Inflammatory Cell Composition Research Articles

Distinct Variations in Gene Expression and Cell Composition across Lichen Planus Subtypes.

Lichen planus (LP) is a highly prevalent inflammatory skin disease. While various clinical subtypes have been defined, detailed comparisons of these variants are lacking. This study aimed to elucidate differences in gene expression and cellular composition across LP subtypes. Lesional skin biopsies from 28 LP patients (classical, oral, genital, and lichen planopilaris) and seven non-diseased skin controls (NDC) were analyzed. Gene expression profiling of 730 inflammation-related genes was conducted using NanoString. Immune cell compositions were assessed by multiplex immunohistochemistry. Gene expression profiles revealed unique inflammatory signatures for each LP subtype. Lichen planopilaris exhibited the most divergence, with downregulated gene expression and upregulation of complement pathway genes (C5-7), along with elevated M2 macrophages. Oral and genital LP demonstrated similar profiles with strong upregulation of TNF-related and Toll-like receptor-associated genes. Oral LP showed the highest upregulation of cytotoxicity-associated genes, as well as high numbers of CD8+ IL-17A+ (Tc17) cells (8.02%). Interferon gene signatures were strongly upregulated in oral and classical LP. The study highlights distinct differences in inflammatory gene expression and cell composition across LP subtypes, emphasizing the need for tailored therapeutic approaches.

Self-report underestimates the frequency of the acute respiratory exacerbations of COPD but is associated with BAL neutrophilia and lymphocytosis: an observational study

RationaleResearch studies typically quantify acute respiratory exacerbation episodes (AECOPD) among people with chronic obstructive pulmonary disease (COPD) based on self-report elicited by survey questionnaire. However, AECOPD quantification by self-report could be inaccurate, potentially rendering it an imprecise tool for identification of those with exacerbation tendency.ObjectiveDetermine the agreement between self-reported and health records-documented quantification of AECOPD and their association with airway inflammation.MethodsWe administered a questionnaire to elicit the incidence and severity of respiratory exacerbations in the three years preceding the survey among current or former heavy smokers with or without diagnosis of COPD. We then examined electronic health records (EHR) of those with COPD and those without (tobacco-exposed persons with preserved spirometry or TEPS) to determine whether the documentation of the three-year incidence of moderate to very severe respiratory exacerbations was consistent with self-report using Kappa Interrater statistic. A subgroup of participants also underwent bronchoalveolar lavage (BAL) to quantify their airway inflammatory cells. We further used multivariable regressions analysis to estimate the association between respiratory exacerbations and BAL inflammatory cell composition with adjustment for covariates including age, sex, height, weight, smoking status (current versus former) and burden (pack-years).ResultsOverall, a total of 511 participants completed the questionnaire, from whom 487 had EHR available for review. Among the 222 participants with COPD (70 ± 7 years-old; 96% male; 70 ± 38 pack-years smoking; 42% current smoking), 57 (26%) reported having any moderate to very severe AECOPD (m/s-AECOPD) while 66 (30%) had EHR documentation of m/s-AECOPD. However, 42% of those with EHR-identified m/s-AECOPD had none by self-report, and 33% of those who reported m/s-AECOPD had none by EHR, suggesting only moderate agreement (Cohen’s Kappa = 0.47 ± 0.07; P < 0.001). Nevertheless, self-reported and EHR-identified m/s-AECOPD events were both associated with higher BAL neutrophils (ß ± SEM: 3.0 ± 1.1 and 1.3 ± 0.5 per 10% neutrophil increase; P ≤ 0.018) and lymphocytes (0.9 ± 0.4 and 0.7 ± 0.3 per 10% lymphocyte increase; P ≤ 0.041). Exacerbation by either measure combined was associated with a larger estimated effect (3.7 ± 1.2 and 1.0 ± 0.5 per 10% increase in neutrophils and lymphocytes, respectively) but was not statistically significantly different compared to the self-report only approach. Among the 184 TEPS participants, there were fewer moderate to very severe respiratory exacerbations by self-report (n = 15 or 8%) or EHR-documentation (n = 9 or 5%), but a similar level of agreement as those with COPD was observed (Cohen’s Kappa = 0.38 ± 0.07; P < 0.001).DiscussionWhile there is modest agreement between self-reported and EHR-identified m/s-AECOPD, events are missed by relying on either method alone. However, m/s-AECOPD quantified by self-report or health records is associated with BAL neutrophilia and lymphocytosis.

Interferon γ Expressing Mucosal Cells in Pediatric Chronic Inflammatory Bowel Disease.

The pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC) is multifactorial and includes aberrations in the composition of gastrointestinal mucosal inflammatory cells. Accurate identification of CD and UC is important as treatment and prognosis differs; however, CD and UC may be difficult to differentiate. Interferon γ (IFNγ) expression appears to be increased in ileal mucosa from CD patients, implying that IFNγ could be a diagnostically useful marker to differentiate CD from UC. This study uses automated assessment of IFNγ immunohistochemical expression in archival GI mucosal biopsies from stomach, duodenum, terminal ileum, and colon in a pediatric population to address this possibility. IFNγ positive mucosal cells are increased in the colon in both CD and UC compared to normal colon and in the ileum of CD compared to normal and UC. The abundance of IFNγ positive cells is not correlated with the presence of active inflammation, indicating that active inflammation is not responsible for the variance in abundance of IFNγ positive cells between cohorts and sites. Overlap between CD, UC, and normal suggests that IFNγ immunohistochemistry may only be clinically useful in select situations such as undetermined inflammatory bowel disease and additional study in these areas is warranted.

Orally administered yeast-derived β-glucan alleviates mast cell-dependent airway hyperresponsiveness and inflammation in a murine model of asthma.

Particulate β-glucans (WGP) are natural compounds with regulatory roles in various biological processes, including tumorigenesis and inflammatory diseases such as allergic asthma. However, their impact on mast cells (MCs), contributors to airway hyperresponsiveness (AHR) and inflammation in asthma mice, remains unknown. C57BL/6 mice underwent repeated OVA sensitization without alum, followed by Ovalbumin (OVA) challenge. Mice received daily oral administration of WGP (OAW) at doses of 50 or 150 mg/kg before sensitization and challenge. We assessed airway function, lung histopathology, and pulmonary inflammatory cell composition in the airways, as well as proinflammatory cytokines and chemokines in the bronchoalveolar lavage fluid (BALF). The 150 mg/kg OAW treatment mitigated OVA-induced AHR and airway inflammation, evidenced by reduced airway reactivity to aerosolized methacholine (Mch), diminished inflammatory cell infiltration, and goblet cell hyperplasia in lung tissues. Additionally, OAW hindered the recruitment of inflammatory cells, including MCs and eosinophils, in lung tissues and BALF. OAW treatment attenuated proinflammatory tumor necrosis factor (TNF)-α and IL-6 levels in BALF. Notably, OAW significantly downregulated the expression of chemokines CCL3, CCL5, CCL20, CCL22, CXCL9, and CXCL10 in BALF. These results highlight OAW's robust anti-inflammatory properties, suggesting potential benefits in treating MC-dependent AHR and allergic inflammation by influencing inflammatory cell infiltration and regulating proinflammatory cytokines and chemokines in the airways.

Poster 150: Immunophenotyping of Synovial Tissue in Adolescents Undergoing ACL Reconstruction Reveals Distinct Inflammatory Responses to Injury

Objectives: Tears of the anterior cruciate ligament (ACL) are common in the pediatric athletic population. In spite of trends towards early surgical reconstruction to minimize the risk of secondary meniscus and cartilage injuries, long term disruption of knee joint homeostasis and progression to post- traumatic osteoarthritis (PTOA) occurs in 50-90% of patients - a particularly challenging problem in this young cohort. The exact pathogenesis of PTOA after ACL injury and reconstruction remains unknown. Evidence from animal models of PTOA suggests a critical role of synovial inflammatory infiltration in both the onset and progression of PTOA. However, there is a scarcity of in vivo human data documenting the synovial inflammatory response to ACL injury. One recent study has looked at immune cell composition in synovial fluid, but there are no comparable publications using synovial tissue. Our primary objective was to use flow cytometry with an advanced immune cell panel to provide a snapshot of the cellular composition of immune infiltrates in the synovium of adolescent patients at the time of ACL reconstruction. Our secondary objective was to look for correlations between demographic and injury parameters and inflammatory cell composition. Methods: Patients between the ages of 12 and 18 years who were undergoing primary ACL reconstruction were enrolled in an IRB-approved prospective study. Exclusion criteria included multiligament knee injuries, revision ACL reconstruction, history of prior knee surgery, and history of inflammatory joint disease. Demographic variables and intraoperative data were recorded. At the time of surgery, an arthroscopic synovial biopsy from the prefemoral synovium was obtained. Synovium tissue was enzymatically digested into a single cell suspension for immune profiling by multicolor flow cytometry. Data was acquired on a BD lsrFortessa flow cytometer and analyzed with FlowJo v10. Statistical analyses were performed with GraphPad Prism including parametric ANOVA (one-way or two- way), simple linear regression, Kruskal Wallis analysis of variance, Mann Whitney T-test, and parallel Principal component analyses (Eigenvalue with Monte Carlo simulation). Heat map variable scaling was calculated relative to the mean and standard deviation of all measurements. Results: Twelve patients were prospectively enrolled in the study (33% male, 67% female, n = 12). Mean age was 15.5 +/- 2.02 years. Median time between injury and surgery was 35.5 days (23.3-67.5). Meniscal tears were present in 58.3% of patients (8.3% medial only, 33.3% lateral only, 16.7% both). Cells isolated from synovium were 36.8% fibroblasts, 9.89% fibrocytes, and 36.0% hematopoietic lineage cells (Fig. 1A). Within the hematopoietic lineage, the dominant cell types were monocytes, macrophages, and T-cells (Fig. 1B). Principle component analysis (PCA) revealed three immunophenotypes. Immunotype 1 had higher mast cell content than other groups and fewer inflammatory cells such as T cells, B cells, and macrophages. Immunotype 2 had strong T/B cell infiltration and lower levels of innate immune cells. These patients also had the highest overall levels of inflammatory cells (Figure 2). Immunotype 3 had a strong innate immune response - in particular higher levels of M2 polarized macrophages - and significantly fewer T/B cells. There were no obvious effects of age, skeletal maturity, or the presence of meniscal tears on immunotype (Table 1). There was a trend towards a correlation between delay in surgery and classification within immunotype 1 (p = 0.09, Table 1). Conclusions: Our study provides the first in vivo analysis of inflammatory cell behavior in the synovium of ACL injured knees. In young patients undergoing primary ACL reconstruction, three immunotypes were discovered. Our pilot data suggest a possible relationship between delay in surgery and a high prevalence of synovial mast cells. This would be of particular interest since mast cells are known to play a role in the development of OA and possibly PTOA. In the more acute setting, two separate immunotypes were encountered, one with heavy inflammation and a predominance of T/B cells, and the other with more moderate inflammation and a higher percentage of innate immune cells / M2 polarized macrophages. These were not clearly tied to patient demographic factors, and could represent intrinsic differences in the response to injury. Future work investigating the correlation between initial immunotype and clinical outcomes – in particular the development of PTOA – may provide insight into the role of synovial inflammation in post-ACL joint health and degeneration, as well as provide strategies for personalized treatments in the prevention of PTOA. [Table: see text]

Differential nasal swab cytology represents a valuable tool for therapy monitoring but not prediction of therapy response in chronic rhinosinusitis with nasal polyps treated with Dupilumab.

Chronic Rhinosinusitis with nasal polyps (CRSwNP) is a common chronic disease with a high impact on patients' quality of life. If conservative and surgical guideline treatment cannot sufficiently control disease burden, biologicals can be considered as a comparably new treatment option that has revolutionized CRSwNP therapy since the first approval of Dupilumab in 2019. With the aim to select patients who benefit from this new treatment and to find a marker for therapy monitoring, we investigated the cellular composition of nasal mucous membranes and inflammatory cells of patients suffering from CRSwNP and undergoing Dupilumab therapy using non-invasive nasal swab cytology. Twenty CRSwNP patients with the indication for Dupilumab therapy have been included in this prospective clinical study. In total, five study visits were conducted with ambulatory nasal differential cytology using nasal swabs starting with the beginning of therapy and followed by visits every 3 months for 12 months. First, these cytology samples were stained with the May-Grunwald-Giemsa method (MGG) and the percentage of ciliated cells, mucinous cells, eosinophil cells, neutrophil cells, and lymphocytes was analyzed. Secondly, an immunocytochemical (ICC) ECP-staining was performed to detect eosinophil granulocytes. Additionally, during each study visit the nasal polyp score, SNOT20 questionnaire, olfactometry, the total IgE concentration in peripheral blood as well as the eosinophil cell count in peripheral blood were recorded. The change of parameters was evaluated over one year and the correlation between clinical effectiveness and nasal differential cytology was analyzed. In both MGG (p<0.0001) and ICC analysis (p<0.001) a significant decrease of eosinophils was seen under Dupilumab treatment. When patients were divided into a Eo-low- (<21%) and Eo-high- (≥21%) group according to the percentage eosinophils in nasal swab catology in the first study visit, the Eo-high-group showed a greater change of eosinophils over time (Δ17.82) compared to the Eo-low-group (Δ10.67) but, however, no better response to therapy. The polyp score, SNOT20 questionnaire, and total IgE concentration in peripheral blood showed a significant decrease during the observation period (p<0.0001). Nasal swab cytology as an easy-to-apply diagnostic method allows detection and quantification of the different cell populations within the nasal mucosa at a given time. The nasal differential cytology showed a significant decrease of eosinophils during Dupilumab therapy and can therefore be used as non-invasvive method for monitoring therapy success of this cost intensive therapy and potentially can allow an optimized individual therapy planning and management for CRSwNP patients. Since the validity of initial nasal swab eosinophil cell count as a predictive biomarker for therapy response was limited in our study, additional studies including larger number of participants will be necessary to further evaluate the potential benefits for clinical practice of this new diagnostic method.

Inflammatory fibroid polyp: A series of 29 cases and a systematic review of the literature

An inflammatory fibroid polyp (IFP) of the gastrointestinal tract is a localized, benign mesenchymal lesion consisting of spindle-shaped stromal cells, eosinophilic granulocytes, and some lymphocytes and plasma cells. The discovery of a frequent mutation of the platelet-derived growth factor receptor A (PDGFRA) gene was the first hint of a gene-regulating process in IFPs. The aim of this study was to investigate the interaction of inflammatory processes and the role of mutation and expression of the PDGFRA gene in the development of IFPs for the first time. We used immunohistochemistry to analyze the composition of inflammatory cells and next generation sequencing (NGS) to provide a broad overview of gene mutations.We report on 29 cases of IFP. The mean age, gender differences, and localization were compatible with the literature. Spindle cell histomorphology was present in 79% of cases showing a typical onion skin-like perivascular arrangement and significantly high CD34 positivity (p = 0.002, Fisher’s exact test). Eosinophilic granulocytes were present in an average density of 60 ± 49/high power field (HPF) (range: 15–200), and there was a significantly higher rate of IFPs larger than 2 cm in size (p = 0.018, Wilcoxon test). All but one cases could be analyzed by NGS. Mutations were observed in 17 cases (60.7%), including 13 (46.4%) mutations in the PDGFRA gene. Among the gastric lesions, mutations were found in exon 18 of the PDGFRA gene with amino acid exchange (Asp842Val) for eight out of 10 cases and in exon 12 in two cases. All three cases in the small intestine revealed mutation of the PDGFRA gene in exon 12. We found no PDGFRA mutation in our colonic cases. PDGFRA expression was significantly correlated with mutations of the same gene (p = 0.005, Fisher’s exact test) and especially with mutations in exon 12 of the same gene (p < 0.001, Fisher’s exact test). Interestingly, three of our cases (10.3%) without mutation or expression of the PDGFRA gene revealed an unusually high concentration of IgG-positive plasma cells (average: 140 ± 26/HPF, range: 110–160) and IgG4-positive plasma cells (average: 87 ± 21/HPF, range: 60–100). For comparison, an IgG4/IgG ratio of more than 0.4 is commonly observed in IgG4-related diseases. Our molecular results were in accordance with 113 genetically analyzed cases published to date. There was a correlation between the IFP site and mutation variants of the PDGFRA gene. IFPs were localized in the stomach in 49.1% of cases, in the small intestine in 47.3%, and in the colon in 3.6%. Exon 12 of the PDGFRA gene was mutated in 41.1% of cases and primarily occurred in the small intestine (82.6%). Exon 18 was mutated in 22.3% of cases and primarily occurred in the stomach (80.0%). The mutated codon interval 566–571 in exon 12 and codon 842 in exon 18 were compatible, as observed in a gastrointestinal stromal tumor. Conclusively, the correlation between mutation and expression of the PDGFRA gene points to different pathways in IFPs. Additionally, our data hint at a morphological but not genetic overlap between IFPs and IgG4-related pseudotumors.

SENESCENCE AND INFLAMMATION

Abstract Cell senescence and inflammation are interconnected mediators of aging and age-related disease. Recent advances in molecular and cellular profiling methods and research models are aiding in our ability to decipher mechanisms through which senescent cells drive inflammatory dysfunction, and inversely, to discover mechanisms through which aging immune cells may drive senescence, inflammation, and pathology. This symposium will feature exciting advances that span the emerging conceptual framework of how senescence and inflammation influence mammalian aging. Dr. Birgit Schilling will discuss the use of advanced mass spectrometric methods for profiling the senescent cell proteome, which reveal new insights into protein pathways and mechanisms of aging and disease. Dr. Matt Yousefzadeh will share how endogenous DNA damage can invoke cellular senescence, which enhances inflammation in both a cell autonomous and non-autonomous manner to drive tissue dysfunction and impact health. Dr. Daniel Tyrrell will discuss discovery of a novel population of age-associated CD8 T-cells that enhance local tissue senescence and inflammation and promote atherosclerosis. Dr. Xu Zhang will share a characterization of senescent cells in skeletal muscle using single-cell RNA-sequencing and the potential recruitment of immune cells by senescent fibroadipogenic progenitors. Dr. Marissa Schafer will discuss cell senescence as a mediator of age-related brain inflammatory cell composition and senescent cell targeting as a strategy to prevent cognitive decline. Importantly, discoveries discussed in this symposium may reveal new avenues for therapeutic development, to ultimately improve human healthspan.

Idiopathic granulomatous mastitis and cystic neutrophilic granulomatous mastitis: two sides of the same coin or distinct entities?

Idiopathic granulomatous mastitis (IGM) is a benign mimic of breast carcinomas. It is defined histologically by the presence of granulomas and inflammation. The closely related cystic neutrophilic granulomatous mastitis (CNGM) shows lipogranulomas, with a reported association with corynebacteria. A large cohort of IGM was reviewed to compare clinical, microbiological and histological features between non-CNGM IGM and CNGM. Cases of IGM were reviewed for histological parameters including the presence of lipogranulomas and composition of inflammatory cells. Clinical data were obtained through hospital records. The cohort included 79 cases, including 51 non-CNGM IGM and 28 CNGM. Comparing non-CNGM IGM and CNGM, there were no differences in clinical or demographical data, other than a younger age of presentation (36.2 vs 41.5 years, p=0.012) for CNGM. Most IGM resolved within the follow-up period (n=57/64, 89.1%), with similar outcomes regardless of treatment (p>0.05). In CNGM, there were more infiltrates of neutrophils (p=0.001), histiocytes (p=0.047), and multinucleated giant cells (p=0.006), but less lymphocytes (p=0.008). Corynebacteria was cultured in two (25%) cases of CNGM, and one non-CNGM IGM (14.3%). Gram-positive bacilli were identified in two cases of CNGM. 'Early' lipogranulomas were observed closely associated to inflamed ducts in three cases of CNGM. Apart from age, there was no distinct clinical or microbiological feature for CNGM. These findings do not support CNGM as a distinct entity. Rather, CNGM-pattern may represent a continuum of IGM, possibly age-related and attributable to ductal inflammation and cystic changes in the breast parenchyma.

The role of P2Y12 in cardiovascular disease beyond atherothrombosis: P2Y12 signaling promotes emergency hematopoiesis after myocardial infarction

Abstract Background Adenosine diphosphate (ADP) plays a pivotal role in platelet activation. The purinergic ADP-receptor P2Y12 has therefore been targeted in the treatment of cardiovascular disease (CVD) to prevent atherothrombosis (1). Beyond P2Y12 expression on platelets, purinergic receptors have also been described on hematopoietic stem and progenitor cells (LSK) (2). After myocardial infarction (MI), accelerated LSK proliferation launches emergency hematopoiesis as the driving force behind the inflammatory response to MI, increasing inflammatory cell production in the bone marrow (BM) and providing leukocyte resupply for local cell recruitment to the infarct (3). The inflammatory cascade after MI covers intricate multilayered interactions between the injured myocardium and the hematopoietic BM that still remain to be fully elucidated and may unearth novel therapeutic strategies. Whereas P2X receptors have recently been found to be involved in cell trafficking (4), the role of P2Y receptors in the hematopoietic BM have not yet been characterized. Purpose This study aims to characterize the influence of P2Y12 signaling on emergency hematopoiesis and cardiac remodeling after MI. Methods Permanent coronary ligation was performed for MI to assess BM activation, inflammatory cell composition, cardiac remodeling and function in murine global and platelet-specific P2Y12 knockout models and under pharmacological P2Y12 inhibition with prasugrel using flow cytometry, qPCR, immunohistochemistry and echocardiography. In vitro studies including colony forming unit (CFU) assays and flow cytometry allowed for investigation of ADP-dependent effects on LSK cells and intracellular pathway analysis. Results We identified ADP as a danger signal for the hematopoietic BM, fueling emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. Detection of P2Y12 expression in LSK implicated a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 deficiency and P2Y12 inhibition with prasugrel decelerated emergency hematopoiesis and consecutively reduced the excessive inflammatory response to MI, translating to lower numbers of hematopoietic progenitors and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition ameliorated chronic adverse cardiac remodeling and preserved cardiac function after MI. Conclusion ADP-dependent P2Y12-mediated activation of hematopoietic stem and progenitor cells in the BM promotes emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel role of P2Y12 antagonists in CVD beyond atherothrombosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)

Spatiotemporal dynamics of macrophage heterogeneity and a potential function of Trem2hi macrophages in infarcted hearts

Heart failure (HF) is a frequent consequence of myocardial infarction (MI). Identification of the precise, time-dependent composition of inflammatory cells may provide clues for the establishment of new biomarkers and therapeutic approaches targeting post-MI HF. Here, we investigate the spatiotemporal dynamics of MI-associated immune cells in a mouse model of MI using spatial transcriptomics and single-cell RNA-sequencing (scRNA-seq). We identify twelve major immune cell populations; their proportions dynamically change after MI. Macrophages are the most abundant population at all-time points (>60%), except for day 1 post-MI. Trajectory inference analysis shows upregulation of Trem2 expression in macrophages during the late phase post-MI. In vivo injection of soluble Trem2 leads to significant functional and structural improvements in infarcted hearts. Our data contribute to a better understanding of MI-driven immune responses and further investigation to determine the regulatory factors of the Trem2 signaling pathway will aid the development of novel therapeutic strategies for post-MI HF.

EMCN knockout leads to increased monocyte infiltration in kidney and albuminuria in mice

Endomucin (EMCN), a type I integral membrane glycoprotein, is specifically expressed in endothelium and enriched in kidney glomerular endothelium. We have previously reported that under resting conditions EMCN functions as an anti‐inflammatory molecule, preventing leukocyte‐endothelial cell adhesion and that EMCN plays an important role in modulating VEGFR2 signaling. The goal of this study is to characterize the phenotype of the kidney in the first‐reported EMCN knockout mouse model. EMCN−/−mice (with a EGFP tag for the knockout allele) were obtained by crossing EMCN‐floxed mice, generated in conjunction with Cyagen Biosciences, with the ROSA26‐Cre mice. The mRNA level of EMCN, CD31, CD34, E‐selection, VCAM‐1, and ICAM‐1 were quantified by reverse transcription PCR and semi‐quantitative PCR using total RNA isolated from the adult mouse kidneys. The mRNA level of EMCN in EMCN−/−mice were not detectable (n&gt;4, p&lt;0.0001) compared to EMCN+/+. Renal CD34 at mRNA level was decreased (fold change, 0.68±0.09 vs 1, n=4, p&lt;0.05) and VCAM‐1 was increased (fold change, 2.2 ±0.45 vs 1, n=4, p&lt;0.01) in EMCN−/− kidneys compared to wildtype (wt) controls. CD31, E‐selection, and ICAM‐1 mRNA levels were unchanged between EMCN−/−and wildtype kidneys. Kidneys from adult EMCN+/+ and EMCN−/− mice were collected, fixed, and sectioned. Expression and localization of EMCN, eGFP, CD45 and podocin in adult kidneys were examined by immunohistochemistry and quantified, which revealed a significant increase in CD45+ cell infiltration in EMCN−/− mice compared to EMCN+/+ mice (72.50±8.13 cells/mm2 vs 39.67±4.91 cells/ mm2, n=4, p&lt;0.001). To characterize the renal inflammatory cell composition, freshly collected kidneys from EMCN+/+ and EMCN−/− were enzymatically digested and the leukocyte markers CD45, CD3, CD4, CD19, CD11b, Ly6G and Ly6C were examined by flow cytometry to identify the proportion of myeloid populations. Flow cytometry analysis suggested a trend of increased CD45+ cells in the kidney from EMCN−/−mice compared to wildtype (n=9, p=0.07), and revealed a significant increase in the percentage of Ly6GhighLy6Chighmyeloid cells in EMCN−/−compared to wildtype kidneys (1.6±0.23 vs 1.01±0.18, n=9, p&lt;0.05). Sporadic albuminuria was detected through Coomassie staining in spot urine samples of EMCN−/− mice. Ultrastructural morphology of glomeruli, examined from both EMCN+/+ and EMCN−/− through transmission electron microscopy, showed the effacement of podocyte foot processes and disorganized endothelial cell fenestrations in glomerular capillaries of EMCN−/− mice compared to wt. The result indicates EMCN plays a key role in the prevention of inflammatory cells infiltration in kidney, presumably by blocking leukocyte adhesion, and is also involves in maintaining normal glomerular filtration barrier structure and function, potentially via its role in VEGFR2 signaling.

P2Y12-dependent activation of hematopoietic stem and progenitor cells promotes emergency hematopoiesis after myocardial infarction

Emergency hematopoiesis is the driving force of the inflammatory response to myocardial infarction (MI). Increased proliferation of hematopoietic stem and progenitor cells (LSK) after MI enhances cell production in the bone marrow (BM) and replenishes leukocyte supply for local cell recruitment to the infarct. Decoding the regulation of the inflammatory cascade after MI may provide new avenues to improve post-MI remodeling. In this study, we describe the influence of adenosine diphosphate (ADP)-dependent P2Y12-mediated signaling on emergency hematopoiesis and cardiac remodeling after MI. Permanent coronary ligation was performed to induce MI in a murine model. BM activation, inflammatory cell composition and cardiac function were assessed using global and platelet-specific gene knockout and pharmacological inhibition models for P2Y12. Complementary in vitro studies allowed for investigation of ADP-dependent effects on LSK cells. We found that ADP acts as a danger signal for the hematopoietic BM and fosters emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. We were able to detect P2Y12 in LSK, implicating a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 knockout and P2Y12 inhibitor treatment with prasugrel reduced emergency hematopoiesis and the excessive inflammatory response to MI, translating to lower numbers of downstream progeny and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition preserved cardiac function and reduced chronic adverse cardiac remodeling after MI. P2Y12-dependent signaling is involved in emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel, non-canonical value for P2Y12 antagonists beyond inhibition of platelet-mediated atherothrombosis.

Inflammatory Cell Composition and Immune-Related microRNA Signature of Temporal Artery Biopsies From Patients With Giant Cell Arteritis.

ObjectivesThe aim of this study was to quantitatively assess distinct immune cell subsets comprising inflammatory infiltrate in temporal artery biopsies (TABs) from patients with giant cell arteritis (GCA), and to link the obtained histopathological data with expression profiles of immune-related microRNAs (miRNAs).MethodsThe study included 68 formalin-fixed, paraffin-embedded TABs from treatment-naïve patients, including 30 histologically positive GCA and 16 negative GCA TABs, and 22 control non-GCA TABs. Quantitative assessment of histological parameters was performed using histopathological and immunohistochemical techniques. miRNA expression analysis was performed by quantitative real-time PCR.ResultsIntense transmural mononuclear inflammatory infiltrates in TAB-positive GCA arteries were predominantly composed of CD3+, CD4+ and CD8+ T lymphocytes, and CD68+ macrophages, accompanied by a strong nuclear overexpression of the nuclear factor of activated T cells, cytoplasmic 1 (NFATC) in the lymphocyte infiltrate fraction. Furthermore, TAB-positive GCA arteries were characterized by significant overexpression of nine pro-inflammatory miRNAs (miR-132-3p/-142-3p/-142-5p/-155-5p/-210-3p/-212-3p/-326/-342-5p/-511-5p) and a significant under-expression of six regulatory immune-related miRNAs (miR-30a-5p/-30b-5p/-30c-5p/-30d-5p/-30e-5p/-124-3p), whose expression levels significantly associated with most evaluated histopathological parameters. Notably, we revealed miR-132-3p/-142-3p/-142-5p/-155-5p/-212-3p/-511-5p as major promoters of arterial inflammation and miR-30a-5p/-30c-5p/-30d-5p as putative regulators of NFATC signaling in TAB-positive GCA arteries.ConclusionOverall, we demonstrated that an altered arterial tissue-specific pro-inflammatory miRNA signature favors enhanced T cell-driven inflammation and macrophage activity in TAB-positive GCA arteries. Moreover, dysregulation of several immune-related miRNAs seems to contribute crucially to GCA pathogenesis, through impairing their regulatory activity towards T cell-mediated immune responses driven by the calcineurin (CaN)/NFAT signaling pathway, indicating their therapeutic, diagnostic and prognostic potential.

Cell Senescence as a Mediator of Age-Dependent Brain Inflammation

Cellular senescence and inflammation are interconnected causes and consequences of tissue aging. Here, we implemented orthogonal approaches to study their interaction in steady-state mature and aged mouse brain. Using single cell sequencing, we identified a putative senescent microglial population, which increased in abundance with age and was characterized by increased expression of p16 and chemotactic senescence associated secretory phenotype (SASP) factors. Using p16-INK-ATTAC transgenic mice to eliminate p16ink4a-positive senescent cells and mass cytometry, we show that p16ink4a-positive cell targeting reduced the abundance of activated inflammatory cells in the aged female brain. Age-dependent declines in executive cognitive function were improved following transgenic p16ink4a-positive cell targeting, and executive function robustly correlated with inflammatory brain cell composition in females. Collectively, our findings demonstrate fundamental differences in the age- and sex-dependent brain inflammatory landscape and implicate p16ink4a-positive senescent cell targeting as a therapeutic strategy to attenuate age-related inflammation and cognitive decline.

Radiographic and High-Throughput Sequencing (HTS)-Based Response Assessment after Brentuximab Vedotin (BV) Plus Ifosfamide, Carboplatin, and Etoposide (ICE) for Relapsed/Refractory (Rel/Ref) Classical Hodgkin Lymphoma (cHL): Updated Results of a Phase I/II Trial

BACKGROUNDThe likelihood of long-term remission following autologous stem cell transplant (ASCT) for rel/ref cHL is predicted by response to pre-ASCT salvage therapy (Blood 2012;119:1665-70). Salvage regimens incorporating the anti-CD30 antibody-drug conjugate BV, administered sequentially and concurrently with chemotherapy, show promise in improving positron emission tomography (PET)-negative complete remission (CR) rates, transplant eligibility, and outcomes (Lancet Oncol 2015;16:284-92; ASH 2015, #293; ASH 2016, #1109; ASH 2016, #1834). While pre-ASCT PET is a strong predictor of outcome, novel HTS-based assays in development imply greater sensitivity than imaging and a potential role in management and surveillance of lymphomas. Here, we report updated clinical results from our ongoing phase I/II trial (#NCT02227199) in the context of HTS-based detection of HL pre- and post-therapy, as well as circulating cytokine and tumor microenvironment markers. METHODSPatients (pts) ≥ 18 years old with first relapse or primary refractory CD30+ cHL were eligible for this IRB-approved prospective clinical trial. Treatment included BV on Days 1 and 8 at either 1.2 or 1.5 mg/kg (based on 3+3 dose-escalation schema; capped at 150 mg), ifosfamide and mesna 5 g/m2 each on Day 2, carboplatin AUC 5 (capped at 800 mg) on Day 2, and etoposide 100 mg/m2 daily on Days 1-3. 2 21-day cycles were given with G-CSF support. BV 1.5 mg/kg was selected as the phase II dose based on reported dose escalation data (ASH 2016, #1834). PET was performed after Cycle 2, with response assigned per Cheson 2007. Peripheral blood (PB) pre- and post-treatment, stem cell (PBSC) product, and (when available) archived formalin-fixed paraffin-embedded tissue (FFPET) from presentation and relapse were collected for correlative studies. DNA for HTS was extracted and analyzed by Adaptive Biotechnologies similar to that described previously (Clin Cancer Res 2014;20:4540-8). Pre-treatment PB cytokine levels were measured by Luminex. Immunohistochemistry (IHC) on FFPET identified components of inflammatory microenvironment. RESULTSTo date, 24 pts have enrolled and 23 are evaluable (1 withdrew consent after 1 cycle). 15 (65%) pts are female. Median age at enrollment was 30 (range, 20-60), with 7 (30%) age ≥ 45. 9 (39%) had advanced-stage disease at initial diagnosis. 14 (61%) did not obtain CR with first-line therapy, all but 1 of which was ABVD-based (the exception received Stanford V). 6 (26%) received prior radiation.Of 23 evaluable pts, 20 (87%) achieved a PET CR by investigator review, while 16 (70%) were in PET CR by central independent review. Of the 4 with discrepant results between investigator and central review, 2 were adjudicated as CR by a 3rd independent reviewer, and 2 were sent for biopsy of residual lesion: 1 biopsy was negative, and 1 noted resolution by imaging guidance so biopsy was aborted. 19 of 22 pts (86%) with adequate follow-up have undergone ASCT. 1 pt in CR after BV-ICE has since relapsed (5%), and no deaths have occurred. The toxicity profile remains similar to that reported previously: 11 (48%) pts experienced Grade 3-4 non-heme toxicity, with sepsis/neutropenic fever (4), hyperglycemia (2), and elevated ALT (2) being the most common; 7 pts (30%) developed neuropathy: 4 Grade 1, 2 Grade 2, and 1 Grade 3; only 1 pt (4%) discontinued study therapy due to toxicity (Grade 3 neuropathy).21 patients had samples tested for disease-specific clonal sequence by HTS. A clone was detectable in PB pre-treatment in only 1 (5%). Post-treatment, none of the PB or PBSC had a detectable clone. Proportions of CD68+ macrophages, granzyme+ cytotoxic cells, and CD20+ B cells by IHC of FFPET from either initial diagnosis or at relapse/enrollment were similar between those who achieved CR and those who did not, as were circulating levels of MIG, HGF, IL2Ra, IL6, and PDL1. CONCLUSIONSBV at a dose of 1.5 mg/kg on Days 1 and 8 per cycle of ICE results in high CR rates, is generally well tolerated, and warrants further investigation in pts with rel/ref cHL. Contrary to previous data (Br J Haematol, 2015;169:689-93), HTS was not able to reliably identify disease-specific clone in PB and thus may not be a useful tool in cHL at present. Inflammatory cell composition and selected serum cytokine levels were not predictive of response to BV-ICE. Enrollment is ongoing, and updated clinical and correlative data will be presented at the meeting. DisclosuresSmith:Dohme Corp: Research Funding; Acerta: Research Funding; Sharp: Research Funding; Janssen: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Merck: Research Funding; Genentech: Research Funding; Portola Pharmaceuticals: Research Funding; Seattle Genetics: Research Funding. Gopal:Seattle Genetics: Consultancy, Research Funding.

Blood and cell infiltrate neutrophilic leucocytes As inflammation markers in chronic endometritis: A prospective non-randomised controlled trial

Background. Inflammation declares itself with the presence of cellular tissue infiltrate, which composition reflects the inflammation type. Chronic inflammation is predominated by mononuclear cell infiltration with a certain amount of neutrophils, which role and significance are not fully understood to date.Objectives. Assessment of the infiltrated neutrophil count at various chronic endometritis severity and its dependency on the functional and metabolic activity in neutrophilic leucocytes in peripheral blood.Methods. This prospective non-randomised controlled trial estimated the CD45+ leucocyte and activated CD16b+ neutrophil counts in inflammation infiltrate using immunohistochemistry protocols. Cell counts per section 1 mm2 were measured with computer morphometry. The content of and NADPH oxidase activity in activated neutrophilic leucocytes in venous blood were estimated with a nitroblue tetrazolium reduction test.Results. The study included 40 women with a history of chronic endometritis (CE) divided in two cohorts by endometrial biopsy data, with inactive (n = 25) and active CE (n = 15). A control cohort comprised 20 women with no signs of CE. The inactive CE cohort had higher counts of CD45+ leucocytes and activated CD16b+ neutrophils in infiltrate compared to control. Higher content of activated neutrophilic leucocytes with higher NADPH oxidase activity were found in peripheral blood. Morphological exacerbation markers of EC were associated with sharper peaks of CD45+ and CD16b+ cell counts in infiltrate and an elevated functional metabolic activity in circulating neutrophilic leucocytes. A strong direct correlation was revealed between blood activated neutrophil and endometrial CD16b+ neutrophil counts, as well as NADPH oxidase activity in blood neutrophils and infiltrate CD16b+ cell counts.Conclusion. Even minor morphological markers of exacerbated endometrial inflammation are accompanied by the elevated infiltrate counts of both total CD45+ leucocytes and activated CD16b+ neutrophils. The functional metabolic activity of peripheral blood neutrophilic leucocytes is interlinked with the inflammatory infiltrate cell composition and reflects severity of chronic endometrial inflammation.

Tick-Borne Encephalitis: A Differential Pattern of Intrathecal Humoral Immune Response and Inflammatory Cell Composition Compared with Other Viral CNS Infections.

To investigate whether and how cerebrospinal fluid (CSF) findings can contribute to distinguish tick-borne encephalitis (TBE) from herpes simplex virus (HSV) and varicella zoster virus (VZV) induced central nervous system (CNS) infections (HSV-I, VZV-I). Chart review and identification of TBE, HSV- I, and VZV-I was carried out, fulfilling the following criteria: (1) clinical signs of encephalitis and/or meningitis, (2) complete CSF analysis and confirmed viral etiology by either PCR or antibody testing in CSF, (3) hospitalized patients, and (4) available brain magnetic resonance imaging (MRI). Fifty-nine patients with 118 CSF/serum pairs were included. These comprised 21 with TBE (35 CSF/serum pairs), 20 (40 CSF/serum pairs) with HSV-I, and 18 (43 CSF/serum pairs) with VZV-I. In contrast to HSV-I and VZV-I, CSF cell differentiation in TBE showed more often an increased (>20%) proportion of granulocytes (p < 0.01) and a more frequent quantitative intrathecal IgM synthesis (p = 0.001 and p < 0.01, respectively), while the second was even more pronounced when follow-up CSF analyses were included (p < 0.001). CSF findings help to distinguish TBE from other viral infections. In cases with CSF pleocytosis and a positive history for a stay in or near an endemic area, TBE antibodies in CSF and serum should be determined, especially if granulocytes in CSF cell differentiation and/or an intrathecal IgM synthesis is present.

Pathological features of COVID-19-associated myocardial injury: a multicentre cardiovascular pathology study.

AimsCoronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been associated with cardiovascular features of myocardial involvement including elevated serum troponin levels and acute heart failure with reduced ejection fraction. The cardiac pathological changes in these patients with COVID-19 have yet to be well described.Methods and resultsIn an international multicentre study, cardiac tissue from the autopsies of 21 consecutive COVID-19 patients was assessed by cardiovascular pathologists. The presence of myocarditis, as defined by the presence of multiple foci of inflammation with associated myocyte injury, was determined, and the inflammatory cell composition analysed by immunohistochemistry. Other forms of acute myocyte injury and inflammation were also described, as well as coronary artery, endocardium, and pericardium involvement. Lymphocytic myocarditis was present in 3 (14%) of the cases. In two of these cases, the T lymphocytes were CD4 predominant and in one case the T lymphocytes were CD8 predominant. Increased interstitial macrophage infiltration was present in 18 (86%) of the cases. A mild pericarditis was present in four cases. Acute myocyte injury in the right ventricle, most probably due to strain/overload, was present in four cases. There was a non-significant trend toward higher serum troponin levels in the patients with myocarditis compared with those without myocarditis. Disrupted coronary artery plaques, coronary artery aneurysms, and large pulmonary emboli were not identified.ConclusionsIn SARS-CoV-2 there are increased interstitial macrophages in a majority of the cases and multifocal lymphocytic myocarditis in a small fraction of the cases. Other forms of myocardial injury are also present in these patients. The macrophage infiltration may reflect underlying diseases rather than COVID-19.

Lung transcriptomic clock predicts premature aging in cigarette smoke-exposed mice

BackgroundLung aging is characterized by a number of structural alterations including fibrosis, chronic inflammation and the alteration of inflammatory cell composition. Chronic exposure to cigarette smoke (CS) is known to induce similar alterations and may contribute to premature lung aging. Additionally, aging and CS exposure are associated with transcriptional alterations in the lung. The current work aims to explore the interaction between age- and CS- associated transcriptomic perturbations and develop a transcriptomic clock able to predict the biological age and the impact of external factors on lung aging.ResultsOur investigations revealed a substantial overlap between transcriptomic response to CS exposure and age-related transcriptomic alterations in the murine lung. Of particular interest is the strong upregulation of immunoglobulin genes with increased age and in response to CS exposure, indicating an important implication of B-cells in lung inflammation associated with aging and smoking. Furthermore, we used a machine learning approach based on Lasso regression to build a transcriptomic age model that can accurately predict chronological age in untreated mice and the deviations associated with certain exposures. Interestingly, CS-exposed-mice were predicted to be prematurely aged in contrast to mice exposed to fresh air or to heated tobacco products (HTPs). The accelerated aging rate associated with CS was reversed upon smoking cessation or switching to HTPs. Additionally, our model was able to predict premature aging associated with thoracic irradiation from an independent public dataset.ConclusionsAging and CS exposure share common transcriptional alteration patterns in the murine lung. The massive upregulation of B-cell restricted genes during these processes shed light on the contribution of cell composition and particularly immune cells to the measured transcriptomic signal. Through machine learning approach, we show that gene expression changes can be used to accurately monitor the biological age and the modulations associated with certain exposures. Our findings also suggest that the premature lung aging is reversible upon the reduction of harmful exposures.