Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.

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.

NLRP1 inflammasome activation induces pyroptosis of hematopoietic progenitor cells.

Cytopenias are key prognostic indicators of life-threatening infection, contributing to immunosuppression and mortality. Here we define a role for Caspase-1-dependent death, known as pyroptosis, in infection-induced cytopenias by studying inflammasome activation in hematopoietic progenitor cells. The NLRP1a inflammasome is expressed in hematopoietic progenitor cells and its activation triggers their pyroptotic death. Active NLRP1a induced a lethal systemic inflammatory disease that was driven by Caspase-1 and IL-1ß but was independent of apoptosis-associated speck-like protein containing a CARD (ASC) and ameliorated by IL-18. Surprisingly, in the absence of IL-1ß-driven inflammation, active NLRP1a triggered pyroptosis of hematopoietic progenitor cells resulting in leukopenia at steady state. During periods of hematopoietic stress induced by chemotherapy or lymphocytic choriomeningitis virus (LCMV) infection, active NLRP1a caused prolonged cytopenia, bone marrow hypoplasia, and immunosuppression. Conversely, NLRP1-deficient mice showed enhanced recovery from chemotherapy and LCMV infection, demonstrating that NLRP1 acts as a cellular sentinel to alert Caspase-1 to hematopoietic and infectious stress.

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-1ß 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-1ß. Administration of IL-1ß 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.

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-1ß 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.

Neutrophils require SHP1 to regulate IL-1ß production and prevent inflammatory skin disease.

The regulation of neutrophil recruitment, activation, and disposal is pivotal for circumscribed inflammation. SHP1(Y208N/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 SHP1(Y208N/Y208N) mice display increased pro-IL-1ß production due to altered responses to MyD88-dependent and MyD88-independent signals. The IL-1R-dependent inflammatory disease in SHP1(Y208N/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-1ß production, neutrophils from SHP1(Y208N/Y208N) mice produce elevated levels of IL-1ß but display reduced caspase 3 and caspase 7 activation. In neutrophils deficient in SHP1, IL-1ß induces high levels of pro-IL-1ß suggesting the presence of a paracrine IL-1ß loop. These data indicate that the neutrophil- and IL-1-dependent disease in SHP1(Y208N/Y208N) mice is a consequence of loss of negative regulation of TLR and IL-1R signaling.

Regulation of interleukin-1beta by interferon-gamma is species specific, limited by suppressor of cytokine signalling 1 and influences interleukin-17 production.

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