Systemic Effects Downstream of NAIP/NLRC4 Inflammasome Activation in Vivo

Systemic Effects downstream of NAIP/NLRC4 Inflammasome Activation in vivo

Randilea Dawn Nichols

A dissertation submitted in partial satisfaction of the

requirements for the degree of

Doctor of Philosophy

Molecular and Cell Biology

in the

Graduate Division

of the

University of California, Berkeley

Committee in charge:

Professor Russell E. Vance, Chair Professor Gregory M. Barton Professor Ellen A. Robey Professor Karsten Gronert

Spring 2017

Systemic Effects downstream of NAIP/NLRC4 Inflammasome Activation in vivo

By Randilea Dawn Nichols

Abstract

Systemic Effects Downstream of NAIP/NLRC4 Inflammasome Activation in vivo

Randilea Dawn Nichols

Doctor of Philosophy in Molecular and Cell Biology

University of California, Berkeley

Professor Russell E. Vance, Chair

To provide the first line of defense against pathogenic microbes, the innate immune system detects infection using pattern recognition receptors, such as Toll-like receptors (TLRs) and cytosolic nucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) which form multi-protein complexes called inflammasomes. Once activated inflammasomes cause lytic cell death (termed pyroptosis) and secretion of interluken-1β (IL-1β) and IL-18. Most in vivo studies of the inflammasome have been conducted in the presence of TLR ligands; thus, it has been difficult to determine whether inflammasome activation is sufficient to induce immunity and inflammation in vivo. Moreover, the question of whether inflammasomes exhibit unique functions in distinct cell types has been greatly ignored. In this dissertation, I will begin with an overview of basic background on inflammasomes and then expand on human autoinflammatory conditions in patients with various inflammasome mutations. In chapter 2, I will describe a novel, knock-in mouse model, iOvaFla. When Cre recombinase is present, the iOvaFla fusion gene, which is the C-terminal 166 amino acids of Legionella pneumophila flagellin fused to ovalbumin, is expressed. The portion of flagellin expressed selectively activates a specific inflammasome called NAIP/NLRC4. NLRC4 activation in Lysozyme2+ cells (monocytes, macrophages, neutrophils via LysM-Cre) in vivo caused a severe systemic inflammatory disease, characterized by systemic neutrophilia, weight loss, and hind limb joint swelling. Disease was entirely NLRC4-dependent and was nearly fully ameliorated on a genetic background that leads to decreased cytokine production, suggesting a dominant role for cytokines and a minimal role for pyroptosis. Consistent with this postulation, neutrophil levels and disease symptoms decreased after therapeutic blockade of the IL-1 receptor. Interestingly, disease was recapitulated by NLRC4 activation selectively in neutrophils (using MRP8-Cre) but the same severe disease symptoms were not induced upon selective inflammasome activation in dendritic cells/tissue macrophages (using CD11c-Cre). Disease that arose after neutrophil- specific NLRC4 activation was similarly ameliorated by therapeutic anti-IL-1 receptor blockade. 1 In chapter 3, I present two more available iOvaFla mouse models: iOvaFla; ER- CreT2+/– and iOvaFla; Vil-Cre+/– mice. iOvaFla; ER-CreT2+/– mice are an inducible, ubiquitous way to activate endogenous NAIP/NLRC4 in any cell type. I developed a protocol to induce iOvaFla expression via tamoxifen without introducing contaminating TLR ligands. This will allow the mice to be used in experiments to study the specific roles of inflammasomes. Next, I describe the function of inflammasomes in intestinal epithelial cells with iOvaFla; Vil-Cre+/– mice. The mice are embryonic lethal. When I crossed them on a genetic background where pyroptosis still occurs but cytokine maturation decreases, the lethality was not rescued. Unlike the iOvaFla; LysM-Cre+/– phenotype caused by cytokines, intestinal epithelial cell pyroptosis appears to be the primary cause for lethality. Lastly in chapter 4, I conclude with a discussion on the lingering questions and future directions of how the iOvaFla mouse models could be used. There are several important mechanistic studies to complete to fully verify consequences of inflammasome activation in neutrophils. I also explore how iOvaFla; ER-CreT2+/– mice can be used to systematically uncover if inflammasomes are sufficient to stimulate the creation of adaptive immune responses. I conclude with a final reexamination of conclusions presented throughout my dissertation.

For

The two people who kept me going and to whom I will be eternally grateful

Bobby, my person,

and

Ashley, my sister.

i Table of Contents

Chapter 1: Innate Immunity and the Inflammasome 1.1 Quick summary of innate immunology 1 1.2 What are inflammasomes? 1 1.2.1 NAIP/NLRC4 inflammasome 1 1.2.2 NLRP1 inflammasome 3 1.2.3 NLRP3 inflammasome 3 1.2.4 AIM2 inflammasome 4 1.2.5 PYRIN inflammasome 4 1.2.6 Other potential inflammasomes 4 1.3 Downstream effects of inflammasome activation 5 1.3.1 IL-1β and IL-18 maturation 5 1.3.2 Pyroptosis and other cell death 6 1.3.3 Other effects 6 1.4 Inflammasomes in health and disease 7 1.4.1 NAIP/NLRC4 inflammasome 7 1.4.2 NLRP1 inflammasome 8 1.4.3 NLRP3 inflammasome 9 1.4.4 PYRIN inflammasome 9 1.4.5 Further links to human disease 10 1.5 Dissertation overview 10

Chapter 2: Selective in vivo activation of NAIP/NLRC4 in myeloid cells 2.1 Introduction 11 2.2 Development of ‘iOvaFla’ transgenic mouse model 13 2.3 Myeloid-specific inflammasome activation leads to systemic inflammation 15 2.4 Inflammasome-driven disease leads to myeloid cell hyperplasia 18 2.5 Increases in systemic cytokines in inflammasome-driven disease 21 2.6 ASC-deficiency relieves inflammatory disease 22 2.7 IL-1R signaling is necessary for inflammatory phenotype 23 2.8 Neutrophil specific inflammasome activation is sufficient to cause disease 25 2.9 Discussion 26

Chapter 3: Expression of iOvaFla transgenic protein using other Cre recombinase drivers 3.1 Introduction 30 3.2 Inducible, ubiquitous expression of iOvaFla using ER-CreT2 30

ii 3.3 Expression of iOvaFla in intestinal epithelium is embryonic lethal 34 3.4 ASC deficiency does not rescue iOvaFla: Vil-Cre+/? Phenotype 35 3.5 Discussion 37

Chapter 4: Conclusions and future directions 4.1 Future directions with iOvaFla and cell specific Cre drivers 39 4.1.1 iOvaFla; LysM-Cre and iOvaFla: MRP8-Cre mice 39 4.1.2 iOvaFla; CD11c-Cre 39 4.1.3 iOvaFla; Vil-Cre 40 4.2 Future directions with iOvaFla: ER-CreT2 mice 40 4.3 Final conclusions 41

Materials and Methods 43 References 46

iii List of Figures and Tables Figures Chapter 1: Innate Immunity and the Inflammasome 1.1 Schematic showing NAIP/NLRC4 oligomerization and activation 2

Chapter 2: Selective in vivo activation of NAIP/NLRC4 in myeloid cells 2.1 Genetic system for inducible NLRC4 inflammasome activation in vivo 12 2.2 Analysis of ‘iOvaFla’ transgenic mouse model 14 2.3 Myeloid-specific inflammasome activation leads to systemic 15 inflammation 2.4 Inflammation develops in the hard and soft tissues 16 2.5 Littermates show no inflammatory phenotype 17 2.6 Basic characterization of inflammasome driven inflammatory pathology 18 2.7 Inflammasome-driven disease leads to myeloid cell hyperplasia 19 2.8 Spleen and lymph node lymphocyte populations relatively unchanged 20 2.9 ANA not required for autoinflammation 21 2.10 iOvaFla: LysM-Cre+/– mice have increased levels of cytokines 22 2.11 ASC-deficiency relieves inflammatory disease 23 2.12 IL-1R signaling is necessary for inflammatory phenotype 24 2.13 iOvaFla+/–; CD11c-Cre+ mice do not develop joint swelling 25 2.14 Neutrophil specific inflammasome activation is sufficient to cause 27 disease 2.15 Final model 29

Chapter 3: Expression of iOvaFla transgenic protein using other Cre recombinase drivers 3.1 Extended tamoxifen treatment lethality in iOvaFla; ER-CreT2+/– mice is 31 NLRC4 dependent 3.2 Shorter tamoxifen treatment resolved lethality 32 3.3 Tamoxifen injection was contaminated with TLR ligands 32 3.4 Tamoxifen treatment by oral gavage was sufficient to activate the 33 inflammasome 3.5 Expression of iOvaFla in intestinal epithelium is embryonic lethal 35 3.6 ASC deficiency does not rescue iOvaFla: Vil-Cre+/? phenotype 37

iv Tables Chapter 2: Selective in vivo activation of NAIP/NLRC4 in myeloid cells 2.1 Averaged pathology scores 17 2.2 Individual pathology scores 18

Chapter 3: Expression of iOvaFla transgenic protein using other Cre recombinase drivers 3.1 Survival of iOvaFla; Vil-Cre mice 34 3.2 Survival of Asc–/–; iOvaFla; Vil-Cre mice 36

v Acknowledgments

First and foremost, thank you to my professor Russell Vance. You took a risk agreeing to let me into your lab when I really knew nothing about immunology or even bacterial pathogenesis. I’m not sure I’ll ever understand what you saw in me, but thank you. I have learned more from you then I probably even realize. You taught me to be more analytical, cautious, and thoughtful with my science, and how to truly read and analyze the literature properly. You may have furthered my pessimism if that’s even possible! Thank you for pushing me when I needed it and rescuing me when I was obviously drowning. Words really cannot describe how grateful I am to have been in your lab.

To the lab past and present, especially Dara Burdette Looney, Kevin Barry, Jeannette Tenthorey, Isabella Rauch, Patrick Mitchell, and Jakob von Moltke– thank you for being my friends, my adopted family, and more importantly my teachers. You taught me new techniques and furthered my understanding of biochemistry, genetics, bacterial pathogenesis, and of course immunology. Dara – thank you for being wonderful friend and letting JT and I annoy the hell out of you sometimes (like when we asked about playing music… Sorry!). Kevin – you guided me through the rollercoaster that is grad school all while keeping me laughing. Also I’m sorry you had to see me break my arm. Jeannette – I don’t know what I would have done without you, my lab wife. I’ve sat next to you for over five years, and I’m not sure how I’m going to science without you in our next steps. Bella – you taught me incredible amounts of immunology and methodology. You also picked up me when I needed it the most. Patrick ‘PandaOtterStarfish’ – thank you for the laughs, hugs, and cute animal videos. Jakob – Thank you for creating the iOvaFla mice but leaving them in my hands!

I thank you Greg Barton for being a science mentor not only during my rotation in you lab but still when I joined the Vance lab. You were always a source of vast wisdom and creative ideas and experiments. I must also thank your lab, especially Meghan Koch, for being such wonderful science siblings and boisterous Friday night drinking adventures. Meghan - As your roton, at first I felt slightly abandoned. What you really did was push me out of my comfort zone. I learned more in my rotation with you then I think I had in all of my other rotations combined. Thank you for handing me a protocol and saying just go do it. I will miss your scientific thoughtfulness and earnest friendship. I cannot wait to read all the papers coming from your soon-to-be lab.

There is still quite a list of scientists that have helped me along my way. I wouldn’t say lab was boring before Heran Darwin joined us for a few months, but things were never the same after she left. Thank you Heran for being such a unique person and mentor. You never held back, which sometimes I definitely needed to hear it. However, my liver is glad you went back to NYC. Sarah Stanley, you may not it, but you were inspiration. I looked up to you not only as a superb scientist but also the way you managed your life and lab. Next I must thank two more fantastic professors I was lucky to have as thesis committee members: Ellen Robey and Karsten Gronert. Your guidance during my

vi committee meetings was invaluable to me being able to complete my Ph.D. in somewhat timely way . Thank you to colloborators like Ann Marshank-Rothstein and Yasmine Belkaid for providing valuable reagents along the way.

Life needs some fun along the way and my MCB 2011 classmates were full of it, like James Nunez, Stefanie Monica, and others. Thanks for having my back and staying close friends. Sometimes you meet people that you instantly connect with and become fast friends with, and that was you Betsy Murdock. We’re going on 10 years of friendship, which has had its share of ups and downs. You’ve really been there for me when things have gotten so dark I wasn’t sure if light even existed anymore. You have been a wonderful sounding board through the years no matter the distance.

When science and lab got too much, I was able to retreat to my sanctuary at The Bar Method Berkeley. Helen Liu, you created my home away from lab. It’s not often someone probably says they count their boss as a valuable friend, but that’s what you are to me. Your guidance, support, and humor have been exceptionally inspiring. Thank you to all the other teachers, front desk, and the clients who have been such a welcoming community. I lived for my Thursday night tacos and Hotsy Totsy with Helen, Yvonne Young, Meghan Fabian, Beth Ellis-Dickson, Shana Rehm, Alicia Mattera, and the others. I cannot and do not want think about how heartbreaking it is going to be leaving you, but I am so very grateful to have gotten to know each of you.

I would not even be here if it wasn’t for my sister Ashley Sewell. From the very beginning, well more like after you went to college, you have been my best friend and biggest supporter. It was never easy being the younger sister to you, since you are so very intelligent and well loved by all. However, it has been an honor trying to follow in and live up to your footsteps. No matter what is going on in my life you were always my first or second phone call. I don’t think I’ve ever really had a secret from you. I love you immeasurably!

I saved the best for last; that’s you Bobby Doyle. The distance was unbearable at times, but you were with me every step of the way even when quals broke me and I broke my own self/arm. You calmed me and talked me through intense anxiety periods and reminded me I was good enough when I didn’t believe in myself anymore. Thank you for being my adventure buddy, my go-to, my love, and my person. I cannot wait for our next adventure of finally being together!

vii

Chapter 1: Innate Immunity and the Inflammasome

1.1 Introduction to innate Immunity The immune system is broadly divided into two branches: innate and adaptive. Innate immunity provides a fast, first line of defense to a pathogen, whereas adaptive immunity is a slower but more specific response that results in immunological memory to a pathogen. To provide this first line of defense, the innate immune system detects infection using pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and cytosolic nucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) which form multi-protein complexes called inflammasomes1, 2. As their name implies, PRRs respond microbial “patterns” by recognizing conserved ligands. These ligands vary greatly in their makeup: nucleic acids, peptides, lipids, etc. Much of innate immunology research has3 focused on determining ligand specificity for the various PRRs and the molecular mechanism by which the PRRs sense their ligands. In addition, some work has been done looking at the bigger picture of whether and how activation of innate immune responses trigger pathogen-specific adaptive immune responses. To date, most of the studies addressing this latter question have focused on TLR-dependent stimulation of adaptive immune responses.

1.2 What are inflammasomes? There are several different inflammasomes, each of which is responsive to unique ligands or stimuli4, 5. Upon pathogen recognition, certain NLRs form cytosolic multi-protein complexes called inflammasomes that function to activate a downstream protease called CASPASE-1 (CASP1)4, 5. CASP1 initiates inflammation by processing IL-1β and IL-18 into their active forms (Figure 1.1). Active CASP1 also cleaves and activates a pore-forming protein called Gasdermin D (GSDMD)6, 7, leading to cytokine release and a lytic cell death called pyroptosis8, 9. Inflammasomes are important in the control of many bacterial pathogens such as Salmonella enterica serovar Typhimurium, Legionella pneumophila, Francisella tularensis, Shigella flexnerii, Pseudomonas aeruginosa, Burkholderia thailandensis, Chromobacterium violaceum, and Yersinia pseudotuberculosis, to name just a few10, 11. Inflammasome responses are also important for responses to viruses. For example, the AIM2 inflammasome is important for the production of a successful response to the DNA viruses murine cytomegalovirus and vaccinia virus11. Conversely, mutations in inflammasome genes have been linked to various autoinflammatory diseases (which will be discussed in chapter 1.4).

1.2.1 NAIP/NLRC4 inflammasomes Activation of the NLRC4 (NLR family, CARD domain containing protein 4) inflammasome requires NAIP proteins (NLR family, apoptosis inhibitory proteins). NAIPs bind to specific bacterial ligands then co-assemble with NLRC412, 13. There are

four NAIP proteins expressed in wild-type C57Bl/6 mice (i.e., NAIP1, NAIP2, NAIP5, and NAIP6) and one NAIP protein in humans. Mouse NAIP1 and NAIP2 recognize the needle protein and inner rod protein of bacterial type III secretion systems (T3SS), respectively. Both NAIP5 and NAIP6 recognize bacterial flagellin10, 14. The ligand for human NAIP has been controversial. One group reported it only recognizes T3SS needle protein, while another group published a splice variant also recognize flagellin14, 15. Both published and unpublished work from the lab has shown NAIP5 detects the C- terminus of flagellin16. Importantly, another innate immune flagellin sensor, TLR5, recognizes different portions of flagellin16. NLRC4 contains a CARD domain, allowing it to directly bind to CASP1. However, NLRC4 also recruits CASP1 via the adaptor protein ASC (Figure 1.1; apoptosis-associated speck-like protein containing a CARD). When ASC is absent, CASP1 does not undergo autoproteolysis, resulting in greatly reduced cleavage of IL-1β and IL-18 into their mature forms (Figure 1.1). Macrophages lacking ASC have been shown to still initiate NLRC4-induced pyroptosis17, 18.

Figure 1.1 Schematic showing NAIP/NLRC4 oligomerization and activation. A phagocytosed bacterium secretes flagellin into a cell’s cytosol through the bacterium’s Type III or IV Secretion Systems (T3SS or T4SS). NAIP5 recognizes flagellin then oligomerizes with 9- 10 NLRC4 monomers. The NAIP/NLRC4 inflammasome can interact with CASP1 and initiate pyroptosis even when ASC is absent. However, ASC is essential for the cleavage of IL-1β and IL-18 into their mature forms.

1.2.2 NLRP1 inflammasome There are a few notable differences between human and murine NLRP1 (NLR family, pyrin domain containing protein 1). Humans have one known gene for NLRP1; whereas, mice have three different genes (Nlrp1a, Nlrp1b, and Nlrp1c) and at least five Nlrp1b alleles. Both human and mouse NLRP1 contain the NBD, LRR, and CARD domains as well as a FIIND domain (‘function-to-find’), which must undergo autoproteolysis for functional NLRP110, 19. Unlike murine NLRP1a-c, human NLRP1 also contains a N-terminal pyrin (PYD) domain. The best characterized NLRP1 is murine NLRP1B. The BALB/129 allele of Nlrp1b is activated by Bacillus anthracis lethal toxin (LeTx). LeTx is a AB toxin containing two proteins: protective antigen (PA) which forms a channel in cell membranes lethal factor (LF) translocates through. Once in the cytosol LF inactivates MAP kinase kinases (MAPKKs) through cleavage. LF also cleaves at the N-terminus of certain NLRP1B alleles10, 19. This cleavage is sufficient to activate the inflammasome. Activators of the other NLRP1B alleles and NLRP1A and NLRP1C are still unknown. Some work indicated that NLRP1B may respond to Toxoplasma gondii19. However, results were mixed once in vivo experiments were performed. If NLRP1b is activated during T. gondii infections, the response is secondary to other activated inflammasomes, likely NLRP3. The mechanism of human NLRP1 activation is still unknown. Thus far, human NLRP1 has not been shown to be activated by LF. In experiments using a cell-free system, a portion of bacterial peptidoglycan, muramyl dipeptide (MDP), was able to induce oligomerization of NRLP1. Oligomerization correlates with CASP1 activation. However, this work has been hard to confirm genetically in cells due to the presence of other punitive MDP sensors19.

1.2.3 NLRP3 inflammasome NLRP3 (NLR family, pyrin domain containing protein 3) is perhaps the least mechanistically understood of the inflammasomes. Both humans and mice have NLRP3, and thus far it has not been found to be constitutively expressed like the NAIPs, NLRC4, or NLRP1. Expression is usually achieved downstream of a TLR stimulation. Because NLRP3 does not have a CARD domain, ASC is required for NLRP3 to recruit CASP1. What makes NLRP3 stand apart from other inflammasomes is its wide range of stimulants, some of which include: crystals particulate matter (uric acid crystals, alum), extracellular ATP through the P2X7 receptor, nigericin, UVB irradiation, viral and fungal products, and bacterial pore-forming toxins4, 10. Models have been proposed in attempt to find a commonality between the known stimuli. Two such examples are that all NLRP3 stimuli converge on potassium efflux or the exposure of cardiolipin from the inner membrane of mitochondria20, 21. Yet a single unifying theory has yet to be proven. Most proposed mechanisms, including the two mentioned above, do not explain all known stimuli4. Thus, much work still needs to be done to fully understand the mechanism of NLRP3 activation.

NLRP3 plays an important role downstream noncanonical inflammasome activation. After lipopolysaccharide (LPS; portion of Gram-negative bacterial cell wall) binding, murine CASPASE-11 (Caspase-4 and Caspase-5 in humans) activates GSDMD leading to pyroptosis11, 22. Before pyroptosis occurs, NLRP3 is activated causing inflammasome formation, CASP1 activation, and cytokine cleavage. Though the mechanism still needs more research, it is thought NLRP3 activation occurs after potassium efflux through the GSDMD membrane pores22.

1.2.4 AIM2 inflammasome The AIM2 inflammasome is unique because AIM2 is not a NLR family protein. Instead it belongs to a family of IFN-inducible PYHIN proteins. AIM2 has a pyrin and HIN-200 DNA-binding domains instead of NBD and LRR domains10. Though structural different than other NLRs, AIM2 still oligomerizes into an inflammasome complex with CASP1 and ASC10. AIM2 responds to cytosolic double-stranded DNA from both bacteria (Francisella tularensis) and viruses (vaccinia)4.

1.2.5 PYRIN inflammasome Like AIM2, PYRIN is also not a NLR protein. It contains a PYD domain, two B- boxes, and a coiled-coil domain. Human PYRIN contains a C-terminal B30.2 domain that mouse PYRIN does not. PYRIN is activated by the loss of negative regulation. Some bacterial have toxins which inactivate the GTPases RhoA11. During homeostasis, RhoA activates the kinases PKN1 and PKN2 which phosphorylate PYRIN. The 14-3-3 proteins bind to phosphorylated PYRIN inhibiting it23. Some hypothesize PYRIN may play a role in negatively regulating other inflammasomes. One report found human PYRIN targets NLRP1, NLRP3, and CASP1 for autophagic degradation. The B30.2 domain is linked to cargo recognition. Mutations in the B30.2 domain that result in reduced cargo recognition and degradation have been linked to patients with autoinflammation (will be further discussed in chapter 1.4)11.

1.2.6 Other potential inflammasomes There have been other NLR and non-NLR proteins reported as inflammasomes: NLRP6 (NLR family, pyrin domain containing protein 6), NLRP12 (NLR family, pyrin domain containing protein 12), IFI16 (IFN–inducible protein 16). The evidence that these proteins are implicated in inflammasome activation has been the observation of inflammasome-dependent cytokine production and pyroptosis via CASP1 activation11. However, the biochemical studies actually showing large multi-protein oligomers is lacking. Research around both NLRP6 and NLRP12 has been contradictory at times. NLRP6 was first published as a potential intestinal microbiota sensor. Nlrp6–/– deficient mice developed dysbiotic microbiota and were more susceptible to colitis24. Taurine was proposed as a ligand while spermine and histamine as negative regulators25. Conversely, another group found that NLRP6 does not sense bacteria but instead

senses viruses leading to a Type I and III Interferon (IFN) response26. More work needs to be done to clarify between these different published functions for NLRP6. NLRP12 was first shown to inhibit noncanonical NF-κB signaling, including during colorectal cancer and also intrinsic NF-κB signaling in T cells during activation27, 28, 29, 30, 31. Later work demonstrated NLRP12 was important for CASP1 to responded to Yersinia pestis and Plasmodium infections22. Recent work. How NLRP12 can play a role in such diverse, and seemingly contradictory functions has yet to be determined. Human IFI16 has been linked to viral recognition. First published back in 2011, IFI16 interacted with ASC and required for CASP1 activation in cells infected with Kaposi's sarcoma-associated virus32. IFI16 was linked multiple times to the recognition of Herpes simplex virus 133, 34, 35, 36, 37. Then in 2014 two paper published IFI16 recognizes incomplete HIV transcripts in ‘bystander’ CD4+ T cells38, 39. This recognition led to CASP1 dependent cell death. The authors hypothesize this is the reason for the decrease in CD4+ T Cells and the development of AIDs38, 39. Since IFI16 is also reported to regulate IFN production, which could indirectly affect CASP1 activation, more studies are needed to prove IFI16 is actually an inflammasome.

1.3 Downstream effects of inflammasome activation There are two major and immediate effects of inflammasome activation: cleavage of IL-1β and IL-18 into their mature forms and pyroptosis. These are ‘small-picture’ immediate effects, but might possibly dictate the bigger roles inflammasomes play in inflammation and immunity. It has previously been proposed that pyroptotic death is a ‘pro-inflammatory’ form of cell death that is associated with the release of inflammatory mediators8. However, the intrinsic inflammatory effects of pyroptosis have been difficult to dissociate from the co-incident release of pro-inflammatory cytokines such as IL- 1β and IL-18.

1.3.1 IL-1β and IL-18 maturation CASP1 cleaves pro-IL-1β and pro-IL-18 into what is considered their mature and active forms. Data indicate pro-IL-18 is constitutively made in quite a few cell types, including non-hematopoietic cells like intestinal epithelial cells. On the other hand, pro- IL-1β is not made as abundantly. Most cells need a ‘priming’ signal, such as a TLR agonist, for pro-IL-1β to be produced40. An intestinal macrophage population has been described to already have pro-IL-1β available41. Data on ImmGen data browser indicates that neutrophils may also express pro-IL-1β42. The mechanism of release after IL-1β and IL-18 are cleaved by CASP1 is highly controversial. Both cytokines lack signal sequences, and their release is independent of both the endoplasmic reticulum and Golgi43. Here are some of the major competing theories of release: 1) release after pyroptosis of the cell9, 44; 2) exocytosis of secretory lysosomes containing IL-1β/-1845; 3) the shedding of microvesicles or exosomes46, 47; 4) secretory autophagy48. All of these theories have evidence both for and against them, including passive release via

pyroptosis. Chen et al demonstrated in 2014 that neutrophils do not appear to undergo pyroptosis yet release active cytokines49. Once released, IL-1β and IL-18 have many roles in inflammation. IL-1β signals through the IL-1R and IL-18 through the IL-18R. Both receptors use the MYD88 signaling pathway to activate canonical NF-κB signaling. NF-κB activation leads to the transcription of hundreds of inflammatory proteins, such as IL-6, IL-12 tumor necrosis factor alpha (TNFα), IFN-gamma (IFNγ), many chemokines, and also pro-IL-1β and pro- IL-18 themselves. IL-1β is a classic pyrogen that induces fever, and activates the hypothalamus-pituitary-adrenal axis to increase cortisol. Signaling via the IL-1R on endothelial cells is important for neutrophil migration to sites of infection and inflammation40. In adaptive immunity, IL-1 is important for development of Th17 T cells and is also important to create a successful adaptive immune response to Influenza infection in mice1, 40. When discovered, IL-18 was first named ‘interferon-γ (IFN-γ)- inducing factor.’ IL-18 stimulates polarized Th1 cell and natural killer (NK) cell responses to produce IFNγ. IL-18 is also crucial for tissue repair, especially to repair the intestinal epithelial barrier 40.

1.3.2 Pyroptosis and other cell death Pyroptosis plays an important role in responding to intracellular pathogens. For bacteria like L. pneumophila or S. typhimurium, pyroptosis is crucial for removing the intracellular niche. Without the availability of this niche, the bacteria cannot replicate as well, and neutrophils are able to find and kill the bacteria8. Pyroptosis occurs after inflammasome activation of murine CASPASE-1/-11 or human Caspase-1/-4/-5. Activated caspase cleaves GSDMD into the N-terminal (GSDMD-N) active and C- terminal (GSDMD-C) autoinhibitory portions6, 11. GSDMD-N localizes to the cell membrane then forms a multimeric pore complex that is 10-16nm wide. Mature (cleaved) IL-1β and IL-18 are able to fit through this pore, consistent with the passive release through cell death theory50. How GSDMD-N forms this pore is still unknown. Many have reported that CAPASE-8 can be recruited into inflammasomes via ASC. CASP8 activation leads to sterile death, apoptosis, instead of pyroptosis. If the apoptotic cells are not cleared in a reasonable time, secondary necrosis can occur. CASP8 has also been shown to have the ability to cleave IL-1β into its mature form51, 52. Most studies have been down in macrophages. Whether or not pyroptosis occurs after inflammasome activation in other cell types is still somewhat unknown. Intestinal epithelial cells, dendritic cells, enterocytes, and some hematopoietic progenitors have all been demonstrated to undergo apoptosis; whereas, monocytes and neutrophils have been shown not to under normal circumstances22, 49, 53, 54.

1.3.3 Other effects Most work understanding the inflammasome’s effects have focused on cytokines or pyroptosis. However, there are likely many effects yet unknown or understudied. One

such example is eicosanoids, inflammatory signaling lipids. Our group was the first to link inflammasome production to eicosanoid production in 201255. The rapid death following FlaTox injection was IL-1β/-18 and pyroptosis dependent. Instead, eicosanoids induced a large and rapid vascular leakage and massive fluid loss. The eicosanoids were produced by a specific cell population, peritoneal tissue macrophages53, 55. One paper published in 2006, linked inflammasome activation to lipid synthesis and membrane biogenesis through activation of sterol regulatory element binding proteins56. Autophagy is a mechanism for cells to remove damaged organelles and recycle cellular metabolites. Through different published mechanisms, autophagy can inhibit inflammasome activation. Cells deficient in autophagosome components have increased inflammasome activation. NLRP3 and NLRC4 both interact with beclin-1 an important autophagy regulator. In addition, NLRC4 inhibits autophagosome maturation through the interaction with class C vacuolar protein-sorting complex. CASP1 too inhibits autophagy after inflammasome activation22.

1.4 Inflammasomes in health and disease A proper inflammasome response can be essential to responding successfully and quickly to various microbial infections. Conversely, an aberrant response is potentially lethal. Many apparent gain-of-function mutations in inflammasome components have been found to correlate with inflammatory disease. Inflammatory disorders have also been linked to disordered IL-1 signaling. Though some IL-1 signaling can occur independently of inflammasomes, it is still important to consider the role inflammasomes play in IL-1 autoinflammation. Most in vivo studies of the inflammasome have been conducted in the presence of TLR ligands; thus, it has been difficult to determine whether inflammasome activation is sufficient to induce immunity and inflammation in vivo. Moreover, the question of whether inflammasomes exhibit unique functions in distinct cell types has been greatly ignored.

1.4.1 NAIP/NLRC4 inflammasome Inappropriate NLRC4 inflammasome activation can result in pathology, or even death, in both mice and humans55, 57, 58, 59, 60, 61. However, the mechanisms by which chronic NLRC4 activation causes pathology remain poorly understood. For example, it is not clear whether IL-1β, IL-18 and/or pyroptotic cell death drive NLRC4-induced disease in vivo. In addition, given that NLRC4 is functional in multiple cell types, including hematopoietic and intestinal epithelial cells, it remains unclear in which cell types NLRC4 activation can drive pathology. A major limitation has been the lack of mouse models that recapitulate NLRC4-driven autoinflammatory disease and/or permit tissue specific activation of endogenous NLRC4 in vivo. There have been three different NLRC4 mutations found in humans suspected be gain-of-function and the cause of the patient’s chronic disease. Two groups published simultaneous articles in 2014 reported two different mutations, T337S and V341A. Though different mutations, the patients had

nearly identical disorders: periodic fevers, enterocolitis, skin rashes, splenomegaly, and other autoinflammatory symptoms59, 60. One patient, when treated with an IL-1R antagonist, anakinra, showed a substantial decrease in symptoms, though her serum IL-18 levels stayed elevated. Anakinra was so effective, the patient was actually able to be taken off corticosteroids and colchicine60. By contrast, a recently reported patient with chronic autoinflammation and the V341A NLRC4 mutation, did not respond well to anakinra alone. Once the patient was given recombinant human IL-18 Binding Protein (IL-18BP), a negative regulator of IL-18, in conjunction with anakinra, she improved61. It is interesting that one patient responded well to anakinra while still having increased IL- 18, while another patient needed both cytokines targeted to improve clinical symptoms. Patients with familial cold autoinflammatory syndrome (FCAS) have been previously identified to have mutations in NLRP3. Kitamura et al.58 reported a Japanese family with FCAS had a missense mutation (H443P) in NLRC4 instead. Though the H443P mutation affects a similar part of the NLRC4 protein as the other mutations listed above, patients with H443P have very different symptoms. When exposed to cold stimuli, patients develop fever, skin rash, and arthritis. Kitamura et al. created transgenic mice overexpressing the H443P NLRC4 variant. Even without the cold stimuli the mice develop a mild autoinflammatory disease. However, since these transgenic mice express a mutant NLRC4 allele under the control of a non-native (invariant chain) promoter along with WT NLRC4, it is not clear whether chronic activation of endogenous NLRC4 might also produce disease, and if so, in which cell types NLRC4 activation drives disease58.

1.4.2 NLRP1 inflammasome There have been quite a few single nucleotide polymorphisms (SNPs) in the Nlrp1 gene associated with human autoimmune or autoinflammatory diseases, such as vitiligo, rheumatoid arthritis, systemic sclerosis, Crohn's disease, and melanoma. Some are in the non-coding regions and could affect transcription. However, this has not been well worked out and needs more evaluation. Some of the mutations found in the protein coding sequence have been experimentally validated. One example is M1184V which has increased FIIND processing and induces more IL-1β. These data come from over- expression in fibroblasts. Using a forward genetic screen Masters et al.62 found a mouse harboring a gain- of-function (Q593P) mutation in Nlrp1a. LPS primed macrophages from the mice secreted IL-1β without any second stimulus necessary. Q953P homozygous mice died within 3 - 5 months of age. The mice had increased neutrophils up to 15 times higher than WT mice and splenomegaly. Even though the mice had increased neutrophils, they also have overall decreased numbers of lymphocytes. The lethal disease was rescued by crossing the mice onto Il-1R–/– or Casp-1/-11–/– backgrounds. However, even on the Il-1R–/– background, pyroptosis of hematopoietic progenitor cells continued, causing chronic leukopenia62.

1.4.3 NLRP3 inflammasome NLRP3 was the first inflammasome to be linked with autoinflammatory diseases in humans called Cryopyrin-Associated Periodic Syndrome (CAPS). Three of the CAPS disorders are FCAS, Muckle-Wells syndrome (MWS), and neonatal onset multisystem inflammatory disease (NOMID). FCAS is the mildest of the three, and NOMID is the most severe. There have been over 182 annotated NLRP3 mutations found to be associated with human diseases63. Inappropriate NLRP3 activation has also been associated with is implicated in arthritis, gout, diabetes, obesity, and Alzheimer’s disease22, 64. Brydges et al. created mice with gain-of-function NLRP3 mutations (A350V or L351P)64, 65. The A350V mutation has been found in MWS patients and the L351P in FCAS patients. Mice expressing these NLRP3 mutant alleles specifically in Lysozyme2+ (hereafter referred to as LysM+; monocytes, macrophages, neutrophils, and select dendritic cell populations3, 66) cells exhibit significant autoinflammation. The mutation associated with FCAS unexpectedly caused the most severe phenotype in mice, resulting in neonatal lethality associated with systemic IL-1β/-18-dependent inflammation. The MWS mutation (A350V) mice still showed an expected chronic, autoinflammatory disease64, 65. In comparison to the NLRC4 H443P mice, an advantage of the NLRP1A and NLRP3 studies is that the gain-of-function mutations were introduced into the endogenous genetic loci. However, since LysM is expressed in monocytes, macrophages and neutrophils, it remains unclear which specific cell types are primarily responsible for driving inflammasome-initiated pathology in vivo. In addition, because the experiments relied on expression of a mutant gain-of-function protein, it is difficult to know whether disease would arise similarly upon chronic activation of wild-type inflammasomes, or whether perhaps wild-type inflammasomes would be subject to homeostatic feedback regulation that is circumvented by the gain- of-function mutation.

1.4.4 PYRIN inflammasome PYRIN is encoded for by the gene MEFV which stands for Mediterranean fever. It was name thus because the gene was first linked to familial Mediterranean fever (FMF). FMF patients experience episodes of fever, sterile peritonitis, pleural inflammation, joint inflammation, and sometimes rash lasting between one to three days67. Most patients are homozygous (though sometimes two different mutations on separate alleles) for mutant PYRIN, but there a few cases of autosomal dominant mutations (only need one allele for disease). FMF is highly penetrant disease with over 150,000 affected patients. Most of the 310 reported mutations fall within the B30.2 domain63. One study reported a mutation at Ser242 resulting in the loss of phosphorylation and thus a 14-3-3 binding motif. Instead of FMF, the patient had a different autoinflammatory disease with neutrophilic dermatosis11.

1.4.5 Further links to human disease IL-1 is vital to the development of various auto-inflammatory human diseases, including inflammasome driven auto-inflammatory disorders. Deficiency of IL-1 receptor antagonist (DIRA) patients have systemic inflammation, including joint swelling and skin lesions. Lesion biopsies have shown excessive neutrophilia68. Both rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (sJIA) patients have increased serum IL-1 and joint neutrophil infiltration69, 70. In a mouse model of RA, neutrophil derived IL-1 is necessary for arthritis to develop70. Anemia, leukocytosis, and arthritis are decreased in sJIA patients treated with anakinra69.

1.5 Dissertation overview In this first chapter, I have introduced relevant background information for my project. I also discussed some of the areas of research lacking in the field to set the stage for what my dissertation research aimed to address. In the next chapter, I introduce the ‘iOvaFla’ mice that will be used throughout my dissertation to characterize inflammasome activation in select sets of cells. Chapter two focuses on the consequences of activating endogenous NLRC4 specifically in LysM+ cells, CD11c+ cells (dendritic cells and tissue macrophages), and even more specifically MRP8+ cells (neutrophils and some myeloid progenitors). In chapter three, I will expand on two more engineered iOvaFla mouse models. The first is iOvaFla to ER-CreT2+/–, which allow for an inducible, ubiquitous way to activate endogenous NAIP/NLRC4. I will present a protocol to induce iOvaFla expression with tamoxifen without stimulating excess PRRs, such as TLRs. Lastly, I explore the role of inflammasomes in intestinal epithelia cells by creating iOvaFla; Vil- Cre+/– mice. iOvaFla Vil-Cre+/– mice die within hours after birth, and I will present the work I undertook to establish cause of death. Lastly, I will conclude by summarizing my research and also discussing how my ‘iOvaFla’ mouse system can be utilized in the future. To better understand the autoinflammatory phenotype of iOvaFla+/–; LysM-Cre+/– mice, more detailed experiments are needed to address if pyroptosis occurs in neutrophils. Controls and more experiments are needed to finish up characterizing lethality in creating iOvaFla; Vil-Cre+/– mice. Lastly, the iOvaFla to ER-CreT2+/– open up a wide breadth of new experimental possibilities. Those mice will allow more careful, elegant work to characterize what, if any, downstream adaptive immune responses are stimulated after NAIP/NLRC4 activation.

Chapter 2: Selective in vivo activation of NAIP/NLRC4 in myeloid cells

2.1 Introduction Most studies have primarily used bacterial infection to activate the endogenous NAIP5/NLRC4 inflammasome [reviewed in10, 71]. Since bacterial infections typically activate multiple PRRs, including TLRs, it has been difficult to separate the effects of NAIP/NLRC4 activation from the downstream pro-inflammatory effects of TLR activation. Indeed, it is not clear whether activation of endogenous NLRC4 alone would be sufficient to drive inflammation in vivo. For example, induction of IL-1β is generally thought to require ‘priming’ signals to induce expression of pro-IL-1β prior to its processing by CASP1 downstream of NAIP/NLRC440, 72, 73. As one approach to elucidate the priming-independent effects of NAIP/NLRC4 activation in vivo, we and others have previously selectively activated NLRC4 using ‘FlaTox’, a recombinant flagellin fusion protein that enters cells and activates NLRC413, 55, 74. Experiments with FlaTox demonstrated that NAIP/NLRC4 activation in vivo can cause significant pathology in the absence of ‘priming’ or inflammasome-inducible IL- 1β/-18 cytokines55. However, FlaTox has acute lethal effects that make it difficult to model the chronic effects of inappropriate NLRC4 activation such as those observed in human NLRC4 gain-of-function patients. Moreover, FlaTox activates most cells that endogenously express NLRC4, so it cannot be used to assess the cell type-specific effects of NLRC4 activation. Furthermore, there is still the question of whether inflammasomes exhibit unique functions in distinct cell types has been greatly ignored. This has meant when humans with autoactivating NLR mutations have been discovered, macrophages are assumed to cell type driving disease60. However, thorough work has not been done to actually verify this. My thesis is focused on looking at both the role of the NAIP/NLRC4 inflammasome in various cell types as well as the broader, ‘big picture’ function of inflammasomes after activation. Before graduating, a previous graduate student in the lab, Jakob von Moltke, generated a genetically targeted ‘iOvaFla’ mouse that expresses, under Cre-inducible control, a gene encoding chicken ovalbumin (Ova) (lacking its signal sequence) fused to the C-terminal 166 amino acids of Legionella pneumophila flagellin (Fla). Ova was included to permit the eventual tracking of adaptive immune responses in these mice, and I will discuss those studies in Chapter 4. The gene encoding the iOvaFla fusion protein was inserted into the ubiquitously expressed Rosa26 locus downstream of a loxP-flanked transcriptional STOP cassette75 to prevent iOvaFla expression until Cre recombinase is expressed (Figure 2.1A). An IRES-GFP reporter was also inserted downstream of the iOvaFla gene fusion to allow us to visualize cells expressing iOvaFla (Figure 2.1A). This mouse model will be used throughout the entirety of my thesis. It allowed me the opportunity to study in vivo inflammasome effects and importantly, I was able to selectively activate the inflammasome by using different Cre recombinase mouse models to express iOvaFla specifically in different tissues.

Figure 1. Genetic system for inducible NLRC4 inflammasome activation in vivo. (A) Schematic showing iOvaFla transgene insertion in the Rosa26 locus. (B) Flow cytometry analysis of bone marrow derived macrophages (BMMs) cultured from WT, Nlrc4–/–; iOvaFla, and Nlrc4–/–; iOvaFla+/–; LysM-Cre+/– mice. (C) Retroviral transduction of B6, iOvaFla+/+, and Nlrc4–/–; iOvaFla+/+ BMMs with either an empty MSCV vector, MSCV vector containing only GFP, or MSCV vector expressing Cre Recombinase.

First to test for proper production of the iOvaFla-IRES-GFP construct, I crossed the iOvaFla mice onto a Nlrc4–/– background (to prevent any pyroptosis) and to Lysozyme2-Cre (called LysM-Cre from here on) transgenic mice to induce iOvaFla- IRES-GFP expression in myeloid cells. Bone marrow derived macrophages (BMMs) were differentiated from wild-type B6, Nlrc4–/–; iOvaFla, and Nlrc4–/–; iOvaFla; LysM- Cre+/– mice. Approximately 44% of Nlrc4–/–; iOvaFla+/–; LysM-Cre+/– BMMs were GFP+, compared to ≤1% of B6 or Nlrc4–/–; iOvaFla+/+ BMMs (Figure 2.1B). There should not

have been GFP expression in the Nlrc4–/–; iOvaFla+/+ BMMs since they had no Cre recombinase present. As further confirmation of Cre-induced iOvaFla-IRES-GFP expression, I transduced B6, iOvaFla, and Nlrc4–/–; iOvaFla BMMs with either an empty retroviral vector, a GFP expression vector as a transduction efficiency control, or a vector expressing only Cre recombinase (Figure 2.1C). As expected, BMMs transduced with empty vector exhibited minimal GFP induction, whereas BMMs transduced with the control GFP vector all exhibited a significant population (27%-33%) of GFP+ cells. Nlrc4–/–; iOvaFla BMMs transduced with the Cre recombinase vector also exhibited a robust GFP+ population (~27%) whereas <5% of (Nlrc4+;) iOvaFla macrophages were GFP+ (Figure 2.1C). The relative lack of GFP expression specifically in NLRC4 inflammasome-competent BMMs is due to loss of GFP expressing cells as a result of NLRC4-induced pyroptotic cell death, as the Vance lab has previously published16.

2.2 in vivo analysis of ‘iOvaFla’ transgenic mouse model I also assessed GFP expression in ex vivo isolated resident peritoneal macrophages from iOvaFla mice. Though NLRC4 inflammasome-competent iOvaFla; LysM-Cre+/– macrophages expressed GFP at a higher median fluorescence intensity (MFI) as compared to their Cre-negative littermates, expression levels were lower when compared to Nlrc4–/–; iOvaFla; LysM-Cre+/– mice (Figure 2.2A, B). The difference in median GFP MFI levels between NLRC4 inflammasome-competent and Nlrc4–/– macrophages is consistent with the expected loss of GFP+ macrophages via pyroptosis after NLRC4 inflammasome activation by iOvaFla55. Unlike macrophages, it has been controversial whether neutrophils undergo pyroptosis upon NLRC4 activation49, 54. Salmonella enterica serovar Typhimurium infected BM neutrophils and ex vivo peritoneal neutrophils release NLRC4-dependent mature IL-1β but were reported not to undergo pyroptosis as measured by lactate dehydrogenase (LDH) release49. Ryu et al.54 reported pyroptosis of lung neutrophils, but only in the absence of NADPH oxidase 2. I therefore sought to examine GFP levels in bone marrow, splenic, and lymph node neutrophils from our iOvaFla mice as an indirect but in vivo assay for pyroptosis. The auto-fluorescence of neutrophils made definitive conclusions difficult. Nevertheless, in all tissues, Nlrc4–/–; iOvaFla; LysM-Cre+/– neutrophils consistently had the highest GFP MFI, whereas Cre-negative littermates had the lowest apparent GFP MFI (green fluorescence in Cre-negative mice is presumably due to background auto-fluorescence) (Figure 2.2C, D). NLRC4-dependent loss of GFP was only significant in neutrophils from spleen and lymph nodes and not in bone marrow neutrophils. One possible explanation for these data is that splenic or lymph node neutrophils, not bone marrow neutrophils, undergo NLRC4-dependent pyroptosis in vivo. However, because measurement of GFP MFI is a highly indirect assay of pyroptosis, other explanations are also imaginable. For example, NLRC4 activation may hinder the development or homing of splenic or lymph node neutrophils. Importantly, I did not observe LysM-Cre-induced GFP expression in other cell types

(e.g., B cells and T cells) (Figure 2.2E). This fits with previously published literature about LysM-Cre expression3, 66. Nevertheless, taken together, the above data demonstrate LysM-Cre-dependent induction of the iOvaFla-IRES-GFP construct in macrophages and neutrophils in vivo.

Figure 2.2. Analysis of iOvaFla transgenic mouse model. (A) Flow cytometry analysis of peritoneal macrophages (CD11b+ F4/80+) from iOvaFla+/–; LysM-Cre–/–, iOvaFla+/–; LysM-Cre+/–, and Nlrc4–/–; iOvaFla+/–; LysM-Cre+/– mice. (B) GFP histograms of peritoneal macrophages (C) Flow cytometry analysis of neutrophils (CD11b+ Ly6GHi, Ly6CLo) from the bone marrow, spleens, and lymph nodes. (D) GFP histograms of neutrophils from the bone marrow, spleen, or lymph nodes, and (E) splenic B cells CD19+ TCRβ–), CD4+ (TCRβ+ C19– CD4+), and CD8+ T cells (TCRβ+ C19– CD8+). Results were analyzed with either a one-way or two-way ANOVA and Bonferroni

2.3 Myeloid-specific inflammasome activation leads to systemic inflammation iOvaFla; LysM-Cre+/– mice were markedly runted prior to weaning (Figure 2.3A), and exhibited significantly lower body weights throughout life compared to their age- matched Cre-negative littermates and the Nlrc4–/– control mice (Figure 2.3B). Unexpectedly, the iOvaFla; LysM-Cre+/– mice also developed severe limb swelling, most

Figure 2.3 Myeloid-specific inflammasome activation leads to systemic inflammation. Spontaneous inflammatory disease manifesting in the tibiotarsal joints (A, B) of iOvaFla+/–; LysM-Cre+/– mice aged 10 -14 weeks. (A), 14 week iOvaFla+/–; LysM-Cre+/– mice are runted and display joint disease compared to age and sex-matched Nlrc4–/–; iOvaFla+/–; LysM-Cre+/– mice. (B) Weight and tibiotarsal joint swelling as the mice age. strikingly in the tibiotarsal (heel) joint (Figure 2.3A, 2.4A). Tibiotarsal joint swelling was 100% penetrant, although the age of onset varied, beginning as early as four weeks or as late as 10 weeks (Figure 2.3B). Damage was more severe in the tibiotarsal joint as compared to the femorotibial (knee) joint (Figure 2.4A, B). In the tibiotarsal joint of iOvaFla; LysM-Cre+/– mice, there was also substantial neutrophilic infiltration leading to inflammation with slight to severe bone and cartilage erosion in the joint (Figure 2.4A). There were no histopathological findings in the joints of Nlrc4–/–; iOvaFla; LysM-Cre+/– or in the LysM-Cre negative littermates (Figure 2.4A, B). In addition to joint damage, the iOvaFla; LysM-Cre+/– mice exhibited significant systemic inflammation amongst soft tissues. Notably, nearly all tissues examined showed significant neutrophilic infiltration. For example, pronounced neutrophilia was observed in sections of iOvaFla; LysM-Cre+/– duodenums (first part of the small intestines). This neutrophilic inflammation was not apparent in the absence of NLRC4 or Cre recombinase (Figure 2.4C). Inflammation was distinguished by neutrophilic 15

infiltration of the submucosa and lamina propria that also extended into the underlying muscularis externa. The inflammation was not accompanied by damage to the surface epithelium (Figure 2.4C). There was also varying degrees of kidney damage in iOvaFla; LysM-Cre+/– mice (Figure 2.4D). Some mice had fibrin and neutrophil accumulation within glomeruli consistent with necrotizing glomerulonephritis, while others had some glomeruli with only thickened membranes. Once again, the Nlrc4–/–; iOvaFla; LysM- Cre+/– control nor LysM-Cre negative mice had no pathological alterations (Figure 2.4D). Similar to the kidney, liver pathology varied amongst the sick iOvaFla; LysM- Cre+/– mice. One surveyed mouse had extramedullary hematopoiesis (EMH), hematopoiesis occurring outside of the bone marrow, in the liver (Figure 2.4E). According to the pathologist, EMH in the spleen of adult mice is a common background

Figure 2.4 Inflammation develops in hard and soft tissues. (A – E) Histology images showing (A) tibiotarsal joint, (B) femorotibial joint, (C) duodenum, (D) kidney, and (E) liver damage in 10 week iOvaFla+/–; LysM-Cre+/– mice compared to Nlrc4–/–; iOvaFla+/–; LysM-Cre+/– mice (tibiotarsal and femorotibial joints at magnification X40 and bar is 500 microns; duodenum at magnification X600 and bar is 20 microns; kidney and liver at magnification X400 and bar is 20 microns).

finding, but EMH in the liver indicates the mouse is undergoing an inflammatory response in other tissues. There was further evidence of joint inflammation when looking at the hind paws of iOvaFla; LysM-Cre+/– mice compared to their littermates (Figure 2.5A). iOvaFla; LysM-Cre+/– mice had expansion of the neutrophil compartment, as evidenced by markedly increased mature and immature neutrophils within the bone marrow, in comparison to the normal mixed myeloid and erythroid lineages of the iOvaFla; LysM- Cre–/– littermate control (Figure 2.5B). Lastly, Lymphatics were dilated in the deep dermal and hypodermal(subcutaneous) plexi of haired skin and also contained large lipid vacuoles in sick iOvaFla; LysM-Cre+/– mice. There were varying degrees of neutrophilic and mononuclear inflammation around the lymphatics (Figure 2.5C). The

Figure 2.4 Littermates show no inflammatory phenotype. (A – C) Histology images of the (A) hind paw, (B) bone marrow, and (C) haired skin in 10 week iOvaFla+/–; LysM-Cre+/– mice compared to iOvaFla+/–; LysM-Cre–/– littermates (hind paw at magnification X10; bone marrow at magnification X600 and bar is 20 microns; skin at magnification X100 and bar is 10 microns).

Table 2.1 Averaged pathology scores.

Hind Tibiotarsal Femorotibial Duodenum Kidney Liver Skin BM - Genotype paw - joint - 20 joint - 20 - 4 - 20 - 8 - 8 4 20 iOvaFla; 14.5 4.5 10 2 8.67 2 2.67 3.67 LysM-Cre+/– Nlrc4–/–; iOvaFla; 0 0 N/A 0 0 0 N/A N/A LysM-Cre+/– iOvaFla; 0 0 0 0 0 1 0 0 LysM-Cre–/– 17

pathology scores are averaged in Table 2.1, while the individual scores are listed in Table 2.2. These data demonstrate that chronic myeloid-specific NLRC4 activation leads to severe joint and systemic inflammation.

2.4 Inflammasome-driven disease leads to myeloid cell hyperplasia To better understand inflammasome-driven disease pathology, I characterized hematopoietic cell populations in sick versus control iOvaFla mice. Complete blood count (CBC) analysis of peripheral blood revealed that iOvaFla; LysM-Cre+/– mice have

Table 2.2 Individual pathology scores. Tibiotarsal Femorotibial Hind Genotype Duodenum Kidney Liver Skin BM joint joint paw iOvaFla; 4, 16, 13, 11, 8, 4, 1, 2, 1, 4, 3, 1, 9, 1, 3, 5, 8 1, 3 10, 8, 8 LysM-Cre+/– 19, 16, 19 11 1 5 4 Nlrc4–/–; 0, 0, iOvaFla; 0, 0, 0 0, 0, 0 N/A 0, 0, 0 0, 0, 0 N/A N/A 0 LysM-Cre+/– iOvaFla; 0 0 0 0, 0 0 1 0 0 LysM-Cre–/–

Figure 2.6 Basic characterization of inflammasome driven inflammatory pathology. (A) Hematocrit concentration and (B) cell numbers from a complete blood count (CBC) performed on the blood of 10 week old iOvaFla+/–; LysM- Cre–/– and iOvaFla+/–; LysM- Cre+/– mice. (C) Spleen and lymph nodes of iOvaFla+/–; LysM- Cre–/– and iOvaFla+/–; LysM- Cre+/– mice and spleen weights. Results were 18

decreased hematocrit levels compared to Cre negative littermates (Figure 2.6A), consistent with anemia of chronic disease (ACD)76, 77. Blood lymphocytes of iOvaFla; LysM-Cre+/– mice were also decreased compared to healthy littermate controls. Although, there were significant increases in both monocytes and neutrophils in the peripheral blood of the sick LysM-Cre positive mice as compared to healthy littermates (Figure 2.6A). Sick iOvaFla; LysM-Cre+/– mice exhibited enlarged spleen and lymph nodes compared to their Cre negative littermates (Figure 2.6B). Flow cytometric analysis of the spleen and pooled peripheral and mesenteric lymph nodes revealed that the tissues were enlarged tissues due to hyperplasia. iOvaFla; LysM-Cre+/– mice had increased levels of monocytes and neutrophils compared to the NLRC4-deficient control (Figure 2.7A, B). Flow cytometry analysis of the bone marrow confirmed the pathologist’s findings of neutropilia (Figure 2.5B). Neutrophil numbers were increased in iOvaFla; LysM-Cre+/– mice compared to

Figure 2.7 Inflammasome- driven disease leads to myeloid cell hyperplasia. (A) Flow cytometry and (B) quantification of monocytes (CD11b+ Ly6CHi Ly6G–) and neutrophils (CD11b+ Ly6GHi Ly6CLo) from mice aged 10-16 weeks. (C) Flow cytometry and (D) quantification of BM neutronphils (CD11b+ Ly6GHi) from 10 weeks old mice. Flow cytometry plots are representative of 2-3 experiments. Results were analyzed as in Figure 2.6

littermate controls (Figure 2.7B, C). Lymphocyte populations were less dramatically affected, with B cell numbers modestly increased in the spleen and decreased in the lymph nodes of iOvaFla; LysM-Cre+/– as compared to Nlrc4–/– control mice (Figure 2.8A, B). iOvaFla expression led to only slight changes in T cells levels in the spleen and lymph nodes, with a slight increase of CD8+ T cells in the spleen as compared to control mice. No significant changes in T cell numbers were observed in the lymph nodes compared to the control Nlrc4–/–; iOvaFla; LysM-Cre+/– mice (Figure 2.8C, D). I assessed whether there was activation of self-reactive B cells by assaying for anti-

Figure 2.8 Spleen and lymph node lymphocyte populations relatively unchanged. (A) Flow cytometry and (B) quantification of B cells (CD19+ TCRβ–). (C) Flow cytometry and (D) quantification of CD4+ (TCRβ+ C19– CD4+) and CD8+ T cells (TCRβ+ C19– CD8+). Results were analyzed with either a one- way or two-way ANOVA and Bonferroni post- tests; * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Figure 2.9 ANA not required for autoinflammation. The positive and negative controls were acquired from Dr. Ann Marshank-Rothstein’s laboratory. The positive control sera is from an F1 of B6 and MZB cross. The negative control sera is from a B6 mouse. Sera from four separate iOvaFla+/–; LysM-Cre+/– mice. All four pictures are from sera diluted 1:1080. nuclear antibodies (ANA). Only one of four assayed iOvaFla; LysM-Cre+/– mice might positive for ANA (Figure 2.9). The reaction was very weak, so it could possibly be a false-positive. Only one mouse testing positive for ANA indicates that ANA is likely not to be the causative agent in this inflammatory phenotype. The overall lack of ANA coupled with minor changes in lymphocyte populations but large changes in myeloid cells furthers the likelihood that the disease is an autoinflammatory not autoimmune disorder.

2.5 Increases in systemic cytokines in inflammasome-driven disease Although pro-IL-18 is constitutively expressed in some cell types78, pro-IL-1β is often suggested to require a ‘priming’ signal (‘signal 1’) for expression, prior to signals that activate CASP1 processing (‘signal 2’), as a safeguard to prevent inappropriate IL- 1β production40, 78. However, exceptions to this simple model have been observed, including instances in which exogenous priming41 or CASP1 processing79, 80, 81 do not appear to be required for IL-1β production. Indeed, once inflammation is initiated, endogenous priming signals (e.g., inflammatory signals released from dying cells) may be sufficient to provide both signals 1 and 2 for IL-1β production. In the iOvaFla; LysM- Cre+/– mice, no exogenous priming signal is provided. This allowed me to assess whether signal 2 alone is sufficient to initiate inflammatory cytokine production. I used a cytokine bead array (CBA) to assay the amounts of serum IL-1α, IL-1β, IFNγ, IL-6, MCP-1 (also known as CCL2), and TNFα. All of the cytokines and chemokines were significantly elevated compared to Nlrc4–/–; iOvaFla; LysM-Cre+/– mice, with IL-1α being 21

Figure 2.10. iOvaFla: LysM- Cre+/– mice have increased levels of cytokines. (A) BD Biosciences CBA and (B) IL-18 ELISA with serum from 16 week old mice. Results were analyzed with either a one-way or two-way ANOVA and Bonferroni post-tests; * = p < the most abundantly increased (Figure 2.10A). Interestingly, levels 0.05, ** = p < 0.01, of IL-1β were also increased, confirming that constitutive expression *** = p < 0.001. or endogenous priming signals are sufficient to drive expression. IL-18 levels, as measured by ELISA, were also significantly increased in iOvaFla; LysM-Cre+/– mice (Figure 2.10B). No cytokines or chemokines were detectable at steady state in Nlrc4–/–; iOvaFla; LysM-Cre+/– mice (Figure 2.10A, B).

2.6 ASC-deficiency relieves inflammatory disease The inflammasome adaptor protein ASC plays a key role in mediating cytokine processing downstream of NLRC4 activation. Cytokine processing is largely dependent on ASC in vitro but less so in vivo18, 82. In order to determine whether ASC is required for disease in iOvaFla; LysM-Cre mice, I generated Asc–/–; iOvaFla; LysM-Cre+/– mice. Interestingly, disease was almost entirely ameliorated on the Asc–/– background, with normal weight gain and no joint inflammation evident through 10 weeks (Figure 2.11A). Some of the Asc–/–; iOvaFla; LysM-Cre+/– mice eventually developed mild tibiotarsal joint inflammation starting at around 18 weeks of age (Figure 2.11A), substantially later than iOvaFla; LysM-Cre+/– mice. ASC deficiency also severely reduced cytokine levels in +/– iOvaFla; LysM-Cre mice, though some cytokines such as IL-6, IFNγ, and TNFα were still slightly elevated on the Asc–/– background (Figure 2.11B). IFNγ was the most statistically significant change between Asc–/–; iOvaFla; LysM-Cre+/– and Nlrc4–/–; iOvaFla; LysM-Cre+/– mice (Figure 2.11B). Levels of GFP in Asc–/–; iOvaFla; LysM- Cre+/– resident peritoneal macrophages were comparable to those observed on (Asc+;) iOvaFla; LysM-Cre+/– macrophages, and significantly lower than those observed on Nlrc4–/–; iOvaFla; LysM-Cre+/– macrophages (Figure 2.11C). These data imply that, as observed previously in vitro, pyroptosis in vivo does not require Asc, and thus raise the possibility that cytokine release rather than pyroptosis is the major driver of disease.

Figure 2.11 ASC deficiency ameliorates disease. (A) Weight and tibiotarsal joint swelling in iOvaFla+/–; LysM-Cre+/–, Nlrc4–/–; iOvaFla+/–; LysM-Cre+/–, and Asc–/–; iOvaFla+/–; LysM-Cre+/– mice of the indicated ages. (B) Quantification and histogram of cellular GFP in peritoneal macrophages (CD11b+ F4/80+) of iOvaFla; LysM-Cre–/–, iOvaFla; LysM-Cre+/–, and Nlrc4–/–; iOvaFla; LysM-Cre+/–, and Asc–/–; iOvaFla; LysM-Cre+/– mice. (C) Serum cytokines measured by a BD Biosciences CBA and IL-18 ELISA from 8-10 week old mice. Results were analyzed with a two-way ANOVA and Bonferroni post-tests; * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

2.7 IL-1R signaling is necessary for inflammatory phenotype After observing significant increases of IL-1 in sick iOvaFla mice (Figure 2.10) and the decrease of IL-1 in the relatively healthy Asc–/–; iOvaFla; LysM-Cre+/– mice (Figure 2.11B), I wanted to determine the importance of IL-1 signaling in the inflammatory phenotype. Once iOvaFla; LysM-Cre+/– mice displayed significant tibiotarsal joint swelling (4.0mm size compared to 2.7-3.0mm in littermates), I treated the mice with an αIL-1R blocking antibody every three to four days for two weeks. After treatment, tibiotarsal joint swelling decreased, and the mice gained weight compared to sick mice treated with an isotype control antibody (Figure 2.12A). The treated iOvaFla; LysM-Cre+/– mice had significantly decreased amounts of IL-1β. The decreases in MCP- 1 and TNFα levels in treated mice were also statistically significant (Figure 2.12B). Humans with auto-activating NLRC4 mutations still have elevated IL-18 after Anakinra treatment58, 60. Consistent with these observations, I did not observe a statistically significant difference in IL-18 levels between untreated and αIL-1R-treated mice (Figure 2.12B). After αIL-1R antibody treatment, the sick iOvaFla; LysM-Cre+/– mice exhibited decreased levels of monocytes and neutrophils in both the spleen and lymph nodes 23

Figure 2.12 Blocking IL-1R relieves disease symptoms. (A) iOvaFla+/–; LysM-Cre+/– mice with joint swelling at 4.0mm were administered either anti-IL-1R blocking antibody or isotype control every three to four days for two weeks. Their weights and ankle size were measured during that time. (B) Post-treatment serum cytokines measured by a BD Biosciences CBA and IL-18 ELISA. (C) Flow cytometry and (D) quantification of monocytes and neutrophils. Results were analyzed with either a one-/two-way ANOVA and Bonferroni post-tests; * = p < 0.05, ** = p < 0.01, *** = p < 0.001. 24

when compared to control antibody treated mice (Figure 2.12C, D). Consequently, IL- 1R signaling appears critical for development of the systemic inflammatory phenotype present in iOvaFla; LysM-Cre+/– mice.

2.8 Neutrophil specific inflammasome activation is sufficient to cause disease Neutrophilia is a prominent clinical feature of inflammasome driven disease in my iOvaFla mice and in other mouse models62, 64 and humans68, 83, 84. Considering IL-1 is a powerful inducer of neutrophil recruitment to tissues, and given that disease is alleviated by blocking IL-1R, it seems likely that neutrophils play a large role in the inflammation. However, it is not clear whether inflammasome activation in neutrophils is sufficient to initiate disease, or whether inflammasome activation in other cell types releases IL-1,

Figure 2.13 iOvaFla+/–; CD11c-Cre+ mice do not develop joint swelling. (A) Weight and tibiotarsal joint swelling as iOvaFla+/–; Cre–, iOvaFla+/–; CD11c-Cre+, and iOvaFla+/–; LysM-Cre+/– mice age. (B and C). Flow cytometry plots and cell number graphs are representative of three separate experiments of mice aged 12 weeks old. (B) Flow cytometry and (C) quantification of monocytes and neutrophils. Results were analyzed with either a two-way ANOVA and Bonferroni post-tests; * = p < 0.05, ** = p < 0.01, *** = p < 0.001. 25

which then leads to neutrophil recruitment. To address whether iOvaFla expression in other myeloid cell types might also drive disease, I also crossed the iOvaFla mice to CD11c-Cre transgenic mice. CD11c-Cre mice express Cre in most dendritic cells, macrophages, and monocytes, but unlike LysM-Cre mice, there is no expression in neutrophils3, 85. This allowed us to focus on the in vivo role of cell-intrinsic NLRC4 inflammasome activation in myeloid cells excluding neutrophils. iOvaFla; CD11c-Cre+ mice exhibited some disease symptoms, but the symptoms were less severe than iOvaFla; LysM-Cre+/– mice. For example, iOvaFla; CD11c-Cre+ mice did not develop joint swelling though they were runted (Figure 2.13A). However, similar to iOvaFla: LysM-Cre+/– mice, monocyte and neutrophil populations were still increased in the spleen and lymph nodes (Figure 2.13B, C). The next step was to determine whether cell intrinsic inflammasome activation in neutrophils was sufficient and possibly necessary for disease development. I crossed the iOvaFla mice to MRP8-Cre transgenic mice, which expresses Cre specifically in neutrophils3, 86, 87. Surprisingly, the iOvaFla; MRP8-Cre+ mice almost fully recapitulated the severe disease phenotype of the iOvaFla; LysM-Cre+ mice. iOvaFla; MRP8-Cre+ mice were runted and develop joint swelling only slightly slower than iOvaFla; LysM- Cre+/– mice (Figure 2.14A, 2B). Moreover, iOvaFla; MRP8-Cre+ mice exhibit increased cytokine levels, with IL-18 being the most significantly increased (Figure 2.14B). IL-1β levels were increased just as in the LysM-Cre mouse line (Figure 2.14B). Flow cytometry analysis demonstrated increased monocytes and neutrophils in the spleen and lymph nodes, again similar to what was observed in iOvaFla; LysM-Cre+/– mice (Figure 2.14C, D). In fact, iOvaFla; MRP8-Cre+ mice have even higher levels of monocytes and neutrophils in the spleen as compared to iOvaFla; LysM-Cre+/– mice. There was no notable difference between the two genotypes in the lymph nodes (Figure 2.14C, D). To determine whether IL-1 drives disease in iOvaFla; MRP8-Cre+ mice, I treated iOvaFla; MRP8-Cre+ with anti-IL-1R blocking antibody using the same protocol we used to treat iOvaFla; LysM-Cre+/– mice (Figure 2.12B). Consistent with my hypothesis, the treatment alleviated joint swelling and the mice gained weight compared to the isotype control treated mice identical to LysM-Cre+/– treated mice (Figure 2.14E). These data indicate that inflammasome activation in neutrophils is sufficient and perhaps even necessary to induce an IL-1-dependent inflammatory disease in vivo. Taken together, our results are consistent with the hypothesis that NLRC4 activation in the neutrophil-lineage is an especially potent driver of disease.

2.9 Discussion The question of whether activation of an endogenous wild-type inflammasome is a sufficient signal to trigger inflammatory disease remains unresolved. Several studies have used bacterial infections to stimulate inflammasome activation in vivo, but these infections are invariably accompanied by robust TLR activation, making it difficult to ascertain the specific and sufficient functions of inflammasome activation in vivo. Other

Figure 2.14 Neutrophil iOvaFla expression is sufficient for inflammatory disease. (A) Weight and tibiotarsal joint swelling as mice age. (B) Serum cytokines measured by a BD Biosciences CBA and IL-18 ELISA of 10-12 week old mice. (C) Flow cytometry and (D) quantification of monocytes (CD11b+ Ly6CHi Ly6G–) and neutrophils (CD11b+ Ly6GHi Ly6CLo). (E) Weight and ankle measurements after OVA-Fla+/–: MRP8-Cre+/– mice were administered either anti-IL-1R blocking antibody or isotype control every three to four days for two weeks. Results were analyzed with either a one-way or two-way ANOVA and Bonferroni post-tests; * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

studies have demonstrated clear pathological effects mediated by gain-of-function mutations in inflammasome genes, but whether chronic activation wild-type inflammasomes can also mediate disease (or are subject to feedback inhibitory control) is unresolved. Thus, to address whether chronic inflammasome activation is sufficient to produce disease, I generated a mouse model to constitutively express cytosolic flagellin, a ligand for the NAIP/NLRC4 inflammasome, in the absence of any other contaminating exogenous stimuli. Selective NAIP/NLRC4 inflammasome activation in LysM+ positive cells is sufficient to cause a systemic inflammatory phenotype. iOvaFla; LysM-Cre+/– mice are runted, and exhibit elevated systemic cytokine levels, ACD, systemic neutrophilia, and obvious joint swelling. Disease could be rescued by therapeutic blockade of the IL-1R. Disease could also be significantly reduced by crossing to the Asc–/– background. Interestingly, ASC is not believed to be essential for NAIP/NLRC4-induced pyroptosis17, 18, and indeed, ex vivo Asc–/–; iOvaFla; LysM-Cre+/– peritoneal macrophages still exhibited reduced expression of a co-expressed GFP, indicative that pyroptosis is still occurring in vivo. Similar to Asc-deficiency, blockade of the IL-1R should also not prevent pyroptosis, yet this treatment significantly ameliorated disease. These lines of evidence raise the possibility that chronic pyroptosis itself is not sufficient to drive inflammatory disease in vivo. LysM-Cre is active in multiple cell types, including macrophages, monocytes, neutrophils and some dendritic cells3. Most studies of the inflammasome have been conducted in macrophages or monocytes, though a few studies have also indicated that neutrophils express functional inflammasomes49, 54, 88, 89. Interestingly, I found that expression of cytosolic flagellin in the neutrophil lineage (mediated by MRP8-Cre) was sufficient to cause severe NLRC4-dependent chronic systemic and joint inflammation, which was also rescued by blocking IL-1R. By contrast, chronic inflammasome activation mediated by CD11c-Cre (expressed in several cell types, including dendritic cells and tissue macrophages3 produced a milder disease with no joint pathology (Figure 2.13). Our results therefore uncover an unexpected function for inflammasome activation in neutrophils in mediating systemic inflammatory disease. Why is inflammasome activation in neutrophils a particularly strong driver of autoinflammatory disease? One possible explanation is that like other unusual cellular subpopulations41, these cells might circumvent the typical requirement for signal one (‘priming’) for expression of pro-IL-1β. Indeed, transcriptional profiling of specific neutrophil populations, including blood and liver neutrophils, indicates these cells express relatively high levels of pro-IL-1β at steady state 42. Another unusual feature of inflammasome activation in neutrophils is that it is reported in some cases to lead to IL- 1β release in the absence of pyroptosis49. Although pyroptosis is typically considered to be a pro-inflammatory form of cell death, it is likely that this lytic cell death is also an important ‘self-limiting’ mechanism to prevent cells from sustained or prolonged release of IL-1β. Indeed, though we could see some evidence for NLRC4-dependent GFP loss from flagellin-expressing neutrophils (indirect measure of pyroptosis), the pyroptotic loss

of GFP in vivo was not nearly as prominent in neutrophils as compared to peritoneal macrophages, a cell population that has been shown to undergo robust pyroptosis55. It could be that inflammasome activation in neutrophils may drive pathology through sustained release of constitutively expressed IL-1β. Though there are few tissue resident neutrophils in most tissues90, 91, 92, once inflammation is initiated, then neutrophil recruitment to tissues may be enhanced and sustained by positive inflammatory feedback loops (Figure 2.15). Lastly, it is also possible that after NLRC4 activation, neutrophils undergo NETosis, a form of neutrophil cell death that is accompanied by the release neutrophil extracellular traps (NETs)93. Work has shown NETosis can stimulate the NLRP3 inflammasome in macrophages94, 95, but it is unclear if inflammasome activation in neutrophils can lead to NETosis. NETs have been linked to chronic inflammatory diseases such as lupus, atherosclerosis, arthritis, and others94, 95, 96, 97, 98. Taken together, my results have identified inflammasome activation in neutrophil lineage cells as a sufficient driver of systemic inflammatory disease. In future studies, it will be of interest to determine whether specific neutrophil sub-populations are especially potent initiators of inflammation, and whether perhaps inflammasome activation in neutrophils plays a role in the initiation of human inflammatory diseases.

Figure 2.15 Final model showing how tissue neutrophils initiate a positive-feed forward loop to drive tissue inflammation and damage.

Chapter 3: Expression of iOvaFla transgenic protein using other Cre recombinase drivers

3.1 Introduction Chapter 2 discussed my effort to characterize in vivo effects downstream of inflammasome activation in multiple cell types, such as macrophages, some dendritic cells, monocytes, and neutrophils. In this chapter, I will develop more mouse models to explore the NAIP/NLRC4 inflammasome in other cells types and what other consequences might occur downstream of activation. Though iOvaFla; LysM-Cre+ mice develop an autoinflammatory disease similar human NLRC4 inflammasomopathies, there is a caveat to using LysM-Cre. The mice are expressing iOvaFla and activating endogenous NLRC4, but this was restricted only to myeloid cells, unlike the human inflammasomopathies. There are many other cells that could be activated in humans but not in the iOvaFla; LysM-Cre+ mouse model. One logical next step is to use a Cre driver that causes ubiquitous iOvaFla expression. There are of course many interesting routes to explore using such a mouse model, for example lose dose iOvaFla expression to mimic human inflammasomopathies or shorter robust activation, more similar to an infection, to study any downstream adaptive response. Once cell type not activated in LysM-Cre mice is intestinal epithelial cells (IECs). IECs are competent for NAIP/NLRC4 inflammasome activation53, 99, 100, 101. The lab first because interested in the role of inflammasomes in IECs because of the observation that Nlrc4–/– –> WT BM chimeras were still sensitive to FlaTox induced lethality. This indicated either radio-resistant cells were the cause of lethality or a non-hematopoietic cell like IECs. There were published papers already examining the importance of inflammasomes in IECs, especially during infections10, 24, 30, 41, 57, 99. Since these initial observations, IEC specific NAIP-, NLRC4-, and CASP1-deficient mice have subsequently been used to carefully examine inflammasome activation in IECs53, 100, 101. Both our lab and another found that once IECs activate their NAIP/NLRC4 inflammasome, the IEC is expulsed53, 100. Others found that NAIPs protect against colonic tumorigenesis, but it is unknown how and if NLRC4 or CASP1 are involved101. In addition, as already previously mentioned, some of the human patients with gain-of- function NLRC4 mutations have severe gut pathology59, 60, 61. It was not addressed which cell type is responsible for the intestinal inflammation. All of these studies use external stimuli to activate NAIP/NLRC4. Often those stimuli are contaminated by TLR ligands,55 making the inflammasome-specific role harder to tease apart. Using the iOvaFla mice to activate NAIP/NLRC4 in IECs would permit analysis of the effect of specific NAIP/NLRC4 inflammasome activation in this cell type.

3.2 Inducible, ubiquitous expression of iOvaFla using ER-CreT2 I crossed ER-CreT2 mice to iOvaFla mice in order to establish a mouse model in which the endogenous NAIP/NLRC4 inflammasome can be activated in any cell type.

Importantly, this is also an inducible system, allowing me control over iOvaFla expression. ER-CreT2 mice express Cre fused to mutated hormone-binding domains of the estrogen receptor (ER)102, 103, 104, 105. ER-CreT2 is inactive until it binds the synthetic ER ligand 4-hydroxytamoxifen (OHT), a metabolite of tamoxifen. Following an established protocol, I treated iOvaFla; ER-CreT2+/–, Nlrc4–/–; iOvaFla; ER-CreT2+/–, and B6 mice with 1mg of tamoxifen or vehicle control for five straight days via intraperitoneal (IP) injection (Figure 3.1)103. The iOvaFla; ER-CreT2+/– mice were stable for the first five days, but starting at day six their weight and temperature both started to decrease (Figure 3.1A). I euthanized them on day seven because of their continued weight loss and temperature decrease. To determine iOvaFla expression, I ran flow cytometry on the Nlrc4–/–; iOvaFla; ER-CreT2+/– mice given tamoxifen to look for GFP positive cells. Between 13.5 – 15.7% cells were GFP+ compared to B6 mouse given tamoxifen (2.7%) (Figure 3.1B).

Figure 3.1 Extended tamoxifen treatment lethality in iOvaFla; ER-CreT2+/– mice is NLRC4 dependent. iOvaFla; ER-CreT2+/–, Nlrc4–/–; iOvaFla; ER-CreT2+/–, and B6 mice were IP injected with 1mg of tamoxifen or vehicle control (5%EtOH, 95% sunflower seed oil) for five straight days. (A) Weight and temperature were recorded for seven days. Arrows indicate tamoxifen injection days. (B) Flow cytometry on peritoneal lavage sample from tamoxifen treated Nlrc4–/–; iOvaFla; ER-CreT2+/– and B6 mice.

The iOvaFla; ER-CreT2+/– response to tamoxifen was promising and problematic at the same time. The system appeared to be working properly expressing iOvaFla and GFP. However, I wasn’t going to be able to study chronic inflammation nor an adaptive immune response if the mice die within seven days. I then decided to modify the protocol to inject the mice for two days instead of five (Figure 3.2). The mice did not suffer any significant weight or temperature loss (Figure 3.2). I kept the mice till day 30 to ensure there were no delayed effects (Figure 3.2). Increased IL-18 in the tamoxifen

Figure 3.2 Shorter tamoxifen treatment resolved lethality. iOvaFla; ER-CreT2 mice were IP injected with 1mg of tamoxifen or vehicle control (5%EtOH, 95% sunflower seed oil) for two days. Weight and temperature were recorded for the first three days then at day 30. Arrows indicate tamoxifen injection days.

Figure 3.3 Tamoxifen injection was contaminated with TLR ligands. WT and MYD88/TRIF (TLR signaling) deficient mice IP injected with either tamoxifen or LPS for one hour then the peritoneal cavity was lavaged with PBS. A BD Biosciences CBA was performed to determine serum cytokine and chemokine levels.

treated mice compared to vehicle control indicated the inflammasome was activated as anticipated (data not shown). Though the survival issue was solved, my next concern was the presence of TLR ligands. One of the most major reasons for developing the iOvaFla mouse was to eliminate any ‘contaminating’ TLR activation, giving us the ability to focus on the role of inflammasomes alone. Tamoxifen is highly hydrophobic and must be dissolved in 5% ethanol and 95% (sunflower seed) oil. To test if TLR ligands were present in the tamoxifen injection, I IP injected WT and MYD88/TRIF (TLR signaling) deficient mice with either tamoxifen or LPS. WT mice given LPS or tamoxifen had increased levels of IL-6 within the peritoneal cavity after one hour of injection (Figure 3.3). IL-6 is one of the predominant cytokines produced downstream of TLRs. Myd88/Trif–/– mice did not have increased IL-6 confirming that the response to tamoxifen injection was likely due to activation of TLRs (which signal via MyD88 and/or TRIF). To resolve the TLR contamination issue, I tested multiple solvents but all still stimulated TLRs (data not shown). A viable solution was to change delivery methods completely. Because the immune system already receives stimulation from intestinal microbiota, administering the TLR-ligand ‘contaminated’ tamoxifen orally should not make a significant difference in intestinal TLR stimulation. Thus, I switched to an oral gavage method in hopes that whatever TLR ligands were in the injection would be lost amongst the many PAMPs already preset in the intestines. I oral gavaged the mice on days zero and two with 4mg of tamoxifen in the same vehicle as before. iOvaFla; ER- CreT2+/– mice had increased serum IL-18 after just one injection (Figure 4). As expected

Figure 3.4 Tamoxifen treatment by oral gavage was sufficient to activate the inflammasome. iOvaFla; ER-CreT2+/–, Nlrc4–/–; iOvaFla; ER- CreT2+/–, and Asc–/–; iOvaFla; ER- CreT2+/– mice were orally gavaged with 4mg on day zero and two. An IL-18 ELISA was performed on day serum from before injections and at day two.

IL-18 was not detected in the serum of tamoxifen treated control Nlrc4–/–; iOvaFla; ER- CreT2+/– and Asc–/–; iOvaFla; ER-CreT2+/– mice (Figure 4). In conclusion, I was able to establish a system for ubiquitously expressing iOvaFla and selectively activating NAIP/NLRC4 inflammasome in a temporal manner.

3.3 Expression of iOvaFla in intestinal epithelium is embryonic lethal Villin-Cre (Vil-Cre) mice express Cre recombinase downstream of the mouse villin 1 promoter leading to expression in villus and crypt epithelial cells in the small and large intestines106. This is a highly specific Cre though there is some expression (<1%) in testes and stomach epithelial lining106. I crossed iOvaFla+/+ mice to Vil-Cre+/? mice (the genotyping protocol did not allow me to determine whether the Vil-Cre mice were heterozygous or homozygous for Vil-Cre). The resulting litters were expected to be 50% Cre positive or 100% Cre positive, depending if the parent was either Vil-Cre+/+ or Vil- Cre+/- (Table 3.1). Remarkably, zero of the 24 weaned mice were Cre positive. In addition, multiple litters were born from one breeder cage with none surviving it to weaning. Taken together, I began to speculate expression of iOvaFla in IECs and activation of NAIP/NLRC4 could be embryonic lethality (Table 3.1).

Table 3.1 Survival of iOvaFla; Vil-Cre mice. iOvaFla; Vil-Cre–/– iOvaFla; Vil-Cre+/– Adults 24/24 0/24 Dead within 0-24hrs 0/8 8/8 After carefully monitoring, I discovered a recently born (<12 hours old) litter of eight pups. Six were already deceased, but two were alive. Within the next 12 hours, the remaining two pups died. All eight pups were genotyped and were found to be Cre positive. I sent four pups for histopathology in hopes a cause of death could be determined (Figure 3.5). The examined tissues appeared largely normal. No cardiac structural abnormalities were seen (Figure 3.5A). The lungs had normal development for neonatal mice (Figure 3.5B). The pathologist noted the brain lacked morphological irregularities and was highly cellular, again a normal finding for neonates (data not shown). There was extramedullary hematopoiesis and glycogen vacuolation present some of the mice (Figure 3.5C). However, both are considered relatively common in neonates. There were no histological alterations in the kidney (Figure 3.5D). Intestines begin to autolysis within five minutes of death. Due to this, no definitive conclusions were drawn from the intestinal histology. There was barrier breakdown, but this could have been due the pup being found already deceased not inflammasome induced pathology (Figure 3.5C, E-F). Since the histology report had no strong conclusions, I moved onto to determining whether or not the lethality was inflammasome dependent.

Figure 3.5 Expression of iOvaFla in intestinal epithelium is embryonic lethal. (A – F) Histology images of the (A) heart (H), (B) lung (Lu), (C) liver (Liv), pancreas (Pan), small intestines (SI) (D) kidney +/– +/– (K), (E) SI, (F) colon (C), and cecum (Ce) in dead neonatal iOvaFla ; Vil-Cre (heart bar is 200 microns; all other tissues at 100 microns).

3.4 ASC deficiency does not rescue iOvaFla; Vil-Cre+/– Phenotype I crossed iOvaFla; Vil-Cre onto an ASC deficient background to separate the roles cell death (via pyroptosis) or cytokine production play in the lethality. As with the crosses on a WT background, the parental Cre zygosity was unknown. Thus, if the parent was heterzygous for Cre, then 50% of the of the pups were expected to be Asc–/–

Table 3.1 Survival of Asc–/–; iOvaFla; Vil-Cre mice. Asc–/–; iOvaFla: Vil-Cre–/– Asc–/–; iOvaFla: Vil-Cre+/– Adults 13/13 0/13 Dead within 0-24hrs 0/36 36/36

Figure 3.6 ASC deficiency does not rescue iOvaFla: Vil-Cre+/– phenotype. Histology images at (A) 100X and (B) 600x of the small intestines from <24 hours old neonatal Asc–/–; iOvaFla; Vil- Cre+/–. (Original magnification x100, bar 100um).

; iOvaFla; Vil-Cre–/– and 50% were expected to be Asc–/–; iOvaFla; Vil-Cre+/-. If the parent was homozygous for Cre, then 100% of the pups should also be Cre+. Much to my surprise, zero of the surviving mice were Vil-Cre positive (Table 3.2). 100% of the dead pups were Cre positive and died at a similar timescale as did the ASC sufficient mice. If indeed Asc is required for inflammasome-dependent release of cytokines (as previously suggested55), then this result would indicate that pyroptosis plays a bigger role in the lethality of IEC inflammasome activation than cytokines. This is in contrast to the IL-1 dependent disease phenotype in iOvaFla; LysM-Cre+/– and iOvaFla; MRP8- Cre+/– mice (Figues 2.11, 2.12 & 2.14). To better characterize the pathological effects of IEC inflammasome activation, litters of newborn pups were euthanized and immediately fixed for tissue dissection and histopathology. Three Asc–/–; iOvaFla: Vil-Cre+/– and one B6 age-matched fixed pups were sent for intestinal imaging. The Vil-Cre pups had villus blunting (shortened height of intestinal villi compared to the control) (Figure 3.6). One pup showed expansion of the submucosa (Figure 3.6A middle row, arrows), and another had expanded lamina propria (Figure 3.6A last row, arrows). Upon closer inspection at higher magnification, one Asc–/–; iOvaFla: Vil-Cre+/– pup had severe villus blunting along with dilation of the submucosal lymphatic channels (Figure 3.6B middle row, asterisk). Vacuolated (lipid- laden) macrophages accumulated in the lamina propria (Figure 3.6B last row, arrow) of another neonate. Villus blunting is consistent with previously published data from us and others53, 100. When ASC is present, the IEC activates NAIP/NLRC4 which will still recruite CASP1 leading to cell sloughing via pyroptosis. Even in the absence of ASC, the phenotype is occurs53.

3.5 Discussion In this chapter, I expanded the available iOvaFla mouse models. By breeding iOvaFla to ER-CreT2+/–, I created an inducible, ubiquitous way to activate endogenous NAIP/NLRC4. After repeated attempts, I established a protocol to induce iOvaFla expression with tamoxifen without stimulating excess PRRs. Oral gavage allowed me to circumvent TLR activation by the TLR ligand contamination in the tamoxifen injection. It was crucial to maintain selectively so that future studies are truly looking at specific role of inflammasomes not inflammasomes and TLRs. There are numerous experiments and uses for this model system which I will cover in chapter four. Lastly, I explored the role of inflammasomes in IECs by creating iOvaFla; Vil- Cre+/– mice. Prior to creating the mice, the NAIP/NLRC4 inflammasome was relatively unstudied in IECs. Later, our lab and others revealed the NAIP/NLRC4 inflammasome is present and very responsive in IECs53, 100. After inflammasome activation, IECs undergo expulsion either through CASP1 and pyroptosis or CASP8 and apoptosis53. Since that was unknown at the time, it was highly unexpected to discover iOvaFla expression in IECs led to embryonic lethality. Zero surviving adults were Cre positive, but unfortunately a clear cause of death in Cre positive pups could not be obtained.

Asc–/–; iOvaFla; Vil-Cre+/– mice were also embryonic lethal. Pyroptosis has been shown to occur even in the absence of ASC, which fits with the lethality not being resolved by ASC deficiency. Pathology of the intestines clearly showed villus which could be a major part of the pathology. Isabella’s recent publication beautifully elucidated the effects of NAIP/NLRC4 activation in IECs53, but more work can still be done.

Chapter 4: Final remarks

4.1 Future directions with iOvaFla and cell specific Cre drivers There are many other cell specific Cre drivers besides the four I talked about in my dissertation. Some examples include Cx3cr1-Cre (tissue macrophages and monocytes), Clec9a-Cre (dendritic cells), and NKp46-Cre (natural killer cells). It would be important to first establish expression of inflammasome components first before breeding the mice. A newly expanding field of research is studying microglia cells in the brain. Cx3cr1-CreER could be used to study what role inflammasome activation would have in microglia.

4.1.1 iOvaFla; LysM-Cre and iOvaFla: MRP8-Cre mice Though I did a great deal of work characterizing and determining the mechanism inducing the inflammatory phenotype of iOvaFla; LysM-Cre mice, more would can be done. There are still quite a few open questions surrounding inflammasome activation in neutrophils. Conflicting reports were published on whether or not neutrophils pyroptosis. Assaying GFP expression loss (Figure 2.2C, D), it looked that tissue neutrophils undergo pyroptosis. However, as stated this is a very indirect in vivo method to determine pyroptosis. More careful studies need to be completed. I am currently working on determine if ex vivo bone marrow neutrophils undergo NLRC4-dependent pyroptosis after FlaTox administration. An undergraduate in lab (Lucian) is taking an imaging approach to determine if there is cell occurs what type of death is it. I also want to confirm in my own hands that neutrophils already express pro-IL-1β transcript. Lastly, lingering questions remain about sufficiency of neutrophils to drive disease. A small subset of BM myeloid progenitor cells have been shown to express MRP8-Cre and thus could express iOvaFla protein. I want to establish whether or not this changes any conclusions we have drawn. On face value, it is unlikely to change my conclusions even if the progenitors are expressing iOvaFla. They are progenitors for neutrophils, so that still fits will the conclusions I am drawing.

4.1.2 iOvaFla; CD11c-Cre mice I was unable to characterize iOvaFla; CD11c-Cre as deeply as I did with the LysM-Cre mice. I did conclusively determine they do not get joint inflammation, but what, if any other differences are there between the two lines. More dendritic cells will express iOvaFla in the CD11c-Cre line and preliminary results revealed some interesting differences in lymphocyte populations compared to LysM-Cre (data not shown). Lymphocyte populations were more significantly disturbed. There were also some differences with cell surface activation markers. It appeared though T cell populations were decreased, most of the remaining T cells were CD44 positive (recently activated). Meticulous, more extensive experiments, such as broad flow cytometry, would illuminate any differences. If adaptive immune populations are changed, this

could indicate the phenotype might be autoimmunity as well as autoinflammation. ANA assay and other autoimmune assays would address that question. Other than possible adaptive immune cell differences, what other differences are there between mice with inflammasome activation in CD11c-Cre+ vs LysM-Cre+ cells? I determined the LysM-Cre and MRP8-Cre phenotype was IL-1 dependent. Though, CD11c-Cre mice do not develop joint swelling, they are runted in size compared to littermates. It would be interesting to determine if IL-1R blocking antibody treatment lead to weight gain and improved health of the CD11c-cre mice as well. Just as with the LysM-Cre and MRP8-Cre phenotype, it is will be important to establish the mechanism causing disease in CD11c-Cre mice.

4.1.3 iOvaFla; Vil-Cre mice The embryonic lethality of iOvaFla; Vil-Cre+/? mice make them impossible to study as comprehensively as the other models. However, I did determine that lethality was an ASC-independent phenotype. In future experiments it would be of interest to cross the iOvaFla;Vil-Cre mice to an NLRC4 deficient background. This is an obvious and important control to demonstrate that neonatal lethality is NLRC4-dependent, as expected. To work around the lethality, I obtained Vil-ER-CreT2 mice. They have the inducible ER-CreT2 downstream of the villin promotor, so pretty much identical to Vil-Cre but inducible. This will allow for experiments on viable adults instead of deceased pups. To further establish the disease (lethality) mechanism, it would be interesting to generate the iOvaFla; Vil-Cre+/? Mice on Casp1–/–, Ripk3/Casp8–/–, and Casp1/Ripk3/Casp8–/– (or Asc/Casp1–/–) or backgrounds. I hypothesize, based on other published work from the lab [cite papers], that lacking either CASP8 or CASP1 would not rescue death. Instead, both CASP1 and CASP8 would need to be deleted for the mice to survive. That would eliminate both compensating pyroptosis and apoptsis pathways downstream of the NAIP/NLRC4 activation in IECs.

4.2 Future directions with iOvaFla: ER-CreT2 mice iOvaFla: ER-CreT2 mice will be an incredibly useful tool to explore whether or not inflammasome activation alone can induce an adaptive immune response. Many studies have tried to reveal the role of inflammasomes, but almost all have been done in the context of TLR ligands. There have been conflicting publications making it hard to conclude inflammasomes role. Both pyroptosis and cytokine production have been hypothesized to provide signals that stimulate adaptive immune responses. However, the involvement of the cytokines has been the focus of most studies. Supporting this notion, IL-1 along with cognate antigen has been shown to cause an expansion in naïve and memory CD4+ T cells107. IL-18 co-absorbed to alum and ovalbumin (OVA) increased the amount of OVA specific IgG1108. Recently, some work has linked the inflammasome to adaptive immune responses. Injecting modified tumor cells expressing full-length flagellin into mice stimulated both TLR and NLR responses. When the

flagellin was mutated to no longer activate the NLRC4 inflammasome, T cell priming was reduced109. Another study demonstrated that during a Salmonella enterica serovar Typhimurium infection, activating the NLRC4 inflammasome in CD8α+ DCs lead to the release of IL-18, which stimulated the release of IFNγ from noncognate memory CD8+ T cells110. Models studying the adaptive immune response to influenza A virus have shown a requirement of ASC, CASP1, and IL-1 receptor (IL-1R)111, 112. Conversely, however, one study using Listeria infection demonstrated that inflammasome activation was detrimental to the development of downstream adaptive responses113. This is just a subset of publications, but indicates a gap whole left in the field’s knowledge. The reason the mice are iOvaFla instead of just ‘iFla’ was to have a clear way to study adaptive immune responses. Ova has been a traditional antigen used to track adaptive immune responses, and there are many established protocols and reagents to track anti-Ova adaptive immune responses. One such example is OT-I and OT-II mice, which are mice that are transgenic for OVA-specific CD8- or CD4-restricted T cell receptors (TCRs). OT-I/-II T cells can be used both in vivo and in vitro assays to look for responses. It is an important open question whether or not a T cell response is stimulated downstream of the inflammasome. Many questions will follow if there is a response. It will be important to establish what type of T cell response. Is it a CD8 or CD4 T cell response? If a CD4 response, what type of helper T cell is it? IL-1β is important for skewing towards Th17 CD4 T cells. Thus, it is likely a Th17 response could be induced. Besides T cells, it will be important to also study B cells. One possible method to track antibody responses in the iOvaFla mice will be an anti-Ova antibody ELISA. This was another method I was developing but stopped to focus on the iOvaFla; LysM-Cre model. This assay is tricky to perform properly due to background variability interfering with results. However, it will be very useful once established to identify conditions in which anti-Ova antibodies are created. Obviously the iOvaFla: ER-CreT2 mice should be used. Nevertheless, for both T and B cell responses, the iOvaFla cell specific Cre models might also be useful. Are different adaptive immune responses created in a iOvaFla; LysM-Cre versus iOvaFla; MRP8-Cre versus iOvaFla; CD11c-Cre mice? If differences are found, it will be interesting to explore what mechanism and cell types are responsible. This work could hold significance for the development of inflammasome stimuli as vaccine adjuvants.

4.3 Final conclusions In this dissertation, I described my characterization of a knock-in mouse, iOvaFla, created by a former graduate student. In chapter two, I presented you the resulting phenotype from the iOvaFla; LysM-Cre cross. iOvaFla; LysM-Cre+/– mice develop a severe autoinflammatory reminiscent of some human autoinflammatory conditions. The mice have chronic, systemic inflammation manifesting in hard and soft tissues. The most striking feature was the increased presence of neutrophils in all assayed tissues.

When ASC was absent, the disease phenotype was greatly diminished. Unexpectedly, this indicated that pyroptosis alone was not sufficient to produce such a severe disorder. This led me to test the hypothesis cytokines we’re the predominant driving force behind disease progression. After treating the mice with a blocking IL-1R antibody, the mice not only gained weight but their joint swelling and decreased in comparison to isotype control antibody treated mice. Lastly, I wanted to determine the initiating cell type behind the disease. Given that the neutrophils were so abundant in my mice and are recognized to play a large role in information, I crossed the iOvaFla mice to a more neutrophil-specific Cre driver, MRP8-Cre. iOvaFla; LysM-Cre+ mice develop an indistinguishable phenotype from LysM-Cre mice. αIL-1R antibody treatment also ameliorated the autoinflammation. This work provides new insights into the role neutrophils play in autoinflammatory disorders. Before, neutrophils were undervalued as inflammation initiating cells. Most work focused on inflammasome activation in macrophages and to a smaller extent dendritic cells. When humans with autoactivating in NLRC4 were discovered, the researchers concluded macrophages were driving the disease60. This was correlative work done ex vivo on patients’ macrophages. Neutrophils did increase in disease flares and should be looked at more closely in the patients. I continued developing iOvaFla mouse models in chapter 3. I engineered mouse that will inducibly express iOvaFla in all cell types after tamoxifen treatment. Tamoxifen is highly hydrophobic and must be dissolved with solvents such as ethanol and oil. Unfortunately, I was unable to find an oil that was not contaminated with TLR ligands. Therefore, I switched to an oral gavage method to give the mice tamoxifen. That way any TLR ligand contamination could be obscured TLR-stimulating intestinal microbiota. Lastly, I expressed iOvaFla in IECs using Vil-Cre. 50-100% of the mice born were expected to be Cre positive. However, zero of the surviving mice were. These results strongly suggest that the phenotype was embryonic lethal. Deleting ASC did not rescue the lethality. This fits with recently published work from the lab describing IEC death and cell expulsion after NAIP/NLRC4 inflammasome activation. All of the iOvaFla Cre mouse models I have described could used to study many topics. More detailed mechanistic studies need to be done in the iOvaFla; LysM-Cre and iOvaFla; MRP8-Cre autoinflammatory phenotype to fully verify effect of inflammasome activation in neutrophils. A major lingering question is do neutrophils undergo pyroptosis. Conflicting reports have been published complicating the ability to draw a singular conclusion. Another poorly studied topic is adaptive immune responses downstream of inflammasomes. The iOvaFla: ER-CreT2 mice are an elegantly engineered tool to carefully, and systemically studied what, if any, adaptive immune responses are stimulated after NAIP/NLRC4 inflammasome activation. The iOvaFla mice yield abundant opportunities to explore inflammasomes in vivo using different selective Cre recombinase systems.

Materials and Methods

Animals All mice are bred and housed under specific pathogen free conditions and fed a standard chow diet (Harlan irradiated laboratory animal diet). Ova-Fla mice were generated by targeting the Rosa26 locus for genomic insertion of a construct encoding a loxP‐flanked transcriptional STOP cassette upstream of the Ova-Fla fusion gene. An IRES–GFP was included downstream of the Ova-Fla insertion to mark cells in which the STOP cassette has been excised and Ova-Fla translated. Founders were crossed to ER-CreT2 (Jax strain 008463), LysM-Cre (Jax strain 004781), MRP8-Cre (Jax strain 021614), CD11c-Cre (Jax strain 008068), and Villin-Cre (Jax strain 004586) transgenic lines. The strains were also crossed to Nlrc4−/− and Asc−/− to generate inflammasome- deficient control mice. Nlrc4−/− and Asc−/− animals were from V. Dixit (Genentech, South San Francisco, CA; Mariathasan et al., 2004).

Experiments were conducted on 10- to 16-week-old age- and generally sex-matched mice. Under isoflurane anesthesia, mice were weighed weekly and calipers were used to measure tibiotarsal joint (heel) size. All animal experiments and endpoints were approved by and performed in accordance with the regulations of the University of California Berkeley Institutional Animal Care and Use Committee.

Bone Marrow Derived Macrophages Bone marrow was harvested from femurs, and cells were differentiated into macrophages by culture in RPMI supplemented with L929 cell supernatant and 10% fetal bovine serum in a humidified incubator (37°C, 5% CO2) for seven days.

Tissue Histopathology Tissues were collected from mice and fixed in 10% neutral buffered formalin. The fixed tissues were shipped to the University of Michigan Unit for Laboratory Animal Medicine for analysis. Soft tissues were processed to paraffin on an automated histology processor using standard IVAC protocols for mouse tissue. Hindlimb samples were decalcified in a commercial formic acid solution (ImmunoCal) for 3 days. Following decalcification, the joints were rinsed and the femorotibial (knee) and tibiotarsal joints were isolated by cutting the bone proximal and distal to each joint. Joints were processed to paraffin as for soft tissues. Following processing, tibiotarsal (heel) joints were embedded in the lateral plane and the femorotibial (knee) joints and distal paw were embedded in the frontal plane, with compression using a manual histology tissue press. 4 micron thickness sections were cut on a rotary microtome and the sections were stained with hematoxylin and eosin on an automated tissue stainer.

Light microscopic evaluation was performed by a board-certified veterinary pathologist blinded to the groups at the time of sample evaluation. Representative photomicrographs were taken using an Olympus DP72 12.5 megapixeldigital camera mounted to an Olympus BX45 light microscope and using the software provided by the manufacturer (cellSens Standard 1.7.1, Olympus Corporation). Photo processing and composite plate construction was performed in Adobe Photoshop CS2, version 9.0. Photo processing was confined to global adjustments of brightness, contrast, sharpness, and image size that did not materially alter the interpretation of the image. Correction of peripheral lens distortion was performed if needed for low magnification photos.

Complete Blood Counts Blood was harvested from mice either by saphenous bleeding or cardiac punctures followed by euthanasia. Blood was kept in EDTA-treated vacutainers (BD product 365974) and ran on a Hemavet 850 at the University of California San Francisco Mouse Pathology Core.

Flow Cytometry Spleens and lymph nodes were harvested from euthanized mice, minced with scissors, and incubated in RPMI with 5% FBS containing collagenase VIII (1 mg/ml, Sigma) at 37°C for 45 minutes. Bone marrow was harvested by cutting the ends of the femur then flushing the marrow out. The digested spleens and lymph nodes were strained. Single- cell suspensions of all the tissues were treated with ACK Lysing Buffer (gibco by Life Technologies) to lyse erythrocytes. Cells were washed and filtered through 40 micron nylon strainers (Fisher Scientific) and counted. Cells were blocked with anti-CD16/32 antibody (2.4G2) and stained for extracellular markers. All antibodies were obtained from either eBiosciences, BioLegend, or BD Biosciences. Data was collected on Fortessa or X20 flow cytometers (BD Biosciences), and analysis was performed using FlowJo 10 Software (Tree Star).

Serum Cytokine Measurements To measure IL-18 by ELISA, Nunc Hi Affinity ELISA plates were coated with anti-IL-18 antibodies (MBL catalog D047-3) at 1μg/mL, blocked with with PBS with 1% BSA (w/v). Serum was diluted 1:5 in PBS with 1% BSA (w/v). Secondary biotin conjugated goat antibodies to IL-18 (MBL catalog D048-6) were used at 1:2000 in PBS with 1% BSA (w/v). Purified IL-18 standard was from Invivogen. Plates were developed with 1mg/mL OPD (Sigma) in Citrate Buffer (PBS with 0.05M NaH2PO4 and 0.02M Citric acid) with a 3M HCl acid stop after 10-15 minutes. Absorbance at 490nm was measured on a SpectraMax M2. IL-1α, IL-1β, IFNγ, IL-6, and MCP-1 were measured with a custom BD Biosciences Cytokine Bead Array according to the BD Biosciences’s guidelines.

Anti-nuclear antibodies (ANA) assay Positive (BALB/c-Faslpr) and negative (BALB/c x B6) control serum came from Dr. Ann Marshank-Rothstein’s lab. I used HEp-2 slides from MBL Bion (AN-1006). Serum was initially diluted 1:40 then 3x (ending with 1:4720) for five more dilutions. All six dilutions were used on a single antigen slide. MBL Bion protocol was followed, and a secondary α-mouse IgG 488 conjugated antibody was used to detect the presence of any antibodies that bound the antigen. The secondary was at 1.5mg/mL and diluted 1:400 when used. Slides were examined and imaged on the lab brightfield microscope.

Antibody Treatments Hamster IgG anti-mouse IL-1R antibody (mIL1R-M147) was obtained from Amgen. Ultra-LEAF Purified Armenian Hamster IgG Isotype Antibody from Biolegend (400940) was used for control injections. Mice were treated with 150μg of each antibody via retro- orbital injections every 3-4 days for two weeks.

Tamoxifen treatment For injection, 200mg of Tamoxifen (Sigma T5468) was measured into a 15mL and 1mL of 100% ethanol added. After shaking for one minute, 9mL of autoclaved sunflower seed oil was added. The 15mL conical was shaken for at least 30 minutes at 37°C. 750μL 2X aliquots were frozen in 1.5mL eppendorf tubes. After thawing, 750μL was added and the tube was again shaken at 37°C for 30 minutes. The vehicle control was made the same way without the tamoxifen of course. Mice were injected with 100μL (1mg).

To gavage the mice with 4mg of tamoxifen, 100mg of Tamoxifen was measured into a 15mL and 0.5mL of 100% ethanol added. After shaking for one minute, 4.5mL of autoclaved sunflower seed oil was added. The 15mL conical was shaken for at least 30 minutes at 37°C. Mice were orally gavaged with 200μL (4mg).

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