Louise H. Cengia

RIPK1 Regulates RIPK3-MLKL-Driven Systemic Inflammation and Emergency Hematopoiesis

Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.

Inflammation and autoimmunity caused by a SHP1 mutation depend on IL-1, MyD88, and a microbial trigger.

A recessive phenotype called spin (spontaneous inflammation) was induced by N-ethyl-N-nitrosourea (ENU) mutagenesis in C57BL/6J mice. Homozygotes display chronic inflammatory lesions affecting the feet, salivary glands and lungs, and antichromatin antibodies. They are immunocompetent and show enhanced resistance to infection by Listeria monocytogenes. TLR-induced TNF and IL-1 production are normal in macrophages derived from spin mice. The autoinflammatory phenotype of spin mice is fully suppressed by compound homozygosity for Myd88poc, Irak4otiose, and Il1r1-null mutations, but not Ticam1Lps2, Stat1m1Btlr, or Tnf-null mutations. Both autoimmune and autoinflammatory phenotypes are suppressed when spin homozygotes are derived into a germ-free environment. The spin phenotype was ascribed to a viable hypomorphic allele of Ptpn6, which encodes the tyrosine phosphatase SHP1, mutated in mice with the classical motheaten alleles me and me-v. Inflammation and autoimmunity caused by SHP1 deficiency are thus conditional. The SHP1-deficient phenotype is driven by microbes, which activate TLR signaling pathways to elicit IL-1 production. IL-1 signaling via MyD88 elicits inflammatory disease.

Fas-mediated neutrophil apoptosis is accelerated by Bid, Bak, and Bax and inhibited by Bcl-2 and Mcl-1

During immune responses, neutrophils must integrate survival and death signals from multiple sources to regulate their lifespan. Signals that activate either the Bcl-2- or death receptor-regulated apoptosis pathways can provide powerful stimuli for neutrophils to undergo cell death, but whether they act cooperatively in parallel or directly cross-talk in neutrophils is not known. Previous studies suggested that Bcl-2 family proteins are not required for Fas-induced cell death in neutrophils, but did not examine whether they could modulate its rapid onset. By monitoring the rate of change in neutrophil viability associated with activation of the Fas-triggered death receptor pathway using real-time cell imaging, we show that the Bcl-2-related proteins Bid, Bax, and Bak accelerate neutrophil apoptosis but are not essential for cell death. Increased Bcl-2 or Mcl-1 expression prevents efficient induction of apoptosis by Fas stimulation indicating that the Bcl-2-regulated apoptosis pathway can directly interfere with Fas-triggered apoptosis. Fas has been shown to initiate NFκB activation and gene transcription in cell lines, however gene transcription is not altered in Fas-activated Bid−/− neutrophils, indicating that apoptosis occurs independently of gene transcription in neutrophils. The specification of kinetics of neutrophil apoptosis by Bid impacts on the magnitude of neutrophil IL-1β production, implicating a functional role for the Bcl-2-regulated pathway in controlling neutrophil responses to FasL. These data demonstrate that the intrinsic apoptosis pathway directly controls the kinetics of Fas-triggered apoptosis in neutrophils.

Regulation of interleukin-1β by interferon-γ is species specific, limited by suppressor of cytokine signalling 1 and influences interleukin-17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.

IL-18 Production from the NLRP1 Inflammasome Prevents Obesity and Metabolic Syndrome

Interleukin-18 (IL-18) is activated by Caspase-1 in inflammasome complexes and has anti-obesity effects; however, it is not known which inflammasome regulates this process. We found that mice lacking the NLRP1 inflammasome phenocopy mice lacking IL-18, with spontaneous obesity due to intrinsic lipid accumulation. This is exacerbated when the mice are fed a high-fat diet (HFD) or a high-protein diet, but not when mice are fed a HFD with low energy density (high fiber). Furthermore, mice with an activating mutation in NLRP1, and hence increased IL-18, have decreased adiposity and are resistant to diet-induced metabolic dysfunction. Feeding these mice a HFD further increased plasma IL-18 concentrations and strikingly resulted in loss of adipose tissue mass and fatal cachexia, which could be prevented by genetic deletion of IL-18. Thus, NLRP1 is an innate immune sensor that functions in the context of metabolic stress to produce IL-18, preventing obesity and metabolic syndrome.

IL-6 promotes acute and chronic inflammatory disease in the absence of SOCS3

The lack of expression of the suppressor of cytokine signalling‐3 (SOCS3) or inactivation of the negative regulatory capacity of SOCS3 has been well documented in rheumatoid arthritis, viral hepatitis and cancer. The specific qualitative and quantitative consequences of SOCS3 deficiency on interleukin‐6 (IL‐6)‐mediated pro‐ and anti‐inflammatory responses remain controversial in vitro and unknown in vivo. Mice with a conditional deletion of SOCS3 in hematopoietic cells develop lethal inflammatory disease during adult life and develop gross histopathological changes during experimental arthritis, typified by elevated IL‐6 levels. To clarify the nature of the IL‐6 responses in vivo, we generated mice deficient in SOCS3 (SOCS3−/Δvav) or both SOCS3 and IL‐6 (IL‐6−/−/SOCS3−/Δvav), and examined responses in models of acute and chronic inflammation. Acute responses to IL‐1β were lethal to SOCS3−/Δvav mice but not IL‐6−/−/SOCS3−/Δvav mice, indicating that IL‐6 was required for the lethal inflammation induced by IL‐1β. Administration of IL‐1β to SOCS3−/Δvav mice induced systemic apoptosis of lymphocytes in the thymus, spleen and lymph nodes that was dependent on the presence of IL‐6. IL‐6 deficiency prolonged survival of SOCS3−/Δvav mice and ameliorated spontaneous inflammatory disease developing during adult life. Infection of SOCS3−/Δvav mice with LCMV induced a lethal inflammatory response that was dependent on IL‐6, despite SOCS3−/Δvav mice controlling viral replication. We conclude that SOCS3 is required for survival during inflammatory responses and is a critical regulator of IL‐6 in vivo.

Neutrophils Require SHP1 To Regulate IL-1β Production and Prevent Inflammatory Skin Disease

The regulation of neutrophil recruitment, activation, and disposal is pivotal for circumscribed inflammation. SHP1Y208N/Y208N mutant mice develop severe cutaneous inflammatory disease that is IL-1R dependent. Genetic reduction in neutrophil numbers and neutrophilic responses to infection is sufficient to prevent the spontaneous initiation of this disease. Neutrophils from SHP1Y208N/Y208N mice display increased pro–IL-1β production due to altered responses to MyD88-dependent and MyD88-independent signals. The IL-1R–dependent inflammatory disease in SHP1Y208N/Y208N mice develops independently of caspase 1 and proteinase 3 and neutrophil elastase. In response to Fas ligand, a caspase 1-independent inducer of IL-1β production, neutrophils from SHP1Y208N/Y208N mice produce elevated levels of IL-1β but display reduced caspase 3 and caspase 7 activation. In neutrophils deficient in SHP1, IL-1β induces high levels of pro–IL-1β suggesting the presence of a paracrine IL-1β loop. These data indicate that the neutrophil- and IL-1–dependent disease in SHP1Y208N/Y208N mice is a consequence of loss of negative regulation of TLR and IL-1R signaling.

Fas regulates neutrophil lifespan during viral and bacterial infection

The regulation of neutrophil lifespan is critical for a circumscribed immune response. Neutrophils are sensitive to Fas/CD95 death receptor signaling in vitro, but it is unknown if Fas regulates neutrophil lifespan in vivo. We hypothesized that FasL‐expressing CD8+ T cells, which kill antigen‐stimulated T cells during chronic viral infection, can also induce neutrophil death in tissues during infection. With the use of LysM‐Cre Fasfl/fl mice, which lack Fas expression in macrophages and neutrophils, we show that Fas regulates neutrophil lifespan during lymphocytic choriomeningitis virus (LCMV) infection in the lung, peripheral blood, and spleen. Fas also contributed to the regulation of neutrophil numbers in the colon of Citrobacter rodentium‐infected mice. To examine the effects of infection on Fas activation in neutrophils, we primed neutrophils with TLR ligands or IL‐18, resulting in ablation of Fas death receptor signaling. These data provide the first in vivo genetic evidence that neutrophil lifespan is controlled by death receptor signaling and provide a mechanism to account for neutrophil resistance to Fas stimulation during infection.

NLRP1a Expression in Srebp-1a-Deficient Mice

The sterol regulatory element binding protein-1a (SREBP-1a) is a lipogenic transcription factor that differentially regulates the expression of inflammation-related genes. One of the most profoundly downregulated genes in Srebp-1a-deficient mice was recently reported to be the inflammasome sensor Nlrp1a (Im et al., 2011). We obtained Srebp-1a-deficient mice to cross with mice containing an Nlrp1a activating mutation to see if the Srebp-1a deficiency could reduce the incidence of their spontaneous inflammatory disease (Masters et al., 2012).