miniMAVS, You Complete Me!

The functional significance of protein diversification through translational regulation in mammals is largely unexplored. Brubaker et al. now describe the generation of two functionally distinct mammalian proteins, MAVS and miniMAVS, from a single bicistronic mRNA and suggest that noncanonical translation may impact multiple players in innate immune regulation.

Caspase-7 activates ASM to repair gasdermin and perforin pores.

Among the caspases that cause regulated cell death, a unique function for caspase-7 has remained elusive. Caspase-3 performs apoptosis, whereas caspase-7 is typically considered an inefficient back-up. Caspase-1 activates gasdermin D pores to lyse the cell; however, caspase-1 also activates caspase-7 for unknown reasons1. Caspases can also trigger cell-type-specific death responses; for example, caspase-1 causes the extrusion of intestinal epithelial cell (IECs) in response to infection with Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium)2,3. Here we show in both organoids and mice that caspase-7-deficient IECs do not complete extrusion. Mechanistically, caspase-7 counteracts gasdermin D pores and preserves cell integrity by cleaving and activating acid sphingomyelinase (ASM), which thereby generates copious amounts of ceramide to enable enhanced membrane repair. This provides time to complete the process of IEC extrusion. In parallel, we also show that caspase-7 and ASM cleavage are required to clear Chromobacterium violaceum and Listeria monocytogenes after perforin-pore-mediated attack by natural killer cells or cytotoxic T lymphocytes, which normally causes apoptosis in infected hepatocytes. Therefore, caspase-7 is not a conventional executioner but instead is a death facilitator that delays pore-driven lysis so that more-specialized processes, such as extrusion or apoptosis, can be completed before cell death. Cells must put their affairs in order before they die.

The RIP1-RIP3 complex initiates mitochondrial fission to fuel NLRP3.

Vesicular stomatitis virus, a single-stranded RNA virus, triggers activation of the serine-threonine kinases RIP1 and RIP3, which damages mitochondria by activating the GTPase DRP1. This results in excessive production of reactive oxygen species and subsequent activation of the NLRP3 inflammasome.

The Prostaglandin E2-EP3 Receptor Axis Regulates Anaplasma phagocytophilum-Mediated NLRC4 Inflammasome Activation.

Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1ß and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum.

Cutting edge: Mouse NAIP1 detects the type III secretion system needle protein.

The NAIP/NLRC4 inflammasomes activate caspase-1 in response to bacterial type III secretion systems (T3SSs). Inadvertent injection of the T3SS rod protein and flagellin into the cytosol is detected through murine NAIP2 and NAIP5/6, respectively. In this study, we identify the agonist for the orphan murine NAIP1 receptor as the T3SS needle protein. NAIP1 is poorly expressed in resting mouse bone marrow-derived macrophages; however, priming with polyinosinic-polycytidylic acid induces it and confers needle protein sensitivity. Further, overexpression of NAIP1 in immortalized bone marrow-derived macrophages by retroviral transduction enabled needle detection. In contrast, peritoneal cavity macrophages basally express NAIP1 and respond to needle protein robustly, independent of priming. Human macrophages are known to express only one NAIP gene, which detects the needle protein, but not rod or flagellin. Thus, murine NAIP1 is functionally analogous to human NAIP.

Production of a p65fl/fl/LysMCre mouse model with dysfunctional NF-κB signaling in bone marrow-derived macrophages.

Here, we describe the production and characterization of a novel p65fl/fl/LysMCre mouse model, which lacks canonical nuclear factor-kappaB member RelA/p65 (indicated as p65 hereafter) in bone marrow-derived macrophages. Cultured bone marrow-derived macrophages that lack p65 protein reveal NF-κB signaling deficiencies, a reduction in phagocytic ability, and reduced ability to produce nitrites. Despite abnormal bone marrow-derived macrophage function, p65fl/fl/LysMCre mice do not exhibit differences in naïve systemic immune profiles or colony forming units and time to death following Salmonella infection as compared to controls. Additionally, p65fl/fl/LysMCre mice, especially females, display splenomegaly, but no other obvious physical or behavioral differences as compared to control animals. As bone marrow-derived macrophages from this transgenic model are almost completely devoid of canonical nuclear factor-kappaB pathway member p65, this model has the potential for being very useful in investigating bone marrow-derived macrophage NF-kappaB signaling in diverse biological and biomedical studies.

Programmed cell death as a defence against infection.

Eukaryotic cells can die from physical trauma, which results in necrosis. Alternatively, they can die through programmed cell death upon the stimulation of specific signalling pathways. In this Review, we discuss the role of different cell death pathways in innate immune defence against bacterial and viral infection: apoptosis, necroptosis, pyroptosis and NETosis. We describe the interactions that interweave different programmed cell death pathways, which create complex signalling networks that cross-guard each other in the evolutionary 'arms race' with pathogens. Finally, we describe how the resulting cell corpses - apoptotic bodies, pore-induced intracellular traps (PITs) and neutrophil extracellular traps (NETs) - promote the clearance of infection.

Detection of pyroptosis by measuring released lactate dehydrogenase activity.

Pyroptosis is a form of programmed, inflammatory cell death that is dependent on the activation of a cysteine protease caspase-1. Following caspase-1 activation via inflammasomes (including NLRP3, NLRC4, Nlrp1b, and AIM2), cells lose membrane integrity and lyse, releasing lactate dehydrogenase (LDH) that is normally maintained within the cell cytosol. Thus, pyroptosis is distinct from apoptosis, which results in cellular contents being enclosed within membrane blebs during cellular demise. LDH is only released from apoptotic blebs after secondary necrosis occurs. Pyroptosis is distinct from necrosis in that it requires the activity of caspase-1. In this chapter, we describe enzymatic assays for the detection of LDH released by pyroptotic cells using a commercially available kit, as well as a simple and cost-effective method adapted from Decker et al. (J Immunol Methods 115:61-69, 1988).

Non-surgical intratracheal instillation of mice with analysis of lungs and lung draining lymph nodes by flow cytometry.

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an appropriate immune response to a variety of immunogens. To model interactions between the lungs and airborne antigens in mice, antigens can be administered intranasally, intratracheally or as aerosols. Delivery by each route involves distinct technical skills and limitations that need to be considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory tract. Hence antigens can access the nasal associated lymphoid tissue (NALT), potentially complicating interpretation of the results. In addition, swallowing, sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

Antagonistic crosstalk between type I and II interferons and increased host susceptibility to bacterial infections.

Type I and II interferons (IFNs αß and γ) have opposing effects on immune resistance to certain pathogenic bacteria. While IFNγ generally plays a protective role, IFNαß exacerbates Listeria monocytogenes and Mycobacterium tuberculosis infections. Our findings provided evidence that this increased susceptibility reflects a novel antagonistic cross talk between IFNαß and IFNγ. Macrophages infected with L. monocytogenes strains that induce IFNαß production responded poorly to IFNγ, as measured by reduced phosphorylation of STAT1 and reduced IFNγ-dependent gene expression. The impaired responsiveness to IFNγ correlated with reduced expression of its receptor, IFNGR, by both infected and bystander macrophages. Down regulation of IFNGR was dependent on responsiveness to IFNγ and mimicked by recombinant IFNß. Mice lacking responsiveness to IFNαß (IFNAR1 (-/-)) retained high IFNGR expression, developed higher expression of MHC-II on macrophages and DCs, and were more resistant to systemic L. monocytogenes infection--but only in the presence of IFNγ. Thus, the ability of IFNαß to down regulate IFNGR provides an explanation for its ability to reduce responsiveness to IFNγ and to increase host susceptibility to bacterial infection. It remains to be determined whether and how such antagonistic interferon crosstalk benefits the host.

Induction of IFN-alphabeta enables Listeria monocytogenes to suppress macrophage activation by IFN-gamma.

Production of type I interferon (IFN; IFN-alphabeta) increases host susceptibility to Listeria monocytogenes, whereas type II IFN (IFN-gamma) activates macrophages to resist infection. We show that these opposing immunological effects of IFN-alphabeta and IFN-gamma occur because of cross talk between the respective signaling pathways. We found that cultured macrophages infected with L. monocytogenes were refractory to IFN-gamma treatment as a result of down-regulation of the IFN-gamma receptor (IFNGR). The soluble factor responsible for these effects was identified as host IFN-alphabeta. Accordingly, macrophages and dendritic cells (DCs) showed reduced IFNGR1 expression and reduced responsiveness to IFN-gamma during systemic infection of IFN-alphabeta-responsive mice. Furthermore, the increased resistance of mice lacking the IFN-alphabeta receptor (IFNAR(-/-)) to L. monocytogenes correlated with increased expression of IFN-gamma-dependent activation markers by macrophages and DCs and was reversed by depletion of IFN-gamma. Thus, IFN-alphabeta produced in response to bacterial infection and other stimuli antagonizes the host response to IFN-gamma by down-regulating the IFNGR. Such cross talk permits prioritization of IFN-alphabeta-type immune responses and may contribute to the beneficial effects of IFN-beta in treatment of inflammatory diseases such as multiple sclerosis.