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Interleukin-17A (IL-17A) is a key mediator of protective immunity to yeast and bacterial infections but also drives the pathogenesis of several autoimmune diseases, such as psoriasis or psoriatic arthritis. Here we show that the tetra-transmembrane protein CMTM4 is a subunit of the IL-17 receptor (IL-17R). CMTM4 constitutively associated with IL-17R subunit C to mediate its stability, glycosylation and plasma membrane localization. Both mouse and human cell lines deficient in CMTM4 were largely unresponsive to IL-17A, due to their inability to assemble the IL-17R signaling complex. Accordingly, CMTM4-deficient mice had a severe defect in the recruitment of immune cells following IL-17A administration and were largely resistant to experimental psoriasis, but not to experimental autoimmune encephalomyelitis. Collectively, our data identified CMTM4 as an essential component of IL-17R and a potential therapeutic target for treating IL-17-mediated autoimmune diseases.

The linear-ubiquitin chain assembly complex (LUBAC) modulates signalling via various immune receptors. In tumour necrosis factor (TNF) signalling, linear (also known as M1) ubiquitin enables full gene activation and prevents cell death. However, the mechanisms underlying cell death prevention remain ill-defined. Here, we show that LUBAC activity enables TBK1 and IKKε recruitment to and activation at the TNF receptor 1 signalling complex (TNFR1-SC). While exerting only limited effects on TNF-induced gene activation, TBK1 and IKKε are essential to prevent TNF-induced cell death. Mechanistically, TBK1 and IKKε phosphorylate the kinase RIPK1 in the TNFR1-SC, thereby preventing RIPK1-dependent cell death. This activity is essential in vivo, as it prevents TNF-induced lethal shock. Strikingly, NEMO (also known as IKKγ), which mostly, but not exclusively, binds the TNFR1-SC via M1 ubiquitin, mediates the recruitment of the adaptors TANK and NAP1 (also known as AZI2). TANK is constitutively associated with both TBK1 and IKKε, while NAP1 is associated with TBK1. We discovered a previously unrecognized cell death checkpoint that is mediated by TBK1 and IKKε, and uncovered an essential survival function for NEMO, whereby it enables the recruitment and activation of these non-canonical IKKs to prevent TNF-induced cell death.

LUBAC components interact with the TLR3 signaling cascade at different levels, thereby tightly controlling TLR3-mediated innate immunity., The linear ubiquitin chain assembly complex (LUBAC), consisting of SHANK-associated RH-domain–interacting protein (SHARPIN), heme-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), and HOIL-1–interacting protein (HOIP), is a critical regulator of inflammation and immunity. This is highlighted by the fact that patients with perturbed linear ubiquitination caused by mutations in the Hoip or Hoil-1 genes, resulting in knockouts of these proteins, may simultaneously suffer from immunodeficiency and autoinflammation. TLR3 plays a crucial, albeit controversial, role in viral infection and tissue damage. We identify a pivotal role of LUBAC in TLR3 signaling and discover a functional interaction between LUBAC components and TLR3 as crucial for immunity to influenza A virus infection. On the biochemical level, we identify LUBAC components as interacting with the TLR3-signaling complex (SC), thereby enabling TLR3-mediated gene activation. Absence of LUBAC components increases formation of a previously unrecognized TLR3-induced death-inducing SC, leading to enhanced cell death. Intriguingly, excessive TLR3-mediated cell death, induced by double-stranded RNA present in the skin of SHARPIN-deficient chronic proliferative dermatitis mice (cpdm), is a major contributor to their autoinflammatory skin phenotype, as genetic coablation of Tlr3 substantially ameliorated cpdm dermatitis. Thus, LUBAC components control TLR3-mediated innate immunity, thereby preventing development of immunodeficiency and autoinflammation.

The linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1 and SHARPIN, is required for optimal TNF-mediated gene activation and to prevent cell death induced by TNF. Here, we demonstrate that keratinocyte-specific deletion of HOIP or HOIL-1 (E-KO) results in severe dermatitis causing postnatal lethality. We provide genetic and pharmacological evidence that the postnatal lethal dermatitis in HoipE-KO and Hoil-1E-KO mice is caused by TNFR1-induced, caspase-8-mediated apoptosis that occurs independently of the kinase activity of RIPK1. In the absence of TNFR1, however, dermatitis develops in adulthood, triggered by RIPK1-kinase-activity-dependent apoptosis and necroptosis. Strikingly, TRAIL or CD95L can redundantly induce this disease-causing cell death, as combined loss of their respective receptors is required to prevent TNFR1-independent dermatitis. These findings may have implications for the treatment of patients with mutations that perturb linear ubiquitination and potentially also for patients with inflammation-associated disorders that are refractory to inhibition of TNF alone.

Summary Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known for specifically killing cancer cells, whereas in resistant cancers, TRAIL/TRAIL-R can promote metastasis via Rac1 and PI3K. It remains unknown, however, whether and to what extent TRAIL/TRAIL-R signaling in cancer cells can affect the immune microenvironment. Here we show that TRAIL-triggered cytokine secretion from TRAIL-resistant cancer cells is FADD dependent and identify the TRAIL-induced secretome to drive monocyte polarization to myeloid-derived suppressor cells (MDSCs) and M2-like macrophages. TRAIL-R suppression in tumor cells impaired CCL2 production and diminished both lung MDSC presence and tumor growth. In accordance, the receptor of CCL2, CCR2, is required to facilitate increased MDSC presence and tumor growth. Finally, TRAIL and CCL2 are co-regulated with MDSC/M2 markers in lung adenocarcinoma patients. Collectively, endogenous TRAIL/TRAIL-R-mediated CCL2 secretion promotes accumulation of tumor-supportive immune cells in the cancer microenvironment, thereby revealing a tumor-supportive immune-modulatory role of the TRAIL/TRAIL-R system in cancer biology., Graphical Abstract, Highlights • TRAIL induces a cytokine cancer secretome in a FADD- and caspase-8-dependent manner • FADD promotes tumor growth along with accumulation of M2-like immune cells in vivo • The TRAIL-induced secretome recruits M2-like immune cells to tumors via CCR2 • TRAIL/CCL2 correlate with a tumor-supportive immune profile in lung cancer patients, Hartwig et al. show that endogenous TRAIL signaling in cancer cells induces a FADD-dependent secretome that promotes the accumulation of M2-like immune cells and tumor growth via host CCR2.

CD148 is a receptor-like protein-tyrosine phosphatase known to inhibit transduction of mitogenic signals in non-hematopoietic cells. Similarly, in the hematopoietic lineage, CD148 inhibited signal transduction downstream of T cell receptor. However, it also augmented immunoreceptor signaling in B cells and macrophages via dephosphorylating C-terminal tyrosine of Src family kinases (SFK). Accordingly, endogenous CD148 compensated for the loss of the main SFK activator CD45 in murine B cells and macrophages but not in T cells. Hypothetical explanations for the difference between T cells and other leukocyte lineages include the inability of CD148 to dephosphorylate a specific set of SFKs involved in T cell activation or the lack of CD148 expression during critical stages of T cell development. Here we describe striking differences in CD148 expression between human and murine thymocyte subsets, the only unifying feature being the absence of CD148 during the positive selection when the major developmental block occurs under CD45 deficiency. Moreover, we demonstrate that similar to CD45, CD148 has both activating and inhibitory effects on the SFKs involved in TCR signaling. However, in the absence of CD45, activating effects prevail, resulting in functional complementation of CD45 deficiency in human T cell lines. Importantly, this is independent of the tyrosines in the CD148 C-terminal tail, contradicting the recently proposed phosphotyrosine displacement model as a mechanism of SFK activation by CD148. Collectively, our data suggest that differential effects of CD148 in T cells and other leukocyte subsets cannot be explained by the CD148 inability to activate T cell SFKs but rather by its dual inhibitory/activatory function and specific expression pattern.

Interleukin 6 (IL-6) is a pleiotropic cytokine that mediates a variety of functions, including induction of the acute-phase response in hepatocytes. IL-6 initiates its action by binding to its cell surface receptor, followed by activation of Janus kinases and tyrosine phosphorylation of the signal transducer and transcription factor (STAT) 3. Although it has been suggested that cholesterol- and sphingolipid-enriched membrane domains, called lipid rafts, and caveolin are involved in this process, their roles in the earliest stages of IL-6-mediated signaling are far from being understood. Here we show that pretreatment of HepG2 hepatoma cells with methyl-β-cyclodextrin (MβCD), which removes cholesterol and destroys lipid rafts, inhibited tyrosine phosphorylation of STAT3 in IL-6-activated, but not PV-activated cells. Furthermore, when the cells were lysed under conditions preserving lipid rafts, no IL-6- or PV-induced phosphorylation of STAT3 was observed. Although most of the STAT3 was found in large MβCD-resistant assemblies in both non-activated and IL-6-activated cells, its association with lipid rafts was weak or undetectable. The extent of IL-6-induced tyrosine phosphorylation of STAT3 was comparable in cells expressing low or high levels of caveolin. Similar STAT3 transducer complexes were observed in freshly isolated rat hepatocytes. The combined data suggest that STAT3 tyrosine phosphorylation occurs in preformed transducer complexes that can be activated in the absence of intact lipid rafts or caveolin.

Tumor necrosis factor (TNF) is a potent cytokine known for its involvement in inflammation, repression of tumorigenesis and activation of immune cells. Consequently, accurate regulation of the TNF signaling pathway is crucial for preventing the potent noxious effects of TNF. These pathological conditions include chronic inflammation, septic shock, cachexia and cancer. The TNF signaling cascade utilizes a complex network of post-translational modifications to control the cellular response following its activation. Next to phosphorylation, the ubiquitination of signaling complex components is probably the most important modification. This process is mediated by a specialist class of enzymes, the ubiquitin ligases. Equally important is the class of dedicated ubiquitin-specific proteases, the deubiquitinases. Together with ubiquitin binding proteins, this ubiquitination-deubiquitination system enables the dynamics of signaling complexes. In TNF signaling, these dynamics translate into the precise regulation of the induction of gene activation or cell death. Here, we review and discuss current knowledge of TNF signaling regulation by the ubiquitin system.

When a BCR on a mature B cell is engaged by its ligand, the cell becomes activated, and the Ab-mediated immune response can be triggered. The initiation of BCR signaling is orchestrated by kinases of the Src and Syk families. However, the proximal BCR-induced phosphorylation remains incompletely understood. According to a model of sequential activation of kinases, Syk acts downstream of Src family kinases (SFKs). In addition, signaling independent of SFKs and initiated by Syk has been proposed. Both hypotheses lack sufficient evidence from relevant B cell models, mainly because of the redundancy of Src family members and the importance of BCR signaling for B cell development. We addressed this issue by analyzing controlled BCR triggering ex vivo on primary murine B cells and on murine and chicken B cell lines. Chemical and Csk-based genetic inhibitor treatments revealed that SFKs are required for signal initiation and Syk activation. In addition, ligand and anti-BCR Ab–induced signaling differ in their sensitivity to the inhibition of SFKs.

Incontinentia Pigmenti (IP) is a rare X-linked disease characterized by early male lethality and multiple abnormalities in heterozygous females. IP is caused by NF-κB essential modulator (NEMO) mutations. The current mechanistic model suggests that NEMO functions as a crucial component mediating the recruitment of the IκB-kinase (IKK) complex to tumor necrosis factor receptor 1 (TNF-R1), thus allowing activation of the pro-survival NF-κB response. However, recent studies have suggested that gene activation and cell death inhibition are two independent activities of NEMO. Here we describe that cells expressing the IP-associated NEMO-A323P mutant had completely abrogated TNF-induced NF-κB activation, but retained partial antiapoptotic activity and exhibited high sensitivity to death by necroptosis. We found that robust caspase activation in NEMO-deficient cells is concomitant with RIPK3 recruitment to the apoptosis-mediating complex. In contrast, cells expressing the ubiquitin-binding mutant NEMO-A323P did not recruit RIPK3 to complex II, an event that prevented caspase activation. Hence NEMO, independently from NF-κB activation, represents per se a key component in the structural and functional dynamics of the different TNF-R1-induced complexes. Alteration of this process may result in differing cellular outcomes and, consequently, also pathological effects in IP patients with different NEMO mutations.

CEACAM1 (biliary glycoprotein or CD66a) is a member of the carcinoembryonic antigen (CEA) subgroup of the CEA family. Eleven RNA isoforms derived from the splicing of a single CEACAM1 gene have been described. Some of the CEACAM1 isoforms have been recognized by the CD66 antibodies in T and B lymphocytes, natural killer cells, granulocytes and epithelial cells in several human tissues. Although it is also present in soluble form in bile and serum, and elevated levels have been found in the serum of patients with liver diseases, it is not known which isoforms are primarily involved. In order to learn more about the distribution and properties of particular CEACAM1 isoforms, we have prepared a monoclonal antibody specific for the A2 domain of CEACAM1, designated TEC-11. This antibody does not cross-react with other members of the CEA family. Immunoblotting analysis revealed that the TEC-11 epitope was present in all cell types expressing CEACAM1 containing the A2 domain [CEACAM1(A2)], including granulocytes (160 000 MW isoform) and sperm cells (140 000 MW isoform). A 115 000 MW isoform of CEACAM1(A2) was present in human serum, bile, saliva and seminal fluid. Human bile, saliva and seminal fluid also contained the 160 000 MW CEACAM1(A2) isoform. Significantly higher serum levels of the 115 000 MW CEACAM1(A2) isoform were detected in patients with obstructive jaundice. The 160 000 MW isoform of CEACAM1(A2) in bile, but not a 115 000 MW isoform in serum and bile, carried the 3-fucosyl-N-acetyl-lactosamine moiety. The combined data indicate that various isoforms of CEACAM1(A2) are present in different body fluids where they could take part in different CEACAM1-mediated functions.

Tetraalkylammonium (TAA) derivatives have been reported to serve as stabilizers of asymmetrical cyanine dyes in aqueous solutions and to increase the yield and efficiency of polymerase chain reaction (PCR) detected by end-point analysis. In this study, we compared the ability of various TAA derivatives (with alkyl chain ranging from 1 to 5 carbons) and some other compounds to serve as enhancers of real-time PCR based on fluorescence detection from intercalating dye SYBR Green I (SGI). Our data indicate that TAA chlorides and some other TAA derivatives serve as potent enhancers of SGI-monitored real-time PCR. Optimal results were obtained with 10-16 mM tetrapropylammonium chloride. The effect of TAA compounds was dependent on the nature of counter ions present and composition of the reaction mixtures used. Based on measurements of SGI-generated fluorescence signal in the presence of PCR-amplified DNA fragments, oligonucleotide primers and/or various additives, we propose that TAA-derivatives reduce the binding of SGI to oligonucleotide primers and thus enhance primer-template interactions during annealing phase. Furthermore, these compounds serve as stabilizers of SGI-containing PCR mixtures. The combined data indicate that TAA derivatives might be a new class of additives contributing to robustness of real-time PCR monitored by asymmetrical cyanine dye SGI.

Summary Recruitment of the deubiquitinase CYLD to signaling complexes is mediated by its interaction with HOIP, the catalytically active component of the linear ubiquitin chain assembly complex (LUBAC). Here, we identify SPATA2 as a constitutive direct binding partner of HOIP that bridges the interaction between CYLD and HOIP. SPATA2 recruitment to TNFR1- and NOD2-signaling complexes is dependent on HOIP, and loss of SPATA2 abolishes CYLD recruitment. Deficiency in SPATA2 exerts limited effects on gene activation pathways but diminishes necroptosis induced by tumor necrosis factor (TNF), resembling loss of CYLD. In summary, we describe SPATA2 as a previously unrecognized factor in LUBAC-dependent signaling pathways that serves as an adaptor between HOIP and CYLD, thereby enabling recruitment of CYLD to signaling complexes., Graphical Abstract, Highlights • SPATA2 bridges the interaction between HOIP and CYLD • CYLD recruitment to the TNFR1-signaling complex requires SPATA2 • Loss of SPATA2 phenocopies absence of CYLD in TNFR1 signaling, Kupka et al. show that the previously demonstrated interaction of CYLD with HOIP, which is required for recruitment of CYLD into signaling complexes, is indirect and mediated by SPATA2. Loss of SPATA2 abrogates recruitment of CYLD to signaling complexes and consequently mimics CYLD deficiency with regards to gene activation and necroptosis induced by TNF.