TAK1 Limits Death Receptor Fas-Induced Proinflammatory Cell Death in Macrophages.

Fas, a member of the death receptor family, plays a central role in initiating cell death, a biological process crucial for immune homeostasis. However, the immunological and pathophysiological impacts to which enhanced Fas signaling gives rise remain to be fully understood. Here we demonstrate that TGF-ß-activated kinase 1 (TAK1) works as a negative regulator of Fas signaling in macrophages. Upon Fas engagement with high concentrations of FasL, mouse primary macrophages underwent cell death, and, surprisingly, Fas stimulation led to proteolytic cleavage of gasdermin (GSDM) family members GSDMD and GSDME, a hallmark of pyroptosis, in a manner dependent on caspase enzymatic activity. Remarkably, TAK1-deficient macrophages were highly sensitive to even low concentrations of FasL. Mechanistically, TAK1 negatively modulated RIPK1 kinase activity to protect macrophages from excessive cell death. Intriguingly, mice deficient for TAK1 in macrophages (TAK1mKO mice) spontaneously developed tissue inflammation, and, more important, the emergence of inflammatory disease symptoms was markedly diminished in TAK1mKO mice harboring a catalytically inactive RIPK1. Taken together, these findings not only revealed an unappreciated role of TAK1 in Fas-induced macrophage death but provided insight into the possibility of perturbation of immune homeostasis driven by aberrant cell death.

2-Deoxy-d-glucose induces deglycosylation of proinflammatory cytokine receptors and strongly reduces immunological responses in mouse models of inflammation.

Anti-proinflammatory cytokine therapies against interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1 are major advancements in treating inflammatory diseases, especially rheumatoid arthritis. Such therapies are mainly performed by injection of antibodies against cytokines or cytokine receptors. We initially found that the glycolytic inhibitor 2-deoxy-d-glucose (2-DG), a simple monosaccharide, attenuated cellular responses to IL-6 by inhibiting N-linked glycosylation of the IL-6 receptor gp130. Aglycoforms of gp130 did not bind to IL-6 or activate downstream intracellular signals that included Janus kinases. 2-DG completely inhibited dextran sodium sulfate-induced colitis, a mouse model for inflammatory bowel disease, and alleviated laminarin-induced arthritis in the SKG mouse, an experimental model for human rheumatoid arthritis. These diseases have been shown to be partially dependent on IL-6. We also found that 2-DG inhibited signals for other proinflammatory cytokines such as TNF-α, IL-1ß, and interferon -γ, and accordingly, prevented death by another inflammatory disease, lipopolysaccharide (LPS) shock. Furthermore, 2-DG prevented LPS shock, a model for a cytokine storm, and LPS-induced pulmonary inflammation, a model for acute respiratory distress syndrome of coronavirus disease 2019 (COVID-19). These results suggest that targeted therapies that inhibit cytokine receptor glycosylation are effective for treatment of various inflammatory diseases.

Cutting Edge: TAK1 Safeguards Macrophages against Proinflammatory Cell Death.

TGF-ß-activated kinase 1 (TAK1) is known to play vital roles for innate and adaptive immunity; however, little is known about its potential role in limiting biological responses such as inflammation. In this study, we report that macrophage TAK1 participates in negatively regulating inflammation by restraining proinflammatory cell death. Macrophages from TAK1-deficient mice underwent cell death in response to LPS and poly(I:C), which took place in a manner dependent on TLR/TRIF-induced active Caspase8-mediated cleavage of gasdermin D, known as an executioner of pyroptosis. Likewise, TNF-α induced Caspase8-dependent gasdermin D processing following cell death in TAK1-deficient macrophages. Importantly, we demonstrated that this type of proinflammatory macrophage death is linked to susceptibility to septic shock in mice lacking TAK1 in macrophages in a TNF-α-independent fashion. Taken together, our data revealed that TAK1 acts as a signaling checkpoint to protect macrophages from unique proinflammatory cell death, ensuring the maintenance of innate immune homeostasis.

IL-15-high-responder developing NK cells bearing Ly49 receptors in IL-15-/- mice.

In mice lacking IL-15, NK cell development is arrested at immature stages, providing an opportunity to investigate the earliest developing NK cells that would respond to IL-15. We show in this study that immature NK cells were present in the spleen as well as bone marrow (BM) and contained IL-15-high-responder cells. Thus, mature NK cells were generated more efficiently from IL-15(-/-) than from control donor cells in radiation BM chimeras, and the rate of IL-15-induced cell division in vitro was higher in NK cells in the spleen and BM from IL-15(-/-) mice than in those from wild-type mice. Phenotypically, NK cells developed in IL-15(-/-) mice up to the minor but discrete CD11b(-)CD27(+)DX5(hi)CD51(dull)CD127(dull)CD122(hi) stage, which contained the majority of Ly49G2(+) and D(+) NK cells both in the spleen and BM. Even among wild-type splenic NK cells, IL-15-induced proliferation was most prominent in CD11b(-)DX5(hi) cells. Notably, IL-15-mediated preferential expansion (but not conversion from Ly49(-) cells) of Ly49(+) NK cells was observed in vitro only for NK cells in the spleen. These observations indicated the uneven distribution of NK cells of different developing stages with variable IL-15 responsiveness in these lymphoid organs. Immature NK cells in the spleen may contribute, as auxiliaries to those in BM, to the mature NK cell compartment through IL-15-driven extramarrow expansion under steady-state or inflammatory conditions.

Critical role of Th17 cells in inflammation and neovascularization after ischaemia.

AIMS: Increasing evidence suggests that CD4(+) T cells contribute to neovascularization in ischaemic tissue. However, the T cell subset responsible for neovascularization after ischaemia remains to be determined. Here, we investigated the role of Th17 cells secreting interleukin (IL)-17, a newly identified subset of CD4(+) T cells, in the neovascularization after murine hindlimb ischaemia. METHODS AND RESULTS: Unilateral hindlimb ischaemia was produced in wild-type (WT) C57BL/6 mice. Depletion of CD4(+) T cells resulted in significantly reduced blood flow perfusion in the ischaemic limbs. The expression of IL-17 and retinoic acid receptor-related orphan receptor γt (RORγt) was up-regulated in the ischaemic limbs. IL-17-deficient mice showed a significant reduction in blood flow perfusion, inflammatory cell infiltration, and production of angiogenic cytokines in the ischaemic limbs compared with WT mice. In bone marrow transplantation experiments, the absence of IL-17 specifically in bone marrow cells diminished the neovascularization after ischaemia. Furthermore, IL-17-deficient CD4(+) T cells transferred into the ischaemic limbs of T cell-deficient athymic nude mice evoked a significantly limited neovascularization compared with WT CD4(+) T cells. CONCLUSION: These findings identify Th17 cells as a new angiogenic T cell subset and provide new insight into the mechanism by which T cells promote neovascularization after ischaemia.

Distinct response in maintenance of human naive and memory B cells via IL-21 receptor and TCL1/Akt pathways.

The molecular mechanisms involving in B-cell survival/proliferation are poorly understood. Here we investigated the molecules affecting the survival of human naïve and memory B cells. Without stimulation, naïve B cells survived longer than memory B cells. Moreover, the viability of memory B cells decreased more rapidly than that of naïve B cells following with Staphylococcus aureus Cowan strain (SAC), anti-immunoglobulin (Ig), or anti-CD40 stimulation, but displayed the same levels of survival following CpG DNA stimulation. We analyzed the transcriptional differences between B-cell subsets by gene expression profiling, and identified 15 genes significantly correlated to survival/proliferation. Among them, IL-21 receptor (IL-21R) and T-cell leukemia 1 (TCL1) proto-oncogene were highly expressed in naïve B cells. IL-21 induced the proliferation of both naïve and memory B cells. Marked phosphorylation of Akt was found in naïve B cells compared with memory B cells. This study suggests that naive and memory B cells are regulated by several distinct molecules, and the IL-21R and TCL1/Akt pathways might play crucial roles in naïve B cells for their maintenance.

STAT6 signalling is important in CD8 T-cell activation and defence against Toxoplasma gondii infection in the brain.

Signal transducer and activator of transcription (STAT) 6 is a molecule involved in interleukin (IL)-4 and -13 signalling. We investigated the role of STAT6 signalling in Toxoplasma gondii-infected mice using STAT6-deficient (STAT6(-/-)) and wild-type (WT) mice. A significantly larger number of cysts were recovered from the brain in STAT6(-/-) than in WT mice on days 28 and 56 post-infection. CD8(+) T cells in cerebrospinal fluid and spleen stimulated with T. gondii antigen produced higher levels of interferon (IFN)-gamma in WT than in STAT6(-/-) mice. CD8(+) T-cell function, estimated by expression of CD25 and cytotoxic activity, was lower in STAT6(-/-) than in WT mice. Transfer of CD8(+) but not CD4(+) T cells, purified from infected WT mice, into STAT6(-/-) mice successfully prevented formation of cysts in the brain. However, transfer of naïve CD8(+) T cells from WT into STAT6(-/-) mice did not show either activation of CD8(+) T cells or a decrease in the number of cysts in the brain. Transfer of splenic adherent cells from WT into STAT6(-/-) mice induced activation of CD8(+) T cells and decreased the number of cysts in the brain. Expression of CD86 on splenic dendritic cells and IL-12 p40 production were weaker in STAT6(-/-) than in WT mice after T. gondii infection. These results indicate that STAT6 signalling is important in CD8(+) T-cell activation, possibly through regulation of antigen-presenting cells, which could suppress T. gondii infection in the brain.

Homeostatic erythropoiesis by the transcription factor IRF2 through attenuation of type I interferon signaling.

OBJECTIVE: Erythrocyte production is tightly regulated by cytokines, particularly erythropoietin (EPO), which affects expansion and viability of erythroid lineage cells via induction of several factors, including Bcl2-like 1 (Bcl-XL). Because type I interferon (IFN) is known to inhibit erythropoiesis, we studied mice deficient in the gene for interferon regulatory factor 2 (IRF2), which functions as a negative regulator of type I IFN signaling, in the context of erythropoiesis regulation. MATERIALS AND METHODS: We performed hematologic analyses and detected normocytic anemia in Irf2-deficient mice. RESULTS: Assessment of the maturation of erythroid progenitors in Irf2-deficient bone marrow by flow cytometry revealed a decreased number of late erythroblasts accompanied by an increased number of early erythroid progenitors. Irf2-deficient mice manifested elevated serum EPO levels, decreased Bcl-XL expression levels and enhanced apoptosis of erythroblasts, which may account for the decreased number of late erythroblasts. We further assessed the role of IRF2 in the regulation of type I IFN signaling during erythropoiesis, and found that additional homozygous mutation of IFNAR1, a subunit of type I IFN receptor complex, led to rescue of the defect of erythropoiesis in Irf2-deficient mice. CONCLUSIONS: Impaired erythropoiesis in Irf2-deficient mice results from excessive type I IFN signaling, which inhibits Bcl-XL expression in erythroid lineage cells. Our present study provides a mechanistic understanding of the potential cross-talk between type I IFN and EPO signaling pathways during erythropoiesis and may offer therapeutic insights into anemia.

IFN regulatory factor-2 deficiency revealed a novel checkpoint critical for the generation of peripheral NK cells.

NK cell development is far less understood compared with that of T and B cells despite the critical importance of NK cells in innate immunity. Mice lacking the transcription factor IFN regulatory factor-2 (IRF-2) are known to exhibit NK cell deficiency. However, the role of IRF-2 in NK cell development has remained unclear. In this study we found that NK cell deficiency in the periphery in IRF-2-deficient mice was due to selective loss of mature NK cells, but not to maturation arrest, and NK cells in these mice exhibited very immature surface phenotypes (CD11b(low)Dx5(low)) with highly compromised NK receptor expression. In contrast, IRF-2-deficient NK cells in bone marrow (BM) showed relatively mature phenotypes (CD11b(low)Dx5(high)) with less compromised NK receptor repertoire. Furthermore, BM NK cells in IRF-2-deficient mice were found to proliferate almost normally, but underwent accelerated apoptosis. These observations indicated that NK cell maturation could advance up to a late, but not the final, stage in the BM, whereas these cells were incapable of contributing to the peripheral NK cell pool due to premature death in the absence of IRF-2. In contrast, NK cell numbers and Ly49 expression were much more severely reduced in BM in IL-15-deficient mice than in IRF-2(-/-) mice. The differential peripheral and central NK cell deficiencies in IRF-2(-/-) mice thus revealed a novel late checkpoint for NK cell maturation, distinct from the early IL-15-dependent expansion stage.

Defective development of splenic and epidermal CD4+ dendritic cells in mice deficient for IFN regulatory factor-2.

Dendritic cells (DCs) play important roles in the initiation and regulation of immune responses. Although several subsets of DCs were identified according to their expression of surface molecules such as CD4, CD8, and CD11b, the regulatory mechanism for the development and homeostasis of these DC subsets remains unclear. Here we show that mice lacking IFN regulatory factor-2 (IRF-2(-/-) mice) exhibited a marked and selective defect in splenic CD4(+)CD11b(+)DCs, instead of CD8 alpha(+)CD11b(-)DCs that were reported to be missing in mice lacking the related transcription factor IRF-8. Furthermore, the numbers of epidermal Langerhans cells in IRF-2(-/-) mice were reduced at least in part because of the lack of the CD4(+)CD11b(+) subset. Studies with radiation bone marrow chimeras as well as in vitro retrovirus-mediated gene transduction showed that IRF-2 was required cell-autonomously for the development of myeloid-related DCs. Notably, these abnormalities in DCs diminished in mice lacking both IRF-2 and the IFN-alpha/beta receptor, indicating that IRF-2 acted through negatively regulating IFN-alpha/beta signals. In contrast, natural killer cells still showed developmental arrest in these double mutant mice, indicating that the mode of action of IRF-2 for CD4(+)DC development is distinct from that for natural killer cell development. Our current findings thus pointed to a previously unknown unique cell-type-selective multimode function of IRF-2 in the regulation of lymphohematopoiesis.

Type I interferons and autoimmunity: lessons from the clinic and from IRF-2-deficient mice.

Type I interferons (IFN-alpha/beta) are produced upon viral and bacterial infections and play essential roles in host defense. However, since IFN-alpha/beta have multiple regulatory functions on innate and adoptive immunity, dysregulation of the IFN-alpha/beta system both in uninfected hosts and during immune responses against infection can result in immunopathologies. In fact, IFN-alpha/beta therapy often accompanies autoimmune-like symptoms. In this regard, we have recently found that mice lacking IFN regulatory factor (IRF)-2, a negative regulator of IFN-alpha/beta signaling, develop spontaneous, CD8(+) T cell-dependent skin inflammation. This unique animal model, together with other animal models, highlights the importance of the mechanism maintaining the homeostasis in the IFN-alpha/beta system even in the absence of infection.

Role for Bcl-6 in the generation and maintenance of memory CD8+ T cells.

Naïve T cells proliferate and differentiate into memory cells after antigenic stimulation or in a lymphopenic environment. We showed here transient increases in memory phenotype CD8+ T cell numbers in the lymphopenic environment of spleens of very young mice. The magnitude of the increase correlated with Bcl-6 expression in the T cells. Bcl-6 controlled the generation and maintenance of antigen-specific memory phenotype CD8+ T cells in the spleens of immunized mice. These data suggest that Bcl-6, which is essential for memory B cell development in germinal centers, is a key molecule for the establishment not only of memory T cells but also of the peripheral T cell compartment in infancy.