The innate immune system contains genome-encoded receptors that provide a first line of defense to infection. Activation of innate immunity triggers adaptive immunity. There are several classes of innate immune receptors, including Toll-like receptors (TLRs), which sense infectious agents in the extracellular/vesicular space; Nod-like receptors (NLRs), which sense microorganisms that penetrate the cytoplasmic space; RIG-like receptors (RLRs), which recognize viral infection and trigger type 1 interferon production; and DNA-sensing receptors. We identified the TLRs for double-stranded RNA (TLR3), single-stranded RNA (TLR7), flagellar protein (TLR5), and lipoprotein (TLR1/2) and have elucidated the roles of many NLRs.
Upon recognition of microbes in the cytoplasm, NLRs can trigger NF-κB activation, interleukin-1b (IL-1b) and IL-18 production, or pyroptosis. In humans, NLR mutations correlate with inflammatory diseases. One example is the NLR Nod2, which recognizes the bacterial cell-wall component muramyl dipeptide (MDP). After activation by MDP, Nod2 undergoes a conformational change that enables the interaction between the CARD domains of Nod2 and the downstream eceptor-interacting protein (RIP) kinase, resulting in activation of NF-κB and the production of antimicrobial peptides. NOD2 is mutated in Crohn's disease (CD), an inflammatory bowel disease (IBD). Our Nod2-deficient mice were more susceptible to infection with intestinal pathogens due to reduced production of antimicrobial peptides. This finding is consistent with the current model that patients with CD may be unable to develop an effective antimicrobial response, causing enhanced infection and severe inflammation.
The family of NLR proteins contains several more recently identified members. We study several of these proteins, including NLRP3 (Nalp3), which senses infection or tissue damage. Activation of NLRP3 initiates the formation of the "inflammasome" through oligomerization with the adapter apoptosis-associated speck-like protein (ASC), and caspase-1, which then leads to the processing and secretion of IL-1β, IL-18, and other substrates.
The elucidation of the activating ligand of NLRP3 has been rather complex; we found multiple stimuli that activate the NLRP3 inflammasome. Jürg Tschopp (University of Lausanne) showed that the NLRP3 inflammasome recognizes uric acid crystals, explaining the inflammatory properties of uric acid in gout; similarly, we found that hydroxyapatite crystals trigger an osteoarthritis-like disease through inflammasome activation. In addition, we found that alum, a crystalline immune adjuvant and the only USA-approved human adjuvant, activates NLRP3 and which triggers inflammatory cytokine production in macrophages and adaptive immunity in vivo. Disruption of the pathway eliminates alum's adjuvant capacity. Likewise, particulate environmental pollutants, including silica and asbestos, also activate the NLRP3 inflammasome to cause devastating chronic inflammatory diseases. Thus, inflammasomes mediate anti-infective immunity, response to necrotic cells, immunopathology to environmental pollutants, and adaptive immunity.
Our genomes encode some 20 or more NLRs, and only a small number of these have been characterized. We identified a new function for one unexplored NLR, known as NLRP6. We sought a mechanism whereby our innate immune system maintains homeostasis with the trillions of microorganisms that coexist with us at all of our surfaces, being most numerous in the intestines. By exposing inflammasome mutant mice to perturbation of gut epithelial integrity, we found that this new NLRP6 inflammasome activates the cytokine IL-18 through the adapter molecule ASC and protease caspase-1. In the absence of this pathway, the mouse is unable to control the microorganisms in the gut and dysbiosis results, leading to an overrepresentation of pathogenic species. This condition led to a heightened susceptibility of these mutant mice to IBD under the conditions tested. Even more surprisingly, the pathogenic bacteria were readily transmitted to normal mice that were simply housed in the same cage or were fostered by a mutant mother carrying this dysbiotic microbiota. The transmission of these microorganisms led to enhanced susceptibility to IBD. We also showed that this susceptibility could be eradicated by treatment with antibiotics or by attenuation of the inflammation that resulted from the microbial dysbiosis. Recently, we and our collaborators found that the NLRP6 system controls the generation of the inner mucus layer, which maintains the relatively microbe-free state of the gut epithelium. It is therefore clear how NLRP6 deficiency can lead to invasion of the epithelial crypts and dysbiosis.
We also sought to determine whether this remarkable inflammasome-regulated dysbiosis could contribute to disease beyond the intestines. Metabolic syndrome, a condition that plagues millions of people in the developed world and is associated with the Western high-fat diet, affects diverse tissues mediating obesity, nonalcoholic fatty liver disease (NAFLD), type 2 diabetes, and heart disease. We found that mice lacking this crucial NLRP6 inflammasome pathway are more sensitive to the development of NAFLD, are more obese, and exhibit the hallmarks of type 2 diabetes. All of these effects can be attenuated by treatment with antibiotics. Moreover, fatty liver disease, obesity, and diabetes could be transmitted from a dysbiotic host to a wild-type mouse. These studies suggest an interaction between our genes, our diet, and the microbiota that inhabit our bodies. Susceptible genotypes, such as deficiency in the NLRP6 pathway, lead to an altered microbiota (which is also impacted by diet), causing, in turn, inflammation. Mice exposed to this kind of double hit develop metabolic syndrome. It will be important to determine to what degree these results are relevant to human disease.
The aforementioned studies, as well as the work of many others, revealed that select members of the intestinal microbiota preferentially drive disease development in mice. However, the identification of disease-driving bacteria in humans has remained challenging and it has therefore been difficult to determine if the changes observed in microbes are a cause or effect of the human disease. We recently developed a new strategy to identify specific members of the human intestinal microbiota that selectively impact disease. In this work, we used the intestinal Immunoglobulin A (IgA) response to the microbiota as a guide to identify members of the microbiota that preferentially interact with and stimulate inflammatory responses in the intestine. After identifying such bacteria in patients with inflammatory bowel disease (IBD), we used gnotobiotic mouse models to demonstrate that bacteria identified in this manner selectively confer susceptibility to colitis. These studies show that a functional classification of the bacterial members of the microbiota based on the immune response can specifically and selectively identify disease-driving and non-disease-driving members of the microbiota. Targeted elimination of such disease-driving bacteria may reduce, reverse, or even prevent disease development. Likewise, these results suggest that specific IgA coated bacteria in human IBD patients may well contribute to morbidity in these diseases.
Our lab has a long-standing interest in the mechanisms by which caspases induce apoptotic cell death. It has long been known that apoptosis is an immunologically silent form of cell death, but the molecular basis of this is unknown. Mitochondria play a central role in the induction of cell death, as well as in immune signaling pathways. We identified a mechanism by which mitochondria and downstream proapoptotic caspases regulate the activation of a cell-intrinsic immune response. Upon permeabilization of mitochondrial outer membrane by Bax and Bak in response to a proapoptotic stimulation, the cGAS/STING pathway is activated by mitochondrial DNA (mtDNA) and induces the expression of type I interferons, resulting in the establishment of a potent state of viral resistance. In parallel, the Bax/Bak-dependent release of cytochrome c in the cytosol induces the well-described activation of caspase-dependent apoptotic cell death. Our results demonstrate that caspase activity is also required to inhibit the mtDNA-dependent induction of type I interferons, thus maintaining the immunologically silent nature of apoptosis. These results demonstrate the pro-inflammatory potential of mtDNA, capable of inducing a highly regulated cell-intrinsic immune response in the context of cell death.
Effector T cells differentiate from naive precursor cells. Recent work has expanded the number of effective T cell subsets from Th1 and Th2—which make, respectively, interferon-γ and the IL-4 family of cytokines—to the Th17 pathway, which develops as a consequence of exposure of the animal to certain microbiota. These Th17 cells make the cytokines IL-17A and IL-17F, as well as IL-21 and IL-22. In recent years, it has become increasingly clear that Th17 cells not only mediate many of the antimicrobial effects against extracellular bacteria and fungi but also mediate the pathogenic effects seen in autoimmune diseases and IBD. We have analyzed the function of cytokines of this lineage and, counterintuitively, have found that IL-17A and IL-22 can play an important protective role under the conditions of IBD, both through inhibiting Th1 CD4 T cells and by providing a protective effect on the gut epithelium, thereby repairing the damage mediated through the inflammatory assault on this structure.
Protective therapies against autoimmunity and autoinflammation are ideally directed toward the establishment of a state of immune tolerance whereby the immune system is returned to homeostasis and autodestruction is halted. We have examined the way in which tolerogenic mechanisms impact these Th17 cells, which can be either pathogenic or protective, depending on the circumstances. We treated mice with therapeutic anti-CD3 mAb, the protein involved in the establishment of the T cell receptor signaling complex, and found that this therapeutic treatment led to the diversion of Th17 cells, which would normally mediate damage, to the duodenum. There, these cells encountered an anti-inflammatory environment and through this, their pathogenicity was controlled. Indeed, two kinds of regulatory T cells are found in the intestine, classical FoxP3+ Tregs and a second class of FoxP3Neg Treg known as Tr1 cells, which are believed to play an important role in maintaining immune homeostasis. Because of their ability to produce IL-10, Tr1 cells are potent regulators of Th17 cells in the gut. However, the origin of Tr1 cells has been unclear. The acquisition of IL-10 expression by some Th17 cells in the gut stimulated us to consider a new hypothesis: Could transdifferentiation into regulatory cells be the physiologic "endgame" for effector T cells and curtail the effector immune response? Thus, in this specific case, Th17 cells might differentiate into Tr1 cells as a way of terminating the Th17 effector program? To test this, we used cytokine "fate" reporter animals to show that Th17 cells, selectively tagged by Rosa26 YFP, convert into Foxp3 Treg cells and Tr1 cells that lost expression of IL–17A and express Foxp3 and IL-10, respectively. This was not simply a switch between expression of two cytokine genes, IL-17A and IL-10, but rather transdifferentiation of one T cell type into another through reprogramming of many genes. This conversion occurred during tolerogenic therapy, infection, and in the steady state. Thus, we believe that transdifferentiation into regulatory cells is a physiologic process that attenuates immunity once the infectious challenge is eliminated; this also provides a conceptual context explaining at least some Tr1 cells as a terminal stage of T cell differentiation. We are interested in the fate of other Th cells and their relationship to regulatory T cells.
In our studies of how these T cell genes are regulated, we identified cis-regulatory elements that are the targets of transcription factors, such as GATA3. In the interleukin-4 (IL-4) locus, the IL-4, IL-13, and IL-5 genes are clustered, and several DNA elements within that region are important for gene expression. IL-4 gene regulation occurs through epigenetic mechanisms that target regulatory elements distal from the IL-4 gene. One of these elements is a previously unrecognized locus control region (LCR) that is found embedded in the introns of the RAD50 gene in the cluster. This LCR, together with these respective promoters and other cis elements of the locus, is in a preassembled complex in naive T cells that serves as a hub from which epigenetic changes in histone acetylation and DNA methylation occur and enables rapid response of the loci. LCRs are members of what are now called “super-enhancers.”
When naive T cells are activated, both the IL-4 locus on chromosome 11 and the interferon-γ (IFN-γ) locus on chromosome 10 are expressed almost immediately, despite the fact that following differentiation these loci are never coexpressed but instead are alternatively expressed in the Th2 and Th1 lineages, respectively. To investigate this rapid coexpression, we examined the physical relationship between these two loci on the different chromosomes. The LCR of the IL-4 locus on chromosome 11 and the IFN-γ gene region on chromosome 10 are associated in the interphase nucleus of the precursor cells but separate upon differentiation into effector cells. Mutation in the LCR on chromosome 11 delays expression of the IFN-γ gene on chromosome 10. We find other such associations and further evidence for their functional roles. Thus, regulatory sequences on one chromosome likely control "in trans" gene expression on other chromosomes. A similar association exists between the LCR and the TNF-α and IL17 loci, on chromosomes 17 and 1, respectively. We showed recently that the association is dependent on CTCF and the transcription factor Oct1.
Studies of mouse immunity have contributed immensely to the understanding of human immunity and disease. Immune systems evolve rapidly, however, and as a result, mouse and human immunity differ in many properties. Humanized mice offer an in vivo system populated with a human immune system where invasive studies of relevance to human immunobiology can be performed. Indeed, we have found that mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models are unable to support development of human innate immune cells, including myeloid cells and natural killer cells. Over the past nine years, we have developed a new approach to humanized mice by tailoring the mouse genome to this purpose by replacing mouse genes with their human counterparts to provide species-specific factors enabling a robust human hematopoietic system. Recently, we have combined several of these engineered genes into a new mouse strain, called MI(S)TRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked in to their respective mouse loci. The human cytokines support the development and function of monocytes/macrophages and natural killer cells derived from human fetal liver or adult CD34+ progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MI(S)TRG mice in a manner resembling that observed in tumors obtained from human patients.
In addition, human cytokines improve the reconstitution of the human immune system derived from adult CD34+ progenitor cells. Humanized MI(S)TRG mice engrafted with bone marrow CD34+ progenitor cells from HIV patients developed a patient-specific immune system. HIV infected patient-derived MI(S)TRG mice supported HIV-1 infection and reflected patient-specific anti-HIV immune responses which interestingly led to viral clearance.
This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and it may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.
Grants from the National Institutes of Health, the Bill and Melinda Gates Foundation, the Blavatnik Family Foundation, the Juvenile Diabetes Research Foundation, and the American Diabetes Association provided partial support for these projects.
As of April 7, 2016