SUMMARY

Although Toll-like receptor 9 (TLR9) has been implicated in regulating cytokine and type I interferon (IFN) production during malaria in humans and mice, the high AT content of the Plasmodium falciparum genome prompted us to examine the possibility that malarial DNA triggered TLR9-independent DNA sensing pathways. Over 6000 ATTTTTAC (“AT-rich”) motifs are present in the genome of P. falciparum, which we show here potently induce type I IFNs. Parasite DNA, parasitized erythrocytes and oligonucleotides containing the AT-r motif induce type I IFNs via a pathway that did not involve previously described sensors including TLR9, DAI, RNA polymerase-III or IFI16/p204. Rather, AT-rich DNA sensing involved an unknown receptor that coupled to STING, TBK1 and IRF3-IRF7 signaling pathway. Mice lacking both IRF3 and IRF7, the kinase TBK1 or the type I IFN receptor were resistant to otherwise lethal cerebral malaria. Collectively, these observations implicate AT-rich DNA sensing via STING, TBK1 and IRF3-IRF7 in P. falciparum malaria.

Inflammasomes regulate the activity of capase-1 and maturation of interleukin-1β and interleukin-18. Recently, AIM2 was shown to bind DNA and engage ASC to form a caspase-1 activating inflammasome. Using Aim2-deficient mice, we reveal a central role for AIM2 in regulating caspase-1-dependent maturation of IL-1β and IL-18, as well as pyroptosis in response to synthetic dsDNA. AIM2 is essential for inflammasome activation in response to Fransicella tularensis, vaccinia virus, mouse cytomegalovirus and plays a partial role in sensing Listeria monocytogenes. Moreover, production of IL-18 and NK cell-dependent IFN-γ production, events critical in early control of virus replication were dependent on AIM2 during mCMV infection in vivo. Collectively, these observations reveal the importance of AIM2 in sensing both bacterial and viral pathogens and triggering innate immunity.

Human Metapneumoviruses (HMPV) are recently identified Paramyxoviridae that contribute to respiratory tract infections in children. No effective treatments or vaccines are available. Successful defense against virus infection relies on early detection by germline encoded pattern recognition receptors and activation of cytokine and type I interferon genes. Recently, the RNA helicase Retinoic acid inducible gene (RIG-I) has been shown to sense HMPV. In this study, we investigated the ability of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I interferons (IFN). Despite the ability of both HMPV-A1 and B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I-mediated sensing of HMPV viral 5’ triphosphate RNA. In contrast to most cell types, plasmacytoid dendritic cells (PDC) displayed a unique ability to sense both the A1 and B1 strains and in this case sensing was via Toll-like receptor (TLR)-7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell-type specific manners.

Interferon regulatory factors (IRFs) are essential in the innate immune response and other physiological processes. Activation of these proteins in the cytoplasm is triggered by phosphorylation of Ser/Thr residues in a C-terminal autoinhibitory region, which stimulates dimerization, transport into the nucleus, assembly with the coactivator CBP/p300 and initiation of transcription. The novel crystal structure of the transactivation domain of pseudophosphorylated human IRF5 reveals a striking dimer in which the bulk of intersubunit interactions involve a highly extended C-terminal region. The corresponding region has previously been shown to block CBP/p300 binding to unphosphorylated IRF3. Mutation of key interface residues supports the observed dimer as the physiologically activated state of IRF5 and IRF3. Thus phosphorylation likely activates IRF5 and other family members by triggering remarkable conformational rearrangements that switch the C-terminal segment from an autoinihibitory to a dimerization role.

While the recognition of microbial infection often occurs at the cell surface via Toll-like receptors, the cytosol of the cell is also under surveillance for microbial products that breach the cell membrane. An important outcome of cytosolic recognition is the induction of IFNα and IFNβ, which are critical mediators of immunity against both bacteria and viruses. Like many intracellular pathogens, a significant fraction of the transcriptional response to Mycobacterium tuberculosis infection depends on these type I interferons, but the recognition pathways responsible remain elusive. In this work, we demonstrate that intraphagosomal M. tuberculosis stimulates the cytosolic Nod2 pathway that responds to bacterial peptidoglycan, and this event requires membrane damage that is actively inflicted by the bacterium. Unexpectedly, this recognition triggers the expression of type I interferons in a Tbk1- and Irf5-dependent manner. This response is only partially impaired by the loss of Irf3 and therefore, differs fundamentally from those stimulated by bacterial DNA, which depend entirely on this transcription factor. This difference appears to result from the unusual peptidoglycan produced by mycobacteria, which we show is a uniquely potent agonist of the Nod2/Rip2/Irf5 pathway. Thus, the Nod2 system is specialized to recognize bacteria that actively perturb host membranes and is remarkably sensitive to mycobacteria, perhaps reflecting the strong evolutionary pressure exerted by these pathogens on the mammalian immune system.

Author Summary

Bacterial and viral infection stimulates production of several cytokines and chemokines that are thought to protect the host against infection. The bacterial strain known to cause tuberculosis elicits production of type I interferons, yet it was unclear how the bacteria isolated within the cell was capable of stimulating this host response. This study reveals that the bacteria use a specialized system to cause damage to these cellular compartments and release bacterial products that activate intracellular innate immune pathways. In this work, we demonstrate that Nod2, Rip2, Tbk-1, Irf3 and Irf5 proteins cooperate to produce type I interferons. Understanding how these pathways are mediated is likely to aid in the design of more effective tuberculosis vaccines.