The All-Encompassing Importance of Innate Immunity

Before I start the President’s Address, I would like to thank the American Association of Immunologists (AAI) members for electing me to serve as President of the AAI. I thank Jeffrey Frelinger, a mentor, friend, and colleague, for the kind introduction. I am also honored to share this session with the Past President of the AAI, Jeremy Boss, who is a long-time friend and colleague.

We all know that this is a historic time and historic meeting. In the 108 years of its history, the AAI annual meeting was canceled only four times: three times during World War II and then last year because of the coronavirus disease 2019 pandemic. At the time of the 2021 virtual meeting, there were over 150 million cases of coronavirus disease 2019 and over 3 million deaths worldwide. The personal, economic, and societal toll is beyond imagination, and the only way to get out of this pandemic is data-driven science, science policy, science knowledge, and competent leadership. I would like to thank all of the health care workers, frontline workers, public health policymakers, immunologists, virologists, and other scientists in the audience for helping us combat this pandemic. I also want to recognize the AAI Executive Office, directed by Michele Hogan, and its staff for carrying on giant responsibilities despite the difficult circumstances. I want to thank the Council members, as well as Past Presidents, the Editors-in-Chief of The Journal of Immunology and ImmunoHorizons, the Course Directors of the AAI Advanced Course and Introductory Course, and finally, the Chairs and members of the AAI committees for devoting their volunteered time to these affairs. Finally, I want to wholeheartedly welcome our international as well as our US-based guest societies. There are 21 guest societies at this meeting.

To start my address, I will first provide some personal background. I grew up in a large family, and my parents and grandmother instilled in us the value of hard work, honesty, and just plain being a good person. My parents went through World War II, as well as the Communist takeover of China, and they took one of the last planes out of China to Taiwan. I was born a few years after they arrived in Taiwan as the last of eight children: six living at the time I was born and two had died during the war. As a diplomat, my dad moved from country to country. During my senior year of high school in the Philippines, my dad asked me, “What would you like to major in in college?” I said, “Literature.” And my father said, “What else are you interested in?” After some thoughts, I replied that I really enjoyed my class in biology, and so that was how my career in biology was decided. My older brother told me about an international scholarship at Illinois State University (ISU), which I applied for and received. I left home for college at the age of 17 and luckily learned that I really liked a lot of the science courses. I worked at the school cafeteria for all of my years at ISU, which taught me the true meaning of hard work. I also became a science tutor, which revealed that I really liked teaching. Later, I applied to Northwestern University as a graduate student in microbiology and immunology in the laboratory of David Ranney. I struggled a lot in graduate school since I had not prepared for a research career, but learned and was fascinated by the burgeoning field of immunology. Similar to many young people, I presented my first oral presentation at an AAI meeting and went for job interviews, where I met Jeff Frelinger, a codiscoverer of class II MHC Ags. I decided to join his laboratory at the University of Southern California. I was comentored by Leslie Weiner, a wonderful neurobiologist and neuroimmunologist.

My postdoctoral project was to identify class II MHC expression in the CNS as a way to identify immune responses in the brain (1). At the time, neuroimmunology was just a nascent field. I had a fulfilling and productive time in Jeff's laboratory and was also fortunate to meet my future spouse, Brian Shigekawa, who was the most supportive partner for a female scientist. Unfortunately, after 14 years of marriage, he died of a chronic illness.

In the early days of our marriage, Brian found a position in the Research Triangle Park, and I did a second postdoctoral fellowship at Duke University with Bernard Amos supported by a National Institutes of Health F32 fellowship that I received while in Jeff’s laboratory. Bernard and I shared an interest in merging the then-novel molecular biologic techniques with immunology, and he gave me free rein to pick any topic to pursue. I was intrigued by gene regulation and decided to study MHC gene regulation as a way to understand cell-specific gene expression in the immune system. I was fortunate in that Russel Kaufman, one of the few molecular biologists at the university then, gave me the opportunity to learn molecular biology in his laboratory, and Peter Cresswell welcomed me to his laboratory meetings. However, the resources were quite limited, and it was a time of great struggle and doubt while I acquired skills in molecular biology and applied them to my own questions in immunology. A couple of years later, Jeff Frelinger moved to a neighboring university, the University of North Carolina. He told me about a faculty opening and encouraged me to apply for it. I practiced my seminar over 50 times, knowing that this might be the only opportunity for me to become an independent investigator. Luckily, the morning after my interview, I got the job offer.

As a faculty member, I stayed focused on unraveling the mechanism of class II MHC gene regulation, and our group published some of the first papers on the functional promoters of this gene family (2, 3). Subsequently, we published extensively on the master regulator of all class II MHCs, CIITA, which was discovered by Viktor Steimle (4). This protein has a nucleotide-binding domain (NBD) and a leucine-rich repeat (LRR). Using both domains, two investigators in my laboratory, Jon Harton and Mike Linhoff, scanned the human genome and discovered 23 human genes that encode proteins bearing the NBD-LRR domains. In the beginning, we named these the CATERPILLER family to represent all of the domains in this family [CARD, transcription enhancer, R(purine)-binding, pyrin, lots of leucine repeats (5)], but this was later changed to NLR, which stands for either NBD-LRR or NOD-like receptor. This family of proteins is preserved from plants to humans (6), and members mediate a plethora of functions, including as master transcription factors, inflammasome sensors/receptors, regulators of signaling pathways, and controllers of cell death, including pyroptosis and autophagy.

In the early days, we did a lot of work on the inflammasome, a macromolecular complex first defined by Jurg Tschopp (7) with disease relevance first discovered by Hal Hoffman a year prior (8). Because of the relatively short time for this address, I won't be able to acknowledge the work by many others in the field who had similar findings as ours. We showed that the NLRP3-ASC (apoptotic speck containing protein with a CARD) inflammasome regulates a programmed cell death process that is not apoptosis, but closer to necrosis (9), which we proposed as pyronecrosis. This form of cell death is most likely what an earlier report referred to as inflammatory cell death or pyroptosis (10), a term that preceded the discovery of the inflammasome. The proclamation of a programmed cell death process other than apoptosis was quite controversial at the time. Our other contributions include studies showing the importance of various inflammasomes in influenza (11), in models of multiple sclerosis (12), neuroinflammation (13), metabolic diseases (14), colitis (15), colon cancer, and other cancers (16). We have also expanded the study of NLR proteins in cancers and found several that play a mitigating role of colorectal cancer and the associated inflammation, including NLRP12 (17), NLRX1 (18), and a non-NLR innate immune receptor, AIM2 (19). Finally, a recent focus of our group has been on defining a subgroup of NLRs as nucleic acid receptors. For example, we showed that NLRX1 intersects with the RNA-sensing pathway (20, 21), while Ian Wilson’s laboratory (22) first showed that it can bind RNA. We found that NLRC3 can bind DNA, as well as RNA (23), and intersects with the STING pathway (24). Others in the field have evidence that NLRP3 may engage oxidized mitochondrial DNA (25, 26), while NLRP1 has been shown to bind dsRNA (27). These findings of nucleic acid binding are important because the ligands for many NLRs are unknown. Finally, on a different topic, we have also studied the encapsulation of a pathogen-associated molecular pattern, cGAMP, which activates the cGAS–STING pathway, in cancer control, vaccination, and autoimmune disease (28–30).

Next, I wish to present in more detail two recent research themes. The first theme is the unexpected finding that intracellular innate immune receptors are important in regulating T cell function through changes in their immune metabolism. The second theme is the link between the microbiota and innate immune receptors and beyond in the context of diseases.

With respect to the first theme, we showed that an NLR protein that is understudied, called NLRC3, can block TNFR-associated factor 6 ubiquitination, therefore leading to a blockade of NF-κB to cause reduced downstream NF-κB–dependent cytokines and immune metabolism, such as glycolysis and oxidative phosphorylation (OXPHOS) (31, 32). This process leads to the attenuation of an overzealous CD4 T cell response in viral infections as well as in autoimmune models. We have two new studies linking innate immune receptors to immunometabolism in T cells.

The first study shows that a DNA-binding inflammasome protein, AIM2, maintains T regulatory (Treg) cells (33). As background, we previously showed that the NLRP3 inflammasome exacerbated experimental autoimmune encephalomyelitis (EAE) (12). Others supported this finding (34) and additionally showed that other inflammasome components, such as the common adaptor molecule, Ab-secreting cell, or caspase-1, also exacerbate EAE (35, 36). These effects are due to the impact of the inflammasome on IL-1 and IL-18, which then affected Th1 and Th17 effector cells. When we studied Aim2^−/− mice, we expected the same phenotype. Instead, the results we found were the opposite of what we expected. Aim2^−/− mice had more severe EAE than wild type mice, while in the same study, Asc^−/− and Casp1Casp11^−/− mice had little disease. Furthermore, Aim2^−/− mice had increased inflammatory foci and demyelination, while Asc^−/− mice had no inflammatory foci and demyelination. We surmised that the function of Aim2 in this disease context is independent of the inflammasome because IL-1β and IL-18 levels were identical between Aim2^−/− and wild type mice.

To explore the alternate function of Aim2, we surveyed its impact on T cells because EAE is an adaptive immune disease. We found that Treg cells were reduced in Aim2^−/− mice, which may explain the exacerbated autoimmunity in these mice. Profiling of AIM2 expression in both humans and mice showed strong expression in T cells. In human cells, AIM2 expression in Treg cells is nearly an order of magnitude greater than its expression in inflammatory macrophages. To understand if this protein is important in Treg cells, we constructed a mouse with a Treg-specific Aim2 deletion, and the strain also exhibited enhanced EAE compared with its wild type control. Foxp3⁺ Tregs were lowered in the spinal cords of Aim2^−/− mice, but IFN-expressing cells were increased, suggesting that there may be a skewing from Tregs into Th1-type cells in the absence of Aim2. To address this point directly, we did a cell fate mapping study and found that Aim2 is important for the stabilization of Treg cells.

To explore the mechanism of action of Aim2 in Treg cells, we next performed RNA sequencing analysis. By gene set enrichment analysis, we revealed that AIM2 reduces mTOR, PI3K, AKT, glycolysis, and MYC, which is consistent with its role in maintaining Treg stability. Because these are immunometabolic pathways, we used the Seahorse assay to show that the presence of AIM2 resulted in decreased glycolysis, increased OXPHOS, and increased fatty acid oxidation. All of these features are consistent with immune metabolisms important for the maintenance of Treg cells. Finally, to define the specific biochemical mechanism, we did a mass spectrometry immunoprecipitation experiment with Dr. Xian Chen and showed that AIM2 in Treg cells interacts with RACK1, which then recruits PP2A to dephosphorylate AKT (37, 38). To show physiologic relevance, we showed that Tregs in Aim2^−/− mice had increased phosphorylated AKT, indicating that AIM2 suppresses the phosphorylation of AKT. Thus, this study shows that AIM2 is expressed by T cells, and it has an inflammasome-independent function achieved by the recruitment of RACK1 and PP2A to cause the blockage of AKT activation. This process leads to reduced glycolysis, increased OXPHOS, and increased fatty acid oxidation, thus increasing Treg cell stability.

Next, I will tell you about another innate immune receptor that affects T cells’ immune metabolism. Several years ago, Sumit Chanda’s laboratory screened a small interfering RNA library and showed that NLRX1 can promote HIV replication (39). We collaborated with his laboratory and showed in both human and mouse myeloid cells that NLRX1 promotes HIV replication by interfering with STING (40).

Since HIV primarily infects T cells and not myeloid cells, and T cells express NLRX1, we next investigated if NLRX1 also affects HIV infection of T cells (41). We performed a proteomics analysis of NLRX1-positive and -negative CD4 T cells. In the presence of NLRX1, the top altered proteins are those involved in OXPHOS. We further showed that HIV infection caused a change in OXPHOS, and this change is dependent on NLRX1, indicating that NLRX1 promotes OXPHOS during HIV infection of CD4 T cells. Furthermore, NLRX1 promotion of OXPHOS is relevant to HIV viral replication since metformin, a Food and Drug Administration–approved drug that inhibits OXPHOS, reduced HIV levels in an NLRX1-dependent fashion. Importantly, in humanized mice that have a human hematopoietic system and are permissive to HIV infection, metformin reduced HIV viral loads and increased CD4 T cells 1 log-fold, suggesting that this drug has the potential as a supplemental therapy for HIV.

To investigate the detailed mechanism, we resorted to a previous paper by Martin Dorf, which demonstrated that NLRX1 interacts with FASTKD5, a mitochondrial protein that is important for cell respiratory response, mitochondrial mRNA processing, and OXPHOS (42). We verified that NLRX1 interacts with FASTKD5 endogenously, and this interaction is important for their coregulation of OXPHOS. To explore the clinical relevance, we collaborated with Rafick Pierre Sekaly’s laboratory at Case Western Reserve University. In an analysis of the Early Capture HIV Cohort Study from the US military HIV research programs, his group interrogated data from patients in the very early stages of HIV infection from both Asia and Africa. These two patient cohorts revealed that increased OXPHOS gene expression is correlated positively with increased set-point viral load. Furthermore, increased NLRX1 is also associated with increased set-point viral load. In summary, the study showed that NLRX1 interacts with FASTKD5 to increase OXPHOS, which promotes HIV replication. Reducing OXPHOS with approved drugs can reduce HIV infection in T cells and may be considered as a supplemental treatment.

I would like to segue into the next theme on the role of microbiota, innate immune receptors, and diseases. I've already told you earlier that NLRP12 is an NLR protein that can attenuate colitis and colitis-associated colon cancer (17, 43). In our hands, NLRP12 inhibits noncanonical NF-κB activation in myeloid cells. By inhibiting this function, NLRP12 reduces the severity of colitis and colitis-associated colon cancer. Later, Liang Chen and Justin Wilson confirmed more severe colitis in Nlrp12^−/− mice compared with wild type controls in specific pathogen-free conditions, but these differences disappeared in germ-free facilities (44). Using Nlrp12^−/− mice and wild type littermates, we found that a group of bacteria called Lachnospiraceae was promoted by the presence of NLRP12. We then reconstituted Nlrp12^−/− mice with Lachnospiraceae and found that the bacteria significantly attenuated colitis, NF-κB and MAPK signaling, and several inflammatory cytokines, such as TNF, IL-6, and IL-1β. In another context, we found that Nlrp12^−/− mice gain weight under a high-fat diet, and Lachnospiraceae can reverse this weight gain such that their body fat is only 10% in bacteria-fed mice (45).

Finally, I will discuss the benefit of Lachnospiraceae in a third context: radiation damage. Radiation therapy is important in cancer treatment, but there are deep concerns regarding the detrimental impact of nuclear accidents and radiation used by terrorists. The National Institutes of Health specifically funded us to find therapies that can protect or mitigate against radiation damage.

During radiation, there are two components of damage during the initial exposure, resulting in the hematopoietic syndrome and the gastrointestinal syndrome. Hao Guo in the laboratory noticed that a selected group of mice survived long term (2 years) after an initial exposure to a high dose of radiation (41). She called these mice “Elite Survivors.” She then compared the microbiota makeup of age-matched aging mice and Elite Survivors and found that the two were distinct. The fecal microbiota from Elite Survivors had a greater diversity of bacteria compared with the age-matched controls. Fecal transplant studies showed that the fecal material from Elite Survivors protected both specific pathogen-free and germ-free recipients from radiation damage. Furthermore, recipients of the fecal material from Elite Survivors also contained bacteria that prevented radiation damage. This group of beneficial bacteria includes Lachnospiraceae, Enterococcaceae, as well as Lactobacillus. The reconstitution of these bacteria individually protected against both hematopoietic and gastrointestinal syndrome. Most impressive, this protection lasted for >300 d.

To link this finding to the clinic, we obtained patient samples from Duke University, Cornell-Weill, and Memorial Sloan Kettering. These samples were from patients exposed to radiation and were followed for the duration of their diarrhea as a side effect. We profiled the presence of Lachnospiraceae, Enterococcaceae, Lactobacillus, or these beneficial bacteria in combination. Interestingly, patients with shorter duration of diarrhea had increased Lachnospiraceae, Enterococcaceae, and Lactobacillus. Furthermore, there was an inverse relationship between Lachnospiraceae and the diarrhea duration, indicating that Lachnospiraceae can mitigate some of the side effects of radiation. Finally, these bacteria have no effect on tumor growth or tumor response to radiation. The last experiment demonstrates that the transferred bacteria do not promote tumor growth or response to radiation.

Next, we explored the mechanism downstream of Lachnospiraceae. Lachnospiraceae can produce short-chain fatty acids (SCFAs). We isolated the bacteria that made high and low amounts of SCFAs, and the former attenuated radiation damage significantly better than the latter. Furthermore, feeding mice SCFAs, specifically propionate, resulted in long-term prevention of radiation syndromes. In addition to SCFA, our collaborators, Kun Lu and Yunjia Lai, used an untargeted, unbiased metabolic study and found that the fecal metabolites of the Elite Survivors and age-matched controls were very distinct. The top metabolite group present in the Elite Survivors belongs to the tryptophan metabolic pathway. We tested two metabolites from the tryptophan pathway. These two tryptophan metabolites mitigated radiation damage and prolonged survival for the duration of the experiment for up to a year. In summary, we showed that there are beneficial bacteria, including Lachnospiraceae, Enterococcaceae, and Lactobacillus, that can mitigate radiation damage. Among these, Lactobacillus was identified by others as a radioprotecting strain (46). Downstream from these bacteria we found SCFAs, specifically propionate and tryptophan metabolites, enhanced both hematopoietic and intestinal recovery. In addition, we found that the protection lasted up to 2 years in some cases.

In conclusion, the encompassing importance of innate immunity is supported by our studies that demonstrate the role of innate immune receptors in infection, inflammation, autoimmune diseases, metabolic diseases, cancer promotion or control, vaccine adjuvant, microbiota determination, and T cell metabolism. We also showed that there are both positive and negative regulators of innate immunity.

Finally, I would like to thank all the people who have spent some time in our laboratory. The list has >150 people, and I just want to thank all of you for contributing to the science. I also wish to acknowledge our collaborators both at the University of North Carolina and outside of the university. I want to thank the research faculty, associate faculty, laboratory managers, technical staff, and administrative staff who keep our laboratory going, and the undergrads who have spent time in our laboratory. I have acknowledged different contributors throughout my talk. For the most recent projects, Weichun Chou from my group and Zengli Guo from Dr. Yisong Wang’s laboratory led the study on Aim2 in Treg cells with help from Xian Chen’s laboratory. Hao Guo led the work on microbiota and radiation damage in collaboration with our colleagues at Duke, Memorial Sloan Kettering, and Cornell. The bacteria were obtained from Vince Young’s laboratory. Haitao Guo led the NLRX1 and HIV work, which was done in collaboration with Qi Wang in Lishan Su’s laboratory and Khader Ghneim in Rafick-Pierre Sekaly’s group. I would also like to acknowledge Elena Rampenelli, who brought us the immune metabolism interest.

Finally, I want to thank my family, my two daughters, their husbands, my adorable grandson, and our extended family for their support. Thank you so much for your attention, and thank you again for electing me to serve.

The author is a cofounder of Immvention Therapeutix and Goldcrest Bio.