Caspase-8 inactivation drives autophagy-dependent inflammasome activation in myeloid cells.

Caspase-8 activity controls the switch from cell death to pyroptosis when apoptosis and necroptosis are blocked, yet how caspase-8 inactivation induces inflammasome assembly remains unclear. We show that caspase-8 inhibition via IETD treatment in Toll-like receptor (TLR)-primed Fadd-/-Ripk3-/- myeloid cells promoted interleukin-1ß (IL-1ß) and IL-18 production through inflammasome activation. Caspase-8, caspase-1/11, and functional GSDMD, but not NLRP3 or RIPK1 activity, proved essential for IETD-triggered inflammasome activation. Autophagy became prominent in IETD-treated Fadd-/-Ripk3-/- macrophages, and inhibiting it attenuated IETD-induced cell death and IL-1ß/IL-18 production. In contrast, inhibiting GSDMD or autophagy did not prevent IETD-induced septic shock in Fadd-/-Ripk3-/- mice, implying distinct death processes in other cell types. Cathepsin-B contributes to IETD-mediated inflammasome activation, as its inhibition or down-regulation limited IETD-elicited IL-1ß production. Therefore, the autophagy and cathepsin-B axis represents one of the pathways leading to atypical inflammasome activation when apoptosis and necroptosis are suppressed and capase-8 is inhibited in myeloid cells.

Promyelocytic leukemia protein targets MK2 to promote cytotoxicity.

Promyelocytic leukemia protein (PML) is a tumor suppressor possessing multiple modes of action, including induction of apoptosis. We unexpectedly find that PML promotes necroptosis in addition to apoptosis, with Pml-/- macrophages being more resistant to TNF-mediated necroptosis than wild-type counterparts and PML-deficient mice displaying resistance to TNF-induced systemic inflammatory response syndrome. Reduced necroptosis in PML-deficient cells is associated with attenuated receptor-interacting protein kinase 1 (RIPK1) activation, as revealed by reduced RIPK1[S166] phosphorylation, and attenuated RIPK1-RIPK3-MLKL necrosome complex formation. We show that PML deficiency leads to enhanced TNF-induced MAPK-activated kinase 2 (MK2) activation and elevated RIPK1[S321] phosphorylation, which suppresses necrosome formation. MK2 inhibitor treatment or MK2 knockout abrogates resistance to cell death induction in PML-null cells and mice. PML binds MK2 and p38 MAPK, thereby inhibiting p38-MK2 interaction and MK2 activation. Moreover, PML participates in autocrine production of TNF induced by cellular inhibitors of apoptosis 1 (cIAP1)/cIAP2 degradation, since PML-knockout attenuates autocrine TNF. Thus, by targeting MK2 activation and autocrine TNF, PML promotes necroptosis and apoptosis, representing a novel tumor-suppressive activity for PML.

Transcriptomic Analysis and C-Terminal Epitope Tagging Reveal Differential Processing and Signaling of Endogenous TLR3 and TLR7.

Toll-like receptor (TLR) signaling is critical for defense against pathogenic infection, as well as for modulating tissue development. Activation of different TLRs triggers common inflammatory responses such as cytokine induction. Here, we reveal differential impacts of TLR3 and TLR7 signaling on transcriptomic profiles in bone marrow-derived macrophages (BMDMs). Apart from self-regulation, TLR3, but not TLR7, induced expression of other TLRs, suggesting that TLR3 activation globally enhances innate immunity. Moreover, we observed diverse influences of TLR3 and TLR7 signaling on genes involved in methylation, caspase and autophagy pathways. We compared endogenous TLR3 and TLR7 by using CRISPR/Cas9 technology to knock in a dual Myc-HA tag at the 3' ends of mouse Tlr3 and Tlr7. Using anti-HA antibodies to detect endogenous tagged TLR3 and TLR7, we found that both TLRs display differential tissue expression and posttranslational modifications. C-terminal tagging did not impair TLR3 activity. However, it disrupted the interaction between TLR7 and myeloid differentiation primary response 88 (MYD88), the Tir domain-containing adaptor of TLR7, which blocked its downstream signaling necessary to trigger cytokine and chemokine expression. Our study demonstrates different properties for TLR3 and TLR7, and also provides useful mouse models for further investigation of these two RNA-sensing TLRs.

Infection-induced inflammation from specific inborn errors of immunity to COVID-19.

Inborn errors of immunity (IEIs) are a group of genetically defined disorders leading to defective immunity. Some IEIs have been linked to mutations of immune receptors or signaling molecules, resulting in defective signaling of respective cascades essential for combating specific pathogens. However, it remains incompletely understood why in selected IEIs, such as X-linked lymphoproliferative syndrome type 2 (XLP-2), hypo-immune response to specific pathogens results in persistent inflammation. Moreover, mechanisms underlying the generation of anticytokine autoantibodies are mostly unknown. Recently, IEIs have been associated with coronavirus disease 2019 (COVID-19), with a small proportion of patients that contract severe COVID-19 displaying loss-of-function mutations in genes associated with type I interferons (IFNs). Moreover, approximately 10% of patients with severe COVID-19 possess anti-type I IFN-neutralizing autoantibodies. Apart from IEIs that impair immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV-2 encodes several proteins that suppress early type I IFN production. One primary consequence of the lack of type I IFNs during early SARS-CoV-2 infection is the increased inflammation associated with COVID-19. In XLP-2, resolution of inflammation rescued experimental subjects from infection-induced mortality. Recent studies also indicate that targeting inflammation could alleviate COVID-19. In this review, we discuss infection-induced inflammation in IEIs, using XLP-2 and COVID-19 as examples. We suggest that resolving inflammation may represent an effective therapeutic approach to these diseases.

HIF-2α is indispensable for regulatory T cell function.

Hypoxia-inducible factor 1α (HIF-1α) and HIF-2α are master transcription factors that regulate cellular responses to hypoxia, but the exact function in regulatory T (Treg) cells is controversial. Here, we show that Treg cell development is normal in mice with Foxp3-specific knockout (KO) of HIF-1α or HIF-2α. However, HIF-2α-KO (but not HIF-1α-KO) Treg cells are functionally defective in suppressing effector T cell-induced colitis and inhibiting airway hypersensitivity. HIF-2α-KO Treg cells have enhanced reprogramming into IL-17-secreting cells. We show crosstalk between HIF-2α and HIF-1α, and that HIF-2α represses HIF-1α expression. HIF-1α is upregulated in HIF-2α-KO Treg cells and further deletion of HIF-1α restores the inhibitory function of HIF-2α-KO Treg cells. Mice with Foxp3-conditional KO of HIF-2α are resistant to growth of MC38 colon adenocarcinoma and metastases of B16F10 melanoma. Together, these results indicate that targeting HIF-2α to destabilize Treg cells might be an approach for regulating the functional activity of Treg cells.

Tumor suppressor death-associated protein kinase 1 inhibits necroptosis by p38 MAPK activation.

Death-associated protein kinase 1 (DAPK1, DAPk, DAPK) is known for its involvement in apoptosis and autophagy-associated cell death. Here, we identified an unexpected function of DAPK1 in suppressing necroptosis. DAPK1-deficiency renders macrophages and dendritic cells susceptible to necroptotic death. We also observed an inhibitory role for DAPK1 in necroptosis in HT-29 cells, since knockdown or knockout of DAPK1 in such cells increased their sensitivity to necroptosis. Increased necroptosis was associated with enhanced formation of the RIPK1-RIPK3-MLKL complex in these DAPK1-deficient cells. We further found that DAPK1-deficiency led to decreased MAPK activated kinase 2 (MK2) activation and reduced RIPK1 S321 phosphorylation, with this latter representing a critical step controlling necrosome formation. Most TNF signaling pathways, including ERK, JNK, and AKT, were not regulated by DAPK. In contrast, DAPK bound p38 MAPK and selectively promoted p38 MAPK activation, resulting in enhanced MK2 phosphorylation. Our results reveal a novel role for DAPK1 in inhibiting necroptosis and illustrate an unexpected selectivity for DAPK1 in promoting p38 MAPK-MK2 activation. Importantly, our study suggests that modulation of necroptosis and p38/MK2-mediated inflammation may be achieved by targeting DAPK1.

Deltex1 suppresses T cell function and is a biomarker for diagnosis and disease activity of systemic lupus erythematosus.

OBJECTIVE: Deletion of Deltex1 (DTX1) in mice caused hyperactivation of T cells and lupus-like autoimmune syndromes, however, the association of DTX1 with human autoimmune diseases is totally unknown. This study investigated the role of DTX1 in human T cell functions and its correlation with disease activity in patients with SLE. METHODS: The influence of DTX1 on T cell function was evaluated using human primary cells. DTX1 expression in peripheral blood mononuclear cells (PBMCs) from healthy controls and SLE patients was measured by quantitative real-time PCR and the SLEDAI was used to assess disease activity. RESULTS: After stimulation with anti-CD3 and anti-CD28, silencing of DTX1 expression enhanced IFN-γ secretion by human T cells. The expression of DTX1 in PBMCs was significantly lower in 100 SLE patients than in 50 age- and sex-matched healthy controls (DTX1/glyceraldehyde 3-phosphate dehydrogenase, 0.452 vs 1.269, P < 0.001). The area under the receiver operator characteristics curve of the model was 0.737 (95% CI 0.658, 0.815). Intriguingly, a low DTX1 level in T cells led to high IFN-γ production in SLE patients and had a correlation with severe disease activity. In addition, low DTX1 expression in SLE patients was associated with active LN, lung involvement or hypocomplementaemia. CONCLUSION: Knockdown DTX1 expression in human T cells reduced IFN-γ secretion. DTX1 expression in the PBMCs was significantly lower in SLE patients and had an inverse correlation with disease activity, indicating that the DTX1 level may be a good disease marker of SLE.

Hypoxia-inducible factor 1α plays a predominantly negative role in regulatory T cell functions.

Hypoxia-inducible factor 1α (HIF-1α) regulates cellular responses to hypoxia. However, conflicting roles for HIF-1α in the functions of regulatory T cells (Tregs) have been reported. In this review, we summarize observations on the requirement for HIF-1α for FOXP3 expression and Tregs development, as well as for HIF-1α-mediated downregulation of FOXP3 and Tregs destabilization. We also examine the association of HIF-1α with Tregs under pathogenic conditions. Based on these findings, we suggest that HIF-1α mainly plays a detrimental role in the function and stability of Tregs and that HIF-1α is disposable for the development and suppressive function of Tregs.

IL-6 receptor blockade corrects defects of XIAP-deficient regulatory T cells.

X-linked lymphoproliferative syndrome type-2 (XLP-2) is a primary immunodeficiency disease attributed to XIAP mutation and is triggered by infection. Here, we show that mouse Xiap-/- regulatory T (Treg) cells and human XIAP-deficient Treg cells are defective in suppressive function. The Xiap-/- Treg cell defect is linked partly to decreased SOCS1 expression. XIAP binds SOCS1 and promotes SOCS1 stabilization. Foxp3 stability is reduced in Xiap-/- Treg cells. In addition, Xiap-/- Treg cells are prone to IFN-γ secretion. Transfer of wild-type Treg cells partly rescues infection-induced inflammation in Xiap-/- mice. Notably, inflammation-induced reprogramming of Xiap-/- Treg cells can be prevented by blockade of the IL-6 receptor (IL-6R), and a combination of anti-IL-6R and Xiap-/- Treg cells confers survival to inflammatory infection in Xiap-/- mice. Our results suggest that XLP-2 can be corrected by combination treatment with autologous iTreg (induced Treg) cells and anti-IL-6R antibody, bypassing the necessity to transduce Treg cells with XIAP.

c-FLIP is a target of the E3 ligase deltex1 in gastric cancer.

The ubiquitin E3 ligase DELTEX1 (DTX1) is specifically downregulated in gastric cancer tissues, and expression of DTX1 is linked to better prognoses and survival in gastric cancer. Cellular FLICE inhibitory protein (c-FLIP) is known for its pivotal role in the resistance of cancer cells to death receptor-induced cell death. Here, we show that DTX1 is an E3 ligase for c-FLIP in gastric cancer cells. DTX1 promoted c-FLIP downregulation. Overexpression of DTX1 sensitized gastric cancer cells to TRAIL-induced apoptosis, whereas DTX1-knockdown attenuated apoptosis induction. DTX1 binds c-FLIPL and directs it into the endosome-lysosomal pathway for proteasome-independent degradation. Moreover, induction of DTX1 in AGS cells by geldanamycin conferred susceptibility of those cells to TRAIL-induced apoptosis. Our results reveal a tumor-suppressive role for DTX1 and suggest a new approach to increasing TRAIL efficacy by raising DTX1 levels in gastric cancer therapy. DTX1 also enhanced c-FLIP degradation and FasL-induced and TRAIL-induced apoptosis in T cells, suggesting that DTX1 constitutes one of the physiological mechanisms regulating c-FLIP stability.

Ubiquitination of tumor suppressor PML regulates prometastatic and immunosuppressive tumor microenvironment.

The tumor microenvironment plays an important role in tumor growth and metastasis. However, the mechanism by which tumor cells regulate the cell and non-cell constituents of surrounding stroma remains incompletely understood. Promyelocytic leukemia (PML) is a pleiotropic tumor suppressor, but its role in tumor microenvironment regulation is poorly characterized. PML is frequently downregulated in many cancer types, including lung cancer. Here, we identify a PML ubiquitination pathway that is mediated by WD repeat 4-containing cullin-RING ubiquitin ligase 4 (CRL4WDR4). Clinically, this PML degradation pathway is hyperactivated in lung cancer and correlates with poor prognosis. The WDR4/PML axis induces a set of cell-surface or secreted factors, including CD73, urokinase-type plasminogen activator receptor (uPAR), and serum amyloid A2 (SAA2), which elicit paracrine effects to stimulate migration, invasion, and metastasis in multiple lung cancer models. In xenograft and genetically engineered mouse models, the WDR4/PML axis elevates intratumoral Tregs and M2-like macrophages and reduces CD8+ T cells to promote lung tumor growth. These immunosuppressive effects were all reversed by CD73 blockade. Our study identifies WDR4 as an oncoprotein that negatively regulates PML via ubiquitination to promote lung cancer progression by fostering an immunosuppressive and prometastatic tumor microenvironment, suggesting the potential of immune-modulatory approaches for treating lung cancer with aberrant PML degradation.

Tumour suppressor death-associated protein kinase targets cytoplasmic HIF-1α for Th17 suppression.

Death-associated protein kinase (DAPK) is a tumour suppressor. Here we show that DAPK also inhibits T helper 17 (Th17) and prevents Th17-mediated pathology in a mouse model of autoimmunity. We demonstrate that DAPK specifically downregulates hypoxia-inducible factor 1α (HIF-1α). In contrast to the predominant nuclear localization of HIF-1α in many cell types, HIF-1α is located in both the cytoplasm and nucleus in T cells, allowing for a cytosolic DAPK-HIF-1α interaction. DAPK also binds prolyl hydroxylase domain protein 2 (PHD2) and increases HIF-1α-PHD2 association. DAPK thereby promotes the proline hydroxylation and proteasome degradation of HIF-1α. Consequently, DAPK deficiency leads to excess HIF-1α accumulation, enhanced IL-17 expression and exacerbated experimental autoimmune encephalomyelitis. Additional knockout of HIF-1α restores the normal differentiation of Dapk(-/-) Th17 cells and prevents experimental autoimmune encephalomyelitis development. Our results reveal a mechanism involving DAPK-mediated degradation of cytoplasmic HIF-1α, and suggest that raising DAPK levels could be used for treatment of Th17-associated inflammatory diseases.

Measuring NLR Oligomerization V: In Situ Proximity Ligation Assay.

The NLRP3 inflammasome is assembled in macrophages and monocytes in response to inflammatory and danger stimuli. The atypical nature of the NLRP3 complex impedes detection of NLRP3 inflammasome formation by conventional biochemical and cell biology methods. In situ proximity ligation assay (PLA) provides an alternative method of detection, localization, and quantification of protein-protein interactions in tissue and cell samples. Two primary antibodies raised in different species detect the two proteins of interest. When the proteins are in close proximity, secondary antibodies conjugated with specific DNA probes hybridize with linking oligonucleotides to form a DNA bridge between the two proteins. Amplification of the DNA bridge then facilitates detection by microscopy using fluorescence probes. Here, we describe application of in situ PLA to detect NLRP3 inflammasome assembly in mouse bone marrow-derived macrophages and human monocyte cell line THP1.

MST3 promotes proliferation and tumorigenicity through the VAV2/Rac1 signal axis in breast cancer.

MST3 (mammalian STE20-like kinase 3) belongs to the Ste20 serine/threonine protein kinase family. The role of MST3 in tumor growth is less studied; therefore, we investigates the function of MST3 in breast cancer. Here, we demonstrate that MST3 is overexpressed in human breast tumors. Online Kaplan-Meier plotter analysis reveals that overexpression of MST3 predicts poor prognosis in breast cancer patients. Knockdown of MST3 with shRNA inhibits proliferation and anchorage-independent growth in vitro. Downregulation of MST3 in triple-negative MDA-MB-231 and MDA-MB-468 breast cancer cells decreases tumor formation in NOD/SCID mice. MST3 interacts with VAV2, but not VAV3, as demonstrated by co-immunoprecipitation and confocal microscopy. By domain mapping of MST3, we determine that the proline-rich region of MST3 (353KDIPKRP359) interacts with the SH3 domain of VAV2. Mutation of the two proline residues in this domain significantly attenuates the interaction between MST3 and VAV2. Overexpression of wild-type MST3 (WT-MST3), but not proline-rich-deleted MST3 (∆P-MST3), enhances the proliferation rate and anchorage-independent growth of MDA-MB-468 cells. Overexpression of MST3 increases VAV2 phosphorylation and GTP-Rac1, whereas downregulation of MST3 or delivery of ∆P-MST3 results in a reduction of VAV2 and Rac1 activation. Knockdown of MST3 inhibits cyclin D1 protein expression. The Rac1 inhibitor EHop-016 attenuates cell proliferation induced by WT-MST3. Finally, Knockdown of MST3 or Rac1 inhibitor decreases cyclin D protein expression, which is important for tumor growth. These results indicate that MST3 interacts with VAV2 to activate Rac1 and promote the tumorigenicity of breast cancer.

Cellular FLIP Inhibits Myeloid Cell Activation by Suppressing Selective Innate Signaling.

Cellular FLIP (c-FLIP) specifically inhibits caspase-8 and suppresses death receptor-induced apoptosis. c-FLIP has also been reported to transmit activation signals. In this study, we report a novel function of c-FLIP involving inhibition of myeloid cell activation through antagonizing the selective innate signaling pathway. We found that conditional knockout of c-FLIP in dendritic cells (DCs) led to neutrophilia and splenomegaly. Peripheral DC populations, including CD11b(+) conventional DCs (cDCs), CD8(+) cDCs, and plasmacytoid DCs, were not affected by c-FLIP deficiency. We also found that c-FLIP knockout cDCs, plasmacytoid DCs, and bone marrow-derived DCs (BMDCs) displayed enhanced production of TNF-α, IL-2, or G-CSF in response to stimulation of TLR4, TLR2, and dectin-1. Consistent with the ability of c-FLIP to inhibit the activation of p38 MAPK, the enhanced activation of c-FLIP-deficient BMDCs could be partly linked to an elevated activation of p38 MAPK after engagement of innate receptors. Increased activation was also found in c-FLIP(+/-) macrophages. Additionally, the increased activation in c-FLIP-deficient DCs was independent of caspase-8. Our results reveal a novel inhibitory role of c-FLIP in myeloid cell activation and demonstrate the unexpected anti-inflammatory activity of c-FLIP. Additionally, our observations suggest that cancer therapy targeting c-FLIP downregulation may facilitate DC activation and increase T cell immunity.

Deltex1 antagonizes HIF-1α and sustains the stability of regulatory T cells in vivo.

Application of regulatory T cells (Tregs) in transplantation, autoimmunity and allergy has been extensively explored, but how Foxp3 and Treg stability is regulated in vivo is incompletely understood. Here, we identify a requirement for Deltex1 (DTX1), a contributor to T-cell anergy and Foxp3 protein level maintenance in vivo. Dtx1(-/-) Tregs are as effective as WT Tregs in the inhibition of CD4(+)CD25(-) T-cell activation in vitro. However, the suppressive ability of Dtx1(-/-) Tregs is greatly impaired in vivo. We find that Foxp3 expression is diminished when Dtx1(-/-) Tregs are co-transferred with effector T cells in vivo. DTX1 promotes the degradation of HIF-1α. Knockout of HIF-1α restores the Foxp3 stability and rescues the defective suppressive activity in Dtx1(-/-) Treg cells in vivo. Our results suggest that DTX1 exerts another level of control on Treg stability in vivo by sustaining the expression of Foxp3 protein in Tregs.

Inability to resolve specific infection generates innate immunodeficiency syndrome in Xiap-/- mice.

Emerging evidence indicates that innate immunodeficiency syndromes are linked to mutations in innate receptors and to specific infections. X-linked lymphoproliferative syndrome type-2 (XLP-2) is associated with deficiency in X-linked inhibitor of apoptosis protein (XIAP), with poorly understood molecular mechanisms. Here we showed that XIAP deficiency selectively impaired B-cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)-mediated innate responses to dectin-1 ligands but did not affect responses to various Toll-like receptor agonists. Consequently, Xiap(-/-) mice became highly vulnerable on Candida albicans infection. The compromised early innate responses led to the persistent presence of C albicans and inflammatory cytokines in Xiap(-/-) mice. Furthermore, priming of Xiap(-/-) mice with the dectin-1 ligand curdlan alone resulted in XLP-2-like syndromes. Restoration of dectin-1-induced Rac1 activation and phagocytosis by resolvin D1, but not up-regulation of nuclear factor-κB, rescued Xiap(-/-) mice from C albicans lethal infection. Therefore, development of XLP-2 in XIAP-deficient patients could be partly due to sustained inflammation as a consequence of defective BCL10-dependent innate immunity toward specific pathogens. Importantly, our results suggest the potential therapeutic value of resolvin D1 in the treatment of XLP-2 and innate immunodeficiency syndromes.

Deltex1 promotes protein kinase Cθ degradation and sustains Casitas B-lineage lymphoma expression.

The generation of T cell anergy is associated with upregulation of ubiquitin E3 ligases including Casitas B-lineage lymphoma (Cbl-b), Itch, gene related to anergy in lymphocyte, and deltex1 (DTX1). These E3 ligases attenuate T cell activation by targeting to signaling molecules. For example, Cbl-b and Itch promote the degradation of protein kinase Cθ (PKCθ) and phospholipase C-γ1 (PLC-γ1) in anergic Th1 cells. How these anergy-associated E3 ligases coordinate during T cell anergy remains largely unknown. In the current study, we found that PKCθ and PLC-γ1 are also downregulated by DTX1. DTX1 interacted with PKCθ and PLC-γ1 and stimulated the degradation of PKCθ and PLC-γ1. T cell anergy-induced proteolysis of PKCθ was prevented in Dtx1(-/-) T cells, supporting the essential role of DTX1 in PKCθ downregulation. Similar to Cbl-b and Itch, DTX1 promoted monoubiquitination of PKCθ. Proteasome inhibitor did not inhibit DTX1-directed PKCθ degradation, but instead DTX1 directed the relocalization of PKCθ into the lysosomal pathway. In addition, DTX1 interacted with Cbl-b and increased the protein levels of Cbl-b. We further demonstrated the possibility that, through the downregulation of PKCθ, DTX1 prevented PKCθ-induced Cbl-b degradation and increased Cbl-b protein stability. Our results suggest the coordination between E3 ligases during T cell anergy; DTX1 acts with Cbl-b to assure a more extensive silencing of PKCθ, whereas DTX1-mediated PKCθ degradation further stabilizes Cbl-b.

Regulation of inflammation by DAPK.

Death-associated protein kinase (DAPK) is a tumor suppressor and negatively regulates several activation signals. Consistent with its potential anti-inflammatory activity, DAPK promotes the formation of IFN-γ-activated inhibitor of translation (GAIT) complex that suppresses the translation of selected inflammatory genes. DAPK has been found to inhibit tumor necrosis factor-α (TNF-α)- or lipopolysaccharides (LPS)-induced NF-κB activation and pro-inflammatory cytokine expression. Inflammation is always associated with T cell activation, while DAPK attenuates T cell activation by a selective suppression in T cell receptor-triggered NF-κB activation. Recent studies, however, also reveal a contribution of DAPK to pro-inflammatory processes. DAPK is shown to mediate pro-inflammatory signaling downstream of TNF-α, LPS, IL-17, or IL-32. In addition, DAPK is required for the full formation of NLRP3 inflammasome, essential for the generation of IL-1ß and IL-18. These results suggest the complicated role of DAPK in the regulation of inflammation that is likely dependent on cell types and environmental cues.