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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-strand 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 probably 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. Thus, 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 other stress that leads to K+ efflux and activates the "inflammasome" through oligomerization with the adaptor 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, including Listeria infection. 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, immunopathology to environmental pollutants, and adaptive immunity.
The immune response sometimes reacts to self-tissues, causing autoimmunity. How can antigenic stimulation of a lymphocyte lead to such different outcomes? During an immune response or in autoimmunity, the lymphocytes divide and differentiate into effector cells. However, when immune tolerance occurs, the cell is either inactivated or dies. How are the decisions made to proliferate, differentiate, be tolerized, or die, and how is this controlled? Regulatory cells producing inhibitory cytokines are critical to prevent autoimmunity. Of these, the CD4+CD25+Foxp3+Treg is the most studied. The functioning, generation, and maintenance of regulatory T cells (Treg) are controlled by cytokines. Both transforming growth factor-β (TGFβ) and IL-10-family cytokines are important. Mice lacking TGFβ develop autoimmunity to several tissues. To elucidate upon which cells TGFβ acts, we expressed a dominant-negative TGFβ receptor (dnTGFβRII) on either T cells or antigen-presenting cells (APCs). Mice displaying the dnTGFβRII on T cells recapitulate the diseases of TGFβ-knockout mice: autoimmunity and IBD. In addition to autoimmunity, such animals have an enhanced anti-infective response, better resistance to infection. Finally, mice carrying the dnTGFβRII on their T cells are resistant to tumors. Thus, tumors use TGFβ to inhibit the antitumor T cell response; but if TGFβ cannot act, immune clearance of tumors occurs.
To determine whether TGFβ controls innate immunity, we expressed dnTGFβRII using the CD11c promoter, which expresses in dendritic (DC) and natural killer (NK) cells, both key mediators of innate immunity. When innate immune cells cannot be inhibited by TGFβ, both NK and DC innate, as well as adaptive, immune responses are enhanced. CD11c dnTGFβRII mice are also more susceptible to autoimmunity, because TGFβ fails to control APC function. Thus, TGFβ controls T cells, APCs, and NK cells.
We revealed additional mechanisms of TGFβ function by studying conditional-knockout mice lacking TGFβRII on T cells. TGFβ is required for Treg homeostasis and function and TGFβRII must be present on a target cell for a Treg to be suppressed. We also found that TGFβ controls the magnitude of T helper 1 cell (Th1) response by setting the level of CD122 β chain of the IL-15 receptor, which controls the pool size of Th1 cells. Many cells make TGFβ. To determine which TGFβ source is important, we first eliminated TGFβ on T cells, using conditional targeting. Mice with T cells that cannot make TGFβ also developed autoimmune disease and IBD, albeit slower than mice lacking the receptor on all T cells. Thus, T cell–produced TGFβ is important in immune response, but other sources must play a role. Regulatory T cells that cannot produce TGFβ poorly control IBD, and T cell–produced TGFβ is essential to generate Th17 cells, which mediate disease in experimental autoimmune encephalomyelitis.
T cells are activated and differentiate into specialized effector cells. How is the effector pathway triggered that is appropriate to the class of infection? We found the Th2 response is activated when parasite antigen induces Notch ligand expression on dendritic cells. This activates Notch in naïve T cells, which in turn induces GATA3, the key Th2 transcription factor, by a Notch-responsive promoter. Thus is a pathogenic signal converted to a signal for T cell differentiation through Notch.
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.
Our laboratory retains a long-standing interest in the underlying mechanisms of apoptosis. The program of cell death is triggered through the activation of cysteine proteases called caspases. Caspase-3 and -7 cleave similar substrates. Caspase-7–knockout mice have only a mild phenotype, but the combination with caspase-3 deficiency results in embryonic lethality. Caspase-3 and -7 are also required for upstream mitochondrial functions in apoptosis, via a positive-feedback loop, in addition to their roles as effector caspases.
Last updated: August 22, 2008
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