CXCL4 synergizes with TLR8 for TBK1-IRF5 activation, epigenomic remodeling and inflammatory response in human monocytes.

Regulation of endosomal Toll-like receptor (TLR) responses by the chemokine CXCL4 is implicated in inflammatory and fibrotic diseases, with CXCL4 proposed to potentiate TLR responses by binding to nucleic acid TLR ligands and facilitating their endosomal delivery. Here we report that in human monocytes/macrophages, CXCL4 initiates signaling cascades and downstream epigenomic reprogramming that change the profile of the TLR8 response by selectively amplifying inflammatory gene transcription and interleukin (IL)-1ß production, while partially attenuating the interferon response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activates TBK1 and IKKε, repurposes these kinases towards an inflammatory response via coupling with IRF5, and activates the NLRP3 inflammasome. CXCL4 signaling, in a cooperative and synergistic manner with TLR8, induces chromatin remodeling and activates de novo enhancers associated with inflammatory genes. Our findings thus identify new regulatory mechanisms of TLR responses relevant for cytokine storm, and suggest targeting the TBK1-IKKε-IRF5 axis may be beneficial in inflammatory diseases.

IKKα-deficient lung adenocarcinomas generate an immunosuppressive microenvironment by overproducing Treg-inducing cytokines.

The tumor microenvironment (TME) provides potential targets for cancer therapy. However, how signals originating in cancer cells affect tumor-directed immunity is largely unknown. Deletions in the CHUK locus, coding for IκB kinase α (IKKα), correlate with reduced lung adenocarcinoma (ADC) patient survival and promote KrasG12D-initiated ADC development in mice, but it is unknown how reduced IKKα expression affects the TME. Here, we report that low IKKα expression in human and mouse lung ADC cells correlates with increased monocyte-derived macrophage and regulatory T cell (Treg) scores and elevated transcription of genes coding for macrophage-recruiting and Treg-inducing cytokines (CSF1, CCL22, TNF, and IL-23A). By stimulating recruitment of monocyte-derived macrophages from the bone marrow and enforcing a TNF/TNFR2/c-Rel signaling cascade that stimulates Treg generation, these cytokines promote lung ADC progression. Depletion of TNFR2, c-Rel, or TNF in CD4+ T cells or monocyte-derived macrophages dampens Treg generation and lung tumorigenesis. Treg depletion also attenuates carcinogenesis. In conclusion, reduced cancer cell IKKα activity enhances formation of a protumorigenic TME through a pathway whose constituents may serve as therapeutic targets for KRAS-initiated lung ADC.

TLR and IKK Complex-Mediated Innate Immune Signaling Inhibits Stress Granule Assembly.

Cellular stress can induce cytoplasmic ribonucleoprotein complexes called stress granules that allow the cells to survive. Stress granules are also central to cellular responses to infections, in which they can act as platforms for viral sensing or modulate innate immune signaling through pattern recognition receptors. However, the effect of innate immune signaling on stress granules is poorly understood. In this study, we report that prior induction of innate immune signaling through TLRs inhibited stress granule assembly in a TLR ligand dose-dependent manner in murine bone marrow-derived macrophages. Time course analysis suggests that TLR stimulation can reverse stress granule assembly even after it has begun. Additionally, both MYD88- and TRIF-mediated TLR signaling inhibited stress granule assembly in response to endoplasmic reticulum stress in bone marrow-derived macrophages and the chemotherapeutic drug oxaliplatin in murine B16 melanoma cells. This inhibition was not due to a decrease in expression of the critical stress granule proteins G3BP1 and DDX3X and was independent of IRAK1/4, JNK, ERK and P38 kinase activity but dependent on IKK complex kinase activity. Overall, we have identified the TLR-IKK complex signaling axis as a regulator of stress granule assembly-disassembly dynamics, highlighting cross-talk between processes that are critical in health and disease.

IFIT5 Negatively Regulates the Type I IFN Pathway by Disrupting TBK1-IKKε-IRF3 Signalosome and Degrading IRF3 and IKKε.

IFN-induced protein with tetratricopeptide repeats (IFITs), known as canonical IFN-stimulated genes (ISGs), play critical roles in regulating immune responses against pathogens and maintaining homeostasis. How the IFIT5 regulates innate immune responses is rarely reported and remains enigmatic. In this study, we discover that human IFIT5 (hIFIT5) functions as a negative regulator of the type I IFN (IFN) pathway in HEK293T cell lines. Our data illustrated that hIFIT5 inhibited the promotor activities of IFN-ß induced by IRF3 and its upstream factors but not by IRF3-5D (activated form of IRF3), suggesting that IRF3 might be a target of hIFIT5. Further investigations revealed that hIFIT5 downregulated the phosphorylation of IRF3 and IKKε and blocked the IRF3 nuclear translocation. Moreover, hIFIT5 impaired the IRF3-TBK1-IKKε complex, accompanied by IRF3 and IKKε degradation. In conclusion, these findings indicate that hIFIT5 is a negative modulator in the type I IFN signaling pathway, opening additional avenues for preventing hyperactivation and maintaining immunity homeostasis.

SARS-CoV-2 ORF9b antagonizes type I and III interferons by targeting multiple components of the RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways.

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to the pathogenesis of coronavirus disease 2019 (COVID-19). The strategy used by SARS-CoV-2 to evade antiviral immunity needs further investigation. Here, we reported that SARS-CoV-2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS-CoV-2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε, rather than IRF3-5D, which is the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of the cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. A mechanistic analysis revealed that the SARS-CoV-2 ORF9b protein interacted with RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS-CoV-2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS-CoV-2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS-CoV-2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID-19.

Deletion of IKK2 in haematopoietic cells of adult mice leads to elevated interleukin-6, neutrophilia and fatal gastrointestinal inflammation.

The IκB kinase complex, consisting of IKK1, IKK2 and the regulatory subunit NEMO, is required for NF-κB signalling following the activation of several cell surface receptors, such as members of the Tumour Necrosis Factor Receptor superfamily and the Interleukin-1 Receptor. This is critical for haematopoietic cell proliferation, differentiation, survival and immune responses. To determine the role of IKK in the regulation of haematopoiesis, we used the Rosa26Cre-ERT2 Cre/lox recombination system to achieve targeted, haematopoietic cell-restricted deletion of the genes for IKK1 or IKK2 in vivo. We found that the IKK complex plays a critical role in haematopoietic cell development and function. Deletion of IKK2, but not loss of IKK1, in haematopoietic cells led to an expansion of CD11b/Gr-1-positive myeloid cells (neutrophilia), severe anaemia and thrombocytosis, with reduced numbers of long-term haematopoietic stem cells (LT-HSCs), short-term haematopoietic stem cells (ST-HSCs) and multipotential progenitor cells (MPPs), increased circulating interleukin-6 (IL-6) and severe gastrointestinal inflammation. These findings identify distinct functions for the two IKK catalytic subunits, IKK1 and IKK2, in the haematopoietic system.

Reduced SULT2B1b expression alleviates ox-LDL-induced inflammation by upregulating miR-148-3P via inhibiting the IKKß/NF-κB pathway in macrophages.

Atherosclerosis is a lipid-driven chronic inflammatory disease in which lipid-laden macrophage foam cells lead to inflamed lesions in arteries. Previous studies have proven that sulfotransferase 2B1b (SULT2B1b) has several roles in the regulation of lipid metabolism and the inflammatory response. However, little is known about the functions of SULT2B1b in ox-LDL-induced inflammation in macrophages. In this study, after treatment with either ox-LDL alone or combined with transfection of siRNAs targeting SULT2B1b, IL-6, TNF-α, NF-κB, IKKß and IκB mRNA and protein expression were determined in Raw264.7 cells by real-time PCR and Western blot, respectively. The proliferative capacity was determined by EdU staining and Cell Counting Kit-8. Our data demonstrated that SULT2B1b knockdown could reduce phosphorylated NF-κB levels and downregulate IKKß protein levels. Additionally, IκB levels were increased and the proliferation of ox-LDL stimulated cells was inhibited after SULT2B1b silencing. Downregulation of SULT2B1b expression was found to upregulate miR-148a-3p expression by microarray assay, while IKKß was a miR-148a-3p target gene. Our study suggests that SULT2B1b knockdown could promote miR148a-3p expression and inhibit activation of the IKKß/NF-κB signalling pathway, which suppressed the inflammatory response in macrophages. Therefore, targeting the SULT2B1b gene might be potentially beneficial for atherosclerosis prevention by decreasing the inflammatory response.

Network- and systems-based re-engineering of dendritic cells with non-coding RNAs for cancer immunotherapy.

Dendritic cells (DCs) are professional antigen-presenting cells that induce and regulate adaptive immunity by presenting antigens to T cells. Due to their coordinative role in adaptive immune responses, DCs have been used as cell-based therapeutic vaccination against cancer. The capacity of DCs to induce a therapeutic immune response can be enhanced by re-wiring of cellular signalling pathways with microRNAs (miRNAs). Methods: Since the activation and maturation of DCs is controlled by an interconnected signalling network, we deploy an approach that combines RNA sequencing data and systems biology methods to delineate miRNA-based strategies that enhance DC-elicited immune responses. Results: Through RNA sequencing of IKKß-matured DCs that are currently being tested in a clinical trial on therapeutic anti-cancer vaccination, we identified 44 differentially expressed miRNAs. According to a network analysis, most of these miRNAs regulate targets that are linked to immune pathways, such as cytokine and interleukin signalling. We employed a network topology-oriented scoring model to rank the miRNAs, analysed their impact on immunogenic potency of DCs, and identified dozens of promising miRNA candidates, with miR-15a and miR-16 as the top ones. The results of our analysis are presented in a database that constitutes a tool to identify DC-relevant miRNA-gene interactions with therapeutic potential (https://www.synmirapy.net/dc-optimization). Conclusions: Our approach enables the systematic analysis and identification of functional miRNA-gene interactions that can be experimentally tested for improving DC immunogenic potency.

Determining the prognostic significance of IKKα in prostate cancer.

BACKGROUND: As the survival of castration-resistant prostate cancer (CRPC) remains poor, and the nuclear factor-κB (NF-κB) pathways play key roles in prostate cancer (PC) progression, several studies have focused on inhibiting the NF-κB pathway through generating inhibitory κB kinase subunit α (IKKα) small molecule inhibitors. However, the identification of prognostic markers able to discriminate which patients could benefit from IKKα inhibitors is urgently required. The present study investigated the prognostic value of IKKα, IKKα phosphorylated at serine 180 (p-IKKα S180) and threonine 23 (p-IKKα T23), and their relationship with the androgen receptor (AR) and Ki67 proliferation index to predict patient outcome. METHODS: A cohort of 115 patients with hormone-naïve PC (HNPC) and CRPC specimens available were used to assess tumor cell expression of proteins within both the cytoplasm and the nucleus by immunohistochemistry. The expression levels were dichotomized (low vs high) to determine the associations between IKKα, AR, Ki67, and patients'Isurvival. In addition, an analysis was performed to assess potential IKKα associations with clinicopathological and inflammatory features, and potential IKKα correlations with other cancer pathways essential for CRPC growth. RESULTS: High levels of cytoplasmic IKKα were associated with a higher cancer-specific survival in HNPC patients with low AR expression (hazards ratio [HR], 0.33; 95% confidence interval [CI] log-rank, 0.11-0.98; P = .04). Furthermore, nuclear IKKα (HR, 2.60; 95% CI, 1.27-5.33; P = .01) and cytoplasmic p-IKKα S180 (HR, 2.10; 95% CI, 1.17-3.76; P = .01) were associated with a lower time to death from recurrence in patients with CRPC. In addition, high IKKα expression was associated with high levels of T-cells (CD3+ P = .01 and CD8+ P = .03) in HNPC; however, under castration conditions, high IKKα expression was associated with high levels of CD68+ macrophages (P = .04), higher Gleason score (P = .01) and more prostate-specific antigen concentration (P = .03). Finally, we identified crosstalk between IKKα and members of the canonical NF-κB pathway in the nucleus of HNPC. Otherwise, IKKα phosphorylated by noncanonical NF-κB and Akt pathways correlated with members of the canonical NF-κB pathway in CRPC. CONCLUSION: The present study reports that patients with CRPC expressing high levels of nuclear IKKα or cytoplasmic p-IKKα S180, which associated with a lower time to death from recurrence, may benefit from IKKα inhibitors.

IKKγ/NEMO Is Required to Confer Antimicrobial Innate Immune Responses in the Yellow Mealworm, Tenebrio Molitor.

IKKγ/NEMO is the regulatory subunit of the IκB kinase (IKK) complex, which regulates the NF-κB signaling pathway. Within the IKK complex, IKKγ/NEMO is the non-catalytic subunit, whereas IKKα and IKKß are the structurally related catalytic subunits. In this study, TmIKKγ was screened from the Tenebrio molitor RNA-Seq database and functionally characterized using RNAi screening for its role in regulating T. molitor antimicrobial peptide (AMP) genes after microbial challenges. The TmIKKγ transcript is 1521 bp that putatively encodes a polypeptide of 506 amino acid residues. TmIKKγ contains a NF-κB essential modulator (NEMO) and a leucine zipper domain of coiled coil region 2 (LZCC2). A phylogenetic analysis confirmed its homology to the red flour beetle, Tribolium castaneum IKKγ (TcIKKγ). The expression of TmIKKγ mRNA showed that it might function in diverse tissues of the insect, with a higher expression in the hemocytes and the fat body of the late-instar larvae. TmIKKγ mRNA expression was induced by Escherichia coli, Staphylococcus aureus, and Candida albicans challenges in the whole larvae and in tissues such as the hemocytes, gut and fat body. The knockdown of TmIKKγ mRNA significantly reduced the survival of the larvae after microbial challenges. Furthermore, we investigated the tissue-specific induction patterns of fourteen T. molitor AMP genes in TmIKKγ mRNA-silenced individuals after microbial challenges. In general, the mRNA expression of TmTenecin1, -2, and -4; TmDefensin1 and -2; TmColeoptericin1 and 2; and TmAttacin1a, 1b, and 2 were found to be downregulated in the hemocytes, gut, and fat body tissues in the TmIKKγ-silenced individuals after microbial challenges. Under similar conditions, TmRelish (NF-κB transcription factor) mRNA was also found to be downregulated. Thus, TmIKKγ is an important factor in the antimicrobial innate immune response of T. molitor.

Porcine Deltacoronavirus Accessory Protein NS7a Antagonizes IFN-ß Production by Competing With TRAF3 and IRF3 for Binding to IKKε.

As an emerging swine enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) not only causes serious diarrhea in suckling piglets but also possesses the potential for cross-species transmission, which has sparked growing interest when studying this emerging virus. We previously identified a novel accessory protein NS7a encoded by PDCoV; however, the function of NS7a was not resolved. In this study, we demonstrated that PDCoV NS7a is an interferon antagonist. Overexpression of NS7a notably inhibited Sendai virus (SeV)-induced interferon-ß (IFN-ß) production and the activation of IRF3 rather than NF-κB. NS7a also inhibited IFN-ß promoter activity induced by RIG-I, MDA5, MAVS, TBK1, and IKKε, which are key components of the RIG-I-like receptor (RLR) signaling pathway but not IRF3, the transcription factor downstream of TBK1/IKKε. Surprisingly, NS7a specifically interacts with IKKε but not with the closely related TBK1. Furthermore, NS7a interacts simultaneously with the kinase domain (KD) and the scaffold dimerization domain (SDD) of IKKε, competing with TRAF3, and IRF3 for binding to IKKε, leading to the reduction of RLR-mediated IFN-ß production. The interactions of TRAF3-IKKε and IKKε-IRF3 are also attenuated in PDCoV-infected cells. Taken together, our results demonstrate that PDCoV NS7a inhibits IFN-ß production by disrupting the association of IKKε with both TRAF3 and IRF3, revealing a new mechanism utilized by a PDCoV accessory protein to evade the host antiviral innate immune response.

Paclitaxel Induces Immunogenic Cell Death in Ovarian Cancer via TLR4/IKK2/SNARE-Dependent Exocytosis.

Emerging evidence shows that the efficacy of chemotherapeutic drugs is reliant on their capability to induce immunogenic cell death (ICD), thus transforming dying tumor cells into antitumor vaccines. We wanted to uncover potential therapeutic strategies that target ovarian cancer by having a better understanding of the standard-of-care chemotherapy treatment. Here, we showed in ovarian cancer that paclitaxel induced ICD-associated damage-associated molecular patterns (DAMP, such as CALR exposure, ATP secretion, and HMGB1 release) in vitro and elicited significant antitumor responses in tumor vaccination assays in vivo Paclitaxel-induced TLR4 signaling was essential to the release of DAMPs, which led to the activation of NF-κB-mediated CCL2 transcription and IkappaB kinase 2-mediated SNARE-dependent vesicle exocytosis, thus exposing CALR on the cell surface. Paclitaxel induced endoplasmic reticulum stress, which triggered protein kinase R-like ER kinase activation and eukaryotic translation initiation factor 2α phosphorylation independent of TLR4. Paclitaxel chemotherapy induced T-cell infiltration in ovarian tumors of the responsive patients; CALR expression in primary ovarian tumors also correlated with patients' survival and patient response to chemotherapy. These findings suggest that the effectiveness of paclitaxel relied upon the activation of antitumor immunity through ICD via TLR4 and highlighted the importance of CALR expression in cancer cells as an indicator of response to paclitaxel chemotherapy in ovarian cancer.

Looking Down on NF-κB.

The diversified NF-κB transcription factor family has been extensively characterized in organisms ranging from flies to humans. However, homologs of NF-κB and many upstream signaling components have recently been characterized in basal phyla, including Cnidaria (sea anemones, corals, hydras, and jellyfish), Porifera (sponges), and single-celled protists, including Capsaspora owczarzaki and some choanoflagellates. Herein, we review what is known about basal NF-κBs and how that knowledge informs on the evolution and conservation of key sequences and domains in NF-κB, as well as the regulation of NF-κB activity. The structures and DNA-binding activities of basal NF-κB proteins resemble those of mammalian NF-κB p100 proteins, and their posttranslational activation appears to have aspects of both canonical and noncanonical pathways in mammals. Several studies suggest that the single NF-κB proteins found in some basal organisms have dual roles in development and immunity. Further research on NF-κB in invertebrates will reveal information about the evolutionary roots of this major signaling pathway, will shed light on the origins of regulated innate immunity, and may have relevance to our understanding of the responses of ecologically important organisms to changing environmental conditions and emerging pathogen-based diseases.

Molecular characterization and functional analysis of IKKα in orange-spotted grouper (Epinephelus coioides).

Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays crucial roles in regulating activation of nuclear factor kappa-B (NF-κB) in response to pathogens infections. Here, we cloned and identified IKKα gene of orange-spotted grouper (Epinephelus coioides), named as EcIKKα. The gene transcript contained a 2262 bp open reading frame, which encoded 753 amino acids. The typically conserved IKKα structure, including serine kinase domain (KD), leucine chain (LZ) structure, helix-loop-helix (HLH) motif and IKKß-NEMO-binding domain, was identified in EcIKKα. Phylogenetic analysis suggested that EcIKKα had the closest relationship with large yellow croaker (Larimichthy crocea) IKKα. Ecikkα was ubiquitously expressed in all tissues tested and the highest expression level was in ovary. After lipopolysaccharide (LPS), flagellin, polyinosinic-polycytidylic acid (poly I:C), polyadenylic-polyuridylic acid (poly A:U), and Vibrio parahaemolyticus stimulation, the expression of Ecikkα increased in grouper spleen (GS) cells. In the luciferase assay, NF-κB-luc activity was significantly up-regulated when human embryonic kidney 293T (HEK 293T) cells were transfected with EcIKKα plasmid. Moreover, overexpression of EcIKKα significantly increased LPS- and flagellin-induced proinflammatory cytokines (interleukin-6 (il-6) and tumor necrosis factor-α (tnf-α)) expression, but did not significantly affect poly I:C- and poly A:U-induced cytokines (il-6 and tnf-α) expression. Overall, these results suggested that EcIKKα functions like that of mammals to activate NF-κB, and it could be involved in host defense against invading pathogens.

Identification of an IKKß inhibitor for inhibition of inflammation in vivo and in vitro.

Targeting on the IKKß to discover anti-inflammatory drugs has been launched for ten years, due to its predominant role in canonical NF-κB signaling. In the current study, we identified a novel IKKß inhibitor, ellipticine (ELL), an alkaloid isolated from Ochrosia elliptica and Rauvolfia sandwicensis. We found that ELL reduced the secretion and mRNA expression of TNF-α and IL-6 and decreased the protein expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in bone marrow derived macrophages (BMDMs) stimulated with LPS. In coincided with the results, ELL suppressed PGE2 and NO production in BMDMs. Underlying mechanistic study showed that ELL inhibited IκBα phosphorylation and degradation as well as NF-κB nuclear translocation, which was attributed to suppression of IKKα/ß activation. Furthermore, kinase assay and binding assay results indicated that ELL inhibited IKKß activity via directly binding to IKKß and in turn resulted in suppression of NF-κB signaling. To identify the binding sites of ELL on IKKß, IKKßC46A plasmid was prepared and the kinase assay was performed. The results demonstrated that the inhibitory effect of ELL on IKKß activity was impaired in the mutation, implying that anti-inflammatory effect of ELL was partially attributed to binding on cysteine 46. Furthermore, ELL up-regulated LC3 II expression and reduced p62 expression, suggesting that autophagy induction contributed to the anti-inflammatory effect of ELL as well. In coincided with the in vitro results, ELL increased the survival and antagonized the hypothermia in the mice with LPS-induced septic shock. Consistently, ELL reduced TNF-α and IL-6 production in the serum of the mice treated with LPS. Collectively, our study provides evidence that ELL is an IKKß inhibitor and has potential to be developed as a lead compound for treatment inflammatory diseases in the future.

Oyster Versatile IKKα/ßs Are Involved in Toll-Like Receptor and RIG-I-Like Receptor Signaling for Innate Immune Response.

IκB kinases (IKKs) play critical roles in innate immunity through signal-induced activation of the key transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factors (IRFs). However, studies of invertebrate IKK functions remain scarce. In this study, we performed phylogenetic analysis of IKKs and IKK-related kinases encoded in the Pacific oyster genome. We then cloned and characterized the oyster IKKα/ß-2 gene. We found that oyster IKKα/ß-2, a homolog of human IKKα/IKKß, responded to challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), and polyinosinic-polycytidylic acid [poly(I:C)]. As a versatile immune molecule, IKKα/ß-2 activated the promoters of NF-κB, TNFα, and IFNß, as well as IFN-stimulated response element (ISRE)-containing promoters, initiating an antibacterial or antiviral immune state in mammalian cells. Importantly, together with the cloned oyster IKKα/ß-1, we investigated the signal transduction pathways mediated by these two IKKα/ß proteins. Our results showed that IKKα/ß-1 and IKKα/ß-2 could interact with the oyster TNF receptor-associated factor 6 (TRAF6) and that IKKα/ß-2 could also bind to the oyster myeloid differentiation factor 88 (MyD88) protein directly, suggesting that oyster IKKα/ßs participate in both RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling for the reception of upstream immune signals. The fact that IKKα/ß-1 and IKKα/ß-2 formed homodimers by interacting with themselves and heterodimers by interacting with each other, along with the fact that both oyster IKKα/ß proteins interacted with NEMO protein, indicates that oyster IKKα/ßs and the scaffold protein NEMO form an IKK complex, which may be a key step in phosphorylating IκB proteins and activating NF-κB. Moreover, we found that oyster IKKα/ßs could interact with IRF8, and this may be related to the IKK-mediated activation of ISRE promotors and their involvement in the oyster "interferon (IFN)-like" antiviral pathway. Moreover, the expression of oyster IKKα/ß-1 and IKKα/ß-2 may induce the phosphorylation of IκB proteins to activate NF-κB. These results reveal the immune function of oyster IKKα/ß-2 and establish the existence of mollusk TLR and RLR signaling mediated by IKKα/ß proteins for the first time. Our findings should be helpful in deciphering the immune mechanisms of invertebrates and understanding the development of the vertebrate innate immunity network.

LL-37 alone and in combination with IL17A enhances proinflammatory cytokine expression in parallel with hyaluronan metabolism in human synovial sarcoma cell line SW982-A step toward understanding the development of inflammatory arthritis.

LL-37 is the only human cathelicidin-family host defense peptide and has been reported to interact with invading pathogens causing inflammation at various body sites. Recent studies showed high levels of LL-37 in the synovial-lining membrane of patients with rheumatoid arthritis, a common type of inflammatory arthritis. The present study aims to investigate the role of LL-37 on mechanisms associated with pathogenesis of inflammatory arthritis. The effects of LL-37 on the expression of proinflammatory cytokines, hyaluronan (HA) metabolism-related genes, cell death-related pathways, and cell invasion were investigated in SW982, a human synovial sarcoma cell line. Time-course measurements of proinflammatory cytokines and mediators showed that LL-37 significantly induced IL6 and IL17A mRNA levels at early time points (3-6 hr). HA-metabolism-related genes (i.e., HA synthase 2 (HAS2), HAS3, hyaluronidase 1 (HYAL1), HYAL2, and CD44) were co-expressed in parallel. In combination, LL-37 and IL17A significantly enhanced PTGS2, TNF, and HAS3 gene expression concomitantly with the elevation of their respective products, PGE2, TNF, and HA. Cell invasion rates and FN1 gene expression were also significantly enhanced. However, LL-37 alone or combined with IL17A did not affect cell mortality or cell cycle. Treatment of SW982 cells with both LL-37 and IL17A significantly enhanced IKK and p65 phosphorylation. These findings suggest that the chronic production of a high level of LL-37 may synchronize with its downstream proinflammatory cytokines, especially IL17A, contributing to the co-operative enhancement of pathogenesis mechanisms of inflammatory arthritis, such as high production of proinflammatory cytokines and mediators together with the activation of HA-metabolism-associated genes and cell invasion.

FcγR-TLR Cross-Talk Enhances TNF Production by Human Monocyte-Derived DCs via IRF5-Dependent Gene Transcription and Glycolytic Reprogramming.

Antigen-presenting cells (APCs) such as dendritic cells (DCs) are crucial for initiation of adequate inflammatory responses, which critically depends on the cooperated engagement of different receptors. In addition to pattern recognition receptors (PRRs), Fc gamma receptors (FcγRs) have recently been identified to be important in induction of inflammation by DCs. FcγRs that recognize IgG immune complexes, which are formed upon opsonization of pathogens, induce pro-inflammatory cytokine production through cross-talk with PRRs such as Toll-like receptors (TLRs). While the physiological function of FcγR-TLR cross-talk is to provide protective immunity against invading pathogens, undesired activation of FcγR-TLR cross-talk, e.g., by autoantibodies, also plays a major role in the development of chronic inflammatory disorders such as rheumatoid arthritis (RA). Yet, the molecular mechanisms of FcγR-TLR cross-talk are still largely unknown. Here, we identified that FcγR-TLR cross-talk-induced cytokine production critically depends on activation of the transcription factor interferon regulatory factor 5 (IRF5), which results from induction of two different pathways that converge on IRF5 activation. First, TLR stimulation induced phosphorylation of TBK1/IKKε, which is required for IRF5 phosphorylation and subsequent activation. Second, FcγR stimulation induced nuclear translocation of IRF5, which is essential for gene transcription by IRF5. We identified that IRF5 activation by FcγR-TLR cross-talk amplifies pro-inflammatory cytokine production by increasing cytokine gene transcription, but also by synergistically inducing glycolytic reprogramming, which is another essential process for induction of inflammatory responses by DCs. Combined, here we identified IRF5 as a pivotal component of FcγR-TLR cross-talk in human APCs. These data may provide new potential targets to suppress chronic inflammation in autoantibody-associated diseases that are characterized by undesired or excessive FcγR-TLR cross-talk, such as RA, systemic sclerosis, and systemic lupus erythematous.

Engagement of Fas differentially regulates the production of LPS-induced proinflammatory cytokines and type I interferons.

Fas (CD95) signalling is best known for its role in apoptosis, however, recent reports have shown it to be involved in other cellular responses as well, including inflammation. Fas and its adaptor protein FADD are known to negatively regulate LPS-induced proinflammatory responses, but their role in LPS-induced type I interferon production is unknown. Here, we demonstrate that Fas engagement on macrophages, using an agonistic Fas antibody CH11, augments LPS-induced NF-κB responses, causing increased production of TNFα, IL-8, IL-6 and IL-12. Conversely, costimulation with both LPS and CH11 causes a significant reduction in the level of interferon-beta (IFNß) production. This differential effect involves the Fas adaptor FADD because while LPS-induced IL-6 production increased in FADD-/- murine embryonic fibroblasts, LPS-induced IFNß production was significantly reduced in these cells. Overexpression of a dominant negative form of FADD (FADD-DD) inhibits LPS-induced IFNß luciferase but not LPS-induced NF-κB luciferase. In contrast, overexpression of full-length FADD inhibited LPS-induced NF-κB luciferase activation but was seen to augment LPS-induced IFNß luciferase. Moreover, FADD-DD inhibits TRIF-, TRAM-, IKKε-, TBK-1- and TRAF3-induced IFNß luciferase production, with coimmunoprecipitation experiments demonstrating an interaction between FADD and TRIF. These data identify FADD as a novel component of the noncanonical Toll-like receptor 4/IFNß signalling pathway and demonstrate that both Fas and its adaptor FADD can differentially regulate the production of LPS-induced proinflammatory cytokines and type I interferons.