Negative Regulator Of Innate Immunity Research Articles

IRAK-M deficiency exacerbates dopaminergic neuronal damage in a mouse model of sub-acute Parkinson's disease.

Emerging evidence has proved that inflammatory responses aggravate the pathological progression of Parkinson's disease. This study aimed to identify the role of Interleukin-1 receptor-associated kinase-M (IRAK-M) as an important negative regulator of innate immunity, in the pathological progression of Parkinson's disease. In the present study, a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection was administered to prepare the acute and sub-acute Parkinson's disease mouse models. Western blot analysis was utilized to examine the protein expressions of tyrosine hydroxylase and IRAK-M. The mRNA expression levels of IRAK-M, interleukin (IL)-6, IL-β, and cyclooxygenase-2 were evaluated via using reverse transcription quantitative PCR (RT-qPCR). The expression of tyrosine hydroxylase-positive neurons in corpus striatum and substantia nigra pars compacta (SNc) tissues was detected using immunohistochemistry. The results showed that the protein and mRNA levels of IRAK-M were considerably upregulated in corpus striatum and SNc tissues in the sub-acute Parkinson's disease model. Furthermore, IRAK-M knockout significantly enhanced the MPTP-induced loss of tyrosine hydroxylase-positive fibers in corpus striatum and tyrosine hydroxylase-positive neurons in SNc, and intensified the effect of MPTP on the activation of microglial cells and the expression of inflammatory cytokines. In addition, sub-acute Parkinson's disease mice with IRAK-M deletion exhibited worse motor abilities than those of wild-type littermates. Overall, the present study suggested that IRAK-M reduces dopaminergic neuron damage in sub-acute Parkinson's disease by suppressing inflammation, which may provide a new therapeutic target for Parkinson's disease treatment.

C. elegans orphan nuclear receptor NHR-42 represses innate immunity and promotes lipid loss downstream of HLH-30/TFEB.

In recent years, transcription factors of the Microphthalmia-TFE (MiT) family, including TFEB and TFE3 in mammals and HLH-30 in Caenorhabditis elegans, have emerged as important regulators of innate immunity and inflammation in invertebrates and vertebrates. Despite great strides in knowledge, the mechanisms that mediate downstream actions of MiT transcription factors in the context of innate host defense remain poorly understood. Here, we report that HLH-30, which promotes lipid droplet mobilization and host defense, induces the expression of orphan nuclear receptor NHR-42 during infection with Staphylococcus aureus. Remarkably, NHR-42 loss of function promoted host infection resistance, genetically defining NHR-42 as an HLH-30-controlled negative regulator of innate immunity. During infection, NHR-42 was required for lipid droplet loss, suggesting that it is an important effector of HLH-30 in lipid immunometabolism. Moreover, transcriptional profiling of nhr-42 mutants revealed wholesale activation of an antimicrobial signature, of which abf-2, cnc-2, and lec-11 were important for the enhanced survival of infection of nhr-42 mutants. These results advance our knowledge of the mechanisms by which MiT transcription factors promote host defense, and by analogy suggest that TFEB and TFE3 may similarly promote host defense via NHR-42-homologous nuclear receptors in mammals.

Heterogeneous Nuclear Protein U Degraded the m6A Methylated TRAF3 Transcript by YTHDF2 To Promote Porcine Epidemic Diarrhea Virus Replication.

Porcine epidemic diarrhea virus (PEDV) belongs to the genus Alphacoronavirus of the Coronaviridae family and can cause fatal watery diarrhea in piglets, causing significant economic losses. Heterogeneous nuclear protein U (HNRNPU) is a novel RNA sensor involved in sensing viral RNA in the nucleus and mediating antiviral immunity. However, it remains elusive whether and how cytoplasmic PEDV can be sensed by the RNA sensor HNRNPU. In this study we determined that HNRNPU was the binding partner of Nsp13 by immunoprecipitation-liquid chromatography-tandem mass spectrometry (IP/LC-MS/MS) analysis. The interaction between Nsp13 and HNRNPU was demonstrated by using coimmunoprecipitation and confocal immunofluorescence. Next, we identified that HNRNPU expression is significantly increased during PEDV infection, whereas the transcription factor hepatocyte nuclear factor 1α (HNF1A) could negatively regulate HNRNPU expression. HNRNPU was retained in the cytoplasm by interaction with PEDV Nsp13. We found that HNRNPU overexpression effectively facilitated PEDV replication, while knockdown of HNRNPU impaired viral replication, suggesting a promoting function of HNRNPU to PEDV infection. Additionally, HNRNPU was found to promote PEDV replication by affecting TRAF3 degradation at the transcriptional level to inhibit PEDV-induced beta interferon (IFN-β) production. Mechanistically, HNRNPU downregulates TRAF3 mRNA levels via the METTL3-METTL14/YTHDF2 axis and regulates immune responses through YTHDF2-dependent mRNA decay. Together, our findings reveal that HNRNPU serves as a negative regulator of innate immunity by degrading TRAF3 mRNA in a YTHDF2-dependent manner and consequently facilitating PEDV propagation. Our findings provide new insights into the immune escape of PEDV. IMPORTANCE PEDV, a highly infectious enteric coronavirus, has spread rapidly worldwide and caused severe economic losses. During virus infection, the host regulates innate immunity to inhibit virus infection. However, PEDV has evolved a variety of different strategies to suppress host IFN-mediated antiviral responses. Here, we identified that HNRNPU interacted with viral protein Nsp13. HNRNPU protein expression was upregulated, and the transcription factor HNF1A could negatively regulate HNRNPU expression during PEDV infection. HNRNPU also downregulated TRAF3 mRNA through the METTL3-METTL14/YTHDF2 axis to inhibit the production of IFN-β and downstream antiviral genes in PEDV-infected cells, thereby promoting viral replication. Our findings reveal a new mechanism with which PEDV suppresses the host antiviral response.

Lacticaseibacillus rhamnosus R0011 secretome attenuates Salmonella enterica serovar Typhimurium secretome-induced intestinal epithelial cell monolayer damage and pro-inflammatory mediator production in intestinal epithelial cell and antigen-presenting cell co-cultures.

Certain lactic acid bacteria (LAB) are associated with immune modulatory activities including down-regulation of pro-inflammatory gene transcription and expression. While host antigen-presenting cells (APCs) and intestinal epithelial cells (IEC) can interact directly with both pathogenic and commensal bacteria through innate immune pattern recognition receptors, recent evidence indicates indirect communication through secreted molecules is an important inter-domain communication mechanism. This communication route may be especially important in the context of IEC and APC interactions which shape host immune responses within the gut environment. We have previously shown that the Lacticaseibacillus rhamnosus R0011 secretome (LrS) dampens pro-inflammatory gene transcription and mediator production from Tumor Necrosis Factor-α and Salmonella enterica serovar Typhimurium secretome (STS)-challenged HT-29 IECs through the induction of negative regulators of innate immunity. However, many questions remain about interactions mediated through these bacterial-derived soluble components and the resulting host immune outcomes in the context of IEC and APC interactions. In the present study, we examined the ability of the LrS to down-regulate pro-inflammatory gene transcription and cytokine production from STS-challenged T84 human IEC and THP-1 human monocyte co-cultures. Cytokine and chemokine profiling revealed that apically delivered LrS induces apical secretion of macrophage inhibitory factor (MIF) and down-regulates STS-induced pro-inflammatory mediator secretion into the apical and basolateral chambers of the T84/THP-1 co-culture. Transcriptional profiling confirmed these results, as the LrS attenuated STS challenge-induced CXCL8 and NFκB1 expression in T84 IECs and THP-1 APCs. Interestingly, the LrS also reversed STS-induced damage to monolayer transepithelial resistance (TER) and permeability, results which were confirmed by ZO-1 gene expression and immunofluorescence visualization of ZO-1 expression in T84 IEC monolayers. The addition of a MIF-neutralizing antibody abrogated the ability of the LrS to reverse STS-induced damage to T84 IEC monolayer integrity, suggesting a novel role for MIF in maintaining IEC barrier function and integrity in response to soluble components derived from LAB. The results presented here provide mechanistic evidence for indirect communication mechanisms used by LAB to modulate immune responses to pathogen challenge, using in vitro approaches which allow for IEC and APC cell communication in a context which more closely mimics that which occurs in vivo.

The RNA helicase DDX5 promotes viral infection via regulating N6-methyladenosine levels on the DHX58 and NFκB transcripts to dampen antiviral innate immunity.

Multi-functional DEAD-box helicase 5 (DDX5), which is important in transcriptional regulation, is hijacked by diverse viruses to facilitate viral replication. However, its regulatory effect in antiviral innate immunity remains unclear. We found that DDX5 interacts with the N6-methyladenosine (m6A) writer METTL3 to regulate methylation of mRNA through affecting the m6A writer METTL3–METTL14 heterodimer complex. Meanwhile, DDX5 promoted the m6A modification and nuclear export of transcripts DHX58, p65, and IKKγ by binding conserved UGCUGCAG element in innate response after viral infection. Stable IKKγ and p65 transcripts underwent YTHDF2-dependent mRNA decay, whereas DHX58 translation was promoted, resulting in inhibited antiviral innate response by DDX5 via blocking the p65 pathway and activating the DHX58-TBK1 pathway after infection with RNA virus. Furthermore, we found that DDX5 suppresses antiviral innate immunity in vivo. Our findings reveal that DDX5 serves as a negative regulator of innate immunity by promoting RNA methylation of antiviral transcripts and consequently facilitating viral propagation.

803 A20 and its repressor DREAM expression govern susceptibility to fibrosis in systemic sclerosis

Recent GWAS have uncovered consistent genetic linkage of SSc fibrotic phenotypes with TNFAIP3, encoding the ubiquitin-editing enzyme A20. A20 has been previously implicated in negative regulation of innate immunity, and hypomorphic A20 variants are associated with autoimmunity in SLE, RA,and others.The transcription factor DREAM binds to the A20 promoter to repress expression. Unbiased transcriptome analysis of skin and lung biopsies from SSc patients showed significantly decreased A20 levels and robust anti-correlation with fibrotic TGF-beta signaling.

Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1-Dependent Induction of SOCS1.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused tremendous economic losses in the swine industry since its emergence in the late 1980s. PRRSV exploits various strategies to evade immune responses and establish chronic persistent infections. Suppressor of cytokine signaling (SOCS) 1, a member of the SOCS family, is a crucial intracellular negative regulator of innate immunity. In this study, it was shown that SOCS1 can be co-opted by PRRSV to evade host immune responses, facilitating viral replication. It was observed that PRRSV induced SOCS1 production in porcine alveolar macrophages, monkey-derived Marc-145 cells, and porcine-derived CRL2843-CD163 cells. SOCS1 inhibited the expression of IFN-β and IFN-stimulated genes, thereby markedly enhancing PRRSV replication. It was observed that the PRRSV N protein has the ability to upregulate SOCS1 production and that nuclear localization signal-2 (NLS-2) is essential for SOCS1 induction. Moreover, SOCS1 upregulation was dependent on p38/AP-1 and JNK/AP-1 signaling pathways rather than classical type I IFN signaling pathways. In summary, to our knowledge, the findings of this study uncovered the molecular mechanism that underlay SOCS1 induction during PRRSV infection, providing new insights into viral immune evasion and persistent infection.

IRAK family in inflammatory autoimmune diseases

Innate immune signaling plays an important role in inflammation, and dysregulation of signaling components within this pathway has been focused as a critical mediator in initiation, progression of inflammatory autoimmune diseases. Toll-like receptors (TLRs) are the most upstream pattern recognition receptors in the immune cells, detecting pathogen associated molecular patterns, initiating signal transduction, by which interleukin-1 receptor-associated kinase (IRAK) family mediates activating signal from TLRs and interleukin-1 receptor. The family comprises of four members, IRAK1, IRAK2, IRAK-M, IRAK4. The family members have a role in either positive or negative regulation of innate immunity, adaptive immunity and inflammation. Accumulated evidence proves that IRAK performs significantly in the pathogenesis of inflammatory autoimmune disorders. On the one hand, both patients and animal modes reported abnormal expression of the family members. On the other hand, functional study in vivo and in vitro demonstrated that the members are implicated in the development of the diseases. Interestingly, IRAK inhibition has potential therapeutic benefits. In this review, we focus on the family, review the physiological roles in different immune cells, and summarize emerging data for highlighting the importance of them in inflammatory autoimmunity.

Differential expression of innate immunity regulation genes in chronic HIV-1 infected adults

ObjectiveChronic activation of the innate immune system plays a central role in HIV-1 disease progression. Negative regulation of innate immunity is critical in preventing the effects of this excessive activation; however, the molecules involved in this process remain to be identified. In this study, we compared the expression of immune regulation genes between HIV-1 infected individuals and healthy control participants to identify genes involved in the regulation of innate immunity in HIV-1 infection. MethodsWe conducted gene expression analysis of a series of immune regulatory genes in viremic treatment-naïve HIV-positive donors, patients receiving highly active antiretroviral therapy (HAART) and HIV-negative healthy control participants. Reverse transcription-quantitative PCR (RT-qPCR) was conducted to determine the expression levels of genes in peripheral blood mononuclear cells isolated from all participants. The spearman correlation test and linear regression analysis were performed to evaluate the correlation between gene expression level and viral load. ResultsThe following differentially expressed genes were identified: A20, CYLD, DDX24, MARCH5, MKRN2, PTP1B, RNF125, S1PR1, SOCS1, IFI35, RBCK1, TTLL12 and USP18. The three most differentially expressed genes were A20, S1PR1, and USP18. USP18 correlated positively with viral load. ConclusionThirteen immune regulation genes were identified in comparisons of viremic treatment-naïve HIV-positive donors, HAART-treated patients and healthy control participants, indicating the potential of these genes as therapeutic targets.

Elevated TNIP3 mRNA Expression in TNF-α-Secreting Cells from Patients with Major Depressive Disorder

Objective: Elevated levels of pro-inflammatory cytokines, in particular tumor necrotic factor alpha (TNF-α), and abnormalities in negative regulation in Toll-like receptor (TLR) signaling pathways are associated with major depressive disorder (MDD). Previous research by our group disclosed lower expression of TNF-α-induced protein 3 (TNFAIP3), one of the negative regulators of the TLR4 signaling pathway, in depressive patients than in healthy controls. Methods: In this study, we assessed the mRNA levels of TNFAIP3, TNFAIP3-interacting proteins (TNIP), including TNIP1, TNIP2, and TNIP3, and TNFAIP3-like proteins, such as cezanne1, cezanne2, trabid, and VCIP135, in TNF-α-secreting cells and examined their association with severity of depression using the 17-item Hamilton Depression Rating Scale (HAMD-17) scores from 30 MDD patients and 30 healthy controls. Twenty-six patients received a second assessment after treatment with antidepressants for 4 weeks. Results: TNF-α-secreting cells displayed higher TNIP3 mRNA expression in MDD patients than in healthy controls before treatment, which was marginally decreased after antidepressant treatment. In addition, the TNIP2 level could be effectively applied to predict changes in HAMD scores after linear regression analysis. Conclusion: Our collective findings suggest that molecules associated with negative regulation of innate immunity are aberrantly expressed in patients with MDD and present potential therapeutic targets.

Abstract 1555: Monoclonal antibodies targeting the TAM family of receptor tyrosine kinases

Abstract The TAM receptors (Tyro3/Axl/MerTK) family of receptor tyrosine kinases (RTKs) are important negative regulators of innate immunity. TAM receptor activation in myeloid cells by its ligands Gas6 or Protein S (PROS) promotes phosphatidylserine-dependent efferocytosis of apoptotic cells, inducing a tolerogenic state and mediating resolution of inflammation. TAM-deficient mice exhibit phenotypes consistent with systemic inflammation and autoimmunity. Importantly, individual ablation of TAM receptors can confer tumor immunity, increased pro-inflammatory cytokines and tumor lymphocyte infiltration, leading to the proposal that TAM receptors act as checkpoints of innate immunity. We hypothesize that pharmacological targeting of this family of receptors with monoclonal antibodies (mAbs) may lead to a similar pro-inflammatory response and recapitulate the antitumor effects observed in TAM-deficient mice. From a panel of human anti-MerTK, Axl or Tyro3 mAbs derived from phage-display libraries or human IgG expressing mice we identified unique mAbs that markedly enhanced cytokine and chemokine release from primary human immune cells, alone or in the presence of inflammatory stimuli. Interestingly, the qualitative and quantitative pattern of cytokine response from dendritic cells was very similar using antibodies targeting the individual TAM receptors, and also similar to activation of dendritic cells using a CD40 agonist mAb, suggesting that TAM-targeting mAbs can promote immune activation. We identified surrogate mAbs targeting mouse TAM receptors that elicited similar responses in vivo, and demonstrated antitumor activity when dosed alone, or in combination with PD-1/L1 blockade in syngeneic tumor models. In addition, human MerTK transgenic, and TAM knockout mice have been generated and characterized in order to establish in vivo proof-of-concept with human TAM mAbs. Overall, pharmacological modulation of TAM receptors with mAbs enhances cytokine production in human and murine model systems consistent with the published role of TAMs as negative regulators of innate immunity. Mechanistic and proof-of-concept studies support further development of these mAbs as novel approaches to overcome these checkpoints of the innate immune response. Citation Format: Diego Alvarado, Laura Vitale, Mike Murphy, Thomas O'Neill, Andrew Proffitt, Jay Lillquist, Gwenda Ligon, Komal Patel, Anna Wasiuk, Jeff Weidlick, Jenifer Widger, Laura Mills-Chen, Andrea Crocker, Colleen Patterson, Russell A. Hammond, Li-Zhen He, Joel Goldstein, Lawrence J. Thomas, Henry C. Marsh, Tibor Keler, Richard Gedrich. Monoclonal antibodies targeting the TAM family of receptor tyrosine kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1555.

Whole transcriptome analysis of the Atlantic cod vaccine response reveals subtle changes in adaptive immunity

Atlantic cod has lost the Major Histocompatibility complex class II pathway – central to pathogen presentation, humoral response and immunity. Here, we investigate the immunological response of Atlantic cod subsequent to dip vaccination with Vibrioanguillarum bacterin using transcriptome sequencing. The experiment was conducted on siblings from an Atlantic cod family found to be highly susceptible towards vibriosis where vaccination has demonstrated improved pathogen resistance. Gene expression analyses at 2, 4, 21 and 42 days post vaccination revealed GO-term enrichment for muscle, neuron and metabolism-related pathways. In-depth characterization of immune-related GO terms demonstrated down-regulation of MHCI antigen presentation, C-type lectin receptor signaling and granulocyte activation over time. Phagocytosis, interferon-gamma signaling and negative regulation of innate immunity were increasingly up-regulated over time. Individual differentially expressed immune genes implies weak initiation of acute phase proteins with little or no inflammation. Furthermore, gene expression indicates presence of T-cells, NK-like cells, B-cells and monocytes/macrophages. Three MHCI transcripts were up-regulated with B2M and SEC61. Overall, we find no clear immune-related transcriptomic response which could be attributed to Atlantic cod's alternative immune system. However, we cannot rule out that this response is related to vaccination protocol/sampling strategy. Earlier functional studies demonstrate significant memory in Atlantic cod post dip vaccination and combined with our results indicate the presence of other adaptive immunity mechanisms. In particular, we suggest that further investigations should look into CD8+ memory T-cells, γδ T-cells, T-cell independent memory or memory induced through NK-like/other lymphoid cells locally in the mucosal lining for this particular vaccination strategy.

Xanthomonas oryzae pv. oryzae XopQ protein suppresses rice immune responses through interaction with two 14-3-3 proteins but its phospho-null mutant induces rice immune responses and interacts with another 14-3-3 protein.

SummaryMany bacterial phytopathogens employ effectors secreted through the type‐III secretion system to suppress plant innate immune responses. The Xanthomonas type‐III secreted non‐TAL effector protein Xanthomonas outer protein Q (XopQ) exhibits homology to nucleoside hydrolases. Previous work indicated that mutations which affect the biochemical activity of XopQ fail to affect its ability to suppress rice innate immune responses, suggesting that the effector might be acting through some other pathway or mechanism. In this study, we show that XopQ interacts in yeast and in planta with two rice 14‐3‐3 proteins, Gf14f and Gf14g. A serine to alanine mutation (S65A) of a 14‐3‐3 interaction motif in XopQ abolishes the ability of XopQ to interact with the two 14‐3‐3 proteins and to suppress innate immunity. Surprisingly, the S65A mutant gains the ability to interact with a third 14‐3‐3 protein that is a negative regulator of innate immunity. The XopQS65A mutant is an inducer of rice immune responses and this property is dominant over the wild‐type function of XopQ. Taken together, these results suggest that XopQ targets the rice 14‐3‐3 mediated immune response pathway and that its differential phosphorylation might enable interaction with alternative 14‐3‐3 proteins.

The Unique Inhibitory IgG Receptor--FcγRIIb.

FcγRIIb is the only inhibitory IgG receptor, which is divided into three subtypes of FcγRIIb1, FcγRIIb2 and FcγRIIb3. It is mainly responsible for the immune balance in vivo by cross-linking with the activated receptor to intracellular transduction inhibitory signals, and it plays an important biological role in the negative regulation of innate immunity and adaptive immunity. An abnormal expression of FcγRIIb on cells would result in autoimmune diseases, infectious diseases, and so forth. FcγRIIb modulates immune responses and further treats related diseases by inhibiting the activation of B lymphocytes, monocytes, mast cells, and basophils induced by activating receptors. It can be used in biotherapeutic methods such as monoclonal antibodies, chimeric recombinant proteins, bispecific antibodies, etc. Our increased understanding of FcγRIIb function also has a foundation for further research and development of FcγRIIb, which also provides potentially farreaching therapeutic implications.

Gasdermin D exerts anti-inflammatory effects by promoting neutrophil death

Abstract Gasdermin D (GSDMD) is considered a pro-inflammatory factor that mediates pyroptosis in macrophages to protect hosts from intracellular bacteria. Here we reveal that GSDMD deficiency paradoxically augmented host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, establishing GSDMD as a negative regulator of innate immunity. In contrast to its activation in macrophages, in which activated inflammatory caspases cleave GSDMD to produce an N-terminal fragment (GSDMD-cNT) to trigger pyroptosis, GSDMD cleavage and activation in neutrophils was caspase independent. It was mediated by a neutrophil-specific serine protease, neutrophil elastase (ELANE), released from cytoplasmic granules into the cytosol in aging neutrophils. ELANE-mediated GSDMD cleavage was upstream of the caspase cleavage site and produced a fully active ELANE-derived N-terminal fragment (GSDMD-eNT) that induced lytic cell death as efficiently as GSDMD-cNT. Thus, GSDMD is pleiotropic, exerting both pro- and anti-inflammatory effects that make it a potential target for anti-bacterial and anti-inflammatory therapies.

The Superimposed Deubiquitination Effect of OTULIN and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Nsp11 Promotes Multiplication of PRRSV.

Linear ubiquitination plays an important role in the regulation of the immune response by regulating nuclear factor κB (NF-κB). The linear ubiquitination-specific deubiquitinase ovarian tumor domain deubiquitinase with linear linkage specificity (OTULIN) can control the immune signaling transduction pathway by restricting the Met1-linked ubiquitination process. In our study, the porcine OTLLIN gene was cloned and deubiquitin functions were detected in a porcine reproductive and respiratory syndrome virus (PRRSV)-infected-cell model. PRRSV infection promotes the expression of the OTULIN gene; in turn, overexpression of OTULIN contributes to PRRSV proliferation. There is negative regulation of innate immunity with OTULIN during viral infection. The cooperative effects of swine OTULIN and PRRSV Nsp11 potentiate the ability to reduce levels of cellular protein ubiquitin associated with innate immunity. Importantly, PRRSV Nsp11 recruits OTULIN through a nonenzymatic combination to enhance its ability to remove linear ubiquitination targeting NEMO, resulting in a superimposed effect that inhibits the production of type I interferons (IFNs). Our report presents a new model of virus utilization of the ubiquitin-protease system in vivo from the perspective of the viral proteins that interact with cell deubiquitination enzymes, providing new ideas for prevention and control of PRRSV.IMPORTANCE Deubiquitination effects of swine OTULIN were identified. The interaction between porcine OTULIN and PRRSV Nsp11 is dependent on the OTU domain. PRRSV Nsp11 recruits OTULIN through a nonenzymatic combination to promote removal of linear ubiquitination targeting NEMO, resulting in a superimposed effect that inhibits the production of type I IFNs.

Gasdermin D Exerts Anti-inflammatory Effects by Promoting Neutrophil Death

SUMMARYGasdermin D (GSDMD) is considered a proinflammatory factor that mediates pyroptosis in macrophages to protect hosts from intracellular bacteria. Here, we reveal that GSDMD deficiency paradoxically augmented host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, which established GSDMD as a negative regulator of innate immunity. In contrast to its activation in macrophages, in which activated inflammatory caspases cleave GSDMD to produce an N-terminal fragment (GSDMD-cNT) to trigger pyroptosis, GSDMD cleavage and activation in neutrophils was caspase independent. It was mediated by a neutrophil-specific serine protease, neutrophil elastase (ELANE), released from cytoplasmic granules into the cytosol in aging neutrophils. ELANE-mediated GSDMD cleavage was upstream of the caspase cleavage site and produced a fully active ELANE-derived NT fragment (GSDMD-eNT) that induced lytic cell death as efficiently as GSDMD-cNT. Thus, GSDMD is pleiotropic, exerting both pro- and anti-inflammatory effects that make it a potential target for antibacterial and anti-inflammatory therapies.

Gasdermin D Exerts Anti-Inflammatory Effects by Promoting Neutrophil Death

Gasdermin D (GSDMD) is considered a pro-inflammatory factor that mediates lytic pyroptotic cell death in macrophages to protect hosts from intracellular bacteria (He et al., 2015; Kayagaki et al., 2015; Shi et al., 2015a). However, GSDMD’s role in clearing extracellular pathogens has not been directly examined. Here we reveal that GSDMD deficiency unexpectedly and paradoxically augmented host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, for the first time establishing GSDMD as a negative regulator of innate immunity. The levels of pro-inflammatory cytokines in the peritoneal cavity were significantly elevated in GSDMD-deficient mice, suggesting that the production of these cytokines was not mainly mediated by macrophage pyroptosis. This further confirmed that in E.coli-induced peritonitis GSDMD acted as an anti-inflammatory factor. In contrast to its activation in macrophages, in which activated inflammatory caspases cleave GSDMD to produce an N-terminal fragment (GSDMD-cNT) to trigger pyroptosis, GSDMD cleavage and activation in neutrophils was caspase independent and mediated by a neutrophil-specific serine protease, neutrophil elastase (ELANE), released from cytoplasmic granules into the cytosol during neutrophil death. ELANE-mediated GSDMD cleavage was upstream of the caspase cleavage site and produced a fully active ELANE-derived Nterminal fragment (GSDMD-eNT) that induced lytic cell death as efficiently as GSDMD-cNT. This is the first description of GSDMD’s role in neutrophil death and the negative regulation of neutrophil-mediated innate immunity. Thus, GSDMD is pleiotropic, exerting both pro- and anti-inflammatory effects that make it a unique target for novel anti-bacterial and anti-inflammatory therapies.

Expression Pattern of Tumor Necrosis Factor-α–Induced Protein 8-Like 2 in Acute Rejection of Cardiac Allograft

BackgroundTumor necrosis factor-α–induced protein-8 like-2 (TIPE2) is a negative regulator of innate immunity and cellular immunity, yet the expression pattern of TIPE2 in acute rejection of cardiac allograft remain enigmatic. MethodsWe developed cardiac transplantation models and divided into 3 groups: a naive group, a syngeneic group, and an allogeneic group. Then, we detected the messenger RNA and protein of TIPE2 in cardiac allografts. Real-time polymerase chain reaction showed expression of CD4 and CD8 in the donor heart, and immunofluorescence assay revealed the association between T cells and TIPE2. ResultsIn our study, we first found that the expression of TIPE2 in cardiac allografts is upregulated compared with the syngeneic control, and increases in a time-dependent manner. The immunocytochemistry of heart grafts revealed a strong expression of TIPE2 in the inflammatory cells, but not in the cardiomyocytes. Finally, we proved that CD4+ and CD8+ T cells infiltrated cardiac allografts abundantly, which express ample TIPE2. ConclusionsThe upregulated expression of TIPE2 in cardiac allografts, mainly came from T cells, which infiltrated the donor heart. This finding indicates that there may be an association between TIPE2 and acute cardiac allograft rejection.

Toll-Interacting Protein, Tollip, Inhibits IL-13-Mediated Pulmonary Eosinophilic Inflammation in Mice

Toll-interacting protein (Tollip) is a key negative regulator of innate immunity by preventing excessive proinflammatory responses. Tollip genetic variation has been associated with airflow limitation in asthma subjects and Tollip expression. Whether Tollip regulates lung inflammation in a type 2 cytokine milieu (e.g., IL-13) is unclear. Our goal was to determine the in vivo role of Tollip in IL-13-mediated lung eosinophilic inflammation and the underlying mechanisms. Tollip-knockout (KO) and wild-type (WT) mice were inoculated intranasally with recombinant mouse IL-13 protein to examine lung inflammation. To determine how Tollip regulates inflammation, alveolar macrophages and bone marrow-derived macrophages from Tollip KO and WT mice were cultured with or without IL-13 and/or IL-33. IL-13-treated Tollip KO mice significantly increased lung eosinophilic inflammation and eotaxin-2 (CCL24) levels compared with the WT mice. IL-13- treated Tollip KO (vs. WT) macrophages, in the absence and particularly in the presence of IL-33, increased expression of the IL-33 receptor ST2L and CCL24, which was in part dependent on enhanced activation of interleukin (IL)-1 receptor-associated kinase 1 (IRAK1) and signal transducer and activator of transcription 6 (STAT6). Our results suggest that Tollip downregulates IL-13-mediated pulmonary eosinophilia in part through inhibiting the activity of the ST2L/IL-33/IRAK1 axis and STAT6.