mitochondrial bioenergetics and function to resolve inflammation (Liu et al., 2012). However, the mechanisms responsible for necroptosis initiated by super-low doses of LPS need to be studied further. We examined the effects of low-dose LPS induced necroptosis in Tollip deficient cells. The involvement of Tollip in cell survival and death in the context of chronic low-grade inflammation has not been examined. In this study, we reveal that Tollip deficient cells have higher cell survival due to the regulation of key apoptotic proteins in murine embryonic fibroblasts, and higher necrotic populations most likely due to lack of macroautophagy completion.

Materials and Methods

Materials

LPS (Escherichia coli 0111:B4), Bafilomycin A, and Lithium Chloride were purchased from

Sigma-Aldrich. BCL2 (no. 3498), MCL1 (no.5453), p-MCL1(Ser159/Thr163) (no. 4579), pSTAT3 (Tyr705) (no. 9131) Antibodies were obtained from Cell Signaling Technology.

Cell Culture

Murine Embryonic fibroblasts were grown in DMEM supplemented with 10% (v/v) FBS, 1%

(v/v) L-glutmaine, 1% (v/v) streptomycin/penicillin and incubated at 37⁰C in a humidified incubator in the presence of 5% CO2. Cells were passaged every 3-4 days to maintain less than

90% confluence.

Cell Transfection

Lipofectamine ratio suggested by the manufacturer’s instructions. The cells were cultured in

Flow cytometry

For in vitro studies, WT, T-/-, and TmCUE mutants were cultured in starving (2% FBS) DMEM overnight before treatments began. Cells were then treated with low dose (10 ng/mL) LPS for 16,

24, or 48 hours. In some experiments, Bafilomycin (10nM) or LiCl (10mM) was also added to

70 the cell culture with LPS for the same time increments. After stimulation with cells were stained using Annexin V Apoptosis Detection Kit APC (Affymetrix) according to the manufacturer’s instructions and subsequently stained with Propidium Iodide, which stains only dead cells with permeable membranes. FACSCanto II (BD Biosciences) was used to analyze the samples.

FACSDiva (BD Biosciences), or Flow Jo (Tree Star, Ashland, OR) processed the data.

Protein isolation and Western Blot Analyses

Cells were harvested after specified treatments and washed with DMEM containing no FBS. The cells were re-suspended in a lysis buffer containing protease inhibitor mixture (Sigma-Aldrich),

Phosphatase Inhibitor cocktail 2 (Sigma-Aldrich), Phosphatase inhibitor cocktail 3

(SigmaAldrich) and subjected to SDS-PAGE. The protein bands were transferred to an immunoblot polyvinylidene difluoride membrane (Bio-Rad) and subjected to immunoblot analysis with the indicated antibodies.

Results Tollip mediates cell survival and apoptosis in murine embryonic fibroblasts with LPS stimulation.

The involvement of Tollip in cell survival and apoptosis was tested utilizing WT and

Tollip deficient fibroblasts that were co-stained with Annexin V/propidium iodide (PI) and analyzed via flow cytomentry post chonic LPS treatment. Cells stained by the Annexin V show signs of early or late apoptosis, while staining with PI show signs of late apoptosis or necrosis.

Tollip deficient cells showed higher live cell populations even at basal levels compared to WT

71 cells (Fig. 1A). Tollip deficient cells continue to show increased populations of live cells with the

16 hour, 24 hour, and 48 hour treatments, thus showing a perpensity to higher survival rates than

WT with LPS challenge (Fig. 1A). Tollip deficient cells show significantly lower populations of early apoptosis and show little to no change with LPS treatment, while WT cells show an increase in early apoptotic populations over time (Fig. 1B). Tollip deficient MEF cells appear to have a slight decrease in early apoptosis populations with chronic LPS exposure over 48 hours

(Fig. 1B). Late apoptosis populations increase drastically in WT fibroblasts with 16 hour LPS challenge and decrease over 24 and 48 hours treatment (Fig. 1C). Tollip deficient late apoptosis cell populations were unaffected by chronic LPS challenge (Fig. 1C). Tollip deficient cells showed elevated percentages of necrotic cells, though not statistically significant due to some variation in samples, when compared to WT cells treated with LPS for 16 hours and at basal levels (Fig. 1D). This data taken together shows that Tollip deficient cells have increased cell survival and less early and late apoptosis occurring with LPS stimulation compared to the WT cells.

Tollip CUE domain is necessary for cell survival in fibroblasts In order to test the importance of the Tollip CUE domain in cell survival, we utilized a

CUE domain mutant containing a M240A/F241A mutation. This mutation causes the ubiquitin interaction patch on Tollip to no longer interact with ubiquitin and removes Tollip’s ability to translocate and localize with the mitochondria (Baker et al., 2015). Over the 16, 24, and 48-hour

LPS treatment the CUE mutant MEFs were unable to sustain high live cell populations and had low live cell populations compared to the WT at basal levels (Fig. 2A). CUE mutant fibroblasts showed lower populations of early apoptotic cells compared to WT fibroblasts (Fig. 2B). WT cells showed increasing early apoptotic cell levels with increased exposure to LPS, while the

CUE mutant fibroblasts showed a decrease in early apoptotic cell levels with increased LPS

72 exposure (Fig. 2B). The CUE mutant appears to have no response to LPS in the late apoptotic population, but it remains consistently high and is significantly higher at basal levels compared to WT fibroblasts (Fig. 2C). The necrotic cell populations were significantly higher in the CUE mutant fibroblasts compared to WT cells (Fig. 2D). Though the necrotic cell populations increased slightly with LPS treatment in the CUE mutant fibroblasts, it was not significant across the time course (Fig. 2D). WT cells had significantly lower necrotic cell populations than the

CUE mutant cells (Fig. 2D). This data taken together shows that the Tollip CUE domain must remain functional in order to regulate cell death in a normal manner. The CUE mutant localizes with the endosome-lysosome and is unable to interact with the mitochondria, which may contribute to dysregulated necrosis. It is also possible that the CUE mutant is over-activating macroautophagy and activating cell death via the autophagy cell death pathway.

Tollip C2 domain necessary for proper regulation of apoptosis and necrosis.

In order to examine the effects a dysfunctional C2 domain had on cell survival with chronic exposure to LPS, a Tollip C2 domain mutant (TmC2) was utilized. This Tollip protein contains a K162A mutation, which has been shown to be key in phosphoinisitide binding. It is proposed to be a key site for assistance in lysosome-endosome fusion, therefore this mutation may cause decreased macroautophagy. WT cells showed significanly higher live cell populations at basal levels compared to TmC2 cells (Fig. 3A). TmC2 cells showed a significant decrease in the live cell population after 48 hours of LPS exposure, but WT and TmC2 live cell populations were not significantly different at 16 and 24 hour treatments with LPS (Fig. 3A). WT cells showed significantly higher early apoptosis populations even at basal levels, while TmC2 early apoptosis cell populations were unaffected by LPS treatment (Fig. 3B). WT cells showed significantly higher late apoptosis populations with LPS challenge compared to TmC2 cells (Fig.

3C). TmC2 cells showed a steady increase in late apoptosis populations with increased LPS exposure and significantly higher late apoptosis populations at basal levels (Fig. 3C). Much like the TmCUE mutant, TmC2 had significantly higher necrotic cell populations compared to WT cells (Fig. 3D). The TmC2 cells had significantly higher necrotic cells populations, though they were unchanged by LPS challenge. This data taken together suggests the C2 domain is necessary for proper regulation of cell surivival and apoptosis in fibroblasts. When either domain is dysfunctional the cells appear to either live or become necrotic with low levels of apoptotic cell populations. It is possible the C2 domain has increased necrotic cell populations due to dysregulated macroautophagy, while the intact CUE domain allows for interaction with the mitochondria to induced cell death through the mitochondrial cell death pathway.

Chemically blocking macroautophagy causes increased necrotic cell populations in WT fibroblasts.

In order to test the effect autophagy inhibition has on cell death of Tollip deficient and

WT fibroblasts, we utilized Bafilomycin A. Bafilomycin inhibits the fusion between endosome/autophagosome and lysosome, similar to Tollip deficiency. WT and Tollip deficient fibroblasts were separately challenged, and co-stimulated with LPS and Bafilomycin for 16 hours. Tollip deficient cells had higher live cell populations than WT cells with 16 hours of LPS treatment (Fig. 4A). WT cells showed a decrease in live cell populations when treated with

Bafilomycin alone, but co-stimulation with LPS and Bafilomycin showed no difference (Fig.

4A). Autophagy inhibition increased WT and Tollip deficient early apoptosis cell populations with or without LPS challenge for 16 hours (Fig. 4B). Late apoptosis populations slightly increased with autophagy inhibition for 16 hours in WT fibroblasts, but no other differences were detected (Fig. 4C). Tollip deficient cells showed significantly higher necrotic populations than

WT cells at basal levels and with LPS treatment (Fig. 4D). This trend is observed in Figure 1, but is illustrated more clearly in Figure 4 with less variation in the samples. WT cells showed significantly increased necrotic cell levels with Baf treatment (Fig. 4D). This increased necrotic cell population with decreased macroautophagy may explain increased necrotic cell levels in

Tollip deficient cells. Tollip deficient cells showed no significant increases in necrotic cell populations with Baf treatments (Fig 4D). Tollip deficiency decreases lysosome-endosome fusion, which bafilomycin mimics. This may imply that the decreased autophagy in Tollip deficient cells is the mechanism by which higher necrotic cell populations exist. These results suggest that late stage autophagy inhibition leads to higher necrosis and begins early apoptosis in both WT and T-/- cells. Thus, possibly explaining the high levels of necrosis seen in Tollip deficient cells due to the naturally decreased inhibition of autophagy in these cells.

Chronic chemical inhibition of autophagy increases apoptosis and cell death

In order to analyze the effects chemically inhibited autophagy had on cell survival with chronic exposure, bafilomycin was utilized and co-stimulated with LPS for 48 hours. WT cells showed a drastic shift in live cell populations when late stage autophagy was blocked both with and without LPS stimulation (Fig. 5A). This is most likely due to the cellular stress caused by lack of clearence to maintain homeostasis. Tollip deficient cells showed a slight decrease in live cell populations with Baf treatment alone (Fig. 5A). As seen with the 16 hour treatment with

Bafilomycin, WT and Tollip deficient cells showed increased early apoptotic cell populations

(Fig. 5B). WT and Tollip deficient cells showed an increase in late apoptosis cell levels with Baf treatment (Fig 5C). WT late apoptosis cell populations nearly doubled from 16 hours to 48 hours, most likely due to the shift of early apoptotic cells to late stage apoptosis over the increased exposure to LPS and Baf (Fig. 4C, 5C). It is important to note that bafilomycin is not a specific

75 inhibitor and not only prevents fusion of the lysosome and endosome/autophagosome, but also blocks the acidification of the lysosome. Thus, chaperone mediated autophagy can also be affected with chronic exposure to bafilomycin. This may account for the effect seen within the

Tollip-/- cells. If these cells are typically compensating with CMA to reduce inflammation this becomes impossible. WT cells showed a drastically increased population of necrotic cells with bafilomycin treatment, while Tollip deficient cells were unaffected by bafilomycin treatment in respect to the necrotic cell population levels (Fig. 5D). This data taken together further illustrates the effects of autophagy inhibition on cell survival and increased necrosis.

Chemically activated macroautophagy decreases cell death when co-stimulated with LPS stimulation, but causes autophagy induced apoptosis without LPS stimulation.

In order to test the effects activated macroautophagy had on cell survival in WT cells,

LiCl was utilized and cells were treated for 16 hours. LiCl is a strong inducer of macroautphagy, which has been shown previously (Motoi et al., 2014). In WT cells macroautophagy activation alone is able to decrease the live cell populations, but when paired with LPS live cell populations increased slightly (Fig. 6A). Macroautophagy activation appears to increase early apoptosis populations without LPS stimulation, and when paired with LPS it increased the effects of LPS induced early apoptosis (Fig. 6B). Late apoptotic cell populations are increased with both LPS and LiCl treatment alone, and when co-stimulated late apoptotic populations are decreased (Fig.

6C). Macroautophagy induction alone causes increased necrosis (Fig. 6D). Stimulation with both

LPS and LiCl reduce the necrotic cell populations in WT cells (Fig. 6D). This data taken together shows evidence that increased macroautophagy can be detrimental. When paired with LPS stimulation, chemically induced macroautophagy has a protective effects. Alternatively,

76 activation of macroautophagy for 16 hours without an inflammatory stimulus decreases cell survival and increases necrosis.

Chemically activated macroautophagy decreases cell survival, but also decreases necrotic populations.

In order to test the effect of chronically activated macroautophagy on WT cells, the cells were treated with LiCl and LPS for 48 hours. Live cell populations were significanly decreased with LPS exposure and LPS treatment paired with activated macroautophagy (Fig. 7A). Cell survival was also effected by macroautophagy activation alone, showing that chronic exposure to activated macroautophagy can cause cell death (Fig. 7A). As expected the apoptotic populations shifted from increased early apoptosis, which was observed at 16 hours, to increased late apoptosis (Fig. 7B, 7C). Macroautophagy activation, however, slightly decreased the necrotic cell population (Fig. 7D). This may be due to the decreased nutrients in the media and may begin to lead to a more beneficial aspect of macroautophagy in these conditions. These results taken together show that macroautophagy can play both beneficail and detrimental roles in cell survival and apoptosis.

Tollip mediates key proteins in the apoptotic and suvival pathway.

In order to analyze the mechanistic aspects of the previously observed phenotypes, key apoptosis regulators were examined. BCL2 plays a large role in survival in cells and is activated through the mitochondrial apoptotic pathway along with Bax. BCL2 acts as an inhibitor and will create a dimer with Bax, a pro-apoptotic protein, in order to exhibit its role in cell survival (Cory and Adams, 2002). Tollip deficient cells showed much higher levels of BCL2 and lower protein levels of Bax (Fig. 8A). Overall MCL1 protein levels were reduced in Tollip deficient cells compared to WT fibroblasts. However, phosphorylated MCL1 had slightly higher protein levels

77 in Tollip deficient cells than in WT cells, MCL1 appears to decrease with increased time of LPS exposure. Tollip deficient cells showed higher levels of activated pSTAT3 (Y705), which is involved in the regulation of cell survival and is a known transcription factor of MCL1 and

BCL2 (Fig. 8A). The Tollip CUE mutant cell line had quite different protein levels of these proapoptotic and pro-survival proteins. BCL2 was diminished considerably in TmCUE fibroblasts compared to WT fibroblasts (Fig. 8B). Bax protein levels were less than WT, but it is important to note that BCL2 is required to create a heterodimer in order to inhibit the pro- apoptotic action of Bax and BCL2 protein levels were nearly undeteactable (Fig. 8B). MCL1 was significantly diminished in TmCUE cells, but unlike the Tollip deficient cells there was no activated MCL1 present (Fig. 8B). The protein levels of pSTAT3 decreased 24 and 48 hour exposure to LPS stimulation in TmCUE cells in contrast to the consistently high levels of pSTAT3 observed in Tollip deficient cells with and without chronic LPS exposure. The low levels of both pMCL1 and BCL2 in TmCUE fibroblasts gives a mechanistic explaination for lower cell survival and higher necrosis in these cells.

Discussion

Tollip plays a pivitol role in the regulation of low-grade inflammation and cell survival.

Low doses of LPS lead to low-grade inflamamtion which can lead to necroptosis, which will then potentiate inflammation through DAMPs. We have shown here that Tollip plays a role in the regulation of necroptosis caused by LPS stimulation. Tollip deficient cells show a much higher live cell population with low-dose LPS stimulation and showed no response to LPS stimulation in the context of cell death. This is most likely due to Tollip’s regulation of key antiapoptotic and pro-apoptotic proteins. Tollip deficient cells will not induce ROS with low-doses of LPS (Maitra et al., 2012b). ROS induction has been shown to upregulate the protein levels of Bax and

78 downregulate the expression of BCL2 in umbilical vein endothelial cells, which correlates with the expression levels observed Tollip defcient and WT fibroblasts (Li et al., 2014). Tollip deficient cells express significantly higher levels of pSTAT3 (Y705), which is a known transcription factor for BCL2 and MCL1 (Bhattacharya et al., 2005). Tollip appears to suppress the expression and activation of STAT3, which would give a mechanistic explaination for the increased live cell populations in Tollip deficient cells. This data taken together suggests that

Tollip upregulates apoptotic proteins with LPS stimulation and down regulates pro-survival proteins, which is most likely induced through ROS production.

Tollip deficient cells exhibited higher necrotic cell populations at basal levels and with 16 hour LPS challenge. It is possible that deficient macroautophagy increases the necrotic cell population through an all or nothing mechanism. To test this WT cells were chonically treated with bafilomycin, which inhibits endosome/autophagosome fusion to the lysosome. At 16 hours

WT cells showed decreased live cell populations, not large changes in early and late apoptosis, but a significant increase in necrotic cells, which mimicked Tollip deficient cells at basal levels.

Therefore, it is possible that Tollip deficient cells have higher necrosis due to the lack of completion of macroautophagy. The higher levels of live cells may be through a separate mechanism in which increased BCL2 protects these fibroblasts from necroptosis.

While examining the importance of the Tollip C2 domain and CUE domain in cell survival, it was revealed that having either domain mutated will cause a drastic increase in necrotic cell populations and decrease cell survival. It was observed that the CUE domain mutant showed a more drastic increase in necrotic cell populations. The TmCUE mutant had lower levels of pro-survival protein BCL2 and higher levels of pro-apoptotic protein Bax when compared to Tollip deficient cells. This correlates with the increased necrotic cell population

79 phenotype seen in these TmCUE fibroblasts. The Tollip CUE mutant has been shown to localize with the lysosome-endosome and will not localize with the mitochondria upon low-dose LPS stimulation like WT Tollip (Baker et al., 2015). It is possible that the CUE mutant will assist with the fusion of the endosome-lysosome, even when it is inappropriate to do so. As shown in the WT cells, chemical activation of macroautophagy caused increased levels of necrotic cells. It is possible that the CUE mutant enhances macroautophagy, therefore increasing necrotic cell populations similar to the WT fibroblasts with chemically enhanced macroautophagy.

In conclusion, Tollip clearly plays a role in cell survival and necroptosis when cells are chonically stimulated with low-doses of LPS. Tollip deficiency increases cell survival through the upregulation of pro-survival proteins and the down regulation of pro-apoptotic proteins. In contrast, Tollip induced ROS may be causing the up-regulation of pro-apoptotic Bax and the down regulation of BCL2 in WT fibroblasts. Cell death regulated through autophagy, however, may facilitate in increasing necrotic cell populations at basal levels. Further studies are required to elucidate the mechanism by which Tollip regulates cell survival. It has been shown in other studies that Bax/Bak -/- mice treated with macroautophagy inhibitor, 3-methyladenine, will show increased cell survival similar to Tollip deficient cells (Ullman et al., 2008). It is possible that

Tollip deficiency causes an inabilty to activate apoptosis through the mitochondria and through the alternative autophagy cell death pathways. When either domain is mutated it caused increased cell death, it is possible the C2 mutant over activated the mitochondrial apoptosis pathway and the CUE domain mutant over activated the autophagy cell death pathway. This resulted in higher cell death for both mutants, and correlates with the increased cell survival observed in Tollip deficient cells.

Further studies should be performed to examine the effects of ROS induction on BCL2 and Bax protein levels in fibroblasts. Tollip deficient cells do not induce ROS, therefore chemical induction of ROS in Tollip deficient cells may show similar BCL2 and Bax protein trends as WT due to the known regulation of these proteins by ROS (Li et al., 2014). The mRNA levels of BCL2 and Bax should also be examined in order to test if these proteins are downregulated through post-transcriptional degradation or lack of gene expression. Further studies on this phenomenon could lead to a better understanding of Tollip’s involvment in diseases such as idiopathic pulminary fibrosis and Crohn’s disease. This data taken together, reveals a novel role Tollip plays in the regulation of cell survival and necroptosis.

Figures

Figure 1. Tollip deficiency increases cell suvival with LPS challenge. Graphical data of WT and T-/- Flow cytometry data. Cells were treated for either 0, 16, 24, or 48 hours with LPS (10ng/mL) treatment. The cells were stained with Annexin V and PI to analyze live cells, early apoptosis, late apoptosis, and necrotic cells populations. A) Live cells (Annexin V-/PI-) percentages of populations for WT verses T-/- MEF cells. B) Early apoptosis (Annexin V+/PI-) percentages of populations. C) Late apoptosis (AnnexinV+/PI+) percentages of populations. D) Necrotic cells (Annexin V-/PI+) percentages of populations. Experiments performed in triplicate. All statistical analysis was performed using student ttest. * p-value<0.05, **p-value<0.01, ***p-value<0.001, ****p-value<0.0001.

Figure 2. Dysfuctional Tollip CUE domain increases necrotic cell population and decreases live cell population. Graphical data of WT and TmCUE Flow cytometry data. Cells were treated for either 0, 16, 24, or 48 hours with LPS (10ng/mL) treatment. The cells were stained with Annexin V and PI to analyze live cells, early apoptosis, late apoptosis, and necrotic cells populations. A) Live cells (Annexin V-/PI-) percentages of populations for WT verses T-/- MEF cells. B) Early apoptosis (Annexin V+/PI-) percentages of populations. C) Late apoptosis (AnnexinV+/PI+) percentages of populations. D) Necrotic cells (Annexin V-/PI+) percentages of populations. Experiments performed in triplicate. All statistical analysis was performed using student t-test. * p-value<0.05 **p-value<0.01 ***pvalue<0.001 ****p-value<0.0001.

Figure 3. Dysfuctional Tollip C2 domain increases necrotic cell population and decreases live cell population. Graphical data of WT and TmCUE Flow cytometry data. Cells were treated for either 0, 16, 24, or 48 hours with LPS (10ng/mL) treatment. The cells were stained with Annexin V and PI to analyze live cells, early apoptosis, late apoptosis, and necrotic cells populations. A) Live cells (Annexin V-/PI-) percentages of populations for WT verses T-/- MEF cells. B) Early apoptosis (Annexin V+/PI-) percentages of populations. C) Late apoptosis (AnnexinV+/PI+) percentages of populations. D) Necrotic cells (Annexin V-/PI+) percentages of populations. Experiments performed in triplicate. All statistical analysis was performed using student t-test. * p-value<0.05 **p-value<0.01 ***pvalue<0.001 ****p-value<0.0001.

Figure 4. Blocking late autophagy causes increased early apoptosis in WT and T-/- cells and necrosis in WT cells. Graphical representation of WT and T-/- Flow cytometry data. WT and T-/- MEF cells were treated with LPS (10ng/mL) and Baf (10nM) for 16 hours prior to cell harvesting for Annexin V and PI labeling. A) Live cells (Annexin V-/PI-) percentages of populations for WT verses T-/- MEF cells. B) Early apoptosis (Annexin V+/PI-) percentages of populations. C) Late apoptosis (AnnexinV+/PI+) percentages of populations. D) Necrotic cells (Annexin V-/PI+) percentages of populations. Experiments performed in triplicate. All statistical analysis was performed using student t-test. * pvalue<0.05 **p-value<0.01 ***p-value<0.001 ****p-value<0.0001

Figure 5. Chronic exposure to autophagy blocker causes increased late apoptosis and Necrosis in WT cells. Bafilomycin increases early apoptosis in Tollip deficient cells. Graphical representation of WT and T-/- Flow cytometry data. WT and T-/- MEF cells were treated with LPS (10ng/mL) and Baf (10nM) for 48 hours prior to cell harvesting for Annexin V and PI labeling. . A) Live cells (Annexin V-/PI-) percentages of populations for WT verses T-/- MEF cells. B) Early apoptosis (Annexin V+/PI-) percentages of populations. C) Late apoptosis (AnnexinV+/PI+) percentages of populations. D) Necrotic cells (Annexin V-/PI+) percentages of populations. Experiments performed in triplicate. All statistical analysis was performed using student t-test. * p-value<0.05 **p-value<0.01 ***pvalue<0.001 ****p-value<0.0001

Figure 6. Activated Macroautophagy promotes cells survival when paired with LPS stimulation, but macroautophagy activation alone causes increased apoptosis and necrosis. Graphical representation of WT Flow cytometry data. WT cells were treated for 16 hours with LPS (10ng/mL) and LiCl (10mM). The cells were stained with Annexin V and PI to analyze live cells, early apoptosis, late apoptosis, and necrotic cells populations. A) Live cells (Annexin V-/PI-) percentages of populations for WT. B) Early apoptosis (Annexin V+/PI-) percentages of populations. C) Late apoptosis (AnnexinV+/PI+) percentages of populations. D) Necrotic cells (Annexin V-/PI+) percentages of populations. Experiments performed in triplicate. All statistical analysis was performed using student t- test. * pvalue<0.05 **p-value<0.01 ***p-value<0.001 ****p-value<0.0001

Figure 7. Activated Macroautophagy decreases cell survival when chronically exposed to LPS, but macroautophagy activation alone causes decreased apoptosis and necrosis. Graphical representation of WT Flow cytometry data. WT cells were treated for 48 hours with LPS (10ng/mL) and LiCl (10mM). The cells were stained with Annexin V and PI to analyze live cells, early apoptosis, late apoptosis, and necrotic cells populations. A) Live cells (Annexin V-/PI-) percentages of populations for WT. B) Early apoptosis (Annexin V+/PI-) percentages of populations. C) Late apoptosis (AnnexinV+/PI+) percentages of populations. D) Necrotic cells (Annexin V-/PI+) percentages of populations. Experiments performed in triplicate. All statistical analysis was performed using student t-test. * pvalue<0.05 **p-value<0.01 ***p-value<0.001 ****p-value<0.0001

( A )

( B )

Figure 8. Tollip mediates apoptosis and survival through differing expression of key proteins. WT, T-/-, and TmCUE MEF cells were treated with LPS (10ng/mL) for amount of times indicated and the levels of proteins indicated and β-actin controls were determined by Western blot analyses. A) WT and T-/- cells were treated for 0, 16, 24, or 48 hours with LPS. B) WT and TmCUE cells were treated for 0, 16, 24, or 48 hours with LPS. Data is representative of three separate experiments.

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