An Immunological Axis Involving Interleukin 1β and Leucine-Rich-α2-Glycoprotein Reflects Therapeutic Response of Children with Kawasaki Disease: Implications from the KAWAKINRA Trial

Purpose A recent phase II open-label study of the interleukin 1 (IL-1) receptor antagonist (IL-1Ra) anakinra in treating IVIG-resistant Kawasaki Disease (KD) patients reported promising results. Here, we aimed to characterize the immunological impact of IL-1 blockade in this unique study population. Methods Patients’ and control sera and supernatants of cells (whole blood, neutrophils, coronary artery endothelial cells) stimulated with recombinant IL-1β were analyzed for single or multiple marker (n=22) expression by ELISA or multiplexed bead array assay. Data were analyzed using unsupervised hierarchical clustering, multiple correlation and multi-comparison statistics and were compared to retrospective analyses of KD transcriptomics. Results Inflammation in IVIG-resistant KD (n=16) is hallmarked by over-expression of innate immune mediators (particularly IL-6>CXCL10>S100A12>IL-1Ra). Those as well as levels of immune or endothelial cell activation markers (sICAM-1, sVCAM-1) declined most significantly in course of anakinra treatment. Prior as well as following IL-1R blockade, over-expression of leucine-rich-α2-glycoprotein 1 (LRG1) associated best with remnant inflammatory activity and the necessity to escalate anakinra dosage and separated inflammatory KD patients from sJIA-MAS (n=13) and MIS-C (n=4). Protein as well as retrospective gene expression analyses indicated tight association of LRG1 with IL-1β signaling and neutrophilia, while particularly neutrophil stimulation with recombinant IL-1β resulted in concentration-dependent LRG1 release. Conclusion Our study identifies LRG1 as known trigger of endothelial activation and cardiac re-modelling to associate with IL-1β signaling in KD. Besides a potential patho-mechanistic implication of these findings, our data suggest blood leukocyte and neutrophil counts to best predict response to IL-1Ra treatment in IVIG-resistant KD.

The Route of Vaccine Administration Determines Whether Blood Neutrophils Undergo Long-Term Phenotypic Modifications

Innate immunity modulates adaptive immunity and defines the magnitude, quality, and longevity of antigen-specific T- and B- cell immune memory. Various vaccine and administration factors influence the immune response to vaccination, including the route of vaccine delivery. We studied the dynamics of innate cell responses in blood using a preclinical model of non-human primates immunized with a live attenuated vaccinia virus, a recombinant Modified vaccinia virus Ankara (MVA) expressing a gag-pol-nef fusion of HIV-1, and mass cytometry. We previously showed that it induces a strong, early, and transient innate response, but also late phenotypic modifications of blood myeloid cells after two months when injected subcutaneously. Here, we show that the early innate effector cell responses and plasma inflammatory cytokine profiles differ between subcutaneous and intradermal vaccine injection. Additionally, we show that the intradermal administration fails to induce more highly activated/mature neutrophils long after immunization, in contrast to subcutaneous administration. Different batches of antibodies, staining protocols and generations of mass cytometers were used to generate the two datasets. Mass cytometry data were analyzed in parallel using the same analytical pipeline based on three successive clustering steps, including SPADE, and categorical heatmaps were compared using the Manhattan distance to measure the similarity between cell cluster phenotypes. Overall, we show that the vaccine per se is not sufficient for the late phenotypic modifications of innate myeloid cells, which are evocative of innate immune training. Its route of administration is also crucial, likely by influencing the early innate response, and systemic inflammation, and vaccine biodistribution.

Severity of the acute phase response in experimental ulcerative colitis under conditions of the use of vitamin D3 in a novel rectal suppository

Цель - изучение влияния витамина D3 в составе оригинальных ректальных суппозиториев на выраженность острофазового ответа при экспериментальном язвенном колите Методика. Эксперимент выполнен на 49 половозрелых крысах-самцах Wistar. Язвенный колит (ЯК) моделировали двухэтапным (накожным, а затем ректальным) применением 3% спиртового раствора оксазолона. Оригинальные суппозитории на основе смеси полиэтиленгликолей массой 300 мг, содержащие 1500 ME витамина D3, вводили per rectum каждые 12 ч в течение 6 сут. Исследования проводили на 2-е, 4-е и 6-е сут заболевания. Клиническую картину оценивали по адаптированной для крыс шкале Disease activity index (DAI) с учетом массы тела, консистенции и наличия крови в каловых массах. Количество лейкоцитов и нейтрофилов определяли на гематологическом анализаторе и в мазках крови. Функциональную активность нейтрофилов исследовали по поглощению частиц монодисперсного латекса в спонтанном и индуцированном НСТ-тесте. Концентрацию в сыворотке крови C-реактивного белка (С-РБ), IL-6 и IL-8 определяли с помощью иммуноферментного анализа, экспрессию TNF-α в стенке толстой кишки - иммуногистохимическим методом. Результаты. При экспериментальном ЯК на 2-е, 4-е и 6-е сут наряду с прогрессивным повышением DAI зафиксированы изменения показателей острофазового ответа (ООФ): увеличивается экспрессия в толстой кишке TNF-α с максимальным уровнем на 4-е и 6-е сут, повышается концентрация в сыворотке крови IL-6 с максимальным уровнем на 4-е, IL-8 - на 6-е сут; в сыворотке возрастает концентрация С-РБ максимально на 6-е сут, в крови увеличивается общее количество лейкоцитов, число палочкоядерных и сегментоядерных нейтрофилов с максимальным уровнем на 2-е и 4-е сут. Возрастает поглотительная и НСТ-редуцирующая способность нейтрофилов крови с максимальными значениями на 4-е и 6-е сут эксперимента. Применение при экспериментальном ЯК ректальных суппозиториев с витамином D3 приводит на 4-е и 6-е сут наблюдения к снижению DAI, экспрессии TNF-α в толстой кишке и количества нейтрофилов, на 2-е, 4-е и 6-е сут - к уменьшению концентрации IL-6 и С-РБ в сыворотке, поглотительной и НСТ-редуцирующей способности нейтрофилов крови, на 6-е сут - к снижению концентрации IL-8 в сыворотке и общего количества лейкоцитов в крови. Заключение. Применение при экспериментальном ЯК оригинальных ректальных суппозиториев с витамином D3 приводит к снижению индекса активности болезни. Выраженность клинических проявлений ослабевает по мере снижения экспрессии в кишке TNF-α, снижения концентрации в сыворотке IL-6, количества в крови лейкоцитов и нейтрофилов, уменьшения НСТ-редуцирующей способности нейтрофилов. Aim. To study the effect of vitamin D3 in rectal suppositories on severity of the acute phase response (APR) in experimental ulcerative colitis (UC). Methods. Experiments were performed on 49 mature male Wistar rats. UC was induced by cutaneous followed by rectal applications of a 3% oxazolone alcohol solution. Novel polyethylene glycol suppositories (300 mg) containing 1500 IU of vitamin D3 were injected per rectum every 12 hrs for 6 days. Data were collected on days 2, 4, and 6 of UC. The clinical picture was assessed according to the Disease Activity Index (DAI) scale adapted for rats, taking into account body weight, consistency of, and the presence of blood in the feces. The number of blood leukocytes and neutrophils was determined on a hematological analyzer and in blood smears. The functional activity of neutrophils isolated from the blood was evaluated by the absorption of monodisperse latex particles and with the spontaneous and induced NBT test. Serum concentrations of C-reactive protein (CRP), IL-6 and IL-8 were measured by enzyme immunoassay. TNF-α expression in the colon wall was measured by an immunohistochemical method. Results. The following changes in APR variables along with a progressive increase in DAI were recorded on days 2, 4, and 6. Expression of colon TNF-α increased, with maximal values on days 4 and 6. Serum IL-6 increased and reached a maximal value on day 4; and serum IL-8 increased, with a maximum on day 6. Serum CRP increased, with a maximum on day 6. The total number of blood leukocytes and the number of stab and segmented neutrophils increased, with maximal values on days 2 and 4. The absorption and NBT-reducing ability of blood neutrophils increased, with maximal values on days 4 and 6. The use of rectal suppositories with vitamin D3 in experimental UC resulted in decreases in DAI, colon TNF-α expression, and the number of blood neutrophils on days 4 and 6; decreases in serum concentrations of IL-6 and CRP, and the absorption and NBT-reducing ability of blood neutrophils on days 2, 4, and 6; and decreases in the serum concentration of IL-8 and in the total number of blood leukocytes on day 6. Severity of clinical manifestations was alleviated with decreases in the expression of colon TNF-α, the serum concentration of IL-6, the number of blood leukocytes and neutrophils, and the NBT-reducing ability of neutrophils decreases. Conclusion. Application of the original rectal suppositories with vitamin D3 every 12 hours in experimental UC leads to a decrease in the disease activity index. The severity of clinical manifestations weakens as TNF-α expression, serum IL-6 concentration, number of leukocytes and neutrophils in blood, and decrease of neutrophil NST-reducing capacity decrease.

Impairment of Neutrophil Functions and Homeostasis in COVID-19 Patients: Association With Disease Severity

Background Emerging data based on analyses of peripheral and pulmonary immune responses to SARS-CoV-2 increasingly suggest that a dysregulated immune response underpins the development of severe disease in COVID-19 patients. Neutrophils are key components of early innate immunity that, if not tightly regulated, contribute to uncontrolled systemic inflammation. We sought to decipher the role of neutrophil phenotypes, functions, and homeostasis in COVID-19 disease severity and outcome. Methods This longitudinal study compares study compares peripheral whole-blood neutrophils from 90 COVID-19 ICU patients with those of 22 SARS-CoV-2 – patients hospitalized for severe community-acquired pneumonia (CAP) and 38 healthy controls. We also assessed correlations between these phenotypic and functional indicators and markers of endothelial damage as well as disease severity. Results At ICU admission, the circulating neutrophils of the COVID-19 patients showed continuous basal hyperactivation not seen in CAP patients, associated with higher circulating levels of soluble E- and P-selectin, which reflect platelet and endothelial activation. Furthermore, COVID-19 patients had expanded aged-angiogenic and reverse transmigrated neutrophil subsets — both involved in endothelial dysfunction and vascular inflammation. Simultaneously, COVID-19 patients had significantly lower levels of neutrophil oxidative burst in response to bacterial formyl peptide, an abnormality that was greater in superinfected than non-superinfected COVID-19 patients. Moreover, patients dying of COVID-19 had significantly higher expansion of aged-angiogenic neutrophil subset and greater impairment of oxidative burst response than survivors. Conclusions These data suggest that neutrophil exhaustion may play a central role in the pathogenesis of severe COVID-19 and identify angiogenic neutrophils as a potentially harmful subset involved in fatal outcome.

Characterising the transcriptome of hypersegmented human neutrophils

Background: Mature human neutrophils are characterised by their multilobed nuclear morphology. Neutrophil hypersegmentation, a pathologic nuclear phenotype, has been described in the alveolar compartment of patients with acute respiratory distress syndrome and in several other contexts. This study aimed to characterise the transcriptional changes associated with neutrophil hypersegmentation. Methods: A model of hypersegmentation was established by exposing healthy peripheral blood neutrophils to the angiotensin converting enzyme inhibitor (ACEi) captopril. Laser capture microdissection (LCM) was then adapted to isolate a population of hypersegmented neutrophils. Transcriptomic analysis of microdissected hypersegmented neutrophils was undertaken using ribonucleic acid (RNA) sequencing. Differential gene expression (DEG) and enrichment pathway analysis were conducted to investigate the mechanisms underlying hypersegmentation. Results: RNA-Seq analysis revealed the transcriptomic signature of hypersegmented neutrophils, with five genes differentially expressed. VCAN, PADI4 and DUSP4 were downregulated, while LTF and PSMC4 were upregulated. Modulated pathways included histone modification, protein-DNA complex assembly and antimicrobial humoral response. The role of PADI4 was further validated using the small molecule inhibitor, Cl-amidine. Conclusions: Hypersegmented neutrophils display a marked transcriptomic signature, characterised by the differential expression of five genes. This study provides insights into the mechanisms underlying neutrophil hypersegmentation and describes a novel method to isolate and sequence neutrophils based on their morphologic subtype.

Aging and interferon gamma response drive the phenotype of neutrophils in the inflamed joint

Objectives: Neutrophils are typically the most abundant leukocyte in arthritic synovial fluid. We sought to understand changes that occur in neutrophils as they migrate from blood to joint. Methods: We performed RNA sequencing of neutrophils from healthy human blood, arthritic blood, and arthritic synovial fluid, comparing transcriptional signatures with those from murine K/BxN serum transfer arthritis. We employed mass cytometry to quantify protein expression and sought to reproduce the synovial fluid phenotype ex vivo in cultured healthy blood neutrophils. Results: Blood neutrophils from healthy donors and patients with active arthritis exhibited largely similar transcriptional signatures. By contrast, synovial fluid neutrophils exhibited more than 1,600 differentially expressed genes. Gene signatures identified a prominent response to interferon gamma (IFNγ), as well as to tumor necrosis factor, interleukin 6, and hypoxia, in both humans and mice. Mass cytometry also found healthy and arthritic donor blood neutrophils largely indistinguishable but revealed a range of neutrophil phenotypes in synovial fluid defined by downregulation of CXCR1 and upregulation of FcγRI, HLA-DR, PD-L1, ICAM-1 and CXCR4. Reproduction of key elements of this signature in cultured blood neutrophils required both IFNγ and prolonged culture. Conclusions: Circulating neutrophils from arthritis patients resemble those from healthy controls, but joint fluid cells exhibit a network of changes, conserved across species, that implicate IFNγ response and aging as complementary drivers of the synovial neutrophil phenotype.

Formation of extracellular traps by circulating neutrophils and monocytes in rheumatoid arthritis patients: a study of new citrullinated autoantigen

Detection of subcellular structures containing typical citrullinated rheumatoid autoantigens in a single compartment presents a special interest, due to importance of anticitrulline autoantibodies for the autoimmune response in RA. Neutrophil and monocyte extracellular traps (NETs and ETs, respectively) may be considered such candidate structures. Our objective was to assess ability of blood neutrophils and monocytes from RA patients to generate NETs and ETs spontaneously and after in vitro induction.32 patients with verified RA and 30 healthy volunteers as controls were included into the study. Circulating neutrophils and monocytes were isolated with one-step density gradient centrifugation using three layers of ficoll-amidotrizoate gradient. Composition of isolated cellular fractions, their viability, and non-specific activation were evaluated microscopically using Trypan Blue exclusion test, as well as Nitro-Blue Tetrazolium test. The NETs were induced by phorbol-12-myristate-13-acetate, and ETs by bacterial LPS. Spontaneous and induced formation of extracellular traps was assessed using fluorescence microscopy. Neutrophil and monocyte fractions contained minute percentages of impurities and low extents of activated and dead cells. Spontaneous NET and ET formation in RA patients was significantly increased comparing to healthy controls. Neutrophils from ACPA-positive RA patients were found to have higher frequency of NET formation, compared to ACPA-negative RA patients. The monocytes did not demonstrate such differences between these subgroups. There were no substantial morphological differences in NETs and ETs patterns between the individuals from both groups. Induced extracellular trap production in RA was significantly higher compared to healthy controls. The level of myeloperoxidase-specific fluorescence in ETs was considerably lower than in NETs. NETs could probably be considered as a source of citrulline autoantigen participating in autoantibody production and stimulation of inflammatory autoimmune responses in RA, whereas ETs may play less important role in this process.

Preoperative Systemic Inflammatory Biomarkers Are Independent Predictors of Disease Recurrence in ER+ HER2- Early Breast Cancer

The host’s immune system plays a crucial role in determining the clinical outcome of many cancers, including breast cancer. Peripheral blood neutrophils and lymphocytes counts may be surrogate markers of systemic inflammation and potentially reflect survival outcomes. The aim of the present study is to assess the role of preoperative systemic inflammatory biomarkers to predict local or distant relapse in breast cancer. In particular we investigated ER+ HER2- early breast cancer, considering its challenging risk stratification. A total of 1,763 breast cancer patients treated at tertiary referral Breast Unit were reviewed. Neutrophil-to-lymphocyte (NLR), platelet-to-lymphocyte (PLR) and lymphocyte-to-monocyte (LMR) ratios were assessed from the preoperative blood counts. Multivariate analyses for 5-years locoregional recurrence-free (LRRFS), distant metastases-free (DMFS) and disease-free survivals (DFS) were performed, taking into account both blood inflammatory biomarkers and clinical-pathological variables. Low NLR and high LMR were independent predictors of longer LRRFS, DMFS and DFS, and low PLR was predictive of better LRRFS and DMFS in the study population. In 999 ER+ HER2- early breast cancers, high PLR was predictive of worse LRRFS (HR 0.42, p=0.009), while high LMR was predictive of improved LRRFS (HR 2.20, p=0.02) and DFS (HR 2.10, p=0.01). NLR was not an independent factor of 5-years survival in this patients’ subset. Inflammatory blood biomarkers and current clinical assessment of the disease were not in agreement in terms of estimate of relapse risk (K-Cohen from -0.03 to 0.02). In conclusion, preoperative lymphocyte ratios, in particular PLR and LMR, showed prognostic relevance in ER+ HER2- early breast cancer. Therefore, they may be used in risk stratification and therapy escalation/de-escalation in patients with this type of tumor.

Plasma proteins elevated in severe asthma despite oral steroid use and unrelated to Type-2 inflammation

RationaleAsthma phenotyping requires novel biomarker discovery.ObjectivesTo identify plasma biomarkers associated with asthma phenotypes by application of a new proteomic panel to samples from two well-characterised cohorts of severe (SA) and mild-to-moderate (MMA) asthmatics, chronic obstructive pulmonary disease (COPD) subjects and healthy controls (HC).MethodsAn antibody-based array targeting 177 proteins predominantly involved in pathways relevant to inflammation, lipid metabolism, signal transduction and extracellular matrix was applied to plasma from 525 asthmatics and HC in the U-BIOPRED cohort, and 142 subjects with asthma and COPD from the validation cohort BIOAIR. Effects of oral corticosteroids (OCS) were determined by a two-week, placebo-controlled OCS trial in BIOAIR, and confirmed by relation to objective OCS measures in U-BIOPRED.ResultsIn U-BIOPRED, 110 proteins were significantly different, mostly elevated, in SA compared to MMA and HC. Ten proteins were elevated in SA versus MMA in both U-BIOPRED and BIOAIR (alpha-1-antichymotrypsin, apolipoprotein-E, complement component 9, complement factor I, macrophage inflammatory protein-3, interleukin-6, sphingomyelin phosphodiesterase 3, RANK, TGF-β1, and glutathione S-transferase). OCS treatment decreased most proteins, yet differences between SA and MMA remained following correction for OCS use. Consensus clustering of U-BIOPRED protein data yielded six clusters associated with asthma control, quality of life, blood neutrophils, hsCRP, and BMI, but not Type-2 inflammatory biomarkers. The mast cell specific enzyme carboxypeptidase A3 was one major contributor to cluster differentiation.ConclusionsThe plasma proteomic panel revealed previously unexplored yet potentially useful Type-2-independent biomarkers, and validated several proteins with established involvement in the pathophysiology of severe asthma.

TAMI-38. TUMOR-ASSOCIATED NEUTROPHILS IN GLIOBLASTOMA PROMOTE THE PERIVASCULAR GLIOMA STEM-LIKE CELL NICHE VIA OSTEOPONTIN SECRETION

Among clinical analyses, elevated neutrophil-lymphocyte ratio has been correlated with poor outcomes of glioblastoma patients independent of other prognostic factors. Additionally, our flow cytometric studies of primary patient samples found neutrophil percentage to be significantly higher in higher-grade glioma versus lower-grade glioma. Tumor-associated neutrophils (TANs) comprise less than 2% of the glioblastoma microenvironment. While TANs were initially considered passive bystanders due to their short-lived nature, investigation of TANs in other cancers revealed distinct pro-tumoral roles. Therefore, we transcriptomically characterized glioblastoma TANs and defined their oncologic effects. Transcriptomic analysis of patient-matched TANs versus peripheral blood neutrophils revealed that functionally quiescent circulating neutrophils infiltrate IDH1-wild type glioblastoma via leukotriene B4 chemoattraction, where tumor cells morphologically and transcriptomically activate them to become TANs. Single-cell RNA-sequencing of patient-matched TANs and peripheral blood neutrophils revealed a subset of tumor-activated neutrophils which adopt a pro-tumoral secretory phenotype, marked by activation of the IL-17 signaling pathway and high osteopontin production. Using immunofluorescence stains of primary patient glioblastoma sections, we demonstrated that activated, myeloperoxidase-positive TANs reside in the perivascular niche of glioblastoma in close proximity to glioblastoma stem-like cells (GSCs) and CD31-positive endothelial cells. Further analysis in culture demonstrated that TAN-secreted osteopontin drives the formation, self-renewal, and proliferation of GSC-containing neurospheres. These results were validated using a syngeneic stem cell-derived IDH1-wild type murine glioblastoma model in vivo. Thus, while TANs are rare in glioblastoma, their enrichment in the glioblastoma perivascular niche uniquely positions them to support the GSCs that are crucial to therapeutic resistance of GBM.