Genetic Approaches to Immune Function and Tolerance
Summary: Richard Flavell uses mouse reverse genetics to study innate and adaptive immunity, T cell tolerance, apoptosis and autoimmunity, and the regulation of T cell differentiation.
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 three classes of innate immune receptors: Toll-like receptors (TLRs), which sense agents in the extracellular/vesicular space; Nod-like receptors (NLRs), which sense microorganisms that penetrate the cytoplasmic space; and RIG-like receptors (RLRs), which recognize viral infection and trigger type 1 interferon production. We identified the TLRs for double-stranded RNA (TLR3), single-stranded RNA (TLR7), flagellar protein (TLR5), and lipoprotein (TLR1/2).
Upon penetration of the cytoplasm, NLRs trigger NFκB activation, interleukin-1 (IL-1) production, or apoptosis. In humans, NLR mutation correlates with inflammatory disease. Nod2 carries a leucine-rich repeat region recognizing bacterial muramyl dipeptide, a nucleotide-binding domain mediating conformational change, thereby enabling oligomerization between CARD domains of Nod2 and downstream receptor-interacting protein (RIP) kinase, causing activation of NFκB and 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 pathogens delivered to the gut, because of reduced production of antimicrobial peptides. This 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 Nalp proteins comprise a second arm of the NLR family. We study several of these, including Nalp3 (NLRP3), which senses infection or tissue damage that activates the "inflammasome" through oligomerization with the adapter apoptosis-associated speck-like protein (ASC), enabling ASC to bind to and activate caspase-1 to process pro-IL-1β and other substrates.
We found that multiple stimuli activate the Nalp3 inflammasome. Jürg Tschopp (University of Lausanne) showed that this inflammasome recognizes uric acid crystals, explaining the inflammatory properties of uric acid in gout. We found that alum, a crystalline immune adjuvant and the only USA-approved human adjuvant, activates the NALP3 inflammasome, which triggers macrophage inflammatory cytokine production 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 Nalp3 inflammasome to cause devastating chronic inflammatory disease. 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 have recently identified a new function for one unexplored NLR known as NLRP6. We sought a mechanism whereby our innate immune system maintains homeostasis with the billions 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 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 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.
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, many of these features of metabolic syndrome, including fatty liver disease and obesity, could simply be transmitted from a dysbiotic host to a wild-type mouse by housing the mice in the same cage and feeding them the same susceptible diet. 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.
Effector T cells differentiate from naïve 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 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 protocols impact these Th17 cells, which can be either pathogenic or protective, depending on circumstances. We treated mice with therapeutic anti-CD3, 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 were converted to a regulatory form of Th17 cells that make the protective anti-inflammatory cytokine IL-10. This molecule is able to control autoaggressive Th17 cells as these cells have the unique property that they express the IL-10 receptor. We showed that this transdifferentiation lent these regulatory Th17 cells the ability to prevent disease both in vitro and in vivo in a variety of model systems. We are now studying the relationships of the individual cell types that develop during disease development and tolerization. Our goal is to determine the relative importance of plasticity on the one hand and lineage commitment of effector T cells on the other.
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 naïve 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.
When naïve 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.
Grants from the National Institutes of Health, the Bill and Melinda Gates Foundation, the Juvenile Diabetes Research Foundation, and the American Diabetes Association provided partial support for these projects.
As of May 24, 2012