Unique Inflammatory Signature Research Articles

IĸBζ is a central modulator of inflammatory arthritis pathogenesis.

Current therapies targeting individual factors in inflammatory arthritis (IA) show variable efficacy, often requiring treatment using combinations of drugs and associated with undesirable side effects. NF-ĸB is critical for production and function of most inflammatory cytokines. However, given its essential role in physiologic processes, targeting NF-ĸB is precarious. Hence, identifying pathways downstream of NF-κB that selectively govern expression of inflammatory cytokines in IA would be advantageous. We have previously identified IĸBζ as a unique inflammatory signature of NF-ĸB that controls transcription of inflammatory cytokines only under pathologic conditions while sparing physiologic NF-ĸB signals. We generated mice harboring myeloid, lymphoid and global deletion of Nfkbiz (the gene encoding IĸBζ). These models were subjected to serum transfer-induced arthritis (STIA). Additionally, pharmacologic inhibitors of IĸBζ were injected intraperitonially. Joint swelling, μCT, immunohistochemistry, flow cytometry, and cytokine measurements were carried out using synovial tissues. Global deletion of Nfkbiz or depletion of neutrophils (vastly IĸBζ+ cells) reduced inflammatory synovial cells and increased anti-inflammatory and regenerative synovial cells, plummeted expression of inflammatory factors and ameliorated experimental mouse IA. Further, expression of Irg1, the enzyme responsible for itaconate production, was increased in synovial cells. Accordingly, the itaconate derivative dimethyl itaconate (DI) inhibited IĸBζ-mediated inflammatory factors. Further, in silico screen identified 8-Hydroxyquinoline (HQ) as putative inhibitor of IĸBζ not affecting physiological NF-ĸB activity. Congruently, systemic administration of either DI or HQ inhibited joint swelling and damage. Our study positions IĸBζ as an inflammation-specific target for therapeutic consideration in RA as its inhibition spares the beneficial functions of NF-ĸB.

229 Comparative immune profiling of four autoimmune skin diseases identifies a mixed type-I and type-II interferon response as a unique inflammatory signature in skin dermatomyositis

229 Comparative immune profiling of four autoimmune skin diseases identifies a mixed type-I and type-II interferon response as a unique inflammatory signature in skin dermatomyositis

Development of an integrated microperfusion-EEG electrode for unbiased multimodal sampling of brain interstitial fluid and concurrent neural activity

Objective. To modify off-the-shelf components to build a device for collecting electroencephalography (EEG) from macroelectrodes surrounded by large fluid access ports sampled by an integrated microperfusion system in order to establish a method for sampling brain interstitial fluid (ISF) at the site of stimulation or seizure activity with no bias for molecular size. Approach. Twenty-four 560 µm diameter holes were ablated through the sheath surrounding one platinum–iridium macroelectrode of a standard Spencer depth electrode using a femtosecond UV laser. A syringe pump was converted to push–pull configuration and connected to the fluidics catheter of a commercially available microdialysis system. The fluidics were inserted into the lumen of the modified Spencer electrode with the microdialysis membrane removed, converting the system to open flow microperfusion. Electrical performance and analyte recovery were measured and parameters were systematically altered to improve performance. An optimized device was tested in the pig brain and unbiased quantitative mass spectrometry was used to characterize the perfusate collected from the peri-electrode brain in response to stimulation. Main results. Optimized parameters resulted in >70% recovery of 70 kDa dextran from a tissue analog. The optimized device was implanted in the cortex of a pig and perfusate was collected during four 60 min epochs. Following a baseline epoch, the macroelectrode surrounded by microperfusion ports was stimulated at 2 Hz (0.7 mA, 200 µs pulse width). Following a post-stimulation epoch, the cortex near the electrode was stimulated with benzylpenicillin to induce epileptiform activity. Proteomic analysis of the perfusates revealed a unique inflammatory signature induced by electrical stimulation. This signature was not detected in bulk tissue ISF. Significance. A modified dual-sensing electrode that permits coincident detection of EEG and ISF at the site of epileptiform neural activity may reveal novel pathogenic mechanisms and therapeutic targets that are otherwise undetectable at the bulk tissue level.

Unique Inflammatory Signature in Hemophilic Arthropathy: Epigenetic Changes Due to Interaction between Blood and Fibroblast-like Synoviocytes

Context: Hemophilic patients bleeding occur mainly into large joints such as knee, elbow or ankle. Severe recurrence of hemarthrosis leads to irreversible arthropathy called hemophilic arthropathy (HA) characterized by a progressive complete destruction of the joint with permanent pain, musculoskeletal disability and poor quality of life. Physiopathology of blood joint disease remains unclear. Some data suggest that HA and rheumatoid arthritis (RA) may share similar pathogenicity including elevation of inflammatory cytokines, chronic proliferative synovitis and cartilage destruction. However, in these two diseases the origin of inflammation differs and is linked to autoimmunity in RA and bleeding in HA. HA is the unique model of arthropathy in which resident cells like fibroblasts-like synoviocytes (FLS) are directly in contact with blood. Therefore, we hypothesized that FLS in HA could have a unique inflammatory signature as a consequence of contact with blood.Materials and methods: Human FLS were isolated from synovial tissues from five different HA patients and two non-HA patients at the time of knee or ankle joint arthroscopic synovectomy after informed consent were obtained from patients. Experiments with FLS cultures were performed between the third and the ninth passage. During that time, cultures were constituted of a homogeneous population of fibroblastic cells. THP-1 cells line has been used as a control.Results: FLS from hemophilic patients have a singular inflammatory cytokines profile- FLS (5x105 cells) were activated or not (medium) with LPS from Salmonella abortus equi (1 µg/ml) for 6 h and activated-cells supernatants were analyzed by ELISA. ELISPOT were performed with a panel of 105 different cytokines. After LPS stimulation, HA FLS expressed a unique profile of cytokines secretion, which differs from non-HA FLS and THP-1 cell line including IL-1α, IL-6 and IL-8 involved in innate immunity, IFN-γ, IL-5, IL-17A and IL-22 involved in adaptive immunity and MCP-1, M-CSF, MIF and MIP-3α involved in macrophages polarization.- Constitutive expression of IL-1 and IL-6 mRNA was detectable in all cells, but in contrast, as shown previously in normal FLS, no TNF-α mRNA was detectable. To investigate mechanisms responsible for the lack of IL-1 protein synthesis in LPS-activated HA FLS, cells were incubated with LPS for 3 h and then with 5 µg/ml actinomycin D. After 2h of actinomycin D treatment, IL-1 mRNA became lower in LPS-activated HA FLS versus LPS-activated non-HA FLS and THP-1 cells. These results indicate that de novo synthesized IL-1 mRNA has a short half-life in LPS-activated HA FLS.- Since cytokines mRNA seems to be unstable, we then investigated microRNA involvement in the regulation of cytokines. A miRNOME (Transcriptome Profiling miRNA, AFFYMETRIX) analysis showed that miRNAs expression was different between HA and nonHA FLS. Five have a very high difference in expression (hsa-miR664b-5p, hsa-miR4516, hsa-miR4289, hsa-miR6719-3p, hsa-miR675-5p), which potentially target mRNA involved in inflammatory pathways (NFKB, MAPK, Wnt/beta-catenin).In conclusion our study demonstrates for the first time that the presence of blood in contact with FLS may induce durable epigenetic changes that may participate to the pathophysiology of hemophilic arthropathy. DisclosuresMignot:Shire: Research Funding. Delarue:Roche: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Frenzel:Shire: Research Funding.

Unique inflammatory profile is associated with higher SARS-CoV-2 acute respiratory distress syndrome (ARDS) mortality.

The COVID19 pandemic has caused more than a million of deaths worldwide, primarily due to complications from COVID19-associated acute respiratory distress syndrome (ARDS). Controversy surrounds the circulating cytokine/chemokine profile of COVID19-associated ARDS, with some groups suggesting that it is similar to patients without COVID19 ARDS and others observing substantial differences. Moreover, although a hyperinflammatory phenotype associates with higher mortality in non-COVID19 ARDS, there is little information on the inflammatory landscape's association with mortality in patients with COVID19 ARDS. Even though the circulating leukocytes' transcriptomic signature has been associated with distinct phenotypes and outcomes in critical illness including ARDS, it is unclear whether the mortality-associated inflammatory mediators from patients with COVID19 are transcriptionally regulated in the leukocyte compartment. Here, we conducted a prospective cohort study of 41 mechanically ventilated patients with COVID19 infection using highly calibrated methods to define the levels of plasma cytokines/chemokines and their gene expressions in circulating leukocytes. Plasma IL1RA and IL8 were found positively associated with mortality, whereas RANTES and EGF negatively associated with that outcome. However, the leukocyte gene expression of these proteins had no statistically significant correlation with mortality. These data suggest a unique inflammatory signature associated with severe COVID19.

Unique inflammatory signature in haemophilic arthropathy: miRNA changes due to interaction between blood and fibroblast-like synoviocytes.

In haemophilia, the recurrence of hemarthrosis leads to irreversible arthropathy termed haemophilic arthropathy (HA). However, HA is a unique form of arthropathy in which resident cells, such as fibroblast‐like synoviocytes (FLS), come into direct contact with blood. Therefore, we hypothesized that FLS in HA could have a unique inflammatory signature as a consequence of their contact with blood. We demonstrated with ELISA and ELISPOT analyses that HA‐FLS expressed a unique profile of cytokine secretion, which differed from that of non‐HA‐FLS, mainly consisting of cytokines involved in innate immunity. We showed that unstable cytokine mRNAs were involved in this process, especially through miRNA complexes as confirmed by DICER silencing. A miRNOME analysis revealed that 30 miRNAs were expressed differently between HA and non‐HA‐FLS, with most miRNAs involved in inflammatory control pathways or described in certain inflammatory diseases, such as rheumatoid arthritis or lupus. Analysis of transcriptomic networks, impacted by these miRNAs, revealed that protein processes and inflammatory pathways were particularly targeted in LPS‐induced FLS, and in particular vascularization and osteoarticular modulation pathways in steady‐state FLS. Our study demonstrates that the presence of blood in contact with FLS may induce durable miRNA changes that likely participate in HA pathophysiology.

Predicting persistence of atopic dermatitis in children using clinical attributes and serum proteins.

Atopic dermatitis (AD) is the most common inflammatory skin disease in children, with 30% of all those diagnosed developing chronic or relapsing disease by adolescence. Such disease persistence cannot yet be predicted. The aim of the present study was to predict the natural course of AD using clinical parameters and serum proteins. Sera of 144 children with AD (age 0-3years) were analyzed for IgE and 33 cytokines, chemokines, and growth factors. Patient disease course until the age of 7years was assessed retrospectively. Unsupervised k-means clustering was performed to define disease endotypes. Identified factors associated with AD persistence at the age of 7years were validated in children with AD in an independent cohort (LISA Munich; n=168). Logistic regression and XGBoosting methods followed by cross-validation were applied to predict individual disease outcomes. Three distinct endotypes were found in infancy, characterized by a unique inflammatory signature. Factors associated with disease persistence were disease score (SCORAD), involvement of the limbs, flexural lesion distribution at the age of 3years, allergic comorbidities, and disease exacerbation by the trigger factors stress, pollen exposure, and change in weather. Persistence was predicted with a sensitivity of 81.8% and a specificity of 82.4%. Factors with a high impact on the prediction of persistence were SCORAD at the age of 3years, trigger factors, and low VEGF serum levels. Atopic dermatitis in infancy comprises three immunological endotypes. Disease persistence can be predicted using serum cytokines and clinical variables.

Identification of an Increased Alveolar Macrophage Subpopulation in Old Mice That Displays Unique Inflammatory Characteristics and Is Permissive to Mycobacterium tuberculosis Infection.

The elderly population is more susceptible to pulmonary infections, including tuberculosis. In this article, we characterize the impact of aging on the phenotype of mouse alveolar macrophages (AMs) and their response to Mycobacterium tuberculosis. Uninfected AMs were isolated from bronchoalveolar lavage of young (3 mo) and old (18 mo) C57BL/6 mice. AMs from old mice expressed higher mRNA levels of CCL2, IFN-β, IL-10, IL-12p40, TNF-α, and MIF than young mice, and old mice contained higher levels of CCL2, IL-1β, IFN-β, and MIF in their alveolar lining fluid. We identified two distinct AM subpopulations, a major CD11c+ CD11b- population and a minor CD11c+ CD11b+ population; the latter was significantly increased in old mice (4-fold). Expression of CD206, TLR2, CD16/CD32, MHC class II, and CD86 was higher in CD11c+ CD11b+ AMs, and these cells expressed monocytic markers Ly6C, CX3CR1, and CD115, suggesting monocytic origin. Sorted CD11c+ CD11b+ AMs from old mice expressed higher mRNA levels of CCL2, IL-1β, and IL-6, whereas CD11c+ CD11b- AMs expressed higher mRNA levels of immune-regulatory cytokines IFN-β and IL-10. CD11c+ CD11b+ AMs phagocytosed significantly more M. tuberculosis, which expressed higher RNA levels of genes required for M. tuberculosis survival. Our studies identify two distinct AM populations in old mice: a resident population and an increased CD11c+ CD11b+ AM subpopulation expressing monocytic markers, a unique inflammatory signature, and enhanced M. tuberculosis phagocytosis and survival when compared with resident CD11c+ CD11b- AMs, which are more immune regulatory in nature.

Chronic rhinosinusitis in elderly patients is associated with an exaggerated neutrophilic proinflammatory response to pathogenic bacteria

Potential effects of aging on chronic rhinosinusitis (CRS) pathophysiology have not been well defined but might have important ramifications given a rapidly aging US and world population. The goal of the current study was to determine whether advanced age is associated with specific inflammatory CRS endotypes or immune signatures. Levels of 17 mucus cytokines and inflammatory mediators were measured in 147 patients with CRS. Hierarchical cluster analysis was used to identify and characterize inflammatory CRS endotypes, as well as to determine whether age was associated with specific immune signatures. A CRS endotype with a proinflammatory neutrophilic immune signature was enriched in older patients. In the overall cohort patients 60years and older had increased mucus levels of IL-1β, IL-6, IL-8, and TNF-α when compared with their younger counterparts. Increases in levels of proinflammatory cytokines were associated with both tissue neutrophilia and symptomatic bacterial infection/colonization in aged patients. Aged patients with CRS have a unique inflammatory signature that corresponds to a neutrophilic proinflammatory response. Neutrophil-driven inflammation in aged patients with CRS might be less likely to respond to corticosteroids and might be closely linked to chronic microbial infection or colonization.

Peripheral Eosinophilia in Patients With Inflammatory Bowel Disease Defines an Aggressive Disease Phenotype.

Peripheral blood eosinophilia (PBE) in inflammatory bowel disease (IBD) is associated with ulcerative colitis (UC) and active disease. Little data exist on the long-term impact of PBE on disease course. We aimed to investigate the multi-year patterns of PBE and its impact on disease severity in a large IBD cohort. We performed a registry analysis of a consented, prospective, natural history IBD cohort at a tertiary center from 2009 to 2014. Demographics, comorbidities, disease activity, healthcare utilization, and time to hospitalization or surgical resection of patients who displayed PBE were compared to patients without PBE. Of the 2,066 IBD patients, 19.2% developed PBE. PBE was significantly associated with UC (P<0.001), extensive colitis (P<0.001), and shorter disease duration (P=0.03). Over six years, PBE patients had more active disease (Harvey-Bradshaw Index P=0.001; ulcerative colitis activity index P<0.001), concurrent C-reactive protein elevation (P<0.001), healthcare utilization (hospitalization P<0.001, IBD surgery P<0.001), and more aggressive medical therapy (prednisone P<0.001, anti-TNF P<0.001). Patients with PBE had a significantly reduced time to hospitalization in both UC (P<0.001) and Crohn's disease (CD) (P<0.001) and reduced time to colectomy in UC (P=0.003). On multivariable modeling, PBE remained significantly associated with hospitalization and surgery in both CD and UC. New diagnosis of UC with PBE was associated with increased steroid (P=0.007) and anti-TNF (P=0.001) requirement. This multi-year study of a large IBD cohort suggests that peripheral blood eosinophilia represents a biomarker of a distinct IBD subgroup, with a unique inflammatory signature, and at risk for worse clinical outcomes.

Cortical Spreading Depression Causes Unique Dysregulation of Inflammatory Pathways in a Transgenic Mouse Model of Migraine

Familial hemiplegic migraine type 1 (FHM1) is a rare monogenic subtype of migraine with aura caused by mutations in CACNA1A that encodes the α1A subunit of voltage-gated CaV2.1 calcium channels. Transgenic knock-in mice that carry the human FHM1 R192Q missense mutation (‘FHM1 R192Q mice’) exhibit an increased susceptibility to cortical spreading depression (CSD), the mechanism underlying migraine aura. Here, we analysed gene expression profiles from isolated cortical tissue of FHM1 R192Q mice 24 h after experimentally induced CSD in order to identify molecular pathways affected by CSD. Gene expression profiles were generated using deep serial analysis of gene expression sequencing. Our data reveal a signature of inflammatory signalling upon CSD in the cortex of both mutant and wild-type mice. However, only in the brains of FHM1 R192Q mice specific genes are up-regulated in response to CSD that are implicated in interferon-related inflammatory signalling. Our findings show that CSD modulates inflammatory processes in both wild-type and mutant brains, but that an additional unique inflammatory signature becomes expressed after CSD in a relevant mouse model of migraine.

Abstract 342: Differences in Inflammatory Dysregulation in TBI Versus Major Trauma

Introduction: Up-regulation of aseptic inflammation is a prominent feature of major trauma, with or without head injury. Observational studies in TBI often lack control groups with major trauma, obscuring the genesis of the reported biomarkers. We hypothesized that patients with TBI would display a unique inflammatory signature that would differ from patients with major trauma without brain injury, due to blood-brain-barrier compromise. Methods: We prospectively enrolled patients with major trauma with and without TBI from a busy level I trauma center ICU. TBI was defined as GCS&lt;13 not related to intoxication, or the presence of intracranial hemorrhage on admission CT scan. Plasma samples were obtained on admission (&lt;8 hours from the trauma), and banked at -80°C for subsequent analysis. Plasma was analyzed by the Stanford Human Immune Monitoring Center using a Luminex human 61-plex analyte platform that screens 61 secreted proteins using multiplex fluorescent immunoassay; samples were run in duplicate for confirmation, and the means reported. Measurements are reported in median [IQR] in pg/mL. P values of 0.05 or less were considered significant, though final data will be adjusted for multiple comparisons. Results: Forty patients were enrolled, 21 had TBI. Total injury severity scores (ISS) (26 [11-27 v. 14 [10-21] P=0.11) and APACHE (14 [12-24] v. 12 [10-14] P=0.07) scores were comparable between groups. Patients in the TBI group had a lower GCS on admission (15 [4-15] v. 15 [15-15]) P=0.04). While both groups had evidence of elevated cytokines, only TNF-alpha (72.23 [63.68-91.27] v. 63.52 [58.36-69.5], P=0.05) and interferon beta (19.31 [14.13-33.8] v. 14.79 [10.71-17.32], P=0.04) were statistically significantly different between the groups, both more elevated in the TBI group. Conclusion: Our data provides preliminary evidence of immunologic dissonance unique to TBI as compared to major trauma. Future studies using biomarkers in TBI must control for major trauma as a confounder.