IL-6 Response Research Articles

Zfp580 inactivation as a new therapeutic target to enhance recovery after stroke in mice

Paracrine cerebral Interleukin 6 (Il6) is relevant for stroke recovery, but systemic Il6 elevation may worsen outcome. Hence, paracrine Il6 response modulation within the neurovascular unit has emerged as an attractive therapeutic approach. Lithium modulates Il6 responses and improves stroke outcome. However, lithium may cause serious adverse effects. Here, we report that Zincfinger protein 580 (Zfp580) mediates the effects of lithium on Il6 signaling. In contrast to lithium, Zfp580 inactivation had no neurotoxic effects, and Zfp580 knock out mice showed no phenotypic changes in cognitive and motor function behavioral tests. We discovered that lithium and hypoxia disinhibited Il6 via Zfp580 suppression and post-translational modification by small ubiquitin-like modifier (SUMO). After transient middle cerebral artery occlusion, loss of Zfp580 reduced paracrine Il6 and increased Il6 trans-signaling. Aside from modulating Il6 signaling, Zfp580 loss improved endothelial resilience to ischemia, was highly neuroprotective resulting in smaller infarcts and enhanced use-dependent neuroplasticity, all of which led to improved functional outcome. In conclusion, inactivation of Zfp580 exerts positive effects on multiple key mechanisms without exhibiting relevant adverse side effects, making it potentially a more specific and effective treatment target for stroke recovery than lithium. To fully assess its potential, Zfp580 inhibitors must be developed.

Brexanolone therapeutics in post-partum depression involves inhibition of systemic inflammatory pathways.

Brexanolone has rapid, long-lasting, and remarkable efficacy in the treatment of post-partum depression (PPD). We test the hypothesis that brexanolone inhibits proinflammatory modulators and macrophage activation in PPD patients, which may promote clinical recovery. PPD patients (N=18) provided blood samples before and after brexanolone infusion according to the FDA-approved protocol. Patients were unresponsive to prior treatment before brexanolone therapy. Serum was collected to determine neurosteroid levels and whole blood cell lysates were examined for inflammatory markers and invitro responses to the inflammatory activators lipopolysaccharide (LPS) and imiquimod (IMQ). Brexanolone infusion altered multiple neuroactive steroid levels (N=15-18), reduced levels of inflammatory mediators (N=11) and inhibited their response to inflammatory immune activators (N=9-11). Specifically, brexanolone infusion reduced whole blood cell tumor necrosis factor-α (TNF-α, p=0.003), and interleukin-6 (IL-6, p=0.04) and these effects were correlated with HAM-D score improvement (TNF-α, p=0.049; IL-6, p=0.02). Furthermore, brexanolone infusion prevented LPS and IMQ-induced elevation of TNF-α (LPS: p=0.02; IMQ: p=0.01), IL-1β (LPS: p=0.006; IMQ: p=0.02) and IL-6 (LPS: p=0.009; IMQ: p=0.01), indicating inhibition of toll-like receptor (TLR)4 and TLR7 responses. Finally, inhibition of TNF-α, IL-1β and IL-6 responses to both LPS and IMQ were correlated with HAM-D score improvements (p<0.05). Brexanolone actions involve inhibition of inflammatory mediator production and inhibition of inflammatory responses to TLR4 and TLR7 activators. The data suggest that inflammation plays a role in post-partum depression and that inhibition of inflammatory pathways contributes to the therapeutic efficacy of brexanolone. The Foundation of Hope, Raleigh, NC and UNC School of Medicine, Chapel Hill.

Toxoplasma gondii microneme protein MIC3 induces macrophage TNF-α production and Ly6C expression via TLR11/MyD88 pathway

Toxoplasma gondii is the most successful parasite worldwide. It is of great interest to understand how T. gondii induce different immune responses in different hosts. In this study, we found that a peptide of T. gondii microneme protein MIC3 induced TNF-α production, NF-κB phosphorylation, iNOS transcription and Ly6C expression in mouse macrophage RAW264.7 cells. MyD88 inhibition, small interfering RNA against Tlr11 and CRISPR/Cas9-mediated knock-out of Tlr11 all reduced MIC3-induced TNF-α production, NF-κB phosphorylation, iNOS transcription and Ly6C expression. Additionally, we determined the location of MIC3 peptide in mouse macrophages using immunofluorescence. MIC3 could both adhere to the cell membrane of mouse macrophages and enter the cells. These results suggest that MIC3 triggered the immune responses in mouse macrophages via TLR11/MyD88/NF-κB pathway. It is known that human macrophages lacking TLR11. We predicted that the immune responses induced by MIC3 in human macrophages were significantly different from those in mouse macrophages. As expected, MIC3 peptide failed to induce TNF-α expression, iNOS expression and NF-κB phosphorylation in human THP-1 derived macrophages. MIC3 induced macrophage immune responses via TLR11. Intriguingly, the amino acid sequence of MIC3 is completely different from the well-known TLR11 ligand profilin, which generates a potent IL-12p40, TNF-α and IL-6 response. In marked contrast to profilin, MIC3 could not induce IL-12p40 expression in both mouse RAW264.7 cells and human THP-1 derived macrophages. Furthermore, the simulated tertiary structure of MIC3 peptide shows poor similarity with the crystal structure of profilin, suggesting that MIC3 might be a different ligand from profilin. These findings about MIC3 and TLR11 will provide us with important insights into the pathogenesis of toxoplasmosis and coevolution during host-parasite interaction.

Caffeine Augments the Lactate and Interleukin-6 Response to Moderate-Intensity Exercise.

The release of interleukin (IL)-6 from contracting skeletal muscle is thought to contribute to some of the health benefits bestowed by exercise. This IL-6 response seems proportional to exercise volume and to lactate production. Unfortunately, high volumes of exercise are not feasible for all people. Caffeine augments the magnitude of increase in circulating IL-6 in response to high-intensity and long-duration exercise. Caffeine also increases circulating concentrations of lactate during exercise. We hypothesized that caffeine, ingested before short-duration, moderate-intensity exercise, would lead to greater circulating concentrations of lactate and IL-6 in a study population comprising both male and female individuals. Twenty healthy adults (10 men and 10 women age 25 ± 7 yr (mean ± SD)) completed 30 min of moderate-intensity cycle ergometer exercise, at an intensity corresponding to 60% peak oxygen uptake, after ingesting either caffeine (6 mg·kg -1 ) or placebo. Arterialized-venous blood was collected throughout each of the exercise sessions. Compared with placebo, caffeine increased circulating concentrations of lactate at the end of exercise (5.12 ± 3.67 vs 6.45 ± 4.40 mmol·L -1 , P < 0.001) and after 30 min of inactive recovery (1.83 ± 1.59 vs 2.32 ± 2.09 mmol·L -1 , P = 0.006). Circulating IL-6 concentrations were greatest after 30 min of inactive recovery ( P < 0.001) and higher with caffeine (2.88 ± 2.05 vs 4.18 ± 2.97, pg·mL -1 , P < 0.001). Secondary analysis indicated sex differences; caffeine increased the IL-6 response to exercise in men ( P = 0.035) but not in women ( P = 0.358). In response to moderate-intensity exercise, caffeine evoked greater circulating lactate concentrations in men and women but only increased the IL-6 response to exercise in men. These novel findings suggest that for men unwilling or unable to perform high-intensity and/or long-duration exercise, caffeine may augment the health benefits of relatively short, moderate-intensity exercise.

Disruption of mitochondrial dynamics triggers muscle inflammation through interorganellar contacts and mitochondrial DNA mislocation

Some forms of mitochondrial dysfunction induce sterile inflammation through mitochondrial DNA recognition by intracellular DNA sensors. However, the involvement of mitochondrial dynamics in mitigating such processes and their impact on muscle fitness remain unaddressed. Here we report that opposite mitochondrial morphologies induce distinct inflammatory signatures, caused by differential activation of DNA sensors TLR9 or cGAS. In the context of mitochondrial fragmentation, we demonstrate that mitochondria-endosome contacts mediated by the endosomal protein Rab5C are required in TLR9 activation in cells. Skeletal muscle mitochondrial fragmentation promotes TLR9-dependent inflammation, muscle atrophy, reduced physical performance and enhanced IL6 response to exercise, which improved upon chronic anti-inflammatory treatment. Taken together, our data demonstrate that mitochondrial dynamics is key in preventing sterile inflammatory responses, which precede the development of muscle atrophy and impaired physical performance. Thus, we propose the targeting of mitochondrial dynamics as an approach to treating disorders characterized by chronic inflammation and mitochondrial dysfunction.

Impaired long-term anti-HBs responses in choronic hepatitis C patients: Results from a five-year follow-up study with healthy control

ABSTRACT Although hepatitis B virus (HBV) vaccination is recommended for hepatitis C virus (HCV)-infected individuals to avoid HBV superinfection, the persistence of their humoral and cell-mediated immunity responses to HBV vaccination is still under investigation. Patients with chronic hepatitis C (CHC) and matched healthy controls, who completed three doses of hepatitis B vaccine (HepB) in 2014, were followed up five years later. One booster dose of HepB was given to those with antibody against hepatitis B surface antigen (anti-HBs) lower than 10mIU/mL. Anti-HBs was tested at follow-up and on the 14th day after the booster dose, as well as HBsAg specific spot-forming cells of interferon γ and interleukin (IL) 2, 4, 5, and 6. At five years, only 56.58% of the CHC patients had sero-protective titers (≥10mIU/mL) of anti-HBs, compared to 70.83% in the controls (P < .05). Similarly, the geometric mean concentration (GMC) of anti-HBs in CHC patients was significantly lower than that in controls (16.95 vs 37.34 mIU/mL, P < .05). After the booster, both GMC and the rate of anamnestic response increased to a very high level in the two groups and the difference between them disappeared (P > .05). Multivariable analysis showed that HCV infection was an independent predictor factor to anti-HBs level at follow-up. HBsAg specific IL-6 was stronger in the CHC patients compared to the controls (P < .05). The data indicate that the durability of protective anti-HBs is poorer in CHC patients compared to healthy individuals, and impaired long-term anti-HBs responses might be associated with the increased HBsAg specific IL-6 responses.

An epithelial-immune circuit amplifies inflammasome and IL-6 responses to SARS-CoV-2

Elevated levels of cytokines IL-1β and IL-6 are associated with severe COVID-19. Investigating the underlying mechanisms, we find that while primary human airway epithelia (HAE) have functional inflammasomes and support SARS-CoV-2 replication, they are not the source of IL-1β released upon infection. In leukocytes, the SARS-CoV-2 E protein upregulates inflammasome gene transcription via TLR2 to prime, but not activate, inflammasomes. SARS-CoV-2-infected HAE supply a second signal, which includes genomic and mitochondrial DNA, to stimulate leukocyte IL-1β release. Nuclease treatment, STING, and caspase-1 inhibition but not NLRP3 inhibition blocked leukocyte IL-1β release. After release, IL-1β stimulates IL-6 secretion from HAE. Therefore, infection alone does not increase IL-1β secretion by either cell type. Rather, bi-directional interactions between the SARS-CoV-2-infected epithelium and immune bystanders stimulates both IL-1β and IL-6, creating a pro-inflammatory cytokine circuit. Consistent with these observations, patient autopsy lungs show elevated myeloid inflammasome gene signatures in severe COVID-19.

Combined Single-Cell Transcriptomics and Mitochondrial Lineage Tracing Identify IL-6 Signaling Responses in TET2 Clonal Hematopoiesis of Indeterminate Potential

Clonal hematopoiesis of indeterminate potential (CHIP) arises from acquired mutations in hematopoietic stem cells, most frequently DNMT3A and TET2. Surprisingly, CHIP is associated with not only hematologic malignancy but also cardiovascular disease and an expanding array of inflammatory conditions with differential pathogenicity due to DNMT3A and TET2 mutations. In mouse models, CHIP increases risk of atherosclerosis in part due to signaling through the IL-6/IL-1β signaling axis. Here, we define the differential signaling response to IL-6 cytokine stimulation in human monocytes from patients with TET2 and DNMT3A CHIP. We stimulated peripheral blood mononuclear cells ex vivo and performed single-cell RNA sequencing (scRNA-seq) on ~150,000 single cells. At basal levels, we observed that genes involved in inflammation, monocyte activation, cell adhesion, and antigen presentation were enriched in monocytes from patients with TET2 CHIP (n=4) compared to control patients without CHIP (n=3). Following IL-6 stimulation, we found inflammatory genes significantly upregulated in TET2 patient monocytes but not in DNMT3A patient monocytes (n=3 patients). We used CellChat to further characterize cell-cell interactions in the IL-6 stimulated CHIP samples. We found that inflammatory ligands such as IL1B, CXCL3, and IL-8 were uniquely enriched in TET2-mutated monocytes after stimulation. We also identified inferred Macrophage Migration Inhibitory Factor (MIF) ligand signaling from TET2 monocytes was significantly higher than DNMT3A monocytes (Figure 1). Larger CHIP clones confer increased disease risk. To test the hypothesis that IL-6 responses are elevated in high TET2 VAF samples, we performed phospho-specific flow cytometry on TET2 mutant samples at baseline and following IL-6 stimulation. We measured pSTAT3 positivity in high VAF samples (TET2hi, >25% VAF, n=3), low VAF samples (TET2lo, <25% VAF, n=3) and controls (n=4). Despite all samples exhibiting response to stimulation, TET2hi monocytes displayed the highest proportion of pSTAT3+, significantly higher than both TET2lo and control samples (Figure 2, ANOVA p= 0.008, Tukey HSD p=0.008 (CTL vs. TET2hi) and p=0.02 (TET2lo vs. TET2hi)). Combining scRNA-seq with genotyping presents the opportunity to compare CHIP mutated and non-mutated cells from within a single patient. To assign clonality within the monocyte population, we performed MAESTER, a mitochondrial lineage tracing technique, in parallel with nuclear DNA genotyping. We then sought to isolate active transcription factor networks using pySCENIC, a tool that derives active transcription factors and their downstream targets from scRNA-seq data. For our analysis, we selected a TET2 mutant sample with high VAF (51%). In the stimulated monocyte condition, we discovered 237 active regulons within the mutant population. Notably, the XBP1 regulon, a known regulator of MHC-II expression, had the highest regulon specificity score (RSS), indicating activation in TET2-mutated cells. The SPI1 regulon, involved in myeloid lineage determination, the pro-inflammatory regulon, REL, and hypoxia-inducible factor HIF1A also had high RSS scores. We demonstrate that TET2 mutant monocytes exhibit unique signaling responses in response to IL-6 stimulation compared to both control samples and non-mutated cells from within the same patient. High VAF TET2 mutant samples displayed an elevated pSTAT3 response compared to lower VAF and control samples, suggesting that IL-6 signaling is a constitutively active, cell-intrinsic property. Our phospho-specific flow cytometry method has potential diagnostic and prognostic utility in stratifying individual risk for inflammation-related consequences of CHIP, such as cardiovascular disease and malignant transformation. Future prospective randomized clinical trials are required to test whether CHIP patients with TET2 mutation status and high VAF would benefit from IL-6/IL-1β interventions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single Cell RNA-Seq Reveals Intra-Tumoral Heterogeneity Relevant to Differentiation States and Outcomes Among Newly Diagnosed Acute Myeloid Leukemia Patients

Background: Intra-tumoral heterogeneity emerging from aberrant differentiation and the accumulation of blasts at different maturation stages underscores AML biology. Importantly, the heterogeneity of AML cells contributes to therapy resistance and poor outcomes. Revealing the underlying biological programs that govern AML differentiation and are shared among patients can translate into effective therapeutic strategies. Methods: We leveraged single cell gene expression profiling (scRNA) of bone marrow mononulear cells from 20 newly diagnosed adult AML patients (median age 73 years; range 52-87 years). After doublet removal and quality assessment, a total of 111,130 single cells passed the filtering criteria. Of those, 56,168 (50.5%) cells (patient range: 293- 9781 cells) were identified as AML cells by integrating flow cytometry, immunohistochemistry, conventional cytogenetic and FISH at time of diagnosis with the scRNA expression profiles. Results: To determine the shared cellular states across different AML patients, we first applied an unsupervised Bayesian hierarchical clustering and identified four AML cell clusters. The four clusters had distinct expression of the lineage and maturation markers: CD34 (primitive), CD33 (myeloid), and CD14 and CD68 (monocytic), suggesting different AML maturation states in these clusters. To further explore the degree of differentiation in each cluster, we applied diffusion mapping, Slingshot pseudotime analysis and CytoTRACE profiling, all of which supported cluster-specific differentiation states. Specifically, Cluster 2 (CD34 high, CD14 negative) was early in the pseudotemporal trajectory, while Cluster 1 (CD14 high, CD34 negative) was towards the end of the trajectory. Clusters 3 and 4 were intermediary. Remarkably, each of the four clusters had contributions from at least 16 patients. These results support our approach to unmask intra-tumoral heterogeneity patterns associated with maturation and shared across different patients, independent of patient characteristics, molecular profiles and cytogenetics. To further explore the biologic pathways correlating with the four clusters that are surrogates of maturation states, we calculated single cell enrichment scores across a comprehensive gene signature collections from curated Molecular Signatures Database and other previously published studies. Cluster 1 was mainly enriched in immune-related pathways including IL6, JAK-STAT3 signaling, and interferon gamma response suggestive of an inflammatory component, and associated with the CD14+ CD68+ CD34- monocytic pattern of differentiation. Cluster 2 had the highest LSC104 stemness score and CD34 expression. Hedgehog signaling pathway was also highly enriched in this cluster and correlated with LSC104 score (r=0.28, p<0.01). This is of interest as hedgehog plays a fundamental role in the leukemic stem cell quiescence. Cluster 3 and 4, harboring lower CD34 expression compared to Cluster 2, but are CD14- CD68- were mainly enriched in metabolic processes including fatty acid metabolism, heme metabolism and erythroid lineage and oxidative phosphorylation, in addition to DNA repair and interferon alpha response. To understand how these defined populations and hierarchies relate to clinical outcomes, we deconvolved bulk RNA signatures in 3 AML cohorts: TCGA, BEAT AML and Abbas et al 2021, using the four clusters as reference. We then assigned each AML patient a cluster membership based on a deconvolution cut-off for cluster abundance. This translated into four distinct clinical groups with significantly different outcomes based on our cluster annotation. Remarkably, AML patients in Cluster 1 (monocytic enriched) had the worst outcomes (Panel B demonstrating TCGA cohort results, p=0.0084). Conclusions: We conducted unsupervised clustering of AML patients and characterized gene and pathway profiles of each cluster revealing a distinct spectrum of differentiation states from primitive to monocytic. Interestingly, AML patients with enrichment to monocytic phenotype characterized by higher inflammatory states demonstrated the worst outcome. Non-monocytic clusters are less variable in marker genes and stemness scores. However, the cluster with the highest CD34 expression are enriched in hedgehog signaling pathway, suggesting a potentially biological programs distinguishing non-monocytic AML cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Type I Interferon Signaling is Required for Oncostatin-M Driven Inflammatory Responses in Mouse Lung

Type I interferons (IFNs) consist of a group of structurally similar cytokines that play an integral role in regulating the immune response to combat lung infections. In certain models type I IFNs have also been associated with suppression of Th2-skewed immune and inflammatory responses. Transient pulmonary overexpression of the gp130 cytokine Oncostatin M (OSM) by Adenovirus vector (AdOSM) induces a robust Th2-skewed cytokine/inflammatory profile in C57Bl/6 murine lungs. In this study we assessed type I IFN function in OSM-mediated inflammation in vivo using Ifnar1-/- C57Bl/6 mice and Ifnar1-deficient cells in vitro. Ifnar1-/- mice showed a significant reduction in AdOSM-induced histopathology (epithelial hyperplasia, alveolar septal wall thickening, cellular infiltration), and levels of IL-6 and chemokine protein (CXCL-1/KC and CCL24/eotaxin-2) in lungs compared with wild-type. Ifnar1-/- murine fibroblasts and human type I IFN receptor (Ifnar)-knockdown fibroblasts were also less responsive to OSM in STAT3 activation and cytokine production compared with Ifnar-sufficient cells in vitro. Exogenous type I IFN induced IL-6 responses in mouse and human fibroblasts and in combination with OSM further stimulated IL-6 production, suggesting a concerted action of type I IFNs and OSM. Taken together, these results demonstrate that cross-talk between IFNAR and OSM signaling enhances cell responses and modulates OSM-driven responses in lung inflammation.

Kai-Xin-San Protects Depression Mice Against CORT-Induced Neuronal Injury by Inhibiting Microglia Activation and Oxidative Stress.

Objective Traditional Chinese medicine formula Kai-Xin-San (KXS) is used to treat psychiatric disorders, especially in anxiety and depression. However, the precise molecular mechanism of action remains unclear. In this study, we investigated the antidepressant effect of KXS on inhibiting inflammation and oxidative stress in corticosterone (CORT)-induced depression. Methods The therapeutic efficacy of KXS was evaluated in a mouse model of depression induced by CORT. Behavioral tests were conducted to evaluate the effectiveness of KXS in treating depressive-like behavior. Nissl staining and β-galactosidase staining were used to assess the effects of KXS on neuronal injury in depressed mice. To screen key potential therapeutic targets of KXS, transcriptome sequences and data analysis were performed. Then, Iba1 immunofluorescence staining and their relative inflammatory factors mRNA expression were conducted to assess the effect of KXS in inhibiting microglial inflammation activation response. Concurrently, the measurement of 4-Hydroxynonenal (4-HNE) immunohistochemistry staining, malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS) were performed to evaluate the effect of KXS on anti-oxidative stress of depression in vivo. Besides, nitric oxide (NO), relative inflammatory factors mRNA expression, JC-1 staining, and ROS were used to evaluate the effect of KXS by lipopolysaccharide (LPS)/interferon-gamma (IFNγ)-induced BV2 cells. Results KXS significantly relieved the depressive-like symptoms induced by CORT, as well as ameliorating the neuronal damage, which decreased microglia inflammatory activation response of IL-1β, IL-6, and tumor necrosis factor α (TNFα) in vivo or in vitro too. Transcriptome Sequencing and Data Analysis showed that KXS mainly by regulating immune system and transduction pathways decreased CORT-induced depression in mice. And showed that there were 19 Principal components and 10 genes in the main regulatory position with the strongest correlation in depression mice. Meanwhile, KXS effectively decreased senescence, the expression of 4-HNE, MDA content, and the production of ROS, while increasing the SOD activity in CORT-induced mice. Besides, KXS significantly reversed the mitochondrial membrane potential loss and excessive ROS production in LPS/IFNγ-induced BV2 cells. Conclusion Our research suggested that KXS might protect depressed mice against CORT-induced neuronal injury by inhibiting microglia activation and oxidative stress.

Low baseline IFN-γ response could predict hospitalization in COVID-19 patients.

The SARS-CoV-2 infection has spread rapidly around the world causing millions of deaths. Several treatments can reduce mortality and hospitalization. However, their efficacy depends on the choice of the molecule and the precise timing of its administration to ensure viral clearance and avoid a deleterious inflammatory response. Here, we investigated IFN-γ, assessed by a functional immunoassay, as a predictive biomarker for the risk of hospitalization at an early stage of infection or within one month prior to infection. Individuals with IFN-γ levels below 15 IU/mL were 6.57-times more likely to be hospitalized than those with higher values (p<0.001). As confirmed by multivariable analysis, low IFN-γ levels, age >65 years, and no vaccination were independently associated with hospitalization. In addition, we found a significant inverse correlation between low IFN-γ response and high level of IL-6 in plasma (Spearman’s rho=-0.38, p=0.003). Early analysis of the IFN-γ response in a contact or recently infected subject with SARS-CoV-2 could predict hospitalization and thus help the clinician to choose the appropriate treatment avoiding severe forms of infection and hospitalization.

A transcriptomics-based meta-analysis identifies a cross-tissue signature for sarcoidosis.

Sarcoidosis is a granulomatous disease of unknown etiology, immunologically characterized by a Th1 immune response. Transcriptome-wide expression studies in various types of sarcoid tissues contributed to better understanding of disease mechanisms. We performed a systematic database search on Gene Expression Omnibus (GEO) and utilized transcriptomic data from blood and sarcoidosis-affected tissues in a meta-analysis to identify a cross-tissue, cross-platform signature. Datasets were further separated into training and testing sets for development of a diagnostic classifier for sarcoidosis. A total of 690 differentially expressed genes were identified in the analysis among various tissues. 29 of the genes were robustly associated with sarcoidosis in the meta-analysis both in blood and in lung-associated tissues. Top genes included LINC01278 (P = 3.11 × 10–13), GBP5 (P = 5.56 × 10–07), and PSMB9 (P = 1.11 × 10–06). Pathway enrichment analysis revealed activated IFN-γ, IL-1, and IL-18, autophagy, and viral infection response. IL-17 was observed to be enriched in peripheral blood specific signature genes. A 16-gene classifier achieved excellent performance in the independent validation data (AUC 0.711–0.964). This study provides a cross-tissue meta-analysis for expression profiles of sarcoidosis and identifies a diagnostic classifier that potentially can complement more invasive procedures.

A systems immunology approach to investigate cytokine responses to viruses and bacteria and their association with disease

Patterns of human immune responses to viruses and bacteria and how this impacts risk of infections or onset/exacerbation of chronic respiratory diseases are poorly understood. In a population-based birth cohort, we measured peripheral blood mononuclear cell responses (28 cytokines) to respiratory viruses and bacteria, Toll-like receptor ligands and phytohemagglutinin, in 307 children. Cytokine responses were highly variable with > 1000-fold differences between children. Machine learning revealed clear distinction between virus-associated and bacteria-associated stimuli. Cytokines clustered into three functional groups (anti-viral, pro-inflammatory and T-cell derived). To investigate mechanisms potentially explaining such variable responses, we investigated cytokine Quantitative Trait Loci (cQTLs) of IL-6 responses to bacteria and identified nine (eight novel) loci. Our integrative approach describing stimuli, cytokines and children as variables revealed robust immunologically and microbiologically plausible clustering, providing a framework for a greater understanding of host-responses to infection, including novel genetic associations with respiratory disease.

Effect of Holstein genotype on ex-vivo cytokine response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) during the periparturient period

Effects of Holstein genotype on innate immune response were assessed with ex-vivo lipopolysaccharide (LPS) and lipoteichoic acid (LTA) stimulation of whole blood from unselected (UH, n = 10) and contemporary (CH, n = 11) Holsteins that differ in production by more than 4,500 kg/lactation. Blood was collected at −14, 7, 28, and 49 days in milk (DIM), mixed with a pathogen-associated molecular pattern (PAMP) molecule (0.01 or 1.0 µg LPS or 10 or 100 µg LTA per mL blood) and incubated (4 h, 37 °C). Plasma cytokines were quantified by ELISA, log10-transformed and analyzed by repeated measures with DIM as the repeated effect. Cytokine responses increased with PAMP dose and decreased as DIM increased. There was a genotype by LPS dose interaction for IL-1β as response to the low dose was greater in UH but did not differ between genotypes for the high dose. The IL-1β response was greater while the IL-6 response to LTA tended to be greater in UH than in CH cows. The more negative energy balance of CH cows did not impact genotype difference in cytokine responses. Results indicate selection since the mid-1960s has decreased ex-vivo, whole blood cytokine response of CH cows to LPS and to LTA.

Natural Exposure- and Vaccination-Induced Profiles of Ex Vivo Whole Blood Cytokine Responses to Coxiella burnetii.

Q fever is a zoonotic disease caused by the highly infectious Gram-negative coccobacillus, Coxiella burnetii (C. burnetii). The Q fever vaccine Q-VAX® is characterised by high reactogenicity, requiring individuals to be pre-screened for prior exposure before vaccination. To date it remains unclear whether vaccine side effects in pre-exposed individuals are associated with pre-existing adaptive immune responses to C. burnetii or are also a function of innate responses to Q-VAX®. In the current study, we measured innate and adaptive cytokine responses to C. burnetii and compared these among individuals with different pre-exposure status. Three groups were included: n=98 Dutch blood bank donors with unknown exposure status, n=95 Dutch village inhabitants with known natural exposure status to C. burnetii during the Dutch Q fever outbreak of 2007-2010, and n=96 Australian students receiving Q-VAX® vaccination in 2021. Whole blood cytokine responses following ex vivo stimulation with heat-killed C. burnetii were assessed for IFNγ, IL-2, IL-6, IL-10, TNFα, IL-1β, IP-10, MIP-1α and IL-8. Serological data were collected for all three cohorts, as well as data on skin test and self-reported vaccine side effects and clinical symptoms during past infection. IFNγ, IP-10 and IL-2 responses were strongly elevated in individuals with prior C. burnetii antigen exposure, whether through infection or vaccination, while IL-1β, IL-6 and TNFα responses were slightly increased in naturally exposed individuals only. High dimensional analysis of the cytokine data identified four clusters of individuals with distinct cytokine response signatures. The cluster with the highest levels of adaptive cytokines and antibodies comprised solely individuals with prior exposure to C. burnetii, while another cluster was characterized by high innate cytokine production and an absence of C. burnetii-induced IP-10 production paired with high baseline IP-10 levels. Prior exposure status was partially associated with these signatures, but could not be clearly assigned to a single cytokine response signature. Overall, Q-VAX® vaccination and natural C. burnetii infection were associated with comparable cytokine response signatures, largely driven by adaptive cytokine responses. Neither individual innate and adaptive cytokine responses nor response signatures were associated retrospectively with clinical symptoms during infection or prospectively with side effects post-vaccination.

Relationship between T-cell-dependent and T-cell-independent vaccines after neurotrauma; is the B-cell response preserved?

ABSTRACT Background After trauma and central nervous system (CNS) injury, trauma-induced immune deficiency syndrome (TIDS) and CNS injury-induced immune deficiency syndrome (CIDS) may negatively affect responses to T-cell-dependent vaccines, such as pneumococcal conjugate vaccine (PCV) recommended after basilar fracture. This study (NCT02806284) aimed to investigate whether there after neurotrauma is a correlation between T-cell-dependent and independent vaccine responses and, thus, if B-cell activity is similarly depressed and whether the T-cell-dependent response is possible to predict. Methods Adult patients with basilar fracture (n = 33) and those undergoing pituitary gland surgery (n = 23) were within 10 days vaccinated with a T-cell-dependent vaccine against Haemophilus influenzae type b (Hib) and a T-cell-independent pneumococcal polysaccharide vaccine (PPSV). Samples reflecting the systemic inflammatory response and pre- and post-vaccination antibody levels after 3–6 weeks against Hib and PPSV were collected and determined by enzyme immunoassays. Results High and significant correlations were detected in the responses to different pneumococcal serotypes, but none between the Hib and PPSV responses. No differences in trauma scores, C-reactive protein, IL-6, IL-10, pentraxin 3, fractalkine or calprotectin plasma concentrations or in ex vivo TNF-α, IL-6 or IL-10 responses to endotoxin were found between Hib vaccination responders and non-responders. Conclusions There was no correlation between the pneumococcal responses and that to Hib, indicating that B-cell function is not similarly depressed as T-cell function. Grading of the trauma or parameters reflecting the innate immune response could not predict the T-cell-dependent vaccine response. There is a need of further studies evaluating the vaccine response after neurotrauma.

An immunometabolic signature of athletes susceptible to respiratory tract illness? Comment on: Evidence of immunometabolic dysregulation and airway dysbiosis in athletes susceptible to respiratory illness.

An immunometabolic signature of athletes susceptible to respiratory tract illness? Comment on: Evidence of immunometabolic dysregulation and airway dysbiosis in athletes susceptible to respiratory illness.

CPEB1 regulates the inflammatory immune response, phagocytosis, and alternative polyadenylation in microglia.

Microglia are myeloid cells of the central nervous system that perform tasks essential for brain development, neural circuit homeostasis, and neural disease. Microglia react to inflammatory stimuli by upregulating inflammatory signaling through several different immune cell receptors such as the Toll-like receptor 4 (TLR4), which signals to several downstream effectors including transforming growth factor beta-activated kinase 1 (TAK1). Here, we show that TAK1 levels are regulated by CPEB1, a sequence-specific RNA binding protein that controls translation as well as RNA splicing and alternative poly(A) site selection in microglia. Lipopolysaccharide (LPS) binds the TLR4 receptor, which in CPEB1-deficient mice leads to elevated expression of ionized calcium binding adaptor molecule 1 (Iba1), a microglial protein that increases with inflammation, and increased levels of the cytokine IL6. This LPS-induced IL6 response is blocked by inhibitors of JNK, p38, ERK, NFκB, and TAK1. In contrast, phagocytosis, which is elevated in CPEB1-deficient microglia, is unaffected by LPS treatment or ERK inhibition, but is blocked by TAK1 inhibition. These data indicate that CPEB1 regulates microglial inflammatory responses and phagocytosis. RNA-seq indicates that these changes in inflammation and phagocytosis are accompanied by changes in RNA levels, splicing, and alternative poly(A) site selection. Thus, CPEB1 regulation of RNA expression plays a role in microglial function.

OP0015 PROINFLAMMATORY MONOCYTES AND MACROPHAGES IN SYNOVIAL FLUID AND BURSAL TISSUE OF PATIENTS WITH POLYMYALGIA RHEUMATICA: POTENT PRODUCERS OF IL-6 AND GM-CSF

BackgroundPolymyalgia rheumatica (PMR) is a common, rheumatic inflammatory disease. Inflammation of bursae and tendon sheaths is a characteristic finding in patients with PMR. Glucocorticoid treatment remains the mainstay treatment for PMR. A study published in 1996 reported that macrophages dominate the inflammatory infiltrates in the glenohumeral synovium of PMR patients1, suggesting the importance of these cells in the immunopathology of PMR. However, the functional and phenotypical heterogeneity of the tissue-infiltrating macrophages in PMR remains obscure. Although treatment with anti-IL-6 receptor (tocilizumab) has shown promising results2, it is unclear whether macrophages contribute to IL-6 production in PMR. Additionally, anti-GM-CSF receptor therapy (mavrilimumab), recently shown to be efficacious in the closely related disease giant cell arteritis3, may also be useful for the treatment of PMR. Knowledge on the functional heterogeneity of monocytes/macrophages in PMR may aid in identifying novel therapeutic targets for this condition.ObjectivesTo determine the phenotype of monocyte/macrophages in peripheral blood, bursal/tenosynovial fluid and bursal tissue of patients with PMR.MethodsPaired peripheral blood (PB), bursal/tenosynovial fluid (SF) and bursal tissue biopsy samples from 11 PMR patients were included in our study. Bursal and tenosynovial samples were obtained from the shoulder. Distribution of the monocyte subsets (classical, intermediate and non-classical monocytes) was determined based on the level of CD14 and CD16 expression by flow cytometry. To study monocyte activation status, markers of ‘M1’ like (CD80 and CD64) and ‘M2’ like (CD206 and FRβ) macrophage polarization were included in the flow cytometry analysis. Immunohistochemistry of bursal tissue biopsies was focused on macrophage markers (CD68, CD86, CD64, CD206 and FRβ) andproinflammatory cytokines (IL-6 and GM-CSF), which were scored semi-quantitatively. Double immunofluorescence stainings were performed to determine the expression of IL-6 and GM-CSF by tissue-infiltrating macrophages in bursal tissue.ResultsMonocytes were detected in the SF of PMR patients. The proportion of classical monocytes was significantly lowered (p=0.001) in SF versus PB, while the proportion of intermediate monocytes was significantly elevated (p=0.001). The expression of CD206 was significantly elevated (p=0.001) but not FRβ in SF monocytes, suggesting GM-CSF skewed phenotype. In bursal tissue, macrophages displayed mixed ‘M1’/’M2’ traits with high expression of all macrophage polarization markers. Proinflammatory cytokines IL-6 and GM-CSF were highly expressed throughout the bursal tissue biopsies. Double immunofluorescence staining confirmed the expression of IL-6 and GM-CSF by the infiltrating macrophages.ConclusionSF monocytes and bursal tissue macrophages show a pro-inflammatory phenotype in PMR. Moreover, tissue-infiltrating macrophages show a prominent IL-6 and GM-CSF response in PMR. Our data add to the rationale of targeting IL-6 and GM-CSF as treatment options in PMR.