"Inhibitory immune checkpoints predict 7-day, in-hospital and 1-year mortality of internal medicine patients admitted with bacterial sepsis".

BACKGROUND: Sepsis is a life-threatening syndrome with complex pathophysiology and great clinical heterogeneity which complicates the delivery of personalized therapies. Our goals were to demonstrate that some biomarkers identified as regulatory immune checkpoints in preclinical studies could 1)improve sepsis prognostication based on clinical variables and 2)guide the stratification of septic patients in subgroups with shared characteristics of immune response or survival outcomes. METHODS: We assayed the soluble counterparts of 12 biomarkers of immune response in 113 internal medicine patients with bacterial sepsis. RESULTS: IL-1 receptor-associated kinase M (IRAK-M) exhibited the highest hazard ratios (HRs) for increased 7-day (1.94 [1.17-3.20]) and 30-day mortality (1.61 [1.14-2.28]). HRs of IRAK-M and Galectin-1 for predicting 1-year mortality were 1.52 (1.20-1.92) and 1.64 (1.13-2.36), respectively. A prognostic model including IRAK-M, Galectin-1, and clinical variables (Charlson Comorbidty Index, multiple source of sepsis, and SOFA score) had high discrimination for death at 7 days and 30 days (area under the curve 0.90 [0.82-0.99]) and 0.86 [0.79-0.94], respectively). Patients with elevated serum levels of IRAK-M and Galectin-1 had clinical traits of immune suppression and low survival rates. None of the 12 biomarkers were independent predictors of 2-year mortality. CONCLUSIONS: Two inhibitory immune checkpoint biomarkers (IRAK-M and Galectin-1) helped identify 3 distinct sepsis phenotypes with distinct prognoses. These biomarkers shed light on the interplay between immune dysfunction and prognosis in patients with bacterial sepsis and may prove to be useful prognostic markers, therapeutic targets, and biochemical markers for targeted enrollment in targeted therapeutic trials.

Methylation of Interleukin-1 receptor-associated kinase-3 and the risk of multiple sclerosis relapse/activity.

This study retrospectively investigated the impact of interleukin-1 receptor-associated kinase-3 (IRAK-3/IRAK-M) silencing by methylation on the likelihood of multiple sclerosis (MS) activity. This cross-sectional study included 90 patients with MS: 45 with active disease (Group 1), 45 in remission (Group 2), and 45 healthy controls. The study included quantitation of IRAK-3 methylation index (MI%), IRAK-3 mRNA, and myeloid differentiation factor88 (MyD88) and assessment of NF-κB activity. IRAK-3 MI% was significantly higher in group 1 compared to group 2, accompanied by lower IRAK-3 mRNA expression, elevated circulating MyD88, and increased NF-κB activity. IRAK-3 MI% correlated negatively with its transcript and positively with MyD88 and NF-κB activity. A logistic regression model was created to predict active demyelination. The C-index was 0.924, which indicates a very strong prediction model. Within the limitations of current work, IRAK-3 methylation level seems to be a promising candidate biomarker for identifying MS patients at risk of relapse.

LXA4 protected mice from renal ischemia/reperfusion injury by promoting IRG1/Nrf2 and IRAK-M-TRAF6 signal pathways.

Excessive inflammatory response and increased oxidative stress play an essential role in the pathophysiology of ischemia/reperfusion (I/R)-induced acute kidney injury (IRI-AKI). Emerging evidence suggests that lipoxin A4 (LXA4), as an endogenous negative regulator in inflammation, can ameliorate several I/R injuries. However, the mechanisms and effects of LXA4 on IRI-AKI remain unknown. In this study, A bilateral renal I/R mouse model was used to evaluate the role of LXA4 in wild-type, IRG1 knockout, and IRAK-M knockout mice. Our results showed that LXA4, as well as 5-LOX and ALXR, were quickly induced, and subsequently decreased by renal I/R. LXA4 pretreatment improved renal I/R-induced renal function impairment and renal damage and inhibited inflammatory responses and oxidative stresses in mice kidneys. Notably, LXA4 inhibited I/R-induced the activation of TLR4 signal pathway including decreased phosphorylation of TAK1, p36, and p65, but did not affect TLR4 and p-IRAK-1. The analysis of transcriptomic sequencing data and immunoblotting suggested that innate immune signal molecules interleukin-1 receptor-associated kinase-M (IRAK-M) and immunoresponsive gene 1 (IRG1) might be the key targets of LXA4. Further, the knockout of IRG1 or IRAK-M abolished the beneficial effects of LXA4 on IRI-AKI. In addition, IRG1 deficiency reversed the up-regulation of IRAK-M by LXA4, while IRAK-M knockout had no impact on the IRG1 expression, indicating that IRAK-M is a downstream molecule of IRG1. Mechanistically, we found that LXA4-promoted IRG1-itaconate not only enhanced Nrf2 activation and increased HO-1 and NQO1, but also upregulated IRAK-M, which interacted with TRAF6 by competing with IRAK-1, resulting in deactivation of TLR4 downstream signal in IRI-AKI. These data suggested that LXA4 protected against IRI-AKI via promoting IRG1/Itaconate-Nrf2 and IRAK-M-TRAF6 signaling pathways, providing the rationale for a novel strategy for preventing and treating IRI-AKI.

IRAK-M Plays A Role in the Pathology of Amyotrophic Lateral Sclerosis Through Suppressing the Activation of Microglia.

Microglial activation plays a crucial role in the disease progression in amyotrophic lateral sclerosis (ALS). Interleukin receptor-associated kinases-M (IRAK-M) is an important negative regulatory factor in the Toll-like receptor 4 (TLR4) pathway during microglia activation, and its mechanism in this process is still unclear. In the present study, we aimed to investigate the dynamic changes of IRAK-M and its protective effects for motor neurons in SOD1-G93A mouse model of ALS. qPCR (Real-time Quantitative PCR Detecting System) were used to examine the mRNA levels of IRAK-M in the spinal cord in both SOD1-G93A mice and their age-matched wild type (WT) littermates at 60, 100 and 140 days of age. We established an adeno-associated virus 9 (AAV9)-based platform by which IRAK-M was targeted mostly to microglial cells to investigate whether this approach could provide a protection in the SOD1-G93A mouse. Compared with age-matched WT mice, IRAK-M mRNA level was elevated at 100 and 140 days in the anterior horn region of spinal cords in the SOD1-G93A mouse. AAV9-IRAK-M treated SOD1-G93A mice showed reduction of IL-1ß mRNA levels and significant improvements in the numbers of spinal motor neurons in spinal cord. Mice also showed previously reduction of muscle atrophy. Our data revealed the dynamic changes of IRAK-M during ALS pathological progression and demonstrated that an AAV9-IRAK-M delivery was an effective and translatable therapeutic approach for ALS. These findings may help identify potential molecular targets for ALS therapy.

Regulation of Inflammation by IRAK-M Pathway Can Be Associated with nAchRalpha7 Activation and COVID-19.

In spite of the vaccine development and its importance, the SARS-CoV-2 pandemic is still impacting the world. It is known that the COVID-19 severity is related to the cytokine storm phenomenon, being inflammation a common disease feature. The nicotinic cholinergic system has been widely associated with COVID-19 since it plays a protective role in inflammation via nicotinic receptor alpha 7 (nAchRalpha7). In addition, SARS-CoV-2 spike protein (Spro) subunits can interact with nAchRalpha7. Moreover, Spro causes toll-like receptor (TLR) activation, leading to pro- and anti-inflammatory pathways. The increase and maturation of the IL-1 receptor-associated kinase (IRAK) family are mediated by activation of membrane receptors, such as TLRs. IRAK-M, a member of this family, is responsible for negatively regulating the activity of other active IRAKs. In addition, IRAK-M can regulate microglia phenotype by specific protein expression. Furthermore, there exists an antagonist influence of SARS-CoV-2 Spro and the cholinergic system action on the IRAK-M pathway and microglia phenotype. We discuss the overexpression and suppression of IRAK-M in inflammatory cell response to inflammation in SARS-CoV-2 infection when the cholinergic system is constantly activated via nAchRalpha7.

Protectin D1 inhibits TLR4 signaling pathway to alleviate non-alcoholic steatohepatitis via upregulating IRAK-M.

Non-alcoholic steatohepatitis (NASH) is a prevalent metabolic disease, characterized by the hepatic steatosis, inflammation, and fibrosis, which is lack of effective treatment currently. Protectin D1 (PTD1), a lipid mediator from omega-3 fatty acid docosahexaenoic acid (DHA), has displayed wide pharmacological actions including anti-inflammation in a variety of diseases, but the role of PTD1 on NASH remains unclear. In this study, using the methionine and choline deficient (MCD) fed NASH model, we explored the effect and underlying mechanism of PTD1 on NASH in mice. Our results showed PTD1 improved MCD-induced steatosis, hepatocellular injury, inflammation and fibrosis. Furthermore, PTD1 inhibited MCD-induced activation of TLR4 downstream molecules (TAK1, p38 and p65) without affecting the levels of TLR4 and phosphorylated IRAK-1. Notably, the levels of IRAK-M protein and the binding between IRAK-M and TRAF6 in the liver were also increased by PTD1 in NASH mice. Moreover, IRAK-M knockout remarkedly reverted the beneficial effects of PTD1 on the NASH in mice. Thus, these results demonstrated that PTD1 could protect mice from NASH by inhibiting the activation of TLR4 downstream signaling pathway, which might be related to the upregulation of IRAK-M, indicating that PTD1 may provide a new treatment for NASH.

IRAK-M Ablation Promotes Status Epilepticus-Induced Neuroinflammation via Activating M1 Microglia and Impairing Excitatory Synaptic Function.

Epilepsy is one of the most common neurological disorders. The pro-epileptic and antiepileptic roles of microglia have recently garnered significant attention. Interleukin-1 receptor-associated kinase (IRAK)-M, an important kinase in the innate immune response, is mainly expressed in microglia and acts as a negative regulator of the TLR4 signaling pathway that mediates the anti-inflammatory effect. However, whether IRAK-M exerts a protective role in epileptogenesis as well as the molecular and cellular mechanisms underlying these processes are yet to be elucidated. An epilepsy mouse model induced by pilocarpine was used in this study. Real-time quantitative polymerase chain reaction and western blot analysis were used to analyze mRNA and protein expression levels, respectively. Whole-cell voltage-clamp recordings were employed to evaluate the glutamatergic synaptic transmission in hippocampal neurons. Immunofluorescence was utilized to show the glial cell activation and neuronal loss. Furthermore, the proportion of microglia was analyzed using flow cytometry. Seizure dynamics influenced the expression of IRAK-M. Its knockout dramatically exacerbated the seizures and the pathology in epilepsy and increased the N-methyl-d-aspartate receptor (NMDAR) expression, thereby enhancing glutamatergic synaptic transmission in hippocampal CA1 pyramidal neurons in mice. Furthermore, IRAK-M deficiency augmented hippocampal neuronal loss via a possible mechanism of NMDAR-mediated excitotoxicity. IRAK-M deletion promotes microglia toward the M1 phenotype, which resulted in high levels of proinflammatory cytokines and was accompanied by a visible increase in the expressions of key microglial polarization-related proteins, including p-STAT1, TRAF6, and SOCS1. The findings demonstrate that IRAK-M dysfunction contributes to the progression of epilepsy by increasing M1 microglial polarization and glutamatergic synaptic transmission. This is possibly related to NMDARs, particularly Grin2A and Grin2B, which suggests that IRAK-M could serve as a novel therapeutic target for the direct alleviation of epilepsy.

Mutations in the Vicinity of the IRAK3 Guanylate Cyclase Center Impact Its Subcellular Localization and Ability to Modulate Inflammatory Signaling in Immortalized Cell Lines.

Interleukin-1 receptor-associated kinase 3 (IRAK3) modulates the magnitude of cellular responses to ligands perceived by interleukin-1 receptors (IL-1Rs) and Toll-like receptors (TLRs), leading to decreases in pro-inflammatory cytokines and suppressed inflammation. The molecular mechanism of IRAK3's action remains unknown. IRAK3 functions as a guanylate cyclase, and its cGMP product suppresses lipopolysaccharide (LPS)-induced nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) activity. To understand the implications of this phenomenon, we expanded the structure-function analyses of IRAK3 through site-directed mutagenesis of amino acids known or predicted to impact different activities of IRAK3. We verified the capacity of the mutated IRAK3 variants to generate cGMP in vitro and revealed residues in and in the vicinity of its GC catalytic center that impact the LPS-induced NFκB activity in immortalized cell lines in the absence or presence of an exogenous membrane-permeable cGMP analog. Mutant IRAK3 variants with reduced cGMP generating capacity and differential regulation of NFκB activity influence subcellular localization of IRAK3 in HEK293T cells and fail to rescue IRAK3 function in IRAK3 knock-out THP-1 monocytes stimulated with LPS unless the cGMP analog is present. Together, our results shed new light on the mechanism by which IRAK3 and its enzymatic product control the downstream signaling, affecting inflammatory responses in immortalized cell lines.

IRAK-M has effects in regulation of lung epithelial inflammation.

BACKGROUND: Epithelial barrier is important for asthma development by shaping immune responses. Airway expressing-IL-1 receptor-associated kinase (IRAK)-M of Toll-like receptor pathway was involved in immunoregulation of airway inflammation through influencing activities of macrophages and dendritic cells or T cell differentiation. Whether IRAK-M has effect on cellular immunity in airway epithelial cells upon stimulation remains unclear. METHODS: We modeled cellular inflammation induced by IL-1ß, TNF-α, IL-33, and house dust mite (HDM) in BEAS-2B and A549 cells. Cytokine production and pathway activation were used to reflect the effects of IRAK-M siRNA knockdown on epithelial immunity. Genotyping an asthma-susceptible IRAK-M SNP rs1624395 and measurement of serum CXCL10 levels were performed in asthma patients. RESULTS: IRAK-M expression was significantly induced in BEAS-2B and A549 cells after inflammatory stimulation. IRAK-M knockdown increased the lung epithelial production of cytokines and chemokines, including IL-6, IL-8, CXCL10, and CXCL11, at both mRNA and protein levels. Upon stimulation, IRAK-M silencing led to overactivation of JNK and p38 MAPK in lung epithelial cells. While antagonizing JNK or p38 MAPK inhibited increased secretion of CXCL10 in IRAK-M silenced-lung epithelium. Asthma patients carrying G/G genotypes had significantly higher levels of serum CXCL10 than those carrying homozygote A/A. CONCLUSION: Our findings suggested that IRAK-M has effect on lung epithelial inflammation with an influence on epithelial secretion of CXCL10 partly mediated through JNK and p38 MAPK pathways. IRAK-M modulation might indicate a new insight into asthma pathogenesis from disease origin.

IRAK-M Regulates Proliferative and Invasive Phenotypes of Lung Fibroblasts.

Lung fibroblasts play an important role in subepithelial fibrosis, one feature for airway remodeling. IL-1 receptor-associated kinase (IRAK)-M was shown to involve fibrosis formation in airways and lung through regulation of inflammatory responses. IRAK-M is expressed by lung fibroblasts, whether IRAK-M has direct impact on lung fibroblasts remains unclear. In this investigation, we evaluated in vitro effect of IRAK-M on phenotypes of lung fibroblasts by silencing or overexpressing IRAK-M. Murine lung fibroblasts (MLg) were stimulated with house dust mite (HDM), IL-33, and transforming growth factor (TGF) ß1. Techniques of small interfering RNA or expression plasmid were employed to silence or overexpress IRAK-M in MLg fibroblast cells. Proliferation, migration, invasiveness, and fibrosis-related events were evaluated. Significant upregulation of IRAK-M expression in MLg cells was caused by these stimuli. Silencing IRAK-M significantly increased proliferation, migration, and invasiveness of lung fibroblasts regardless of stimulating conditions. By contrast, IRAK-M overexpression significantly inhibited proliferation and motility of MLg lung fibroblasts. IRAK-M overexpression also significantly decreased the expression of fibronectin, collagen I, and α-SMA in MLg cells. Under stimulation with TGFß1 or IL-33, IRAK-M silencing reduced MMP9 production, while IRAK-M overexpression increased MMP9 production. Modulation of IRAK-M expression affected cytokines production, either decreased or increased expression of TNFα and CXCL10 by the cells regardless of stimulation. Our in vitro data reveal that IRAK-M directly impacts on lung fibroblasts through modulation of cellular motility, release of inflammatory, and fibrotic cytokines of lung fibroblasts. These might suggest a new target by regulation of IRAK-M in slowing airway remodeling.

The IRAK-M death domain: a tale of three surfaces.

The anti-inflammatory interleukin-1 receptor associated kinase-M (IRAK-M) is a negative regulator of MyD88/IRAK-4/IRAK-1 signaling. However, IRAK-M has also been reported to activate NF-κB through the MyD88/IRAK-4/IRAK-M myddosome in a MEKK-3 dependent manner. Here we provide support that IRAK-M uses three surfaces of its Death Domain (DD) to activate NF-κB downstream of MyD88/IRAK-4/IRAK-M. Surface 1, with central residue Trp74, binds to MyD88/IRAK-4. Surface 2, with central Lys60, associates with other IRAK-M DDs to form an IRAK-M homotetramer under the MyD88/IRAK-4 scaffold. Surface 3; with central residue Arg97 is located on the opposite side of Trp74 in the IRAK-M DD tetramer, lacks any interaction points with the MyD88/IRAK-4 complex. Although the IRAK-M DD residue Arg97 is not directly involved in the association with MyD88/IRAK-4, Arg97 was responsible for 50% of the NF-κB activation though the MyD88/IRAK-4/IRAK-M myddosome. Arg97 was also found to be pivotal for IRAK-M's interaction with IRAK-1, and important for IRAK-M's interaction with TRAF6. Residue Arg97 was responsible for 50% of the NF-κB generated by MyD88/IRAK-4/IRAK-M myddosome in IRAK-1/MEKK3 double knockout cells. By structural modeling we found that the IRAK-M tetramer surface around Arg97 has excellent properties that allow formation of an IRAK-M homo-octamer. This model explains why mutation of Arg97 results in an IRAK-M molecule with increased inhibitory properties: it still binds to myddosome, competing with myddosome IRAK-1 binding, while resulting in less NF-κB formation. The findings further identify the structure-function properties of IRAK-M, which is a potential therapeutic target in inflammatory disease.

Modulation of Inflammatory Cytokine Production in Human Monocytes by cGMP and IRAK3.

Interleukin-1 receptor-associated kinase-3 (IRAK3) is a critical checkpoint molecule of inflammatory responses in the innate immune system. The pseudokinase domain of IRAK3 contains a guanylate cyclase (GC) centre that generates small amounts of cyclic guanosine monophosphate (cGMP) associated with IRAK3 functions in inflammation. However, the mechanisms of IRAK3 actions are poorly understood. The effects of low cGMP levels on inflammation are unknown, therefore a dose-response effect of cGMP on inflammatory markers was assessed in THP-1 monocytes challenged with lipopolysaccharide (LPS). Sub-nanomolar concentrations of membrane permeable 8-Br-cGMP reduced LPS-induced NFκB activity, IL-6 and TNF-α cytokine levels. Pharmacologically upregulating cellular cGMP levels using a nitric oxide donor reduced cytokine secretion. Downregulating cellular cGMP using a soluble GC inhibitor increased cytokine levels. Knocking down IRAK3 in THP-1 cells revealed that unlike the wild type cells, 8-Br-cGMP did not suppress inflammatory responses. Complementation of IRAK3 knockdown cells with wild type IRAK3 suppressed cytokine production while complementation with an IRAK3 mutant at GC centre only partially restored this function. Together these findings indicate low levels of cGMP form a critical component in suppressing cytokine production and in mediating IRAK3 action, and this may be via a cGMP enriched nanodomain formed by IRAK3 itself.

IRAK-M deletion aggravates acute inflammatory response and mitochondrial respiratory dysfunction following myocardial infarction: A bioinformatics analysis.

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide. Interleukin-1 receptor associated kinase (IRAK)-M is a regulator of Toll-like receptor mediated inflammatory responses and plays an important role in the pathophysiologic processes of acute MI. We aimed to explore the effect of IRAK-M on regulating biological function and molecular interactions post-MI through bioinformatics analysis. Datasets from the Gene Expression Omnibus database were used to identify characteristics of IRAK-M expression in MI patients. The expression of IRAK-M was upregulated in MI patients and altered in a time-dependent manner during MI progression. Enrichment analysis showed that biological processes related to inflammatory response and leukocyte activation were markedly activated in MI patients with upregulated IRAK-M. Furthermore, we constructed MI model using wildtype and IRAK-M-/- mice and performed proteomics analysis of infarcted hearts. Functional enrichment of proteomics data indicated that IRAK-M deletion aggravated a series of pathophysiologic functions, such as acute inflammatory response, macrophage activation and mitochondrial dysfunction. S100A8/A9 acted as the central molecule in the above functions based on the protein-protein interaction network and was significantly elevated in IRAK-M-/- infarcted hearts at both the protein and mRNA levels. In conclusion, IRAK-M functioned as an essential regulator in pathophysiologic processes post-MI, exerting effects not only on controlling acute inflammatory responses but also on mediating mitochondrial respiratory function based on integrated bioinformatics analysis. SIGNIFICANCE: In this study, we combined microarray datasets and a proteomics approach to explore the effect of IRAK-M on mediating biological processes and systemic molecular interactions following MI. Our data firstly showed that IRAK-M is involved in ATP synthesis and mitochondrial respiratory chain complex during MI progression. S100A8/A9 acted as the central molecule in above regulatory network and displayed a tight connection with IRAK-M. The findings provide novel evidence and clues for understanding the complex roles and molecular mechanisms of IRAK-M in the development of MI.

IL-23 production in human macrophages is regulated negatively by tumor necrosis factor α-induced protein 3 and positively by specificity protein 1 after stimulation of the toll-like receptor 7/8 signaling pathway.

The IL-23/IL-17 axis plays an important role in the development of autoimmune diseases, but the mechanism regulating IL-23 production is mainly unknown. We investigated how TNFAIP3 and Sp1 affect IL-23 production by human macrophages after exposure to resiquimod, a TLR7/8 agonist. IL-23 production was significantly upregulated by resiquimod but only slightly by LPS (a TLR4 agonist). Interestingly, IL-23 levels were significantly attenuated after sequential stimulation with LPS and resiquimod, but IL-12p40 and IL-18 levels were not. TLR4-related factors induced by LPS may regulate IL-23 expression via TLR7/8 signaling. LPS significantly enhanced TNFAIP3 and IRAK-M levels but reduced Sp1 levels. After exposure to resiquimod, RNA interference of TNFAIP3 upregulated IL-23 significantly more than siRNA transfection of IRAK-M did. In contrast, knockdown of Sp1 by RNA interference significantly attenuated IL-23 production. Transfection with siRNA for TNFAIP3 enhanced IL-23 expression significantly. After stimulation with resiquimod, GW7647-an agonist for PPARα (an inducer of NADHP oxidase)-and siRNA for UCP2 (a negative regulator of mitochondrial ROS generation) enhanced TNFAIP3 and reduced IL-23. siRNA for p22phox and gp91phox slightly increased Sp1 levels. However, after exposure to resiquimod siRNA-mediated knockout of DUOX1/2 significantly enhanced Sp1 and IL-23 levels, and decreased TNFα-dependent COX-2 expression. Concomitantly, TNFAIP3 levels was attenuated by DUOX1/2 siRNA. TNFAIP3 and Sp1 levels are reciprocally regulated through ROS generation. In conclusion, after stimulation of the TLR7/8 signaling pathway IL-23 production in human macrophages is regulated negatively by TNFAIP3.

Structural anomalies in a published NMR-derived structure of IRAK-M.

Signaling by Toll-Like Receptors and the Interleukin-1 Receptor (IL1-R) involves intracellular binding of MyD88, followed by assembly of IL1-R Associated Kinases (IRAKs) into the so-called Myddosome. Using NMR, Nechama et al. determined the structure of the IRAK-M death domain monomer (PDBid: 5UKE). With this structure, they performed a docking study to model the location of IRAK-M in the Myddosome. Based on this, they present a molecular basis for selectivity of IRAK-M towards IRAK1 over IRAK2 binding. When we attempted to use 5UKE as a homology modeling template, we noticed that our 5UKE-based models had structural issues, such as disallowed torsion angles and solvent exposed tryptophans. We therefore analyzed the NMR ensemble of 5UKE using structure validation tools and we compared 5UKE with homologous high-resolution X-ray structures. We identified several structural anomalies in 5UKE, including packing issues, frayed helices and improbable side chain conformations. We used Yasara to build a homology model, based on two high resolution death domain crystal structures, as an alternative model for the IRAK-M death domain (atomic coordinates, modeling details and validation are available at https://swift.cmbi.umcn.nl/gv/service/5uke/). Our model agrees better with known death domain structure information than 5UKE and also with the chemical shift data that was deposited for 5UKE.

SARS-CoV-2/ACE2 Interaction Suppresses IRAK-M Expression and Promotes Pro-Inflammatory Cytokine Production in Macrophages.

The major cause of death in SARS-CoV-2 infected patients is due to de-regulation of the innate immune system and development of cytokine storm. SARS-CoV-2 infects multiple cell types in the lung, including macrophages, by engagement of its spike (S) protein on angiotensin converting enzyme 2 (ACE2) receptor. ACE2 receptor initiates signals in macrophages that modulate their activation, including production of cytokines and chemokines. IL-1R-associated kinase (IRAK)-M is a central regulator of inflammatory responses regulating the magnitude of TLR responsiveness. Aim of the work was to investigate whether SARS-CoV-2 S protein-initiated signals modulate pro-inflammatory cytokine production in macrophages. For this purpose, we treated PMA-differentiated THP-1 human macrophages with SARS-CoV-2 S protein and measured the induction of inflammatory mediators including IL6, TNFα, IL8, CXCL5, and MIP1a. The results showed that SARS-CoV-2 S protein induced IL6, MIP1a and TNFα mRNA expression, while it had no effect on IL8 and CXCL5 mRNA levels. We further examined whether SARS-CoV-2 S protein altered the responsiveness of macrophages to TLR signals. Treatment of LPS-activated macrophages with SARS-CoV-2 S protein augmented IL6 and MIP1a mRNA, an effect that was evident at the protein level only for IL6. Similarly, treatment of PAM3csk4 stimulated macrophages with SARS-CoV-2 S protein resulted in increased mRNA of IL6, while TNFα and MIP1a were unaffected. The results were confirmed in primary human peripheral monocytic cells (PBMCs) and isolated CD14+ monocytes. Macrophage responsiveness to TLR ligands is regulated by IRAK-M, an inactive IRAK kinase isoform. Indeed, we found that SARS-CoV-2 S protein suppressed IRAK-M mRNA and protein expression both in THP1 macrophages and primary human PBMCs and CD14+ monocytes. Engagement of SARS-CoV-2 S protein with ACE2 results in internalization of ACE2 and suppression of its activity. Activation of ACE2 has been previously shown to induce anti-inflammatory responses in macrophages. Treatment of macrophages with the ACE2 activator DIZE suppressed the pro-inflammatory action of SARS-CoV-2. Our results demonstrated that SARS-CoV-2/ACE2 interaction rendered macrophages hyper-responsive to TLR signals, suppressed IRAK-M and promoted pro-inflammatory cytokine expression. Thus, activation of ACE2 may be a potential anti-inflammatory therapeutic strategy to eliminate the development of cytokine storm observed in COVID-19 patients.

Lipoteichoic Acid from Staphylococcus aureus Activates the Complement System via C3 Induction and CD55 Inhibition.

Staphylococcus aureus inhibits complement activity by secreting a variety of toxins. However, the underlying mechanism of complement component regulation by lipoteichoic acid (LTA), a cell wall component of S. aureus, has not been elucidated. In this study, we observed that aLTA (LTA of S. aureus) increased C3 expression in THP-1 cells. The mechanism of aLTA-mediated C3 induction includes an aLTA-toll-like receptor (TLR) 2 interaction, interleukin 1 receptor associated kinase (IRAK) 2 recruitment, and nuclear factor kappa B (NF-kB) activation. In HepG2 cells, C3 protein production begins to increase from 3 h and increases steadily until 48 h. On the other hand, CD55 levels increased up to 6 h after aLTA treatment and started to decrease after 24 h and levels were decreased at 48 h by more than 50% compared to untreated cells. The expression of CD55 in HepG2 cells was shown to be regulated by IRAK-M induced by aLTA. Serum C3 levels increased in mice injected with aLTA, which resulted in an increase in the amount and activity of the membrane attack complex (MAC). We also observed that CD55 mRNA was increased in the liver 24 h after aLTA injection, but was decreased 48 h after injection. These results suggest that aLTA increases complement levels via induction of C3 and inhibition of CD55, which may cause associated MAC-mediated liver damage.

IRAK-M knockout promotes allergic airway inflammation, but not airway hyperresponsiveness, in house dust mite-induced experimental asthma model.

BACKGROUND: IL-1 receptor associated-kinase (IRAK)-M, expressed by airway epithelium and macrophages, was shown to regulate acute and chronic airway inflammation exhibiting a biphasic response in an OVA-based animal model. House dust mite (HDM) is a common real-life aeroallergen highly relevant to asthma pathogenesis. The role of IRAK-M in HDM-induced asthma remains unknown. This study was aimed to investigate the effect of IRAK-M on allergic airway inflammation induced by HDM using IRAK-M knockout (KO) mice and the potential underlying mechanisms. METHODS: IRAK-M KO and wild-type (WT) mice were sensitized and challenged with HDM. The differences in airway inflammation were evaluated 24 hours after the last challenge between the two genotypes of mice using a number of cellular and molecular biological techniques. In vitro mechanistic investigation was also involved. RESULTS: Lung expression of IRAK-M was significantly upregulated by HDM in the WT mice. Compared with the WT controls, HDM-treated IRAK-M KO mice showed exacerbated infiltration of inflammatory cells, particularly Th2 cells, in the airways and mucus overproduction, higher epithelial mediators IL-25, IL-33 and TSLP and Th2 cytokines in bronchoalveolar lavage (BAL) fluid. Lung IRAK-M KO macrophages expressed higher percentage of costimulatory molecules OX40L and CD 80 and exhibited enhanced antigen uptake. However, IRAK-M KO didn't impact the airway hyperreactivity (AHR) indirectly induced by HDM. CONCLUSIONS: The findings indicate that IRAK-M protects allergic airway inflammation, not AHR, by modifying activation and antigen uptake of lung macrophages following HDM stimulation. Optimal regulation of IRAK-M might indicate an intriguing therapeutic avenue for allergic airway inflammation.

Lipoteichoic acid of Enterococcus faecalis interferes with Porphyromonas gingivalis lipopolysaccharide signaling via IRAK-M upregulation in human periodontal ligament cells.

Periodontitis is a chronic inflammatory disease of the gum caused by infection with multispecies oral bacteria. Since the periodontopathic bacteria, Porphyromonas gingivalis together with Enterococcus faecalis are frequently detected in patients with a severe form of periodontitis, interactions between their virulence factors might play an important role in progression of the disease. P. gingivalis and E. faecalis possess lipopolysaccharide (Pg.LPS) and lipoteichoic acid (Ef.LTA), respectively, as the major virulence factors inducing inflammatory responses. However, the combinatorial effect of these virulence factors on chemokine expression was poorly understood. Here, we examined the interaction between Ef.LTA and Pg.LPS on IL-8 induction in human periodontal ligament (PDL) cells. Pg.LPS, but not Ef.LTA, induced IL-8 expression at both mRNA and protein levels, which was suppressed in the presence of Ef.LTA. Although Ef.LTA and Pg.LPS could stimulate Toll-like receptor 2 (TLR2), Ef.LTA did not interfere with Pg.LPS induced-TLR2 activation. However, Ef.LTA decreased Pg.LPS-induced phosphorylation of ERK, JNK, and p38 kinase. Furthermore, Ef.LTA suppressed Pg.LPS-induced IL-8 promoter activity as well as AP-1, NF-IL6 and NF-κB transcription factors, which are indispensable for IL-8 expression. Interestingly, Ef.LTA enhanced only IL-1 receptor-associated kinase-M (IRAK-M) expression among the tested negative regulators of TLR intracellular signaling cascades in the presence of Pg.LPS. In addition, silencing IRAK-M restored the decreased IL-8 expression by Ef.LTA in the presence of Pg.LPS. Collectively, these results suggest that Ef.LTA inhibits Pg.LPS-induced IL-8 expression in human PDL cells via inducing the expression of a negative regulator of TLR signaling cascades, IRAK-M.

Neutrophils Deficient in Innate Suppressor IRAK-M Enhances Anti-tumor Immune Responses.

Tumor-associated immune-suppressive neutrophils are prevalent in various cancers, including colorectal cancer. However, mechanisms of immune-suppressive neutrophils are not well understood. We report that a key innate suppressor, IRAK-M (interleukin-1 receptor-associated kinase M), is critically involved in the establishment of immune-suppressive neutrophils. In contrast to the wild-type (WT) neutrophils exhibiting immune-suppressive signatures of CD11bhighPD-L1highCD80low, IRAK-M-deficient neutrophils are rewired with reduced levels of inhibitory molecules PD-L1 and CD11b, as well as enhanced expression of stimulatory molecules CD80 and CD40. The reprogramming of IRAK-M-deficient neutrophils is mediated by reduced activation of STAT1/3 and enhanced activation of STAT5. As a consequence, IRAK-M-deficient neutrophils demonstrate enhanced capability to promote, instead of suppress, the proliferation and activation of effector T cells both in vitro and in vivo. Functionally, we observed that the transfusion of IRAK-M-/- neutrophils can potently render an enhanced anti-tumor immune response in the murine inflammation-induced colorectal cancer model. Collectively, our study defines IRAK-M as an innate suppressor for neutrophil function and reveals IRAK-M as a promising target for rewiring neutrophils in anti-cancer immunotherapy.